Entire Book

Forensic Medicine - From Old Problems to New Challenges
Edited by Prof. Duarte Nuno Vieira
ISBN 978-953-307-262-3
Hard cover, 382 pages
Publisher InTech
Published online 12, September, 2011
Published in print edition September, 2011
Forensic medicine is a continuously evolving science that is constantly being updated and improved, not only
as a result of technological and scientific advances (which bring almost immediate repercussions) but also
because of developments in the social and legal spheres. This book contains innovative perspectives and
approaches to classic topics and problems in forensic medicine, offering reflections about the potential and
limits of emerging areas in forensic expert research; it transmits the experience of some countries in the
domain of cutting-edge expert intervention, and shows how research in other fields of knowledge may have
very relevant implications for this practice.
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Ljiljana Vasović, Milena Trandafilović, Ivan Jovanović, Slađana Ugrenović, Slobodan Vlajković and Jovan
Stojanović (2011). Types and Subtypes of the Posterior Part of the Cerebral arterial Circle in Human Adult
Cadavers, Forensic Medicine - From Old Problems to New Challenges, Prof. Duarte Nuno Vieira (Ed.), ISBN:
978-953-307-262-3, InTech, Available from: http://www.intechopen.com/books/forensic-medicine-from-old-
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1
Avoiding Errors and Pitfalls in Evidence

Sampling for Forensic Genetics
B. Ludes and C. Keyser
Laboratoire d’anthropologie moléculaire, Institut de médicine légale,
Université de Strasbourg
France
1. Introduction
DNA fingerprinting or DNA profiling (as it is now known) was first developed by Alec
Jeffreys in 1985 (Jeffreys et al., 1985), who found that in the human genome, some regions
contained DNA sequences that were repeated over and over again, next to each other. He
also discovered that the number of repeated unit could differ from individual to individual
allowing human identity testing. Since that time, DNA typing methods has been commonly
used in criminal cases (to identify a suspect or a victim or to absolve an innocent individual)
as well as in the identification of missing persons or in paternity testing. Today, the most
commonly used DNA repeat regions used are microsatellites also known as Short Tandem
Repeats (STR). These loci in which the repeat unit is at least two bases but no more than
seven in length, are amplified by PCR (Polymerase Chain Reaction) in a multiplex fashion
(multiple primers) reducing sample consumption. Today, for the majority of forensic cases
where DNA of preserved quality is available, the identification procedures of biological
samples are performed by commercially well-validated kits incorporating 15-16 highly
variable STR loci (plus amelogenin) such as PowerPlexR (Promega) and AmpFlSTRR
(Applied Biosystems). With highly automated equipment, STR profiling can process
hundreds of samples each day and became the cornerstone of forensic DNA testing,
including national DNA databases with STR-profiles of convicted felons. Nevertheless, it is
of great importance to make the distinction between the samples containing large quantities
of high quality DNA and those containing minute amounts of DNA and/or poor quality
molecules. If for the first type of samples, the occurrences of errors or pitfall are rare, in the
second type, the interpretation of the allelic profiles should be done with care and caution.
In this article, the authors will focus on the analysis of challenging samples, in other words,
samples containing either (i) minute amount of DNA or (ii) degraded DNA or (iii) mixture
of DNA or (iv) DNA polymerase inhibitors or (v) contaminating DNA molecules. Indeed,
DNA is stable and remains intact when stored in a dry or frozen state but will be degraded
when stored under inappropriate or bacterially contaminated conditions. Two types of
damage are mainly likely to affect DNA over time: hydrolytic and oxidative damage.
Hydrolytic damage results in deamination of bases and in depurination and
depyrimidination, whereas oxidative damage results in modified bases (Lindahl, 1993). Both
mechanisms reduce the number as well as the size of the fragments that can be amplified by
PCR. Failure to amplify DNA may also result from the presence of inhibitors that interfere
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2 Forensic Medicine – From Old Problems to New Challenges
with the PCR such as low-molecular-weight compounds, supposedly derived from the
crime scene environment, which coextract with the target DNA molecules and potently
inhibit the activity of the DNA polymerase ( Keyser-Tracqui C. and Ludes B., 2005).
Contamination by DNA coming from outside the case represents one of the major
limitations to DNA analysis. The authors will describe the strategies developed to overcome
the difficulties which begin with the biological sample collection.
2. Biological sample collection

2.1 Samples
Various kinds of samples can be typed with the PCR-based methodologies such as:
 Blood samples and blood stains
 Cigarette buts (Hochmeister et al., 1991)
 Human hairs with a special mention of the possibility of analysis of single hair (Higuchi
et al., 1991)
 Urine samples and urine stains (Brinkmann et al., 1992)
 Fingernail scraping (Wiegand et al., 1993)
 Bite marks (Sweet et al., 1997)
 All kinds of touched objects (Van Oorschot and Jones, 1997) such as tools, clothing,
firearms, parts of vehicle, food, condoms, glass, bottles, lip cosmetics, wallets, jewellery,
paper, cables, stones and construction material (Van Hoofstat et al., 1999; Webb et al.,
2001; Wickenheiser, 2002; Rutty, 2002; Polley et al., 2006; Petricevic et al. 2006; Sewell et
al., 2008; Horsman-Hall et al., 2009)
 FTA cards can be used to collect blood or saliva in order to assure a better preservation
of the DNA molecules by the specific fixation on the treated card paper
 Teeth and bone tissues as well as burnt tissues
Touched objects provide a wide scope for revealing the offender’s DNA profile in
investigations of offences including theft, burglary, vehicle crimes, street robbery, drug
cases, homicide, rape and sex offences, clandestine laboratories, armed robbery, assaults,
crime. The positive DNA identification from those samples allowed the creation of national
offender databases ( Harbison et al., 2001; Gunn, 2003; Walsh and Buckleton, 2005; Gill et al.,
2000; Whitaker et al. 2001) to identify serial offenders and criminals.
2.2 Collecting methodologies

One of the best methods to collect trace samples is the use of swabs after having identified
as precisely as possible the areas to target. The first step is to swab the hole defined surface
by one or several moistened swab multiple times with some pressure and rotation given to
the swabs. The second step is to complete the swabbing by the application of dry swabs to
recapture the moisture containing hydrated cells. Co-extraction of these swabs to enhance
overall retrieval of DNA is recommended (Castella and Mangin, 2008; Sweet et al., 1997;
Pang and Cheung, 2007).
The moistening agent can be sterile water, 0, 01% sodium dodecyl sulphate (Wickenheiser,
2002) or isopropanol (Hansson et al., 2009). The quantities of cellules retrieved depend also
of the physical characteristics of the surface (Wickenheiser, 2002) and the use of different
moistening agents for different surfaces may facilitate collection. The quality of the swabs is
also important, the quality should be DNA-free; cotton swabs are the most frequently used
but other types such as foam may also be considered (Wickenheiser, 2002; Hansson et al.,
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Avoiding Errors and Pitfalls in Evidence Sampling for Forensic Genetics 3
2009; 57, 111, 112). It has been shown that the yield of DNA from moist or frozen swabs are
higher that from dried swabs. After collecting the biological material from a surface it is
recommended to process the swab in the laboratory. If these conditions are not available, the
swabs must be frozen immediately after collection.
According to some authors, tape is the best way to retrieve DNA containing material from
worn clothing or from touched surfaces without collecting in the same time inhibitory
factors present on this material (staining chemicals and/or color denim). By pressing a strip
of tape multiple times over a target area, the most recently deposited material , with fewer
inhibitory factors, are collected. In our experience, this method is not often used and should
be replaced by a easiest way to collect DNA such as cutting away stain fragment samples.
To isolate relevant target cells from other over-whelming cell types, laser microdissection
techniques were used. The different cell types can be recognized by morphological
characteristics, various chemical staining or fluorescence labeling techniques. These
methods allow to establish a clear DNA profile from few cells present in a mixture samples
that otherwise had not be detected while swabbed by the major component and not
detectable in the profile ( Elliott et al., 2003; Anslinger et al., 2005; Anoruo et al., 2007 ;
Sanders et al., 2006). With laser micro dissection techniques ( Anslinger et al., 2007;
Vandewoestyne et al., 2009), it has been shown that cells derived from a male contributor
can be analyzed separately from those derived from a female contributor after
morphological or fluorescent labeling identification. For this method, coated glass slides are
required and a sample must be transferred from the collection material to the slide. As cells
could be lost during this transfer, it would be preferable to use actually laser
microdissection methodology is directly used on the initial collection material.
3. DNA analyses
3.1 DNA extraction
The classical ways of DNA extraction from forensic routine case work were the organic
methods and sometimes the use of resin like Chelex 100R Bio-Rad (Walsh et al., 1991) which
may induce the molecule degradation during long storage periods. Actually, in cases of
degraded samples or when only minute amounts of DNA are available, the use of silica-
coated magnetic beads to capture the molecules from the rest of the lysed cells is
recommended. These extraction procedures are also performed in some laboratories by
robotic systems (Greenspoon et al, 2004; Frégeau et al., 2010). The loss of DNA during the
extraction step could be linked to the substrate sustaining the sample. Nevertheless, this loss
is principally linked to the used methodologies namely the organic extraction techniques.
The majority of samples submitted for analyses contain relatively large amounts of DNA,
above the 0.1-0.5ng minimum required by most common STR profiling systems. Below this
amount, specific methods like those used by molecular anthropologists on ancient DNA
samples must be developed.
The optimization of the extraction methods involves:
 The extraction of all the available DNA;
 To remove all amplification inhibiting elements without the loss of DNA;
 To amplify all the extracted molecules with adding the amplification reagents to the
device containing the DNA rather to add the DNA to the amplification tube and to
loose molecules in pipette tips or on the tube walls ;
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3.2 DNA quantitation

It seems not necessary to quantitate all the samples in particular highly degraded samples or
trace samples given the expected low concentration of DNA. The only advantage lay in
having an indication of the approximate quantity present in order to prevent repeat analyses
of over-amplified samples and when interpreting the profile. It must be emphasized that a
negative quantitation result should not prevent to process the samples. With the real-time
quantitation method applied on low template samples, the results should be taken as an
indication of the concentration and not as an absolute measurement as with higher DNA
amounts. In criminal cases, it is of common practice to retain a certain amount of the
samples for the future further typing by a second laboratory as a cross examination.
3.3 DNA amplification

For samples containing enough DNA of high molecular weight, the classical technics of
DNA extraction can be performed without pitfall, appropriate technologies were developed
to increase the chance to obtain useful profiles from very minute DNA samples such as the
low copy number (LCN) procedure with extra cycles or low template DNA (LTDNA)
methods. Minute samples or trace DNA refers to samples where only 100pg to 200pg of
DNA could be extracted according to different authors. These methods increased the
possibility to amplify successfully DNA from trace scene samples (McCartney, 2009;
Budowle et al., 2009). Difficulties can be raised in the interpretation of those profiles where
the peak heights may be below a validated threshold level.
During this step, the exponential amplification of DNA results in the production of billions
of copies of the template molecule. So every DNA contamination will be also amplified and
can false the result and on the other hand the excess of DNA produced by the PCR will be
present either on the machines used but also in the surrounding environment such as the air
and the work surfaces. To avoid these contaminations, all the steps of the analyses (pre-PCR,
PCR itself, post-PCR) must be performed in physically separated laboratories.
The step of amplification is a very critical one and was optimized for low level template
amounts. Amplification is the main field where the biologists must have control of the
quality of the molecule. To enhance the success of trace DNA amplification, it was proposed
to increase the number of cycles (Gill et al., 2000). The number of cycles used during the
PCR of the STR loci is increased to 34 compared to the standard 28 cycle reactions. In
molecular anthropology and in ancient DNA work, the number of cycles could be increased
up to 60 in order to maximize the success of amplification (Rameckers et al., 1997).
Numerous authors have described the efficacy of increasing cycle numbers ((Gill et al., 2000;
Whitaker et al., 2001; Kloosterman et Kersbergen, 2003). Complete profiles with substantial
increases in peak heights have been described (Gill et al., 2000) but contaminating DNA may
also be amplified through enhancing the number of cycles. When the sensitivity is increased,
more sporadic contamination will be detected and the laboratories must enhance the
stringency of contamination prevention. “Mini-STR” kits were developed containing
redesigned primers which had significantly higher success rates with degraded DNA due to
smaller amplicons. The minifiler STR kitR produced by Applied Biosystem showed a higher
success rate with degraded or inhibited DNA than the classical kits and requires also a
lower template input approximately 0.125 ng compared to 0.5ng (Mulero et al., 2008). The
optimization of the multiplex with the increased priming and amplification efficiency of the
new primers can explain the better sensitivity of the amplification.
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The efficiency of the amplification reaction can also be increased by the addition of chemical
adjuvants such as bovine serum albumin (BSA). BSA is known to prevent the inhibition of
the activity of Taq polymerase by sequestering phenolic compounds which otherwise
scavenge the polymerase (Kreader, 1996).
3.4 Detection of amplified product

To increase the detection of amplified product , methods have been developed to purify the
PCR amplicons, to remove salts, ions and unused dNTPs and primers from the reaction by
using filtration (Microcon filter columns), silica gel membranes (Quiagen MinElute) or
enzyme hydrolysis (ExoSAP-IT) (Forster et al., 2008; Petricevic et al., 2010; Smith and
Ballantyne, 2007)). This purification step is performed to remove negative ions such as Cl-
which prevents inter-molecular competition occurring during electrokinetic injection
allowing a maximum amount of DNA to be injected into the capillary of the sequencer. To
enhance the quantity of DNA available for the detection, it is also possible to concentrate the
PCR product during the purification process.
3.5 Difficulties of the typing of trace DNA

The side effect of increasing the ability to amplify the DNA molecule and in particular
minutes amounts of material is the increased likelihood of contamination being detected
and of artifacts of the amplification process due to stochastic effects.
Four major cases of interpretation difficulties can be summarized:
 Allele drop-out is due to a preferential amplification of one allele at one or more
heterozygous loci. This kind of pitfall is relatively frequent when very low quantities of
DNA are amplified (Whitaker et al., 2001; Gill et al., 2000; Gill et al., 2005; Lucy et al., 2007).
The interpretation of profiles obtained from minutes amounts of DNA must in each
case take in account the possibility of an allele drop out.
 Allele drop in, this occurrence is due to amplification artifacts such as stutter. This
artifact may be also frequently seen in the analyses of trace DNA amounts (Whitaker et
al., 2001). When stutter alleles are present in a STR profile it is rather difficult or
impossible to characterize the number of individuals having their DNA in the sample
and assigning of alleles within a mixture.
 Allele drop is due to sporadic contamination occurring from various origins such as
crime scene, sampling, non DNA free material or at the laboratory work.
 A decreased heterozygote allele balance within a locus and between loci. In this feature,
the peak height imbalance within and between loci are due to the same amplification
effects that cause drop-out. In those cases, the evaluation of the zygosity at a particular
loci may be extremely difficult.
No methods can actually eliminate completely artifact product during the amplification step
in particular when the DNA is degraded or present in minute amounts but their occurrence
should be statistically evaluated. To be able to develop such an approach it is of importance
to understand the factors that may cause each type of artifact and the accurate data
regarding the frequency and scale of their occurrence. Benschop et al. (2010) present one of
the first large-scale efforts to characterize artifacts generated by different trace DNA
amplifications. These authors showed also their investigations to highlight an effective
method to generate a useful consensus profile.
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3.6 Pitfall at the interpretation step

For each profile interpretation, the sampling of biological material found at the crime scene
must be replaced into context and the possibility of pitfalls should be taken into account
such as the possibilities of material transfer, the difficulties of the amplification process and
the possibility of artifacts affecting the true result. This interpretation carefulness is of
particular importance when the analyses are performed on degraded or very low quantities
of DNA and has to consider imperatively the four most common features which can occur in
those cases: allele drop-out, allele drop-in, stutter bands, contamination and decreased
heterozygote balance. Strict interpretation guidelines can give reliable and robust result and
minimize these pitfalls.
The introduction of detection thresholds may give a reliability of DNA profiles
interpretations in particular for degraded DNA or minutes amounts of DNA. The
background noise is generally eliminated by the establishing a threshold of 50 RFU. In order
to avoid false homozygote by allelic drop-out , separate thresholds were established referred
to as the low-template DNA threshold T, the match interpretation threshold (Budowle et al.,
2009), the limit of quantitation (Gilder et al., 2007) is set at 150-200 RFU. The allele peaks
should be above this limit to be sure that it is a true homozygous but even the respect of this
limit may not prevent allele drop-out in all cases. Other authors (Gill and Buckleton, 2010)
have recommended that instead of thresholds, a more continuous measure should be used
which is modeled on the risk of dropout based on peak heights.
One of the most used methods to eliminate incorrect genotypes is to replicate the
amplifications reactions and to generate consensus profiles (Whitaker et al., 2001; Gill et al.,
2000; Benschop et al. , 2010; Taberlet et al., 1996). But currently, no consensus has been
found on either the minimum number of replicates needed or how frequently one needs to
observe an allele within the number of replicates conducted to be sure that the found allele
is a true one. Benschop et al., (2010) consider that four replicates for degraded or very low
amounts of DNA may be the most appropriate rules for considering a profile as a true one.
Gill et al. (2000) proposed a statistical model, mentioned by other authors (Balding and
Buckleton, 2009; Gill and Buckleton, 2010; Curran, 2005), which provides the necessary
probabilistic methods where the probability of observing the evidence profile can be
combined with prior knowledge regarding dropout, the number of potential contributors,
the possibility of contamination and other factors (Van Oorschot et al., 2010).
3.7 Mixture interpretation

A particular mention must be made for DNA mixture interpretation. In fact mixed samples
are by definition composed of one or more major contributors with high quantities of DNA
and with a minor contributor present only at trace levels, in other cases, the contributors are
all present at trace levels. A profile can be falsely identified as a false mixed samples when
high stutter peaks are present indicating that the DNA is coming from multiple individuals
although it truly derive from a single source. In mixed samples, the high probability of
drop-in, drop-out and increased stutter bands avoid the precise determination of the
number of contributors and the separation of the genotypes at any given locus. This is
frequently the case in degraded DNA or when the DNA is present in very few amounts
(Walsh et al., 1996; LeClair et al., 2004; Gibb et Huell, 2009).
In such cases, the amplification reaction is also source of bias and pitfalls in over-
amplification of some alleles and allowing a dropping-out of minor contributor’s alleles at
some loci.
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Recommendations were published by the International Society of Forensic Genetics on

mixture sample interpretation (Gill et al, 2006). A likelihood ratio (LR) approach was
proposed for the interpretation for low template level mixture with the incorporation of an
assessment of the probability of allele drop-in and drop-out in such cases.
Bright et al. (2010) proposed the use of the heterozygote balance and average peak heights at
each locus to calculate the mixture ratio and distinguish among the contributors’ genotypes
(Van Oorschot et al., 2010).
For all these reasons, interpretation of mixture samples must be done very carefully
particularly in cases where DNA is degraded or present in few quantities.
4. Contaminations issues
Contaminations are the major pitfall in the analyses of DNA in the forensic field either in
producing valuable profiles or in accurate interpretation of the results. This is a major issue
when the samples are degraded or when the DNA molecules are present in minute
amounts. Contaminations may appear in every step of the analysis process from the
sampling on the crime scene to the laboratory work. Rutty and Graham (2005) highlight
that the contaminations can occur on the body itself or during the sampling of the evidences,
at the scene of the crime, during the transportation of the body to the mortuary, at the
autopsy room and after, of course, during the laboratory procedures.
At the crime scene, one of the more frequent situation where contaminations of the crime
scene can occur if the individuals who entered the scene speak or caught and handle
evidences over the corps before the arrival of the forensic investigative team. Rutty and
Graham (2005) described airborne DNA contamination in mortuaries.
Methods were described in order to avoid the possibility of contaminations:
 To perform analyses about the persistence of DNA on different kinds of surfaces in
various environmental conditions (Toothman et al., 2008; Rutty et al., 2003; Cook et
Dixon, 2007);
 To improve and standardize the sample collection methodologies in order to improve
the targeting of the samples and to decrease unwanted underlying DNA;
 To collect the profiles of all the persons involved in the collecting and laboratory steps
to recognize a contamination coming from these professionals;
 Some laboratories require samples from the area immediately adjacent to the target area
to have a so called “blank sample”.
The operating procedures on the crime scene must be precisely fixed to minimize the
possibility of contaminations (Rutty et al., 2003):
 To avoid breathing, talking and of course coughing during the sampling step in
restricting the access of non specialist investigators to the scene;
 The use full-body scene suit (to avoid contamination by cell shedding coming from
exposed areas of skin), hood, hair net, gloves and mouth masks by all the investigators
in charge of the sampling step;
 To avoid direct touching of the evidences containing the DNA and changing gloves and
masks regularly at the crime scene and obviously in the laboratories;
 All the results are compared against the database containing the DNA profiles of all the
persons who were involved in all the steps of the sampling and laboratory processing of
the evidences in order to detect contaminations coming from them;
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 To use DNA-free disposable equipment to collect the DNA on the target surfaces (Van
Oorschot et al., 2005), and to systematically decontaminate thoroughly all the devices
which would be in physical contact with the sample.
 For victims taken to a hospital in attempt to seek treatment, the different surfaces
(stretcher, hospital beds, tables), the instruments which will be used (scissors to cut
away the clothing, electrocardiogram leads, other medical equipment).
Methods to minimize the possibility of contamination in the laboratory have been largely
developed. Some of the guidelines are:
 Use of DNA-free plastic ware and consumables, recommendations for manufacturers
and laboratories were made by several scientific societies (Gill et al., 2010), Scientific
Working Group on DNA Analysis Methods [SWGDAM], European Network of
Forensic Science Institutes [ENFSI], Biology Specialist Advisory Group [BSAG];
 Shortwave (254 nm) UV exposition of the working surfaces when nobody is working
and frequent and thorough cleaning of work areas within laboratories. The top of doors
of each room are also equipped with UV source. All appliances, containers, pipets,
racks, laboratory coats and work areas (laminar airflow surfaces, PCR box) are cleaned
and irradiated by UV during the non-working hours (Keyser-Tracqui et Ludes, 2005).
 Periodic assessment of the level and location of DNA within the work place and on
relevant tools;
 All the different steps of the analysis process going from the sample examination step to
the extraction procedure, the DNA amplification reaction and at the end, the
interpretation of the profiles must be conducted in dedicated laboratory rooms. The
analyses of traces samples are also performed a part of the high DNA quality and
quantity DNA samples. A “one-way traffic” rule is also observed in the laboratory, once
the technician has entered the PCR or the post-PCR rooms, they are not allowed to
return to the extraction or pre-PCR rooms until the next day or a complete cloth
changing in order to prevent contamination by aerosol particles. All general equipment
and apparatus, pipets as well as reagents are dedicated to the analysis area (Extraction,
pre-PCR, post-PCR rooms) ;
 Cross comparison of results obtained from different cases (having recorded at which
locations the analyses were performed by whom and at what time) to detect
unexpected contaminations;
 Analysis of reference samples and extraction (blank) as well as amplification controls at
each step of the procedure are a major help to highlight inter-case contamination. The
extraction control checks the purity of the extraction reagents and the amplification
control indicates the purity of the PCR reagents with no DNA added.
The possibility of the presence of contaminations should be taken in mind at every profile
interpretations in particular in cases of degraded DNA or if the molecule is present in very
few quantities. As described before the difficulty of the interpretation of a mixed sample
must be emphasized, in fact the profile can contain background DNA, crime-related DNA,
post-crime contamination.
5. Conclusions
Since the method of DNA fingerprints has been described two majors goals have been
followed, first to obtain highly discriminating genetic profiles from minute amounts of DNA
and for highly degraded samples, second to avoid the possibility of contaminations due to
the crime scene work, the sampling step or the laboratories procedures.
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Swabbing and taping a touched area for retrieval of DNA seems simple but experience in
case works showed how easy it is to get wrong. The scene crime technicians should be
trained and wear appropriate scene clothing to protect the crime scene and its environment.
The interpretation of the results should take in account these contamination possibilities by
a LR framework incorporating the criminal aspects of DNA evidence (Raymond et al., 2008).
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2
Death Scene Investigation from

the Viewpoint of Forensic Medicine Expert
Serafettin Demirci and Kamil Hakan Dogan
Selcuk University,
Turkey
1. Introduction
Medical expertise is crucial in death investigations. It begins with body examination and
evidence collection at the scene and proceeds through history, physical examination,
laboratory tests, and diagnosis – in short, the broad ingredients of a doctor’s treatment of a
living patient. The key goal is to provide objective evidence of cause, timing, and manner of
death for adjudication by the criminal justice system. Death investigation has been
performed for centuries in all societies, although not always by medical professionals
(Committee, 2003). The association of law and medicine dates back to the Egyptian culture
as early as 3000 B.C. The English coroner system was mentioned in documentations around
the 12th century B.C. (Spitz, 2006).
Although the primary goal of a death investigation is to establish the cause and manner of
death, the role of the death investigation extends much further than simply answering these
two questions. A common question asked is, “Why does it matter? The person is dead.”
While it is true that the dead cannot benefit, the value in death investigation is to benefit the
living and future generations. In a culture that values life, explaining the death in a public
forum (the meaning of “forensic”) is crucial for many reasons. And this interest goes beyond
simple curiosity (Wagner, 2009).
In homicide, suspected homicide, and other suspicious or obscure cases, the forensic
medicine expert should visit the scene of the death before the body is removed. Local
practice varies but any doctor claiming to be a forensic medicine expert should always make
himself available to accompany the police to the locus of the death. This duty is often
formalized and made part of a contract of service for those forensic medicine experts who
are either full-time or substantially involved in assisting the police, in England and Wales,
the 'Home Office Pathologists' are permanently on call for such visits and in many other
jurisdictions, such as the medical examiner systems in the USA, and the European State and
University Institutes of Forensic Medicine, there is usually a prearranged duty roster for
attendance at scenes of death (Saukko & Knight, 2004). In many cases, the scene
investigation is more important than the autopsy. A thorough and complete investigation
commonly leads to the proper diagnosis of the cause and manner of death prior to an
autopsy (Avis, 1993; Dix & Ernst, 1999).
Why go to the scene? The purpose of having the forensic medicine expert attend the death
scene is severalfold. By viewing the body in the context of its surroundings, the forensic
medicine expert is better able to interpret certain findings at the autopsy such as a patterned
imprint across the neck from collapsing onto an open vegetable drawer in a refrigerator. The
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forensic medicine expert is also able to advise the investigative agency about the nature of
the death, whether to confirm a homicide by a specific means, evaluate the circumstances to
be consistent with an apparent natural death, or interpret the blood loss from a deceased
person as being more likely due to natural disease than to injury. This preliminary
information helps the investigative agency to define its perimeter, structure its approach,
organize its manpower, secure potentially important evidence, and streamline its efforts.
Nonattendance at death scenes has been regarded as one of the classical mistakes in forensic
pathology. Hospital pathologists performing forensic autopsies who are not trained to, or
able to, attend death scenes should be provided with information on how, when, and where
the body was found, by whom, and under what circumstances. In some deaths, the
immediate environment does not contribute to death, such as in cases of metastatic breast
carcinoma. In other cases, the environment plays a role although it does not cause the death;
for example, consider a case in which a person with marked coronary atherosclerosis
collapses with a dysrhythmia while shoveling snow. On the other hand, the scene
description and scene photographs are critical in documenting that the physical
circumstances and body posture are indicative of death due to positional asphyxia because
the autopsy in these cases may yield very few findings. The most meticulous autopsy in all
academia will provide only a speculative cause and manner of death in a 30-year-old man
with a negative history, negative toxicology, and autopsy findings of visceral congestion.
Yet at the scene, a screwdriver is next to an uncovered electrical outlet on a rain-soaked
patio at the decedent's house, which is undergoing renovation. The cause and manner of
death are provided by the scene (Lew & Matshes, 2005).
The examination of a death scene and subsequent collection of potential evidential material
requires special skill, knowledge, aptitude, and attitude. The manner in which a death scene
investigation is conducted may be a critical factor in determining the success of an
investigation. The thorough examination of a death scene requires a disciplined and
systematic approach to recording the various observations made and collection of potential
evidential material. This must be combined with the analysis of various observations and
the interrelationship of potential evidentiary material (Horswell, 2005a).
If resources are sufficient and the circumstances of death so dictate, it is ideal for a forensic
medicine expert to perform a scene investigation. This is particularly relevant if the body
remains at the scene of death, and has not been transported to the hospital during attempts
at resuscitation; however, a scene investigation can be vitally important and provide
valuable information even if the body has been transported to the hospital. If a body is
pronounced dead at the scene (as opposed to after transport to the hospital), many death
investigation systems require a scene investigation. Others have various protocols as to
which case types absolutely require a scene investigation (whether or not the body is
present at the scene). Case types that should always have a scene investigation include all
confirmed or suspected homicides, suicides, accidents, child deaths, traffic-related deaths,
in-custody deaths, and workplace-related deaths (Prahlow, 2010).
Death scene investigation may include a combination of the following types of incidents and
examinations:
 Accidental deaths, which include a multitude of circumstances, including misadventure
 Suicidal deaths, which include a multitude of circumstances
 Homicidal deaths, which include a multitude of circumstances
 Sudden deaths, with or without suspicious circumstances
 Difficult victim identification, which includes mummification and putrefaction
 Disaster victim identification dealing with multiple casualties (Horswell, 2005a)
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Death Scene Investigation from the Viewpoint of Forensic Medicine Expert 15
This chapter will focus on the steps of death scene investigation and some real cases will be
analyzed.
2. Crime scene & death scene

In some “incidents,” it may be readily apparent that a crime has indeed been committed and
it is a “crime scene.”
The primary crime scene is an area, place, or thing where the incident occurred or where the
majority or a high concentration of physical evidence will be found, for example, where
there has been a sudden suspicious death.
Secondary crime scene(s) are areas, places, or things where physical evidence relating to the
incident may be found. The potential physical evidence will usually be transported away
from the primary crime scene.
Some examples include: The deceased, the get-away vehicle in crimes of armed robbery, the
suspect, the suspect’s environment, the suspect’s vehicle, the weapon used in the crime
(Horswell, 2005a). This classification does not infer any priority or importance to the scene,
but is simply a designation of sequence of locations (Miller, 2003).
If a deceased person is at the scene we call it the death scene. One of the initial and primary
tasks is to determine whether a crime has been committed at the death scene.
Every death scene is a potential crime scene. It is important to carefully examine the scene
for evidence or unusual circumstances that may indicate the death of the person is other
than by natural causes (Moldovan, 2008).
3. Investigative tools and equipment

The forensic medicine expert should always have appropriate equipment ready to take to a
scene investigation at a moment's notice. Further equipment may be carried if autopsies
have to be carried out in places where good mortuary facilities are not available. Most
forensic medicine experts carry a 'murder bag' in their car and though every expert has his
own choice of equipment, the following is a reasonable inventory:
 Waterproof apron and rubber gloves.
 Writing implements (pens, pencils, markers).
 Disposable (paper) jumpsuits, hair covers, face shield, etc.
 Thermometer, syringes and needles, sterile swabs.
 Autopsy dissection set, including hand-saw.
 Cutting needles and twine for body closure.
 Swabs and containers for blood and body fluids.
 Formalin jars for histological samples.
 Plastic bags, envelopes, paper, spare pen and pencil.
 Printed body charts for recording external injuries.
 Hand lens, electric torch, mini-tape recorder.
 Foul-weather gear (raincoat, umbrella, etc.).
 Personal comfort supplies (insect spray, sun screen, hat, etc.).
 Camera, usually 35 mm single-lens reflex with electronic flash (with extra battery). The
recent advent of compact digital cameras or digital video cameras with the facility to
take still pictures has made instant reviewing possible.
The thermometer can be either a long chemical mercury type, reading from 0 to 50°C, or the
more modern electronic digital variety with a probe carrying a thermocouple. The amount
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of equipment varies with the facilities likely to be available. In developed countries there are
likely to be good mortuary facilities available in a hospital or municipal mortuary and the
police forces will have extensive scenes-of-crime expertise with photography, specimen
containers and so on. In developing countries and the more remote areas of other states, the
forensic medicine expert may have to be virtually self-sufficient in respect of both crime
investigation and the subsequent autopsy.
In addition to medical kit, the experienced forensic medicine expert will always have
appropriate clothing such as rubber boots and rain or snow-wear ready to hand for any call
(Clark, 1999; Saukko & Knight, 2004).
4. Steps of death scene investigation

The deceased is the most valuable piece of potential evidence at any death scene. Hence, a
systematic and thorough examination of the deceased should be undertaken at every death
scene. Blood spillage or spatter should be noted and will remain after the removal of the
body. Weather conditions, location, and poor lighting may mask some faint injuries and
trace evidence on the body, therefore the death-scene investigator should document in
writing, by sketch, and by photography all information about the body that can be gathered
at the scene (Horswell, 2005b). The forensic medicine expert should focus on the physical
condition of a body at a scene. Without a scene investigation, much initial, valuable body
information can be lost. The following points will serve as a guide.
4.1 Pre-planning the death scene investigation

When initially notified, a forensic medicine expert should determine as much information as
possible from the caller. Approximate age and gender places a subject in a certain "medical
category." An attempt should be made to ascertain if there is any evidence of foul play or if
any instruments are available that might have played a role in the subject's death. By
gathering these data, a forensic medicine expert is able to anticipate additional infonnation
that may be needed upon arrival at a scene (Dix et al., 1999). The first rule in performing a
death scene investigation is to make certain that the scene is safe and secure. Usually, this
requires police involvement but in some instances, it will require other professionals, such
as fire department personnel or utility workers. The second rule is to not contaminate or
disturb the scene. At the very least, death investigators should wear disposable examination
gloves and it is also advisable to wear shoe covers and hair nets. Occasionally, full body
covering is desirable. When touching items at a scene, examination gloves should always be
worn and care should be taken not to sit on furniture or lean against or brush against walls
or furniture (Prahlow, 2010). The death-scene investigator must seek answers to the
following questions: is trace evidence at the scene consistent with the death having occurred
at this location? Does the body contain any trace evidence that is unusual for this location,
for example, mud on soles of shoes, grass, or seed material embedded in or found on the
clothing when the deceased was located inside a building? Is the death one that can be
attributed to natural causes? Are there any external signs of violence? Is there anything
amiss or out of the ordinary regarding the scene? (Horswell, 2005b).
4.2 Cooperation among investigators

A successful death investigation, involving more than one individual, requires cooperation
and coordination. Any potential conflicts should be worked out (Dix et al., 1999). The
opportunity to meet at the scene initiates the collegial working relationship between the
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forensic medicine expert and the detective/investigator, and promotes interagency rapport
as both professionals strive to solve the medical mystery of why that particular person died
at that particular time, under those particular circumstances. This is not melodrama, just
intellectual satisfaction for exploring an extremely important, educational, and fascinating
aspect of death investigation. After all, a gunshot wound is a gunshot wound: it is the
circumstances behind that gunshot wound that are frequently so compelling and always so
instructive about human nature (Lew & Matshes, 2005).
4.3 Documentation of the scene

All death scenes should be secured and recorded photographically and diagrammatically. If
the information to hand, backed by the postmortem, suggests that the death was due to
natural causes then the scene should not be processed any further. However, if there are
signs at the scene, and other information suggests that the deceased died in suspicious
circumstances, and this is reinforced by signs of a struggle or anything unusual, further
processing for latent impressions and trace evidence should take place (Horswell, 2005b).
The four major tasks of documentation are note taking, videography, photography, and
sketching. All four are necessary and none is an adequate substitute for another. For
example, notes are not substitutes for photography.
Documentation, in all its various forms, begins with the initial involvement of the inves-
tigator. The documentation never stops; it may slow down, but the need for documentation
remains constant. Death scene documentation will be discussed below in the sequence it
should follow at a death scene. The systematic process presented will maintain the
organized nature of scientific death scene investigation.
4.3.1 Taking notes at the death scene

Effective notes as part of an investigation provide a written record of all of the crime scene
activities. The notes are taken as the activities are completed to prevent possible memory
loss if notes are made at a later time. Accurate crime scene note taking is crucial at sider the
who, what, when, why, and how, and specifically include:
 Notification information. Date and time, method of notification, and information received.
 Arrival information. Means of transportation, date and time, personnel present at the
scene, and any notifications to be made.
 Scene description. Weather, location type and condition, major structures, identification
of transient and conditional evidence (especially points of entry), containers holding
evidence of recent activities (ashtrays, trash cans, etc.), clothing, furniture, and weapons
present.
 Victim description. Position, lividity, wounds, clothing, jewelry, and identification
(presence or absence).
 Crime scene team. Assignments to team members, walk-through information, the
beginning and ending times, and the evidence-handling results (Miller, 2003).
The forensic medicine expert should observe a great deal, but do very little. He or she
should note the position of the body in relation to nearby objects and establish the plan of
the premises if indoors. A sketch or his own photograph is sometimes useful, and some
forensic medicine experts use a Polaroid, digital or video camera for instant recording of the
death scene.
Any obvious cause of death should be observed, and any blood pools or splashes noted in
relation to the position of the corpse. The shape of such splashes should be observed, as
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blood striking perpendicularly to a surface leaves a circular mark, whilst that landing
obliquely is pear-shaped, with the sharper end towards the direction of flight. If the scene is
one of apparent violence then the blood flow patterns may indicate the type of weapon and
how it was used (Horswell, 2005b; Saukko & Knight, 2004). Both natural and unnatural
deaths can produce abundant blood at a scene. Traumatic deaths that involve arterial or
venous bleeding, such as stabbing, can produce abundant blood at the scene with spattering.
Gunshot wounds can cause extensive external bleeding, but some wounds can cause
minimal external bleeding and massive internal bleeding. In short, the amount of blood
perceived at a scene does not indicate the severity of the trauma (Wagner, 2009).
4.3.2 Videotaping the death scene

Videotaping a death scene has become a routine documentation procedure. Its acceptance is
widespread, due to the three-dimensional portrayal of the scene and increased availability
of affordable equipment with user friendly features like zoom lens and compact size. Jury
acceptability and expectation have also added to the recognized use of videography in death
scene investigations.
Videography of the crime scene should follow the scene survey. The videotaping of death
scenes is an orientation format. The operator should remain objective in recording the death
scene. Videotaping of death scenes is a valuable tool that allows clear perception that is
often not possible with the other documentation tasks. It is not an adequate substitute for
any of the other tasks (Miller, 2003).
4.3.3 Photographing the death scene

The purpose of still photography documentation of the death scene is to provide a true and
accurate pictorial record of the death scene and physical evidence present. Still photography
records the initial condition of the scene. It provides investigators and others with a record
that can be analyzed or examined subsequent to the scene investigation, and serves as a
permanent record for legal concerns. Photography of a death scene is normally done
immediately following the videography of the scene or after the preliminary scene survey. A
systematic, organized method for recording the death scene and pertinent physical evidence
is best achieved by proceeding from the general to specific guideline. Adherence to this
guideline allows orientation of the entire death scene, orientation of the evidence within the
scene, and provide; examination quality photographs of specific items of evidence that may
be used for analysis away from the scene. The number of photographs that should be taken
at a death scene cannot be predetermined or limited (Miller, 2003). Information such as body
location and unique circumstances at the death scene may help a forensic medicine expert. It
is important to keep in mind the legal implications of the photographs. Will the
photographs be subpoenaed? (Dix et al., 1999). The scene and body are photographed before
anything is moved or removed. Treat the body with respect. Never remove the clothing in
full view of onlookers. If it is not feasible to move the body to a secure area of the scene,
police officers may hold up sheets around the body, mobile panels may be used, or police
vehicles may be used to block visibility from the public (Lew & Matshes, 2005).
4.3.4 Sketching the death scene

The final task in documentation of a death scene is sketching. All of the previous tasks for
documentation record the death scene without regard to the size or measurement of the
scene and its physical evidence. Sketching the death scene is the assignment of units of
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measurement or correct perspective to the overall scene and the relevant physical evidence
identified within the scene (Miller, 2003). The deceased's location relative to other objects
and structures within the scene is very important. The position of the deceased is plotted:
the head and groin of the deceased are good points on the body to use for plotting its
position. Accurate measurements should be noted to place the items within the scene in the
sketch in the same locations as they appear in the scene (Horswell, 2005b).
4.4 Identification of the deceased

Positive identification of the decedent is crucial in all death inquiries. The family should be
notified. Information such as medical history, work, and social history can only be obtained
after an identification is established. Care must be taken to insure that the identification is
absolutely correct (Dix et al., 1999).
4.5 Examination of the body

A systematic, thorough inspection and evaluation of the decedent should be performed by a
forensic medicine expert. If he/she always begins at the top of a subject's body and moves
toward the feet, the possibility of missing important injuries or evidence is lessened (Dix et
al., 1999). The body should be prone (face up) during the examination, if possible. Photos of
the original position of the body must be taken before the body is moved. One begins with a
general assessment and progresses from head to toe, pushing clothing aside but not
removing it. Some find it easier to assess rigor, livor, and algor mortis initially. The purpose
of the assessment of the body at the scene is to provide some insight into the nature of the
case and a working cause of death (Wagner, 2009).
One of the most important questions that needs answering is: did death occur at this
location? The position in which the deceased was discovered is of particular importance as it
will provide an indication as to whether the deceased was moved or not before being
discovered. The presence or absence of rigor mortis or stiffness of the body, whether absent,
minimal, moderate, advanced or complete, will help the death-scene investigator determine
if the person died at that locus in the position as found. Some death-scene investigators with
relevant training and experience may feel they are in a position to evaluate rigor mortis and
hypostasis. A pink-purple discoloration is usually present at the lowest point of the body.
This is due to the settling of the blood by gravitation and the location and state of fixation
should be noted and photographed. For example, unfixed livor blanches white when
moderate pressure is applied, as opposed to fixed livor mortis, which remains the same
color when pressure is applied. If livor mortis is noted on the deceased in areas not
consistent with forming in the lowest parts of the body then the death-scene investigator
should consider the possibility that the deceased was moved after death (Horswell, 2005b).
Victims may be found in contorted or apparently uncomfortable positions on the floor,
commonly the bedroom or bathroom. Generally, the more contorted the body, the more
sudden the death. The person appears to have “fallen in his tracks.” However, this does not
mean the decedent lying apparently comfortably in bed did not also die suddenly. Bodies
found in awkward positions that compromise breathing can die of positional asphyxia. The
chest wall must be able to rise and fall for respiration to occur. If one is wedged too tightly
in a position, the chest wall cannot rise and fall (Wagner, 2009) (Fig. 1) .
Many inexperienced investigators focus on a major injury and neglect to evaluate the rest of
the individual. This can lead to important oversights such as fingernail marks, bruises, and
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Fig. 1. Seventy two-year-old man had lost the key of the door of his house in his vineyard
and he tried to go in from a small hole which he made on the roof. He was stuck and found
dead in the hole due to positional asphyxia.
abrasions. Documentation of this inspection should be made noting the presence and
absence of unusual markings or abnormalities. Descriptions of the state of rigor and livor
mortis as well as the body temperature of a subject helps a forensic medicine expert to
estimate the time interval since death. Environmental assessment, including temperature,
heating or cooling systems, moisture, and wind conditions must be made at a death scene so
that the environmental influence on a decedent can be determined. The assessment should
also include the types of clothing and jewelry. This information may be needed to assist in
determinating the time a subject was last seen alive. Clothing should be appropriate for the
weather and location found. If not, it needs to be explained. One should also determine if
the clothing fits an individual. If a subject is decomposing, then clothing may appear too
small due to body swelling. If the clothing is the incorrect size, one must determine why.
Was the person wearing someone else's when death occurred? Or, was the decedent
redressed by another person after death? Note the cleanliness of the clothing. A variance in
the clothing or body cleanliness may indicate that he was handled by another individual
after death (Dix et al., 1999). General uncleanliness such as lack of bathing, very dirty
clothes, urine -or feces- stained clothes, long and dirty nails, and poor oral hygiene may be
due to alcoholism, drug abuse, or a mental disorder (Wagner, 2009). Is the clothing worn
properly? Are buttons fastened and zippers closed? It is common to find opened zippers in
intoxicated males or some elderly persons living alone. If the clothing is inconsistent with
normal dressing techniques, consider whether a subject had a disability contributing to this
behavior. Jewelry should be carefully noted and reported as to its type, style, color and body
location. All jewelry must be listed, regardless of its apparent value. Obvious "missing"
jewelry should also be noted, such as only one pierced earring, or no wedding ring on a
married individual. Currency and credit cards should be handled as valuable items.
Currency should be counted in the presence of another and credit card details noted. If an
investigator decides that these items may be given to the next-of-kin at the death scene, he
must be certain that the relative has the legal right to such items. No analyses should be
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performed on a decedent's body at a scene, such as gunshot residue or fingerprinting,

without the expressed consent of the forensic medicine expert responsible for the
postmortem examination. Clothing should not be removed, a body should not be cleansed,
and liquids or powders should not be placed on the deceased as these might interfere with
radiographs or chemical testing. If more than one hour has elapsed since the initial body
assessment and the decedent is still at the scene, a second assessment should be recorded. A
thorough body visualization by a forensic medicine expert gives him/her the capability to
differentiate between injuries noted at a scene and any bodily injuries sustained during
conveyance to the morgue (Dix et al., 1999).
A common misconception among laypeople is that a “painful” expression on the face or a
contorted position means the person suffered during the process of dying. Generally, there
is no correlation between facial expressions, body positions, and suffering. Pain and
suffering can be assessed before and during the dying process, but it is done carefully and
generally by the forensic medicine expert after evaluating the autopsy and investigative
information. This information can be useful to the family, and can become arguable in civil
court cases (Wagner, 2009).
4.6 Other scene information collection

An investigator must also gather information that relates to cause and manner of death.
Each type of death requires specific scene information. For instance, questions to be asked in
a motor vehicle fatality would not be the same as those asked in an autoerotic asphyxia
death. Since different questions need to be asked, an investigational guide for each specific
type of death can be very useful. For example, it is critical in suicides resulting from a
handgun that investigators determine the handedness of a subject (Dix et al., 1999). The
scene should be searched for a medical history in nearly all death investigations. This search
may be as simple as finding an inhaler for asthma nearby a gunshot wound victim or as
complicated as going through cabinets full of medication at a residence. The deceased’s
physician can always be called and the hospital records will be available tomorrow, but one
has only a single chance to explore the scene to find out what is really going on with the
person’s diseases and treatment. Many people do not take the treatments the doctor ordered
and reject advice given at the hospital. Only interviewing witnesses and searching the scene
will reveal this information (Wagner, 2009).
4.7 Determining what information has already been developed

Prior to a forensic medicine expert's arrival, law enforcement officers, paramedics, and other
support personnel probably have communicated with individuals or witnesses at the scene.
A forensic medicine expert needs to know this initial information so that he can compare it
with the decedent's body data and determine if there are any discrepancies. It is better to ask
the question twice and get the same answer, than to accept as fact information that has been
checked by one source. A forensic medicine expert needs to determine, for instance, if the
body data (rigor, livor, temperature, clothing, injuries, etc.) are different from the witness
information (Dix et al., 1999).
4.8 Collecting evidences which may be found at the death scene

Forensic medicine experts and law enforcement agents work cooperatively in a team effort.
Although the medical expert has jurisdiction over the body, law enforcement has
jurisdiction over the entire scene. The forensic medicine expert is invited to the scene and, as
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a guest, must comply with house rules. In Britain, for example, several teams converge on a
scene of crime, including photographers and video operators, and Scene of Crime Officers
(SOCOs) whose function is to collect trace evidence. Scientists from the nearest forensic
laboratory often attend with their police liaison officers, as well as fingerprint officers and,
of course, the investigating officers from the Criminal Investigation Department. The lead
detective will walk the forensic medicine expert through the scene, relaying information and
pointing out salient features. The forensic medicine expert should realize that the area
within the perimeter of the scene is one giant piece of evidence, and restrict his or her
physical contact to the body and items immediately touching the body (Lew & Matshes,
2005; Saukko & Knight, 2004).
Where no such backup is available, the forensic medicine expert must try to collect trace
evidence himself, but he should remain within the limits of his own expertise. The forensic
medicine expert should accept the instructions of police officers in relation to the approach
to the body so as to preserve the immediate environment as much as possible. Out-of-doors
access is often limited to a single pathway marked by tapes, and in a building a track to the
corpse is usually pointed out by the detective in charge. The doctor should not touch
anything unnecessarily and certainly not smoke or leave any object or debris of his or her
own. Increasingly, those visiting the scene of a crime are given disposable overalls and
overshoes to wear, so that fibers, hairs and so on from the visitor are not spuriously
transferred to the scene (Saukko & Knight, 2004). The Locard Exchange Principle states that
whenever two objects come into contact, a mutual exchange of matter will take place
between them. Linking suspects to victims is the most important and common type of
linkage accomplished by physical evidence in criminal investigations. Linking victims and
suspects to objects and scenes can also be accomplished by use of the physical evidence
(Miller, 2003) (Fig. 2).
Fig. 2. The 18-year-old murderer killed his employer in his workplace as he did not pay his
salary. On the death scene investigation, a horror mask (on the top) and footprints of sports
shoes of the murderer (on the bottom) were found. These evidences were used to determine
the murderer.
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After surveying the overall death scene, it should be easy to recognize the sequence in
which evidence is to be collected and areas to be searched and in what order. The collection
and search should be systematic, ensuring absolutely nothing is overlooked.
Priority in collection should be given to:
 any items that are in danger of being removed or destroyed by wind, rain, vehicles,
animals, tides, and the movement of individuals at the scene
 the collection of any evidence which will enable access to the deceased or any critical
area of the death scene, such as along entry and exit paths
 those critical areas of the crime scene which may render the most evidence, or once
processed, enable the removal of a body, or the remainder of the examination to be
carried out
 areas which may give a quick indication as to the identity of any suspect(s)
 areas which when processed will permit the release of scene guards and other resources
 the general examination of the remainder of the death scene for potential evidence.
In establishing the manner and sequence of collecting potential evidence by death scene
investigators, consideration must be given to the possible destruction of evidence and which
approach will yield the best result in terms of useful information (Horswell, 2005b).
Clues about the cause and manner of a death and who committed a crime may be found at a
scene. The following list includes different types of evidence and how they are usually
collected and preserved.
Blood - Dried particles should be scraped into a drycontainer. Some dried areas may be
sampled with a wet swab. A specimen should be dried before sealing it in a container.
Articles of clothing or other objects containing blood may be submitted to a laboratory for
sample removal by a technician.
Semen - An article of clothing containing semen should be collected or the specimen on the
clothing can be lifted with water or saline.
Fingerprints - Soft objects that leave an impression may be collected in their entirety. Prints
on hard objects like glass or furniture should be lifted at the scene.
Firearms and other weapons - These should be submitted to a lab without special treatment at
a scene. A technician must ensure proper handling so that fingerprints are not smudged or
ruined.
Bullets and cartridges - These should not be grasped with metal forceps because points of
comparison may be damaged.
Hairs and fibers - These should be placed in separate containers and should not be crushed
with hard objects such as metal tweezers.
Suspicious foods and pills - Each item should be placed in separate containers or bags to
prevent contamination.
Footprints and tire marks - At the scene, casts should be made and close-up photographs
should be taken.
Tool marks - There should be close-up photographs of the marks made by tools and, if
possible, the damaged material should be removed for analysis by a lab technician.
Blood spatters - These should be photographed and described for analysis as to distance and
angle of spatter. Samples may be removed for testing and preservation.
Other - Glass, soil, documents, cigarette butts, tobacco, and all items thought to be involved
in arson should be collected and submitted to a lab.
Each item submitted to a lab should be referenced by either a photograph or written
description as to its location in the scene. All containers with items submitted to the lab
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must be labeled on the lid and side of the container, with a case number, date, time, type of
specimen, and name of the person who collected the specimen. A "chain of custody" begins
at this point and continues until a disposition of the specimen is completed (Dix et al., 1999).
Methods of searching critical areas include grids that are larger in less critical areas and
smaller in critical areas, or searching in a clockwise or counterclockwise direction from a
fixed point, or conducting a line strip search. All these form part of conducting a
professional systematic search of a death scene. A systematic approach to the searching of
death scenes reduces stress and fatigue and ensures a more comprehensive search and
recovery operation, minimizing the chance of losing potentially valuable evidentiary
material (Horswell, 2005b). Any weapon or other item possibly related to the death and
found at a scene should be brought to the morgue for analysis by a forensic medicine expert.
Often, substances are the causative agent in the death. All medication and alcoholic
beverage containers should be confiscated as these will be invaluable to the toxicologists.
Note the location where each item was found. Studies have shown that a fatal intoxicant is
likely to be found in the same location as a decedent. Any drug paraphernalia, notes, or any
unusual item that might have been used by the subject should be confiscated (Dix et al.,
1999).
4.9 Interviewing persons regarding the death

Interviews should include basic information such as the subject's identification, clothing,
time, date, state of health, date and time the body was discovered, and medical,
employment, and social history. Any recent events that may have a bearing on the death are
also important. A death investigator should always ask if a decedent had recently been
involved in any potential harmful situations. This information may be extremely helpful if
later attempts are made to make a prior incident a contributing factor in the death. If suicide
is suspected, it is preferable to interview family members and close friends as soon as
possible after the death is discovered. This may preclude guilt-related, subconscious,
erroneous statements made by loved ones several days later (Dix et al., 1999).
4.10 Estimating the post-mortem interval at the scene

The general warmth or coolness of the hands and face can be assessed by touch, and the
degree of rigor mortis felt by gently testing the limbs. The ambient (environmental)
temperature must be taken as soon as possible after the discovery of the body, preferably by
police scene of crime officers who usually arrive at the locus before the forensic medicine
expert. The ambient temperature should be taken as near to the body as possible, as
microenvironments can exist, even inside buildings or rooms. Information should be sought
as to how much disturbance of the ambient temperature might have occurred, such as
opening doors and windows, or turning fires or central heating on or off, so that some idea
of post-discovery distortions of temperature can be estimated later. The insertion of a
thermometer into the rectum at this stage in the investigation, as advocated by some
textbooks, is controversial.
At a scene of death, this usually means either pulling down trousers or pants, and otherwise
disturbing clothing, often in cramped and ill-lit places, frequently out in the open. It also
risks contaminating the rectum and perineum, by introducing seminal fluid from the anal
margin into the rectum, making subsequent examination of that area (and taking swabs for
semen) of reduced value. As so many violent crimes now have sexual or homosexual
overtones, the practice of taking rectal temperatures at the scene should be performed only
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if the forensic scientists or police scene of crime officers are satisfied that trace evidence from
the clothing, swabs from the vulva, vagina and anus, etc., can be obtained satisfactorily
before rectal thermometry is performed.
In other words, a cost-benefit analysis must be made at the scene, to decide if the difficulties
of taking a rectal temperature are worth the small potential advantage of an earlier
measurement. In many cases, where the body has obviously been there long enough for the
core temperature to have reached ambient - or where other circumstantial evidence has
indicated that the time of death is known to a greater degree of accuracy that can be hoped
for by thermometry - then nothing is lost by postponing the procedure until the body
arrives at the mortuary for autopsy, which, in British practice, is usually directly after the
body is moved from the scene.
If the autopsy is to be delayed for many hours owing to difficulties with transport or lack of
facilities, then much more must be done at the scene and temperature measurements are
justified.
An alternative is to use a place other than the rectum. The axilla and mouth give low
readings, which cannot reliably be correlated with the deep temperature because of variable
exposure to the air temperature. More useful is the auditory meatus or nostril, the
thermometer or thermocouple probe being inserted as deeply as possible. Reliable,
reproducible readings can be obtained from these sites, which have the great advantage of
being easily accessible without moving clothing, as well as not being required for swabbing
to investigate possible sexual assaults (Saukko & Knight, 2004).
Using scene markers to determine when an individual died, though unscientific, is often
more accurate than determinations made by scientific means. This is especially true in badly
decomposed bodies. Scene markers include:
 Uncollected mail or newspapers.
 Whether the lights are on or off.
 A TV schedule opened to a time and date.
 How the individual is dressed.
 Any food that is out or dirty dishes in the sink.
 Sales receipts or dated slips of paper in the deceased’s pockets.
 When the neighbors last saw the individual or observed a change in his habits. Thus, if
he typically went for a walk every evening and suddenly is no longer seen, then one
might conclude that death occurred on or about the day he failed to take his walk
(DiMaio & DiMaio, 2001). Different clues from a scene also must not be overlooked:
Was food being prepared? Was a major appliance on? Were there indicators as to a
decedent's activities just prior to or at the time of death? A forensic medicine expert
may use the answers to such questions to arrive at an estimation of the time of death
(Dix et al., 1999).
4.11 Ending the death scene investigation

When the forensic medicine expert has made the best examination possible in the
circumstances, his next function is to ensure that the corpse is removed to the mortuary for
autopsy with the least disturbance and loss of evidence. He should supervise the removal
himself or at least delegate the duty to another person whom he knows is careful and
competent. Each hand should be enclosed in a bag, secured at the wrist by adhesive tape or
string. A similar bag should be placed over the head. The packaging medium may vary, but
generally paper bags are recommended.
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The body should be placed gently in a 'body-bag', which has a zip closure, or moved on to a
large, new plastic sheet, at least 2 metres square. If a sheet is used, the edges should be
wrapped over the body and secured with adhesive tape. The object of the exercise is to
retain any loose objects, hairs and fibres that may be adhering to the body or the clothing.
The sheet or bag is taken by the forensic laboratory after the body is removed in the
mortuary so that they may screen it for trace evidence. The transport of the body is the
responsibility of the police or other agency such as the coroner or his officer. The body in its
plastic wrapping should be placed in a rigid fibreglass 'shell' or ordinary coffin, and taken
by hearse, van or police transport to the chosen mortuary.
Physical damage during the removal should be avoided as much as possible, though in
difficult or inaccessible sites this is easier said than done. In fires, the body may be seriously
damaged before or during recovery, sometimes because its presence is not suspected in the
smoke-filled, often waterlogged, debris of a conflagration. Handling brittle, charred, bodies
can easily cause the splits at joints that may mimic ante-mortem injuries.
In summary, the function of a forensic medicine expert at any scene of suspicious death is to
observe the situation, to conserve any fragile evidence, to supervise the removal of the body
and offer an opinion, based on experience, about the nature of death where this can
reasonably be done. He is not there to act as a latter-day Sherlock Holmes, voicing
unsubstantiated theories on non-medical matters, nor attempting to overinterpret the
situation from the flimsiest of facts. The forensic medicine expert is part of a team of
specialists, all experts in their own field, and it is as a member of such a cooperative,
coordinated group that his best contributions can be made (Saukko & Knight, 2004).
5. Homicide
In the community, the most serious crime is that of the intentional killing of one person by
another and it is therefore necessary that each of these events be thoroughly investigated by
a team of specialists (Horswell, 2005b).
Death scenes may be indoors or outdoors. The death may have occurred at the scene or the
body may have been “dumped.” The death scene may be untouched since the crime was
committed or it may have been contaminated by the untrained or the unwary. The murderer
may have intentionally altered the scene in an effort to mislead investigators or make a
statement, usually a defiant one. A crime scene altered in this manner is said to have been
staged.
The forensic medicine expert’s focus is mainly on the body. What is the position of the
body? What clothes are on the body and are they intact, dirty, torn, or rearranged? If there is
blood, is it spattered or pooled? Detailed photographs of the body and the surroundings are
critical. What is the temperature of the body? What is the ambient temperature? What
injuries are visible? What is the state of rigor mortis? Are there any signs of a struggle? Does
anyone know the identity, or presumptive identity, of this person? If there are bullet
wounds, the forensic medicine expert determines where the entrance wound or wounds are.
If there are exit wounds, the forensic medicine expert notes the presence of bullet holes in
the walls or other objects to help determine the position of the victim when the shots were
fired. Here, the expertise of the ballistics or firearm expert is crucial (Adelman, 2007).
Homicide victims need to be examined front and back to determine the nature and extent of
injuries. For example, once the nature of the injuries is confirmed (gunshot wounds with no
casings on the scene), the police will be able to focus their efforts on finding a shooter with a
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revolver, as opposed to searching for an assailant with another type of weapon such as an
ice pick. Once the extent of injuries is seen, the forensic medicine expert will know how
many radiographs are required. A beating death will alert the team that a struggle may have
ensued, and scalp hair and fingernail scrapings/clippings are required, in addition to a
blood standard obtained during the autopsy. Whenever sexual assault/battery is a
possibility, specimens for a sexual battery kit must be obtained from the deceased victim
prior to cleaning the body. Bodies with patterned injuries from an object or weapon still at
the scene should be photographed with the object close to, but not touching, the injured part
of the body. The patterned injury and the object should be photographed separately with a
scale. A weapon may be brought to the autopsy for comparison with the wounds only after
the weapon has been processed for trace evidence, DNA, and fingerprints to prevent
allegations of contamination at the autopsy (Rogers, 2004; Lew & Matshes, 2005).
It is always advantageous for the forensic medicine expert to visit the death scene of a
possible homicide. By visiting the scene and actually seeing the position of the body and the
pattern of injuries to the deceased and the arrangement of objects in the surrounding areas,
the forensic medicine expert can put the pieces of the puzzle together and attempt to
reconstruct the circumstances that led to the event (Fig. 3). The autopsy becomes a major
item in the solution of this puzzle (Adelman, 2007).
Fig. 3. The murderer sometimes binds the victim’s hands and mouth before killing the
victim. 65-year-old man was found dead in his bed, his hands and mouth were bound. The
cause of death was strangulation and blunt head trauma.
Always be professional―remember that onlookers, including the decedent's family, and
news media may be at the perimeter of the scene, so do not say or do anything that would
reflect poorly on yourself and the organization you represent. Trash (discarded gloves, etc.)
should be placed in bags designated for investigators' refuse, and not in the garbage cans
that are part of the scene because in actuality, they are evidence. Never remove items from a
scene for souvenirs (Rogers, 2004; Lew & Matshes, 2005).
In any given case of suspected homicide, it is self-evident that the forensic medicine expert
who performs the autopsy should visit the death scene because all injuries must be
examined within the context of the event. There are still far too many cases where this does
not occur, thus making it impossible to carry out an exact reconstruction of the sequence of
events in later stages of the criminal investigation. In numerous cases, however, the initial
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situation is inadvertently changed by police forces or rescue teams that first arrive at the
crime scene. As a result, the initial scene is often not sufficiently documented, and changes
may lead to misinterpretation in the future analyzing process (Schröer & Püschel, 2006).
Don’t forget: The victim himself or herself is the most important crime scene (Trestrail, 2007)
(Fig. 4) .
Fig. 4. The murderer killed his 36-year-old brother by strangulation and blunt head trauma.
The victim was found on the floor in prone position near his bed. The belt buckle of the
killer was found inside the hand of the victim (arrows) and this belt buckle helped in
identifying the killer. Strangulations should be presumed to be homicidal unless proved
otherwise. In order to determine the origin of ligature strangulation, it is necessary to
perform a detailed investigation of death scene and examine the type of ligature on the neck
of the victim carefully (McMaster et al., 2001; Verma & Lal, 2006).
In some murders, after killing the victim, the murderer uses a very sharp cutting weapon (a
saw, axe, etc.) to sever the limbs and cut the body into small pieces. The operation is
generally carried out immediately after the crime, although more rarely a long time may
pass between the two events. Dismemberment of the corpse allows the murderer to clear the
scene of the crime to delay investigations until the body is found. It also makes it easier to
transport the body even for long distances, during times of day when possible witnesses
could be about, without raising suspicion (Di Nunno et al., 2006). In a case which authors
visited the death scene, a 57-year-old woman’s corpse was found between the bed and
wardrobe in her house in a prone position. The victim’s severed head, right arm and both
hands were found in a cardboard box near the bed. After death scene investigation and
autopsy the murderer was determined as her 33-year-old schizophrenic daughter (Dogan et
al., 2010a) (Fig. 5) .
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Fig. 5. The 57-year-old woman was found between bed and wardrobe. Her severed head,
right arm and both hands were in a cardboard box. On the right, bloody sports suits of the
assailant in the washing machine.
Fig. 6. A homicide victim found in a well (on the left) and a victim who was burned (on the
right, note the unburned parts of the clothes which are useful for identification).
Sometimes the assailants, after killing the victims, try to hide their crimes by disposing the
corpses by burying, by burning, by throwing them into water or wells, or concealing them
in distant places (Figs. 6-8).Corpses found in wells or lime pits must be identified, and the
cause and manner of death must be determined. There are several circumstances that may
lead to the presence of corpses in wells. People may accidentally fall in wells where safety
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measures have not been taken. Other people may use wells for the purpose of committing
suicide. Also, the victims of a homicide may be thrown into a well for concealment. A
murdered victim may be thrown into a well to prevent the body from being found. In the
cases of homicide, the wells chosen to dispose of the body are often distant from the victim’s
district and close to the killer’s district. Wells can provide a means for concealing a corpse
and that the corpse can sometimes only be found upon a confession by the killer (Dogan et
al., 2010c).
Fig. 7. A buried homicide victim in a desolate land.
Fig. 8. The victim was killed by blunt head trauma first, then 20 kg iron (arrows) was bound
to his legs and thrown into a lake.
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Homicide-suicide (HS) events are defined by a perpetrator killing one or more victims
before killing him or herself. The term ‘‘dyadic death’’ has also been used for these
incidents, because deaths often involve a pair of persons (Milroy et al., 1997). In most of the
HS cases, the perpetrator knows the victim (Dogan et al., 2010e) (Fig. 9).
Fig. 9. Death scene of a homicide-suicide. Twenty two-year-old man killed his 16-year-old
lover (illegitimate relationship), then killed himself with his handgun. The man was married
with another woman. Note the handgun between victims.
5.1 Homicide by poisoning

Our ability to detect poisons has greatly improved over the last 100 years, but our ability to
suspect poisoning in the first place has not improved, and may have actually gotten worse.
Some things that might come up in an investigation that should send up a red flag are as
follows:
 The death occurred in a normally healthy individual. Certainly a person can die
without warning, but when this type of death occurs, a deeper look into the cause is
called for, including an autopsy.
 An individual interfered with the victim receiving proper medical attention. This may
lead one to wonder if that person does not want educated eyes and minds delving into
the possible cause of the condition in question.
 There is no sign of violence to the body. This is always an indication that the death
could have been the result of a poisoning misadventure.
 The affliction appeared as a natural disease yet failed to respond to normal treatment
methods.
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 An illness reoccurred in cycles; that is, the victim became ill at home, went to a medical
facility and seemed to recover, then went home and became ill again, and so on. This
would indicate that there is something in the home environment that is proving
unhealthy for the individual. Could it be the chronic administration of heavy metals
(e.g., arsenic) in the person’s meals? There certainly have been recorded criminal cases
in which this has happened, and the poisoner is often not caught in the initial stages of
the homicide attempt.
 There are common mysterious symptoms in a common group of people. This could
indicate that there has been a mass tampering, or that the supposed specific target was a
off the mark of the poisoner.
 There is an individual who is anxious to dispose of food, drink, or medicine of which
the victim partook. In this case, it is clear that the person is attempting to foil the
investigation by destroying critical evidence.
 An individual prevented friends or relations from being sent for during the victim’s
illness. The criminal investigator should question what that person did not want others
to witness.
 There is an insistence on no autopsy. The criminal investigator should clearly state that
one will take place. Once again, the desire not to have educated minds look at the
problem comes to the forefront.
 There is an insistence on a rapid cremation. This could be construed as an attempt to
burn the primary evidence of the crime and foil the investigation. The criminal
investigator should clearly state that an investigation must take place before cremation
can proceed.
 While grieving over the loss of a close family member or friend, a certain individual
does not freely begin to offer an explanation for the cause of death. Neither will the
person attempt to guide the investigation in any way. If the person does, it could very
well be an attempt to divert investigators’ attention from his or her crime, and
investigators must be aware of this.
 An individual shows a familiarity with poisons and possesses literature about poisons.
In this case, not just a red flag should go up, but a whole sky full of mental fireworks
(Trestrail, 2007).
It has been stated that poisoning is the least used method of homicide, accounting for only
3–6% of known homicide cases (Adelson, 1974, as cited in Trestrail, 2007). Because of the
complexity of poisoning homicide, it is one of the most difficult homicides to prove.
Regarding the death scene, with poisoning multiple locations may have come into play
during the planning and execution of the murder. Each location can yield important clues
that must be included in the complete case investigation. Some of the locations and the
items to look for to yield clues are as follows:
 Where the victim was found (vomited material, clothes containing poison residue).
 Where the poison was administered (medicine bottles, food/beverage containers).
 Where the poison was disposed of (storage areas, trash containers, sink traps, vacuum
cleaner bags).
 Where the poison was prepared (tools with poison residues, utensils, clothes, containers).
 Where the poison was procured (stolen items, receipts of purchase, signature on a poison
register, computer files) (Trestrail, 2007).
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6. Suicide
The finding of suicide as a manner of death may impact families differently. Suicide carries
a stigma on the family name in many cultures. Consequently, objections are often raised by
survivors when suicide is documented on the death certificate. Additionally, the finding of
suicide can influence the payment of life insurance policies, as most exclude suicide within
the first two years after issuance of the policy in order to prevent profit from an individual’s
death (Moldovan, 2008).
Fig. 10. Thirty seven-year-old woman hanged herself in her house. There was a farewell
letter on the floor.
Fig. 11. Two items found at a suicide death scene. The packing of a rodenticide and the last
SMS message in the victim’s phone which was sent to his friend containing a suicide note.
An experienced death investigator would recognize several ingredients in the scene. A
plastic bag, a large rubber band used to hold the bag in place, and drugs or alcohol are often
present in suicide scenes. In his book Final Exit, Derek Humphry described in detail how a
person can take their own life using the equipment and procedure described here
(Humphry, 1991, as cited in Moldovan, 2008). Moldovan (2008) had observed this death
scene frequently in the many suicide scenes he investigated. He often found the book Final
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Exit near the body, indicating that the decedent used it as a reference for the final act of self
destruction.
Fig. 12. Seventy eight-year-old woman hanged (partial) herself in the garden of her house.
Fig. 13. The father hanged himself to the same place where his daughter hanged herself (as
he loved his daughter so much, he couldn’t stand up to the pain). There were farewell letters
in both of their clothes’ pockets.
Hanging is one of the most preferred methods for suicide, but homicidal hangings were also
reported (Vieira et al., 1988; Sauvageau, 2009). So it is important to visit a death scene in
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hanging deaths. To determine the cause of death in hanging cases, while the corpse is still at
the death scene and in the suspended position, a detailed investigation should be performed
by a team including a forensic medicine expert. Further evidence from the death scene
investigation, statements from witnesses, the presence of a suicide note, and autopsy
findings can all help to determine whether the victim was responsible for his or her own
death (Figs. 10-16).
Fig. 14. The hand of the suicide victim is still gripping the handgun (cadaveric spasm).
Fig. 15. The death scene of a 13-year-old boy’s suicide. The shotgun is in front of the victim,
and the entry wound is under the chin. According to the witnesses, after he argued with his
father in the garden of his father’s office, he had ran into the office of his father and took his
father’s shotgun and killed himself.
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Fig. 16. The death scene of a 75-year-old woman who stabbed herself in the neck. There is a
basin which she collected her blood (right top) and there are hesitation wounds on the neck
(right bottom). She had psychiatric problems.
The tying together of the wrists in hanging cases is rare, but may not indicate a homicide, so
long as the hanging ligature could not have been self-applied. At first glance, a hanging
body found with their hands tied together would give the impression of a homicide but
some suicidal people try to avoid being rescued by others or themselves. The closing of the
mouth with a plastic bag or a scarf was thought to have removed the possibility of calling
out for help during the hanging. Both the tying together of the hands and closing of the
mouth were regarded as precautions taken by the victims to prevent any change in mind
and an indication of their resolve to go through with the suicide (Fig. 17). In addition,
placing soft material against the ligature loop was thought to be an attempt to lessen the
feeling of pain (Demirci et al., 2009a) (Fig. 18).
Fig. 17. Fifty two-year-old man who hanged himself. Both hands were tied limply behind
his back with clothesline and a plastic bag was tied around the mouth.
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Fig. 18. A soft piece of cloth against the ligature.

In some cultures, religious books and findings indicating praying before suicide may be
found at death scene. Demirci et al. (2008a) reported that in investigating medicolegal death
cases believed to be of suicidal origin, evidence showing that this action was committed by
the victim, the presence of a suicide note at the death scene, and a history of a previous
suicidal attempt, the presence of daily axillary and pubic shaving on the external
examination of the victim’s body, when of the Muslim faith, may also be considered a
feature of suicide (Fig. 19).
Fig. 19. A death scene of a 42-year-old woman’s suicide. There was a razor and cut axillary
hair in the sink of the bathroom (arrows on right up). Also a prayer rug, pictures of herself,
her husband and two daughters, and her ring were on the carpet of the room (right down).
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Fig. 20. Forty one-year-old man went to the woodland with his motorcycle (arrow) and
hanged himself on a tree. A suicide note about his familial problems was found in his
pocket.
Fig. 21. The corpse of a 70-year-old woman who threw herself into the well in the garden of
her house. A farewell letter was found in her house.
Suicidal acts carried out in places open to public can be highly traumatic for witnesses (Owens
et al., 2009; Reisch & Michel, 2005; Tranah & Farmer, 1994). Moreover, they are considered
more newsworthy than those occurring at home, and media reporting may encourage further
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suicides (Michel et al., 1995; Pirkis et al., 2007). It was suggested that nearly a third of all
suicides occur in public places (Owens et al., 2009). The association of bridges and high
buildings with suicide by jumping is well-known, but many other public places offer means or
opportunity for suicide. Hanging, car exhaust poisoning and burning involve elaborate
preparations and require seclusion. For these deaths, woods and isolated rural car parks
provide the perfect opportunity (King & Frost, 2005) (Fig. 20). Wells are a preferred locality for
suicides, which is the one reason why individuals may jump into a well, regardless of whether
there is water or not. A suicide by drowning, although seen in all age groups, seems to be a
preferred method for the elderly individuals (Dogan et al., 2010c) (Fig. 21).
7. Accident
An accidental death scene investigation launches after someone is dead in an automobile or
other such accident. The investigation evaluates evidence, usually immediately, as to how
the accident occurred. These are some types of accident scenes:
Auto Accident - An auto accident scene investigation may include an accident reconstruction
if liability is in dispute. An investigator diagrams and photographs the auto accident scene
and evaluates several factors, including points of impact to the vehicles, skid marks,
roadway conditions and witness statements.
Fire - The investigator photographs, diagrams and examines the scene. The person who first
discovered the fire and the participating fire personnel are interviewed. Physical evidence
may be collected for further examination. A report may be drafted about the investigator's
conclusions.
Slip and Fall - If a patron slips and falls, the slip and fall accident scene investigation usually
begins with one of the employees of the establishment. What kind of fall occurred? Was
there a defect in the ground or flooring? Was there a hazardous condition? How long were
these conditions exposed? Photographs of the scene may be taken, and available witnesses,
including the store employee or manager, are interviewed.
Fig. 22. A 17-year-old worker was dead due to electric shock in his workplace. The scene
investigation revealed electrical leakage from the defect of the plastic sheath of the cable of
carpet washing machine.
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It is sometimes difficult to determine the manner and cause of death, if a detailed death
scene investigation is not performed. In a case reported by Demirci et al. (2008b), a 30-year-
old man’s death was due to throat-cutting. They reported that although the cut in the neck
initially suggested homicide, it was found to have occurred as a result of an accident in his
workplace after the death scene investigation and autopsy. This case emphasizes the
importance of the examination of incident scene and autopsy in determining the origin.
Similar cases are deaths due to electric shock. The forensic medicine expert should visit the
death scene before the autopsy if it is possible (Fig. 22).
Carbon monoxide (CO) is a colorless and odorless gas, and is lighter than air. It is an
incomplete combustion product of hydrocarbons. About 600 accidental deaths due to CO
poisoning are reported every year in the United States. CO usually causes accidental deaths,
because it is pure and odorless (Thom & Keim, 1989; Cobb & Etzel, 1991; Saukko & Knight,
2004) (Figs. 23,24).
Fig. 23. A family (father, mother and child) was found dead in their bed due to carbon
monoxide poisoning. There was a coal stove in the room and soot traces (arrows) were
observed at the entry point of the stovepipe on the wall which indicating leakage of smoke
of the stove.
Carbon monoxide can affect drivers of a moving vehicle, usually owing to a defective
exhaust system that allows gas to percolate through the floor or engine bulkhead into the
interior. Rarely, a strong following wind can blow the external exhaust-gas through the
open doors of a van or truck. Another cause is a leak in the heat exchanger in vehicles that
use a direct air supply from around the exhaust manifold to provide passenger heating
(Saukko & Knight, 2004). In motor vehicles in which persons must remain for a long period
of time while the vehicle is parked, for example, trucks with sleeping cabs, road service
vehicles and mail trucks, a separate heater (working independently of the engine of the
vehicle) may be used to heat the vehicle. The engine of the apparatus works with diesel fuel
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or gasoline. Combustion products burning in the pre-combustion chamber heat the fins of
the engine. The air passing through the fins is heated and is transferred into the cabin.
Malfunction of such an apparatus may be the cause of CO poisoning or fire. So the
supplementary heater in the truck might be the cause of fatal CO poisoning and of the fires
in the cabins of the trucks (Demirci et al., 2009c) (Fig. 25).
Fig. 24. A 42-year-old man was found dead due to carbon monoxide poisoning in his
bathroom. There was an LPG water heater which had not a smoke pipe in the bathroom.
Fig. 25. A 48-year old male truck driver (ellipse) was found dead due to a fire in a truck
parked in an open area of the truck garage. Scene investigation revealed that the cause of
fire was broken supplementary heater (arrows) in the truck.
Decapitation may be suicidal, homicidal, and accidental. Accidental decapitations can result
from traffic accidents, or occupational accidents. Decapitation by industrial trauma can
occur at any age, and is often associated with heavy machinery in workshops or farm
equipment being towed behind a tractor (Sharma et al., 1995). The helix elevator is an
appliance connected to a tractor. It is used for loading grains from a field to any vehicle,
such as a trailer, for transportation. In a case Demirci et al. (2009b) reported, the victim was a
41-year-old male farmer. In the stackyard, a helix elevator machine was loading a trailer
with barley while the victim was distributing the loaded barley with a shovel in the trailer.
He had tied a scarf loosely over his face and neck because he was allergic to the barley dust.
When the victim’s head and neck were level with the turning helix elevator shaft, the scarf
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was pulled up and wrapped around the shaft. The scarf then slid around the victim’s neck
and tightened, causing the head to separate from the body (Fig. 26).
Fig. 26. A 41-year-old male farmer was found decapitated in the stackyard. He was working
with a helix elevator machine. He had scarf tied loosely over his face and neck but the scarf
was pulled up and wrapped around the shaft of the machine. The scarf then slid around the
victim’s neck and tightened, causing the head to separate from the body.
Possession of firearms is limited because of the technological requirements in production
and strict laws. However, anyone can manufacture a handmade firearm by following some
simple instructions and these actions do not a carry any legal liability. A mole gun is an
unusual weapon used to kill moles in agricultural areas. Mole guns are primitive weapons
produced for the purpose of trapping and are capable of firing a standard shotgun cartridge.
Injuries and deaths caused by mole guns are generally a result of an accident while the
victim is setting or controlling the gun (Demirci et al., 2008c) (Fig. 27).
Fig. 27. A 42-year-old man was injured on the right thigh region while he was setting a mole
gun to kill moles which were damaging the vegetables in his field, and died shortly after at
the incident scene. The mole gun was found at the scene.
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In some cases, it is important to distinguish accidental manner from suicidal or homicidal

ones. For example, if a ligature mark is present on the neck, this is usually suicide or
homicide. But sometimes the death may be accidental origin. In a case reported by Dogan et
al. (2010d), a 53-year-old woman who had been working in the laundry of a hospital sat on
the counter of the ironing machine to heat her back and leaned her back closer to the
machine in a cold winter day. At that point, her coworkers left the room. When they re-
entered the room 15 min later, they found her dead and observed that her scarf was caught
in the roller cylinder of the ironing machine (Fig. 28).
Fig. 28. The chief physician of the hospital showing the victim who was found strangulated
with her scarf by the roller cylinder of the ironing machine in the laundry of the hospital. On
the right side the ligature mark is seen on the neck.
Children have an increased risk for injury or death from accidents for a variety of reasons
compared to adults. Perhaps the greatest reason is their natural curiosity, which leads them
to explore their environment and investigate situations where they often do not recognize
potential hazards (Byard, 1996). Accidental asphyxia can occur in childhood as a result of a
variety situations (Dogan et al., 2010b) (Fig. 29).
Fig. 29. The one-year-old child’s neck was entangled in a tight cable of the electric heater
while he was crawling on the floor of the living room.
Farm accidents are a frequent occurrence in many countries; for example, in the United
States, farming is rated second only to mining in terms of occupational danger (Rivara,
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1985). Unguarded agricultural power take-off (PTO) drivelines and the related components,
including secondary drivelines powered by the PTO, have been historically recognized as
serious farm-related hazards that can cause severe, permanently disabling injuries and
death when entanglement occurs (Beer & Field, 2005). PTOs are rapidly rotating shafts that
transfer power from the tractor attached at one end to a piece of farm machinery at the other
end. Clothing or body parts can become entangled, resulting in amputation or avulsion of
body parts, strangulation, and massive crushing injuries (Karlson & Noren, 1979) (Fig. 30).
Dogan et al. (2010f) reported that 5.8% of the farm tractor-related fatalities involved deaths
resulting from PTO entanglement. In these cases, there were extensive crush injuries to the
chest, abdomen, and extremities. None of the turning shafts in these cases had safety
shields.
Fig. 30. Fatality involving power take-off (water pump) entanglement.
8. Natural deaths
An important portion of the deaths investigated by forensic medicine experts involve
natural diseases, the most common being cardiovascular disease. Natural diseases processes
alter the way the body reacts to and repairs from injuries. The older the person, the more
likely that natural disease has a role in the death (Figs. 31,32). This concept can work in
reverse. One can erroneously assume that because the person is young, natural disease is
not a factor in the death. Many people have unknown or undiagnosed natural diseases that
manifest in sudden, unexpected death. A common history in these cases is that “he hadn’t
seen a doctor in years” or “he didn’t believe in doctors.” The result is that the first doctor he
sees is the forensic medicine expert, who diagnoses what was a treatable natural disease
such as a cardiovascular disease. “Sudden death” is a term used frequently in death
investigation but its meaning can be ambiguous. In some situations, death can literally be
instantaneous, such as with a massive pulmonary embolus. In others, such as a myocardial
infarction, the death can be instantaneous, or take minutes to hours or longer. Sudden
cardiac death is a sudden, unexpected death from cardiac causes within one hour of onset of
symptoms (Wagner, 2009). Investigating natural deaths might not be very exciting to some
but can be interesting and rewarding. For example, Wagner (2009) reported that he found an
aortic aneurysm in a 14-year-old girl who died suddenly while running. Knowing this
condition to be genetic, a study of 12 family members showed the same abnormality in
three, thus saving those individuals the same fate as their relative.
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Fig. 31. Seventy six-year-old woman who had been living alone was found dead in her
home in sitting position and holding a glass in her hand. Note the livor mortis on face,
hands and left foot due to the position of the deceased.
Fig. 32. A 74-year-old man was found dead in half-naked position lying alongside his car.
The investigation revealed that when the man and a young woman were having sex, the
man suddenly deteriorated and died. The cause of death was determined as myocardial
infarction at the autopsy.
9. Sudden and unexplained infant death

Sudden unexplained infant death (SUID) is the sudden and unexpected death of an infant
due to natural or unnatural causes. SUID applies to the death of an infant less than 1 year of
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age, in which investigation, autopsy, medical history review, and appropriate laboratory
testing fails to identify a specific cause of death. Sudden infant death syndrome (SIDS) is one
of several causes of SUID. However, SIDS, unlike the other SUID causes, is a diagnosis of
exclusion. Even with a thorough death scene investigation, review of the clinical history,
and autopsy, SIDS is difficult to distinguish from other SUIDs, such as accidental suffocation
and asphyxia. In the world of death investigation, infant death investigation is unique. From
scene through certification, these investigations require skill and knowledge drawn from
disciplines outside those typically considered a part of medicolegal education (Corey et al.,
2007; Hanzlick, 2001; Shapiro-Mendoza, 2006). The post mortem examination, ideally should
include a history of the gestation, delivery and postnatal development, a death scene
investigation, a family psycho-social history, a complete autopsy, and a confidential case
conference (Bajanowski et al., 2007).
Having knowledge about the many causes of SUID, in addition to SIDS, is of utmost
importance for the death scene investigator. At the scene, the investigator will gather
evidence as well as information from the parents or caregivers who were with the infant and
who may be in a great deal of distress. All of this information is crucial for distinguishing
between a natural death, an accidental death, or a homicide.
The following is a brief overview of known causes of infant death that are oftentimes
overlooked during investigation, resulting in the cause of death being listed as SIDS on the
death certificate.
Asphyxia or suffocation is caused by the inability to breathe. This condition leads to a lack
of oxygen in the body, which can lead to loss of consciousness and death. Asphyxia can be
caused by choking, constriction of the chest or abdomen, strangulation, narrowing of airway
passages (severe allergic reaction or reactive airway disorders), or the inhalation of toxic
gases. Common objects that are involved with asphyxia or suffocation include plastic bags,
soft pillows, and soft materials such as bedding or stuffed animals. These objects can
occlude the mouth and nostrils, causing suffocation. The most commonly reported cause of
asphyxia in infants is accidental suffocation and strangulation in bed.
If the investigator is very observant, knows what to look for, and is particularly careful in
talking with the caregiver, he/she may pick up some clues that will help determine the
specific cause of asphyxia or suffocation and determine whether the manner of death was
accidental or intentionally inflicted. A thorough death scene investigation can help answer
questions about environmental factors that may have interfered with breathing (e.g.,
covering of the nose and mouth) or hazards related to aspiration, choking, electrocution,
excessive heat or cold, and other external factors.
There are a number of risk factors associated with asphyxia and suffocation. The following
is a list of the typical causes of infant asphyxia and/or suffocation.
 Overlaying or accidental suffocation on a shared sleep surface.
 Accidental strangulation from unsafe surroundings.
 Wedging or entrapment.
 Immersion in water or drowning.
 Choking.
 Neck compression (Shapiro-Mendoza, 2006).
Accidental asphyxia can occur in younger children and infants, who may move into
positions in which their airways become occluded, their bodies become wedged so that they
are unable to breathe, or they become suspended from their clothing or restraining
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harnesses (Gilbert-Barness et al., 1991; Nixon et al., 1995; Byard, 1996). Many houses in
Turkey (especially those situated in the villages and slums) are built with metal rings
mounted in the ceilings, so that the occupants can set up swing-like cradles, which are
hammock-like in nature. The cradles are constructed by tying two ropes between the two
metal rings and connecting them with cloth. Infants are placed in these cradles on top of
cushions, and ligatures (e.g., scarf, rope, or sash) are tied around the cradles to prevent them
from falling out. However, the ligature can wrap around the neck and asphyxiate the infant
if it leans out of the cradle (Dogan et al., 2010b) (Fig. 33).
Fig. 33. The position of dead infant in the swing-like cradle. As the victim leaned out of the
cradle, the ligature that was tied around the swing-like cradle wrapped around the child’s
neck, resulting in asphyxia.
There are a number of risk factors associated with the infant's environment that may be
connected with the death. The following is a list of causes typically associated with the
environment or death scene.
 Poisoning or intoxication.
 Electrocution.
 Hypothermia.
 Hyperthermia.
Inborn errors of metabolism are rare genetic disorders that stop or prevent the body from
turning food into energy. These disorders are usually caused by defects in the enzymes that
help break down foods in the body. When the body cannot process these foods, a buildup of
toxic substances or a deficiency of substances needed for normal body function can occur.
This buildup can be fatal if not controlled with diet or medication. Some metabolic diseases
are inherited.
Injuries can be fatal or nonfatal, and they can occur unintentionally or intentionally (because
of purposeful acts of harm). It is often difficult to determine whether an infant’s injury was a
result of an unintentional or intentional act. Examples of unintentional injuries include the
infant choking on a small toy or rolling over in bed onto the infant.
Shaken baby syndrome (SBS) is one form of abusive head trauma that occurs when an infant
or young child is violently shaken or struck against a hard or soft surface. Shaking may
cause bleeding over a large portion of the brain. SBS can cause severe brain damage as well
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as death. In cases where a child receives a head injury from a fall or other impact, there may
be external signs of injury, such as bruising or abrasions on the scalp. In SBS, there may be
no signs of injury on the infant (Shapiro-Mendoza, 2006).
9.1 SUID scene investigation

The physical environment of the death scene may play an important role in the cause and
manner of the infant’s death. Some research has indicated that the change of seasons, which
requires turning on or off heating or cooling devices (furnace, fireplace, air conditioner,
ceiling fan), might precipitate an apneic event. Therefore, it is important to determine,
describe, and document the specific environmental conditions of the scene such as room
temperature and other factors that may affect the microenvironment of the infant at the time
of death (e.g., air current from ceiling fan, humidity levels in a spa, water temperature in a
hot tub).
The forensic medicine expert should personally inspect the death scene to gain a thorough
understanding of the possible environmental hazards to which the infant might have been
exposed. He or she, should observe and document the furnishings in the room/area where
the infant was found dead or unresponsive. In addition, the investigator should describe the
general state of the room/area; if there is evidence of rodent, insect, or animal activity or a
generally unkempt situation, this should be documented as accurately and objectively as
possible. The scene should be documented with photographs, diagrams, and descriptions.
Fumes that are noticed at the scene might have contributed to or been the cause of the
infant’s death and should be noted in the investigative report. A description of the fumes
might provide forensic scientists with clues that will assist them in ordering laboratory tests.
The investigator should describe the fumes and their intensity and attempt to ascertain the
source of the fumes. If necessary, local fire department personnel should be contacted to
ensure that the scene’s air is clear of harmful substances.
The smell of smoke may indicate a live-fire situation or tobacco use at the scene. Smoke
might have contributed to or been the cause of the infant’s death. A description of the smoke
smell may provide forensic scientists with clues that will assist them in ordering laboratory
tests. The investigator should describe the smoke smell, its intensity, and its possible
sources.
Mold growth at the scene may have exposed the infant to dangerous airborne pathogens. A
description and location of mold growth may provide forensic scientists with clues that will
assist them in ordering laboratory tests. The investigator should describe the mold growth
and its location in relation to the infant’s sleeping/activity area. Photographs of any
suspicious material should be taken at this time.
The observance and documentation of peeling paint at the scene may indicate an infant’s
exposure to dangerous lead-based materials. A description of the room and the location and
size of the peeling paint area can provide forensic scientists with clues that will assist them
in ordering appropriate laboratory tests. The location and size of the peeling paint and its
location in relation to the infant’s sleeping/activity area should be described as accurately as
possible. The investigator should contact the local health department if the problem presents
safety concerns to persons in the vicinity (Ernst et al., 2006).
10. Conclusion
The forensic medicine expert should visit the death scene before the autopsy if it is possible.
Although, investigation and legal systems differs from country to country, there is always a
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crime scene investigation team. If the forensic medicine expert does not have the
opportunity to visit the death scene him/herself, he/she would check the documents (notes,
sketches, photographs, etc) which crime scene investigation team prepared. Many
medicolegal deaths may be resolved by death scene investigation. A forensic medicine
expert should never forget: If the death scene investigation is not performed before the
autopsy, that autopsy will be an imperfect autopsy.
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Demirci, S., Dogan, K.H., Erkol, Z. & Gunaydin, G. (2009c). Two death cases originating
from supplementary heater in the cabins of parked trucks. J Forensic Leg Med, Vol.
16, No. 2, Feb. 2009, pp. 97-100
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Di Nunno, N., Costantinides, F., Vacca, M. & Di Nunno, C. (2006). Dismemberment: a

review of the literature and description of 3 cases, Am JForensic Med Pathol, Vol. 27,
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3
Diagnostic of Drowning
in Forensic Medicine
Audrey Farrugia and Bertrand Ludes
Institute of Legal Medicine,
11 rue Humann 67085,
Strasbourg Cedex
France
1. Introduction
The diagnostic of drowning is described in the literature as one of the most difficult in the
field of forensic medicine (Piette & De letter, 2006). In fact, the external examination and the
autopsy findings are in most of the cases not specific and the laboratory investigations are
controversially appreciated by the scientific community. The main goal in this field is to
differentiate a death by submersion from a immersion of a body. Death of a victim found in
water should not always be related to drowning (Knight, 1991).
It is important to remind that the death by drowning is defined as a death due to
submersion in a liquid and the mechanism in acute drowning is hypoxemia and irreversible
cerebral anoxia (D.J. Di Maio & V.J.M. Di Maio 1989).
2. Physiopathology
Considering the pathophysiology of human drowning, the role of mechanical airways
obstruction and the washing out of alveoli surfactant as well as the shifts of fluid and
electrolytes are still debated. In fact, several phases were described during the drowning
process, first a breath-holding phase, followed by involuntary inspiration, gasping for air
and loss of consciousness. The death is secondary of the development of cerebral hypoxia
leading to irreversible brain damage. The duration of the phases is dependent on
various factors, such as age, previous diseases, breath holding tolerance of the victims
and the temperature of the water. Consciousness is usually lost within 3 minutes of
submersion.
The inhaled water enters the alveolar spaces of the lungs and destroys the surfactant
inducing pulmonary edema with the transudation of protein-rich fluid into the alveolar
spaces. The surfactant washout decreases the lung compliance and ventilation-perfusion
mismatch resulting to an hypoxemia secondary of non oxygenation of blood flowing
through underventilated portions of the lung. A non cardiogenic pulmonary edema will
result with secondary metabolic acidosis. This is the main pathophysiological mechanism of
drowning and the fluid and electrolyte shifts are quite secondary.
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It was stated that fresh water is hypotonic and hyponatremic relative to blood inducing,
after inhalation, a movementt of water from the alveoli into the blood and movement of
sodium from the blood into the alveoli. These changes induce haemodilution, hypervolemia,
hypnonatremia, hyperkalemia and haemolysis (Jeanmonod et al., 1992). As the sea water is
very hypertonic relative to the blood, the water movement goes from blood into the alveoli
and the electrolytes (sodium, chloride, magnesium) from the alveoli into the blood. The
consequences of the sea water drowning should be haemoconcentration, hypovolemia and
hypernatremia. The biochemical tests that proposed to assess the diagnostic of drowning
are based on these fluids and electrolytes shifts. It is during the phases where water is
penetrating from the alveoli into the blood circulation that particles like diatom passing
through the alveolar-capillary interface before reaching internal organs.
A vagal reflex may be also induced by inhalation of water, it will increase peripheral airway
resistance with pulmonary vasoconstriction, decreased lung compliance and reduction of
ventilation – perfusion ratios (Ornato, 1986).
An intense stimulation of nerve endings at the skin, the mucosa of the ear drum, the
pharynx or the larynx by cold water can lead to a cardiac reflex arrest. It was assumed that
10% of the drowned humans die after laryngospasm or breath-holding without actually
aspirating fluid (Ludes & Fornes, 2003). A discussion was also hold about the volumes of
inhalated water and the effect on the circulation. In drowning, the inhalated volume of
water can range, from relatively small to very large. It has been showed that small amount
of water, particularly cold water, may induce vaso vagal reflex or cardiac arrest reflex.
When great amounts of water are inhalated and pass through the alveolar-capillary interface
and enter the circulation, the phenomenon of destruction of surfactant and of the alveoli
architecture leads to asphyxia. During the entering of water into the blood stream, the
diatoms present in the drowning fluid may reach the internal organs.
To establish the diagnosis of drowning it is of particular importance to correlate
informations about the circumstances preceding the death, the past medical history of the
victim if known, the circumstances of the body recovery from the water, the external
examination, the autopsy findings and the results of the complementary analyses
(histologic, biochemical, toxicological analyses and diatom test).
3. Autopsy findings
The majority of the autopsy findings are related to asphyxia and have no specific link to
drowning. The signs of drowning depend on the delay in recovering the body and on the
development of the putrefaction phenomenon which alter the positive signs of drowning.
One of the signs of drowning would be large amounts of froth present around nostrils and
mouth in freshly drowned bodies. This froth is also present in the upper and lower airways.
Froth can also be observed in cases of edema of left ventricular failure but in drowning cases
the volume of froth is generally much more abundant than in other origins.
It is admitted that lung weights are higher in drowning cases but it was shown that normal
weights are possible in the drowning cases after cardiac arrest reflex or vaso vagal reflex.
After water inhalation, the lungs may be over inflated, filling the thoracic cavity, generally
water logged referred to as “emphysema aquosum”. So the surfaces of lungs have a marbled
appearance with dark red areas linked with collapsed alveoli, interspersed with more
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Diagnostic of Drowning in Forensic Medicine 55
aerated tissues areas. The fluid is trapped in the lower airways and blocks the passive
collapse of the bronchi that normally occurs after death. Subpleural bullae of emphysema,
sometimes hemorrhagic may be found and are related to the rupture of the alveolar walls
(Pounder, 2005). Even if these signs are mostly evocating of drowning, none of them is
pathognomonic of water inhalation.
The body having sunk to the bottom of the site of drowning, will show a pattern of
post mortem injuries such as post-mortem abrasion over the forehead, the prominent points
of the face, the anterior trunk, the backs of the hands and the fronts of the lower legs.
Injuries may also inflicted by passing watercraft in navigable waters by stumbling against
rocks or by animal activities. Accidental or suicidal injuries due to the way the person falls
or enters into the water may also be observed. Post-mortem injuries linked to the way of
recovery of the body using ropes and hooks can also be seen. These kinds of post-mortem
injuries can mimic ante-mortem wounds and the differentiation between ante and post-
mortem injuries is quite difficult because of the lack of the usual criteria of ante-mortem
wounds.
It can also be found sand, silt, seashells and weeds in the airways, lungs, stomach and
duodenum of drowned victims. If this material is fund in abundance within the alveoli, it
can be related to an immersion during life as long as it concerns a freshly drowned body.
This material may also enter the upper airways during the post-mortem immersion period
and it is possible that small quantities may enter the oesophagus and stomach but it is
unlikely that it will reach the alveoli to any significant extent if the post-mortem submersion
is short.
One of the sign of immersion is skin maceration becoming visible after various time interval
depending on the temperature of the immersion water. The skin becomes wrinkled, pale
and sodden like a “washer woman’s skin”. These changes appear at the finger tips, palms,
backs of the hands, and later, the soles. The next step is the detachment of the thick keratin
of hands and feet which pull off in “glove and stoking fashion”. Nails and hair become
loosened after a few days. Other signs of immersion are cutis anserine and post-mortem
distribution of hypostasis. The presence of mud, silt or sand on the body was described but
has no diagnostic value.
4. Complementary investigations
4.1 Histology
The microscopic investigations must be performed on all the organs of non putrefied bodies
in the aim to make the difference between a death by drowning and other causes of death.
The lung examinations can show over-distension of the alveoli, thinning of the alveolar
septa and compression with narrowing of the capillary network (Pounder, 2005).
The modifications in lungs are heterogeneous distributed and multiple sections must be
performed to assess the diagnostic. In fact the microscopic appearance may be entirely
normal in some part of the lungs.
Several staining techniques must be performed such as the staining for elastic fibers (orcein )
and reticulin fibers (Fornes et al., 1988 ; Ludes & Fornes, 2003). The examination of other
organs (brain, heart, liver) shows none specific histological changes indicative of hypoxia
such as acute congestion and swelling of the capillary endothelia.
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4.2 Biological tests

The chemical changes in plasma after drowning were based on the fluid and electrolyte
shifts after the penetration of either sea or fresh water in the alveoli and in the blood stream
(Modell & Davis, 1969). It was proposed the measurement of the specific gravity of blood, of
the concentration of sodium, chloride and potassium. For the electrolytes, the diagnosis of
drowning was based on changes of these electrolytes between the blood samples taken from
the right versus left ventricle (Bray, 1985; Couteselinis & Boukis 1976; Karkola &
Neittaanmaki, 1981). Such electrolyte shifts were described in many other causes of death
and do not provide reliable evidence of drowning.
A special mention must be made for the blood strontium analysis. The toxicological analysis
are performed to show the presence of medicaments or alcohol, taken before death in
suicidal or accidental conditions and to determine the serum level of strontium which is
described as a good parameter of drowning in sea water (Piette & Timperman, 1989). In case
of fresh water drowing, the water concentration of strontium must be higher than the serum
concentration to be a valuable parameter in favour of drowning.
Authors such as Kane et al. (1996, 2000) and Nübel et al. (1997) proposed the detection by
molecular biology techniques of the 16S rRNA subunits of ribosomal RNA for plankton
detection in tissues samples indicating an active water inhalation and may assess the
diagnostic of drowning. According to these authors, the sequence comparison of the
variable regions of 16S rRNA could provide sufficient information to allow the
discrimination of both close and distant phylogenetic relationships.
Abe et al. (2003) and Suto et al. (2003) proposed the detection of chlorophyll-related genes of
Euglena gracilis and Skeletonema costatum to identify plankton in the victim’s tissues. It is
important to emphasize that these methods give only qualitative results (He et al., 2008) but
the quantitative approach can only be achieved by the diatom test which may also give an
indication of the site of drowning.
In fact, diatoms can be considered as particles present in the submersion water which are
inhalated during drowning and once in the blood stream which reach the closed organs.
Under strict extraction and identification conditions, these particles are good markers of
drowning.
Diatoms are unicellular algae belonging to the class of bacillariophycae which includes more
than 15 000 species living in fresh, brakish or sea water. The skeleton of these algae is called
a frustule which is constituted by two valves fitting together to enclose the cytoplasm
(Ludes & Fornes, 2003) and made of hard silice.
Due to this hard silicaceous skeleton, diatoms can be recovered from putrefied or burnt
tissues by either enzymatic or acid digestion (Ludes et al., 1994). The identification of these
algae is based on the structure of their valves showing different symmetry allowing the
distinction of two main groups namely the centric diatoms and the elongated or pennate
diatoms.
After a long period of time where the use of the diatom test was very controversial due to
false positive results linked to the presence of diatoms in closed organs of non drowned
victims (Foged, 1983; Gylseth & Mowe, 1979; Schellmann & Sperl, 1979; Schneider, 1980,
1990; Schneider & Kolb, 1969), it was stated by several authors that under strict defined
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analytical conditions this test could discriminate between drowning and none drowning
cases (Auer & Möttönen, 1988 ; Neidhard & Greedyke, 1967; Peabody, 1977; Pollanen,
1997,1998; Pollanen et al., 1997). Auer & Möttönen (1988) were one of the first authors to
propose that 20 diatoms per microscope slide obtained from lung samples should be a
sufficient concentration to exclude false positive due to contaminations. We also proposed
qualitative and quantitative criteria for a positive drowning diagnostic with the diatom
test.
For us, an analysis will be considered as positive when at least 20 diatoms are identified per
100 µl of a pellet sediment extracted from a 2 g lung sample and the identification of more
than 5 complete diatoms (with exclusion of fragments) per 100 µl of a pellet sediment
extracted from a 2 g tissue samples such as brain, kidney, liver and bone marrow. Bone
marrow is described as a sanctuary organ and if diatoms reach this tissue, the diagnostic of
drowning could be assessed.
In controlled samples belonging to non drowned victims, we newer find a number of
diatoms above the fixed criteria. When diatoms were found in closed organs of drowned
victims, the results in lung samples were in each case also above the 20 algae per 100 µl
pellet. To assess the diagnostic of drowning, it is of high importance to perform a qualitative
analysis of the found diatoms and the comparison of the diatoms present in the closed
organs and the microflora of the presumed site of drowning.
In this aim, water samples must be collected at the drowning site (two samples of 100 ml) as
well as algae scraped from stones present in the water.
The samples are disposed in clean containers and the extraction and identification protocols
on water and tissue samples were described by our group (Ludes et al., 1999). All reagents
and glass containers must be checked for the absence of diatoms before use, and
contamination from exogenous diatoms must be avoided by using diatom-free water and by
protecting the organs during autopsy from the clothes of the victims and from the skin
surface.
At each step of the analyses and the identification of diatoms, a potential contamination
must be considered. This test cannot be proposed to assess the diagnostic of drowning in
bathtub or in water containing very few algae, for example in iced water.
If water samples are not available, it is possible to compare the diatoms found in the organs
with data collected in the rivers by a continuous water monitoring which can be set up by,
for example, the Agencies in charge of the survey of water quality.
We set up a continuous water monitoring of the main rivers of our area (Ludes et al., 1996).
Seasonal variations of the concentration of diatoms and the diatom profile are determined at
a given month by the five most frequent species. The relative abundance of each diatom
may also vary along the course of the river. So, the site of drowning may be determined by
comparison between the water microflora with the diatoms found in the lungs. In fact,
diatoms of more than 50 – 60 µm in size rarely pass the alveoli-capillary barrier even after
the rupture of the alveoli by the inhalation of water.
The diagnostic of drowning can be achieved when the qualitative analysis shows that the
algae found in the organ belongs to the water microflora and the quantitative criteria are
fulfilled (Hendey, 1973, 1980 ; Ludes et al., 1999).
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5. Conclusion
The diagnostic of drowning may be achieved after having considered all the forensic
investigations performed in those cases, i.e: external examination, autopsy findings,
histological and toxicological analysis, blood strontium determination, biochemical analysis
and diatom test. The diatom test was still considered controversial by the by the literature
but we defined qualitative and quantitative criteria which could exclude false positive
results. It is of particular interest in case of putrefied bodies where the other investigations
have failed.
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K (2003). Phytoplankton gene detection in drowned rabbits. Leg. Med. 5: S142—
S144.
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4
Forensic Investigation in Anaphylactic Deaths

Nicoletta Trani, Luca Reggiani Bonetti, Giorgio Gualandri1,
Giuseppe Barbolini and Margherita Trani2
1Azienda
USL of Reggio Emilia,
2Nuovo
Ospedale di Sassuolo (MO)
Department of Diagnostic Services, Pathology and Legal Medicine, section of Legal
Medicine, University of Modena and Reggio Emilia, Modena
Italy
1. Introduction
Deaths anaphylaxis related have always been very difficult in objectification in autopsies. So
they are object of study for pathologists and legal doctors. In this work we want to propose
a methodological protocol based on the various available diagnostic tools to use when an
anaphylaxis related death is suspected.
2. Pathogenesis
The anaphylaxis is an allergic reaction. An allergic reaction is a spontaneous and exaggerate
reaction of the body to a particular substance. These substances, called allergens, cause
production of antibodies when enter the body. The exposition with the substance happens
by inhalation, ingestion, contact or inoculation of the allergen. Every substance can act as
allergen. Among the more frequent substances we remember the heterologous proteins
(hormone like insulin, vasopressin, parathormon; enzymes like trypsin, chemotrypsin,
penicillinases; pollens, food like eggs, fish, hazel-nuts, cereals, beans, chocolate; antiserums,
hymenoptera venom); polysaccharides (iron dextran); drugs (antibiotics like penicillins,
cephalosporins, Amphotericin B, nitrofurantoin, local anesthetics like procaine and
lidocaine; vitamins like thiamine and folic acid); diagnostic substances (iodated means of
contrast, sodium dehydrocoled, sulfobromoftaleine); industrial chemical products (ethylene
oxide). (Fauci et al., 2009)
The concept of anaphylaxis comes from the study of the actinotoxins on the dogs’ arterial
blood pressure (Richet, 1902).
It is well known that the anaphylactic shock is an example of the immediate type of
hypersensitivity reaction inducing a diffuse organ hypoperfusion. It has been defined
(Delage & Irey, 1972) as the failure of the peripheral circulation induced by an antigen
(allergen)-antibody reaction in already sensitized subjects for a foreign substance. Whenever
the hypoperfusion is complicated by increased capillary permeability, a rapidly irreversible
circulatory fatal damage occurs (anaphylactic death).
Delage and Irey (Delage & Irey, 1972), in their clinico-pathological study of 43 validated
cases of drug-induced fatal anaphylactic shock, the predominant role of penicillin,
subsequently confirmed (Di Maio & Di Maio 2001; Menchel et al., 1987; Weeden, 1988), is
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reported. In these as well as in other cases immunoglobulin E (IgE) antibodies have been
tested to various allergens, present in contrast media (Di Maio & Di Maio 2001; Lang et al.,
1995; Pumphrey & Roberts, 2000; Risgaard et al., 2008), sera (Vance & Strassmann, 1942;
Johann-Liang et. Al., 2011), insect venom (Pumphrey, 2000; Riches et al., 2001; Yunginger et
al., 1991), and food (Di Maio & Di Maio 2001; Pumphrey, 2000; Pumphrey & Roberts, 2000;
Yunginger et al., 1991) are thought to initiate anaphylactic reaction in patients previously
sensitized towards that allergen (often unknowingly).
A register including all fatal anaphylactic reactions in the UK is operative since 1992
(Pumphrey, 2008). In France and Belgium since 2001 a university research team has founded
the Allergy Vigilance Network, that in addition to reporting cases of severe anaphylaxis, to
determine the prevalence of sensitization to risk allergens and screening and long-term
monitoring of dangers related to new foods, ingredients and adjuvant sensitizing factors,
with the French National Institute for Food Safety (AFSSA) and the Ministry of Consumer
Affairs (DGCCRF) and various patient associations, also analyzes dangers related to the
allergenicity of natural and modified food proteins (Moneret-Vautrin et al., 2002; Moneret-
Vautrin, 2007).
Allergy depends on the individual “predisposition”. In certain people the contact between
the allergen and the human body causes an abnormal immune reaction that clinically
appears with the wide spectrum of manifestations of the allergic reaction (Crane, 2006;
Liccardi et al., 2006)
Hypersensitivity reaction can be classified according the four type of immuno-pathological
reaction of Gell and Coombs (Fauci, 2009):
- type I: they are the result of an IgE-mediated reaction that leads to an immediate
hypersensitivity
- type II: IgG or IgM mediated. These antibodies are directed against cellular surface’s
antigens altered by the drug that provoke a complement-mediated cytotoxicity.
- type III: immuno-complexes mediated. The immunocomplexes’ dimensions determine
the site of deposition and the consequent immunological damage.
- type IV: retarded hypersensitivity. They come out by the interaction of the antigen with
T lymphocyte and determine a cell-mediated reaction.
Type I reaction is characterized by a rapid activation (in few minutes)of vasoactive and
spasmogen substances by antibodies that are on the surface of the mastcells and basophils.
It’s composed of three phases:
1. sensitization, when the immune system come into contact with the allergen for the first
time and stimulates IgE antibodies by B cells (the IgE production is under the control of
TH2 CD4+ that increases its production and under the control of TH1 that reduces the
production). The IgE bind mastcells’ and basophils’ receptors making them sensible to a
next exposition to the antigen.
2. initial reaction: Immune system has a memory; so at the second exposition to the
allergen there’s a binding between the antigen and the IgE antibodies localized on the
mast-cells and basophils (sensitized). High affinity receptors (IgE) are almost
exclusively on the mast-cells and on the basophils while low affinity receptors are also
in other cytotypes (eosinophils, macrophages, platelets).
When the allergen binds to the high affinity receptor there’s the activation of the
mastcells that leads to the degranulation of the mastcells and release of primary
mediators (preformed) such as histamine, adenosine, chemiotactic mediators (e.g. the
ones for the eosinophils), enzyme (tryptases, kynases), proteoglicans. There’s also the
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Forensic Investigation in Anaphylactic Deaths 63
release of secondary mediators of ‘de novo’ synthesis such as leucothrienes,

prostaglandines, platelets’ aggregation stimulation factors, cytokines, chemokines. In
the first 30-60 minutes after exposition symptoms happen. Histamine is characterized
by a very short half-life in the circulation, tryptase and chymase, are stable post-mortem
(Edston et al., 2007; Nishio et al., 2005) and respectively used in post-mortem diagnosis
of acute anaphylaxis (Edston, 2007; Nishio, 2005; Pumphrey, 2000; Riches et al., 2001;
Schwartz, 1987; Yunginger et al., 1991). Tryptase is a serine protease stored mainly in
mast cell granules, not found in circulating basophils, eosinophils, platelets or any other
cell, represented by two varieties: an active free form () and an inactive tetramere ()
(Ansari et al., 1993; Schwartz et al. 1995).
The former is a protein released from mast cell granules during anaphylactic reactions,
the latter is a similar protein secreted by resting mast cells and raised in mastocytosis
(Pumphrey & Roberts, 2000; Schwartz et al. 1995).
Chymase is a mast cell-derived serine protease, characterized as an angiotensin II-
generating enzyme (Nishio, 2005) and used to determine mast cells and thus to assess
the timing of wounds after deaths (Bonelli et al., 2003; Urata et al., 1990).
It is quite stable in serum and a significant positive correlation between serum chymase
and tryptase levels was found in post-mortem diagnosis of anaphylaxis (Nishio, 2005).
Heterogeneity of human mast cells is known (Irani & Schwartz, 1994; Weidner &
Austen, 1993) and recently different subsets of mast cells (MC) are distinguished by
immunohistochemistry (Perskvist & Edston, 2007), as follows:
MC-TCs (formerly connective tissue mast cells) mainly composed of histamine, heparin,
tryptase, chymase, cathepsin G and carboxypeptidase, preformed and stored in
granules.
 MC-T (formerly mucosal mast cells) lacking or containing only small amounts of
chymase, carboxypeptidase and cathepsin.
 MC-C lacking tryptase and not further characterized
3. Late phase: after 2 hours from the initial response the presence of antigen is not
necessary and the tissue infiltration begins by inflammatory cells (neutrophils,
eosinophils, basophils, monocytes) with consequent tissue lesions (in particular
epithelia and mucosas).
This is the typical allergic reaction that usually brings to vasodilatation, skin rash, edema,
itching; but the clinical spectrum is very wide and the allergic disturbs can be poor or get
the death for anaphylactic shock.
Anaphylaxis is the most dangerous among the allergic reaction and it is a severe systemic
reaction, with an often sudden and important beginning, with an acute response that
happens in a variable time from few seconds to few minutes after the antigen exposition.
Anaphylaxis can be elicited for every concentration of the antigen (also minimal, sometimes
it happens during skin test for drugs, etc.) (Bernstein et al., 2004; Blanton & Sutphin, 1949;
Eleuterio Gonzàlez et al., 1997; Harris & Sure, 1950; Liccardi et al., 2006; Lockey et al., 1987;
Riezzo, 2010; Weber-Mani & Pichler, 2008).
The typical anaphylaxis consists of sudden weakness, itching and urticaria, chest
oppression, respiratory distress (wheezing) followed by cardio-circulatory collapse.
Symptoms maybe very variable and could be involved almost all the functions/apparatus.
Could be involved: cardio-vascular system(tachycardia, hypotension, arrhythmias,
ischemia/ myocardial infarction, heart arrest, symptoms from hypoperfusion are constant),
nervous system (vertigo, asthenia, syncope, convulsions), eye (conjunctival injection,
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lachrymation), upper airway (nasal congestion, sneezing, hoarseness, stridor, pharyngeal or

laryngeal edema, cough, obstruction, laryngospasm), lower airway (dyspnea,
bronchospasm, tachypnea, involvement of the accessory respiratory muscles, cyanosis,
respiratory arrest), skin (rash, erythema, itching, urticaria or urticarial reaction, edema,
maculo-papular rash), gastrointestinal apparatus (nausea, vomiting, abdominal pain,
diarrhea)(Crane et al. 2006; Fauci, 2009; Rovere-Querini, 2010).
Lethal cases are mainly due to: acute respiratory distress derived by the glottis edema or by
bronchial obstruction/bronchospasm; cardio-vascular collapse also without an important
respiratory distress.
In the lethal cases between the contact with the allergen and the anaphylaxis there’s a very
short time. The anaphylaxis shows immediately or in few minutes after the exposition; in
the most of the cases by 15-20 minutes. Reactions after 60 minutes from the exposition are
very rare. As soon the reaction occurs as easier the death is; sometimes death can be
immediate and, however by 1-2 hours. More rarely death occurs by 24 hours.
3. Proposal of a methodological protocol

In most of the cases the diagnosis of anaphylactic death represents a challenging deal. So it’s
very important that the anatomo-pathological and/or medico-legal investigations must be
very scrupulous and must analyze:
 medical history of the deceased and eventual investigations on the spot;
 necropsy with:
 lab tests, for which it’s better to use peripheral blood sample and not central ones;
 histological tests
 histochemical and immuno-histochemical tests.
3.1 Medical history and investigations on the spot

To make diagnosis of anaphylactic death it’s important to make a correlation between the
symptoms and an insect bite, the ingestion of food, drugs or other substances.
So we should collect anamnesis by family and general practitioner, especially if related to an
history of allergy. Some patients, however, doesn’t know to have allergies and anaphylaxis
is the first (and last) allergic reaction they have in their life.
Especially in the cases with medical history negative for past allergic reaction it’s important,
when possible, going on the spot where the death occurred to get the eventual syringes used
for injection and/ or to evaluate the presence of nests of wasps. It’s important hearing to
witnesses that could tell the symptoms of the victim. Sudden weakness, itching and
urticaria, chest oppression and respiratory distress (wheezing) followed by cardio-
circulatory collapse may occur. Symptoms maybe very variable and could be involved
almost all the functions/apparatus as we remembered before.
3.2 Complete necroscopic exam

It’s very important beginning with an accurate external exam to verify the presence of signs
such as rash, urticaria or angioedema; to verify the skin integrity finding out eventual site of
inoculation: it’s important to investigate also the sites covered by hair. If there a positive
finding it’s opportune to proceed, during the successive autopsy, also to get a skin sample
after the examination of the route in the case of subcutaneous or intramuscular injection.
During the autopsy the pathological findings are often aspecific.
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Usually we find multivisceral congestion, aspecific finding in various different types of death.
(Barnard, 1967; Da Broi & Moreschi, 2011; Delage & Irey, 1972; Di Maio & Di Maio, 2001;
Edston & van Hage-Hamsten, 2005; James & Austen, 1964; Low & Stables, 2006; Lu et al., 2006;
Menchel et al, 1987; Pumphrey & Roberts, 2000; Shen et al.,2009; Yilmaz et al., 2009).
You can find:
 glottis edema and/or of the pharyngo-laryngeal districts;
 congestion and/or pulmonary edema;
 hyperinflation of the alveoli with acute emphysema;
 endo-luminal bronchial secretions- this finding is more frequent if there’s an asthmatic
factor and it’s usually related to a almost immediate death;
 hemorrhagic petechiae: it’s suggestive of an asphyxial component of the death and it’s
usually associated with an almost immediate death.
These findings can change according to the allergen type, to the way of administration and
to the time passed between the exposition and the death (Edston & van Hage-Hamsten,
2005; Low & Stables, 2006; Pumphrey & Roberts, 2000). If the death is very fast the only
macroscopic finding is an important multivisceral congestion associated or not with the
petechial hemorrhages (Edston & van Hage-Hamsten, 2005; Low & Stables, 2006; Pumphrey
& Roberts, 2000; Roberts & Pumphrey, 2001).
In the table n. 1 there are the results of different studies present in literature (Barnard, 1967;
Delage & Irey, 1972; Greenberger et al, 2007; James & Austen, 1964; Low & Stables, 2006;
Pumphrey & Roberts, 2000; Shen et al.,2009; Yilmaz et al., 2009).
Study
Autopsy findings James & Austen
Delage & Irey (1972) Barnard (1967)
(1964)
Number of cases 40 6 50
3 cases
penicillin, 1 case
guinea-pig
haemoglobin; 1 Insect-Stings
case bee venom;
1 case ragweed
extraxt
Erythematous skin
rash/cutaneous 35
edema
Pulmonary
congestion and 36 5 35
edema
Upper airway
15 4 14
edema
Hyperinflation of
the lungs and/or
18 5 16
mucous plugging
of airways
Petechial
10
hemorrahages
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Study
Autopsy findings Pumphrey & Roberts
Low & Stables (2006)
(2000)
Number of cases 56 18
Venom Food Drugs Venom Food Drugs Undetermined
(19) (16) (21) (4) (2) (10) (2)
Erythematous skin
rash/cutaneous 1 2 0 0 0 0 2
edema
Pulmonary
congestion and 14 9 18 3 0 5 2
edema
Upper airway
6 10 7 3 0 0 1
oedema
Hyperinflation of
the lungs and/or
7 5 3
mucous plugging of
airways
Petechial
4 5 1
hemorrahages
Study
Autopsy findings
Greenberger et al. (2007) Shen et al. (2009) Yilmaz et al. (2009)
Number of cases 25 28 36
Erythematous skin
rash/cutaneous 3 4 2
edema
Pulmonary
congestion and 18 28 29
edema
Upper airway
16 15 11
edema
Hyperinflation of
the lungs and/or
3 11 5
mucous plugging of
airways
Petechial
6 3
hemorrahages
Table 1. Autopsy findings.
A complete autopsy, with histo-pathological and chemical-toxicological investigations, is
mandatory in every case.
3.3 Laboratory tests

A very useful first investigation is the research of the total and specific IgE for specific
substances: The Igs are very stable also after death (Hieda et al, 1991).
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The finding of total IgE doesn’t demonstrate the anaphylaxis but indicates that the subject
was sensible for particular substances (e.g. insect venom, antibiotics, etc..). However , in
there’s a positive history or suspect for allergies for specific substances, every suspected
substance must be tested with specific IgE. If there isn’t an accurate anamnesis or an history
of allergy it’s a good idea testing the most common allergens. (Calvani et al., 2007; Hamilton
& Adkinson, 2003; Horn et al., 2004).
A second investigation on the cadaverous blood sample is the dosage of beta-tryptase. As
we already said, the degranulation of the mast-cells releases powerful chemical mediators
(histamine, tryptase, etc..) (Ansari et al., 1993, Carson et al., 2009; Way & Baxendine 2002).
The tryptases, instead, are relatively stable post-mortem (values can remain high for some
days in a serum sample kept at room temperature and for some months if freezed) (Joint
Task Force on Practice Parameters et al., 1998; Horn, 2004) and their dosage is very useful in
the diagnostics of acute anaphylaxis. As already said, in addition to mast cells also the
basophiles produce tryptases but fewer than 300-700 times compared to skin and lung mast
cells. So the serum concentration of tryptase is considered an index of mast cells activation.
In particular we must determine the beta-tryptases that are usually released by mast cell
degranulation (while the alfa-tryptase is secreted constitutively by mast cells and represent
an index of the mast cells mass and so it is present in the mastocytosis) (Kanthawatana et al,
1999; Schwartz 2004).
For this reason the ratio between total tryptase (alfa + beta) and beta-tryptase is important
to distinguish between an episode of anaphylaxis and patients with systemic mastocytosis: a
ratio less than 10 is usually indicative of an anaphylactic reaction while a ratio <20 suggests
a systemic mastocytosis (Joint Task Force on Practice Parameters et al., 2005; Lieberman et
al, 2010; Schwartz et al., 1995, Schwartz & Irani, 2000). Serum levels of tryptase quickly
increase and are detectable by 30 minutes (the concentration peak is reached in the first 2-3
hours) and remain high for about 5 hours (Joint Task Force on Practice Parameters et al.,
2005; Lieberman et al, 2010). High levels of beta-tryptase point out a degranulation and, so,
support the diagnosis of anaphylaxis.
Usually the increase of the serum level of tryptase is bigger as much as the anaphylaxis has
been severe. It’s important underline that the negativity of this test doesn’t exclude an
anaphylactic death. In fact Sampson has demonstrated that the rise of this enzyme could be
absent in the anaphylaxis by food, maybe because of the involvement of other cells such as
basophils or monocytes/macrophages (Sampson et al., 1992).
Therefore tryptase concentrations in femoral blood (not influenced by position at death or
resuscitation efforts) (Edston et al., 2007) and serum chymase and tryptase levels (Nishio et
al., 2005; Shen et al., 2002) have been suggested in postmortem cases to validate the
diagnosis of anaphylactic deaths.
The histamine is another product of mast cell degranulation. This mediator, even if is very
valid in vivo (it’s an index of mast cell activation even though not specific of the mastcells
alone) , isn’t an effective indicator after death because has a very short half-life (2 minutes).
So the N-methylhistamine, that is a product of histamine degradation and is stable in the
urine, but in the cases of anaphylactic death the time is too short to find it into the urine
(Sthephan et al. 1990; Edston et al, 2005, 2007).
Among the other possible tests we remember the serum titration of a mastcell-specific
chymase (Nishio et al., 2005; Osawa et al. 2008), that is a serum protein mainly kept into the
mastcell granules.
It’s important to note that the positivity to total IgE or of the serum tryptase cannot be
considered , by the forensic profile, as a sure indication of a death by anaphylaxis because
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the positivity of one or both the markers has been found also in other pathologies (Randall
et al., 1995; Horn et al., 2004) such as traumatic death or the sudden infant death syndrome
(Buckley et al., 2001; Edston et al., 1999; D’Errico et al., 2008; Holgate et al, 1994; Nishio &
Suzuki, 2004; Schwartz, 2001) but must be integrated with the results of other investigations
that must be done in every case of death.
3.2 Histology
Finally the histo-pathological diagnosis is very important and may show eosinophilia
(Delage ande Irey, 1972) especially in the upper and lower airway, in the liver and in the
spleen (Voigt, 1966); the presence of glottis edema and/or pharyngo-laryngeal edema that,
histologically, could be associated with a wide dissociation of collagen fibers and of the
glandular elements, eosinophilic infiltration and vascular congestion (Pumphrey and
Roberts, 2000).
Sometimes, using hematoxylin-eosin stain, there’s lung hyperinflation with emphysema,
endo-luminar mucous and peri-bronchial congestion, edema and eosinophilic infiltrate.
Another method is the mast cell count in the various organs and tissues: unfortunately
specific stainings for mast-cells are based on the metachromatic properties of the
cytoplasmic granules and showed limited:
1. the positivity of the mast cells varies according the technique used to fix and stain
(Strobel et all., 1981);
2. the counts in the tissue ‘post-mortem’ after the anaphylaxis is underestimated because
of the mast cells’ degranulation during anaphylaxis. The staining can’t put in evidence
the degranulated mast cells; so, because the number of mast cells varies from each one
it’s impossible decide how many cells have degranulated.
3. the base –level of mast-cell concentration after death is strongly underestimated.
In literature, however, there is a case (Heard et all., 1989) where the authors compare the
pulmonary concentration of mast-cells in the allergic subject pre- (biopsy) and post-mortem
showing a diminution in the latter sections.
3.3 Auxiliary techniques: histochemical and immuno-histochemical

Since histology alone cannot give absolute results, it have been studied more complex
techniques such as histochemistry and immuno-histochemistry.
In 1960 Glenner and Cohen (Glenner & Cohen, 1960) identified the proteases of mast cell
granules using histo-chemical procedures. The main morphologic characteristic to
distinguish mast cells is the presence, in cytoplasm, of many roundish granules,
homogeneous in man, soluble in water, that stain methacromatically with basic dyes such as
Toluidine blue, or with dyes for glycosaminoglycans polymerized such as Alcian blue. The
granules are coated with membrane and contain heparin and histamine. In particular, the
presence of heparin, an anticoagulant glycosaminoglycan, accounts for the staining of these
granules. In the anaphylaxis the massive degranulation could be emphasized with this
technique with the highlight of the granules next to mast cells. Furthermore it has been
identified antibodies against histamine but they are not useful for post-mortem evaluation
since histamine is poorly stable (Johansson et al., 1992)
Pagoda red stain is another histo-chemical procedure successfully employed for the
histological demonstration of several substances with fibrillar periodical structure, like
amyloids (Battaglia et al., 1985; Yanagihara et al. 1984), cellulose, siloxanes, polysiloxanes
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and polyethylene polymers and more rarely to put in evidence eosinophilia in various
tissues (Kyono et al, 1982). This technique displays a mixing of cytotypes,
contemporaneously on the same slide, easily identifiable, since degranulated mast cells and
their outside granules appear brilliant red over a pale blue background (Trani et al., 2008).
Cytotypes EMBP Chymase Tryptase CD117 Pagoda red

Eosinophils ++ - - - +++
Mast cell - +++ +++ - +++
- negative reaction ++ moderate reaction +++ strong reaction
Table 2. Panel of identification of eosinophils and mast cells: a comparative evaluation
(Trani et al. 2008).
Pagoda Red stain is a dye originally employed in the industrial field to dye clothes and
occasionally carried out in cytopathology in case of nasal (Rivasi & Bergamini, 1988) or
ocular (Rivasi et al., 1992) allergic processes possibly related to the presence of airborne,
nonhuman elements.
Immuno-histo-chemical investigations although more expensive than histo-chemical ones
allow to characterize the immuno-phenotype of the various cells in the inflammatory
infiltrate associated with anaphylaxis; especially specific antibodies can bind superficial
antigens in these cells such as tryptases and chymases (Akin et al. 2007; Carson & Cook,
2009; Irani et al., 1989; Perskvist & Edston, 2007).
Human mast cell tryptases comprise a family of trypsin-like neutral serine proteases that are
predominantly expressed in mast cells. This antibody is useful for the identification of very
atypical or immature mast cells (MC) in mast cell leukemia, and for the detection of small,
even minute, dense focal MC infiltrates in staging procedures in patients with known
cutaneous mastocytosis. Using an avidin-biotin enhanced immunoperoxidase procedure,
with monoclonal antibodies (AA1, AA3, and AA5) directed against human mast cell
tryptase, it’s possible to obtain an intense staining of mast cells in paraffin-embedded tissue.
It represents an highly specific and sensitive means for the detection of mast cells in
routinely processed tissues.
Chymases belong to a family of serine proteases like intracellular granule and it’s involved
in regulating extracellular matrix proteolysis and promoting tissue remodelling (Doggrell &
Wanstall, 2004). This substance is mainly found in mast cell cytoplasm but also outside the
mast cells in the connective tissues surrounding vascular walls and, in less concentration, in
basophils. (Hamada et al. 1999). Chymase antibodies is available for formaldehyde-fixed
tissue and can be used simultaneously to tryptase by a sandwich technique applying the
two antibody (Buckley et al. 1999).
The eosinophilic major basic protein, also known as MBP, PRG2, a proteoglycan 2, BMPG or
bone marrow natural killer cell activator, is a constituent of the eosinophilic granules. High
levels of the pro-EMBP are present in pregnancy serum and in placenta where it develops a
complex with other proteins. It may influence antiparasitic defense mechanisms as
cytotoxin and helminthotoxin and immune hypersensitivity reactions (Trocme et al. 1989).
The role of EMBP is to modulate inflammation and lead to tissue destruction resulting in
cytotoxic effects (Butterworth & David, 1981). It has been demonstrated to be elicited in
mast cell and basophil (Trocme et al. 1989) degranulation.
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4. Conclusions
Post-mortem diagnosis of anaphylactic death is very difficult and it’s possible only
excluding every other cause of death and taking into considerations the results of other
exams: accurate medical history, complete necroscopic examination integrated with
histological examination, lab tests, and auxiliary techniques of histochemistry and immuno-
histo-chemistry. This diagnosis, in particular for the medico-legal aspect, cannot be based on
the positivity of one only type of investigation (e.g. bioumoral tests) because that positivity
can be found also in other pathologies (Horn, 2004) but must be integrated with the results
of more investigations.
5. Acknowledgment
Many thanks to Aldo Ferrucci and Mr Vidic for their kind patience and to everyone that
supported the authors in writing this chapter.
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5
Forensic Age Estimation in Unaccompanied

Minors and Young Living Adults
Andreas Schmeling1, Pedro Manuel Garamendi2,
Jose Luis Prieto and María Irene Landa4
1ForensicPhysician Institute of Legal Medicine of Münster
2ForensicPhysician Council of Forensic Medicine of Istanbul
3Forensic Physician Complutense University of Madrid
4Forensic Phsycian Institute of legal Medicine of the Basque Country
1Germany
2Turkey
3,4Spain
1. Introduction
1.1 FAE a need in nowadays everyday forensic practice
Forensic Age Estimation (FAE) defines an expertise in forensic medicine which aims to
define in the most accurate way the chronological age of person of an unknown age
involved in judicial or legal proceedings. The term “estimation” defines more precisely than
other as “diagnosis” the real limits inherent to this expertise. The state of the art in FAE is
such that nowadays there is no medical test or a group of tests that absolutely and
accurately let us know the exact chronological age of a human being (Ritz Timme et al.,
2000).
Nevertheless, in everyday practice, Justice Courts and other Public Institutions require this
type of experts reports to forensic physicians and Legal Medicine Institutes. In this expertise
it´s needed a collaborative knowledge of very diverse disciplines like Forensic and Physical
Anthropology, Odontology and even some general medical specialities like Radiology of
Pediatrics all of them ideally centered in Legal Medicine Institutes.
FAE is not at all a recent field of expertise in Forensic Sciences and Judiciary History. In old
Roman Empire, the eruption of second molar indicated the moment in which a young male
was considered for military service (Schmeling, 2008). During XIX century age estimation
was mainly performed by dentists. In 1837, Edwing Saunders published “The Teeth a Test
of Age” and British Parlament decided to teeth eruption as an accurate method to determine
the age of kids. At those ages, minimum criminal age was 7 years old in Britain and also in
1883 minimum required age for mining workers was 9 years. Nevertheless, at that time,
also some voices expressed their criticism with this practice for an age estimation. In 1846,
Dr. Pedro Mata in his text book of Legal Medicine expressed his concern on assuring
without any kind of a doubt an age estimation based solely on teeth eruption (Mata, 1846).
In 1895, Röntgen discovered X rays. His discover opens a new dimension for age estimation
in living subjects. The applications of his discovery in Legal Medicine were nearly
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immediate and age estimation in living subjects rapidly changed to include these new tests
based on radiographies of the skeleton as a complement to the teeth eruption classical
methods. In 1886 in Munchen, Angerer was first to suggest that radiographies from carpus
could be indicator of an age in young persons (Brogdon, 1998). In 1887, Behrendsen
published the first systematic review of age variations in carpus (Schmeling, 2008).
During the next 40 years, different researches focused in defining the standard radiological
maturation of human skeleton with age (Stevenson,1924; Flecker,1933; Galstaun, 1937;
Sidhom & Derry, 1931; Pryor, 1908; Borovansky & Hnévkovsky, 1927; Davies & Parsons,
1927; Paterson, 1929; Meenes & Holly, 1932; Adair & Scammon, 1921). The lack of
consciousness on the harms inherent to X-rays exposure lead to a massive amount of X-rays
expositions only on investigation purposes. Between decades of 50 to 80 of XX century, main
definitive methods of age estimation based on radiological analysis of carpus (Greulich &
Pyle, 1959; Tanner et al, 1983) and dental maturation (Nolla, 1960; Demirjian, 1975) were
defined.
Until recent years, forensic physicians in European Countries weren´t usually required for a
FAE in living subjects. Census and Birth Registration in Europe was really accurate and only
exceptionally an expert report was needed to assess an age of a European citizen. But in the
last two decades European countries have received a flood of immigrants from
extracommunitarian countries. In many cases these non European citizens don´t have any
documents that ascertain their chronological age or there are doubts on the certainty of
chronological age alleged. This situation is specially complex in a case of an immigrant
supposedly minor of age. European Legislation and International Traits assure an special
treatment of these immigrant minors when they are unaccompanied and they must be
consider under the trusteeship of European authorities. This special treatment must also be
assured in case of criminal proceedings in which minors have a special protection as victims
and also as responsible of criminal activities. In all those cases, Justice Courts and Public
Institutions require forensic doctors a FAE expert report (European Migration Network,
2010).
It´s very difficult to ascertain the total number of immigrants supposedly minors
unaccompanied with an known chronological age in European Countries. In our legal
framework, unaccompanied minors is the term to define this population. There are many
different reasons in each European country to determine the uncertainty of a statistics on the
total number of unaccompanied minors. In countries like Germany or Spain, the division of
the Government in Lander or Autonomous Communities turn difficult to maintain a central
register of minors unaccompanied. In most European Countries it was also observed that
many minors under trusteeship of authorities suddenly disappear from the asylum
institutions. Some studies also stress the fact that there is a group of minors unaccompanied
who don´t seek for asylum and they keep as immigrants in many European cities unknown
to the authorities (European Migration Network, 2010).
During 2008 in Germany the census of minors unaccompanied was a total of 763 subjects.
From these 324 were up to 15 years old and 438 were aged 16 to 17. Most of them were
detected at the airports and they came mainly from Iraq and Afghanistan during 2007 and
2008. During 2003 to 2006, the most usual nationalities were Turkish and Chinese, and other
nationalities more unusual were Russian, Serbian, Vietnamese and Nigerian (Federal Office
for Migration and Refugees, 2009).
In Spain, the census of unaccompanied minors by the end of 2008 was an estimation of 6000
minors under trusteeship of Spanish authorities. Nearly all of them came from Magreb and
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Forensic Age Estimation in Unaccompanied Minors and Young Living Adults 79
Sahel African Subsaharian countries, being most usual nationality Moroccan. Until 2003
most of them arrived at Spain inside trucks and industrial vehicles crossing Mediterranean
Sea in Algeciras (Andalucia). Since 2003, this changed and most of the minors detected
arrived at Southern Spanish and Canary Islands coast by sea in “pateras” or “cayucos”,
different light and dangerous boats that illegally cross Atlantic and Mediterranean sea
waters (Bravo, 2005; PICUM, 2008).
In both cases, Spain and Germany, nearly all of the unaccompanied minors are male. Only
in Somalian nationality girls are more frequent than boys (Bueno & Mestre, 2006; European
Migration Network, 2010).
Nowadays, there is still no consensus in European countries on which methods must be
applied when an age estimation of a supposedly minor is needed. All Member States
attempt to determine this using a variety of techniques. European Statistical data are also
uncertain about the methods really applied in every country. Nevertheless, official data
from EU indicate that in some countries like United Kingdom authorities usually only apply
an interview with the minor by a social worker without a medicine doctor examination. UK
Border Agency accepts the “Merton compliant age assessment” carried out bt two specially
trained social workers when assessing the individual´s asylum claim, unless there is
evidence to the contrary. On the other side, Austria since 2010 applies the so called
“multifactorial examination methodology” that consists of three elements: an inspection by
a doctor, a dental analysis and X-ray examinations, the latter performed only with the
consent of the minor. If the age of the minor cannot be determined exactly, the benefit of the
doubt is given to the individual concerned. In France, also a psychological interview is used
for age estimations of unaccompanied minors. In six out of all Member States no skeletal
development estimation by radiographies was applied and in other eight ones no dental
analysis was performed (European Migration Network, 2010).
ACNUR has recently recommended EU authorities to unify methodology applied in age
estimation to ensure the protection of children and the defense of immigrants Human Rights
in Member States. Different international groups of experts have published guidelines and
recommendations on FAE, like American Board of Forensic Odontologists (ABFO),
International Organization for Forensic Odonto-Stomatology (IOFOS) or Study Group of
Age Estimation of the German Society of Legal Medicine (AGFAD). This chapter aims to
spread these recommendations on FAE by these international groups of experts (UNHCR,
2000,2001,2002).
2. Methods
2.1 Selection of the methods
In the infant and juvenile stage the use of morphological methods based on radiological
examination of dental and skeletal development is recommended (Ritz Timme et al., 2000).
However, the methodology employed varies, while the criteria followed internationally for
official application of these techniques are disparate, with some countries arguing the
dubious validity that much scientific research currently accords this type of proof, whose
margins of error do not permit the diagnostic reliability required in such cases (UNHCR,
2000, 2001, 2002). For this reason, in certain countries X-ray tests are only used in criminal
cases (Solari & Abramovitz, 20202). Despite this, the programs developed by official
institutions and NGOs have established protocols of good practice which include the
elements corresponding to age assessment (UNHCR, 1997).
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In accordance to the guidelines proposed by Ritz-Timme et al for an age assessment method

to be considered acceptable, it must fulfill the following requirements:
1. The method must be transparent and provable, presented to the scientific community,
as a rule by publication in peer-reviewed journals.
2. Clear information concerning accuracy of age assessment by the method should be
available.
3. The method needs to be sufficiently accurate to fulfill the specific demands of the single
case to solve the underlying questions.
4. In cases of age assessment in living individuals principles of medical ethics and legal
regulations have to be considered, especially if medical intervention is involved.
Reference material used must fulfill certain requirements (Solari & Abramovitch, 2002):
 Adequate simple size. The number of subjects of each sex and age group should be ten
times the number of the examined features.
 The age indicated by the subjects should be verified.
 An even distribuition of subjects across all age groups.
 All data have to be collected separately for both sexes.
 The time of the examination should be recorded.
 The examined features should be defined unambiguously.
 The technique used in the examination should be described precisely.
 Information on genetic-geographic origin, socio-economic status and health of the
reference population is indispensable.
 The simple size, mean value and statistical parameters of deviation should be provided
for every feature examined.
 Information on inter- and intra-observer error is desirable.
Meinl et al stand out in questioning the terms “genetic-geographic origin” “socio-economic
status” and “state of health”, given the difficulty of establishing a definition of these
parameters and the ethical aspects of these terms, which would condition their use in a
reliable manner in a study (Meinl et al. 2007).
Many papers have been published concerning age estimation, and numerous different
methods developed, although some of them have been considered inaccurate. The choice of
a particular method will depend on the specific conditions of each case and mainly on the
accuracy required.
In the opinion of the mentioned authors (Ritz-Timme et al. 200), the published data which
fulfill the demands listed above in childhood and adolescence, are those based on the
examination of dental and/or skeletal development, applied by trained personnel. In
childhood (0-14 years) radiological examination of dental development includes all tooth
types of teeth. In adolescence (14-21 years), thirds molars are the only teeth undergoing
maturation, resulting to a lesser degree in accuracy. In both cases, sex and race influence
tooth development, so those factors have to be into account.
The German age study group considers that age diagnosis examination should include
(AGFAD, 2001):
 A physical examination which also records anthropometric data, signs of sexual
maturation and any age-relevant developmental disorder.
 An X-ray examination of the left hand.
 A dental examination which records dentition status and evaluates an
orthopantomogram.
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It also recommends that these methods are used in combination for the purpose of
increasing accuracy in age assessment and to facilitate the identification of age-relevant
developmental disorders (Schmeling et al. 2004).
Each part of the examination is recommended to be performed by a specialist experienced in
setting up expert reports and participating in regular ring experiments (see below) for
quality assurance, with a coordinating expert giving a comprehensive assessment on the
basis of the different parts of the evaluation performed by the respective specialists
(Garamendi et al.2011).
3. Physical examination
Physical examination in cases of age determination should include measurements such as
body height and weight, body type and body mass index, as well as any visible signs of
sexual maturity and the results of a general physical examination, and should describe any
signs suggestive of a pathological condition which may interfere with the maturation rate of
the child.
There seems to be general agreement among authors that the interpretation of results
obtained from anthropometric variables is an imprecise factor for the prediction of
chronological age. Some studies have shown that individuals of greater height and weight
and those with an athletic body type and an above-average BMI are among those who, in a
specific population, may exhibit a more advanced bone age in relation to actual
chronological age (Bueno et al. 1996).
Signs of sexual maturation are examined by evaluating the stage of development of the
penis and scrotum, pubic hair growth, axillary hair growth, facial hair growth, and
laryngeal prominence in male subjects; and breast development, axillary hair growth, and
shape of the hip in female subjects.
The most widely used method for the study of secondary sexual characteristics is the
staging described by Tanner (Marshall & Tanner, 1969, 1970). The method was devised to
estimate the stage of development or physiological age for medical, educational or sports
purposes, and to identify delayed or advanced sexual maturation when the chronological
age of the subject is known. The method uses a five-stage scale to evaluate the status of
pubic hair growth and breast development in girls, and pubic hair growth and development
of the penis, scrotum and testes in boys.
Breast development stages (girls) (Marshal & Tanner, 1969):
 Stage 1: Prepubertal, papilla elevation only.
 Stage 2: Breast bud stage, elevation of breast and papilla as a small mound, enlargement
of areola diameter.
 Stage 3: General enlargement of breast and areola.
 Stage 4: Projection of areola and papilla as secondary mound.
 Stage 5: Mature stage, adult contour with areola in same contour as breast and only
papilla pojecting.
Pubic hair growth stages (girls) (Marshal & Tanner, 1969) :
 Stage 1: Prepubertal, no pubic hair.
 Stage 2: Sparse growth of long, slightly pigmented, downy hair, straight or only
slightly curled, chiefly along the labia.
 Stage 3: Considerably darker, coarser and more curled, with an increase in amount. The
hair spreads sparsely over the junction of the pubes.
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 Stage 4: Hair resembles adult type, but no spread to the medial surface of the thighs.
 Stage 5: Adult in quantity and type, spread to medial thighs.
Genital development stages (boys) (Marshal & Tanner, 1970):
 Stage 1: Prepubertal, no change in size or proportion of testes, scrotum and penis from
early childhood.
 Stage 2: Enlargement of scrotum and testes, reddening and change in the texture of the
scrotal skin.
 Stage 3: Increase first in length then breadth of penis, further growth of testes and
scrotum.
 Stage 4: Enlargement in lenght and breadth of penis and development of glans, further
growth of testes and scrotum, darkening of the scrotal skin.
 Stage 5: Genitalia adult in size and shape.
Pubic hair stages (boys) (Marshal & Tanner, 1970):
 Stage 1: Prepubertal, no pubic hair.
 Stage 2: Sparse growth of long, slightly pigmented, downy hair, straight or slightly
curled, chiefly at the base of the penis.
 Stage 3: Considerably darker, coarser and more curled, with an increase in amonut. The
hair spreads sparsely over the junction of the pubes.
 Stage 4: Hair resembles adult type, but no spread to the medial surface of the thighs.
 Stage 5: Adult in quantity and type, spread to medial thighs.
Axillary hair growth, facial hair growth and laryngeal prominence development may also be
assessed using the four-stage classification proposed by Nezy et al (Neizy et al, 1975).
Of the forensic methods recommended for age determination, assessing age on the basis of
physical traits is the least precise. Evaluating sexual maturity has the greatest margin of
error and should be used for age determination only in conjunction with an evaluation of
skeletal maturity and tooth development. Multiple pathological conditions and non-
pathological, idiosyncratic conditions cause a large range of variation in the onset of external
changes associated with sexual maturation in different subjects. Therefore, age
determination cannot be made on the basis of these examination data alone.
Moreover, irrespective of the difficulty in interpreting the results due to interobserver and
intraobserver differences, there are few series analysing the progression of these parameters
with chronological age in different populations, and the few available are mainly focused on
developed countries (Koc et al, 2001; Cameron, 1993).
However, the physical examination is extremely useful for evaluating the potential impact
of pathological factors on the maturation status estimated using other methods. The great
discrepancy between height, weight and external signs of maturation and the bone and
dental age estimated using radiographic methods should guide the examiner on the
potential interference of pathological conditions and to a weighted estimation of age.
Most diseases delay development and are thus conducive to underestimation of age. Such
underestimation of age would not disadvantage the person concerned in the judicial
framework. By contrast, overestimating age due to a disease that accelerates development
should be avoided at all costs. Certain diseases which occur very rarely, in particular
endocrine disorders, may affect not only the attainment of height and sexual development,
but also skeletal development. Endocrine diseases that may accelerate skeletal development
include precocious puberty, adrenogenital syndrome, and hyperthyroidism.
Similarly, a general physical examination may show symptoms such as exophthalmos,
virilisation of girls, acromegaly and gigantism, which are indicative of pathological
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disorders and must also redirect the estimation of age. Another indication of a possible
endocrine disorder is a discrepancy between skeletal age and dental age, as dental
development normally remains unaffected by endocrine disorders (Schmeling et al. 2007).
4. Bone age in carpus

The most studied anatomical region for age diagnosis, in particular before full maturity is
reached at the age of 18, is the carpus and the hand (Garamendi & Landa, 2003).
Among the primitive series which analyzed epiphyseal maturation of the carpus as the main
object of study or in the context of a general series of long bones are those by Stevenson in
1924, Galstaun G in 1930, Sidhom G and Derry DE in 1931, Borovansky L and Hnevkovsky
O in 1929, Davies DA and Parsons FG in 1927, Paterson RS in 1929, Meenes TO and Holly
LE in 1932, Adair FL and Scammon RE in 1921, Francis CC and Werle PB in 1939, and Pryor
JW in papers published between 1908 and 1933. The latter author was among the first to
note that ossification occurs earlier in females than in males, even during the foetal period.
However, it was not until large longitudinal population studies were carried out in the early
20th century under the auspices of private foundations in both Europe and the U.S. that the
study of the carpus took a prominent role among the anatomical regions studied for age
determination, in particular in adolescence (Bañón, 2004).
In 1937, Todd compiled hand radiographs taken of individuals during a study conducted by
the Western Reserve University in Cleveland, Ohio, and together with comparisons made
against the examination of a series of skeletons, he published his Atlas of Skeletal Maturation
(Todd, 1937) based on the selection of a representative radiograph of the hand for each age
and sex. The radiograph had to meet two requirements: have the same degree of
development for the 28 bones and ossification nuclei which were the subject of the study,
and be from an individual with a degree of body development which was within the
average for their age and sex.
Todd’s Atlas was used as a standard of reference until it was revised by WW Greulich and
SI Pyle in 1950, who used it to compile their well known Atlas still widely used today,
Radiographic atlas of skeletal development of the hand and wrist, published in two editions in
1951 and 1959 (Greulich & Pyle, 1950-1959). The Greulich and Pyle series was based on a
total sample of 6,879 healthy middle-upper class North American children. Essentially, the
method evaluates a “mean” bone age by matching an X-ray against the bones of a standard
atlas, and normality estimates based on a range of results are made using standard
deviation values.
During World War II the Oxford Health Survey was started and conducted by John Ryle on
470 children. The measurement data included radiographs of the carpus, which were
analysed by Roy Acheson. Acheson looked to improve the Todd system by increasing the
accuracy of the assessment. Under his proposal:
 Each bone or ossification nucleus was studied individually.
 Each bone or ossification nucleus was pre-assigned an identifiable stage as it matured,
and each stage was given a progressive score.
 A final maturity score was calculated by adding up all of the partial scores obtained for
each individual bone.
The skeletal maturity stage then yields a continuous value which can be used for growth
measurements, such as height or weight.
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Acheson’s procedure was later refined by Tanner and Whitehouse into the Tanner-
Whitehouse charts (Tanner et al, 1983). With this method, the study of each hand yields a
total score which can be used to calculate an overall maturity stage which is then matched to
distribution tables by age and sex based on percentile distributions.
Therefore, there are two main types of methods for bone age assessment based on carpal
bones: atlas methods, with the Greulich and Pyle atlas being the main international standard
of reference, and numerical methods, with the three editions of the Tanner-Whitehouse
method being the main reference. There are some other mixed methods, such as the
Thieman-Nitz method based on a German population. There are adaptations of these
methods to virtually all populations in practically every country.
In principle it would appear that a numerical method such as the Tanner-Whitehouse
method should be more reliable, however, in practice it is subject to intra- and interobserver
errors similar to the GP method, and is negatively affected by technical problems arising
from an incorrect positioning of the hand while the X-ray is taken, which can be better
solved by graphical methods. Some authors recommend the application of the GP method
instead of the TW3 for clinical purposes, on the basis of economy of means criteria after
ascertaining that the TW3 method is far more costly in terms of time and the results are
similar to those obtained through the GP method (Garamendi & Landa, 2003).
Several attempts have been made to develop TW2 and TW3 numerical system software for
automated evaluation of X-ray plates. In theory, this would allow for a uniform
quantification of results without interference from distortion factors derived from the
observer. However, the results are not yet comparable to those obtained through the manual
method, and collaboration between radiologists and IT experts is still needed to improve
hardware and software systems.
The interpretation of bone age results obtained by any of the available methods must be
adapted to the characteristics of the population of the subject of the study. The factors which
may modify bone age progression in a particular subject are not perfectly defined, although
several studies have identified differences associated with pathological, racial or socio-
economic factors. The overall impression from the most recent research is that socio-
economic factors, which affect the nutrition patterns and hygiene and health conditions of
the subjects, are the most significant in terms of their capacity to modify results (Schmeling
et al, 2000, 2001). Racial factors are discussed by several authors in different studies, and
while they alone do not seem to justify significant differences in bone age, there are no
unquestionable data allowing one to categorically affirm or deny the specific impact of this
factor. On the contrary, the studies conducted are conclusive that certain pathological
conditions may affect the results of bone age assessment, albeit the list of those pathological
conditions cannot be considered in any way exhaustive.
There is a large number of studies in the context of ethnic and racial impact, some of them of
questionable methodological basis and at times contradictory results, mainly conducted on
populations of European Caucasians, North American Caucasians, other North American
ethnic groups (including the genetically questionable racial group of Hispanics), different
Mongoloid and Caucasian populations from Asia, and some incomplete studies on central
and southern African Negroid populations.
The most recent studies in Europe appear to indicate that maturation rates for European
Caucasians are close to those described in the GP and TW2 systems or are slightly delayed
or advanced in relation to them. In some cases, the differences with the original methods
were small yet so statistically significant that the need to create charts and atlases specific to
these populations has been proposed.
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Classical studies conducted in the seventies and eighties on Mongoloid Asian populations
showed that the bone ages of the Chinese and Japanese groups were delayed in relation to
chronological age during the prepubertal period; however, accelerated growth in the
postpubertal period resulted in maturity being reached at a similar age as for European and
American Caucasians. The most current series on modern populations with better socio-
economic status show a trend to adjust results even further to the rate of bone age
maturation of western populations. Similar findings have been reported in India and
Pakistan, where studies have shown advances in bone age in relation to chronological age
during the postpubertal period, more obvious in subjects from an upper social class.
In the U.S., studies indicate that Caucasian subjects either closely fit the GP and TW2
standards or often show a certain advance in maturation. By contrast, studies on Negroid
subjects show contradictory results in the series. For Gross et al. the black race fits the GP
standard better than the white race (Gross et al, 1995). In the series conducted by Ontell et al.
and Lodler et al. the black race is advanced in relation to the GP standard (Lodler et al, 1993;
Ontel et al, 1996). The series obtained by Marshall WA on black Jamaicans compared to the
TW2 UK60 indicates delayed bone age from age 13 irrespective of socio-economic factors
(Marshall et al, 1970). Lastly, in a study of black and white subjects in the U.S., Gilsanz V
found no significant differences between bone age and chronological age in both races after
the socio-economic factors were made equal (Gilsanz et al, 1988).
As far as our knowledge extends, the inhabitants of Muslim countries in the Near East and
northern Africa, and populations of these countries who have migrated to developed
countries have not been systematically studied and it is not known whether their rate of
bone maturation is in keeping with the progression described for other populations
(Souguir, 2002). The only studies available are those by Koc et al (Koc et al, 2001), Büken et
al (Büken et al, 2007) and Garamendi et al (Garamendi et al., 2005). The study conducted by
Koc A et al. on a modern Turkish population showed delayed bone age up until the age of
13, and a discreet advance after that age in relation to the GP atlas. A major objection to this
study is that the population sample had a chronological age of only up to 17. This problem
was later solved in a recent study reported by Büken et al., who examined carpal
radiographs of 409 Turkish boys and girls of Caucasian background aged 11 to 19. Similarly
to Koc et al., their study indicated that their population cohort exhibited advanced bone age
between ages 13 and 17, and a relative delay in the 18 to 19 age group. In 2005, Garamendi et
al. published a study of 114 Moroccan immigrants with a confirmed age of 12 to 25,
presenting a joint analysis of the variations from the Greulich and Pyle standard for carpal
X-rays as well as of the dental age assessed by orthopantomography.
Some authors consider that the socio-economic characteristics of each population are the
most significant factors affecting variation in the rate of bone age maturation. Other studies,
on the contrary, fail to confirm this hypothesis. A study on a black Jamaican population
reported by Marshall WA found no variations according to the upper or lower social class of
the subjects (Marshall et al., 1970). However, modern specific studies of this variable by
authors such as Jahari AB et al on an Indonesian population (Jahari, 2000), Fleshman AK on
an African population (Fleshman, 2000), and Melsen B et al on a population of adopted
foreign minors in Denmark (Melsen et al, 1996) clearly identify socio-economic factors and
poverty as causing significant delay in the rate of the bone maturation sequence during the
prepubertal period.
Pathological factors clearly identified as altering bone age maturation rate include, among
others, nocturnal enuresis (Dundaroz et al., 2001), GH deficit (Vallejo-Bolaños et al., 1999),
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obesity (Bueno et al, 1996), high-level competitive sport activities (Theintz et al., 1993) or
bone malformations, and exposure to physical agents causing injury such as frostbite
(Freyshmidt et al., 2001).
5. Dental age
Dental age can be assessed accurately in childhood, because many of the teeth are
developing simultaneously.
5.1 Dental eruption

Visual inspection of dental eruption was the first and most usual method for dental age
assessment. In a work entitled “The Teeth, a Test of Age”, Edwin Saunders in 1837 proposed
to the English parliament the use of the degree of dental eruption as a method to determine
the age of child workers in factories, where the age limit was nine years (Bang, 1989). For a
long time, and even up to the present day in many parts of the world where birth registers
do not exist, dental development is used as a child age indicator, adopted as a biological-
legal indicator.
However, although this method is fast, cheap and not very influenced by intra- and
interobserver error, eruption is not a good age indicator when used alone, due to factors like
interindividual or populational variation (Garn et al., 1959; Moorees et al, 1963), systemic or
local diseases (Ungar, 1937) or the elapsed time without changes (Teivens & Mörnstad,
2001).
Numerous authors have investigated the chronology and sequence of eruption in different
populations (Foti et al., 2003; Fulton & Price, 1954; Giles et al., 1963; Logan & Kronfield,
1933; Olze et al, 2007; Planells et al, 1993; Saunders et al 1993; Tanguay et al. 1984; Van der
Linden, 1980). Some of this research examines the correlation between dental eruption and
other development parameters; for example Lewis and Garn (Garn & Lewis, 1959) which
evaluates parameters such as somatic and sexual growth, personality and state of health, or
Green (Green, 1961) which attempts to establish correlations between dental, skeletal and
chronological age and weight and height, finding a stronger correlation between dental and
chronological age even than that existing between dental and bone development. Hagg and
Taranger (Hagg & Taranger, 1980) study the relationship between dental eruption and
maximum puberal growth, finding a low correlation between somatic and dental
development. Baume and cols (Baume et al., 1954) have shown changes in dental eruption
related to hypophyseal hormonal levels.
5.2 Dental maturation

Mineralization of deciduous tooth crowns begins at around 3 or 4 months of intrauterine
life, with calcification continuing after birth during the neonatal period (Burdi, 1992). Root
formation is generally completed between 18 months and 3 years of age.
Mineralization of adult teeth meanwhile takes around nine years, beginning with the first
permanent molar around the moment of birth (Evans & Knight, 1981).
The dental development process correlates with different morphological stages of
mineralization that can be observed with radiographic techniques, and undergo much more
uniform and gradual changes than eruption; more controlled by genetics and less influenced
by external factors than all other measurable criteria of maturity (Frucht et al., 2000). This is
the reason why several methods of dental age assessment have been developed.
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All the age assessment methods based on dental maturation follow the same procedure.
First the stage of development of each of the teeth is evaluated from radiographic records,
the method of choice being panoramic radiography or orthopantomography (OPT). Next the
stage of development is related to the age of each tooth, derived from study of a sample of
known age. This estimation method is based on subjective evaluation of the stages and has
many defects. The biological variation in development is also wide for all teeth.
Due to the differences existing between methods and populations of different origin, these
elements must be expressed as well as the confidence interval. Numerous studies have
provided maturation scales in both deciduous and permanent teeth populations,
identifying the successive states of development, though they show differences in the
methodology employed (longitudinal versus transversal methods, definition of the
development stages, etc)(Demirjian et al., 1973; Moorrees et al., 1963; Nolla, 1960). During
the infant period where simultaneous development of several teeth can be observed the
majority of these age assessment methods show variations of around 2 years to the average
for confidence intervals of 90-95%, indicating rather low accuracy. Studies carried out on
samples of known ages (Liversidge, 1994; Saunders et al., 1993), show differences of some 6
months to the real ages.
Nolla classified dental development into 10 calcificación stages from crypt state to closure of
the root apex (Nolla, 1960). The Nolla study warned that mineralización development began
and ended earlier in females, though there appear to be no differences in the sequence of
development finalization. The Nolla method is one of the most widely used clinically as a
reliable procedure for dental development estimation in permanent teeth. Diverse studies
(Bolaños et al, 2003; Haavikko, 1974; Staaf et al, 1991), applying the Nolla method find an
average assessment error of around 2 years for a 95% confidence interval.
One of the systems most universally used to evaluate the degree of permanent dental
development is that proposed by Demirjian, Goldstein and Tanner (Demirjian et al., 1973),
based on analysis of a sample of French-Canadian children. The original method evaluates
the degree of calcification of the seven teeth in the left mandibular hemiarch, excluding the
third molar, from radiographic records. 8 maturation stages (A to H) are established for each
tooth, from the start of crown calcification to the root apex close, in a similar way to the
Nolla method. Each tooth is attributed a formation stage, then converted into a score
depending on the sex, following the same mathematical technique used to evaluate skeletal
development by the Tanner-Whitehouse method (Tanner et al., 1975). The scores of the
seven teeth are added to obtain the so-called dental maturity score on a scale of 0 to 100.
This score is transformed through the corresponding tables into dental age. The method has
the disadvantage that it does not include a valuation of the third molars, so can only be used
for preadolescent ages.
Subsequently the same author has produced updates to the original method, proposing a
valuation system for four teeth (both premolars and molars) with different standards
(Demirjian, 1976).
In all cases, given that maturation development is different for the two sexes, the sex must
be determined beforehand (Levesque et al., 1981).
The widespread use of this method as an infant age assessment procedure has meant that
the results of the Demirjian study have been tested in other populations. Numerous studies
over recent decades show a slight delay in maturation of the original French-Canadian
population, causing overestimation when the original results of the method are applied to
other populations (Bolaños et al., 2003; Davis & Hagg, 1994; Eid et al., 2002; Frucht et al.,
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2000; Koshy & Tandom, 1998; Liversigde etl., 1999; Loevy & Goldberg, 1999; MacKenna et
al., 2002; Nykanen et al., 1998; Nystro et al., 1986; Prabhakar et al., 2002; Staaf et al., 1991;
Willems et al., 2001). The aforesaid overestimation varies between some months and several
years in age, and it is recommended that standard values based on studies of the same
population to which the method is applied are used.
Seeking a more accurate statistical model that explains as well as possible the correlation
between the degree of dental development and chronological age, Teivens and Mönstard
(Teivens & Mönstard, 2001) have recently produced diverse mathematical functions
modifying the original Demirjian method, obtaining the best results when a cubic regression
model is used (R2 = 0.95). This model has been tested in a comparative study between Swiss
and Korean subjects, showing statistically significant differences, with earlier development
in the Swedish sample of 2 months for males and 6 months for females.
5.3 The third molar in age assesment.

Age assessment becomes more complicated once the root apex of the second permanent
molar has closed (at around 14 years of age) due to the variablilty of third molar
development. The third molar is the tooth showing greatest frequency of agenesia (Garn &
Lewis, 1962), the most irregular in its maturation sequence (Kieser, 1990) and, in contrast to
the rest of dentition, tends to appear earlier in males than females (Levesque et al., 1981).
However, due to the scarcity of alternative indicators maturity evaluation in this tooth is one
of the prime tools in age assessment in these cases
Recent years have seen a proliferation of studies focusing on third molar maturation as an
age assessment method, with the purpose of contributing data enabling us to better
understand the factors influencing the maturation of this tooth and establish more specific
reference values providing more reliable diagnosis.
Despite this, the continual increase in immigration of young people from third world to
industrialised countries, and the need to have a reliable and sound age assessment
procedure in the absence of documents showing the date of birth, have for some years now
prompted studies based on third molar development (Harris & Nortjé, 1984; Micci &
Buzzanca, 1998; Robetti et al., 1982; Robetti et al., 1993). The third molar is the only tooth
undergoing maturation during juvenile years, and is especially attractive as a study subject
because the degree of mineralization can be easily ascertained through non-invasive
methods such as radiology.
Of the numerous methods for third molar maturation evaluation, there seems to be a broad
current consensus that the Demirjian method (Demirjian et al., 1973), is the most suitable, for
several reasons:
The Demirjian stages are defined by morphological changes more objectively valued than
speculative estimates of length (Olze et al, 2005, 2006). Stages of root formation are more
clearly defined and show the highest values for inter-observer and intra-observer agreement
and for correlation between the defined stages and true age (Dhanjal et al., 2006).
In comparison with other age diagnosis methods, those used in estimating legal age or
minor status should minimise false positives with the aim of avoiding mistaken
classification of a minor as of legal age.
Intra-observer and inter-observer agreement are reported to be high (Dhanjal et al., 2006;
Orhan et al., 2007; Prieto et al., 2007) with best agreement for mandibular third molar when
Demirjian’s method is employed (Arany et al., 2004; Dhanjal et al., 2006).
In a study carried out by the Research Committee of the American Board of Forensic
Odontology Mincer et al (Mincer et al., 1993) evaluate precision in age assessment from the
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development state of the lower third molar, valued according to the Demirjian method.
According to this study, stages A to D (up to complete crown formation) and stage H
(complete apex closure) would show respectively a strong probability that the individual is
younger or older than 18. Accuracy, established based on the difference between the real age
and that calculated from the degree of dental development, is situated at 4.8 years for the
95% range (two standard deviations). Using the same valuation system in a Swedish
population sample Thorson and Hägg (Thorson & Hägg, 1991) observed a weak relation
between chronological and dental age (underestimation of chronological age), with a mean
difference between the estimated and the chronological age of about ±4.5 years in girls (95%
confidence interval) and ±2.8 years in boys, and intra-observer error of ±0.8 years (95%
confidence interval) which for the authors rules out this method for age assessment.
Kullman et al (Kullman et al., 1992) obtain similar results using a system based on
classifying root development into 7 development stages, with standard deviations of 1 to 2
years to the average age in the different development stages.
In the review carried out by Rirz-Timme et al (Rotz Timme et al., 2000) the third molar gives
standard error values (SEE) varying in a range of 1-2.5 years, with correlation ratios (r)
between 0.32 and 0.85.
The use of standard error as a measure of accuracy has been criticised by diverse authors
(Aykroyd et al., 1997; Giles & Klepinger, 1988) and recent papers propose the use of
Bayesian probability as an alternative to regression analysis (Braga et al., 2005), though more
studies with respect to these items are desirable.
Perhaps due to the fact that a good part of the third molar formation process occurs once
puberty is reached, sexual dimorphism contrary to the rest of the dental maturation
processes can be observed, with males reaching the different development stages earlier
than females, independently of their ethnic origin. Studies published to date agree in this
respect (Arany et al., 2004; Garn et al., 1959; Gunst et al., 2003; Haavikko, 1974; Harris, 2007;
Harris & Nortjé, 1984; Köhler et al., 1994; Levesque et al., 1981; Meinl et al, 2007; Orhan et
al., 2007; Prieto et al., 2007; Solari & Abramovitch, 2002; Throson & Hägg, 1991;
Willerhausen et al., 2001).
Studies evaluating third molar development in the maxillary and mandibular arch appear to
agree in the greater advance in maturation of maxillary molars versus mandibular ones
(UNHCR, 2002; 6),(14),(37),Meinl et al., 2007; Orhan et al., 2007; Solari & Abramovitch, 2002;
Willerhausen et al., 2001). For Mincer (Mincer et al., 1993) this could reflect different control
mechanisms in the process of development of the two arches. He also observes that
combining the results of the teeth of both arches seems to improve accuracy slightly.
Though impaction of the third molars has been put forward as a cause of delay in root
formation (Köhler et al., 1994), a recent work by Friedrich (Friedrich et al., 2005) concludes
that the topography of the wisdom teeth did not influence the timing of root development.
All studies coincide in the absence of significant differences depending on the side (left or
right) (Meinl et al., 2007; Orhan et al., 2007; Prieto et al., 2007; Willerhausen et al., 2001).
Dental development can be altered by long term conditions, congenital syndromes, nutrition
deficiencies or hormonal disorders, among others. Meanwhile, the factors influencing dental
formation are difficult to identify.
Meinl et al (Meinl et al., 2007) call attention to the consequences of exclusion of subjects
showing certain pathologies or irregular development, so that age assessment methods
would be only applicable to individuals with a pretended normal dental state. In their study
they decide not to carry out exclusions for such motives and include all the subjects of the
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original sample, observing that none of the outliers had characteristics associated with
altered growth, and also that individuals with severe development alterations did not show
precarious findings. In accordance with these results they assume the hypothesis that dental
development underlies strong regulation mechanisms which seem difficult to alter, even
under pathological conditions.
As in all anthropological analysis, the characteristics of the reference population are a very
significant element. The influence of genetic, nutritional, and geographical factors must be
taken into account, when standards are developed. The applicability of standards generated
to members of ethnic groups that are different from the reference population has been the
subject of controversial discussion. Up to now several studies have been undertaken in
different populations, with the aim of observing the usefulness of the third molar as a
reliable indicator of age(Arany et al., 2004; Blankenship et al., 2007; Bolaños et al., 2003;
Garamendi et al., 2005; Gunst et al., 2003; Gunst et al., 2003; Harris, 2007; Harris & Nortjé,
1984; Kullman, 1992; Martín, 2007; Meinl et al., 2007; Micci & Buzzanca, 1998; Mincer et al.,
1993; Nambiar, 1996; Olze et al., 2004, 2006, 2007; Prieto et al. 2007; Robetti et al., 1982; Solari
& Abramovitch, 2002; illerhausen et al., 2001; Yaacob et al., 1996). These studies have proved
that dental development shows slight variations among different populations, making
specific studies necessary.
Although certain heterogeneity is observed when comparing the results of studies carried
out on different populations, the results of a work (Prieto et al., 2007) performed on a
sample of 1050 ortopantomographs of young Spaniards aged 14 to 21 show that the Spanish
population undergoes maturation development of the lower third molars faster than the
North American, French-Canadian , Austrian and Scandinavian populations, while more
similar to the Hispanic population of the United States.
Garamendi et al (Garamendi et al., 2005) studied a sample of 114 illegal Moroccan
immigrants, whose real age was subsequently obtained. The examination included
radiográphical dental study to estimate third molar maturation following the Demirjian and
Goldstein method previously commented. The results show that this constitutes a good age
diagnosis method –though the standards used were those obtained from the original
French-Canadian population – while efficiency was increased by combining bone
maturation valuation methods.
The lack of data on the influence of the ethnic factor in mineralization represents a
restriction in the reliability of age assessment and therefore in the value of forensic
information essential to legal soundness. This has come about for diverse reasons, such as
the use of different assessment methods, small sample sizes, or African population birth
data not checked, which prevent data being directly comparable.
In various studies together with South African and Japanese colleagues, Olze et al (Olze et
al., 2004, 2006, 2007) evaluate the possible influence of the ethnic factor in third molar
mineralization and eruption, observing a slower maturation process in the mongoloid
population, which could reach the predominant stages of mineralization 1-2 years older and
Africans 1-2 years younger, than Caucasoids and similar behaviour as regards third molar
eruption. These authors think that these differences could be owed to the difference in the
palate dimensions, smaller in mongoloids, which could cause a delay in eruption and
because of this in mineralization.
Blankenship et al (Blankenship et al. 2007) and Harris (Harris, 2007) evaluate the differences
between black and white North Americans. The results coincide with those of Olze et al.
showing greater maturation speed in the black population.
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The scientific studies performed, though representing an important contribution to our

knowledge of third molar maturation, do not resolve the problem facing us in determining
the age of a young illegal immigrant. In the case of Spain and the majority of European
countries, these immigrants come in different proportions from Morocco, the sub-Sahara,
Latin America and Eastern Europe.
Martín de las Heras et al (Martín, 2007) evaluates the development of the third molar in in
the northern Spanish population and the Spanish and Moroccan population resident in one
of the Spanish cities of North Africa (Ceuta). The interest of this work centres on the fact that
the Maghreb population is one of the most frequent illegal immigrant groups in Spain and
other Mediterranean European countries.
The lack of maturation standards for African countries and the difficulty of carrying out
studies in the countries of origin makes it necessary to perform studies enabling us to
evaluate dental maturation in these countries to arrive at more reliable data.
The inter-populational differences shown by these studies underline the need for specific
work in this area. More research is needed in this field.
The results of the work published agree in stating that once the third molar reaches the H
stage of Demirjian, the probability that an individual is of legal age is greater than 90%,
independently of ethnic origin, sex or the tooth evaluated (Arany et al., 2004; Meinl et al.,
2007; Mincer et al, 1993; Solari & Abramovitch, 2002). For this reason this element can be
considered a useful marker to resolve the question of whether an individual of unknown
age can be considered an adult for legal effects.
Only in criminal cases is the use of radiography justifiable.
In practice it is not habitual to request the express consent of a minor to carry out the
exploration and complementary examinations, including radiological tests. Given that the
examination will affect their privacy and that radiological tests mean exposure to radiation
with potentially harmful effects, as we understand it it should be obligatory to obtain the
consent of the subject, and to suspend examination in the absence of consent. This aspect is
included in the guide of good practice of the program for unaccompanied minors in Europe.
It must further be appreciated that using techniques developed for clinical purposes - to
seek possible pathologies - in evaluating the degree of maturation for a specific age
represents a perversion, as it inverts the application in the search for evidence to deduce the
age of an individual from their maturation level when there is no clinical reason justifying
such use.
Attempts to standardisation, calibration and evaluation procedures have been scarce up to
now, pointing to the need for guidelines on this and all other aspects.
The difficulty involved hence in diagnosis and the potential sources of evaluation makes it
necessary to draw up common guidelines for action, based on scientific evidence and
unifying the criteria to be followed, such as those of the Study Group on Forensic Age
Diagnosis (AGFAD), founded in Berlin in 2000. The group has published guidelines on age
diagnosis on living individuals for criminal, civil and asylum proceedings among others
(Schmeling et al., 2007).
The International Organization for Forensic Odonto-Stomatology (IOFOS) has published
recommended procedures for quality assurance in forensic dental age estimation
http://www.odont.uio.no/foreninger/iofos/quality/Age-IOFOS.htm
Regarding these recommendations, Solheim and Vonen(86) highlight the discrepancies of
criteria among experts over the steps to follow between those who only wish to apply a
statistical method and report on the results and those who prefer to express the expert
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opinion taking into consideration the life conditions of the individual such as state of health
and nutrition, clinical findings and of course the results of the statistical scientific methods.
These authors coincide with the German Interdisciplinary Working Group for Age
Diagnostic (34) that the conclusions should end with a complete assessment of the most
likely chronological age.
Just as Ritz-Timme et al (Ritz-Timme et al., 2000) recommend, efforts should be made to
develop external quality control, something perfectly possible in the field of age assessment,
with the aim of guaranteeing quality standards enabling an adequate response given the
important role played by forensic medicine in the legal and social fields of age assessment.
5.4 Age estimation in the clavicle

To answer the question of whether a person has reached the age of 18 it is particularly
helpful to evaluate the ossification status of the medial epiphysis of the clavicle, because all
other examined developmental systems may already have completed their growth by that
age.
A number of studies have been conducted on the time frame for the ossification of the
medial clavicular epiphyseal cartilage in the age group concerned for forensic age
diagnostics in living individuals. One group of studies adopted an anatomical perspective,
assessing ossification by means of autopsy or direct skeletal inspection (Todd & D’Errico,
1928; McKern & Stewart, 1957; Owings Webb & Myers Suchey, 1985; MacLaughlin, 1990; Ji
et al., 1994; Black & Scheuer, 1996; Shirley, 2009; Singh & Chavali, 2011), while the other
group took a radiological approach.
Several authors pointed out, that data from dry bone material are not directly comparable
with data from radiological studies. Krogman and Isçan (Krogman & Isçan, 1986) as well as
Kreitner et al. (Kreitner et al, 1998), for example, argued that commencement of fusion can
be detected in radiographs before any union of epiphysis and metaphysis can be visible on
dry bone. Therefore reference values from dry bone studies should not be applied to
assessments based on radiographs.
Radiological methods to examine the medial clavicular epiphysis in living individuals are
conventional radiography (CR) (Flecker, 1933; Galstaun, 1937; Jit & Kullkarni, 1976;
Schmeling et al., 2004; Garamendi et al. 2011), computed tomography (CT) (Kreitner et al.,
1997, 1998; Schulz et al., 2005; Schulze et al., 2006; Bassed et al., 2010; Kellinghaus et al.,
2010a, b), as well as new approaches using magnet resonance imaging (Schmidt et al., 2007;
Hillewig et al., 2011) and ultrasoundsonography (Schulz et al., 2008b; Quirmbach et al.,
2009; Schulz et al., 2010).
While traditional classification systems differentiate between four stages of clavicle
ossification (stage 1: ossification centre not ossified; stage 2: ossification centre ossified,
epiphyseal plate not ossified; stage 3: epiphyseal plate partly ossified; stage 4: epiphyseal
plate fully ossified), Schmeling et al. (Schmeling et al., 2004) divided the stage of total
epiphyseal fusion into two additional stages (stage 4: epiphyseal plate fully ossified,
epiphyseal scar visible; stage 5: epiphyseal plate fully ossified, epiphyseal scar no longer
visible). Figure 1 shows the stages of clavicular ossification for CR and CT.
There is only one study referring to conventional radiography that meets the requirements
of a reference study as stated by the Study Group on Forensic Age Diagnostics (Schmeling et
al., 2008). In this study the earliest age at which stage 3 was detected in either sex was 16
years. Stage 4 was first observed in women at 20 years and in men at 21 years. Stage 5 was
first achieved by both sexes at age 26 (Schmeling et al., 2004). It was concluded that plain
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chest radiographs can essentially provide a basis for assessing clavicular ossification. If
overlap in posterior-anterior views impedes evaluation additional oblique images
(Bontrager & Lampignano, 2009) should be taken to facilitate age estimation.
In 1997 and 1998, Kreitner et al. published the first CT-based studies in which the medial
epiphyseal ossification of the clavicle was evaluated applying a four stage scheme. Since
these studies did not discriminate results by sex, their forensic value is limited. In a CT
study conducted by Schulz et al. in 2005, presenting more cases and results discriminated by
sex, the five stage classification by Schmeling et al. (Schmeling et al., 2004) was used. The
earliest occurrence of stage 3 in females was noted at age 16 and in males at age 17. Stage 4
was first achieved by both sexes at age 21. Stage 5 was first noted in females at age 21 and in
males at age 22, which is 4 or 5 years earlier than is reported in the conventional
radiographic studies. Schulz et al. (Schulz et al., 2005) raised the question whether CT scans
with a slice thickness of > 1 mm could cause misinterpretation of clavicle ossification status
and recommended examining the influence of slice thickness on the age intervals of
ossification stages in additional studies.
In a study on the influence of the slice thickness on the ability to assess the stages of
clavicular ossification Mühler et al. (Mühler et al., 2006) retrospectively analysed the CTs of
40 individuals which have been examined within the scope of age diagnostics. Scans with
slice thicknesses of 1, 3, 5, and 7 mm have been reconstructed from the obtained data. Seven
out of 80 clavicular epiphyseal plates showed differences depending on the slice thickness in
the particular stages of ossification. In 1 case a slice thickness of 1 mm led to a different
diagnosis of the ossification stage than a slice thickness of 3 mm, in 3 cases the diagnoses
differed between the slice thicknesses of 3 mm and of 5 mm, and in another 3 cases between
5 and 7 mm. The authors therefore concluded that for age estimation purposes the slice
thickness should be 1 mm in order to ensure maximum accuracy and diagnostic reliability.
The findings of this study were confirmed by Kaur et al. (Kaur et al., 2010).
Recently, Kellinghaus et al. (Kellinghaus et al., 2010) published data from a thin-slice CT
study. In this study stage 3 was first achieved by male individuals at age 17 and in females
at age 16. The occurrence of stage 4 was first found in both sexes at the age of 21. In either
sex, the earliest observation of stage 5 was at age 26. These findings are consistent with the
data from the conventional study of the clavicle (Schmeling et al., 2004).
A further improvement of age diagnostics based on clavicular ossification was the
subdivision of stages 2 and 3 by Kellinghaus et al. (Kellinghaus et al., 2010). The sub-
classification stages were defined as follows (see figure 2):
Stage 2a: The lengthwise epiphyseal measurement is one third or less compared to the
widthwise measurement of the metaphyseal ending
Stage 2b: The lengthwise epiphyseal measurement is over one third until two thirds
compared to the widthwise measurement of the metaphyseal ending
Stage 2c: The lengthwise epiphyseal measurement is over two thirds compared to the
widthwise measurement of the metaphyseal ending
Stage 3a: The epiphyseal-metaphyseal fusion completes one third or less of the former gap
between epiphysis and metaphysis
Stage 3b: The epiphyseal-metaphyseal fusion completes over one third until two thirds of
the former gap between epiphysis and metaphysis
Stage 3c: The epiphyseal-metaphyseal fusion completes over two thirds of the former gap
between epiphysis and metaphysis
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Stage 3c first appeared at age 19 in both sexes. If stage 3c is found, it is therefore possible to
substantiate that an individual has already reached the legally important age threshold of 18
years.
For forensic age estimations in living individuals, non-ionising procedures for the
presentation of the medial clavicular epiphyseal cartilage would be desirable, as the
radiation exposure from the necessary imaging examination could be decreased
considerably. Against this background Schmidt et al. (Schmidt et al., 2007) prospectively
evaluated magnetic resonance (MR) scans of 54 sternoclavicular joints of bodies aged
between 6 and 40 years. All of the examined medial clavicular epiphyseal cartilages
permitted an assessment of the degree of ossification. Stage 2 was first observed at the age
of 15 years, the earliest age at which stage 3 was observed was 16 years, and stage 4 was
first observed at the age of 23 years. Very recently Hillewig et al. (Hillewig et al., 2011)
published a four-minute approach for MRI of the medial clavicular epiphysis in living
individuals.
In a comparative study using CR, CT and MRI for staging of 15 sternoclavicular joints the
ossification stage was in agreement in each of the three imaging methods used in 6 cases
(Vieth et al., 2010). In the remaining cases the ossification stage was assessed either one stage
higher or lower in one of the applied imaging methods than in the other two techniques. In
five cases (compared to CT), respectively seven cases (compared to MR), CR showed a
higher ossification stage than CT and MR imaging. In two cases CR resulted in the
determination of a lower stage than in CT and MR imaging. Twice the MR scans showed a
less advanced ossification stage than CT-based images. The authors concluded that in age
estimation practice, modality-specific reference studies are to be applied in order to
guarantee an adequate assessment of the ossification stage of the medial clavicular
epiphysis.
Another radiation free approach to evaluate the ossification stage of the medial clavicular
epiphyses is ultrasound sonography. Schulz et al. (Schulz et al., 2008) prospectively
evaluated 84 right clavicles of test subjects 12–30 years of age by means of ultrasound. For
the sonographic assessment of the clavicle ossification, the traditional classification had to be
modified as follows (see figure 3):
Stage 1: The medial end of the clavicle is configured acute-angled. A bony center of
ossification is not representable.
Stage 2: The medial end of the clavicle is separated from the bony center of ossification by a
sound gap.
Stage 3: Both an ultrasound gap with a bony center of ossification and a fully ossified
epiphyseal plate with a convex curved end of the clavicle are representable.
Stage 4: The medial end of the clavicle is convex curved. A bony center of ossification is not
representable.
The earliest ages at which the respective ossification stages were observed were 17 years for
stage 2, 16 years for stage 3, and 22 years for stage 4. Another pilot study by means of
sonography was performed by Quirmbach et al. (Quirmbach et al., 2009). In this study stage
4 was first observed at 20 years. These results were reviewed in a reference sample of 601
healthy volunteers aged between 10 and 25 years by Schulz et al. (Schulz et al., 2010). The
earliest observation of stage 4 in women was at 19 years in both sexes. Based on these
findings it can reliably be stated that an individual with stage 4 has already accomplished 18
years of age.
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Fig. 1. Schematic drawings and pictures of the stages 1-5 of clavicular ossification as
revealed by conventional radiography (CR) and computed tomography (CT)
Fig. 2. Schematic drawings and pictures of the stages 2a-3c of clavicular ossification as
revealed by means of thin-slice CT
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Fig. 3. Schematic drawings and pictures of the stages 1-4 of clavicular ossification as
revealed by ultrasound
6. Interpretation of results
6.1 Interpretation of results from combined methods. The clavicle and the twilight
zone
AGFAD Guidelines on Age Estimation in Living Subjects recommend that when performing
a FAE different methods must applied in the same subject. These recommendations mean
that in the same subject different FAE results will be available (AGFAD, 2001).
Previous series (Garamendi et al., 2005) proved that the accuracy of FAE improves when
different age estimation methods are simultaneously applied. The under ROC curve area
increase when both bone age and dental age methods are applied when assessing if a subject
could be older or younger than 18.
In cases of combined analysis of age estimation based on dental age and bone age in carpus,
the lowest result of both methods should be used for a final estimation because of ethical
reasons and legal accuracy.
Fig. 4. Simulacra of distribution of results of bone age in carpus and in clavicle in relation
with complete fusion of distal radius and complete union of medial clavicle.
More complex is the application of results in case of doubt about an age estimation over or
under 18 years or 21 years old based on results of an age estimation in the clavicle. Positive
results (clavicle stage 4 of Schmeling´s method) indicate positively that the age of the subject
will be most probably older than 21,6 in males and 21,3 in females (Kellinghaus et al., 2010).
Nevertheless, stage 3 is observed in subjects up to 26 years old and aritmetical mean of stage
4 is 29,6 +/- 4,2 in males and 8,2+/-4,2 in females. 100% of the sample in stages 4 or 5 of
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Schmeling method is observed only in subejcts older than 26 (Kreitner et al., 1997;
Schmeling et al., 2004). These statistical results are graphically explained in figure 4 in which
it´s clear that a negative result of stage 4 in a subject older than 21 years and younger than 26
is in many cases most probable than a positive result. It turns the range between 18 and 26
years old a twilight zone in which a positive result is definitive but a negative result is the
most probable one and suppose the imposibility to assess a FAE of being older than 21 years
old in most cases of subjects younger than 26.
Nevertheless, more research is needed in this specific field to define more precisely the best
standarized approach to FAE based on simultaneous different methods.
6.2 Ethnical and socioeconomic factors: sexual maturation, ossification, dental

development
Since the subjects whose age has to be estimated mostly belong to populations for which no
reference studies are available that could be used for forensic purposes, the question arises
whether there are significant developmental differences between various ethnic groups
which would prohibit the application of relevant age standards to members of ethnic groups
other than the reference population. In this respect the term 'ethnicity' shall be used only to
identify the affinity of various populations in terms of origin.
Comprehensive studies of the relevant literature revealed that the major ethnic groups of
interest to forensic age estimation achieve defined stages of ossification, dentition and
sexual maturity in the same natural sequence, so that it is generally possible to apply the
relevant reference studies also to other ethnic groups (Schmeling et al., 2001).
6.2.1 Sexual maturation

Data on sexual maturation is relatively scarce. Comparative studies on sexual maturation
were conducted by Harlan et al. (Harlan et al., 1979, 1980), Channing-Pearce and Solomon
(Channing-Pearce & Solomon, 1987), Wong et al. (Wong et al., 1996) and Huen et al. (Huen
et al., 1997). Between 1966 and 1970, Harlan et al. (Harlan et al., 1979) analysed the sexual
development of 6,768 male Americans aged between 12 and 17 years. They found no
significant differences between blacks and whites. In 1980 Harlan et al. (Harlan et al., 1980)
published a representative study examining the sexual maturation of a female American
population of the same age group. This study observed relatively faster rates of maturation
for blacks compared to whites. Channing-Pearce and Solomon (Channing-Pearce &
Solomon, 1987) examined sexual development in a study involving 362 black and 355 white
girls in Johannesburg, South Africa. Unlike Harlan et al. (Harlan et al., 1980), they came to
the conclusion that black girls on average reached full sexual maturity later than white girls.
Wong et al. (Wong et al., 1996) examined sexual maturation in a 1993 study involving 3,872
boys from southern China. They found that the time pattern of sexual maturation was
comparable to that of Europeans, with the exception that Asians developed pubic hair later.
Huen et al. (Huen et al., 1997) published a similar study including 3,749 girls from southern
China. They found that, according to the mean values for the individual stages of maturity,
the examined girls were among the earliest to reach sexual maturity worldwide.
6.2.2 Ossification
Numerous studies are available on skeletal maturation of all major ethnic groups (Africans,
Australians, Caucasoids and Mongoloids) (Schmeling et al., 2000a). Because there are
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several potential factors of influence and their simultaneous action makes assessment of
population differences a difficult exercise, all the more as the validity of some of those
investigations seems to be limited to small sample size, the exclusive consideration of non-
relevant age groups, lack of information on health, ethnic identity and socioeconomic status
and absence of confirmed data on proband age. Hence, for the problem at hand, greatest
relevance may be claimed for studies on various ethnic groups of similar socioeconomic
status and living in one and the same region or populations of one and the same
socioeconomic status living in different regions. Such studies are available from the USA
where research has been conducted on descendants of Caucasoids, Mongoloids and
Africans as well as from numerous ethnic groups of the former Soviet Union.
In a comparison with the Greulich-Pyle standards, Sutow (Sutow, 1953) discussed racial
differences as one of the causes of retarded skeletal maturation of Japanese children living in
Japan. His findings were checked by Greulich (Greulich, 1957) who referred to Japanese
individuals living in the USA. He studied hand bone development in 898 children of
Japanese descent aged between 5 and 18 years living in the San Francisco Bay area of
California. While retarded skeletal maturation, in comparison with the Greulich-Pyle
standards was recorded by Sutow for all age groups of Japanese living in their own country,
such retardation was detected by Greulich only in boys aged between 5 and 7 years. Boys
aged between 13 and 17 and girls between 10 and 17 years even exhibited comparative
acceleration. Greulich concluded that the significant retardation, in comparison with the
Greulich-Pyle standards, recorded for children living in Japan was attributable to less
favourable nutritional and environmental conditions rather than to racial differences.
Improved living standards in recent decades resulted in accelerated skeletal maturation
even in Japanese living in Japan (Kimura, 1977a, 1977b) which, in the meantime has come to
lie within the range of socioeconomically advanced European populations (Beunen et al.,
1990; Wenzel et al., 1984).
Whereas some authors (Massé & Hunt, 1963; Garn et al., 1972) reported comparatively
accelerated skeletal development in Africans in early childhood, ethnic origin obviously has
no significant impact on the bone growth rate in later childhood and adolescence. Platt
(Platt, 1956) studied skeletal maturation in 100 black inhabitants of Florida, 143 blacks in
Philadelphia and 100 whites in Philadelphia aged between 5 and 14 years. In none of these
three groups was skeletal age, as determined by identical X-ray standards, significantly
different from chronological age. Platt compared his results with studies on black residents
of Africa. Mackay (Mackay, 1952) recorded retardation by 1.5 to 2 years for East Africans,
while Weiner and Thambipillai (Weiner & Thambipillai, 1952) recorded an average
retardation of 16 months for West Africans. The assumption of an ethnic impact on skeletal
maturation would justify expectation of a continuous series of phenomena ranging from
severe retardation in blacks in Africa to moderate retardation in black Americans who had
mixed with whites to absence of retardation in whites. Such continuous series do not exist,
and consequently Platt, postulated that health and nutrition are the major factors
influencing skeletal maturation.
Skeletal maturation in 461 black and 380 white Americans in the Lake Erie region was
studied by Loder et al. (1993) between 1986 and 1990. Using the atlas method of Greulich
and Pyle on the age group of 13-18 years, they recorded comparative acceleration of 0.45
years for white boys, 0.16 years for white girls, 0.38 years for black boys and 0.52 years for
black girls. Johnston (Johnston, 1963) studied the same age group of white Americans in
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Philadelphia by the same method and found acceleration values of 0.39 years for boys and
0.58 years for girls. Johnston's data for white Americans were almost identical with Loder's
findings for black Americans, which seems to clearly underline that in the populations of the
age group studied there were no ethnic differences with regard to skeletal maturation.
Roche et al. (Roche et al., 1975, 1978) investigated skeletal maturation in the context of race,
geographic region, family income and educational standards of parents in a representative
cross-section of the US population aged between 6 and 17 years. They found no consistent
black-white differences, no significant differences between regions and no urban-rural
differences.
Comprehensive studies were conducted on skeletal maturation in different ethnic groups of
the former Soviet Union, and 16 studies of 17 ethnic groups in different climatic and
geographic zones of the former Soviet Union were evaluated by Pashkova and Burov
(Pashkova & Burov, 1980). Included were Russians, Ukrainians, Georgians, Armenians,
Azerbaidjanis, Balkarians, Cabardines, Kazakhs, Tadchiks, Uzbeks, Ingushi, Chechenians,
Udmurtians, Chukchen, Koryaks, Intelmenians and Evenkians. The range of variation at all
stages of skeletal maturation was less than one year in all populations studied. However, the
causes of those variations were attributed by the authors to relatively small samples,
different methods and techniques used in the studies or undiagnosed clinical conditions of
probands but were not attributed to ethnic, regional or climatic differences.
Studies evaluated so far seem to suggest that there is a genetically determined element to
skeletal maturation which does not appear to depend on ethnicity and may be exploited
under optimum environmental conditions (i.e. high socioeconomic status), whereas a less
favourable environment may lead to retardation of skeletal maturation. Applying X-ray
standards to individuals of a socioeconomic status lower than that of the reference
population, usually leads to underestimating a person's age. In terms of criminal
responsibility, this has no adverse effect on the person concerned (Schmeling et al., 2000,
2006).
6.2.3 Dental development

Few comparative studies are available on the subject of third molar mineralization. Gorgani
et al. (Gorgani et al., 1990) examined 229 black and 221 white US citizens aged 6-14 years.
Among the black subjects crown mineralization of the third molars was completed 1 year
earlier. Harris and McKee (Harris & McKee, 1990) studied 655 white and 335 black US
citizens aged 3.5-13 years. Whereas the black subjects reached the earlier stages of third
molar mineralization about 1 year earlier, the gap appeared to narrow for later stages. This
trend is confirmed by the work of Mincer et al. (Mincer et al., 1993). They examined 823 US
citizens (80% white, 19% black) aged 14-25 years but did not establish any significant
differences in the time frame for third molar mineralization. Daito et al. (Daito et al., 1992)
addressed third molar mineralization in 9111 Japanese children aged 7-16 years and
compared their data with the values provided by Gravely (Gravely, 1965), Rantanen
(Rantanen, 1967) and Haavikko (Haavikko, 1970) for Caucasoid populations. No significant
differences were discovered. These studies only lend themselves to limited comparison due
to small sample sizes, varying methods and assessment by different observers. A further
problem lies in the fact that the age data for subjects of black African origin often was not
verified. Moreover, most available studies focus on the earlier stages of mineralization.
A comparative study of third molar mineralization (Olze et al., 2004) was carried out on
three population samples: one German, one Japanese and one South African. To this end,
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3652 conventional orthopantomograms were evaluated on the basis of Demirjian’s stages.

Statistically significant differences between the samples investigated were established for the
age at which stages D-G of third molar mineralization were achieved. Significant differences
between German and Japanese males were noted for stages D-G of mineralization. Significant
differences between Japanese and German females were observed for stages D-F. According to
these findings, Japanese males and females were approximately 1-2 years older than their
German counterparts when they reached stages D-F. Significant age differences between South
African and German males applied to stages D-E. Significant age differences between South
African and German females were observed for stages E and G. The South African subjects
were approximately 1-2 years younger than the German subjects upon achieving these stages
of mineralization. Significant age differences between the South African and Japanese samples
were ascertained for both sexes at stages D-G. The South African subjects were approx. 1-4
years younger than the Japanese subjects upon reaching these stages.
The population differences observed here may be due to differences in palatal dimensions
between the ethnic groups surveyed. The largest palatal dimensions are observed in
Africans and the smallest in Mongoloids, with Caucasoids assuming the middle rank (Byers
et al., 1997). Inadequate space in the maxillary crest causes delay in third molar eruption, if
not retention (Fanning, 1962). In turn, retained third molars mineralize later than teeth
whose eruption has not been impeded (Köhler et al., 1994). This would explain why
Caucasoid populations occupy the middle position in relative terms when it comes to third
molar mineralization, while Mongoloid populations display a comparative delay and
African populations a relative acceleration.
With regard to the eruption of third molars, some studies have found significant differences
between specific populations (Schmeling et al., 2001). While in Caucasian populations third
molars generally do not erupt before age 17 (Müller, 1983), Brown (Brown, 1978), Chagula
(Chagula, 1960), Otuyemi et al. (Otuyemi et al., 1997), and Shourie (Shourie, 1946) describe
cases of eruption starting in African, Australian and Indian populations already at age 13.
Comparative studies on the relation between age and third molar eruption are available for
black and white Americans, Africans, and Asians. Garn et al. (Garn et al., 1972) studied the
dentition of all permanent teeth in 953 black and 998 white Americans. In black Americans,
the maxillary third molars developed 3.7 years earlier, and the mandibular third molars 5.6
years earlier, than in white Americans. Hassanali (Hassanali, 1985) compared the eruption
times of third molars in 1,343 Africans and 1,092 Asians in Kenya. He found that in Africans
third molars appeared two to three years earlier. The forensic applicability of these studies is
limited, since age data for subjects of African origin are often not verified.
Olze et al. (Olze et al., 2007) analyzed and compared the chronological course of third molar
eruption in German, Japanese, and South African populations with proven age of subjects.
They found that their German sample had an intermediate rate of dental development as
determined by comparing the different ages of third molar eruption. The defined eruption
stages occurred at earlier ages in the investigated South African sample, and at later ages in
the Japanese sample. Statistically significant population differences were observed in males
at stages A and B. The South African males were on average 3.0 to 3.2 years younger than
the German males at these stages of development, and the Japanese males were on average
3.1 to 4.2 years older than their South African counterparts for the same developmental
stage. The females exhibited statistically significant population differences at stages A, B and
C. The South African females reached the target stages on average 1.6 to 1.8 years earlier
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than the German females, whereas the Japanese females were on average 0.9 to 3.3 years
older than their German counterparts. It was concluded that population-specific reference
data should be used when evaluating third molar eruption for the purpose of forensic age
estimation.
6.3 Pathological factors that produce bone age retardation and acceleration
Pathological conditions alter FAE based on methods of bone and dental age maturation.
Albeit being less affected by pathological and ambient conditions than bone age, biomedical
literature proves that dental age can be altered by some entities not necessary pathological,
like delayed puberty or obesity.
It has been repeatedly demonstrated in the literature that bone age is affected by a wide
range of pathological conditions. Main pathological conditions altering bone age maturation
are those endocrinological conditions that modify the hypothalamus - hypophysis- gonads
axis. Nevertheless other pathological entities affect bone age maturation: some clinical
syndromes (as Soto's syndrome or Weaver's syndrome), bone dysplasias (some of them
accelerating and other decelerating bone age maturation) and some drugs intake (table 1
and 2) (Taybi & Lachman.1990).
Forensic examiner must be aware of these conditions. The existence of such pathological
conditions or drugs intake must must elucidated, as they evidently affect the bone age and
dental age maturation. The results of a FAE must include corrections in relation with the
existence of such accelerating or decelerating factors.
7. Ethical questions
7.1 Exposition to radiation without clinical indication
When an X-ray examination is carried out exclusively with FAE purposes and without a
clinical indication the question arises on the possibility of detrimental effects due to the
radiation exposure (European Commision 2004)
The effective dose from an standard X-ray examination of the hands is 0,1 microSievert
(µSv), 26 µSv in case of Orthopantomograms, 220 µSv in X-ray examination of the proximal
epiphyses of the clavicle and 600 to 800 µSv in case of TC of the sternoclavicular joints. The
effective dose in case of a complete thorax TC is 6,6 mSv (Rammstahler et al.2009).
Some authors indicate that an amount of effective annual doses for X-ray examinations of
less than 10 µSv are negligible. Other authors have stressed the insignificance of these usual
FAE examination doses in comparison with naturally-occurring and civilizing radiation
exposure. The effective dose from naturally-occurring radiation exposure has been
calculated as an average in Germany at about 1,2 mSv per year and in The Netherlands at
about 2,0 mSv. Fly staff of airplanes receive an average of 2000 mSv per year as a result of
staying high in air (cosmic radiation). The radiation exposure from intercontinental flight at
an altitude of 12000 meters is 0,008 mSv per hour (Schmeling.2008).
It follows that the radiation dose effective in case of an intercontinental flight is equivalent
to 2 orthopantomograms and a CT of sternoclavicular joints is equivalent to 3,5 months of
naturally occurring radiation exposure. On the basis of these comparisons the health risk as
a result of usual X-ray examinations for FAE is negligible (Schmeling.2008)
Nevertheless, radiation exposure produce not only stochastic but non-stochastic damages
the physicians must be aware of. Non-stochastic effects appear above 100 mSv and are
therefore irrelevant in usual radiological diagnosis. But non-stochastic effects don't have
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such a threshold and are not dose related, so their eventual appearance in case of X-rays
examinations must be cautiously considered. Some authors minimize and other maximize
the harm inherent to these non-stochastic effects (Garamendi&Landa.2010)
Bone Age Advanced Drugs altering bone age

Adrenal Hiperplasia Gigantism Amiodarone
Acrodysostosis Hypertiroidism Budenoside
Adrenocortical Tumour Idiopatic isosexual Buclometasone, Dipropionate
precocious puberty
Aldosteronism, primary Lipodistrophy Phenitoine
Arthrogryposis Marshall Smith Syndrome Metilphenidate
Beckwith-Wiedemann, McCune Albright Syndrome
syndrome
Cerebral tumour Neurofibromatosis
Cockayne syndrome Obesity
Congenital Adrenal Peripheral dystosis
Hyperplasia (Brailsford)
Cushing Syndrome Sotos syndrome
Ectopic Gonadotropine Trisomy 8 Syndrome
Production
Familar Advanced Bone Age Weaver syndrome
Table 1. Pathologic factors associated with bine age acceleration and drugs intake that alter
bone age process.
Anyway, albeit apparently negligible the possible detrimental effects due to radiation
exposure must be considered. A best practice in FAE on the use of X-ray exams without
other medical indications should avoid unnecessary repetition of the exams to minimize the
radiation dose effective received by probands, should include the practice of the minimum
necessary exams to avoid unnecessary exams and should include the elaboration of National
Registers of unaccompanied minors to avoid the unnecessary repetition of all tests by
different physicians at different dates (Garamendi et al.2011)
7.2 Ethical dimension of the expert report

Forensic physicians involved in FAE case analysis must be conscious of the ethical
dimension of the conclusions written on their expert reports. Dr. Mata in 1842 pointed out
that expressing an expert opinion in a Justice Court must be cautious (Mata, 1842). This old
professor reminds future forensic physicians that writing a report expressing complete
reliability in a question without a solid scientific basis could be an error. Some physicians
understand that expressing themselves with complete certainty at Justice Court helps judges
to decide on questions like FAE more clearly. Oppositely, this is not an advisable attitude as
it can give the Court a false impression of certainty in questions not completely certain in
the state of the art for the scientific community. This very same principle is the one
precluded by Evidence Based Medicine.
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Bone Age Retarded

Addison Disease Dubowitz syndrome Juvenil Idiopatic Other chronic
Osteoporosis infections, as Malaria
Amonopterin Elite sports Juvenil Rhemtaoid Papillon-Lefevre
fetopathy Arthritis Syndrome
Anemias Endemic Diarrhea KBG syndrome Parasitosis
Aspartylglucosa- Extreme weight loss Kocher-Debre- Patterson Syndrome
minuria Semelaigne Syndrome
C Syndrome Fetal rubella Laron Dwarfism Phenylketonuria
syndrome
Celiac Disease Freeman-Seldom Larsen Syndrome Pleonosteosis
syndrome
Cistis fibrosis Fucosidosis Legg, Calvé, Perthes Prader- Willi
Disease Syndrome
Cleiodocraneal GAPO syndrome Lenz-Manjewski Raquitism
dysplasia hiperostotic dwarfism
Coffin-Lowry Geophagia-dwarfism Lesch-Nyhan Renal Failure
Syndrome hipogonadism syndrome
syndrome
Coffin-Siris Syndrome Glycogenosis tipe I Malnourishement Renal tubulra acidosis
Crohn Disease Hipoadrenalism Marinesco-Sjögren Rickets
syndrome
Cupper Deficiency Hipogonadism Mauriac syndrome Rubinstein-Taybi
syndrome
De Lange Syndrome Hipoparatiroidism Melnick-Needles Silver-Russel
osteodysplasty syndrome
De Morsier syndrome Hipopituitarism Metartropic dysplasia Talasemia
De Sanctis-Cacchione Hipotiroidism Meyer dysplasia of Thalassemia
syndrome femoral head
Deaf mutism-goiter Histiocitosis X Mucolipidosis II The 3 M syndrome
euthyroidism
syndrome
Deprivation dwarfism HIV infection Mucopolysacharidosis Trichohesis nodosa
syndrome
Deprivation Syndrome Hypogonadysm Nephrotic syndrome Trisomy 21
Diabetes Mellitus Incontinentia Nervous Anorexia Weill-Marchesani
pigmenti Syndrome
Diabetes Mellitus Intrauterine growth Noonan syndrome Wilson disease
retardiation
Drugs Johanson-Blizzard Osteoporosis Zellweger Syndrome
syndrome idiopatic juvenile
Table 2. Pathologic factors associated with bone age process retardation.

Case reports like the one published by Nambiar et al in 1998 warns forensic physicians of
the real ethical dangers of not correctly expressing the degree of certainty of the results
included in their expert reports (Nambiar et al., 1998).
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8. Expert report
According to the recommendations of the Study Group on Forensic Age Diagnostics
(Schmeling et al., 2008) the collected findings and the determined stages are to be presented
in detail in the expert report. The used stage classifications and reference studies are to be
mentioned.
Reference studies used for forensic age estimation should meet the following requirements:
 Adequate sample size
 Proven age of subjects
 Even age distributions of subjects
 Analysis separately for both sexes
 Information on the time of examination
 Clear definition of the examined features
 Detailed description of the methods
 Data on the reference population regarding ethnicity, socioeconomic status, state of
health
 Data on the sample size, mean value, and range of scatter for each examined feature
Examples of reference studies are Greulich and Pyle (Greulich & Pyle, 1959), Gunst et al.
(Gunst et al., 2003), Kahl and Schwarze (Kahl & Schwarze, 1988), Kellinghaus et al.
(Kellinghaus et al., 2010a, b), Mincer et al. (Mincer et al., 1993), Olze et al. (Olze et al., 2003,
2004b, 2006), Ruhstaller (Ruhstaller, 2006), Schmeling et al. (Schmeling etl., 2004), Tanner et
al. (Tanner et al., 2001), Thiemann et al. (Thiemann et al., 2006).
For each examined feature, the report must indicate the most likely age and the range of
scatter of the reference population. While mean values and medians show the most likely
age for a certain age characteristic standard deviations and interquartile differences are
common measurement data for the ranges of scatter. Mean values and standard deviations
are valid only for normally distributed features. 68% of the test persons of the reference
population with a certain feature lie between the mean value plus/ minus one standard
deviation and 95% of the test persons lie between the mean value plus/ minus two standard
deviations. Medians and percentiles are distribution independent parameters. 50% of the
test persons with a special feature lie within the 25th and the 75th percentiles. This difference
is also called interquartile difference.
It has to be pointed out that means and medians can not be used for the last stage of the age
characteristics because they depend on the upper limit of the examined age group. Instead
of mean values the 50% probability value should be used for the last stage of an age
characteristic. This value can be calculated by means of logistic regression (Knell et al.,
2009).
The results of the individual examinations should be compiled in a final age diagnosis. The
individual’s most likely age is estimated on the basis of all partial diagnoses and a critical
discussion of the individual case. This final age diagnosis should include a discussion of the
age-relevant variations resulting from application of the reference studies in an individual
case, such as different ethnicity, different socioeconomic status or diseases that may affect
the development of the individual examined.
However, for age diagnoses obtained with a combination of methods there is still no
satisfactory way to scientifically determine the margin of error. While a number of reference
studies collected data on individual features and some studies both on skeletal maturation
and tooth mineralization (Grön, 1962; Lacey, 1973; Lamons & Gray, 1958; Pfau & Sciulli,
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1994), there is still no reference study available analysing all required features for one single
reference population. If independent features are examined as part of an age diagnosis that
combines several methods, it may be assumed that the margin of error of the combined age
diagnosis would be smaller than that for each individual feature. So far it has not been
possible to quantify this difference. Since combining methods also makes it possible to
identify statistical outliers, the margin of error of the combined age diagnosis should
decrease to a certain extent, which unfortunately is also not quantifiable.
Indirect conclusions about the range of scatter of the summarizing age diagnosis were
possible after verifying age estimates carried out at the Institute of Legal Medicine in Berlin.
To this effect, the court's case files of the persons originally examined for age estimation
purposes at the institute were consulted to see whether the actual age of these persons was
established during the court proceedings. In all cases where the age of the person concerned
could be verified beyond doubt deviation between estimated and actual age ranged between
plus or minus 12 months (Schmeling et al., 2003).
Furthermore the expert report should give the degree of probability that the stated age is the
actual age or that the individual’s age is above the relevant age limit. To this purpose the
following probability ratings can be used:
 “almost certain probability (beyond reasonable doubt)”: probability > 99.8% (This
probability refers to the threefold standard deviation).
 “very probable” or “high probability”: probability > 90%
 “probable”: probability > 50%
 “undecided”: probability = 50%
 “less probable” or “improbable”: probability < 50%
 “very improbable”: probability < 10%
 “almost certainly improbable”: probability < 0.2%
The following phrasing gives an example for an adequate conclusion:
“Summarizing all test results the following can be established:
 There is a very high probability that the given date of birth is not correct but that an
earlier date can be assumed,
 there is a very high probability of an age of above 14 years and a high probability of one
above 16,
 there is but very low probability that the 18th year of age has been reached,
 and there is almost certain probability that the 21st year of age has not been reached.”
9. Practical cases
Case 1
The accused, of Afghan origin, was under investigation on murder charges. According to his
own statement he was 13 years and 5 months old at the time of examination. As the
investigative authorities had considerable doubts relative to the age given by the person
concerned, a court order was issued for forensic age estimation with inclusion of X-ray
examinations.
In the course of physical examination, a body length of 168 cm was measured, body weight
was 55 kg. The upper lip and chin were covered with 5 mm-long beard growth, the cheeks
manifested an after-shave condition. The larynx was prominent. The armpits displayed 5
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mm-long hair stubble. The genital region manifested a dense area of curly hair rising up
sparsely to the navel. Genital development was to be placed in the second phase of puberty.
The physical stature and the pattern of body hair corresponded to those of an adolescent.
No indications of developmental disorders were detected.
Fig. 5 shows the hand radiograph of the person concerned. The X-ray morphology of the
carpal bones appeared normal. The former epiphyseal plates of the metacarpals and
phalanges were completely ossified and in part still slightly marked. The distal epiphyseal
plates of the radius and ulna were for the most part open and presented incipient
ossification only in the middle section. On the basis of the morphological findings of the X-
rays, a skeletal age of 16-17 years according to Thiemann et al. (Thiemann et al., 2006)
emerged. In the reference study by Schmeling et al. (Schmeling et al., 2006), for a skeletal age
of 16 years a mean age of 15.3 years with a standard deviation of 0.8 years was reported. For
a skeletal age of 17 years, the mean age is 16.8 years, whilst standard deviation amounts to
1.1 years. A reference study which gathered data from a socioeconomically highly
developed population was used. For this reason, it may be assumed that the actual age of
the person concerned does not lie below the estimated age. As the development of the hand
skeleton was not complete, no X-ray examination of the clavicles was performed.
Fig. 5. Case 1: orthopantomogram

In the course of dental examination it was ascertained that the third molars had not erupted
into the oral cavity. An evaluation of the orthopantomogram revealed that tooth 18 was
congenitally absent and that impacted tooth number 28 presented a mineralisation stage D
according to Demirjian whilst teeth 38 and 48 presented a stage E (fig. 6). In the reference
study by Olze et al. (Olze et al., 2003), a mean age of 16.3 years was given for mineralisation
stage D of tooth number 28 with a standard deviation of 3.2 years. The mean age for stage E
of the lower third molars is 16.7 years with standard deviations of 2.1 to 2.3 years. On the
basis of the ethnicity of the person concerned, a reference study for Caucasians was used.
In the synopsis of the findings it was determined that the person concerned was most
probably 16-17 years old at the time of examination. At the time of examination the 14th year
of life had been completed beyond reasonable doubt. The age stated by the person
concerned was not consistent with the examination findings. In the course of the court case
it came to light that the actual age of the person concerned at the time of examination was 16
years and 4 months.
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Fig. 6. Case 1: hand radiograph

Case 2
The person to be examined was under investigation for a drug offence. According to his
own statement the accused came from Guinea-Bissau and was 17 years and 8 months old at
the time of examination. As the investigative authorities had considerable doubts relative to
the age given by the person concerned, a court order was issued for forensic age estimation
with inclusion of X-ray examinations.
In the course of physical examination, a body length of 178 cm and a body weight of 68 kg
were measured. The upper lip, chin and cheeks manifested an after-shave condition. The
larynx was prominent. The armpits displayed an area of dense curly hair. The genital region
was covered with a horizontally restricted area of dense curly hair. The external genitalia
were mature. The physical stature and the pattern of body hair corresponded to those of an
adult. No indications of developmental disorders were detected.
Fig. 7 shows the hand radiograph of the person concerned. The X-ray morphology of the
carpal bones appeared normal. The former epiphyseal plates of the metacarpals and
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phalanges were completely ossified and no longer detectable. The former epiphyseal plate
of the radius was completely ossified and only very discretely marked in the middle third.
The former epiphyseal plate of the ulna was completely ossified and no longer detectable.
Ossification of the hand skeleton was thus complete. In line with this, skeletal age according
to Thiemann et al. (Thiemann et al., 2006) was 18 years. In the reference study by Schmeling
et al. (Schmeling et al., 2006), for a skeletal age of 18 years a mean age of 18.2 years with a
standard deviation of 0.7 years was reported. The minimum age was 16.7 years. A reference
study which gathered data from a socioeconomically highly developed population was
used. For this reason, it may be assumed that the person concerned was not younger than
16.7 years at the time of examination.
Fig. 7. Case 2: hand radiograph

As development of the hand skeleton was complete, a CT examination of both
sternoclavicular joints was performed with a slice thickness of 1 mm. The former medial
clavicular epiphyseal plate was completely ossified on both sides. In the region of the former
epiphyseal plates, remnants of the epiphyseal scar still remained both on the right and the
left (fig. 8 and 9). Thus, a stage 4 was present on both sides according to Schmeling et al.
(Schmeling et al., 2004). In the reference study by Kellinghaus et al. (Kellinghaus et al., 2010)
the mean age for stage 4 was 29.6 years with a standard deviation of 4.2 years. The
minimum age was around 21.6 years.
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Fig. 8. Case 2: CT scan of the sterno-clavicular joints, the arrows show remnants of the
epiphyseal scar of the right clavicle
Fig. 9. Case 2: CT scan of the sterno-clavicular joints, the arrows show remnants of the
epiphyseal scar of the left clavicle
In the course of dental examination it was ascertained that the person concerned had an
incomplete set of teeth. Teeth numbers 11, 12, 21, 22 and 37 were missing. All four third
molars had erupted into the oral cavity and had reached the occlusal plane. An evaluation
of the orthopantomogram revealed that all the third molars presented a mineralisation stage
H according to Demirjian (fig. 6). In the reference study by Olze et al. (Olze et al., 2006) a
mean age of between 22.7 and 22.9 years was given for mineralisation stage H of the third
molars with standard deviations of between 2.3 and 2.5 years. The minimum age was given
as 17 years (Olze et al., 2004). On the basis of the ethnicity of the person concerned a
reference study for black Africans was used.
In the synopsis of the findings it was determined that the absolute minimum age of the
person concerned was 21.6 years. At the time of examination both the 18th and the 21st year
of life had been completed beyond reasonable doubt. The age stated by the person
concerned was not consistent with the examination findings.
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Fig. 10. Case 2: orthopantomogram
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6
Epidemiology and Diagnostic Problems of

Electrical Injury in Forensic Medicine
William Dokov and Klara Dokova
Medical University of Varna,
Bulgaria
1. Introduction
The first fatal electrical injury reported in scientific literature was in France in 1879 (Jex-
Blake, 1913). A stage carpenter was killed at Lyon by the alternating current of a Siemens
dynamo giving a voltage of about 250 volts at the time. The first electrocution death in UK
was in 1880, close to Birmingham (Jex-Blake, 1913). Samuel W. Smith was the first person in
the United States to die after electrocution by a generator in Buffalo, New York, in 1881
(Daley, 2010). Since those first cases the annual number of electrical injuries and deaths from
electric shock have steadily increased as a result of the widespread use of electricity and the
application of electrically powered machinery.
Although electricity is a relatively recent invention, humans have always been exposed to
the devastating electrical power of lightning and understandably attributed it to
supernatural powers (Koumbourlis, 2002). Beginning around 700BC the ancient Greeks
depicted lightning as a tool of warning of their god of thunder Zeus (O’Keefe Gatewood,
2004). In Roman mythology Jupiter used thunderbolts as a tool of vengeance and
condemnation, thus those stuck by lighting were denied burial rituals. For the Vikings,
lightning was produced by the hammer of Thor the Thunderer as he rode through the
heavens. In the East, early statues of Buddha show him carrying a thunderbolt with arrows
at each end. In Chinese mythology the goddess of lightning, Tien Mu, used mirrors to direct
bolts of lightning. African tribes, the Native American Navajo culture and many others also
have specific beliefs about lightning.
Benjamin Franklin is generally regarded as the father of electrical science, the person who
proved that lightning is an electric phenomenon and that thunderclouds are electrically
charged with his famous kite experiment (O’Keefe Gatewood, 2004). He constructed a kite
and flew it during a storm. When the string became wet enough to conduct, Franklin, who
stood under a shed and held the string by a dry silk cord, put his hand near a metal key
attached to the string, causing a spark to jump.
Today it is known that lightning is a phenomenon not restricted to the Earth planet only. It
is observed in the atmosphere of Jupiter (Little, 1999), and in this sense lightning presents
danger to flying craft and their crew as well (Uman, 2003).
1.1 Definitions and terms

Electrical injury, electrical shock, electrocution are often used as synonyms when trauma
caused by electric current is being discussed. In this text “electrical injury” is used as the
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term with the broadest meaning. The existing various definitions of “electrical injury” are
principally similar. The Russian Bolshaya medicinskaya encyclopedia defines electrical
injury as an injury caused by electric current or a result from contact with lightning. K. Duff
and McCaffrey distinguish between electrical injury and lightening injury (Duff, 2001). The
former they define as the sequelae due to accidental contact with man-made or generated
electrical power and the later as a sequelae of naturally occurring lightening strike.
According to the Merck manual electrical injury is a damage caused by generated electrical
current passing through the body (Cooper, 2009).
1.1.1 Terms
Information for the following terms is presented as a basic explanation of electricity and the
effects of electrical energy (CDC, 1998).
 electricity (electric current) – is the directed flow of an atom’s electrons (the negatively
charged outer particles of an atom) through a conductor such as wire. Its main
characteristics which determine the hazard effect of electricity on the human body
are:
 voltage – the force or pressure that causes electricity to flow through a conductor,
measured in volts (V). Usually household current is 110 to 220V. Anything over 500V is
considered high voltage. Life threatening levels of voltage are above 50-60V. Death
occurs in 25% of cases in contact with electricity of 127-380V; in 50% of contacts with
1000V; and in 100% if the voltage is 3000V. A more important characteristic is the
difference of the voltage at the entrance and exit of the electric chain. A difference up to
24 V is considered acceptable according to international safety norms;
 power/strength –is the flow of electrons from a source of voltage through a conductor
and is measured in amperes (Amps). The contact with a current with more than 60 mА
per 1 sec is life threatening , and above 100 mA is usually lethal. Current up to 50 mA
is accepted as less dangerous for direct current and up to 10 mА for alternating current.
 type of current - electrical current is categorized as direct current (DC) or alternating
current (AC). Direct is the current which flows in one direction only (as in a car battery).
Sources of direct current are batteries, solar cells, dynamo, etc. Alternating current (AC)
is the current which flows back and forth (a cycle) through a conductor. It is more
dangerous than the direct current.
 rate – the rate of the cycles (back and forth) of the alternating current per second is
measured in Hertz. The normal rate in Europe is 50 cycles per second or 50 Hertz. In the
United States it is 60 cycles per second [or 60 Hertz (Hz)]. Most dangerous is electricity
with a rate of 40-60 Hertz; electricity with a rate of approximately 500 kilohertz is not
dangerous.
 resistance - is the ability to impede the flow of electricity. Most of the body's resistance is
concentrated in the skin. The thicker the skin is, the greater its resistance. A thick,
callused palm or sole, for example, is much more resistant to electrical current than an
area of thin skin, such as an inner arm. The skin's resistance decreases when broken (for
example, punctured or scraped) or when wet. If skin resistance is high, more of the
damage is local, often causing only skin burns. If skin resistance is low, more of the
damage affects the internal organs. Thus, the damage is mostly internal if people who
are wet come in contact with electrical current, for example, when a hair dryer falls into
a bathtub or people step in a puddle that is in contact with a downed electrical line
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Epidemiology and Diagnostic Problems of Electrical Injury in Forensic Medicine 123
 duration of exposure - the longer the person is exposed to the current, the worse the
injury
 pathway of current - the path that the electricity takes through the body tends to
determine which tissues are affected. Because alternating current continually reverses
direction, the commonly used terms “entry” and “exit” are inappropriate. The terms
“source” and “ground” are more precise. The most common source point for
electricity is the hand, and the second most common is the head. The most common
ground point is the foot. A current that travels from arm to arm or from arm to leg
may go through the heart and is much more dangerous than a current that travels
between a leg and the ground. A current that travels through the head may affect the
brain
 electric arc – continuous, high-density electric current between two separated
conductors in a gas or vapour with a relatively low potential difference, or voltage,
across the conductors. According to the power the current can jump from centimetres
up to a meter. Electric arcs across specially designed electrodes can produce very high
heats and bright light.
 lightning – an abrupt, discontinuous natural electric discharge in the atmosphere,
characterised with a high strength in the range of 100 000 amps and voltage of several
millions volts for a very short period – less than 0,0001 sec. A lightening has thermal,
light, acoustic and mechanic damaging influence.
 electric sign / burn mark – a specific skin damage at the point of contact of the current
with the skin. It is a coagulating necrosis. Their typical macroscopic characteristics are
relatively small size - diameter up to 1cm or less, craterlike, round or with a groove
form, grey-whity colour, thick bottom and shaft-like edges. Their existence is a
morphological proof for the influence of electrical current. Most often they appear at the
point of entrance of the current in the human body, so the mechanism of connection
between the body and the chain can be clarified.
 metallization – a process of coating metal on the surface of non-metallic objects; in the
case electrical injury the metal from the current conducing object is coated on the point
of contact with the skin. The colour of the metal depends on the type of the metal
contained in the conducting object. This is a morphological sign for the influence of
electric current. It determines the point of contact between the body and the current; it
can provide information for the conducting object.
 electroshock weapons – a group of incapacitating weapons used for subduing a person
by administering electric shock aimed at disrupting superficial muscle functions.
They achieve continuous, direct, or alternating high-voltage discharge 20 000V-80
000V, causing pain, shock, muscle spasms. Duration of the electroshock for more than
sec can cause loss of orientation, coordination , and sometimes sleeplessness
(insomnia).
 „lightning figures” – paralytic dilatation of subcutaneous blood vessels with specific
form: tree or fern - like, occurring on the path of the current along the body in the cases
of a person affected by atmospheric electricity.
2. Classification of electrical injuries

Electrical injuries can be classified in different ways:
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2.1 According to the sources of electricity

2.1.1 Injuries from natural sources of electricity
- Injuries from atmospheric electricity – lightning and globe lightning
- Injuries from biological electricity – mostly fish
- Static electricity
2.1.2 Injuries from technical sources of electricity

- domestic electricity – 110 -250 V
- technical electricity – up to tens of thousands volts
- weapons using electricity – electro-shockers, electro-guns
2.2 According to the severity of the damages electrical injuries are principally
2.2.1 Fatal injuries – also called electrocution
2.2.2 Non-fatal subdivided as (CDC, 1998)
- electric shock
- electrical burn
- electrical falls
2.3 According to the circumstances at which electrical injuries occur

2.3.1 Forensic cases
- homicides with electricity
- suicides with electricity
2.3.1 Accidents
- domestic accidents
- occupational accidents
- leisure accidents
3. Epidemiology of electrical injuries

Despite significant improvements in product safety, electrical injuries are still the cause of
considerable morbidity and mortality.
The frequency of non–fatal electrical injuries is usually presented based on routinely
collected, easily accessible hospital or other medical records. Data from a Survey of
Occupational Illnesses and Injuries (SOII) for the period 1992-2002 suggest that rates for
electrical shock in USA for the 10 year period remained steady at 2 per 100 000 workers. The
electrical burn rate remained steady at 1 per 100 000 workers (Cawley J, 2006). Data of this
kind depends largely on the severity of trauma and on the accessibility of health services.
Adequate analysis of incidence of nonfatal – electrical injuries would require prospective
population studies.
Epidemiology of fatal electrical injuries can be more adequately studied based on vital
statistics and national death registers. Electrical injuries fall in the class “External causes of
death”. In the 10th revision of ICD this is class XX with codes W85, W86 and W87. In the
older 9th version of ICD, electrical injuries are included in class 18 under code 925 –
accidents caused by electric current. Data from the detailed mortality database of WHO
reveals that mortality from electrical injuries in the European region varies more than
hundred times (figure 1.) (WHO, 2010).
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NL,2008 0.2
Norw ay,2008 0.4
Ireland,2008 0.4
Israel,2007 0.7
Austria, 2008 0.8
UK,2007 1.0
Germany,2006 1.0
Denmark,2006 1.3
Italy,2007 1.5
Sw eden,2007 1.8
France,2007 1.9
Belgium,2004 2.2
Spain,2005 2.4
Malta,2008 3.3
Czech Rep.2008 0.3
3.4
Portugal,2003 3.6
Croatia,2008 0.2
4.5
Slovenia,2008 4.6
Estonia,2008 0.9
4.8
Cyprus,2007 5.1
Lithuania,2008 1.3
6.1
Poland,2007 0.5
6.3
Hungary,2008 2.4
6.7
Serbia,2008 1.3
6.9
Georgia,2001 2.0
8.7
Slovakia,2005 2.0
9.8
Latvia,2007 1.1
13.6
Azerbaijan,2007 2.2
13.9
Bulgaria,2006 1.2
15.3
Romania,2008 2.6
16.9
Moldova,2008 3.9
18.5
Kyrgyzstan,2006 5.7
19.8
Uzbekistan,2005 4.4
25.2
0.0 5.0 10.0 15.0 20.0 25.0 30.0
male female
Fig. 1. Age-adjusted mortality rates from electrical injuries per 1 million population,
European region
There is a clear East – West gap in relation to fatal electrical injuries mortality in Europe.
Rates are much higher in Eastern European countries like Moldova, Romania, Bulgaria,
Uzbekistan. This fact clearly needs attention and explanation. While the discrepancy affects
both genders men living in Eastern countries are the most affected group.
Because severe electrical injuries tend to occur primarily in the workplace, they usually
involve adults between 40 - 50 years of age (figure 2). In Western European countries, where
mortality rates are lower, children up to the age of ten years are almost not affected. In
eastern European countries with higher mortality rates all age groups are affected including
youngest children.
Electrical injuries (excluding lightning) are responsible for approximately 500 deaths per
year or 0.63 per million people in the United States (CDC, 1998). For Australia and New
Zealand mortality from electrocution for 2007-2008 is also less then 1 per million
population (ERAC, 2008). Generally, trends of mortality rates from electrical injuries are
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decreasing in most of the countries and especially in the developed parts of the world.
(WHO, 2010)
3.5
2.5
1.5
0.5
0
< 1 year
1-4
5-9
10-14
15-19
20-24
25-29
30 - 34
35 - 39
40 - 44
45 - 49
50 - 54
55 - 59
60 - 64
65 - 69
70 - 74
75 +
Azerbaijan, 2007 Bulgaria, 2006 Italy,2007
Kyrgyzstan, 2006 Netherlands,2008 Poland,2007
Moldova,2008 Romania,2008 UK,2007
Fig. 2. Age-specific mortality rates from electrical injuries for the European region, per 1
million population, European region (WHO EDMDB)
A substantial number of papers add to the information from routine mortality statistics by
reporting numbers of electrical injuries for different geographic areas (table 1).
First Source of data Type of el. Area Period Cases Details

author, injury
year
Akcan, Retrospective cases of Adana, 1999– 37 cases; mean age 31 (83.8%) cases
2007 review of autopsy electrocution Turkey 2004 11.35; male; all deaths -
report among age range 18 accidental
children months - 18 years;
Dokov, Retrospective electrocution Central 1980– 485 cases, 413 24% occupational
2008 review of autopsy and 2006 cases in men; injuries; increase
reports Northeaste mean age 37.3 in summer
rn Bulgaria years, rate of
fatalities 1,29 per
100 000 per year
Lindstrom Retrospective cases coded Sweden 1975 - 285 cases; 269 151 of fatalities in
, 2006 review of National with ICD- 2000 men; mean age 38 leisure time; 132
Cause-of-Death codes E925 years, age range 10 in an occupational
Register; and W85–87; months– 92 years situation;
suicides and
deaths by
lightning
excluded
Lucas, retrospective electrocution Northern 1982 – 59 cases; age range 50 cases
2009 review of autopsy Ireland 2003 17 months - 80 accidental, 9
reports at the years; rate of suicides; increase
Northern electrical fatalities in summer
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Ireland State , 1,4 per 1 000 000

Pathologist’s per year
Department
Nguyen, Retrospective fatalities in Canada 1991– 21 cases median
2004 review of 10 children 0– 1996 age 13.2 years; 5 of
provincial and 2 19 these were cases of
territorial years from lightning strikes
coroners’ offices electrocution
across Canada (no , including
data for Nova lightning
Scotia)
Pointer, Retrospective Cases coded Australia 1 Jan. 162 cases; 2 per 1 42% domestic
2007 review of death with T75.4, 2001 mln population for accidents
certificates fromT75.0, W85, - 31Dec. 4 years;
Austarlian BureauW86, W87, 2004 7 cases of deaths
of Statistics or X33, in from lightning- all
underlying males from 16-57
cause or years
additional
cause of
death,
Rautji, Review of autopsy electrocution South 1996- 153 cases 3 cases without
2003 reports and Delhi 2001 burn marks, 1
hospital records suicide
Sheikhaza Retrospective electrocution Tehran, 2002 - 295 cases, age 285 accidents ( 188
-di, 2010 review of autopsy Iran 2006 range, 11 months - occupational) 10
report 75 years with a suicides; no burn
mean age of 28.99; marks in 16 cases;
279 male cases increase in
summer
Taylor, Bureau of Labor fatal USA 1992 - 2525 cases, most among 20–
2002 Statistics Census occupational 1999 98.6%in males 34 yrs, whites and
of Fatal electrocution indians; increase
Occupational s in summer,
Injuries
Tirasci, Retrospective electrocution Diyarbekir 1996- 123 accidental 31% of cases b/w
2006 review of autopsy ; lightning , Turkey 2002 cases, mean age 0-10 yrs. of age;
reports excluded 20,7, range 2-63 lack of burn
years of age; 86 marks in 11,4%;
male 56 cases domestic;
increase in
summer;.
Turkmen, Retrospective electrocution Bursa city, 1996- 63 cases (59 Most in 30-39 yrs
2008 review of autopsy Turkey 2003 males); mean age of age; 63,5%
reports 32.5; age range 5 occupational
to 62 years accidents; usually
in summer
Wick, Retrospective electrocution Adelaide, 1973- 96 cases in total, of
2006 review of autopsy Australia 2002 whom 87 males
reports
Table 1. Studies of fatal electrical injuries based on forensic records for the period 2000-2010
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The diversity of these studies not only in terms of time periods but also age groups, types of
electrical injuries covered makes their direct comparison impossible. Generally these studies
confirm that electrical injuries are much more common among men. Almost everywhere in
the world electrical injuries are more common in the summer season. The reasons for this
repeated observations are that during the wormer months of the year people dress lightly
and loose the protective effect of clothes, the skin is wet and the threshold for electrical
stimulus of the heart is much lower. (Ajibaev, 1978)
Approximately half of the total number of electrocutions are occupational accidents, and
constitute the fourth leading cause of work-related traumatic death (5–6% of all workers’
deaths). The other half of electrical fatalities are domestic or leisure accidents, mostly
associated with malfunctioning or misuse of consumer products.
3.1 Suicide by electrocution

While most deaths due to electrocution are accidental in nature, the forensic specialist
should be familiar with electrocution as a method of suicide. Such cases are rare and usually
described as casuistical. We have conducted one of the largest studies covering a period of
41 years (1956-2006) and eight regions (from 28 in total) in Bulgaria, a country with a high
rate of fatal electrical injuries in Europe (Dokov, 2009).
From 63 825 reviewed autopsy reports 945 were cases of electrocution deaths and 59 of the
later were suicides by electrocution. This accounts for 0,09% of all reviewed autopsies and
6,2% of all electrical fatalities. Males prevail definitely (54 of the victims) over females. The
mean age of the victims was 45 ± 6 years (ranging from 14 to 75years). The methods used for
suicide were quite diverse (figure 3) with high and low voltage electricity used with a similar
frequency. This finding contrasts with the reports from Nothern Ireland (Lucas, 2009) where
all nine victims identified for a 21 year period had used the domestic electricity supply,
usually by removing the insulating sleeving of electrical flex so as to expose the wires.
contact with cable 28
unspecif ied 12
climbing up high-v olltage transmission line 8
contact with liv e wire 7
contact with power transf rormer 2
specially constructed dev ice 1
touching railway wires 1
0 5 10 15 20 25 30
Number
Fig. 3. Methods of suicide by electrocution

In addition we have identified a somewhat cyclical pattern of suicides by electrocution with
peaks in the middle of the weak and in September, with summer the typical season.
3.2 Epidemiology of lightning strikes

Lightning strikes cause serious injuries in 1000-1500 individuals each year
(Adukauskakeine, 2007). European countries with higher mortality from electrocutions have
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also a higher rate of lightning fatalities. Romania, Moldova, Bulgaria, have more than 1 fatal
lightning strike per million population per year (figure 2).
Lightning mortality European region
Germany, 2006
United Kingdom,2007
France, 2007
Belgium, 2004
Spain,2005
Hungary, 2008
Austria, 2008
Poland,2007
Italy, 2007
Uzbekistan,2005 Female
Denmark, 2006 Male
Ireland, 2008
Serbia,2008
Lithuania,2008
Czech Rep. 2008
Slovakia,2005
Republic of Moldova,2008
Romania2008
Bulgaria, 2006
Kyrgyzstan, 2006
0 0.5 1 1.5 2 2.5 3 3.5 4
Fig. 2. Lightning mortality in the European regions, by gender, per 1 million population
In a review of lightning strike deaths for the USA, based on data from both the National
Centers for Health Statistics (NCHS), the Census of Fatal Occupational Injuries (CFOI), the
authors find a total of 374 struck-by-lightning deaths occurring during the period 1995-2000
(an average annualized rate of 0.23 deaths per million persons) (Adekoya, 2005). The
numbers of lightning deaths are highest in Florida and Texas.
Between 75% and 85% of all lightning deaths are to men in the age group 25-45 years. Thirty
per cent of all deaths involve people who work out of doors and 25% involved people
participating in outdoor recreations.
Investigations of lightning strikes around the world demonstrate that the predominance of
strikes is in summer months in mid-afternoon in moist atmospheric tropical and
mountainous environments (Uman, 1971).
Data from WHO database regarding the European countries (for a fifteen year period)
indicates that lightning fatalities trends are stable. At the same time one of the studies
covering the longest - 41 year period (1965-2006) from Bulgaria indicates that lightning
fatalities might exhibit a cyclic trend (Dokov, 2009). For Bulgaria it is suggested to be around
30years.
While lightning fatalities can be successfully analyzed based on available mortality data, it is
much more difficult to obtain figures on lightning injury. The ratio of deaths to injuries is
likely to be between 2 and 10. Those who are fortunate enough to recover from lightning
strike frequently suffer severe and prolnged psychological damage, characterized -by
withdrawal, depression, fatigue, sleep disturbance, difficulty with fine mental and motor
functions, paraesthesias, headaches and storm phobias (Andirews, 1992).
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4. The process of forensic medical diagnosis in case of electrical injury

4.1 Examination at the scene of death
In a case of electrical injury the examination at the scene of death has to be carried out from
a team of a policeman, forensic medical specialist and a technical expert (power engineer).
The tasks of the engineer are to provide evidence for the sources and reasons of electrical
flow and to assure all necessary precautionary and safety measures during the inspection.
The forensic medical expert needs to pay special attention to the following problems during
the inspection:
In cases of an accident with technical electricity: The forensic expert needs to determine is there
a contact with source of electricity – wires, devices etc., and the position of the victim in
relation to them. The specialist has to look for circumstances facilitating the accident, such as
increased dampness, wet clothes, lack of protective clothing, gloves, shoes, etc. On the
clothes can be found signs of electrical influence (burns, other electrical sings); on the shoes
there might be breaks at the points of entrance or exit of the electric chain; burns; melted
nails; magnetized metal objects. Electrical burn-marks should be looked for carefully on the
body, but in up to 20% of cases they might be missing. The outer inspection of the body can
provide evidence of mechanic injuries - a result of falling from electrical pylon or a roof, or
other not typical burns. In the case of a suicide, uncovered wires can be wrapped or fixed in
some way to the body, and a letter might be left.
Information for the beginning and the course of the accident should be collected from
witnesses during the process of inspection, together with information on the clinical picture
before the time of death of the victim.
In cases caused by high voltage technical electricity or electric arc – deep local burns or even
carbonization at the point of contact can be found, metallization, stings or burns of hairs,
external traumatic injuries due to throwing back of the body. Such cases do not cause
diagnostic difficulties.
In cases of an accident from electric weapons can be found changes identical with electrocution
from low voltage electricity – round, point like burns or hyperemia 5-7 mm in a diameter.
In cases of an injury with atmospheric electricity there is a specific surrounding situation. The
victim is most often in the open, after a lightning storm, under a tree. Signs of atmospheric
electrical influence in the surrounding environment can be found – burns or tree splitting,
melted or magnetized metal objects or parts of constructions. Very often the clothes of the
victim are severely torn and the body might be denuded. Hairs on the head might be singed,
hairs on the chest or genitals might be intertwined, and the typical for electrical influence
sequelae as burns of different stages can be observed including carbonization of parts of the
body.
4.1.1 Practical tasks of the forensic medical expert during the examination at the
scene of death
 To check that all necessary safety measures are in place before the begging of the
inspection;
 To prove the fact of death;
 To make a detailed description of the position of the body in relation to the sources of
electricity (outlet, wires, devices);
 To describe the status of the clothes (wet/dry), presence of protective gloves, shoes,
condition of instruments with which the victim had worked;
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 To look for evidence for electrical influence (electric burn signs, non-specific burns,
metallization);
 To look for traumatic injuries, their character and relation to the death;
 To estimate the time of death;
 To describe all observations and facts; to inform the leader of the inspection about the
observations and assure their existence in the inspection protocol.
4.1 Post-mortem examination in the autopsy room

4.1.1 Outer inspection of the body
The first task of the forensic expert is to look for evidences for the influence of electricity
such as the presence of electrical burn, electrical burn-marks, metallization on the skin etc.
In the case of contact with high voltage electricity – wide burn areas on the skin and deeper
tissues can be caused and observed reaching to carbonization (Pictures 1-2).
a) b)
Picture 1. High voltage/20КV/ injuries
High voltage electricity can cause damages from distance – so called electrical arc. It also
causes burns. Diagnostics in such cases is not a problem.
a) b)
Picture 2. High voltage injuries
In the case of influence of electricity with low voltage, electrical burn marks appear. Their
usual macroscopic view is with round-like or oval shape, sometimes they are an imprint of
the form of the electro-conductive object (Pictures 3-4).
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a) b)
Picture 3. Low voltage injuries on feet
a) b)
Picture 4. Low voltage injuries on palm (a) and metalization of skin of the leg (b)
The central area of the damage is hollow and the edges are above the level of the
surrounding skin. The skin in the damaged area is dry, thick grey-yellowish in color. In
areas without horn layer of the skin they look like chafes.
Microscopic view: multiple cavities in the horn layer with various shape can be observed.
Often fissures are formed on the borderline with the epidermis, reaching sometimes to
complete tearing off of the horn layer. After colouring with haematoxylin eosin some
focuses with basophilic colour can be found. Deposition of metal particles from wires can
often be observed in such focuses. In the cell layers of the epidermis, cells and their nuclei
are with elongated form. Vortex, chains and similar figures are formed at some spots. Blood
vessels in the derma reveal various changes – spasm, paresis of some vessel, others are
empty, without blood or with haemolysed blood. Such a complex of morphological changes
in skin in the zone of contact with electro-conductive surface can be viewed as specific for
the influence of technical electricity.
Important:
 The presence of electrical burn marks does not necessarily mean that the cause of death
is electrical injury. Electrical burn marks often can be observed for a period of months in
people who have survived electrical injury.
 Electrical burn marks can appear after the moment of death.
 A contact with electroconductive object with low voltage can cause death without burn
marks which happens in as much as 20% of the cases.
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4.2.2 Changes in the internal organs as a result of electricity

In cases of death from domestic or technical electricity a picture of sudden death is usually
observed. Broken bones, formation of bone purls and other traumatic injuries of internal
organs are possible in cases of high voltage injuries. A specific feature which appears in
some cases of death from atmospheric electricity is perforation of the tympanum.
Microscopic changes in internal organs (Naumenko, 1980, Nazarov, 1992):
In the brain - oedema around vessels and cells, focal hemorrhages around vessels,
vacuolization and karyolysis in the pyramidal cells. No specific changes have been
described in neurons.
In myocardium - dilation of blood vessels, cyanosis to stasis, focal haemorrhages, interstitial
oedema, fragmentation of muscle fibres. Often the cross striation of fibres is missing. There
are small but multiple focal necrosis in myocardiocytes.
In lungs – spasm of the bronchi with epithelial swelling, interstitial oedema, focal
haemorrhages, circulatory disturbances.
Walls of blood vessels – areas with destruction of the intima might be observed, together with
necrotic changes of the smooth muscle fibers from medium layer, tendency for thrombosis.
(Xuewei, 1992)
Kidneys – oedema of the renal pelvis mucosa, swelling of the epithelium of the kidney
channels, homogenization and descvamation, circulatory changes similar to those observed
in all other internal organs.
Liver – focal necrotic changes in hepatocytes. Cyanosis in blood vessels with focal
haemorrhages around vessels.
Death from electrocution is a result of several different mechanisms – heart ventricle
fibrillations, paralysis of respiratory muscles, paralysis of the respiratory center, shock and
late sequels (as a result of burns or injury ion the cases of longer survival). In the cases of
electrocution the so called delayed death is possible to occur – as a result of fatal arrhythmia,
thrombosis or myocardial infarction after two-three hours, sometimes several days after the
accident.
4.3 Practical tasks of the forensic medical expert during the post mortem examination
 Repeated examination of the clothes and the body of the victim.
 Description of all external injuries and fixing those through schemes and/or pictures.
Finding specific features for the influence of electricity and adequate description of the
electrical burn marks in terms of localization, distance from main body lines and
points, distance from the feet basis, shape, size, color (gray, black, shades in the case of
metallization of the skin), relief of the surface (craterlike, uneven etc.), edges (exfoliated,
raised, uneven, thick, friable, burned) is of greatest importance most .
 Performing a full autopsy and exclusion of other causes for death.
 Taking materials for laboratory tests /histological, chemico-spectroscopic etc/
During the autopsy the expert should try to answer the following questions:
 Has death occurred as a result of electricity ?
 What was the position of the victim at the point of the electrical injury?
 Which part of the body was in contact with the source of electricity?
 Which were the entrance and exit points of the electrical current?
 Is there evidence for metallization providing information on the characteristics of the
conductor with which there was a contact?
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 Are there circumstances facilitating the electrical injury (condition of clothes and
surrounding environment, changes as a result of diseases.)?
 Is there evidence for self inflicted electrical injury
5. Conclusion and future research directions in the area

Until the present moment almost all scientific enquiries related to electrical injuries were
focused on the changes at the point of contact of the skin with the electric current.
Future work should redirect its attention towards the search for specific changes in target
organs as a result of the influence of electric current – such as heart, brain and blood vessels.
These are the organs whose damage is directly related to the process of death. It is our deep
conviction that such specific changes occur and should be possible to be proved with
histological, histochemical, electro-microscopic or other methods.
At present the diagnosis “death from electrical injury” quite often is based on indirect
criteria. Strict, definitive unambiguous diagnosis is still awaiting its discoverers.
6. References
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7
Child Deaths
Gurol Canturk, M. Sunay Yavuz and Nergis Canturk
Ankara University, Department of Forensic Medicine,
Celal Bayar University, Department of Forensic Medicine,
Ankara University, Institute of Forensic Sciences
Turkey
1. Introduction
Childhood is the development process which starts with birth and continues till
adolescence. According to Convention on the Rights of the Child, every individual is a child
till the age of 18. A major proportion of childhood mortality is associated with trauma.
Natural mortality during childhood is also high. Accidents have an important place with
regards to manner of death; however, child homicide is also standing out with its causes
and outcomes.
There is a consensus that children less than 10 has lower tendency to commit suicide. The
incidence of child suicides increases during adolescence and young adulthood. Deaths
during each and every term of childhood possess some unique differences and features. The
aim of this section is to assess deaths due to medico-legal reasons during childhood.
Childhood mortality may be analyzed under diverse titles according to age groups and
manners of death.
2. Fetal and perinatal deaths

Fetal death is defined as death before 22 completed weeks of gestation when the conceptus
exhibits no sign of life after complete separation from the mother. Perinatal infant deaths
include deaths over 28th gestational week (late fetal period) and the deaths during
postpartum first week. Neonatal deaths occur within 28 days after the delivery; the first
week includes early neonatal deaths and the following 2-4 weeks include late neonatal
deaths. Neonatal deaths comprise 67% of deaths occurring during the first 1 year of life. The
most common causes of death are congenital abnormalities, infections, perinatal asphyxia
and metabolic disorders. Perinatal period is of great importance in the perinatal medicine
due to higher rate of infant mortality. Natural mortalities, accidents and homicides might
occur during this period. Medico-legal investigations are initiated for some perinatal deaths
and the cause of death is determined during post-mortem examination. Perinatal deaths
include still births, abandoned infants and suspected infanticide (Keeling, 2009a, Pakis &
Koc, 2010b, Ozkara et al., 2009).
Childhood mortality globally decreases thanks to socioeconomic development and
interventions that keep the child alive. Despite this fact, every year 8.8 million children less
than 5 die throughout the world. Infectious diseases (pneumonia 18%, diarrhea 15%, and
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malaria 8%) comprise 68% of these deaths. 41% (3.6 million) of these die during the first 4
weeks of life (neonatal deaths). Preterm birth complications, birth asphyxia, sepsis and
pneumonia are the most important reasons of neonatal deaths. 49% of deaths among
children less than 5 occurred in five countries: India, Nigeria, Democratic Republic of the
Congo, Pakistan and China. Birth asphyxia, sepsis, preterm birth complications, and
congenital abnormalities are to blame as global totals for neonatal causes of death in these
five countries. Most of (83%) neonatal deaths occurred in the African region and in the
Southeast Asian region (Black et al. 2010, Lawn et al., 2010).
All relevant information with regards to the event before post-mortem examination should
be acquired. There are actions to be taken in cases of concealed pregnancy, unattended
delivery and abandonment. Fetal maturity, time of death, was the baby born alive, cause of
death and evidence of trauma should be scrutinized. Concealed pregnancy and unattended
delivery have higher neonatal mortality risk when compared to in-hospital deliveries. Some
findings might be helpful in order to learn whether the baby was born alive or not.
Maceration of the baby is a sign of stillbirth. Cutaneous maceration occurs six hours after the
death. Deep-red discolouration of the umbilical cord at its fetal insertion is a finding which
reveals that fetal death occurred at least six hours before the delivery. Inflammatory change
on the umbilical cord is a significant finding. Air in stomach and middle ear, food in the
stomach, macroscopic and microscopic findings in the inspection of lungs are assessed in
terms of live birth. These questions will be difficult to answer if the body decays. Most of the
babies defecate a few minutes after the delivery. Meconium within the colons is also a
positive sign indicating full time birth (Keeling, 2009a, Pakis & Koc, 2010b).
Lowest gestational ages and birth weights, congenital malformations and SIDS are the
factors playing a role in mortality rates. Moreover, socioeconomic status, ethnicity/race,
pregnancy at extreme advanced maternal age (≥45 years), obese women, multiple
pregnancy, prematurity, diabetic pregnancy, delivering an infant outside the normal
working week are reported as risk factors for prenatal, perinatal and neonatal mortality rate
(Mathews & MacDorman, 2007, Alexander et al., 2003, Dudenhausen & Maier, 2010, Yogev
et al., 2010, Teramo, 2010, Pasupathy et al., 2010, Flick et al., 2010).
Antenatal and perinatal deaths, premature and intrauterine growth retardation should be
taken into consideration as risk factors. Placenta insufficiency is the most important cause of
intrauterine growth retardation. Some of the studies report that placenta and umbilical cord
pathologies, congenital abnormalities, infections, hyaline membrane disease and trauma at a
lower degree are the causes of antenatal and perinatal mortality. Detailed anamneses and
post mortem examination are crucial especially for cases in which there is a claim of medical
malpractice. Cases with undetermined cause of death are more common among intrauterine
and neonatal mortalities when compared to adulthood mortality. This rate reaches up to
50% in some of the studies (Pakis & Koc, 2010b).
A study from Istanbul which includes 184 cases reported that; based on the examinations of
lesions and the findings from autopsies, there was no sign to indicate that the neonates were
battered with a tool in 96.2% of the cases (Ozkara et al., 2009). Although traumatic cases are
rare, it should be remembered that fetal and perinatal mortality might be associated with
trauma.
3. Sudden infant death syndrome

Sudden infant death syndrome (SIDS) is defined by Beckwith in 1970 as follows: "The
sudden death of any infant or young child, which is unexpected by history, and in which a
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Child Deaths 139
thorough post-mortem examination fails to demonstrate an adequate cause for death"

(Beckwith, 2003). Infant is a term used to describe babies under 1 year of age. All infants
who died sudden, unexplained, or unexpectedly (SUDI) are not SIDS. According to a
research report, only 39% of SUDI’s are SIDS cases (Mitchell et al., 2000). SIDS is one of the
major causes of infant deaths between 1 month and 1 year old (Moon et al., 2007). SIDS is a
status of not being able to determine the cause of death situation in sudden, unexpected
death cases of an infant aged between 1 month and 1 year, although crime scene
investigation, family history, microbiological, toxicological and histological studies are
made. SIDS is very hard to be diagnosed when it is not possible to reach a diagnosis despite
the fact that all the investigations are made in sudden and unexpected infant deaths and in
cases of exclusion of other possible diagnoses. Such deaths are natural deaths, the causes of
which cannot be determined (Keeling, 2009b).
In sudden, unexpected infant deaths, a detailed history, especially the detailed history of
death and crime scene investigation, is the most important part of the research (Willinger et
al., 1991). Diagnosis cannot be accessed only by external examination. Bajanowski et al
reported unnatural death in 17 of 339 sudden and unexpected death cases which have no
evidence at external examination (Bajanowski et al., 2005). In the Istanbul study carried out
in all children mortality cases under the age of 18, deaths between the ages of 1 month and 1
year were reported as 21.9% (Canturk et al., 2007).
Although pathogenesis is not clear, the number of SIDS reduced in recent years (Van
Norstrand & Ackerman, 2010). Possible mechanism is re-breathing carbon dioxide by
infants who are thought to have been stayed in a small unventilated area (Patel et al., 2003).
(Figure 1). Epidemiological studies trying to identify genes and genetic factors are
associated with SIDS. Central nervous system pathways, cardiac channalopathies, immune
dysfunction, differences in response to nicotine metabolism and energy pathways are
investigated (Van Norstrand & Ackerman, 2010).
Fig. 1. Infant is re-breathing carbon dioxide.
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Prone and side sleeping position, soft bed and sleeping pad, beret, and bonnet use during
sleep, bed sharing and too hot sleep environment are risk factors (Kinney & Thacch, 2009).
Similarly, being male infant, preterm delivery, high risk pregnancy, twin pregnancy, being
multi-parity mother’s baby, living within large family are other risk factors. Single and
young mothers, mothers who smoked during pregnancy, and who give birth to children
with low birth weight are risky. Low socio-economic level is known to be risky (Daltveit et
al., 1997, Blair et al., 2006). And it is reported that low socio-economic level also causes other
risk factors (Fleming et al., 2003). In a study it is reported that SIDS babies are sharing beds
in 70% cases (Brixney et al., 2011). In SIDS cases, most infants die while sleeping in their
bedrooms. Death usually occurs in the morning while asleep. (Fleming et al., 2003). Babies
are usually 2-4 months old, and usually not followed-up in the prenatal period. Low
parental education level, short time drug usage of mothers during pregnancy and winter
months are also risky (Byard & Krous, 2003). Bottle feeding and avoiding breast feeding are
risk factors, too (Blackwell et al., 2005). Inflammatory changes are often in SIDS cases and
infection is a moderate risk factor (Highet, 2008). Staphylococcus aureus, Streptococci and
Escherichia coli have been reported to be related pathogens (Blackwell et al., 2005). Also,
smoking at home is a risk factor and is associated with the number of cigarettes (Blair et al.,
1996). An apparent life-threatening event (ALTE) is a state of emergency, characterized by
central or obstructive apnea, skin color change (pallor or cyanosis), decreased muscle tone
(hypotonia) and a combination of choking or gagging (National Institutes of Health
Consensus Developmental Conference on Infantile Apnea and home monitoring, 1987).
ALTE identified in the siblings is thought to be caused by obstruction of the upper airway
(Southall et al., 1997). Even if a single episode of unexplained ALTE exists in the anamnesis,
SIDS should be suspected (Romanelli et al., 2010, Rosen et al., 1986). Alcohol consumption
within family and sleeping together has been reported to be risky in previous studies
(Phillips et al., 2011).
History is very important in cases of SIDS. Most of the time before the scene investigation,
there may have been an intervention in the scene. The previous death of a baby, who
belongs to the same family, the history of pregnancy and the baby's medical history up until
death are important. The presence of a dead sibling may cause the investigator to suspect
about the possibility of hereditary disease and death as a result of abuse (Meadow, 1999).
In unexpected infant mortality, sleeping environment should be examined carefully in terms
of asphyxia and extreme heat (Figure 2). The exact position of the baby, the proximity
degree of the bodies during sleep with mother and father, blankets, pillows, quilts and
blankets should be investigated as to whether appropriate for use or not. Alcohol and
substance abuse in the family, level of parents’ fatigue should be questioned (Keeling,
2009b). SIDS, which cannot be diagnosed without detailed main aims in autopsy,
differentiates natural and unnatural death, and determines the cause of death (Ozkara et al.,
2009). Radiological, microbiological and pathological studies including toxicology and
neuropathology contribute to the autopsy. For toxicological examination, blood, urine, and
intraocular fluid, if the eyes cannot be examined pathologically, should be sampled
(Keeling, 2009b). Genetic analysis should be done in the SIDS cases. (Klaver et al., 2011).
When diagnosed ones are subtracted from the SUDI cases, cases with specific criteria for
SIDS should be classified as SIDS and others should be classified as deaths of unspecified
cause. (Cologlu & Cakalir, 1999). Multidisciplinary, detailed and meticulous research is very
important in sudden unexpected child deaths (Inanici et al., 2001).
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Child Deaths 141
Fig. 2. Broken bed should not be used.

In evaluation of outward appearance, well-groomed infant child can have minor
malformations or dysmorphic features. Frothy secretions in nose holes are common.
Occurrence of dead marks on the front of the face and chest indicates the prone position of
the baby. Hyperemia of internal organs and non-specific changes are seen in asphyxia
(Cologlu & Cakalir, 1999). Thymus should be examined carefully. Generally, size of the
thymus is found in normal range. A large number of thymic petechiae which is the most
important and most common manifestation of SIDS is found especially in the thoracic region
and mostly seen on the posterior face (Keeling, 2009b). Small and numerous petechiae also
can be found on subpleura (Goldwater, 2008). On epicard and pleural face of diaphragma,
petechiae can exist (Cologlu & Cakalir, 1999). Petechiae attend to more than 80% of
Becwith's cases (Beckwith, 1988). 61% of SIDS cases in Kleemann’s study had petechiae
(Kleemann et al., 1995). At this age, mesenteric lymph nodes are often expanded as a reflex
to the level of environmental antigenic stimulation (Keeling, 2009b).
The origin of sudden infant death can be natural illness or trauma. This can also be valid for
adults (Pakis & Koc, 2010a). Infection is one of the major causes of death in this age group
and should be considered in the differential diagnosis (Canturk & Canturk, 2001). In this
case, microorganisms can be detected by molecular methods (Baasner et al., 2003).
Cardiovascular system-based deaths are common. Cardiomegaly should be warning for
researchers about cardiovascular origin. Cardiac malformations, ventricular septal defect
(Cohle et al., 1999), coronary artery anomalies should be investigated (Rowley & Shulman,
2010, Lipsett et al., 1994). Although rare, myocardial infarction in the neonatal period is
defined (Canturk et al., 2006), severe cardiomyopathy can also be seen (Dettmeyer &
Kandolf, 2010). It is important to diagnose these cases, because many of them are familial
(Pakis & Koc, 2010a). Dysrhythmias are varied, but complete bundle branch block is
common. This diagnosis is important in terms of pregnancies of infant’s mothers and close
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relatives. Postmortem genetic analysis shows that cardiac ion channel mutations like
Brugada syndrome, long QT syndrome and short QT syndrome are associated with SIDS
(Goldwater, 2008). Other possibilities are genetic-metabolic diseases, and the beta-oxidation
defects are considered in this group. Presence of hypoglycemia and hyperammonemia
which are common in these diseases can trigger infections (Keeling, 2009b). Sudden
unexpected death in epileptic children can be seen (Sillanpää & Shinnar, 2010).
Definitive histological evaluation is important in SUDI cases. Pulmonary edema and
congestion are common findings in SUDI cases. Round-cell infiltration is often located on
alveolar wall and there are peribronchial lymphoid aggregates (Keeling, 2009b). In 60% of
SIDS, focal acute inflammation exists in the upper and lower respiratory tract (Krous et al.,
2003). In many SIDS cases, arcuate nucleus hypoplasia, periventricular leukomalacia and
brain nucleus subtle gliosis can be found (Keeling, 2009b). Relatively, gliosis in the brain is a
common finding (Kinney, 2005). Also continuing hematopoiesis in the liver is one of the
findings of SIDS and found to be significantly higher, compared with the control group
(Töro et al., 2007).
4. Natural deaths in infants and children

Sudden natural deaths in childhood constitute about 5% of all deaths (Aleszewicz-
Baranowska, 2002). Causes of natural death in infants and children relevant with all
systems, especially related to the cardiovascular system (Variend, 2009). The most common
and mortal cause of childhood cardiovascular system diseases are myocarditis, hypertrophic
cardiomyopathy, long QT syndrome and Preexitation syndromes with aortic stenosis,
tetralogy of Fallot, transposition of great arteries, Ebstein's syndrome, congenital heart
defects, such as coronary artery anomalies (Aleszewicz-Baranowska, 2002, Vetter, 1985). The
adolescent period, cardiomyopathies are reported as the most common cause of sudden
cardiac death (Thiene et al., 1988).
Causes of natural death in children and infants vary from country to country and due to
living in urban or rural areas and age. While in developed countries, congenital anomalies,
premature birth, birth trauma, malignancies are the causes of death, in developing
countries, preventable causes like infectious diseases, nutritional disorders, etc. are seen
primarily. Diseases-related deaths are seen more often in urban than rural areas. While,
children between the ages 1-4 infections are main cause, cardiovascular causes, epilepsy,
intracranial hemorrhage, and asthma are prevalent in children elder than 14 (Neuspiel &
Kuller 1985). In developing countries, sepsis and other infections are among the leading
causes of deaths under the age of five. Neonatal tetanus, malaria, measles in Nigeria,
congenital syphilis, measles, AIDS in Papua New Guinea and tetanus in India has an
important role among the causes of death (Bamgboye & Familusi, 1990, Aikhionbare et al.,
1989, Duke et al., 2002, Choudhury et al., 1991). In Tokdemir et al’s study; under the age of
18, 178 case of whom autopsies evaluated in Elazıg between 2001-2007, in 7.8% of them
death cause were resulted as natural , 92.8% of cases were determined in the 0-5 age group,
and the cardiovascular system took the second place, after the respiratory system diseases
(Tokdemir et al., 2009).
Mortality rates under 5 years old also vary from country to country. According to 2009,
World development indicators of the World Bank, Mortality rates under 5 years old is 7,8 in
USA, , 4.2 in Germany, 3.20 in Turkey and 198.6 (for 1000 new-born baby) in Afghanistan
(World Bank, World Development Indicators).
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Child Deaths 143
5. Asphyxial deaths
Death from asphyxia is common among childhood deaths. Except drowning, entrapment
asphyxia, foreign body inhalation, plastic bag asphyxia overlaying and wedging,
strangulation, hanging by a ligature, imposed airways obstruction, abuse of inhalants,
chemical asphyxia can be considered among the causes of deaths from asphyxia (Byard,
2000, Busuttil, 2009b).
According to ICD asphyxia is defined as follows:
- - Accidental drowning and near drowning
- - Obstruction of the airways due to inhalation or any foodstuff or suffocation
- - Obstruction of the airways due to inhalation or any foreign body or suffocation
- - Accidental mechanical suffocation.
Petechiae is still accepted as the pathognomonic finding of asphyxia. It is not rare to see
such haemorrhage in a single zone or only in the eyes. It can be seen on the anterior chest
wall and on the body as well in early neonatal deaths and stillbirths due to retroplasental
haemorrhage (Busuttil, 2009b).
5.1 Entrapment asphyxia

The curiosity of children related to various objects and spaces can result in difficult
situations and they cannot escape from such difficulties and might die. Box type freezers,
refrigerators, old cabinets, large chests, suitcases that are left idle within children’s reach
may lead to problems. To be locked in the trunk of a car can have a similar effect.
There is an accidental asphyxia case reported in the literature, in which the person’s head got
entrapped in the car window (Byard & James, 2001). In the USA between the years 1987-1998
11 pediatric cases were reported. The children reported were at the age of 6 or younger and
kept locked in the trunk of automobiles and died due to hyperthermia and asphyxia (Centers
for Disease Control and Prevention,1998). A study conducted in Australia indicated that 13
(31%) out of 47 non-intentional asphyxiation cases were due to head and neck entrapment
(Altmann & Nolan 1995). It was also reported that a 19 month old girl died due to the neck
compression since her neck got entrapped in the shopping cart (Jensen et al., 2008).
When a child is kept closed in the car in a hot day, heat stroke may develop due to the
asphyxial changes together with the heat effect. If the ambient temperature is more than
29.5° C, such deaths may happen. If the ambient temperature is more than 29.5° C, the
temperature in the vehicle will be more than 55 ° C (Busuttil, 2009b).
5.2 Plastic bag asphyxia

As plastic bags are common in our daily life, children play games with plastic bags. They
put plastic bags over their heads and may die accidentally while they are playing with
plastic bags. Such deaths are not rare. Moreover such deaths are common among the
children who are solvent abusers (Saint-Martin et al., 2009).
In many countries it is legally obligatory to make holes on plastic bags that will enable air
flow (Busuttil, 2009b).
5.3 Hanging by a ligature

Hanging by a ligature is not common among the children under the age of 14. The studies in
the literature are mainly case reports and epidemiological studies are missing. It is more
common among boys. Cervical spine, hyoid, or thyroid fractures are not frequently seen.
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In the USA hanging/suffocation rate among the individuals between the ages 10 and 24 was
increased significantly from 1992 to 2006 (Jones et al., 2000). In Australia there is also an
increase in hanging by both males and females between the years 1998 and 2007, when
compared with the previous decade (Bridge et al., 2010).
In the background there is misery or depression. Post-mortem psychological evaluation
shows failure at school and pretension behaviors among the peers. These children might try
to hurt themselves before. Autoerotic accidental deaths are reported among children at the
age of 9 and above (Busuttil, 2009b,Large & Nielssen 2010).
5.4 Traumatic asphyxia

Traumatic asphyxia is rare in children. It is generally associated with crush injuries. The
pathophysiology is different from adults (Large & Nielssen 2010). In the literature there
some case reports concerning crush under car or jeep tires, or under some objects or garage
door (Wyatt et al., 1998, Nishiyama & Hanaoka 2000). When thorax is stable, but there is no
respiratory movement, traumatic asphyxia can occur. Central cyanosis and petechial
heamorrhage are classical with congestion findings end up at the level of clavicles at the
superior part of the obstruction. In children such findings are observed at the superior part
of the obstruction, in case the children are crushed in the crowd or under the walls or any
other object due to explosion, conflicts, or natural disasters such as earthquakes and oil
explosions.
Another type of accidental asphyxia is related to traffic accidents, in which the child is
crushed under the vehicle. Mostly internal organ damage and diffuse soft tissue injuries are
seen in children. The abrasions frequently seen on the body of the child show the direction
of the car passing over the child in line with the dragging direction. The majority of these
children are under the age of 3 and they are male (Busuttil, 2009b).
5.5 Foreign body inhalation

Aspiration of foreign bodies can be fatal particularly in the first year of life. Anatomical and
physiological characteristics and behavioral factors cause higher risk in terms of foreign
body aspiration among the children under the age of 3 (Hurtado & Della-Giustina 2003).
Frequently toys or foodstuff cause foreign body aspiration in children. Although the
majority of these cases show immediate symptoms while the child is eating, there are also
some cases that result in the death of the child in the sleep without showing any immediate
symptom. Foreign body caused for the death can be identified in the airways during the
autopsy.
Aspirated solid or semi-solid foreign body may setin the main bronchi, trachea or larynx of
the child. If the foreign body is large enough to close the air way totally, then there will be
an immediate asphyxia due to lack of air through the lungs (Busuttil, 2009b).
There is more risk in laryngeal spasm and death due to laryngeal foreign body, when
compared to foreign bodies in the trachea and bronchi (Hurtado & Della-Giustina 2003).
It is reported that the foreign bodies mostly go to the right bronchus due to the anatomical
position (Hurtado & Della-Giustina 2003).
The inhaled vegetable particles might swell up in the forthcoming hours and even days and
cause cough, stridor, wheeziness, short of breath and cyanosis. Peanut and other organic
foreign bodies aggravate asphyxia through tissue edema due to acute inflammatory
response. The American children between the ages 1 and 3 are under a higher risk. It results
in 0.7 deaths per 100.000 annually. Death from foreign body inhalation is due to the
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Child Deaths 145
tendency of children to put everything into their mouths. Such young children do not have
molar teeth. They tend to chew foodstuff with their incisors. When any foodstuff is sent
back they are inclined to inhale due to a reflex reaction (Busuttil, 2009b).
Toddlers possess higher risk in terms of foreign body aspiration. If there is any mastication
problem or disphagia in older children, food aspiration might be seen. The fatality risk is
more in mentally retarded children or in children suffering from neurological disorders such
as cerebral palsy. On the labels of the packaged foodstuff the appropriate consumer group
should be stated for the safe consumption of the foodstuff (Byard, 2000).
5.6 Overlaying and wedging

The baby is overlaid by an adult during the deep sleep phase or the baby is accidentally
suffocated due to sleep induced extraneous intoxication. The risk of overlaying is the
highest in the babies under the age of 5-months. However overlaying can be seen in children
until the age of 2. An adult or an older child who is overlaid the child, cause him/her to be
kept under the bed or pillow. The child cannot cry due to the pressure in his/her chest and
cannot take attention. In some of these babies expected clinical findings and even petechia
cannot be seen. In some cases, unusual lividity indicating the pressure zone can be observed
besides contusions and abrasions (Busuttil, 2009b).
For many years accidental suffocation cases of infants are considered as the cause of SIDS
when infants sleep with their parents. Recently it is being debated that overlaying is a cause
of SIDS. The reason for the debate is that the autopsy findings, crime scene investigation,
family history and epidemiological findings are not different in SIDS and overlaying (Byard,
2000).
Kirchner reported 515 mortality cases under the age of 2 in a period of 7 years. 121 out of
515 cases died due to overlaying by their parents, siblings or other adults. Kirchner also
reported that 77% of these cases were under the age 3-months. 394 out of 515 deaths
happened due to entrapment in the bed. 296 of them died in the beds of their parents.
According to Kirchner’s report 79 cases died in the waterbeds. 2 cases died due to alcohol
and substance use. 10 cases died in the adult sunbeds and finally 9 cases died in adult beds
with rails (Busuttil, 2009b).
5.7 Strangulation
Although strangulation is a homicide and suicide-related cause of death in adults, it is an
accident-related cause of death due to asphyxia in children. It is the 4th most common cause
of unintentional injuries for infants under the age of 1 after traffic accidents, drowning and
burns (Chinski et al., 2010).
Unintentional or accidental self-strangulation is quite commonly reported in young children
mostly by use of a loose wire, rope and other potential ties typically around the house and
frequently close to the bed. Tangled death cases account for 14,3% of all childhood mortality
in the US. (Busuttil, 2009b).
5.8 Airway obstruction

One way of child abuse is to cover infant’s mouth by soft materials such as pillows and to
press the infant against the chest by parents or other care givers. The infant may not seem to
have tried hard but there might be signs of convulsions due to cerebral hypoxia following
hypoxia and cyanosis without the presence of significant traumatic findings; respiratory and
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cardiac arrest may follow. This situation may be caused by a mother previously diagnosed
with Munchausen by Proxy willing to draw attention due to her psychosocial problems.
External examination may not indicate any finding, but presence of haemosiderin
deposition in lungs both in alveolar cells and interalveolar septa is a quite critical finding.
(Busuttil, 2009b).
5.9 Solvent abuse

Inhalable hydrocarbons generally create an impairing effect on mental functions similar to
alcohol or substance abuse. They contain petroleum and petroleum products that are
present in the composition of many household products such as cleaning and decorating
materials, paints, polishers, lacquers, adhesives, room sprays, hair straighteners, dry
cleaning solvents, shoe polishes, labels or stain removers. Death may happen in various
ways. Reports indicate use of such substances by older children for arousal during
autoerotic activity.
Autopsy may reveal limited information in such suspected abuse cases. The body may
present rashes and vesicles around the mouth orifice due to the effect caused by the solvent
on the skin. Solvent may be olfactive during post-mortem examination. One of the lungs
should be taken inside a plastic bag for analysis. Blood, liver and kidneys may also be
examined toxicologically (Busuttil, 2009b).
5.10 Reverse suspension

This rare condition presents a situation, where the organs of a child changes position in the
upward direction during a game activity resulting in shifting of the diaphragm and air
depletion ultimately causing death. Death happens slowly when respiratory efforts are
consumed (Busuttil, 2009b, Kurtzman et al., 2001).
5.11 Chemical asphyxia

This is a term used in the presence of non-inhalable gases around the child. For instance,
kerosene and paraffin as a fuel, carbon monoxide liberated from fires or from exhaust
smoke or barbecue coal in closed spaces or chlorine effusing from swimming pools,
hydrogen sulphide and methane emitting from outdated mines and gases dispersing from
catch basins may cause mortality. These conditions generally affect elderly individuals,
mobile kids and mostly boys in environmental accidents. (Busuttil, 2009b, Meyer et al.,
2007).
6. Drowning
Drowning happens due to aspiration of water into upper and lower airways by the reflex at
the end of the apnea time (Yorulmaz & Cakalir, 1999). Drowning is the second most
common cause of traumatic death for children between the ages of 1-14 (Gilchrist et al.,
2004, Bener et al., 2011). In most of the drowning cases, death happens due to hypoxemia
and subsequent cerebral hypoxia following inhalation of water down to alveoli (Gok,
1983a). 40% of all drowning cases involve children (Canturk et al., 2009). A study conducted
in Istanbul on child mortality reports drowning as the cause of death in 79 out of 736
children (10.73%) (Canturk et al., 2007). Drowning may happen in the bathroom, toilet,
buckets and jerry cans, swimming pools, ponds, decorative pools, building sewerage
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Child Deaths 147
systems, farming reservoirs and tanks, canals, lakes, streams, creeks and sea (Pearn, 2009).
The origin of childhood drowning is generally accidental (Brüning et al. 2010). Children
between the ages of 1-4 represent the high-risk age group (Iqbal et al., 2007). Summer
months are quite risky for drowning events since the weather tends to be warmer than the
rest of the year (Canturk et al., 2009). Male gender, African race and adolescent age are the
other risk factors (Bener et al., 2011, Hyder et al., 2008). Drowning cases are mostly reported
in weekends and between 14.00-19.00 hours (Tyebally & Ang, 2010).
All dead bodies taken out of water should not necessarily be considered to have died of
drowning and the person might have died because of
1. A natural disease before he/she has fallen into the water,
2. A natural disease when he/she was in the water,
3. Trauma before having been thrown into the water,
4. Traumatic reasons when in water,
5. Hypothermia and sympathetic inhibition, parasympathetic stimulation in cold water,
6. Drowning (Yorulmaz & Cakalir, 1999,Knight, 1996d).
Every drowning is a forensic case. One should always remember that child abuse or non-
accidental injury, homicide, euthanasia and negligence may go along with the anatomical –
pathologic characteristics of drowning. A detailed crime scene investigation and witness
statement taking process should continue with a thorough anamnesis taking and post-
mortem examination, if necessary to be complemented with radiography, chemical
examination, diatomeae analysis and photograph taking (Canturk et al., 2009). Drowning
does not have any specific laboratory finding to help with the diagnosis and therefore it is
one of the most challenging diagnostic works of forensic pathology (Arslan et al., 2005).
Since there is no specific histopathological finding to diagnose drowning, other possible
causes of death should be ruled out by way of autopsy, histopathological examination,
chemical and toxicologic examination. Lung alterations are not specific in drowning cases
(Yorulmaz & Cakalir, 1999, Knight, 1991). Diagnosis is even harder in pediatric cases. Since
the time of staying in water is only minutes in 99% of pediatric cases, the amount of ingested
water may be very small, which challenges the diagnosis (Pearn, 2009).
Mortality due to cardiac arrest that develops by laryngospasm or vasovagal mechanism in
absence of fluid in airways is defined as dry drowning (Yorulmaz & Cakalir, 1999). The
macroscopic appearance of lungs may differ depending on fresh or salty nature of the
drowning water. Since the salty water coming into the alveoli is hypertonic, the water in the
vascular bed passes onto the alveoli causing hemoconcentration in blood, hypovolemia and
severe pulmonary edema presenting with bloody-fluidy wet appearance in lung cross
sections. Drowning in fresh water, on the other hand, presents with hypotonic water coming
in the alveoli passing the water from vascular system to the blood and consequently causing
hemodilution, hypervolemia and hemolysis creating a drier look in the cross sections of the
lung than in drownings in salty water (Demirci & Dogan, 2010). There are age-related risk
factors for drowning of children. The most common drowning spot for children is the
swimming pools. Buckets, washbowls and Jacuzzis also constitute risky zones for children
aged 4 and under. (Tyebally & Ang, 2010). Toddlers may drown when left unattended in the
bathtub or when left alone with full buckets, whereas adolescents mostly drown outdoors,
in which case possible alcohol intake may be the case (Byard, 2000).
Drowning zones mostly depend on communities and geographical position of their water
resources (Byard, 2008, Wang et al., 2010). Nevertheless, the most common drowning zone
for childhood events is the swimming pools. (Tyebally & Ang, 2010). In as much as
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swimming is the fun and healthy way of refreshing in summer months, it brings along the
risk of drowning (Schwebel et al., 2007). Most of the drowning cases in swimming pools and
sea involve victims in the age group of 5-15. Boys tend to drown more than girls in
swimming pools and sea (Tyebally & Ang, 2010, Pelletier & Gilchrist 2011).
80-90% of child drownings in bathtub are accidental. Particularly, infants under 12 months
are under risk (Somers et al., 2006). Drowning cases have been reported by use of bathtub
seats and rings when bathing infants in bathtubs (Rauchschwalbe et al., 1997). Inefficient
adult attendance and bathing of more than one infant constitute risk factors (Somers et al.,
2006). As a classical story, the tired mom starts bathing her children during which the
telephone rings or the door knocks causing a sudden and unexpected interruption of the
family routine. The mom lets her children stay in the bathtub, when older children get out of
the bathtub leaving the younger child alone. The victim is generally the youngest or the
second youngest child of the family (Pearn, 2009). Although drownings in buckets or
washbowls are generally accidental, the possibility of homicide should not be overlooked in
these cases (Pearn, 1992). Most of the victims are younger than 12 months with an age
interval of 7-15 months. (Mann et al., 1992)
Children may also drown in rivers, lakes, creeks, sewerage systems and trenches. 90% of
cases are boys mostly in the age group of 8-12. They generally drown when playing or
swimming in prohibited areas, in which case their friends can’t help or more than one child
may die (Pearn, 2009).
Presence of fungal foam in external examination would be the strongest finding to indicate
drowning in water. Localization of post-mortem stains is consistent with the body position.
Goose skin look, wet skin, launderer’s hand and foot are the findings that can manifest
themselves only in long time stay in water. In drowning cases that present with both non-
specific asphyxia symptoms during internal examination (hyperemia, Tardieu’s spots,
edema and hyperemia in internal organs) and fluid aspiration, lungs are hyperemic, bright
and swollen. Furthermore, materials belonging to the drowning site such as algae or sand
may be present in the respiratory system. Post-mortem radiologic examination should also
be performed to rule out the possibility of child abuse. Besides, alcohol and drug analysis
should also be performed as a part of toxicologic workup. Toxicologic workup may reveal
content of the drowning fluid from the lung tissue. Although false positive or negative
results should be reassessed, diatom analysis from bone marrow is especially important for
drowning in the sea. (Yorulmaz & Cakalir, 1999,Canturk et al., 2009, Pearn, 2009, Knight,
1996d,Demirci & Dogan, 2010, Geserick et al. 2010).
7. Poisoning
In parallel with technological developments, there has been an increase in the risk of
poisoning due to the increase in the number of chemical substances and drugs. Poisonings
are important causes of pediatric emergency service applications and of morbidity and
mortality in children and adolescents (Andiran & Sarikayalar 2004, Cheraghali & Taymori
2006).
Reasons for intoxication show variability in a wide range. Among these; drugs, various
chemicals, pesticides, solvents, toxic gases or smokes, a variety of metals and minerals,
animal bites or stings, and some poisonous plants and foods can be counted (Gurpinar &
Asirdizer 2006). Epidemiological features of childhood poisonings differ from country to
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Child Deaths 149
country and vary depending on socio-economic and cultural conditions of communities

(Senanayeke & Karalliedde 1998, Paritsis et al. 1994).
Based on data from United States of America Poison Control Center, it is indicated that
approximately two and a half million poisonings occurred in 2008, 38,7 percent of which
were under 3 years of age and more than half of which were under six years of age
(Bronstein et al., 2008).
The pattern and risks of intoxication vary depending on age (Marchi et al., 1991, Soyucen et
al., 2006). Poisonings in children under one year old include therapeutic errors such as false
drug dose adjustment of doctors or families, or prescribing mistakes, while in children
between the ages of 1-5 accidental poisonings and over the age of 10, suicidal poisonings are
common (Andiran & Sarikayalar 2004).
Food poisoning, which occurs as accidental ingestion of toxic substances, is one of the most
important causes of poisoning in children and is most frequently seen among boys between
the ages of 1-5 (Marcdante, 2006, Mert & Bilgin 2006, Dart et al., 2007,Busuttil, 2009a,
Hoffman & Osterhoudt 2002). Since children can be curious and tend to bring everything in
their mouths without noticing that they can be harmful, food poisonings are common in
these ages (Roidgers & Matyunas 2002). Intentional (voluntary) poisoning is another cause
of poisoning seen in children and is more common among adolescent girls (Soyucen et al.,
2006, Marcdante, 2006, Dart et al., 2007, Busuttil, 2009a, Bana, 1997). Compared to
adolescents, children are more sensitive to environmental stresses and their emotional
reactions are higher due to hormonal changes, and suicide attempts are seen more
commonly.
Substances causing intoxication differ from country to country. It is reported that poisonings
mostly occur as a result of oral intake of substances and drugs are the most common causing
agents of acute toxicity (Andiran & Sarikayalar 2004, Marchi et al., 1991, Soyucen et al., 2006,
Dart et al., 2007, Yavuz & Ozguner 2003, Ucar et al., 1993). In Western Europe and North
America, domestic products, drugs, carbon monoxide and volatile substances take the first
place, while in developing countries, causes such as pesticides, household products,
medicines, animal or insect bites are in the forefront (Ellenhorn, 1997, Cardozo & Mugerwa
1972, Jamil, 1990).
The majority of childhood poisonings occur at home and approximately 45% of home
accidents are acute poisonings (Asirdizer et al., 2005). It is notified that, in Andiran et al’s
study performed in Ankara, 93.3% third of poisoning cases occurred at home (Andiran &
Sarikayalar 2004); in Petridou et al’s study realized in Athens, 88.7% of scene was home and
living room, bedroom or kitchen (Paritsis et al. 1994); in Soyucen et al’s study performed in
Sakarya, 92.7% of poisonings occurred in the house (Marchi et al., 1991). According to the
World Health Organization, toxicity-related death rates in children between the ages of 1-14
are 0.05 in Denmark, the 0.12 in USA and Canada, 0.75 in Korea (Busuttil, 2009a).
8. Road traffic accidents

Death due to road traffic accidents is one of the major reasons of childhood mortality
(Durkin et al., 1999). The global economic cost of motorized vehicle accidents and injuries of
pedestrians is around USD 500 billion.
WHO reports that injuries due to road traffic accidents is still an important public health
problem. 1.2 million people die and 50 million people are injured due to traffic accidents in
the world annually (Chakravarty et al., 2007). In 27 Member States of the EU around 50000
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people, 8500 of whom are pedestrians, die due to traffic accidents (Arrequi-Dalmases et al.,
2010).
Road traffic accident issue is a major public health problem for particularly low and
medium income countries. Contrary to the low and medium income countries, pedestrians
constitute the largest group in the traffic accident related injuries and mortalities in the high
income countries (Mabunda et al., 2008). It is reported that low socio-economic status is an
increasing risk factor for child pedestrian deaths, and that the children with low socio-
economic status has 4-5 folds more mortality risk when compared the children with the
highest socio-economic status (Desapriya et al., 2011, Busuttil, 2009d).
In accordance with the National Highway Traffic Safety Administration (NHTSA) report
4641 pedestrians died in 2004 in USA. This number corresponds to 11% of the traffic
accident related deaths. Pedestrians generally make fatal mistakes in the afternoon or
evening time when crossing a street. Pedestrians at the age of 12 and below are found faulty
in the accidents in 90% (Ulfarsson et al., 2010).
The number of in-vehicle deaths in traffic accidents particularly in the developed countries
has a decreasing trend in recent years. The ratio of mortality between the ages 0 and 14 due
to traffic accidents in Europe is 48 %. The ratio of pedestrians between the ages of 15 and 17
died in traffic accidents is 21 %. Although the pedestrian mortality is high in the less
developed countries, this problem is not rare in the developed countries. For instance the
number of pedestrian deaths in the USA was 4675 in 2004 and increased up to 4881 in 2005.
Recently around 5000 pedestrians die and 600000 pedestrians are injured in the USA
annually (Chakravarty et al., 2007, Arrequi-Dalmases et al., 2010). The ratio of pedestrian
deaths to the traffic accident related deaths in Hong Kong is more than 50%. This ratio is
higher than the ratio in the USA, Japan and many western countries. Children possess high
injury and mortality risk in traffic accidents. The risk is highly correlated with driver
characteristics, socio-economic environment and demographic characteristics of victims (Sze
& Wong 2007). The following factors have a serious impact on the injuries in road traffic
accidents: type of the vehicle, speed of the vehicle, size of the vehicle, impact angle of the
vehicle, center of gravity of the pedestrian when the vehicle comes into contact with the
body of the pedestrian, driver characteristics and alcohol intake (Chakravarty et al., 2007).
The researchers focus on the socio-economic characteristics of children, particularly the
pedestrians, undergone traffic accidents. Types of injury depend on the age, gender and
socio-economic status of the individual. The risk of death for child pedestrians is found
associated with the socio-economic status, economic conditions of the family and ethnicity
(Chakravarty et al., 2007, Mabunda et al., 2008, Busuttil, 2009d, Graham et al., 2005,
Newbury et al., 2008, Presley et al., 2007).
Pedestrian injuries and deaths have an increasing trend in the world. Children constitute the
most sensitive group among the pedestrians in traffic accident related injuries and deaths.
Pedestrian injuries are the second most common cause of death among the involuntary
injuries between the ages of 5 and 14. Children below the age of 15 have a ratio of 8% in
overall pedestrian deaths due to road traffic accidents in the USA. On the other hand this
age group comprises 30% of the overall pedestrian injuries (Chakravarty et al., 2007). In-
vehicle injuries due to road traffic accidents are mostly seen at the age group of 10-19.
Injuries related to bicycle accidents are mostly seen at the age group of 10-14, and the
injuries related to motorbike accidents are commonly seen at the age group of 15-19 (Agran
et al., 2001). In Africa the traffic accident related pedestrian deaths constitute the most
significant group. In four provinces of South Africa 7433 pedestrian deaths were reported
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Child Deaths 151
between the years 2001 and 2004. 18.8% of the deaths in question were children and
adolescents under the age of 20 (Mabunda et al., 2008).
It is reported that children in adverse circumstances are inclined to traffic accidents. It is
thought that environmental characteristics play a role in traffic accidents (Durkin et al.,
1999). It is stated that around 85% of the deaths due to traffic accidents happen in medium
and low income countries (Hyder et al., 2006).
The majority of the injuries due to road traffic accidents happen generally in the afternoon
or evening time or on weekends. According to some studies deaths mostly happen in
summer. The majority of deaths are seen between 6 p.m and 12.00 p.m. as per the American
national data. When it gets dark, the sight distance of drivers may be shortened. Together
with alcohol intake and pedestrian traffic, it might affect the occurrence of traffic accidents.
It is reported that mostly boys get injured and die due to traffic accidents (Durkin et al.,
1999, Chakravarty et al., 2007, Mabunda et al., 2008, Desapriya et al., 2011, Newbury et al.,
2008).
Different studies are concluded with different results concerning the scene of traffic
accidents – whether in the city center, suburban or rural areas – that resulted in injury or
death of children. Some studies indicate that road traffic accidents mostly happen in the city
center, whereas some others state that road traffic accidents are mainly seen in rural areas
(Chakravarty et al., 2007, Mabunda et al., 2008, Desapriya et al., 2011). However it is
generally asserted that accidents happen at the vicinity of the child’s home (Busuttil, 2009d).
Alcohol plays an evident role in motorized vehicle accidents likewise in pedestrian injuries.
Alcohol intake is common among the drivers and the pedestrians. In 2005 the number of the
pedestrians died in alcohol related traffic accidents in USA was 2180. This number
comprises around 45% of the overall pedestrian deaths. The pedestrian or the driver might
take alcohol (Chakravarty et al., 2007). In accordance with the study conducted on road
traffic accidents in four provinces of South Africa, alcohol intake was confirmed in 58%
(2326) of the cases tested for alcohol (Mabunda et al., 2008).
The children’s body parts injured due to traffic accidents were studied in various studies.
Head trauma is the most common injury. The ratio of head trauma differs on the basis of the
fact whether the child is a pedestrian, or in the vehicle, or riding bicycle (45.4% among
pedestrians, 40.2% among riders, 38.9% in vehicle). Besides head trauma, spinal, thorax and
abdominopelvic traumas also result in severe injuries and deaths (Durkin et al., 1999,
Arrequi-Dalmases et al., 2010).
According to a study conducted in Manhattan, USA, the number of injuries among the
school age children per 100.000 persons is 127.2 for pedestrians, 37.4 per riders and 25.5 for
children in the vehicle. The number of the children having accident – whether pedestrian (6-
10 ages) or rider (9-15 ages) – peaks in summer and in the afternoon time. The number of
the children having accident in vehicle (12-16 ages) shows little variation on the basis of
seasons and tends to increase in the evening and night hours. Traffic accidents peak at the
age of 15. 22.1% of the severe injuries of the school children between the ages 5 and 16 are
related to traffic. Around 2/3 of the individuals severely injured and ¾ of the individuals
died in the road traffic accidents are the pedestrians (Durkin et al., 1999).
Riding bicycle is a popular childhood activity. The injuries and deaths among the bicycle
riders peak between the ages 9 and 15. Boys are exposed to accidents more than girls. The
mortality rate due to bicycle accidents are subjected to the extension of bicycle use on the
roads and to the conditions of the roads. The conditions of motorbike accidents are similar
to bicycle accidents. The risk factors for the bicycle riders are listed as follows: not to use
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helmet, crash with motorized vehicles, to ride bicycle at an unsafe environment, male
gender and alcohol and substance use. It is asserted that nonuse of helmet would lead to
more severe injuries. The most common injury mechanism is reported as fall from the
bicycle. The body parts most frequently injured are the upper extremities. They are followed
by the head and neck region. And finally comes the lower extremities. Among severe
injuries contusion of brain or intracerebral haematoma due to head trauma, blunt abdominal
trauma with laceration or rupture of internal organs are reported. It is indicated that the
number of chest and abdominal injuries is increasing recently. Similar to road traffic
accidents, bicycle accidents also happen in the afternoon time most frequently (Busuttil,
2009d, Agran et al., 2001, Hyder et al. 2006, Kiss et al., 2010, Acton et al., 1995, Linn et al.,
1998, Puranik et al., 1998, Klin et al., 2009, Rivara et al., 1997).
The time spent until the injured reach to the trauma center is critical. The percentage of the
nonfatal injuries is striking. Millions of people get hurt in the traffic accidents every year in
the world. The data suggest that this number increases dramatically until 2020 in the
countries particularly, where the number of vehicles increases rapidly (Chakravarty et al.,
2007).
During the investigation of fatal road traffic accidents, the evaluation of factors such as the
scene of the collision, witness statements, clothes of the dead, vehicles involved in the
accident and the laboratory analysis of the autopsies will be helpful to clarify the accidents
(Busuttil, 2009d).
Death due to trauma has a significant place among the childhood mortality. Traffic accident
is the most common cause among the mortality due to trauma. Children are exposed to road
traffic accidents mostly as pedestrians. Mostly the boys generally in summer are seen as the
victims of the traffic accidents due to head trauma. In such traffic accidents socio-economic
and ethnic factors play an important role (Newbury et al., 2008, Hyder et al. 2006, Mazurek,
1994). Infant seats, toddler seats and safety seats are found helpful in minimizing the
injuries and deaths in the vehicle. It is hard to estimate the next move or behavior of the
children when they are on foot (Busuttil, 2009d). The children should not be allowed to play
on the roads or among the vehicles. The children should wear helmet, while they are riding
bicycle or motorbike. Children should fasten seat belts, while seated in vehicles, or they
should use infant seats, toddler seats or safety seats. We can only save our children from
traffic accidents by taking such precautions. Furthermore the children and drivers should be
more careful in the afternoon time when children are off the school, since traffic accidents
happen more frequently in the afternoon time. It is also important to train the children both
in the school and at home on traffic.
9. Home accidents
Home accidents, occur at home or around the house (Gailerd & Herve 1991). Although
home injuries are seen in all age groups, due to being preventable and often, leading up to
mortality and morbidity, they are important cause of death for children in both developed
and developing countries (Scholer et al., 1997, Harris & Kotch 1992). Among children and
adolescents, home accidents are the second most common cause of death after traffic
accidents (Jacobsson & Schelp, 1987, Laffoy, 1997).
Home accidents seen in children under 18 years old, are classified as deaths due to
poisoning with solid/liquid and gas compounds, falls and blunt trauma, burn, scald or
electric shock, drowning and asphyxia, fire arm wounds and stabbing on the basis of "Home
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Child Deaths 153
Accident Prevention Inventory" proposed by Tertinger (Tertinger et al., 1984), and has been
modified by Asirdizer et al (Asirdizer et al. 2005). Home accidents, are seen in all age
groups, especially in children between the ages of 0 and 6 (Asirdizer et al. 2005).
0-6 age group of children, because they spend more time at home, usually in the kitchen, the
living room and the bathroom, may face numerous injuries (Bourget & McArtur 1989,
Gallagher & Hunter 1995, Kilic et al., 2006). Among children under one year old, drowning
and foreign body aspiration and between the ages of 1-4 falling, multiplication, water scalds,
flame burns and poisonings are more common. At ages of 2-4, when cleaning agents and
drugs are left around; after 5 years old poisonings with drugs stored in the fridge and high
places are increasing (Yildirim, 2008).
Home accident types vary according to geographical regions. The most common type of
accident is falls and this type of accident is the major cause of childhood injuries and deaths
in many regions of the world (Peden et al., 2002, Pomerantz et al., 2001).
9.1 Other accidents

Sports injuries, bicycle accidents and some game accidents are among other childhood
accidents. Studies indicate that bicycle accidents usually occur in boys and school-age
children (Thompson et al., 1990, Cushman et al., 1990).
If accidents and emergency department visits were examined, bicycle accidents are seen as
the frequent cause of multiple system injury and also head and brain injuries are the most
common cause of death in children accidents (Ji et al., 2006, Guzel et al., 2006, Clarke &
Sibert 1986). In order to prevent head trauma, in many countries of the world's and
especially in developed countries, helmet use is made compulsory by law for motorcycle
and bicycle drivers, but still helmet use of bicycle drivers in developing countries is at a very
low level and the rate has been reported to be around 8% (Brown et al., 2002). Mortality
rates of bicycle accidents are indicated between 1.2-4.6% (Ji et al., 2006, Heng et al., 2006).
Sports and game injuries in childhood rarely result with sudden death. Evaluation of these
cases necessitates the identification of activities prior to death, medical history and detailed
autopsy of the deceased (Byard et al. 2002).
10. Fall from height

Falls from height are important causes of morbidity and mortality in childhood traumas.
According to the reports of the World Health Organization, falls from height rank second
among the causes of death in children (Sala et al., 2000, Taviloglu et al., 2001, Peden, 2009).
The important factors that affect mortality are patient age, drop shape, floor structure,
position of the fall, fall height, the injured tissue and the pathology developing in these
organs (Sala et al., 2000, Mathis et al., 2003, O’Neill, 2000, Lallier et al., 1999, Chalmers et al.,
1996).
In the United States, each year, more than three million children visit emergency
departments because of falling (Committee on Injury and Poison Prevention, 2001). In
childhood, falls from height hold an important place in home accidents and occur in the
form of falling from staircases, balconies, windows or roof (Yagmur et al., 2004, Lallier et al.,
1999, Istre et al., 2003).
Etiology of falls from height may vary due to structural and seasonal characteristics of the
countries, regions or cities (Yagmur et al., 2004, Lallier et al., 1999). In the United States, in a
study investigating the epidemiology of deaths due to trauma in rural areas, deaths due to
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falls from height have been determined as such a large percentage of %20 (Campbell, 1988).
In the South Eastern Anatolia region of Turkey, falling from the roof of houses is quite
common. In this region, in a related study about falling from the roof, %49,4 of the patients
were noted as under 10 years of age and mortality rate was noted as %5,8 (Yagmur et al.,
2004).
The risk of accidental fall from height is most common among preschool boys and younger
children because their neural control mechanisms, sensory systems and cognitive abilities
associated with hazard awareness and avoidance skills are insufficient (Chang & Tsai 2007).
According to some studies, the head region is the mainly affected region in the body system
(Cassidy et al., 2003, Champion et al., 1989, Potoka et al., 2001, Osmond et al., 2002, Hall et
al., 1989). Deaths mostly develop in the early period due to multiple trauma or fatal head
trauma (Mosenthal et al., 1995, Buckman & Buckman 1991, Velmahos et al., 1997). In the
necropsy studies of Hall et al, head trauma is reported as the most common reason of death
in children falls (%70,5) (Hall et al., 1989); because children have a higher head/body ratio
than adults (Champion et al., 1989, Buckman & Buckman 1991).
According to the studies about falls from height in childhood, mortality rates are noted
between %1,3 and %5,9 (Velmahos et al., 1997, Murray et al., 2000).
11. Deaths due to neglect, starvation and physical agents (heat, cold,
electricity)
11.1 Neglect- starvation
Is the situation where fundamental needs of a child like nutrition, health, shelter, clothing,
protection and supervision are not properly met by his/her parents (Can et al. 2010).
Neglect should be repetitive and should impair the health and development status of the
child (Nathanson, 2000).
Accidents rank the first in most cases of child mortality due to neglect. Street accidents
(traffic accidents, bicycle accidents, fall from a height), domestic accidents (entrapment in
anaerobic places, burns, poisoning, drowning in water), in-car traffic accidents (failure to
use child restraints or to put on safety belts, neglecting car maintenance) are the most
common ones (Cologlu & Cakalir 1999). It is hardly possible to distinguish between
accidental and non-accidental injuries. Post-mortem examination should be performed with
relevant post-mortem radiological examination (Nathanson, 2000, Cologlu & Cakalir 1999).
The second most common type of neglect is starvation or forced starvation. Starvation can
be due to insufficient delivery or non-delivery of food, delivery of improper food, severe
loss of appetite or social hunger (Cologlu & Cakalir 1999) Starvation is the condition where
there is severe loss of vitamins, minerals, nutrients and energy (Altun & Altun 2010). During
the early days of starvation, the glycogen stored in the liver and muscles are used as energy
source but glycogen may be understored in children (Altun et al., 2004). As carbohydrates
deplete, proteins and fats become the main source of energy. Abundance of fatty acids are
formed and transformed into ketone bodies (Altun & Altun 2010, Milroy & Parai 2011).
Body mass index drops down. The child gets slim or cachectic. Shortness in height,
dehydration, growth and development retardation are noticeable. The skin is dry, atrophic,
cracked and hyperkeratotic in appearance. Post-portem examination indicates
subcutaneous and internal adipose tissue depletion and atrophy in organs other than the
brain. Gastrointestinal organs shrink. Digestive tract is empty; dilatation may be observed in
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Child Deaths 155
the gall bladder; hepatic adiposity may be observed (Cologlu & Cakalir 1999, Altun et al.,
2004). Diabetes mellitus findings are absent in mortality due to starvation but ketoacidosis
may be present in blood (Milroy & Parai 2011). Most of the neglected death cases are under
the age of 1 and measurements such as height-weight, head circumference, femur length
and sitting height should be done properly and meticulously (Knight, 1996e).
11.2 Hypothermia
Hypothermia is the condition when the internal body temperature drops under 35⁰C. Due to
limited thermoregulation in children, tissue oxygenation is reduced in prolonged
hypothermia resulting in cardiac arrhythmia (Cologlu & Cakalir 1999, Turk, 2010).
Hypothermia mostly occurs in infants during delivery at home. This is also called sklerema
neonatorum. In the case of premature infants or infection or congenital heart disease, the
infant gets more prone to hypothermia. Since infants have a smaller body mass with higher
surface area/mass proportion, they suffer from a rapid temperature loss. Subcutaneous
adipose tissue is smaller in infants with underdeveloped vasomotor reflexes. Adolescents,
on the other hand, may suffer from failure to feel the cold following sports activities and
alcohol intake or may be subject to hypothermia in case of fatigue (Cologlu & Cakalir 1999,
Eke & Soysal 1999). Hypothermia and hypoglycemia are mostly the cause of death for
infants left out in mosque or church gardens or rich neighborhoods. Post-mortem stains are
light pink in autopsy with erosion and hemorrhage in the digestive tract mucosa
accompanying hemorrhagic pancreatitis and multi-infarct zones in organs (Cologlu &
Cakalir 1999).
11.3 Hyperthermia
Hyperthermia is the condition when the body temperature is above 40⁰C. Hyperthermia
may be due to either external reasons (such as heat stroke) or internal reasons (such as
infections) (Eke & Soysal 1999). In closed spaces with a high humidity ratio and lack of air
flow (such as entrapment in the car in hot weathers) there is a high risk of hyperthermia.
Children have a limited thermoregulation capacity when compared to adults. Post-mortem
examination indicates non-specific alterations, hyperthermia in internal organs as well as
edema in the brain and lungs accompanied with petechial hemorrhages. It is important that
both ambient temperature and body temperature are measured during crime scene
investigation (Cologlu & Cakalir 1999).
11.4 Burns
Mammalian tissues can preserve viability in a relatively narrow window of temperature
between 20- 44⁰C (Eke & Soysal 1999). Child’s skin is softer than adults and is more sensitive
to heat (Busuttil, 2009c).
Width and depth of the burnt skin surface as well as its proportion to entire body surface
affect morbidity and mortality. Burn surface area is determined according to the classical
Rule of Nines. Each arm 9, each leg 18, anterior chest 18, posterior chest 18, head 9 and
genitalia 1.
1st degree: is the burn that affects the epidermis. Presents with erythema and mild pain. Sun
burns are a good example.
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2nd degree: is the burn that affects the epidermis and dermis in varying degrees. Superficial
2nd degree burns only invade upper 1/3 of the dermis, and are characterized with blister
formation. Deep 2nd degree burns, on the other hand, penetrate into lower 1/3 of the dermis.
3rd degree burns: are the ones covering the entire epidermis and dermis. (Busuttil, 2009c,
Knight, 1996a).
Burns may occur as a result of contact with dry – hot or wet – hot substances or chemicals.
The heat damage caused by hot fluids is mostly referred to as scalding. (Busuttil, 2009c,
Duke et al., 2011). Burns in children are mostly accidental typically occurring as scalding,
but suicide and homicide possibilities should never be overlooked (Hilal et al., 2008). Scalds
are severe injuries. Victims generally get to be scalded at home by exposure to hot fluids
(Figure 3). There are reports of scalding with boiled milk when making cheese (Cekin et al.,
2010). Approximately 10% of abused children have burns and scalds with about 25% of
scald injuries resulting from non-accidental reasons (Chester et al., 2006, Jacobi et al., 2010).
Abuse-related burns should be differentiated from accidental burns (Figure 4). Arms, face,
anterior trunk and neck are risky spots for accidental burns. These are generally asymmetric
burns with irregular limits and irregular depth. Abuse-related burns mostly manifest with
burns on the thigh and legs, genitalia, hands and feet as glove or sock type burns. These are
burns with regular and symmetric limits and depth (Maguire et al., 2008, Maguire, 2010).
(Figure 5). Post-mortem examination of a burn case should seek to identify the dead body,
indicate cause of death and should try to answer whether the person was trapped alive in
fire or not.
Fig. 3. Children are usually scalding accidentally.
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Child Deaths 157
Fig. 4. Accidental burns.
Fig. 5. Abusive burns.

External examination may indicate Pugilistic attitude due to contraction of flexor muscles in
upper and lower extremities as a result of denaturation of muscle proteins in exposure to
extreme heat. Tissues are dried and toughened due to loss of fluid. There may be bone
fractures. Identification of the body may be difficult due to blackened and disintegrated
facial features, skin contraction and tightness, scorching of hair and other heat-related
changes. Internal examination reveals scalded organs, but the skull may present with
extradural hematoma-like, soft, fragmented clots in light chocolate color referred to as heat
hematoma (Eke & Soysal 1999, Busuttil, 2009c, Knight, 1996a).
11.5 Electric shock

It is the condition when the electric current (electrons) travels over the child’s body as they
move from one point to another or in other words, when the body becomes a part of the
electricity circuit. The biggest obstacle against the electric current is the skin, which has a
stronger resistance than the internal tissues of the body (Eke & Soysal 1999). As dry skin has
a resistance of 40.000-100.000 ohms, wet skin manifests a resistance of approximately 1000
ohms and callous skin of 2.000.000 ohms (Busuttil, 2009c). Since the child has a thinner skin,
it would have a weaker resistance. It generally develops as a result of accidental contact
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with an electricity-connected cable. Entry point is generally the hand or the foot, while the
exit point is the other hand or the other foot that contacts the ground (Eke & Soysal 1999,
Gupta et al., 2009). The most dangerous flow path is the one that goes between the left arm
and the right leg. Electricity current may of course run into the body from any spot on the
body (Gupta et al., 2009). When it enters the body from the hand, the most important impact
that it creates would be the spasm on flexor muscle groups causing a ‘‘hold on’’ effect. As a
result, grasping the conductor during electric shock becomes inevitable prolonging the time
of electricity to pass the body and worsening the severity of injury (Knight, 1996b). Muscle
contractions and spasms may throw away the victim causing extra injuries (Eke & Soysal
1999, Fodor et al., 2011). The origin of the event is mostly accidental in electric shocks that
frequently develop as a domestic or occupational accident, and yet the possibility of child
abuse or homicide should never be overlooked. It is important to keep the possibility of
suicide in mind in adolescent cases (Canturk et al., 2008, Shetty et al., 2010). Death due to
electric shock usually develops as a result of ventricular fibrillation or respiratory failure. If
the electric current travels through the chest and the abdomen, spasm in intercostal muscles
and the diaphragm would cause respiratory paralysis. If it travels through the heart, the
result would be arrhythmias, ventricular fibrillation and cardiac arrest. But when it travels
through the head and the neck, death would develop as a result of paralysis in the
respiratory and circulatory center in the brain stem (Eke & Soysal 1999). There are cases
reported with death due to air embolism resulting from electrical injury of neck veins
(Kitulwatte & Pollanen 2009). External examination may not indicate any finding in death
cases due to electric shock but may as well present with a carbonated body. Electric entry
and exit wounds may not always be easily detectable. Entry wound may be in the palms,
between the digits, in the oral cavity or in the internal wall of the lips and should be looked
for meticulously. Electric burns may present with a grey-white colored parchment look with
a typical crater appearance with folded edges. Internal examination indicates non-specific
alterations (Eke & Soysal 1999, Polat, 2006).
11.6 Lightening strike

Lightening is the discharge of the electrical potential of the atmosphere between clouds and
the earth (Gok, 1983b). With lightening, a DC of 2000- 2 billion Volts gets discharged on
earth in an extremely short period of time (in 0,1-1ms) (Busuttil, 2009c). Although rare, it
may cause an injury as big as crush injuries (Rash, 2008). Lightening strikes generally occur
out in the meadows or forests during spring and summer times (Gok, 1983b). Farmers in
small settlements and their children as well as swimmers are under risk (Demirel et al., 2007,
Kilbas et al., 2008). Death occurs due to burns, respiratory and circulatory arrest. There may
be wide burns on the clothing and on the individual. Metallic parts of clothes have gained a
magnetization feature. Clothes may be torn apart or shredded (Eke & Soysal 1999). External
examination indicates erythema and fumigation and scorching on burn wounds as well as in
the hair. Lichtenberg figures may be present on the skin resembling tree branches
originating from dilatation and tearing of small blood vessels. (Eke & Soysal 1999, Polat,
2006, Domart & Garet, 2000, Whitcomb et al., 2002). The most common cause of death is
cardiopulmonary arrest (Kilbas et al., 2008). Pulmonary edema, contusion, hemorrhage and
ARDS may be seen in the respiratory system. Prolonged Q-T, myocardial infarction and
Takotsubo’s cardiomyopathy may develop in the heart. Intracranial hemorrhage may be
present in the central nervous system (Whitcomb et al., 2002, McIntyre et al., 2010).
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Child Deaths 159
12. Wounds
All kinds of damage caused on the body by physical or chemical substances are defined as
wounds. The features of the damage on the tissue depend on the energy transferred on the
tissue, transfer period to the tissue, width of the transfer area, structure of the substance
causing injury, impact angle and the status during the impact, the structure of the body part
affected and its status during impact. Wounds caused by substances and tools with diverse
features display different characteristics. These wounds can be grouped into five. 1- Blunt
traumatic wounds, 2- incised wounds (sharp object wounds), 3- Penetration wounds, 4-
penetrating stab wounds, 5- incised & crush wounds (Cetin, 1999, Knight, 1996f).
All the physicians involved with forensic medicine should be familiar with the
characterization and images of the wounds. Fatal childhood wounds alone will be assessed
here. Blunt traumatic wounds: might be observed as abrasions, ecchymosis, hematoma,
laceration and bone fractures (Cetin, 1999). This group of wounds is commonly observed
and coexist in deaths due to general body trauma related wounds such as traffic accidents
and falling down from height. Internal body, major vessel and medulla spinalis wounds
might be fatal (Cetin, 1999, Bilgen et al., 2009). These might be caused by an accident or
occur as a result of homicide and neglect. Ecchymosis is the most common abuse wound
(Maguire, 2010). Existence of many abrasions, bruises and hematomas can not be assessed as
an abuse finding alone. Toddlers and active school age children might have plenty of
ecchymosis especially on knees, pretibial area and forehead (Nathanson, 2000). Abrasions
with a particular shape and ecchymosis might be helpful in terms of understanding the
cause of the wound (Gok, 1983b).
Bite marks, ecchymosis and laceration or combination of three might be observed. Marks
should be measured and teeth structure should be defined via the remaining teeth marks in
order to assess the possibility that marks may be caused by the bites of another child or an
animal (help of the forensic dentist is of value) (Nathanson, 2000).
Existence of many fractured bones is a strong evidence of abuse. Medium shaft fractures,
spiral or oblique fractures of long bones, metaphyseal and epiphyseal fractures and
smashes, periosteal thickening, numerous rib fractures, linear fractures on skull especially
on parietal bone and compression fractures are associated with abuse (Maguire, 2010,
Nathanson, 2000, Knight, 1996c).
Incised wounds, penetration wounds, penetrating stab wounds, sharp incised & crush
wounds associated mortality might be caused by an accident or a homicide. Suicide is also a
possibility for the adolescent groups. Localization and number of the wounds and wound
age are important in understanding the cause of the wound. Crime scene investigation,
social and medical history of the child and post mortem examination findings should be
interpreted together (Ekizoglu & Arican 2010).
13. Firearm fatalities

Injuries are leading causes of mortality throughout the world both for childhood and other
age groups (Fraga et al., 2010, Meel, 2007). Firearm fatalities are the most common causes of
traumatic death together with deaths from motor vehicle crashes in countries like USA. In
1992, 5367 children and adolescents aged 1 to 19 years were killed by firearms. 63% were the
victims of homicides, 27% of suicides and 9% of unintentional injuries. In contrast, during
that same year in Britain, firearms were involved in a total of 46 deaths (Mazurek, 1994).
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There is a steady nationwide increase in the death toll from firearms and the USA still lacks
effective gun control legislation (Mazurek, 1994, Powell et al., 1996). For example, firearms
are easily available in the USA and Scandinavian countries (Canturk et al., 2010). Firearm
fatalities are common in countries like Columbia, South Africa and Brazil while it is quite
low in Japan. Firearm fatalities are more common in countries which legally ensure easy
access to firearms and have low-to-mid income level (Meel, 2005). Firearm fatalities are
higher among men and the age group 15-44. Minority youth are disproportionately affected
by firearm homicides. Availability of firearms at home is reported to increase the risk of
firearm fatalities for children and adolescents. Firearm fatalities among children are rarer
under the age of 10. The incidence is progressively higher for the age groups 10-14 and 15-19
(Fraga et al., 2010, Meel, 2007, Mazurek, 1994, Powell et al., 1996, Canturk et al., 2010, Meel,
2005, Presley et al., 2007, Grossman et al., 2005, Canturk et al., 2007, Agran et al., 2001).
Fatalities might occur due to negligent behavior with firearms, children playing with
firearms and during hunting or firearm cleaning process. Children might shoot themselves
or might be shot by a family member or a friend. Pistols are the most common causes of
death among other firearms. Shotgun and rifle follow the pistols as a fatality cause
(Heninger & Hanzlick 2008, Barber et al., 2002, Bhattacharya et al., 1998). Firearms are
mostly to blame for homicides and suicides among children (Mazurek, 1994, Canturk et al.,
2007).
Firearm fatalities manner of death is mostly homicide for children. (Meel, 2005, Heninger &
Hanzlick 2008, Byard et al., 2009, Sorenson & Berk 1999, Eber et al., 2004). A study which
compares the pediatric firearm fatalities in Australia and USA reports that the ratios vary;
however, fatalities are more frequently seen among men, the cases are more common
respectively as manner of death, homicide, suicide and accidental deaths; the most
frequently injured body part was reported to be the head (Byard et al., 2009).
Easy legal access to firearms in some developed countries increases the risk of suicide
committed with firearms especially for the adolescents (Canturk et al., 2010, Meel, 2005,
Portzky et al., 2005, Grossman et al., 1999). The pediatric suicide risk increases together with
the increase in age. The fatal and violent forms of suicide are preferred by boys (Canturk et
al., 2010). Hanging is reported to be the most common manner of pediatric suicide. Suicides
committed via firearms increase during adolescence and young adulthood (Canturk et al.,
2010, Madge, 1999).
Unintentional firearm fatalities are experienced less among children when compared to
homicides and suicides. These are sometimes taken as suicides. Children might accidentally
die as a result of a bullet fired out of the firearm by family members or aggressors. Access to
firearms at home increases the pediatric risk of injury or death. Children or adolescents may
lead to an accident while trying to impress others or children might die during the firearm
cleaning process or hunting (Grossman et al., 2005, Barber et al., 2002, Karger et al., 2002,
Hemenway et al., 2010).
In a study from Istanbul, most of the cases (n=36, 53.4%) were aged 16-20 years (Asirdizer et
al., 2010). Firearms other than handgun, rifle, shotgun may cause childhood mortality.
Nonpowder firearms are not toys. These may cause serious injuries or fatalities among
children and adolescents. 32 of 39 nonpowder gun-related (ball-bearing guns, pellet guns,
air rifles, paintball guns) deaths reported in USA between the years 1990 and 2000 were
among children less than 15. It is estimated that 3.2 million nonpowder guns are sold every
year in USA (Laraque, 2004, O’Neill et al., 2009). A study reports that 16 of 59 simply
modified blank cartridge gun related deaths within 4 years occurred among the ages of 11-
www.intechopen.com
Child Deaths 161
20 (Uzun et al., 2009). Nonpowder firearms and blank cartridge guns related child deaths
most frequently occur among men as it is the case for other firearm deaths; head injuries are
the most common type while most of the cases are suicide cases(Laraque, 2004, O’Neill et
al., 2009, Uzun et al., 2009). Celebratory gun shooting injuries are mostly seen among men
in metropolitans and crowded places during celebrations or festivals and holidays.
However, there are rare cases of women or child injuries or deaths (Ozdemir & Unlu 2009).
14. Acknowledgment
The authors wish to thank Miss Emine Tug for her drawings our figures.
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8
Child Abuse and the External

Cause of Death in Estonia
Marika Väli1,2, Jana Tuusov2, Katrin Lang3 and Kersti Pärna3
1Institute of Pathological Anatomy and Forensic Medicine, University of Tartu
2Estonian Forensic Science Institute
3Department of Public Health, University of Tartu
Estonia
1. Introduction
Violence against children cuts across boundaries of geography, race, class, religion and
culture. Injury and violence are serious threats to the health and well-being of children
worldwide. Children are at high risk from injuries that can lead to death or disability.
A small proportion of violence against children leads to death, but most often the violence
does not even leave visible marks. Violence can have severe implications for children’s
development and in the most severe cases, it can lead to death or injury. However, it can
also affect children’s health, their ability to learn or even their willingness to go to school at
all.
The World Health Organization (WHO) estimates that 40 million children below the age of
15 suffer from abuse and neglect, and require health and social care. In 1998, a UNICEF
report quantified the large East-West gap in European child mortality from external causes
(injuries and violence). In the past decade, much has changed in central and Eastern Europe,
economically, politically and socially. Some positive changes are seen in child injury rates in
this region, and hopefully the East-West gap in European child mortality will diminish.
Violence against children is defined as any form of violence, whether physical, mental and
sexual, abandonment or negligence, ill-treatment or exploitation that puts their lives in
danger or negatively impacts their lives, physical or psychological health dignity, or
development.
In this chapter we present recent trends and current situation of child injury mortality in
Estonia. We also describe the forensic medical system, examination of the child and the
expert report, and give an overview of cases physical and sexual child abuse in Estonia.
In Estonia forensic medical doctors are a medical experts in physical and sexual abuse
assisting Law Enforcement, but they are also involved in investigating all child deaths due
to external factors.
In all cases including child abuse, an examination by a forensic medical doctor is done only
when requested by a police officer, prosecutor or court. In cases where the child is less than
16 years of age, a parent, police officer, teacher, social worker or careworker must be present
during the examination. The examination of a child is carried out at the forensic department
or at the hospital if the child is admitted for inpatient treatment.
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2. The examination of the child and the expert report

In suspected child abuse cases an examination is performed. The aim of the examination is
to find out the primary injury, but also to carry out physical examination of the child and
record.
During the examination the forensic medical doctor describes the signs and symptoms that
could point primarily to the presence of injuries or the complication of injuries. Often the
expertise is not timely as the child requires medical attention. In these cases the doctors
must document and describe the injuries and sometimes the full examination is performed
at the hospital.
As in other countries, the medical doctor may lack knowledge to detect and record injuries,
so the forensic examination is needed. From incorrectly completed medical documents the
forensic doctor can not decide what kind of injuries the victim has, nor the timeline or the
cause of injuries.
The task of the forensic medical doctor is to find the injuries, timing and causes of injuries
and answer all questions that may rise during the proceedings. In all cases involving child
abuse, an examination by a forensic medical doctor is done only when requested by a police
officer, prosecutor or court. In case the child is less than 16 years of age, a parent, police
officer, teacher, social worker or careworker must be present during the examination. The
examination of a child is carried out at the forensic department or at a hospital if the child is
there.
The forensic medical experts report details the existence or the absence of injury to health;
the nature of the injury (diagnosis); the way the injury was incurred; assessment to the link
between the person’s previous state of health and injury to health (if applicable); the injury’s
threat to life; the duration of the injury; other conclusions related to the assignment. A
forensic doctor can use also the opinion of other medical specialists and this will be also
mentioned in the examination. In physical abuse cases involving children often a
paediatrician is involved in the examination.
In recent years, special rooms have been created for questioning and examining children at
the police stations throughout Estonia. Police officers with special training, prosecutors and
other specialists use these rooms when dealing with children.
3. Child abuse
Child abuse is a worldwide problem affecting children from birth to 18 years of age. Each
year, hundreds of thousands of children suffer abuse or neglect. Many studies have shown a
consistent pattern regarding the abuse and neglect inflicted on children of both genders.
Approximately 75% of sexual abuse is inflicted upon girls. Girls also are more likely to
suffer from emotional abuse and neglect. Boys, on the other hand, are more likely to
experience physical trauma (other than sexual abuse). When focusing solely on cause of
death, studies indicate fathers are more likely to kill their child via physical abuse, while
mothers kill by neglect (for example, starvation).
In most cases, the abuser is someone known to the child – a parent, family member, teacher,
or regular careworker. The issue of abused children is an important public health problem
since intra-family violence, including child abuse, is a so-called inside-family problem that is
usually not discussed in public. The risk of child abuse is higher in families where there are
often conflicts between family members, low parental involvement in the family and cold or
hostile relationships between children and their parents. Those parents who had been
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Child Abuse and the External Cause of Death in Estonia 179
abused during their own childhood were more likely than others to abuse their own
children. We found that family sociopathy (alcohol problems) and some family members
disability or handicap problems might predict child maltreatment; low family income and
poor parental warmth are associated with risk for child neglect. Therefore the number of
cases concerning child abuse is relatively low in comparison with other countries.
In Estonia the issue of abused children has been under discussion since 1990. Child abuse in
Estonia is probably far more prevalent than generally thought. As elsewhere, national
statistics are not available, as the nature of the problem makes it hidden in the society and
difficult to detect and record. The pupils from different types of schools who have
participated in such studies confess that they have encountered emotional, physical, and
sexual abuse as well as negligence. The most common types of abuse according to these
inquiries were verbal sexual abuse, negligence of education, emotional abuse, mental sexual
abuse, and negligence of health. Physical abuse, physical sexual abuse and physical
negligence were less common. Most abused children suffer greater emotional than physical
damage. An abused child may become depressed. He or she may withdraw, think of suicide
or become violent. An older child may use drugs or alcohol, try to run away or abuse others.
However, when comparing the findings of studies performed in pupils from ordinary
Estonian schools with those for children with special needs, the incidence of negligence and
sexual abuse are far more common in the latter ones. It can be also said that the problem of
abused children has gained more attention in the recent years. For example, this is reflected
in the discussions about whether to hit children is acceptable or not. The attitude towards
hitting a child has changed in recent years. Still, these discussions have not reached the
point as to where to draw the line between an accident and child abuse. This to a large
extent is a matter of definition. When a two-year-old child drowns in the pond – is it an
accident or negligence? In Estonia such cases are usually considered accidents.
3.1 Physical abuse

Physical abuse is physical aggression directed at a child by an adult, but this is very often
neglected and without adequate attention. The reason for this is that the specialists of
different fields do not cooperate. It is of paramount importance that the specialists of
different fields think in the same way in the event of child abuse, and also understand the
ways of acquiring injuries in the same way.
The physical signs of child abuse is sometimes called battered child syndrome. Physical
abuse tends to occur at moments of great stress. Physical child abuse or non-accidental child
trauma refers to fractures and other signs of injury that occur when a child is hit in anger.
According to the data by the Estonian Forensic Institute about 50 children less than 14 years
of age annually need physical examination. Most of the cases are related to violence at
school or at home, but children are also injured in traffic accidents. During recent years the
number of detailed examinations of physically abused children has decreased by 50%. The
number of children injured in traffic accidents has also decreased (with only a handful of
cases each year). From 2001 to 2009 most of the children examined were between 7 and 14
years old, boys incurred injuries three times more often than girls, and only 10 children
under one year old did so.
According to the literature data, the injuries acquired in association with child abuse
comprise 7–27% of the total number of injuries to children (Overpeck et al., 1999), and
children are most frequently assaulted at the age of less than five years (Laursen & Nielsen,
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2008). Among the prevailing injuries are bruises, abrasions and other mild injuries; head,
face and extremities are the most frequently affected regions.
Who causes the injuries? Fathers, mothers, adoptive parents, other members of the family.
Why are children abused? Often the reason is discord in the family, single mothers,
underaged pregnancies, low educational level of the parents and poor living conditions
(Lang et al., 2010). Many people who commit physical abuse were abused themselves as
children. As a result, they often do not realize that abuse is not appropriate discipline. Often
people who commit physical abuse also have poor impulse control. This prevents them from
thinking about what happens as a result of their actions.
The causes of hospitalisation have been studied in the case of traumas to children in Estonia,
and it appears that the main cause of hospitalisation for children of this age are contusions,
bone fractures and wounds associated with a fall. Apart from the falls the others cases of
suspected abuse include burns, the occurrence of different objects in throat, and
unclear cases.
According to the questionnaire study carried out in Estonian schools during 2001–2009, 45% of
children suffer from school violence but most of them do not inform their parents or the police
about it. One fifth (20%) of children get hurt at school, one fourth (25%) of children are injured
by their schoolmates and 1/10 (10%) of children suffer from domestic violence. One of the
causes of physical abuse in small children is definitely the shaking of babies, i.e. Shaken Baby
Syndrome (SBS). In Estonia Shaken Baby Syndrome was first diagnosed in 1999.
Shaken Baby Syndrome is most common in children below one year of age, and it is known
to occur as a result of child abuse - it is caused by vigorous shaking and/or swinging of the
infant. In most cases, an angry parent or caregiver shakes the baby to punish or quiet the
child. Such shaking usually takes place when the infant is crying inconsolably and the
frustrated caregiver loses control. Many times the caregiver did not intend to harm the baby.
When an infant or toddler is shaken, the brain bounces back and forth against the skull. This
can cause bruising of the brain (cerebral contusion), swelling, pressure, and bleeding in the
brain. The large veins along the outside of the brain may tear, leading to further bleeding,
swelling, and increased pressure. This can easily cause permanent brain damage or death.
Excessive shaking causes the rupture of cortical and bridging veins in the brain, possibly
resulting in subdural haematoma, or less frequently subarachnoid haematoma and brain
oedema. Subdural haematoma is the most common intracranial pathology observed in cases
of SBS, and it is seen in approximately 80% of children with this syndrome. In the United
States 750–3750 cases of SBS are diagnosed each year, whereas in Estonia only 2–3 cases per
year. The incidence rate of SBS is 40.5 cases per 100 000 children below one year of age in
Estonia. The study performed by Talvik and co-authors revealed that the majority of the
families of these children had economical difficulties (75% of the families received only
social benefits, but no salary at the time of injury) (Talvik et al., 2002). These facts suggest
that a poor socio-economic situation is one important factor contributing to violence against
children. This is confirmed by the data from other research that the people who abuse
children have frequently low educational status and more frequently drug and alcohol
abusers.
A unique form of physical child abuse is Munchausen syndrome by proxy. In this situation,
a parent will purposely either invent symptoms and falsify records (for example, fever)
resulting in unnecessary tests, hospitalizations, and even surgical procedures. This
psychiatric illness of the parent(s) requires a high index of suspicion, and its consideration is
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part of the investigation of any child with recurrent complaints that are not supported by
physical or laboratory findings.
3.2 Sexual abuse

Sexual abuse of children is forcing or persuading a child to participate in sexual acts without
the child’s understanding of the situation. It does not necessarily mean sexual intercourse or
physical contact. It also includes incest, paedophilia, exhibitionism and molestation, but also
sexual intercourse – urogenital, anogenital or vaginal intercourse with a child. It is difficult
to determine how often child sexual abuse occurs, because it is more secret than physical
abuse. Children are often scared to tell anyone about the abuse. Many cases of abuse are not
reported.
Children become the victims of sexual violence usually at home and from people, who they
actually know, most often stepfathers and fathers. Child sexual abuse occurs in all social
and economic classes of people. It has the same type of risk factors as physical child abuse,
including: alcohol and drug abuse and family troubles. Abusers often have a history of
physical or sexual abuse themselves (Johnson, 2007).
In sexual abuse cases, the most important factor is a timely and correct gynaecological
examination, but also a correctly taken analysis (sperm). The importance of interviewing the
child cannot be underestimated, what they say should be recorded in their own words.
When it is possible, the nature of sexual contact should be ascertained. All other parts of the
examination are the same as in the case of physical abuse including a complete general
examination, recording growth and sexual maturity.
The colposcopic investigation of the anogenital region in girls and anal region of boys is
very important, as injuries to the mucosa are not easy to see and with the attached camera, it
provides documentation of the examination’s findings. The possibility to use a colposcope is
available in all four forensic departments in Estonia. Both specialists (gynaecologist, forensic
doctor) attend the examination if this is possible, but this is not mandatory. In Estonia
forensic doctors are capable of carrying out gynaecological examinations without the
presence of a gynaecologist.
If the child is less than 16 years the consent of the parent of carer is needed. If the parents
want to have the examination but the child is against this, then the child’s wish is taken into
account. The child has the right to refuse or accept the parent’s presence during the
examination.
It is advised that the physical and gynaecological examination is performed by a forensic
doctor, but if it is not possible, the doctor on duty has to do it following the same principals.
In cases of sexual abuse, cooperation between the police, social worker, paediatrician and
the forensic doctor is very important.
Similarly to the data reported in published papers, the victims of sexual abuse in Estonia are
usually younger than 12 years of age, most frequently between three and seven years of age,
and two to three times more likely to be handicapped children (Kvam, 2000). During the
past two years (2008–2009) Estonian forensic medical doctors performed in total 27
examinations on sexually abused children aged 0-14 years, and the majority of them were
girls (girls vs. boys ratio 22 : 5). In Estonia the cases of vaginal and anogenital intercourse are
the most frequent, and the cases of incest are also not uncommon. In the cases of sexual
abuse it is often hard to evaluate the examination’s findings, because injuries usually heal
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within a short time period and abnormal findings of the anogenital region may be caused by
other factors (blunt force trauma, infection). The problem concerning Estonian forensic
doctors is the small number of reported cases giving them very little experience of the
problem.
4. External causes of death

In every single industrialized country, injury has now become the leading killer of
children.Taken together, traffic accidents, intentional injuries, drownings, falls, fires,
poisonings and other accidents kill more than 20 000 of 1–14-year-old children every year in
the OECD countries.
Deaths from injury in Estonia form about one third of all deaths to children aged up to 14,
this is considerably more than in neighbouring countries. The most pronounced difference
between Estonia and other countries in child deaths resulting from injuries are in infant
deaths. External causes of death form about one third of all deaths in children 0–14 years
old in Estonia. The reduction in childhood mortality shows some progress has been
achieved over the recent years.
Injury deaths (V01–Y89) All deaths, Total mortality rate per

Year
N % N 1000
2001 56 28.6 196 0.82
2002 40 28.4 141 0.61
2003 44 27.5 160 0.71
2004 31 23.1 134 0.62
2005 40 30.1 133 0.64
2006 31 27.7 112 0.55
2007 29 23.2 125 0.63
2008 27 22.3 121 0.61
2009 17 12.5 87 0.43
Table 1. Total mortality rate per 1000 for all deaths (including injury related deaths) among
children, 2001–2009 (Statistics Estonia, 2011)
The annual total mortality rate in Estonia was 0.62 in 2001–2009. During the last few years a
decrease has been observed both in the general mortality and injury-related mortality of
children (Table 1; Figure 1). In 2007–2009 injury mortality rate decreased from 14.9 to 4.9 per
100 000 among 10–14-year-old children children in Estonia (Figure 1).
From the beginning of 2006 infant mortality has decreased, but less significant progress has
been observed for childhood and adolescent deaths. During 2001–2009, 391 children aged 0–
14 were autopsied by forensic doctors at the Forensic Science Institute and 310 (79.2%) of
causes of death were attributed to the external causes.
The primary external causes of child death in Estonia are various kinds of mechanical
suffocation (strangulation, aspiration of foreign bodies or gastric content, drowning,
compression). In Estonia asphyxia formed 40.3% of unintentional deaths, followed by
mechanical injuries (transport and falls) and poisonings. Drowning and aspiration were the
most frequent cause of asphyxia. Strangulation was registered as the cause of death in six
cases, with an additional 18 other cases in which the intent was impossible to identify. This
group included for example obstruction of the airways with a foreign body, being struck by
a blunt object and others.
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16 14.9
14
12.5
12
Mortality rate per 100 000
10.5
10 9.0
7.5
8
6.3
6 4.9
0
2003 2004 2005 2006 2007 2008 2009
Fig. 1. Injury mortality rates per 100 000 among 10–14-year-old children, 2003–2009 (Statis-
tics Estonia, 2011)
The major problem in Estonia is a high mortality rate from unintentional injuries (accidents)
that formed 64.2% of all deaths of children aged 10–14 in 2001–2009 (Table 2).
Intent of death Number Column %

Disease 81 20.7
External causes (injuries) 310 79.3
Unintentional injuries 251 64.2
Asphyxia 125 32.0
Drowning 56 14.3
Aspiration 45 11.5
Strangulation 6 1.5
Other 18 4.6
Transport accidents 80 20.5
Poisoning 24 6.1
Intentional injuries 29 7.4
Homicides, suicides 16 4.1
Intent unknown 29 7.4
Total 391 100.0
Table 2. Deaths from diseases and injuries among 10–14-year-old children, 2001–2009
(Statistics Estonia, 2011)
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Traffic accidents are the leading health threat to children in many countries (Törõ et al.,
2011; Durkin et al., 1999). Although the number of traffic accidents has decreased
considerably in Estonia in recent years, including also the number of accidents involving
children, transport accidents still prevail among mechanical injuries, constituting 25.8% of
external causes of death. Most of the victims were from the oldest age group (between 15
and 19 years of age), and 78% were passengers in motor vehicles, 19% pedestrians (most of
the cases represent accidents in the home environment where the car reversed over a child)
and only one child died as the result of a bike accident. Similar results were also reported in
an article comparing the injuries to children who died from traffic accidents in three capital
cities (Budapest, Vilnius, and Tallinn) (Törõ et al., 2011).
Poisonings constitute about 7.7 % of all cases of unintentional deaths, and they are mainly
caused by carbon monoxide (CO) and medicinal products. Poisonings with medicinal
products are usually observed in children one to four years of age who happen to get access
to the drugs at home. These include three cases of poisoning with aethazine tablets, and
poisoning with dimedrole and amitriptyline. In addition opiate poisoning occurred in a 14-
year-old boy and one case of poisoning with unknown gas (presumably butane) was also
registered. The rest of the cases represent poisoning with CO in association with fires (seven
boys and ten girls).
When looking at the causes of unintentional death by age group, it can be concluded that
the decrease in children’s mortality has mainly occurred on account of the age group of
children below one year of age (Figure 2). In 2005–2009 unintentional infant mortality rate
decreased steadily from 85.1 to 31.6 per 100 000. Mortality rates in other age groups have not
changed significantly. Analysing the cause of death and manners of death, the main causes
of death in children below one year of age include head traumas and suffocation, although
the manner of death remains unclear in many cases.
Unclear causes are also apparent in children aged one to four years, but the prevailing
causes of death are accidents. The reason for this is a limited availability of accompanying
data and therefore forensic doctors have not enough medical data to decide about the form
of violence used.
Accidents prevail also in the age groups five to nine years and 10–14 years, the latter group
also includes the occasional case of suicide. Similar to other countries a big problem in the
case of deaths among children below one year of age is the high rate of deaths from an
unknown cause.
Thus, 29 cases of death with an unknown cause were registered between 2001–2009,
including nine cases of putrefaction, eight cases of suffocation, one case of poisoning, one
fatality in a fire, and 10 cases of mechanical injury, including three unclear cases where the
child died during birth.
The task of a forensic doctor is to try to find out whether the injuries detected at the autopsy
represent intentional injuries, or whether the child could develop these as the result of an
accident. Similar to other countries such accidents happen mostly at home. According to
Sengoelge (Sengoelge et al., 2010) home injuries were the leading cause of injury death in
children under five years of age in 16 European countries.
Brain traumas are prevailing among the cases of deaths of unknown manner (in nine cases
out of ten and one case represented a combined head and chest trauma). In 2001–2009 brain
traumas constituted 17.1% (63 cases) of all causes of death in children, first of all in children
between one and five years of age.
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120
115.8
100
85.1
80
62.7
60
47.1
40 41.2 32.8 37.9
26.7 24.5 31.6
18.1 29.8
20.1 15.5 18.8 19.2
20 14.9
17.8 12.9 8.4
19.4 13.9 14.7 13.9 13.0
11.0 9.4 6.2
11.8 11.4 9.1
4.7 10.3 6.4 4.9
0
2001 2002 2003 2004 2005 2006 2007 2008 2009
0 1-4 5-9 10-14
Fig. 2. Unintentional mortality rates per 100 000 by age group, 2001–2009 (Statistics Estonia,
2011)
Forensic doctors are usually able to find out the cause of death in the case of a head trauma,
but this is not enough to establish the exact mechanism of injury. For example: A two-and-a-
half month old infant was found in a pram, the forensic doctor has found an epidural and
subdural haematoma. Question: could the child have fallen by itself?
Second example: a five-month-old infant: linear fractures of both temporal bones,
comminuted fracture of the right occipital bone, epidural, subdural and subarachnoid
haematoma. Question: mechanism of injury (beating)?
Third example: two-month-old infant: subarachnoidal haematoma in the right frontal lobe,
cerebral contusion, major haematoma of the aponeurosis and haematomas of the face.
Question: mechanism of injury (caused by forceful blows with a wide-surfaced object, e.g.
hands)?
Injuries from traffic accidents and those caused by blunt objects are commonly the cause of a
brain trauma. A forensic doctor finds as the cause of death either asphyxia or head trauma,
but this is not enough to establish the exact cause of the injury and determine the manner of
death, because of insufficient preliminary data.
The number of youth suicides has decreased in recent years but Estonia still is among the
countries with the highest risk of suicide in the world. Suicides were registered in 16 cases in
the study years, and the majority of cases were boys (12 cases). Most of the children
committing suicide were aged between 10–14 years, but some children were younger
(Figure 3). In 2001–2009 the highest suicide mortality rate was 7.5 per 100 000 among 10–14-
year-old girls in the first study year.
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8 7.5
7 6.2
5.8
6
4 3.2
3 2.2 2.5 2.7
2.1 2.4
3.1
2
0
2001 2002 2003 2004 2005 2006 2007 2008 2009
boys girls
Fig. 3. Suicide mortality rates per 100 000 among 10–14-year-old boys and girls, 2001–2009
(Statistics Estonia, 2011)
The most common way of committing suicide was hanging, in addition to this one shooting
injury and one case of jumping from height were registered. 13 cases of homicide were
registered in the study years, and most of these were children below one year of age (eight
cases). In 2001–2009, the highest homicide infant mortality rate was 15.4 per 100 000 in 2003
(Figure 4).
18
15.4
16
14.2
14
12
10
7.8
8 6.6 6.3
6
4 3.2
1.5 1.8
2 1.1
0
2001 2002 2003 2004 2005 2006 2007 2008 2009
0 1-4 5-9 10-14
Fig. 4. Homicide mortality rates per 100 000 by age group, 2001–2009 (Statistics Estonia,
2011)
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Head injury and choking were the main ways of carrying out the homicide. Two cases
include knife injuries, where also the other members of the family were killed (father and
one other relative in one case). One shooting injury occurred also in the study years.
5. Conclusion
Reduction of death through injury should be included in the current public health agenda
and given a high priority in many countries, including Estonia. It is important to identify the
potentially preventable cases and detect risk factors (single parents, young mothers, low
educational level, bad living conditions etc), as well as to analyze the incidence and
circumstances of different types of violence.
Deaths related to child abuse are preventable and it is therefore important to estimate the
amount of such deaths, but also to study the circumstances leading to these deaths. Deaths
related to child abuse occurred more often in families that had problems with alcohol abuse,
unemployment etc. and/or mothers who had a low level of education.
More information is needed on the circumstances of the violent deaths among children. This
would enable not only to correctly classify the manner of death in suspicious cases, but also
to eventually reduce the numbers of violent deaths among children.
For adequate assessment of the level of child abuse, it is important to know, who and how
evaluates the injuries inflicted on a child, as well as who are the members of the
investigation team. Close cooperation between various specialists is essential for the correct
diagnosis.
Rates of child mortality from injuries have fallen across Europe. In the former Soviet
countries, this is likely to reflect improvements in living conditions since the transition.
Child deaths from injuries are avoidable and measures to reduce them would have a
significant impact upon the overall burden of child mortality in Europe.
It is important to identify the potentially preventable cases and detect risk factors (single
parents, young mothers, low educational level, bad living conditions etc), as well as to
analyze the incidence and circumstances of different types of violence. According to
published figures more than 50% of cases can be prevented.
6. Acknowledgment
The study was supported by the Estonian Science Foundation (grants no 6592 and 8256).
7. References
Durkin, M.S., Laraque, D., Lubman, I. & Barlow, B. (1999). Epidemiology and the prevention
of traffic injuries to urban children and adolescents. Pediatrics, Vol.103, No.6, pp.
1273–1274, ISSN 0031-4005
Johnson CF. Abuse and neglect of children. In: Kliegman RM, Behrman RE, Jenson HB,
Stanton BF. (2007). Nelson Textbook of Pediatrics. 18th ed. Philadelphia, Pa: Saunders
Elsevier, chap 36
Kvam, M.H. (2000). Is sexual abuse of children with disabilities disclosed? A retrospective
analysis of child disability and the likelihood of sexual abuse among those
attending Norwegian hospitals. Child Abuse Negl, Vol. 24, No. 8, pp. 1073–1084,
ISSN 0145-2134
www.intechopen.com
Lang, K., Pärna, K., Grjibovski, A.M. & Väli, M. (2010 ). Deaths of infants subject to a
forensic autopsy in Estonia from 2001 to 2005: what can we learn from additional
information? Popul Health Metr, Vol. 8, pp. 27, ISSN 1478-7954
Laursen, B. & Nielsen, J.W. (2008). Influence of sociodemographic factors on the risk of
unintentional childhood home injuries. Eur J Public Health, Vol. 18, No. 4, pp. 366-
370, SSN 1464-360X
Overpeck, M.D., Brenner, R.A., Trumble, A.C., Smith, G.S., MacDorman, M.F. & Berendes,
H.W. (1999). Infant injury deaths with unknown intent: what else do we know? Inj
Prev, Vol. 5, No. 4, pp. 272–275, ISSN 1475-5875
Sengoelge, M., Hasselberg, M. & Laflamme, L. (2011). Child home injury mortality in
Europe: a 16-country analysis. Eur J Public Health, Vol. 21, No. 2, pp. 166–170, ISSN
1464-360X
Smith, S.L. & Andrus, P.K. (1998). Infant rat model of the shaken baby syndrome: pre-
liminary characterisation and evidence for the role of free radicals in cortical
hemorrhaging and progressive neuronal degeneration. J Neurotrauma, Vol. 15, No.
9, pp. 693–705, ISSN 0897-7151
Statistics Estonia. (2011). Statistical database. Available from http://www.stat.ee/ (16. March
2011, date last accessed).
Talvik, I., Vibo, R., Metsvaht, T., Männamaa, M., Jüri, P., Heidmets, L.-T., Hämarik, M. &
Talvik, T. (2002). Raputatud lapse sündroom. Eesti Arst, Vol. 81, No. 1, pp. 23–29,
ISSN 0235–8026
Törõ, K., Szilvia, F., György, D., Pauliukevicius, A., Caplinskiene, M., Raudys, R., Lepik, D.,
Tuusov, J. & Väli, M. (2011). Fatal Traffic Injuries Among Children and Adolescents
in Three Cities (Capital Budapest, Vilnius, and Tallinn). J Forensic Sci, [Epub ahead
of print], ISSN 1556-4029
Väli, M., Lang, K., Soonets, R., Talumäe, M. & Grjibovski, A.M. (2007). Childhood deaths
from external causes in Estonia, 2001–2005. BMC Public Health, Vol. 7, pp. 158, ISSN
1478-7954
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9
Sexual Assault in Childhood and Adolescence

Hakan Kar
Mersin University, Faculty of Medicine, Department of Forensic Medicine
Turkey
1. Introduction
1.1 Definition of sexual assault
Although definition of violence may vary in different societies and cultures, it can be
defined as all behavior that affects bio-psycho-social status of individuals.
In the United Kingdom the Sexual Offences Act 2003 defines "sexual assault" as when a
person (A)
1. intentionally touches another person (B),
2. the touching is sexual,
3. B does not consent to the touching, and
4. A does not reasonably believe that B consents (Official text of the Sexual Offences Act,
2003).
In the United States the definition of sexual assault varies widely between the individual
states. The Rape, Abuse & Incest National Network defines "sexual assault" as unwanted
sexual contact that stops short of rape or attempted rape. This includes sexual touching and
fondling. (Rape, Abuse, and Incest National Network [RAINN], 2005) According to the U.S.
Department of Health & Human Services, "sexual assault can be verbal, visual, or anything
that forces a person to join in unwanted sexual contact or attention." Sexual assault is
therefore somewhat of an umbrella term, and can describe many things, including:
 rape, including partner and marital rape
 unwanted sexual contact (touching or grabbing)
 unwelcome exposure of another's body, exhibitionism, or voyeurism
 child sexual abuse
 incest or molestation
 sexual harassment
 sexual exploitation of clients by therapists, doctors, dentists, or other professionals
(U.S. Department of Health & Human Services,2011).
Sexual violence is described as sexually motivated behavior that exerted against one’s
privacy despite one’s resistance. Furthermore, all sexually motivated behavior directed to
low aged or mentally retarded individuals included in scope of the term of sexual violence
(Christian et al., 2000; Chu&Tung, 2005; Herbert et al., 1992).
1.2 Incidance and prevelance sexual assault

Sexual assault is significantly underreported worldwide. Most of the rape victims do not
disclose the assault because of being accused or exposed to repeated assaults.
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Underreporting of sexual assault "might arise from the fear of being re-victimized in the
criminal justice system, of not being believed, from self-blame and from failure by rape
victims to equate their experience with the legal definition of rape."
Women may fear that they would be blamed for the assault, or believe that reporting would
place them or their families in danger of retaliatory violence. A recent publication by the
Open Society Institute's Network Women's Program states: “Rape goes largely unreported
across the region. The act of rape is surrounded by pejorative stereotypes: women ask for it,
they provoke it by their dress or behavior, or they cry rape to take revenge on a man; normal
men do not commit rape, and so on. In addition, reporting procedures, at the police station
and again in the courts, are complicated and degrading. In most cases, if a woman reports
being raped, she is regarded with suspicion and rarely believed; she lacks any form of police
or court protection, leaving her vulnerable to retaliation—either from the offender or, in
some cases, from members of her family who feel she has brought them dishonor.”
Approximately 700.000 women in the reproductive age group are victims of sexual assault
in the United States and, 25,000 women are raped per year in France. Unfortunately only
16% of rapes are reported to police, however 50% of victims of rape have expressed that,
they would report the rape after a warranty of secrecy about their identity
(Bechtel&Podrazik, 1999; Santiago et al., 1985). Most of the rape victims do not disclose the
assault because of being accused or exposed to repeated assaults (Ledoux&Hazelwood,
1995, Crowley, 1999).
Despite this underreporting, available statistics indicate that sexual assault is a pervasive
problem in all societies. Charlotte Bunch, in an article included in UNICEF's 1997
publication, The Progress of Nations, has stated that "statistics on rape from industrialized
and developing countries show strikingly similar patters: Between one in five and one in
seven women will be victims of rape in their lifetime."
Special Rapporteur on Violence Against Women, Radika Coomaraswamy, in her 1997 report
on violence against women, detailed the following statistics:
 A Canadian study reports that 23.3% of women had been victims of rape or attempted
rape.
 22% of adult women in Seoul had been the victims of rape or attempted rape.
 In Jakarta, Indonesia, city police recorded 2,300 cases of sexual violence against women
in 1992, 3,200 cases in 1993, and 3,000 in the first half of 1994.
 Out of 331,815 reported crimes against women in 1993 in the Russian Federation, 14,000
were rapes.
 A survey in the United Kingdom found that 19.4% of women had been victims of
sexual violence.
 Adolescents constitute 20-50% of all rape victims in the United States.
 In a study conducted at a university in the United States, one of six female students
reported having been the victim of rape or attempted rape in the past year. One out of
fifteen men reported having committed rape or attempted to commit rape
(Coomaraswamy, R. 1997).
According to estimates from the World Health Organization:
 In some countries, almost one in four women may experience sexual violence by an
intimate partner, and that almost one-third of young girls report that their first sexual
encounter was forced.
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Sexual Assault in Childhood and Adolescence 191
 The percentage of women who reporting having been sexual assaulted in the past five
years in Tirana, Albania in 1996 was 6%.
years in Budapest, Hungary in 1996 was 2%.
years in Ðiauliai, Kaunas, Klaipeda, Panevežys, and Vilnius in Lithuania in 1997 was
4.8%.
years in Ulaanbaatar and Zuunmod, Mongolia in 1996 was 3.1%.
 In a survey of women in the Czech Republic, 11.6% of women reported experiencing
forced sexual contact in their lifetime, and 3.4% reported that they had experienced this
on more than one occasion. (World Health Organization [WHO], 2002)
2. Child Sexual Assault

2.1 Definition and classification
Child sexual abuse is a form of child abuse in which an adult or older adolescent uses a
child for sexual stimulation. It is generally defined as contacts between a child and an adult
or other person significantly older or in a position of power or control over the child, where
the child is being used for sexual stimulation of the adult or other person. The World Health
Organization has defined child sexual abuse and exploitation as the involvement of a child
in sexual activity that he or she does not fully comprehend, is unable to give informed
consent to, or for which the child is not developmentally prepared and cannot give consent,
or that violates the laws or social taboos of society.
The United Nations Convention on the Rights of the Child (CRC) is an international treaty
that legally obliges states to protect children's rights. Articles 34 and 35 of the CRC require
states to protect children from all forms of sexual exploitation and sexual abuse. This
includes outlawing the coercion of a child to perform sexual activity, the prostitution of
children, and the exploitation of children in creating pornography. States are also required
to prevent the abduction, sale, or trafficking of children (United Nations, 1989).
Forms of child sexual abuse include asking or pressuring a child to engage in sexual
activities (regardless of the outcome), indecent exposure of the genitals to a child, displaying
pornography to a child, actual sexual contact against a child, physical contact with the
child's genitals (except in certain non-sexual contexts such as a medical exam), viewing of
the child's genitalia without physical contact (except in nonsexual contexts such as a medical
exam), or using a child to produce child pornography.
Child sexual abuse can be classified as:
 Sexual assault – a term defining offenses in which an adult touches a minor for the
purpose of sexual gratification; for example, rape (including sodomy), and sexual
penetration with an object. Most U.S. states include, in their definitions of sexual
assault, any penetrative contact of a minor’s body, however slight, if the contact is
performed for the purpose of sexual gratification.
 Sexual exploitation – a term defining offenses in which an adult victimizes a minor for
advancement, sexual gratification, or profit; for example, prostituting a child, and
creating or trafficking in child pornography.
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 Sexual grooming - defines the social conduct of a potential child sex offender who
seeks to make a minor more accepting of their advances, for example in an online chat
room. (APA Board of Professional Affairs , 1999; Child Welfare Information Gateway,
2009; Finkelhor & Ormrod, 2001; WHO, 1999)
2.2 Incidance and prevelance

Sexual assault is a sociological problem affecting individuals in all age groups. According to
the Convention on the Rights of the Child, a child means every human being below the age
of eighteen years unless under the law applicable to the child, majority is attained earlier
(Office of the United Nations High Commissioner for Human Rights,1989).
Different studies reports different percentages of child and adolescent sexual assault. For
example it’s reported that % 43 of 766 cases of sexual assault were under 18 age in a study of
Michigan State University (Jones et al., 2003). It’s mentioned that 178 of 405 victims were
adolescent in another research. Teenagers 16-19 are reported to be victims of rape or sexual
assault more than twice as likely as any other age group in USA. Children and especially
adolescent females are sexually assaulted more frequent comparatively to adults according
to many studies (Navratil, 2003; Jones et al., 2003; Peipert&Domagalski, 1994). In author
series most of the sexual assault victims (%87.65) were less than 18 years of age.
Estimates of child sexual abuse rates vary for many reasons. Less than 10 percent of set
abuse is reported to the policed (Finkelhor et al,1988). Even in self-reporting surveys, abuse
may be underreported because many people are afraid or ashamed to reveal victimization,
have repressed memories of abuse, refuse to participate in studies or deny that what
happened was "real" abuse.
Definitions of both abuse and the age of maturity affect frequency rates. Some researchers
have estimated that over 50% of the female child population will experience some form of
sexual abuse before the age of 18 (Russell, 1984; Wyatt, 1985), while others have reported
rates of 11% and lower (Fritz et al., 1981; Kercher & McShane, 1984). Similarly, while a meta-
analytic study by Rind and Tromovitch, (1998) reported mean prevalence rates of 17% and
28% for males and females respectively, the range for males was 3% to 37%, and for females
8% to 71%. The results of the systematic review of 11 studies on the prevalence of child
sexual assault in Switzerland shows that the percentage of participants that ever
experienced any form of child sexual assault, prevalence rates assessed by a single general
item are considerably lower (up to 18.1% for girls and 3.0% for boys) than when rates were
calculated on the basis of several items assessing specific forms of child sexual assault (up to
39.8% for girls and 10.9% for boys) (Schönbucher et al., 2011). Such wide variation in the
prevalence rate is due to differences in the definition of child sexual abuse, the type of
sample used, design, and measurement techniques.
Sarafino (1979) estimated the national incidence of reported and unreported child sexual
abuse to be over 336,000 cases per year. Sarafino arrived at this figure by calculating the
rates of reported sexual offences per 100,000 children in each of the four locales, and then
applying this rate to the national total of 61 million children. This led to an estimated 74,725
cases of child sexual abuse in a one-year period. The rate of unreported cases was calculated
by multiplying 74,725 by 3.5 (assuming that the number of unreported cases is at least 3 or 4
times higher than the reported cases as believed by several experts in the field). The number
of reported cases was added to the estimated number of unreported cases. Consequently, it
was estimated that approximately 336,200 sexual offences are committed against children
every year in the United States.
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In this respect, the prevalence of sexual assaults, especially among children and adolescent,
is thought to be extremely higher than in literature.
2.3 Characteristic features of child sexual assault

Few people are aware of the true state of the science on child abuse. Instead, most people's
beliefs have been shaped by common misconceptions and popular myths about this hidden
crime. Societal acceptance of these myths assists sex offenders by silencing victims and
encouraging public denial about the true nature of sexual assaults against children.
Common Myths about Child Sexual Abuse:
 Child sexual abuse is a rare experience.
 Children make up stories or lie about sexual abuse.
 A child is most likely to be sexually abused by a stranger
 Child sexual abuse is always perpetrated by adults.
 Normal-appearing, well educated, middle-class people don't molest children
 Children who are being abused would immediately tell their parents.
 Boys can't be sexually abused.
 Sexual abuse of a child is usually an isolated, one-time incident.
 Child molesters are all, 'Dirty old men.
Boys abused by males are or will become homosexual
 Boys are less traumatized as victims of sexual abuse than girls
 Children will naturally outgrow the effects of sexual abuse or incest
 People are too quick to believe an abuser is guilty, even if there is no supporting
evidence.
 Children who are being abused will show physical evidence of abuse.
 Acts like fondling, kissing, or touching, for example, are not really sexually abusive,
and don't really harm the young person
 Children and youth are sexually abused because their parents/caregivers neglected to
care for, or supervise them properly.
 Preschoolers do not need to know about child sexual abuse and would be frightened if
educated about it.
However children of all ages, races, ethnicities, and economic backgrounds are vulnerable to
sexual abuse. Child sexual abuse affects both girls and boys in all kinds of neighborhoods
and communities, and in countries around the world.
Even the mean age of child sexual assault varies in different studies, most authors agree
upon the reality of “children are sexually assaulted in every ages of childhood and
adolescence”. Many researches represent data about the age of child sexual assault between
infancy and 18 years of age (De Jong et al., 1982; Mian et al., 1986; Riggs et al., 2000). Even
sexual assault of girls especially adolescents are more frequent; boys are also at significant
risk of sexual abuse, often at younger ages than girls. (De Jong et al., 1982)
Female dominancy of the victims is described in many descriptive researches which focused
on child and adolescence sexual assault (Peipert&Domagalski, 1994; Jones et al.; 2003,
Navratil, 2003). It was found 254 female (86%) and 40 male (14%) children in South Africa,
85.5% of the victims were female and 14.5% were male in Canada, 113 girls and 17 boys in
Minessota, USA and 77% of girls and 23% of boys in England (Bentovim, 1987; Dubé&
Hébert, 1988; Cox et al., 2007; Tilelli et al., 1980).
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Most often, sexual assault victims are assaulted by an acquaintance not stranger. A number
of studies revealed the percentages of acquaintance assailants as changing from 56% to 78%
(Christian et al., 2000; Csorba et al., 2005; Dube&Hebert, 1988; Grossin et al., 2003;
Lauritsen&Meldgaard, 2000; Muram et al., 1995; Peipert,&Domagalski, 1994; Sahu et al.,
2005). Most children are abused by someone they know and trust, although boys are more
likely than girls to be abused outside of the family (American Medical Association, 1992;
Courtois, 1988 A study in three states found 96 percent of reported rape survivors under
age 12 knew the attacker. Four percent of the offenders were strangers, 20 percent were
fathers 16 percent were relatives and 50 percent were acquaintances or friends. Among
women 18 or older, 12 percent were raped by a family member, 33 percent by a stranger and
55 percent by an acquaintance. (Langan&Harlow, 1994) In another study it was found that
fifty-nine percent (398) of the children were sexually abused by an acquaintance, 21% (145)
of the children were sexually abused by a relative, and 5% (33) of the children were sexually
abused by a stranger. (Murphy et al., 2010) Abuse typically occurs within a long-term, on-
going relationship between the offender and victim, escalates over time and lasts an average
of four years. Offenders often develop a relationship with a targeted victim for months
before beginning the abused (Courtois, 1988) In author’s series 73 cases were acquaintance
sexual assault, stranger assault were only in 4 cases. (Table 1)
Husband
Types of Non
religiously
acquaintance Relative Friend or Step Total
Relative Neighbor Adjunct Fiancé but not Stranger
ship / Age , acquain- beloved father (%)
registry
sex tance
married
M 1
0-6 2
F 1 (2.6)
M 2 1 1
7-12 11
F 3 1 2 1 (14.28)
M 1 1
13-15 22
F 3 5 2 8 1 1 (28.58)
E
16-18 42
K 1 7 8 1 2 5 18 (54.54)
Total
(%) 5 14 16 1 2 7 26 4 2 77
(100)
Table 1. Relationship between victims and perpetrators with respect to sex and age groups
This finding might be connected with characteristics of these age groups such as physically
weakness, low comprehension about the abusive acts.
The place of sexual assault are indoor especially victims own home and outdoor sexual
assault is rare in most studies. The location of assault are reported as Own home inside 25%,
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other home inside 19%, own home outside 9%, other home outside 5%, other 11%, public
place 6%, school 4%, unknown 21% in a study performed in South Africa. (Cox et al. 2007)
The incidents most often took place in the victim’s or assailant’s home (76.7%). A
total of 11.9% of the incidents occurred in another closed place, while 8.8% occurred
in an open public place in another study (Dube&Hebert, 1988). The place of sexual assault
was perpetrators’ home in 39.74 % of the cases, followed by outdoor in only 23.08 % of the
cases in authors’ series.
Children are mostly assaulted during the day rather than night. In a study 60% of the cases
were seen during the day, 34.9% between 18:00 and midnight, and 5.1% between
midnight and 6:00 (Dube&Hebert, 1988). Another author mentioned that 49% of sexually
assaults occur in broad daylight (Firsten, 1990).
Child abuser is young rather than an old person in generally. Adolescent sexual offenders
report having approximately two paraphilias with onsetbetween ages 15 to 18 years of age
(Abel et al., 1987). It is typical that they act upon these deviant impulses in adolescence.
Nearly half of adult convicted rapists and child molesters committed their first offense
between 8 and 18 years of age, with model age being 16 (Groth et al., 1982). The disparity
between the age of victims’ and perpetrators’ was detected to be 1-2 years in 14.29% of the
cases, 3-5 years in 25.97%, 6-10 years in 32.47%, and 11 years and over in 27.27% cases in
authors’ series.
More offenders are male than female, though the percentage varies between studies. The
percentage of incidents of sexual abuse by female perpetrators that come to the attention of
the legal system is usually reported to be between 1% and 4% (Denov, 2003). Other studies
shows that women commit 14% to 40% of offenses reported against boys and 6% of offenses
reported against girls (Dube et al., 2005; Finkelhor, 1994).
A number of studies have stated that, victims of child sexual assault are generally do not
disclose the assault. However, most of victims applied to legal authorities disclose the
assault because of secondary psychiatric problems and fear, and 55.6% of these had noticed
to be assaulted many years before reporting (Safran, 1998; Jones et al., 2003). There are many
factors that may have influenced the rate at which children were referred for medical care
following the sexual abuse, including delayed disclosures. However, abuse by strangers is
often treated with more seriousness by other disciplines than abuse by family members or
others known to the child. Similarly, 76.92% of cases referred to sexual assault evaluation
unit later than three days after assault, in authors’ study. The main cause of delay in 19.23%
of the cases explained the cause of delay as, they were anxious about being accused or
punished, which support the idea of “victims might conceal the assault because of the fear
of being accused, punished or injured by perpetrators”.
The American Academy of Pediatrics Committee on Child Abuse and Neglect recommends
forensic evidence collection when sexual abuse has occurred within 72 hours of the
examination (Kellogg, 2005). Adams recommends evidence collection within 24 hours for
prepubertal children (Adams, 2008; Christian et al., 2000) suggest that the best evidentiary
material obtained from children post-abuse is found in the first 24 hours.
3. Forensic investigation of child sexual abuse victims

Anyone evaluating children for suspected sexual abuse must have an education and
working knowledge of forensic interviewing, child bio-psycho-social development,
prepubertal and postpubertal anatomy, and the ability to identify and interpret physical
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findings, including those which are normal, indicative of trauma, or unclear or uncertain
(base on our understanding of these issues to date)
3.1 Forensic interviewing

First step of child sexual abuse evaluation is the interviewing the victim. Obtaining an
unbiased history from a child who may have been sexually abused may be the most
important part of the evaluation, particularly since diagnostic physical findings are
frequently absent. Interviewing the child, steps must be taken by the examiner to prepare
the child for the interview and examination, such as and to explain why.
The conversation should begin with topics that are interesting and not “threatening” for the
child. The examiner spend enough time getting acquainted with the child before
examination and should be patient and friendly in order to establish the desired level of
relationship. Children are frightened by a hurried or demanding examiner, but they
generally respond sufficiently to and cooperate with a pleasant one. It is not necessary for
the examiner to wear a lab coat or other hospital and medical suit; such apparel may be
frightening for younger children.
Then the history regarding concerns about sexual abuse should be obtained from the parent
or caregiver separately from the child. Many parents are understandably worried and
appreciate an opportunity to share their concerns privately. The history should be
comprehensive and include the child's current and past medical problems, as well as social
and family histories. Parents should be asked how the abuse came to light or, if the child has
not disclosed abuse, why the parents suspect it.
The interview and examination room must be designed child-friendly. It would be helpful
to perform this interview with multidisciplinary approach at once in sexual evaluation units
for children to avoid retraumatization of the victim. The interview should be designed in
multidisciplinary assessment involving skilled forensic interviewing of the child and a
medical examination done by a medical provider with specialized training in sexual abuse.
In order to minimize child interviews, these assessments are frequently held in settings such
as child advocacy centers, where forensic interviewers and medical clinicians, child
protective service workers, and police and district attorneys can work jointly to address the
legal and protective issues in a coordinated fashion. Therefore Children’s Advocacy Centers
was found and have spread rapidly in USA. One of the primary goals of Children’s
Advocacy Centers (CACs) is to improve child forensic interviewing following allegations of
child sexual abuse. They aim to coordinate law enforcement, child protective, medical, and
other agencies, and typically use a single interviewer to provide information to every
investigator involved in the case. Traditional methods for interviewing children have often
been criticized as ineffective in assessing the truth and unnecessarily stressful for children.
Three specific criticisms of these methods are that (1) investigation activities and decision-
making are not coordinated across the multiple agencies involved, (2) children are
interviewed too many times by too many interviewers and have to “tell their story over and
over again,” and (3) children are interviewed in stressful or compromising locations that
disturb them further and make it difficult to talk. Sexual evaluation units or centers for
children like CACs must aim to coordinate multiple investigations, to limit the number of
interviews and interviewers children have, and to provide “child friendly” locations for
interviews (Cross, 2007)
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Interview should be structured and protocol-guided rather than standard interview

practices. There are many useful structured interview protocols frequently used just like
National Institute of Child Health and Human Development's Structured Interview Protocol
or Investigative Interviewing Practice Guideline of American Professional Society on the
Abuse of Children etc. Researchers examining the National Institute of Child Health and
Human Development (NICHD) interview protocol, inparticular, have found that the
recommended open-ended, free narrative questioning techniques are effective in eliciting
information about abuse in forensic settings, at least with children who are forthcoming in
disclosing the abuse. Lamb et al. (2003) also showed that open-ended invitations are just as
effective with the younger as with the older children, although younger children report over
all less information than older children (Lamb, 2003).
Children should be asked if they know why they have been brought to the doctor and to
relate what happened to them. Open-ended questions such as “Has anyone ever touched
you in a way that you didn’t like or in a way that made you feel uncomfortable?” should be
asked. The child’s statement should be recorded in its own words. Whenever possible, the
nature of the sexual contact, including pain, penetration and ejaculation, should be
ascertained. Careful documentation of questions and responses is critical.
When an incident is disclosed, the following information must be obtained with a gentle and
non-threatening manner, using language that the child can understand
 Who was the person who did this?
 With what part of his/her body?
 What part(s) of the patient's body was (were) touched?
 How many times was the child touched?
 When was the last time that it happened?
 At what location did the abuse occur?
 Was there any exposure to blood or body fluids?
 Did the child experience pain to the affected body part?
 For male assailants, was there ejaculation?
 Did the child tell anyone about the incident? (Adams, 2004)
The history must be recorded using the exact words used by the child to describe the event,
particularly when such language is unique, for example, “He put his finger in my coochie.”
Words to describe the genitalia can be singular to the vocabulary of a child, giving credence
to his or her testimony in a court of law (American Academy of Pediatrics. Committee on
Child Abuse and Neglect., 1999; Giardiano, 2005; Sternberg, 2001).
3.2 Physical examination

3.2.1 Informed consent
Prior to physical examination, written, witnessed informed consent to examination,
collection of specimens, release of information to authorities, and taking photographs
should be obtained by parents, relatives or acquaintances. The law officer is not present at
the examination.
Before examination some steps must be taken to prepare the child for the examination, such
as explaining its comprehensive nature “The doctor will examine your entire body,
including your private parts”, to empower the child “Nothing will be done to hurt you, but
if it does hurt or you feel uncomfortable, say stop and we will find another way”, and to
explain its purpose “I need to check you to make sure your body is okay”.
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3.2.2 Physical examination of the body

The first step is the complete physical examination of the body with careful recording of any
trauma away from the genital area. There is a spectrum of injuries from incipient bruises,
fresh abrasions and lacerations, up to evidence of prolonged physical abuse of the child with
healing injuries of various types and ages and old scars. In some assaults, restraining force is
severe enough to leave “fingertip” and other bruises on the limbs or strangling marks on the
neck (Fig 1). Bite marks are common in sexual assaults and it is important to measure and
photograph them carefully to allow matching or exclusion of the teeth of the alleged
assailant (Fig 2). If there is a bite mark on the patient or if the patient gives a history of the
perpetrator’s licking a portion of her body (e.g., the nipples), these areas should be swabbed
in an attempt to recover saliva. These swabs can then be analyzed for DNA. Positive DNA
identification has been made in a number of cases from saliva on the body of the victim.
After the swabbing of the bite mark, photographs should be taken. A metric ruler should be
included in the photographs. Ideally, one should have a forensic odontologist on call so that
they can examine and document the bite mark. They might take casts of the bite mark in
addition to photographing it. The medical examiner should carefully search for fibers, hair,
glass, paint, or any foreign material that might have been transferred to the victim’s body
from the assailant (Laraque, 2006)
All clothing also should be examined for stains, tears, missing buttons, dirt, gravel, grease,
leaves, etc. The examiner should examine the hands to see if the fingernails are broken. Is
the pubic hair matted? Are there any foreign hairs mixed with the patient’s pubic hair? After
examining the hands for trace evidence, the fingernails are clipped and placed in marked
containers. The fingernail clippings and foreign materials or pubic hairs can subsequently be
examined by the Crime Laboratory for foreign material that might have come from the
assailant.
Oral cavity should be examined and should include evaluation for evidence of forced oral
penetration such as bruising or petechiae of the hard or soft palate and/or tears of the
frenulum.
Fig. 1. Fingertips on the inner side of the thigh
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Fig. 2. Bite marks
3.2.3 Genital examination

Knowledge of normal ano-genital anatomy is crucial for all forensic examiners. This
knowledge will provide a framework for understanding that a normal ano-genital
examination does not negate the possibility of sexual abuse and most importantly will allow
the examiner to recognize deviations from normal that may be concerning for sexual abuse.
One of the most challenging aspects of the female genitalia examination is evaluation of the
hymen. The appearance of the hymen changes with age and in response to hormonal
influences. The prepubertal hymen is characterized as thin, translucent, and sensitive to touch.
It becomes thickened, elastic, redundant, and accommodating in puberty, as the result of a
physiologic increase in estrogen exposure (Fig 3). Common normal variations in the
appearance of the hymen include imperforate, microperforate, cribriform, and septate forms.
Fig. 3. Prepubertal hymen
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An infant or a very young girl can be examined either on the examining table or while on a
parent’s lap. During the genital and anal examination, the mother positions the child and
the assisting nurse separates the child’s thighs so that the examiner can inspect the genital
and the anal areas. The use of labial traction can greatly enhance visualization of the hymen.
The labia majora are gently retracted between the thumb and forefinger with force applied
downward and outward. Locations of abnormalities should be described as on a clock face
with the urethra in the 12-O’clock position and the anus at the 6-O’clock position. In
pubertal girls, estrogen causes the hymenal tissue to become thicker and more compliant;
therefore, detection of trauma can be more challenging (Lahoti et al., 2001). Any abnormal
findings should be confirmed in the knee-chest position. The examiner should take
particular note of vulvar inflammations, eruptions, open lesions, tears, lacerations, pain, and
discharge (Fig 4). The patency of the hymenal orifice is determined, the size of the introital
opening measured, and the form and thickness of the hymen is recorded. In the prepubertal
girl, vaginal penetration usually results in tearing of the hymen in the posterior 180º. These
lacerations may be associated with bruising or abrasions both ventrally and towards to the
posterior fourchette and lateral introital tissues. The colposcopy will improve injury
detection and provide more details including small lacerations, inlamation and scars (Fig 5).
If there is a discharge, the character, consistency, and color should be noted. The presence of
any odor should also be recorded. If there is evidence of infection, dry smears for
bacteriologic studies, cultures, and wet slide preparations should be prepared. Fresh wet
smears must be examined for Trichomonas vaginalis, clue cells, and Candida albicans. A
speculum examination may be necessary for pubertal adolescent females. The vaginal walls
as well as the cervix should be visualized to detect any evidence of trauma and to obtain
samples from fluid collections.
Fig. 4. Hymenal laseration at 7-O’clock Fig. 5. Colposcopic filter view of Fig4

position (Inflammation of the laceration wall and
ecchymosis at 10-O’clock is obvious)
Clinical examination of the anus is often disappointing in the sense, first, that little is to be
found and, secondly, that the correct interpretation of abnormalities remains a matter of
serious debate. Vaginal injuries or abnormalities are more often recognized as possible signs
of abuse, while anal and perianal injuries may be dismissed as being associated with
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common bowel disorders such as constipation or diarrhea. Penetration by a larger object

may result in injury varying from a little swelling of the anal verge to gross tearing of the
sphincter, or even bowel perforation. If lubrication is used and the sphincter is relaxed,
perhaps no physical evidence will be found. Even penetration by an adult penis can occur
without significant injury. After penetration, sphincter laxity, ecchymosis, swelling, and
small mucosal tears of the anal verge may be observed as well as sphincter spasm (Fig 6, 7).
Within a few days the swelling subsides and the mucosal tears heal. Skin tags can form
because of the tears. Repeated anal penetration over a long period may cause a loose anal
sphincter and an enlarged opening (Fig 8) Perianal region should also be examined for
genital warts (Condyloma accuminata) (Fig 9). DNA typology of Human Papilloma Virus
must be identified to compare with the suspected perpetrators.
Fig. 6. Perianal ecchymosis
Fig. 7. Mucosal tears
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Fig. 8. A loose anal sphincter and an enlarged opening
Fig. 9. Anal warts (Condyloma accuminata)

In performing an anal examination in children, the examiner carefully notes the symmetry
and tone of the anus when the buttocks are separated. This can be performed with the child
supine or in the more traditional lateral position. Again, positive findings need to be
confirmed in the prone position. In addition to symmetry, the physician should note the
presence of tags, fissures, or scars. With the exception of a bleeding laceration after are port
of sodomy (Fig 10), the presence of anal tags, bumps, and scars may be nonspecific findings
that do not confirm whether a child has been sexually abused unless they can be positively
related to previously identified acute findings. Evidence of acute anal trauma may be seen if
the child is evaluated soon after the assault; however, anorectal changes are rarely definitive
indicators of abuse. Swelling of the anus with blue discoloration is suggestive of trauma and
may be present up to 48 hours after the event. It is important not to confuse this finding
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with hemorrhoids. Perianal erythema is suspicious for trauma. It may also be seen in
children with encopresis, poor hygiene, pinworms, or Group A streptococcal or
staphylococcal infection. Documented anal injury after sexual assault is distinctively
uncommon, and any injuries that do occur can heal quickly and often without visible
residua (Hobbs&Wynne, 1989).
Fig. 10. Deep lacerations of the anus

The physician should examine the penis, testicles and perineum for bite marks, abrasions,
bruising or suction ecchymosis in the anogenital examination of the male child victim.
Evaluation of the anus may be performed with the patient in the supine, lateral recumbent
or prone position with gentle retraction of the gluteal folds. The anal examination of the
male is the same as in the female.
Current practice dictates that positive findings be recorded photographically or with video,
and colposcopic or digital imaging should be of diagnostic quality. As it is clear that
utilization of a colposcope improves injury detection colposcope should be the standard of
care for examining children who may have been sexually abused.
The American Academy of Pediatrics Committee on Child Abuse and Neglect recommends
forensic evidence collection when sexual abuse has occurred within 72 hours of the
examination (Kellogg, 2005). Christian et al. (2000) suggest that the best evidentiary material
obtained from children post-abuse is found in the first 24 hours. Adams et al.’s (1994) study
showed a significantly higher incidence of abnormal genital findings in girls examined
within 72 hours of the abuse as compared to those examined a month or more from the
event.
Different studies repot various percentages of genital injuries depend on time of
examination after assault. Percentage of anogenital injuries was reported 84%, and average
interval of medical evaluation after assault is 17 hours in a study of Michigan State
University (Jones et al., 2003). Some form of forensic evidence was identified in 24.9% of
children, all of whom were examined within 44 hours and over 90% of children with
positive forensic evidence findings were seen within 24 hours of their assault, presence of
genital injury was %23 and in another child sexual assault series (Christian et al., 2000).
Nongenital trauma was found in only 5.5% and anomalies were found in the genital
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examination of 25.4% of the victims in another review of 511 cases (Dubé&Hébert, 1988).
However, the absence of these findings is common in girls who have suffered perceived
genital penetration. For example, an observational study of 506 girls age 5 to 17 years, who
disclosed penile-genital penetrative abuse, found that most girls did not have definitive
physical findings of abuse regardless of the number of reported penetrations. Specifically,
no findings were seen on expert review of photocolposcopy in all of the girls less than 10
years of age (N=74), 87 percent of girls ≥10 years of age with no history of consensual sex
(358 of 410 patients), and 82 percent of girls ≥10 years of age with a history of consensual sex
(18 of 22 patients) (Anderst, et al., 2009).
3.3 Forensic evidence sampling for laboratory examinations

Rates of recovery of forensic evidence from prepubertal children evaluated for sexual
assault vary from 6 to 42%. Early clinical examination within 24 to 72 hours to assault is the
key point in determining traumatic changes and forensic evidences. Forensic studies should
be performed especially when the examination occurs within 72 hours of acute sexual
assault or sexual abuse (Christian et al., 2000).
Forensic evaluation requires collection of numerous specimens. Providers should use
evidence collection kits with careful attention to guidelines for specimen collection.
Collected samples include
 The victim's clothing
 Swabs and smears from the buccal mucosa, vagina, and rectum and from other areas
highlighted by ultraviolet light
 Combed specimens from the scalp and pubic hair
 Fingernail scrapings and clippings
 Control samples of the victim's scalp and pubic hair (ideally, at least 20 to 25 pulled
hairs per site)
 Whole blood sample
 Saliva sample
Obviously the most important identifying element for the examiner and the pathologist is
the documented presence of an ejaculate, so that the retrieval of the spermatozoa is more
critical than ever. It should be stressed that the lack of evidence of ejaculation by no
means refutes a complaint of sexual assault. Many of the men convicted for sexual assault
may suffer from some form of sexual dysfunction that impaires their ability to ejaculate. If
the abuse has occurred within the last 72 hours the presence of sperm should be
investigated. Detection of acid phosphatase is another technique used to detect semen,
acid phosphatase can, however, normally be found in very low levels in the adult female
vagina, so quantification of the enzyme is important to verify ejaculation. The p30 protein
is a semen glycoprotein of prostatic origin. The p30-enzyme is linked with an
immunosorbent assay. This protein is semen-specific and is not found in vaginal fluids. It
is thus a more sensitive and specific method of semen detection. Acid phosphatase and
p30 protein test should be helpful when perpetrator is suffered from asospermia or
aspermia (Stefanidou et al., 2005).
Identification of genetic markers in blood, saliva and serum (ABO typing and other blood
enzyme systems) should be performed within 72 hours of acute sexual assault or sexual
abuse. DNA fingerprinting can, nowadays, establish the identity of a perpetrator with a
high degree of certainty.
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The victim should also be evaluated for pregnancy and sexually transmitted infections by a
gynecologist in multidisciplinary approach. The diagnosis of sexually transmitted diseases
is important not only to the care of the victim but also in determining the fact of sexual
contact. This evidence may be prima facie, or confirmatory. Gonorrhea or syphilis infections
are diagnostic of sexual abuse after perinatal transmission has been ruled out. Herpes type
2, Chlamydia, Trichomonas, and condyloma infections are extremely unlikely to be due to
anything but abuse, particularly in children beyond infancy. HIV, and herpes simplex virus
type 2 should also be tested (Kawsar et al., 2008).
Finally, toxicological analysis of blood and urine should be performed in case that the child
has been abused while under the influence of drugs.
3.4 Interpretation of findings

The interpretation of findings in children with suspected sexual abuse depends upon the
constellation of historical, physical, and laboratory findings. The history is often the most
important part of the evaluation. The provision by the child of a spontaneous, clear,
consistent, and detailed description of sexual molestation is specific for sexual abuse and
should be reported to legal authorities and/or child protective services.
Adams et. al. published a series of criteria about interpreting physical and laboratory
findings in suspected child sexual abuse in 2007. (Table2).
A Product of an ongoing collaborative process by child maltreatment physician specialists,

under the leadership of Joyce A. Adams, MD
Findings documented in newborns or commonly seen in non-abused children:
(The presence of these findings generally neither confirms nor discounts a child's clear
disclosure of sexual abuse)
Normal variants
1. Periurethral or vestibular bands
2. Intravaginal ridges or columns
3. Hymenal bumps or mounds
4. Hymenal tags or septal remnants
5. Linea vestibularis (midline avascular area)
6. Hymenal notch/cleft in the anterior (superior) half of the hymenal rim (prepubertal girls),
on or above the 3 o'clock – 9 o'clock line, patient supine
7. Shallow/superficial notch or cleft in inferior rim of hymen (below 3 o'clock – 9 o'clock
line)
8. External hymenal ridge
9. Congenital variants in appearance of hymen, including: crescentic, annular, redundant,
septate,cribiform, microperforate, imperforate
10. Diastasis ani (smooth area)
11. Perianal skin tag
12. Hyperpigmentation of the skin of labia minora or perianal tissues in children of color,
such as Mexican-American and African-American children
13. Dilation of the urethral opening with application of labial traction
14. “Thickened” hymen (May be due to estrogen effect, folded edge of hymen, swelling
from infection, or swelling from trauma. The latter is difficult to assess unless follow-up
examination is done)
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Findings commonly caused by other medical conditions:

15. Erythema (redness) of the vestibule, penis, scrotum or perianal tissues. (May be due to
irritants, infection or trauma *
16. Increased vascularity (“Dilatation of existing blood vessels”) of vestibule and hymen.
(May be due to local irritants, or normal pattern in the non estrogenized state)
17. Labial adhesions. (May be due to irritation or rubbing)
18. Vaginal discharge. (Many infectious and non-infectious causes, cultures must be taken to
confirm if it is caused by sexually transmitted organisms or other infections.)
19. Friability of the posterior fourchette or commissure (May be due to irritation, infection,
or may be caused by examiner's traction on the labia majora)
20. Excoriations/bleeding/vascular lesions. These findings can be due to conditions such as
lichen sclerosus, eczema or seborrhea, vaginal/perianal Group A Streptococcus, urethral
prolapse, hemangiomas)
21. Perineal groove (failure of midline fusion), partial or complete
22. Anal fissures (Usually due to constipation, perianal irritation)
23. Venous congestion, or venous pooling in the perianal area. (Usually due to positioning
of child, also seen with constipation)
24. Flattened anal folds (May be due to relaxation of the external sphincter or to swelling of
the perianal tissues due to infection or trauma*
25. Partial or complete anal dilatation to less than 2 cm (anterior-posterior dimension), with
or without stool visible. (May be a normal reflex, or may have other causes, such as
severe constipation or encopresis, sedation, anesthesia, neuromuscular conditions,)
INDETERMINATE Findings: Insufficient or conflicting data from research studies: (May

require additional studies/evaluation to determine significance. These physical/laboratory
findings may support a child's clear disclosure of sexual abuse, if one is given, but should be
interpreted with caution if the child gives no disclosure. In some cases, a report to child
protective services may be indicated to further evaluate possible sexual abuse.)
Physical Examination Findings
26. Deep notches or clefts in the posterior/inferior rim of hymen in pre-pubertal girls,
located between 4 and 8 o'clock, in contrast to transections (see 41)
27. Deep notches or complete clefts in the hymen at 3 or 9 o'clock in adolescent girls.
28. Smooth, non-interrupted rim of hymen between 4 and 8 o'clock, which appears to be less
than 1 millimeter wide, when examined in the prone knee-chest position, or using water to
“float” the edge of the hymen when the child is in the supine position.
29. Wart-like lesions in the genital or anal area.
(Biopsy and viral typing may be indicated in some cases if appearance is not typical of
Condyloma accuminata)
30. Vesicular lesions or ulcers in the genital or anal area (viral and/or bacterial cultures, or
nucleic acid amplification tests may be needed for diagnosis)
31. Marked, immediate anal dilation to an anterior-posterior diameter of 2 cm or more, in
the absence of other predisposing factors
Lesions with etiology confirmed: Indeterminate specificity for sexual transmission (Report
to protective services recommended by AAP Guidelines2 unless perinatal or horizontal
transmission is considered likely)
31. Genital or anal Condyloma accuminata in child, in the absence of other indicators of
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abuse.
32. Herpes Type 1 or 2 in the genital or anal area in a child with no other indicators of sexual
abuse.
Findings Diagnostic of Trauma and/or Sexual contact (The following findings support a
disclosure of sexual abuse, if one is given, and are highly suggestive of abuse even in the
absence of a disclosure, unless the child and/or caretaker provide a clear, timely, plausible
description of accidental injury. (It is recommended that diagnostic quality photo-
documentation of the examination findings be obtained and reviewed by an experienced
medical provider, before concluding that they represent acute or healed trauma. Follow-up
examinations are also recommended.)
Acute trauma to external genital/anal tissues
33. Acute lacerations or extensive bruising of labia, penis, scrotum, perianal tissues, or
perineum (May be from unwitnessed accidental trauma, or from physical or sexual abuse)
34. Fresh laceration of the posterior fourchette, not involving the hymen.
(Must be differentiated from dehisced labial adhesion or failure of midline fusion.
May also be caused by accidental injury
or consensual sexual intercourse in adolescents
Residual (healing) injuries
(These findings are difficult to assess unless an acute injury was previously documented at
the same location)
36. Perianal scar (Rare, may be due to other medical conditions such as Crohn's Disease,
accidental injuries, or previous medical procedures)
37. Scar of posterior fourchette or fossa. (Pale areas in the midline may also be due to linea
vestibularis or labial adhesions)
Injuries indicative of blunt force penetrating trauma (or from abdominal/pelvic
compression injury if such history is given)
38. Laceration (tear, partial or complete) of the hymen, acute.
39. Ecchymosis (bruising) on the hymen (in the absence of a known infectious process or
coagulopathy).
40. Perianal lacerations extending deep to the external anal sphincter (not to be confused
with partial failure of midline fusion)
41. Hymenal transection (healed). An area between 4 and 8 o'clock on the rim of
the hymen where it appears to have been torn through, to or nearly to the base, so there
appears to be virtually no hymenal tissue remaining at that location.
This must be confirmed using additional examination techniques such as a swab,
prone knee-chest position or Foley catheter balloon (in adolescents), or prone-
knee chest position or water to float the edge of the hymen (in prepubertal girls). This
finding has also been referred to as a “complete cleft” in sexually active adolescents and
young adult women.
42. Missing segment of hymenal tissue. Area in the posterior (inferior) half of the hymen,
wider than a transection, with an absence of hymenal tissue extending to the base of
the hymen, which is confirmed using additional positions/methods as described above.
Presence of infection confirms mucosal contact with infected and infective bodily secretions,
contact most likely to have been sexual in nature:
43. Positive confirmed culture for gonorrhea, from genital area, anus, throat, in a child
outside the neonatal period.
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44. Confirmed diagnosis of syphilis, if perinatal transmission is ruled out.

45. Trichomonas vaginalis infection in a child older than 1 year of age, with organisms
identified by culture or in vaginal secretions by wet mounts examination.
by an experienced technician or clinician)
46. Positive culture from genital or anal tissues for Chlamydia, if child is older than 3 years
at time of diagnosis, and specimen was tested using cell culture or comparable method
approved by the Centers for Disease Control.
47. Positive serology for HIV, if perinatal transmission, transmission from blood products,
and needle contamination has been ruled out.
Diagnostic of sexual contact
48. Pregnancy
49. Sperm identified in specimens taken directly from a child's body.
Table 2. Approach to Interpreting Physical and Laboratory Findings in Suspected Child
Sexual Abuse. (Adams et. al., 2007)
4. Management of sexual abuse in children and adolescents

The management of sexual abuse involves prevention of sexually transmitted infections
(STI) and pregnancy. Psychosocial support and anticipatory guidance should be offered to
the victims and their nonoffending caregivers.
4.1 Sexually transmitted infections prophylaxis

A prepubertal child will acquire a sexually transmitted infection (STI) as the result of sexual
abuse is low and varies according to the local prevalence of STIs. Thus asymptomatic
children do not generally require STI prophylaxis. However, cultures must be obtained
prior to treatment whenever prophylaxis is prescribed (Siegel et. al, 1995). For adolescents,
STI prophylaxis is recommended for those who present within 72 hours of the event
(because of the high prevalence of preexisting asymptomatic infection, the risk of pelvic
inflammatory disease, and the possibility of loss to follow-up). For pubertal females who
present after 72 hours, STI prophylaxis should be prescribed if the assailant is known to be
infected, the victim has signs or symptoms of infection, or at the victim's request. (American
Academy of Pediatrics, 2006).
4.2 Pregnancy prophylaxis

The highest risk of pregnancy occurs during the three days preceding and including
ovulation. Knowing the timing of the event in relation to the patient's ovulatory period is
helpful in further assessing risk. Emergency contraception (postcoital contraception) should
be offered to all pubertal female patients and should be strongly advised to females at
highest risk for pregnancy.
4.3 Psychological support

The child's physical and emotional well-being are of primary concern. The child should be
reassured that what happened was not the child's fault and that he or she did nothing
wrong. Children in whom sexual abuse is confirmed or suspected should be referred to a
mental health professional for evaluation and counseling. The family of the victim may also
need treatment and support to cope with the emotional trauma of their child's abuse
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Short-term sequelae of sexual abuse include fear, disturbances in sleep and eating, phobias,
guilt, shame, anger, depression, school problems, delinquency, aggression, hostility,
antisocial behavior, inappropriate sexual behavior, and running away. Longer-term effects
include depression, sleep problems, eating disorders, obesity, feelings of isolation,
stigmatization, poor self-esteem, problems with interpersonal relationships, negative effect on
sexual function, revictimization, substance abuse, and suicidal behavior. Psychosocial follow-
up is the key point to avoid these short-term or longer-term sequelaes (Hymel& Jenny 1996)
4.4 Social support

Social support is one of the most important steps in the management of abused children.
The case should be reported to the social services or child protective services after forensic
investigation completed. Social support should be maintained to prevent further harm to
children from sexual or other types of abuse or neglect. When the case is reported to social
services, they must look into it if they think there is a real risk to the safety or well-being of
the child. Social services will decide if the child needs protection and what needs to be done
to protect them.
They must decide, or contribute to decision-making in some key areas. Is a child safe?
Should a child remain at home, or be removed? What type and level of services does this
child/family need? Can these services be offered while the child is living with the alleged
abuser? Of the myriad problems presented by this family, which one(s) should be
addressed, and which ones should be addressed first? How therapeutically accessible are
the members of this family? At what stage of change are they? What is the level of future
risk to the child (as opposed to immediate safety)?
 Social services accomplish all these services through:
 Assessing suspected cases of abuse and neglect
 Assisting the family in diagnosing the problem
 Providing in-home counseling and supportive services to help children stay at home
with their families
 Coordinating community and agency services for the family
 Petitioning the court for removal of the child, if necessary
 Providing public information about child abuse, neglect, and dependency.
5. Conclusion
 Child Sexual Abuse defined as contacts between a child and an adult or other person
significantly older or in a position of power or control over the child, where the child is
being used for sexual stimulation of the adult or other person.
 The United Nations Convention on the Rights of the Child (CRC) is an
international treaty that legally obliges states to protect children's rights. Articles 34 and
35 of the CRC require states to protect children from all forms of sexual exploitation and
sexual abuse.
 The prevalence of sexual assaults, especially among children and adolescent, is thought
to be extremely higher than in literature.
 Children and especially adolescent females are sexually assaulted more frequent
comparatively to adults.
 Children are sexually assaulted in every ages of childhood and adolescence.
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 Sexual assault victims are mostly female and assaulted by an acquaintance not stranger.
 Children are mostly assaulted indoor and during the day.
Most of the child abuser is male and young rather than an old person.
 Victims of child sexual assault generally do not disclose the assault.
 During forensic investigation, obtaining an unbiased history from a child who may
have been sexually abused may be the most important part of the evaluation,
particularly since diagnostic physical findings are frequently absent.
 In order to minimize child interviews, these assessments should be held in settings such
as child advocacy centers, where forensic interviewers and medical clinicians, child
protective service workers, and police and district attorneys can work jointly to address
the legal and protective issues in a coordinated fashion.
 Prior to physical examination, written, witnessed informed consent to examination,
collection of specimens, release of information to authorities, and taking photographs
should be obtained by parents, relatives or acquaintances.
 All clothing, the body, oral cavity and genitalia should be examined for any evidences
of sexual assault.
 Early clinical examination within 24 to 72 hours to assault is the key point in
determining traumatic changes and forensic evidences.
 Examiners should use evidence collection kits which guidelines for specimen collection
for laboratory analysis.
The interpretation of findings in children with suspected sexual abuse depends upon the
constellation of historical, physical, and laboratory findings.
 The management of sexual abuse involves prevention of sexually transmitted infections
(STI) and pregnancy. Psychosocial support and anticipatory guidance should be offered
to the victims and their nonoffending caregivers.
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10
Cannabinoids: Forensic
Toxicology and Therapeutics
Helena M. Teixeira1,2,3 and Flávio Reis4
1National Institute of Legal Medicine – North Branch and
CENCIFOR – Forensic Sciences Center, Portugal
2Medicine Faculty, University of Coimbra
3Medicine Faculty University of Porto
4Laboratory of Pharmacology and Experimental Therapeutics,
IBILI, Medicine Faculty, University of Coimbra

Portugal
1. Introduction
Marijuana, hashish and other psychoactive products obtained from Cannabis sativa are the
most produced and trafficked illicit drugs around the world (CND, 2006).
It is difficult to estimate the moment when man began to use some of the preparations from
cannabis sativa. Thus, the reported consumption of the plant and its derivatives appears as an
ancient practice, in many parts of the globe, from India to China, extending from the Middle
East (Persia, Asia Minor and Egypt) to Africa, through the Christian culture, until the West
(Ellenhorn & Barceloux, 1988; Ladrón de Guevara & Moya Pueyo, 1995; Rodríguez-Vicente
et al., 1995). The effects that these compounds have on an individual brain have been
addressed in several instances, such as religious practice, or simply in the search of
pleasurable sensations. Cannabis sativa has been used in China nearly for five thousand
years, being its cultivation related to fiber, oil and seeds production (Camp, 1936). However,
Asians also knew its narcotic action in the seventh century BC, incorporating cannabis in
their religious rituals and as a therapeutic agent, in neurological and psychiatric diseases
(Mechoulam, 1991).
Cannabis consumption came to the Iberian Peninsula across North Africa, once conquered by
the Arabs. But its presence was ephemeral, not achieving a significant presence on all the
Christian kingdoms (Nahas, 1982). Ramazzini, an eighteenth century physician, studied its
potential toxic effects, but it was O'Shaughnessy, an Irish surgeon who lived in India as a
British colonial army doctor, the first scientific researcher to carry out pharmacological
studies with the plant and the promoter of its application in therapy (Nahas, 1973;
O’Shaughnessy, 1842). Thus, in 1842, after reviewing its therapeutic use in India and after
some experimental research on animals, he introduced Cannabis in Europe (Robson, 2001).
Indeed, this doctor was impressed with the outstanding application of this drug as a muscle
relaxant, anticonvulsant, analgesic and anti-emetic. However, due to its uncontrollable
power, there was a rapid decline in its therapeutic value. Thus, in 1840, the French physician
Jacques-Joseph Moreau, considered as the father of psychopharmacology, described in his
book "Du Haschisch et de l'alienation mentale, Psychologiques études" (1845), the toxic effects of
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216 Forensic Medicine - From Old Problems to New Challenges
hallucinogens, calling attention to the danger of its use, since it could produce individual
and social deterioration, and also cause addiction. Since 1971, the use of cannabis was
controlled by the so-called "Drug Abuse Act”, which forbade the use of both medical herbs
and their active constituents, cannabinoids. Its use had already been removed from medical
practice since 1932, the year it was eliminated from the British Pharmacopoeia. Ten years
later, it was removed from the United States and 34 years later, from the Indian
Pharmacopoeia. The controversy over its hallucinogenic actions on the brain has eclipsed its
possible medical uses (Evans, 1997).
Cannabis consumption as a drug of abuse begins to spread in some European countries in
the 60's and was popularized in the '70s and '80s. It is estimated that, presently, cannabis
world production mainly occurs in America (46%), followed by Africa (26%) and by Asia
(22%) (UNODC, 2007).
2. Botany: Cannabis sativa L

The genus Cannabis is composed of a single plant species, Cannabis sativa Linn., classified by
Linnaeus in 1753, based on specimens from India but with different shapes. The
morphological characteristics, fiber production, oils or resins, and even the size, are so
varied that botanical classification becomes very difficult (Astolfi et al., 1979; Ellenhorn &
Barceloux, 1988; Rodríguez-Vicente et al., 1995). Some botanists argue for the existence of
three species: Cannabis sativa, Cannabis indica and Cannabis ruderalis, while others admit the
existence of only two. Cannabis sativa, hemp common name (Cannabis means hemp; sativa
means sowing or cultivating), is a plant endemic to a geographic area between the Caspian
and Black Seas, passing through Persia and India, growing up in the Far East since ancient
times. Under normal conditions it can have one or two meters height reaching, in its highest
development stage, up to four meters. Cannabis sativa has been cultivated, for centuries, due
to the hemp present on the stems, seeds and oil and due to its biological active substance
(9-Tetrahydrocannabinol) in the higher parts with flowers, varying its chemical
composition according to the different parts of the plant. The plant growth favourable
conditions are a moist soil with high nitrogen content, being the clayey soils the worse
conditions to its growth (Wilsie & Reddy, 1946). This plant has been adapting to different
climates, adaptation accompanied by morphological changes, especially related to the leaf
(Eckler & Miller, 1912). In fact, plant cannabinoids chemistry is much more complicated
than the pure 9-THC and thus, multiple effects can be expected due to the additional
cannabinoids presence as well as other chemicals (Turner et al., 1980).
3. Consumption patterns
The potency of cannabis products is determined by its 9-THC content, usually given as a
percentage of 9-THC. ElSohly et al. (2000) estimated, in a study performed between 1980
and 1997 in confiscated marijuana samples, that 9-THC percentage was between 1.5 and
4.2%, being, however, sometimes higher. The highest percentages were found in marijuana
samples (29.9%), hashish (52.9%) and oil (47.0%). In 2005, the average or typical level of 9-
THC Cannabis resin at the retail level ranged between 1% and 17%, being this variation
range difficult to explain given the common origin of most European resin.
Over the past 20 years, more modern farming methods and crops increasingly sophisticated
have been developed, leading to increased potency on Cannabis products. In the so-called
"flower power" days from the 60s and 70s, every marijuana cigarette contained about 10 mg
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Cannabinoids: Forensic Toxicology and Therapeutics 217
of 9-THC. Currently, a cigarette can have about 150 mg 9-THC (9-THC between 6 and
20%, corresponding to 60-200 mg/cigarette) or 300 mg, if mixed with hashish oil. Thus,
nowadays, a cannabis consumer is frankly exposed to higher doses than in previous 60 or 70
decades (Gold, 1991; Mendelson, 1987; Schwartz, 1991). However, the 9-THC content also
varies extraordinarily, depending on the different Cannabis sources and preparations. In
fact, there are several Cannabis preparations, leading to different consumption forms and
different names (even according to different countries), as well as different power degrees
(Astolfi et al., 1979; Ellenhorn & Barceloux, 1988; Rodríguez-Vicente et al., 1995): the herb,
consisting of several parts of the plant, presents a variable active ingredients quantity
depending on the part of the plant used for its preparation. It is usually smoked alone, but it
may be mixed with tobacco. It has different names according to the country (among others,
in Portugal and in Mexico, marijuana; in Morocco and Spain, grifa or marihuana; in South
Africa, dagga; in Great-Britain, hemp; in Brazil, maconha); the resin, also called haxixe, hachis,
hashis, hash, charas or chira, is five to eight times more potent than the herb, being the product
spontaneously secreted by plants in small drops, thus corresponding to the resin of the
plant. It can also be extracted from the plant through organic solvents. It can be smoked in
special pipes or in cigarettes, being the resin, after burning, mixed with tobacco; the hash
oil, obtained by hot extraction of the plant or by hashish extraction with organic solvents
and consequent evaporation, has, as a concentrated resin, a high power.
Nº
Nº compound
Class Class compound
in the plant
in the plant
Cannabinoids 61 Simple ketones 13

Cannabigerol (CBG) 6 Simple acids 20
Cannabichromene (CBC) 4 Fatty acids 12
Cannabidiol (CBD) 7 Simple esters and lactones 13
1(9)-THC 9 Steroids 11
1(8)-THC 2 Sugars and similar 34
Cannabiciclol (CBL) 3 Monosaccharides 13
Cannabielsoin (CBE) 3 Disaccharides 2
Cannabinol (CBN) 6 Polysaccharides 5
Cannabinodiol (CBND) 2 Cyclitols 12
Cannabitriol (CBT) 6 Amino-sugars 2
Other Cannabinoids 13 Terpenes 103
Nitrogen compounds 20 Monoterpenes 58
Quaternary bases 5 Sesquiterpenes 38
Amides 1 Diterpenes 1
Amines 12 Triterpenes 2
Alkaloids spermidines 2 Mixture of terpenoid 4
Amino Acids 18 Non-cannabinoid phenols 16
Proteins, glycoproteins & enzymes 9 Flavonoid glycosides 19
Hydrocarbons 50 Vitamins 1
Simple alcohols 7 Pigments 2
Simple aldehydes 12 Total 421
Table 1. Compounds classes found in Cannabis sativa (Honório & Silva, 2006).
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4. Chemical structure and properties

Cannabis sativa present a large number of different chemicals, as illustrated in Table 1, being
cannabinoids the main class. These compounds vary in number and quantity, according to
the climate, soil type, variety cultivated and the way the crop was performed.
The observed variations also depend on the part of the plant used for their extraction, the
drugs preparation method for the consumption, as well as its storage conditions (Waller,
1971). Cannabis contains about 421 different chemical compounds, including 61
cannabinoids (Turner et al., 1980). During the consumption by smoking, more than 2000
compounds can be produced by pyrolysis. Eighteen different classes of chemicals, including
nitrogen compounds, amino acids, hydrocarbons, sugars and fatty acids can contribute to
the single known pharmacological and toxicological properties of cannabinoids (Huestis,
2002). The term "cannabinoid" was attributed to the compound’s group with 21 carbon
atoms present in Cannabis sativa, also added to their carboxylic acids, analogs and possible
transformation products (Honório & Silva, 2006). They are usually formed by three rings,
cyclohexene, benzene and tetrahidropiran. Some are the responsible for the power of the
various psychoactive plant preparations (Mendelson, 1987).
Fig. 1. Cannabinoid 3D chemical structure (a) and linear structures of two numbering
systems used for cannabinoid compounds (b) and (c) (Honório & Silva, 2006).
The chemical structure of a cannabinoid type is shown in Figure 1, indicating the main
numbering systems in the literature. The first report of proven isolation of the cannabis
active ingredient in its pure form, 9- Tetrahydrocannabinol or simply 9-THC, dates from
1964, by Gaon and Mechoulam. Due to the great interest in the effects caused by the
compounds extracted from cannabis, several studies have been conducted with the aim of
identifying possible relationships between their chemical structure and their biological
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activity. Cannabinol (CBN) was the first known cannabinoid, a phenolic compound resin
obtained by Wood and collaborators in 1896. Canh, in 1932 got its cleansing in the
crystalline acetate form, demonstrating that it is a phenolic derivative of dibenzopiran.
Later, thanks to Cahn (1933) and Adams (1940), the structure of the CBN was established.
In 1940, Adams and Baker isolated another cannabis resin principle, which designated as
Cannabidiol (CBD). Both CBN and CBD have lack active and enhancer effects. Later, in
1970, Mechoulam demonstrated that in the plant, there is a cannabinoids biosynthesis cycle
that relates them, proving that the different components isolated by different authors were
intermediate products. Although it is known that 9-THC has six isomers (as an
isomerization result), only the isomers 9-THC e 8-THC were isolated from the natural
product. Of all the natural cannabinoids, 9-THC is the most active compound, existing in
the two forms mentioned above.
5. Cannabinoids properties
In fact, 9-THC is the psychoactive cannabinoid with higher potency. Concerning the other
cannabinoids present in the plant and about which there is some information:
i. 8-THC presents a very similar pharmacological profile to that of 9-THC, although
lower, and thus, it has been studied for its possible use in drugs without psychoactive
effects. This compound is only present is some plant varieties, being its concentration
much lower when compared to 9-THC (Mechoulam et al., 1992).
ii. CBN (Canabinnol) also has some psychoactive properties, among which are those
related to the 9-THC discriminative stimuli (Järbe & Mathis, 1992). This activity is, in
animals, about one tenth of the described for 9-THC. However, the results in humans
have been quite contradictory, since some authors found that, by its intravenous
administration, the CBN produces similar effects to those described for 9-THC (Pérez-
Reyes et al., 1973), contrary to those observed by Hollister (1974), who didn’t detect any
effect when administration was performed orally.
In fact, when comparing with 9-THC, CBN has a higher affinity for the CB2 cannabinoid
receptors than for the CB1 cannabinoid receptors (Munro et al., 1993). Being CB2 a
peripheral receptor, CBN seems to participate in the immune system modulation, having
been attributed, a long ago, to cannabinoids.
iii) CBD (Cannabidiol) is a cannabinoid practically devoid of psychoactive properties, since
it is not capable to disconnect, from a CB1 receptor, neither an agonist nor an antagonist
(Thomas et al., 1998). Since it is not a psychoactive substance, a detailed research has been
developed in order to evaluate its possible clinical effects, and it has been described at least
one case where its oral administration resulted in an effective long-term treatment of a
psychosis framework (Zuardi et al., 1995).
We can, thus, say that cannabinoids properties depend on their chemical structure. Minimal
variations in the THC molecule components can cause major changes in its activity.
6. Toxicokinetics
6.1 Absorption
9-THC absorption varies depending on the administration route. Inhalation is the most
common administration route among cannabis consumers (smoke inhalation from water
pipes or cigarettes), although there are also references to its use either orally (beverages or
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food ingestion) or parenteral, providing a rapid and efficient method for drug distribution
in the body. Cannabis sativa preparations (hashish, marijuana) are mainly consumed in
cigarettes, and approximately 30% of the 9-THC present in marijuana cigarettes or hashish
are destroyed by pyrolysis during smoking (Huestis, 2002). The combustion heat leads to
THC acids transformation to 9-THC, as well as 8-THC synthesis from CBD.
Simultaneously, the existent 9-THC is largely destroyed by smoking, originating CBN. This
suggests that the maximum amount of 9-THC absorbed during smoking does not exceed
70% of the 9-THC content existent in the cigarettes. The intense pleasure effects can be
produced due to the almost immediate CNS exposure to the drug. In fact, after smoking,
there is a rapid absorption of 9-THC through the respiratory tract into the bloodstream.
However, about 18% of an inhaled 9-THC dose is absorbed (Ohlsson et al., 1980), being an
oral dose significantly less effective (Nahas, 1979). Moreover, 9-THC bioavailability after
inhalation is highly variable (Barnett et al., 1982, Lindgren et al. 1981; Ohlsson et al. 1980;
Ohlsson et al., 1982, Pérez-Reyes et al., 1982), because it is affected, not only by the specific
characteristics of the cigarette and its corresponding combustion, but also by the inhalation
intensity, administration duration, among other factors. Experienced smokers inhale more
efficiently than inexperienced people, being the 9-THC bioavailability, in a marijuana
cigarette with approximately 1-2% of the drug, between 16 and 40% for chronic users and
between 13 and 14% for occasional users (Ohlsson et al., 1982). Cannabinoids oral ingestion
leads to lower plasma 9-THC levels than by inhalation, i.e., gastrointestinal absorption
represents, approximately, one third of the achieved via inhalation. Orally, its bioavailability
is reduced due to the gastric fluid acidity, the intestinal metabolism, as well as the first-pass
enterohepatic system effect (Agurell et al., 1986). It has been observed that in acidic
conditions (at a pH above 4.0), 9-THC isomerizes, giving rise to 8-THC or 9-
hidroxihexahidrocanabinol. At a more acidic pH, the rupture of the pyran ring occurs,
leading to the formation of several replaced cannabinoids. These changes could possibly
explain the loss of 9-THC activity after oral administration due to the acidic pH of the
stomach (Garret et al., 1978). However, large intra-and inter-individual differences may also
contribute to uncertainty in the effective dose distribution (Agurell et al. 1986; Ohlsson et al.,
1982).
9-THC can be detected in plasma within seconds after inhaling the smoke of a marijuana
cigarette (aHuestis et al., 1992), with plasma peak levels reached about 7 to 8 minutes after
starting smoking, with euphoria and a maximum heart acceleration at about 20 minutes after
(Perez-Reyes et al., 1982). However, after an oral administration, absorption is slow and
irregular (Blaw et al. 1984; Ohlsson et al., 1980, Wall et al., 1983), reaching the highest 9-THC
plasma levels about 45 minutes after ingestion and remaining relatively constant for 4 to 6
hours (Wall et al., 1983). The clinical effects begin 30-60 minutes after oral consumption,
reaching a peak 2-3 hours after ingestion (Isbell et al., 1967) and it can hardly be correlated
with plasma levels. Bioavailability is reduced in about 20-40% after oral administration
(Ohlsson et al., 1980, Wall et al., 1983) due to the drug degradation within the gastrointestinal
tract (Perez-Reyes et al., 1973). We can, thus, say that a greater oral amount of 9-THC is
required to achieve the same physiological effects as by inhalation. Moreover, after oral
administration, a gradual increase of its plasma concentration is produced and it can last for
several hours, delaying the onset of their psychoactive effects (Cone and Huestis, 1993).
6.2 Distribution
Studies on 9-THC bioavailability showed considerable differences between pulmonary and
oral routes. Smoking seems to be the most effective method for drug administration. 9-THC
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entrance in the blood and its subsequent distribution to the tissues is very rapid, with very
similar kinetics to the ones obtained after an intravenous administration.
Only 3% of the 9-THC detected in the blood is in its free form. About 97 to 99% is bound to
plasma proteins, primarily (60%) to lipoproteins ( and ) (Hunt & Jones, 1980; Wall et al.,
1983) and the remain to albumin at a 6: 4 ratio. For this reason, the free concentration in
plasma is actually very low (Klansner et al., 1975), being in the erythrocytes in only about
10% (Garret & Hunt, 1974; Widman et al., 1974). With a large distribution volume (10L/Kg),
high 9-THC lipid solubility leads to increased concentration and prolonged retention of the
drug in fatty tissues (aJohansson e col., 1989), like the nervous tissue. Indeed, concerning its
effective distribution in the tissues, 9-THC is pulled out from the plasma to the tissues in
about 70% (Hunt and Jones, 1980), although the distribution (which only occurs for the free
fraction) is limited by the low concentration of its free form in the blood. Therefore, this
distribution will depend on each organ blood flow. Consequently, given the greater
distribution through more vascularized organs, and due to its high lipid solubility, brain is
the organ where higher 9-THC concentrations are achieved: 3 to 6 times higher than in
plasma and just in 30 minutes.
Initial studies in animals, after 9-THC administration, marked with 14C, showed that 9-
THC concentrations in the tissues (in many cases of 9-THC and metabolites) were higher in
the lung, liver, kidney, heart, stomach, spleen, fat gray, placenta, thyroid, pituitary and
mammary gland, when compared with brain or blood (Kreuz & Axelrod, 1973; Leighty,
1973; Ryrfeldt et al., 1973, Siemens et al., 1979). Later studies in rabbits also suggested that
the highest 9-THC concentrations can be detected in fat and in the heart, but not in the
brain (Leuschner et al., 1986). The relatively low 9-THC levels found in the brain can be,
mainly due to its strong irrigation and consequent rapid and constant 9-THC
transportation from the blood into and out of the brain. Afterwards, it distributes by adipose
tissue, which is, together with the spleen, its major deposit, three days after administration
(Rawich et al., 1979). Several weeks are needed for the drug to be completely eliminated,
even after discontinuing the administration (Kreuz & Axelrod, 1973). A slow cannabinoids
release from fatty tissues and a significant enterohepatic recirculation contributes to the long
half-life in plasma, which has been estimated to be around 4 days, or even more in chronic
marijuana users (Johansson et al., 1988). Moreover, cannabinoids may remain the double of
the time, in the plasma of regular smokers (Mason et al., 1983). In fact, this gradual
cannabinoids release from the tissues to the bloodstream extends its presence in the blood
and subsequent entry into the brain, being this one possible explanation for the absence of a
withdrawal syndrome when the administration is suspended (Agurell et al., 1986).
6.3 Metabolism
Only a minimal amount of 9-THC is eliminated from the body in its original form (with
less than 1% excreted by the kidneys in its unchanged form), and most appear as
metabolites in faeces (68%) or in urine (12%). The drug is also present in other tissues and
biological fluids such as saliva, hair and sweat. 9-THC is almost completely metabolized in
the liver, although metabolism can also occur in the lung and intestine (Agurell et al., 1986).
In man, 9-THC metabolism involves allylic oxidation, epoxidation, aliphatic oxidation,
decarboxylation and conjugation reactions. The allylic oxidation at C-8 and C-11 and
aliphatic oxidation at the side chain lead to the formation of hydroxylated metabolites. The
mono- and di-hydroxy metabolites are then oxidized to form acids and hydroxy acids.
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Thus, in studies performed in vivo and in vitro, it was been shown that 9-THC is primarily
metabolized in its hydroxylated compound by the hepatic microsomal enzymes
(cytochrome P450) by allylic hydroxylation at carbon 11. 9-THC is metabolized in 11-
hydroxy-9-tetrahydrocannabinol (9-THC-OH) (Iribarne et al., 1996, Matsunaga et al.,
1995), considered to be his true active metabolite (Lemberger et al., 1970). Similarly, 8-THC
follows a very comparable degradation pathway (Agurell et al., 1981), being rapidly
hydroxylated to 11-hydroxy-8-tetrahydrocannabinol in the liver (Matsunaga et al., 1995).
Hydroxylation at position 11 is the most important 9-THC metabolism reaction in most
species, including humans. The 11-hydroxy-9-tetrahydrocannabinol (9-THC-OH) has a
similar pharmacological activity and potency than 9-THC. 9-THC-OH is quantifiable in
plasma 10 minutes after a 9-THC intravenous administration. However, by oral
administration, the relationship between this metabolite and the main drug is about 5 times
higher than the one measured after an intravenous administration (Lemberger et al., 1971).
Even so, plasma concentrations achieved after oral administration can range from 50 to
100% when compared to the detected 9-THC concentrations (Wall et al., 1983). After
smoking marijuana, the detected 9-THC-OH concentrations are lower, about 10% of the 9-
THC concentrations (aHuestis et al., 1992; Wall et al., 1983), reaching the maximum 9-THC -
OH peak (Cmax) in approximately 13 minutes after smoking, with maximum concentrations
of, around, 7 ng/ml, after a single marijuana cigarette.
After 9-THC administration, its psychological effects begin to occur about 10 to 20 minutes
after, although the effects caused by 9-THC-OH are only evident about 3-5 minutes later
(Lemberger et al., 1973). This difference may be due to their pharmacokinetic properties,
particularly different distribution and transfer into the nervous system, since their effects are
equivalent. This can also explain the fact that, after an oral administration, the
pharmacodynamic effects are higher than those induced with the same 9-THC
concentration, but reached after smoking (Ohlsson et al., 1980). However the
biotransformation process continues, and the active metabolite 9-THC-OH may oxidize,
giving rise to the corresponding carboxylic acid (9-THC-COOH) or return to hydroxylate
itself. In this second case, it converts to 8, 11-dihydroxy-9-THC, i.e., 11-hydroxy derivative
transformation occurs in the liver into dihydroxy-9-THC. These compounds are then
transformed into other hydroxylated metabolites, more polar, inactive, which are then
excreted in urine and faeces. The 9-THC-OH oxidation leads to the production of an
inactive metabolite, 11-nor-9-carboxy-9-tetrahydrocannabinol (9-THC-COOH) (Lemberger
et al., 1972), identified in plasma, urine and faeces (Wall & Perez-Reyes, 1981, Wall et al.,
1983). Subsequently, conjugation with the glucuronic acid can occur, being 9-THC-COOH
and its glucuronide conjugates the main end biotransformation products in many species,
including humans (Halldin & Widman, 1983). Renal clearance of these polar metabolites is
always slow due to its extensive plasma protein binding (Hunt & Jones, 1980). After
smoking, 9-THC-COOH plasma concentrations gradually increase, becoming higher than
9-THC concentrations shortly after smoking, whereas 9-THC plasma concentrations
decrease very rapidly (aHuestis et al., 1992). Hence, the 9-THC-COOH detection time is
much higher than for 9-THC or for 9-THC-OH. The CBD (Cannabidiol) metabolism is
quite complex, with the possible production of almost 83 metabolites (Harvey, 1991). The
proportions of these compounds also vary between species (Harvey & Mechoulam, 1990).
The metabolism of CBN (cannabinol) is less complex than for other cannabinoids. In most
species, the hydroxylation at C-11 predominates, although there is also an important side
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chain hydroxylation. The excreted metabolites are mainly 11-hydroxy-CBN, the CBN acid-
11-oic acid and its hydroxylated side chain analogues (Brown and Harvey, 1990).
6.4 Elimination
Over 65% of the drug is excreted in faeces (68%), with approximately 13% excreted in urine
(Wall et al., 1983). A total of 80-90% is excreted in 5 days, mainly in the hydroxylated and
carboxylated metabolites forms. Only minimal amounts are excreted in their free forms
(Hunt & Jones, 1980, Wall et al., 1983). Therefore, we can say that both 9-THC and 8-THC
are not eliminated in their free form but in the form of metabolites (THC-OH and/or THC-
COOH) or by glucuronic acid conjugation, producing different glucuronides. This process
takes place in the liver involving several enzymes. The glucuronides formed are highly
hydrophilic and therefore easily eliminated in the urine. 9-THC-COOH metabolite has been
detected in either urine or faeces (Wall & Perez-Reyes, 1981), while 9-THC-OH
predominates in the faeces. In fact, biliary excretion, and the consequent elimination
through the faeces is the major route of unconjugated metabolites elimination (Wall et al.,
1983), although most of the metabolites are reabsorbed from the gut. This enterohepatic
circulation, which leads to more than 15% of the metabolites (Nahas, 1979), is responsible for
the delay in the final active metabolites disposition, contributing to a prolonged excretion
and to its accumulation among different body tissues.
In urine a total of 20 9-THC metabolites were identified, two glucuronic acid and 18
unconjugated acids forms. Indeed, the 9-THC-COOH glucuronide conjugate is the primary
urinary metabolite formed (Williams & Moffat, 1980). All other unconjugated acids
metabolites identified in urine, excepting the 11-nor-9-9-THC-COOH, undergo oxidation or
are degraded, forming varied carboxylated or hydroxylated metabolites. The average life of
the inactive metabolites is about seven days, staying in the body for up to thirty days
(Sutheimer et al., 1985). Some authors even accept the metabolites presence in urine within
72 days after use (Ellis et al., 1985), despite having an estimated 9-THC plasma elimination
time of 4 days (aJohansson et al., 1989).
7. Action mechanism
For a long time ago, some hypotheses had been proposed to explain 9-THC action
mechanism, suggesting that 9-THC may exert its actions through a nonspecific drug
interaction with cell membranes and intracellular organelles (Hillard et al., 1985; Martin,
1986). However, it is notoriously difficult to delineate the precise action mechanisms of
cannabinoids, given the evident 9-THC activity in several places, including the receptors
for opiates and benzodiazepines, as well as marked effects on prostaglandins synthesis and
protein metabolism (Burstein et al. 1982; Welch & Eads, 1999). Cannabinoids inhibit
macromolecular metabolism according to the dose, presenting a wide effects range on the
enzyme systems, neurotransmitters and hormone secretion (Bloom, 1982, Chakravarty et al.
1975; Dalterio et al. 1977; Dalterio et al. 1987; Dill & Howlett, 1988; Pertwee, 1988). These
numerous and diffuse effects supported the hypothesis of a nonspecific interaction.
However, with cannabinoids pharmacology knowledge advance, it became obvious that
some structural aspects would be required for the cannabinoids activity, including the
receptor binding in the target cells (Mechoulam, 1991).
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7.1 Cannabinoid receptors

Nowadays it is clearly known that cannabinoids act through its interaction with specific
endogenous receptors, discovered by Devane et al. (1988) and later colonized. Indeed, a
specific protein receptor was discovered, named CB1 (central receptors) (Matsuda et al.,
1990, Munro et al., 1993), in the mouse nerve cells, being now known that it can also be
found in the brain of the mouse, guinea pig, dog, monkey, pig and man, and peripheral
nerves. The biology and behaviour associated with brain areas are consistent with the
behavioural effects produced by cannabinoids (Table 2). The highest density of receptors is
found in the basal ganglia cells, involved in coordinating body movements.
A) - Brain regions where cannabinoid receptors are abundant

Brain regions Tasks associated with the region
Basal ganglia Motion control
Cerebellum Coordination of body movements
Hippocampus Learning, memory, stress
Cerebral Cortex Cognitive functions
B) - Brain regions where cannabinoid receptors are moderately concentrated
Brain regions Tasks associated with the region
Hypothalamus Maintenance of body functions (temperature, electrolyte
balance, reproductive function)
Amygdale Emotional response, fear
Backbone Peripheral sensation, including pain
Brainstem Sleep, temperature regulation, motor control
Table 2. Brain regions where cannabinoid receptors are abundant or moderately
concentrated and functions associated with these areas (Honório & Silva, 2006).
CB1 receptors mediate the majority of the cannabinoids responses in the central nervous
system (CNS), being abundant in the cerebral cortex, hippocampus, amygdale, basal
ganglia, cerebellum, and thalamus (Ashton, 2001; Robson, 2001). The high receptors density
in the caudate nucleus and cerebellum are consistent with the marked cannabinoids effects
in motor behaviour (Romero et al. 1995; Sanudo-Pena et al., 2000). The significant link
within the cerebral cortex and hippocampus explains the marked cannabinoids effects on
perception, cognitive aspects, memory, learning, endocrine function and body temperature
regulation (Chait & Perry, 1994, Compton et al. 1993; Hampson & Deadwyler, 1999).
The CB1 receptors location in the hippocampus supports that cannabinoids play an import
role in appetite and energy regulation. This supposition is strengthened by its presence in
important peripheral organs, as the GI tract, liver, skeletal muscle and adipose tissue.
Despite being primarily viewed as a central receptor (presence in the nervous tissue)
(Herkenham et al., 1990), this CB1 receptor was also found in the adipose tissue (Bensaid et
al., 2003), myocardium (Bonz et al. 2003), vascular endothelium (Liu et al., 2000) and in
sympathetic nerve terminals (Ishac et al., 1996).
In 1993, Munro et al. identified a second cannabinoid receptor, the CB2 receptor, present,
preferentially, in the immune system cells, outside the CNS (peripheral receptor). Efforts
have been made to improve the chemical manipulation of cannabinoids, to maximize the
selectivity for these receptors CB2, avoiding the psychoactive effects (Robson, 2001). This
distinctive peripheral cannabinoid receptor seems to play a major role in
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immunomodulation (Lynn & Herkenham, 1994; Reggio et al., 1997), showing a significant
anti-inflammatory and immunosuppressive activity. It has already been postulated the
existence of a third receptor - CB3 - (Fride et al., 2003), but the receptor itself has not been
colonized yet. It is believed now that the two cannabinoid receptors - CB1 e CB2 - are
responsible for many biochemical and pharmacological effects produced by the majority of
cannabinoid compounds (Matsuda et al., 1990). The functional differences existent between
the two receptors types are still not known, but structural differences raise that possibility.
In 1986, Howlett et al. showed that 9-THC inhibited the intracellular enzyme adenylyl
cyclase (AC) and that this inhibition occurred only in the presence of a G-protein complex,
this is, in the presence of a cannabinoid receptor, which is a typical member of the largest
known receptors family: G-coupled protein, containing seven transmembrane domains
(Glass & Northup, 1999; Howlett et al., 1991). The body's cells respond in different ways
when a ligand interacts with the cannabinoid receptor. The receptor intracellular surface
interacts with G proteins that regulate effectors’ proteins such as AC, or calcium and
potassium channels, and via a protein kinase activated by mitogen (Bayewitch et al. 1996;
Bidaut-Russell et al. 1990).
7.2 Endogenous cannabinoids

The cannabinoid receptors discovery prompted the search for an endogenous ligand to
which the receptors naturally interact. For each biological receptor (in this case, a brain
receptor) there is probably an endogenous agonist, i.e., a compound naturally produced by
the body that interact with the receptor. The first endogenous cannabinoid discovered was
arachidonoylethanolamine known as anandamide, derived from the Sanskrit word ananda,
meaning "happiness." This substance was isolated from pig brain, by Devane et al. (1992),
and these authors observed that, being chemically distinct from the cannabinoid plant,
compared with 9-THC, it has a moderate affinity for CB1 receptors, but with most of the 9-
THC actions, although having a short action period. In general, the affinity of anandamide
for cannabinoid receptors is only ¼ to ½ the affinity displayed by 9-THC, these differences
being related to the cells or tissues used in those studies as well as with the other
experimental conditions (Smith et al. , 1994). Anandamide mimics 9-THC action since it
binds to both receptor subtypes, CB1 and CB2, and has a similar pharmacological activity,
despite having little power to exert some effects (Fride & Mechoulam, 1993; Howlett , 1995;
Mechoulam & Hanus, 1996; Pertwee 1995). This ligand was found in several regions of the
human brain (hippocampus, striatum and cerebellum) where CB1 receptors are abundant,
suggesting the involvement of endogenous cannabinoids in the brain functions controlled
by these areas. However, substantial concentrations of anandamide are also found in the
thalamus, a brain area with few CB1 receptors (Di Marzo et al., 1994). An interesting fact is
that anandamide was also found in small amounts in other parts of the body such as the
spleen, where there are high concentrations of CB2 receptors, and heart (Ameri, 1999;
Fowler, 2003; Howlett, 1998; Pop, 1999). It may, therefore, be concluded that the molecule
anandamide has both central and peripheral impact (Di Marzo et al., 2000). Although
Deutsch and Chin (1993) have proposed a biosynthetic pathway for anandamide, it appears
that this gets rid of cell membranes followed by depolarization due to a calcium influx (Di
Marzo, 1998, Evans et al., 1992). Also Sugiura et al. (1995) examined the effects of
anandamide and another endogenous compound, 2-araquidonil glycerol (2-AG) in
connection with a specific cannabinoid receptor, assuming that the latter substance can also
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be an endogenous ligand with relevant role in the brain. Under normal conditions, the
endocannabinoid system appears to be tonically active; instead, the endocannabinoids are
produced as needed, act locally and are rapidly inactivated by cellular uptake and
enzymatic hydrolysis (Giuffrida et al., 2001).
In addition to the identification of these ligands there were also synthesized some specific
CB1 receptors antagonists (Rinaldi-Carmona, 1994) and CB2 (Portier et al., 1999). The
SR141716 (now under the name of Rimonabant) was the first specific CB1 antagonist
receptor, with high affinity, blocking the acute effects of 9-THC and other CB1 agonists in
vitro and in animals (Adams et al. 1998; Rinaldi-Carmona, 1994). In 2001, Huestis et al.,
conducting the first clinical studies on the pharmacokinetic and pharmacodynamic effects of
Rimonabant, administered orally, in combination with cannabis smoking, demonstrated that
cannabis administration alone produced the expected physiological responses with the
consequent intoxication reflection and, when combined with Rimonabant, a dose-dependent
blocking effect of cannabis was observed. Significant advances in cannabinoid research have
opened new frontiers leading to an increasing interpretation of their effects and the role of
endogenous cannabinoids in man.
8. Pharmacodynamic effects
Due to their action mechanism, cannabinoids exert multiple conducts, acting in almost all
biological systems. Its activity is multiple and complex due to the variety of psychoactive
products present in the plant whose toxic and pharmacological actions may overlap or be
additive. The main factors influencing the toxicity of these substances are: dose,
administration route, individual's personality, dependence degree, concomitant
administration of other substances and chronological stage of the administration.
The behavioural and physiological cannabinoids effects have been increasingly reported
over the recent decades (Adams & Martin, 1996; Dewey, 1986; Jones, 1987), including
euphoria and relaxation feelings, times reaction changes, lack of concentration, learning and
memory changing or mood states (such as panic reactions and paranoia). The spectrum of
cannabinoids behavioural effects is unique, leading to a consequent classification of these
drugs as stimulants, tranquilizers, or hallucinogenic (Benowitz et al. 1979; Hollister, 1986;
Law et al., 1984). Other common physiological effects include increased appetite, dry mouth,
vasodilation and decreased respiratory rate. Cannabinoids may affect the immune and
endocrine systems, producing lung damage and influencing neonatal and child
development of (Chandler et al., 1996, Day et al., 1991, Fried et al. 1999; Fried & Smith, 2001;
Tashkin et al., 1991). The physiological effects of cannabinoids are most relevant for the
main systems are:
a. Effects on Cardiovascular System
The 9-THC effects of on the cardiovascular system depend on the dose, with a decrease in
heart rate with low doses and increased at higher doses (which may exceed 160 beats/min).
This substance may also lead to a decrease in contractile force and lead to progressive
reduction in coronary blood flow (Tashkin et al., 1978). Acute administration of
cannabinoids in humans produces vasodilation and tachycardia, resulting in an overall
effect on systemic blood pressure (Huestis et al., 1992). However, the unrelenting use of 9-
THC results in hypotension mediated by CB1 receptors and bradycardia (Benowitz et al.,
1975, Lake et al., 1997). Endocannabinoids induce vasodilation by acting, directly, on CB1
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receptors located in the arterial smooth muscle of the brain (Gebremedhin et al., 1999).
Moreover, the same occurs at other vessels, through an increased synthesis of nitric oxide
(NO) endothelium-dependent (Deutsch et al., 1997). These and many other effects on CV
can be an increased risk for individuals with pre-existing heart disease (especially in
patients with heart failure and coronary), as already reported, for example, in acute cardiac
accidents cases, often fatal for Cannabis consumers (Ashton, 2001).
b. Metabolic Effects
The endocannabinoid system appears to play a crucial role in regulating metabolism and
body composition. The appetite stimulation (especially for sweets) and dry mouth due to
decreased salivary secretion are usually adverse reactions produced in cannabis consumers.
The consequent weight loss may suppose that there is a changing in glucose metabolism.
However, several studies show that there is no agreement on the glucose levels change
(Hollister et al. 1968; Lindemann, 1933, Weil et al., 1969). However, its effect on other
metabolic processes is of great significance. Thus, control of food intake and body
composition results from complex interactions between the adipocytes, the mesolimbic
system, hypothalamus and gastrointestinal tract. The hunger feeling often existent in
consumers is mediated by an intestinal hormone, ghrelin, which is produced in most
circumstances of weight loss. Moreover, leptin, an endogenous hormone, can reduce food
intake. The serum concentration of this hormone is directly proportional to the degree of
adiposity, but obese people have lower sensitivity to the hormone. A protein produced in
adipose tissue, adiponectin stimulates fatty acid oxidation and body weight decrease, being
its concentration lower in obese individuals (Considine et al., 1996, Cummings et al. 2002;
Fruebis et al. 2001).
Both cannabinoid receptors and their endocannabinoids ligands are present in tissues
related to food intake. The concentration of endocannabinoids in the hypothalamus
decreases after leptin administration (Di Marzo et al., 2001). Studies in animals (rodents)
showed that the CB1 receptor agonists are potent inducers of hyperphagia (Jamshidi et al.,
2001, Kirkham et al., 2002, Williams et al., 1999), while their antagonists prevent such effect
(Di Marzo et al., 2001). In another study, it was found that mice do not express CB1
receptors resulting in spontaneous calories reduction (Cota et al., 2003). For all these
interferences at the cannabinoid system, metabolic modulation of this pathway has been
considered to be a greater possibility for therapeutic intervention in obesity (Feliciano et al.
2007; Francischetti et al., 2006).
c. Effects on Pulmonary System
Inhaling the smoke of marijuana cigarettes (or 9-THC) produces acute bronchodilation in
healthy subjects and asthmatics, bronchodilation that may last at least one hour (Tashkin et
al. 1977; Vachon et al., 1973). It is important to note that cannabis smokers have a higher lung
cancer risk than tobacco consumers because of the high aromatic hydrocarbons content in
marijuana smoke, which has higher concentrations of irritant substances, such as sterols,
terpenes, among others (Fehr and Kalant, 1972). Comparing a normal about five times
tobacco cigarette with a marijuana cigarette, it is estimated that the latter produces more
carboxyhemoglobin, with consequent maintenance increase in the respiratory tract (Benson
& Bentley, 1995, Wu et al., 1988). Chronic use of cannabis cigarettes is associated with
bronchitis and emphysema. It is estimated that 3-4 cannabis cigarettes per day equals more
than 20 tobacco cigarettes a day, with subsequent evidence of acute and chronic bronchitis
(Benson and Bentley, 1995).
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d. Effects on Vision
The administration of cannabis cigarettes in normal individuals causes a slight constriction of
the pupil, preserving light reflection, marked congestion of conjunctiva vessels, tearing and
intraocular pressure reduction related to dose. In fact, vasodilatation and redness of the
conjunctiva is a characteristic sign of cannabis use (Paton & Pertwee, 1973). Other changes
include colour perception and light adaptation changes (Ohlsson et al., 1980).
9. Cannabinoids and driving influence

Experimental studies have been repeatedly demonstrating 9-THC effects on an individual
cognitive function and psychomotor skills, influencing learning and information acquisition,
changing the individual's memory capacity, coordination and reaction times (Chait & Pierri,
1992; Kurzthaler et al. 1999; Leire et al., 1989). The biggest concern with cannabinoids acute
effects is related to road traffic or labour accidents (Hall, 2001). Indeed, 9-THC acute effects
on cognitive function and psychomotor skills have been subject of extensive study, noting
that, at doses between 40 and 300 mg/kg, cannabinoids can cause a dose-dependent
reduction in tasks that require memory use, reaction times, in motor functions and
coordination (Ameri, 1999; Curran et al. 2002; D 'Souza et al. 2004; Hall & Solowij 1998;
Hampson & Deadwyler 1999; Lewek et al. 1998; Lichtman et al. 2002; Ramaekers et al.,
2004).
The impaired state induced by cannabinoids has been studied by several authors in tests
performed on drivers (Lamers & Ramaekers, 2001; Ramaekers et al., 2000), demonstrating
that their driving risk effects increase with the dose, being more extensive and persistent in
activities that require more careful attention. There is an enormous concern, both in the
European Union and in the United States of America (USA) regarding the link between
cannabinoids consumption and traffic accidents. However, from a legal standpoint, the
evaluation and interpretation of the corresponding accuracy is still a big challenge, since the
association between 9-THC levels and the accidents risk is not perfectly clear. Currently,
some authors claim that there is very little scientific evidence demonstrating that 9-THC or
9-THC-COOH detection in body fluids can be used as impairment evidence in any
circumstance. They assume, for example, that 9-THC or its metabolite can be detected in
the body for days after smoking cannabis and thus, their presence may be indicative of a
previous consumption and not of a recent use, not being certain that the presence of the
drug in the body indicates an impairment state (Drummer et al., 2003). Papafotiou et al.
(2005), through experimental studies in volunteers, acknowledged that the negative
relationship between driving performance and 9-THC levels found in blood is due to the
fact that the 9-THC peak concentrations are achieved in the CNS a few time after being
achieved in blood. Similarly to what occurs with benzodiazepines, where the maximum
influenced state is observed one hour after peak plasma concentrations are reached (Rush &
Griffiths, 1996), the maximum influenced state after 9-THC consumption occurs after
achieving the maximum blood concentration. On the other hand, it has been shown that the
influence and accident risk due to recent cannabis increases with dose, with an influenced
state already present at low doses, being even worse at higher concentrations (Ramaekers et
al., 2004). Note, however, that it is not perfectly acknowledged how to correlate the plasma
9-THC levels variation with driver's behaviour, although this relationship has been
simulated in experimental behavioural cannabinoid pharmacodynamics and
pharmacokinetics studies. Ramaekers et al. (2006) developed, thus, a study in cannabis
consumers, concluding that the impairment sate was progressively higher with increasing
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9-THC concentrations. They admitted that already with 9-THC concentrations between 2
and 5 ng/ml there was significant influence state; when 9-THC was detected between 5
and 10 ng/ ml, about 75 to 90% of the individuals were under the influence and over 30
ng/ml, there was a were 100% influence. Cone and Huestis (1993) also conducted a similar
research and concluded that the ability to drive may be influenced one hour after
consumption, during the 9-THC elimination phase, even when the concentrations decrease
to 13 ng/ml. Berghaus et al. (1995) go even further, stating that with 6 ng/ml of 9-THC the
information processing is already affected, with attention and vision changes at 9 ng/ml
and 12 ng/ml, respectively. They demonstrated that the 9-THC driving influence is mainly
evident in the first two hours after consumption, leading to performance changes, with
higher prevalence on attention, psychomotor and cognitive capacities.
10. Therapeutic perspectives of the endocannabinoid system modulation

Endocannabinoids (EC) are involved in several physiological functions, among which,
special attention has been given to the regulation of appetite by central mechanisms and its
influence on obesity (van Thuijl et al., 2008; Kirkham, 2009). Considering these innovative
findings, the research for new pharmacological agents has drastically increased and the
discovery of rimonabant, a synthetic antagonist of CB1 receptors, has confirmed the
important role of endocannabinoid system on the modulation of food ingestion and
energetic balance (Butler et al., 2009). These facts led to the first clinical studies using
rimonabant as a new tool against obesity and its associated metabolic disorders. However,
psychiatricside-effects, namely central, which include increased risk of depression and even
suicide, US Food and Drug Administration declined permission for rimonabant, and in
October 2008, rimonabant was also suspended across the EU. After rimonabant withdrawal,
other CB1 antagonist drugs have also tested, including the taranabant, which was associated
with weight loss in rats and in humans (Fong et al., 2007; Addy et al., 2008). However, due
also to central side effects, including anxiety and depression, the clinical trials were stopped
in October 2008 (EMEA. The European Medicines Agency recommends suspension of the
marketing authorisation of Acomplia: http://www.emea.europa.eu 2008).
Although several other different influences of endocannabinoids have been discussed
during the last years, including in inflammation, diabetes, cancer, affective and
neurodegenerative diseases, and epilepsy (Izzo et al., 2009), the most recent findings are
related to their cardiovascular actions (Durst & Lotan, 2011), which seem to be very ample
bust also complex. In vivo experiments with rats have demonstrated the action of
anandamide and 2-AG on the development of atherosclerotic plaque, as well as an effect on
heart rate, blood pressure, vasoactivity and energy metabolism (action in dyslipidemia and
obesity). Recent studies with an antagonist of CB1 receptors showed that the modulation of
ECS can play an important role in reducing cardiovascular risk in obese and dyslipidemic
patients. Similarly, studies in rats have demonstrated the action of CB2 receptors in
adhesion, migration, proliferation and function of immune cells involved in the
atherosclerotic plaque formation process. The ECS have been implicated in hypotensive
stages associated with hemorrhagic chock, both endotoxic and cardiogenic, and even to
advanced liver cirrhosis; on the other hand, recent evidence suggests that ECS plays an
important role in cardiovascular regulation associated with hypertension, as well as a
protective role in ischemia grafting. The development of atherosclerotic plaque and the
metabolic stages associated to obesity are also matter of study of possible ECS
pharmacomodulation.
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Effects on myocardial ischemia/reperfusion and preconditioning

Initial studies used isolated preparations of heart to study the role of ECS in myocardial
ischemia/reperfusion (I/R) and preconditioning. The involvement of ECS in
preconditioning induced by the endotoxin (lipopolysaccharide: LPS) against the injury
induced by I/R on myocardium has been implicated for the first time in 2001, based on the
hypothesis that LPS could increase the production of endocannabinoids in inflammatory
cells (Varga et al., 1998). A 90 minutes of low flow ischemia followed by 60 minutes of
reperfusion with normal flow in isolated rat hearts pretreated with LPS was compared with
a saline solution. The pretreatment with LPS reduced the infarct size and improved
functional recovery after reperfusion when compared with control group, which could be
attenuated by SR144528 (CB2 antagonist), but not by rimonabant (CB1 antagonist),
suggesting the involvement of myocardium CB2 in the cardioprotection induced by LPS
(Lagneux & Lamontagne, 2001). In a subsequent study, in which the preconditioning was
triggered by heat stress, the SR144528 also abolished the effect of reducing the infarct size,
unlike rimonabant (Joyeux et al., 2002).
These early studies suggested that the protection created by the preconditioning induced by
heat stress or by LPS was mediated by the action of endocannabinoids in the CB2 receptors.
In contrast, when preconditioning was induced by a brief period of ischemia (5 minutes), the
blockade of CB1 and CB2 receptors did not raise the abolition of protection, and both
receptors have been implicated in preserving endothelium-dependent vasodilatation
induced by serotonin (Bouchard et al., 2003). The palmitoylethanolamide or the 2-AG, but
not anandamide, when added to perfused isolated rat hearts offer protection against
ischemia by reducing myocardial damage and infarct size and by improving the functional
recovery of myocardium (Lepicier et al., 2003). The SR144528 completely blocked the
cardioprotective effect of palmytoylethanolamide and 2-AG, whereas rimonabant only
inhibited, partially, the effect of 2-AG (Lepicier et al., 2003). Similarly, ACEA and JWH015
(CB1 and CB2 agonists) also reduced the size of the infarct in this model (Lepicier et al.,
2003). In contrast, it was found that anadamida’s effect of reducing infarct area could also be
antagonized by CB1 and CB2 antagonists; however, the same could not be mimicked by
selective CB1 and CB2 agonists, suggesting an involvement of a different site of CB1 and
CB2 receptors. Another recent study, which used a model of delayed preconditioning in
rats, induced by transdermal treatment of nitroglycerin (as an NO donor) for 24 hours,
suggested that the protective effect of nitroglycerin against myocardial infarction is
mediated trough CB1 receptors. Nitroglycerin increased the concentration of 2-AG in
myocardium, but did not increase anandamide (Wagner et al., 2006). These pioneer studies
implicated a possible contribution of CB2 functional receptors in cardiomyocytes and the
endothelial cells responsibility, at least in part, on the protective effects of preconditioning.
Indeed, subsequent studies showed the presence of CB2 receptors in myocardium, in
cardiomyoblast cells and in endothelial cells with different origins ((Mukhopadhyay et al.,
2003; Blazquez et al., 2003). Concurrently with the beneficial effect of the activation of CB2
receptors in cardiomyocytes, a recent study showed that THC protected cardiomyoblast
cells H9c2 submitted to hypoxia in vitro, presumably trough the activation of CB2 receptors
and increased NO production (Shmist et al., 2006).
In an ischemia/reperfusion injury model in rats, both anandamide and HU-210 decreased
the incidence of ventricular arrhythmias and reduced the size of the infarct, presumably
trough the activation of CB2 receptors but not CB1 receptors (Krylatov et al., 2001). In an
myocardic I/R injury model induced by ligation of coronary artery in rats, the reduction of
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the second myocardic injury depending on leucocytes subsequent to the initial I/R injury
was attributed to the activation of CB2 receptors, since the protection given by WIN 55.212-2
could be prevented by AM630, but not by AM251 (a CB1 antagonist) (Di Filippo et al., 2004).
Two recent studies in myocardial infarct models, acute and chronic, in rats, showed that
cannabinoids contribute to hypotension and cardiac depression associated to cardiogenic
acute shock, which could be attenuated by antagonists of CB1 receptors (Wagner et al.,
2003).
Overall, despite the role of CB1 receptors and of endocannabinoids in the protection given
by the preconditioning against myocardic I/R, the issue remains controversial,
recommending further investigation namely using mice with deletion of genes and more
selective agonists of CB2 receptors. However, the findings that imply CB2 receptors’
importance, presumably in both endothelial and inflammatory cells and perhaps in
cardiomyocytes, are quite encouraging.
Cerebral ischemia/reperfusion (cerebrovascular accident)
The ECS may constitute an essential mechanism of neuroprotection, in both acute forms of
neuronal injury, such as stroke or brain trauma, and in several chronic neurodegenative
disorders, including multiple sclerosis, Parkinson’s disease, Hungtington’s disease,
Alzheimer’s disease and amyotrophic lateral sclerosis (Pacher et al., 2006a). Although the
exact mechanisms of this neuroprotection are not yet completely understood, several
processes dependent and independent of CB receptors seem to be involved: 1) modulation
of the immune responses and release of inflammatory mediators by CB1, CB2 and not
CB1/not CB receptors in neurons, astrocytes, microglia, macrophages, neutrophils and
lymphocytes (Klein, 2005); 2) modulation of synaptic plasticity and excitatory glutamatergic
transmissions via presynaptic CB1 receptors (Freund et al., 2003); 3) activation of
cytoprotective signaling pathways (Pacher et al., 2006a); 4) modulation of calcium
homeostasis and excitability trough interactions with calcium channels, potassium and
sodium, gap junctions and intracellular calcium reserves, and with NMDA receptors
(Freund et al., 2003); 5) central hypothermia mediated by CB1 receptors, presumably by the
reduction of the metabolic rate of needed oxygen; 6) antioxidant properties of cannabinoids
(Hampson et al., 2000); 7) modulation of endothelial activity and inflammatory response,
leucocytes mobilization, adhesions to the endothelium, transmigration and activation
presumably through CB2 receptors. The first evidence of a neuroprotective effect of
cannabinoids has emerged in research studies on cerebrovascular accident, in which was
used the non psychoactive cannabinoid dexanabinol/HU-211 that exerts its effect through
CB1/CB2 independent mechanisms, in cerebral ischemia models in vivo in rats and gerbils
(Pacher et al., 2006a). Further studies also investigated the neuroprotective effects of CB1
receptors stimulation with synthetic agonists. The synthetic cannabinoid WIN 55.212-2
attenuated the neurological damage in the hypothalamus resulting from cerebral global and
transient ischemia in rats and reduced infarct size after permanent focal cerebral ischemia
induced by cerebral middle artery occlusion, when it was administrated 40 minutes before
or 30 minutes after occlusion, in a dependent way from CB1 receptors, since the protective
effect was prevented by rimonabant (Nagayama et al., 1999). WIN 55.212-2, as well as
anandamide and 2-AG, did also confer protection to cultured cortical neurons submitted to
hypoxia and glucose deprivation in vitro, but these effects proved to be insensitive to
antagonists of CB1 and CB2 receptors (Nagayama et al., 1999).
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In models of cerebral middle artery occlusion in rats, the agonist BAY38-7271 reduced the
size of the infarct, even when administered intravenously 4 hours after the occlusion
(Mauler et al., 2002). The pre-treatment with rimonabant partially attenuated the effect of
HU-210, indicating the involvement of CB1 receptor. However, the protective effect of HU-
210 could be completely abolished by warming the animals’ body until the controls
temperature, showing that hypothermia mediated by CB1 receptors was responsible for the
beneficial effects observed (Leker et al., 2006). Similarly, hypothermia mediated by CB1
receptors was responsible for the neuroprotective effects of THC in a model of cerebral
ischemic injury in rats (Hayakawa et al., 2004), and in a model of global cerebral ischemia
injury in rats (Louw et al., 2000). Concurrently with the neuroprotection mediated by CB1
receptors, mice without CB1 receptors showed an increased neurotoxicity to NMDA and
high mortality levels in permanent focal cerebral ischemia, and an increased infarct area,
with neurological deficits more severe after transient focal cerebral ischemia and decreased
blood flow in brain in ischemic penumbra during reperfusion, when compared with
controls under the same aggressions (Parmentier-Batteur et al., 2002). In contrast, several
recent studies do not support the neuroprotective role of endocannabinoids in the activation
of CB1 receptors. In fact, rimonabant and LY320135 (CB1 receptors antagonist) reduced the
size of the infarct and improved the neurological function in a cerebral ischemia model in
rats, induced by brain middle artery occlusion (Muthian et al., 2004), while low doses of
WIN 55,212-2 showed no protective effects (Muthian et al., 2004). Recent studies have
evaluated the effect of selective CB2 agonists (O-3853, O-1966) in a model of cerebrovascular
accidents. CB2 agonists significantly decreased cerebral infarct and improved motor
function after cerebral middle artery occlusion for one hour, followed by 23 hours of
reperfusion in rats, by attenuation of the increased mobilization of leucocytes and their
adherence to vascular endothelial cells induced by transient ischemia (Zhang et al., 2007).
The role of CB2 receptors in I/R injury was also supported by the increased accumulation of
CB2-positive macrophages derived from resident microglia and/or from invading
monocytes resulting from I/R cerebral injury (Ashton et al., 2007).
In general, it seems clear that both agonists and antagonists of CB1 receptors may play a
neuroprotective effect on cerebral I/R injury. The reason for the contradictory effects of the
pharmacological blockade versus genetic “knockout” of CB1 receptors is still unclear, but
could be related with effects that are independent from CB1 antagonists, and that’s the
reason why this subject requires further clarification. In the case of CB2 agonists, the most
likely protection mechanism is the reduction of increased leukocyte infiltration, mobilization
and adhesion to vascular endothelial cells and consequent activation, in a process induced
by I/R transient injury.
Circulatory shock (organ/body ischemia and/or ischemia/reperfusion)
In addition to its well-known immunologic and neurobehavioral actions, cannabinoids and
their synthetic endogenous analogs exert complex cardiopressant and vasodilator effects,
which were implicated in the mechanisms underlying hypotension associated to
hemorrhagic shock, cardiogenic and septic, advanced liver cirrhosis, cirrhotic
cardiomyopathy, heart failure induced by doxorubicin and shock associated to necrotizing
pancreatitis (Lamontagne et al., 2006; Ashton & Smith, 2007; Ribuot et al., 2005; Moezi et al.,
2008; Sarzani, 2008). These depressant effects of the cardiovascular system could be
prevented or reversed by the pretreatment with CB1 receptor antagonists, and they have
been analyzed in many recent studies. CB receptors antagonists (eg. rimonabant, AM281,
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AM251 and SR144528) prolonged the survival in septic shock or in necrotizing pancreatitis
(Varga et al., 1998), increasing mortality in hemorrhagic (Wagner et al., 1997) and
cardiogenic (Wagner et al., 2001) shock, despite the increase in blood pressure. One possible
explanation for this intriguing controversy is the hypothesis that vasodilatation mediated by
endocannabinoids can provide a survival value by increasing tissues oxygenation,
neutralizing the excessive sympathetic vasoconstriction triggered by hemorrhage or by
myocardium infarct, which could be avoided by blocking CB1 receptors. In contrast, the
blockade of CB1 receptors could increase survival in endotoxic shock by preventing the
primary hypotensive response to LPS (Pacher et al., 2006a). Even more complicated is the
fact that, in hemorrhagic shock, both cardiogenic and septic, UH-210, WIN 55,212-2 and
THC (CB agonists) are able to improve endothelial function and/or survival (Varga et al.,
1998). Since cardiovascular failure and dysfunction in many of the cited studies are
triggered by I/R injury and/or ischemia, and consequently oxidative/nitrosative stress and
inflammatory response associated to the activation of several cell death pathways
downstream (Pacher et al., 2007), another explanation for the different beneficial effects of
agonists and antagonists in circulatory shock could reside in their various anti-inflammatory
and/or antioxidant properties (Klein, 2005). These could be attributed to their inverse
agonist properties or to mechanisms independent from CB1 and CB2 receptors (Pertwee,
2006).
In global terms, it seems clear that both cannabinoids and antagonists of CB receptors may
exert several beneficial effects in shock models in rats; however, the specificity of these
effects and their importance for the circulatory shock in humans requires further
investigation.
Role of endocannabinoid system in hypertension
The potential use of cannabinoid ligands as antihypertensive agents was even considered
since 1970 (Archer, 1974; Birmingham, 1973), and were further reviewed (Sarzani, 2008;
Pacher et al., 2005). Cannabinoids decrease blood pressure in hypertensive rodents
primarily because of decrease cardiac contractility, suggesting that could have a therapeutic
role on hypertension and cardiac hypertrophy. Rimonabant, the CB(1) receptor blocker
induced a significant increase in cardiac contractility and blood pressure in hypertensive
rats but, on the contrary, contributed to decrease blood pressure in weight-loss clinical trials
especially in obese patients with hypertension, which suggests that the overactivation of the
ECS in intra-abdominal obesity could be a deleterious effect, in particular from a
cardiometabolic opinion (Sarzani, 2008). In addition to the studies in animal models that
were already mentioned, it was found that inhalation of THC causes a greater and more
lasting fall in blood pressure in hypotensive subjects when compared with normotensive
subjects (Crawford et al., 1979). Although the mechanism underlying this increased
sensitivity is not cleared yet, it suggests a role of endocannabinoid system in the regulation
of cardiovascular functions in hypertension. In a recent study, using three different
experimental models of hypertension to explore this possibility, the authors found a
significant endocannabinergic tone in hypertension that limits the blood pressure rise and
cardiac contractility through the activation of cardiac and vascular CB1 receptors (Bátkai et
al., 2004b). It was also found, that over-regulation of these same receptors contributes to
potentiate of this tone, maybe trough the inhibition of the activation of endogenous
anandamide, stabilizing blood pressure and the contractility of the heart in hypertension.
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These findings contribute to the interesting possibility of using inhibitors of fatty acid amide
hydrolase in the treatment of hypertension. More clinical studies will be needed to clarify
this interesting possibility in a near future.
Role of endocannabinoid system in atherosclerosis
Cannabinoids, endogenous and synthetic, have complex cardiovascular actions through the
activation of CB1 receptors (vascular and myocardial) (Steinberg et al., 2007). The decline of
cardiac function associated with age and the changes in inflammation genes expression,
nitrative stress and apoptosis in rats FAAH-/- compared with wild type rats was analyzed
(Batkai et al., 2007; Mach & Steffens, 2008). The authors found that increased levels of
anandamide in FAAH-/- rats have a protective effect, which is consistent with the protective
role of cannabinoids in inflammatory disorders, such as atherosclerosis. Besides that,
anandamide demonstrated its capacity to attenuate, in a dose-dependent manner, the
expression of ICAM-1, induced by TNF-α, and of VCAM-1 in endothelial cells of human
coronary arteries, and also THP-1 monocytes adhesion in a process dependent on CB1 and
CB2 receptors (Batkai et al., 2007). Contrary to the potential beneficial effect in
cardiovascular disease, the endocannabinoids may exhibit some prothrombotic effects. In
fact, both anandamide and 2-AG were described as activators of human and rodent
platelets. The platelets are cellular anucleated fragments that circulate on blood stream.
Besides their recognized role in homeostasis and in thrombus formation, platelets may also
have proinflammatory properties and be growth regulators, contributing to the progression
of atherosclerosis (Leite et al., 2009). Endothelial cells, macrophages and platelets may, by
itself, increase their synthesis of endocannabinoids during the formation of atherosclerotic
plaque, leading to the activation of platelets. Alternatively, these cells are able to metabolize
2-AG and anandamide, which can offset the increased levels of cannabinoids. CB1 blockade
with rimonabant, besides reduce weight and abdominal adiposity, improves
cardiometabolic profile, due to multiple influences, including increased levels of high
density lipoprotein (HDL)-cholesterol and reduced triglycerides (Despres et al., 2005; Van
Gaal et al., 2005). A possible role for CB2 receptors on the progression of atherosclerosis was
suggested in an experimental model. The authors found that oral low-doses THC treatment
could inhibit the development of atherosclerostic plaque, which was reversed using
SR144528, an antagonist of CB2 receptors (Klein et al., 2003). The progression of
atherosclerosis was associated with a reduced infiltration of macrophages in the
atherosclerotic lesions. The mobilization, adhesion and trans-endothelial migration of
leukocytes are triggered by the local production of chemokines, its receptors and adhesion
molecules (Braunersreuther & Mach, 2006). Cannabinoids, endogenous or synthetic, have
shown to modulate the migration of several cell types, including immune cells trough
activation of CB2 receptors (Miller & Stella, 2008). In overall, despite some interesting
findings, a specific role of endocannabinoid signaling during atherosclerosis remains to be
better elucidated.
New therapeutic opportunities of ECS in cardiovascular disorders
Obesity remains a continuous healthy problem and research issue, which is explained by the
serious consequences associated with it, as well as by the increasing incidence of type 2
diabetes and associated obesity, including in younger individuals. In this way, the ECS, due
to its well known properties of weight control and energy balance, appeared as a promising
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target for the treatment of obesity, namely by blocking its receptors. The blockade of these
receptors was effectively done by rimonabant, which was viewed as a promising drug for
the treatment of obesity. Besides its action on obesity, rimonabant has also proved to be
efficient in controlling vascular diseases in several clinical trials and, therefore, this drug
was presented as an effective therapeutic approach for treating obesity and cardiovascular
disease. However, despite the proven effectiveness in weight loss, rimonabant clinical use
was associated with several side effects, which mainly includes the following three groups:
the first one includes psychiatric disorders such as depression and anxiety; the second one is
associated with gastrointestinal disturbances such as nausea; and finally the third group
with regard to neurological changes that are reflected in headaches and vertigo. Despite
these adverse effects, which originated its removal from the marker, since the blockade of
CB1 receptors continues to prove an asset in the management of obesity and its associated
risks (such as reduction of lipogenesis, decreased waist circumference, insulin resistance and
dyslipidemia), research in the modulation of the ECS has continued.
ENDOCANNABINOIDS
(ANANDAMINE and/or 2-AG)
(Local effect of CB1 and/or CB2 activation)
Cardiac contractility Adesion of inflammatory cells

Platelet activation Lynfocyte and neutrophils activation
VSMC proliferation and migration Monocyte recruitment/transmigration
Endothelial cell activation Macrophages inflammation
Release of inflammatory cytokines T-Cell recruitment/activation
Protection Against Reduction of Antiarrhthmic Reduction of Anti-atherogenic

Ischemic Injuries Blood Pressure Effect Shock Episodes Activity
(Mainly CB2) (Mainly CB1) (Mainly CB1) (CB1 and CB2) (Mainly CB2)
Fig. 3. Endocannabinoids pleiotropic effects on cardiovascular physiology, demonstration

protection activity on several tissues, together with positive impact on various other cells
that contribute to cardiovascular/atherosclerotic pathologies, such as the monocytes,
macrophages, lymfocyttes, leucocytes, neutrophils and other inflammatory cells.
Although CB1 receptors seem to be the main target for new therapeutic interventions, CB2
receptors are also involved in several mechanisms and, since they are present in immune
cells and are, apparently, involved in modulating immune responses, they are extremely
important and may be seen as a therapeutic target as well. Several line of evidences have
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been clearly suggesting that cannabinoids and their endogenous and synthetic analogs can
promote important cardiac effects, which includes hypotension and cardiodepression. The
actions seem to be mediated by complex mechanisms, including both direct and indirect
effects both on the vasculature and on the myocardium. Furthermore, the ECS, including
endocannabinoids and cannabinoid receptors, have been implicated in the myocardial and
cerebral ischemia/reperfusion, in hypotensive state associated with hemorrhagic, endotoxic
and cardiogenic shock, and in advanced liver cirrhosis. There is also promising evidences
hypothesizing a key role for the endocannabinergic system in the cardiovascular regulation
in hypertension, as well as a beneficial action on atherosclerotic plaque. Resuming,
cannabinoids are able to modulate a countless number of physiologic functions,
demonstration a pleiotropic protective action on the cardiovascular physiology (Figure 3)
and therefore, endocannabinoid system is a potential target for the treatment of several
diseases and the research about this subject still have a long way to go. The evidence so far
gathered shows that the modulation of ECS (as agonism or antagonism of its receptors) is an
enormous potential field for research and intervention in multiple areas of human
pathophysiology. The development of selective drugs for the CB1 and CB2 receptors may
open a door to new therapeutic regimens, in particular in several cardiovascular disorders.
11. Conclusion
The recreational use of the plant Cannabis sativa and the attempt to exploit their potential
therapeutic use have been described over the centuries. The popularity of marijuana, one of
the most common forms of consumption as a recreational substance and as a drug, reflects
its ability to later sensory perceptions and to reduce anxiety. Experimental studies have been
repeatedly demonstrated 9-THC effects on an individual cognitive function and
psychomotor skills, influencing learning and information acquisition, changing the
individual's memory capacity, coordination and reaction times. The biggest concern with
cannabinoids acute effects is related to road traffic or labour accidents. Indeed, 9-THC
acute effects on cognitive function and psychomotor skills have been subject of extensive
study, noting that, at doses between 40 and 300 mg/kg, cannabinoids can cause a dose-
dependent reduction in tasks that require memory use, reaction times, in motor functions
and coordination. It has been shown that the influence and accident risk due to recent
cannabis increases with dose, with an influenced state already present at low doses, being
even worse at higher concentrations. 9-THC plasma concentrations can be very variable,
with values between 1 and 35 ng/ml in suspected impaired drivers and between 1 and 100
ng/ml in fatal road traffic drivers.
Non-psychoactive actions of marijuana, like pain relief, were also described in ancient texts.
However, the biochemical and pharmacological study of this substance has a fairly recent
start. Endocannabinoids are involved in several physiological functions, among which,
special attention has been given to the regulation of appetite by central mechanisms and its
influence on obesity. Considering these innovative findings, the research for new
pharmacological agents has drastically increased and the discovery of rimonabant, a
synthetic antagonist of CB1 receptors, has confirmed the important role of endocannabinoid
system on the modulation of food ingestion and energetic balance. Although several other
different influences of EC have been discussed during the last years, including in
inflammation, diabetes, cancer, affective and neurodegenerative diseases, and epilepsy, the
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most recent findings are related to their cardiovascular actions, which seem to be very
ample bust also complex. The ECS, which includes the endocannabinoids and its receptors,
have been implicated in hypotensive stages associated with hemorrhagic chock, both
endotoxic and cardiogenic, and even to advanced liver cirrhosis; on the other hand, recent
evidence suggests that ECS plays an important role in cardiovascular regulation associated
with hypertension, as well as a protective role in ischemia grafting. The development of
atherosclerotic plaque and the metabolic stages associated to obesity are also matter of study
of possible ECS pharmacomodulation.
The continued approach of biophysics and molecular characterization of ligands for the
cannabinoid receptor will contribute decisively to the success of cross-level research of ECS.
Those advances will be pivotal for the development and definition of the profile of new
chemical entities as therapeutic endocannabinoid modulators. They may also facilitate the
identification of new dynamics of the ECS to be used as predictive and/or diagnostic
orientation biomarkers for the patients, as well as therapeutic based on ECS
pharmacomodulation. The therapeutic approach of cardiovascular system starting from the
modulation of ECS appears to be a promising and multidisciplinary issue of study that is
still in its early stages but that could be a field for better therapeutic intervention in several
disorders, including of cardiovascular and cardiometabolic nature.
12. References
Adams IB, Compton DR, Martin BR. Assessment of anandamide interaction with the
cannabinoid brain receptor: SR141716 antagonism studies in mice and
autoradiographic analysis of receptor binding in rat brain. J Pharmacol Exp Ther.
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11
Pharmacogenetics Role in Forensic Sciences

Loredana Buscemi and Adriano Tagliabracci
Institute of Legal Medicine, Department of Neuroscience,Università Politecnica delle
Marche, Ancona,
Italy
1. Introduction
The completion of Human Genome Project and advancement of analytical technology with
the large-scale identification of genome polymorphisms have contributed to the field of
forensic science, especially in the studies on genetic basis of most important inherited
arrhythmia syndromes responsible to sudden cardiac death, a major cause of death
worldwide, and of individual differences in response to potential toxicants, with a new
emerging area of interest, the so called pharmacogenetics.
The term pharmacogenetics was first used in the late 1950s (Clayman, 1952) and can be
defined as the study of variability in drug responses as a function of genetic differences
among individuals; applied to nontherapeutic foreign substances, collectively referred to
as xenobiotics, the equivalent term toxicogenetics is used (Nebert, 1999; Mancinelli, 2000;
Park and Pirmohamed, 2001; Roses, 2002; Wolf, 2000). The later coined term
'pharmacogenomics' usually refers to changes in gene expression as a consequence of
drug exposure. However, the two terms, pharmacogenetics and pharmacogenomics, are
often used synonymously.
One of the goals of pharmacocogenetics is the identification of the molecular genetic bases
for interindividual variations in susceptibility to the anticipated effects of a drug or of
xenobiotics (Pirmohamed and Park, 1999). If we want to provide a modern interpretation of
the famous assertion of Paracelsus (1493-1541), it is true that "the dose makes the poison",
but in different degrees, depending on the genetic characteristics of individuals.
There is no doubt that adverse drug reactions (ADRs) are a common cause of morbidity and
mortality, despite extensive and well-regulated registration processes for proving drug
efficacy and drug safety, and are associated with substantial costs of medical care (Lazarou,
1998; Pirmohamed, 1999).
Genetic studies could also clarify the origins of addictions, a diverse set of common,
complex diseases, that are to some extent tied together by shared genetic and environmental
etiological factors (Goldman, 2005; Kendler, 2003). The use and abuse of legal and illegal
substances is a worldwide public health priority with repercussions extending from the
level of the individual to the family, community, and society.
This chapter will focus on: a) adverse drug reactions; b) drug addiction; c) variability in the
human genome; d) pharmacogenetic variability in drug response; e) genetic approaches to
understand the individual differences in susceptibility to drugs/xenobiotics responses; f)
ethical issues relating to the collection of genetic data.
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2. Adverse drug reactions

Any substance that is capable of producing a therapeutic effect can also produce unwanted
or adverse effects; the risk of such effects ranges from near zero to high (Edwards, 2000). An
adverse drug reaction (ADR), according to the World Health Organization definition, is “a
response to a drug that is noxious, unintended, and undesired effect of a drug, which occurs
at doses used in man for prophylaxis, diagnosis, or therapy of disease, or for modification of
physiological function”. This definition excludes therapeutic failures, intentional and
accidental poisoning (ie, overdose), and drug abuse. Also, this does not include adverse
events due to errors in drug administration or non-compliance (taking more or less of a
drug than the prescribed amount); using this narrow definition avoids overestimating the
ADR incidence (Lazarou, 1998). The terms adverse reaction and adverse effect are
interchangeably, but must be kept in mind that is the “drug” that has an adverse effect
whereas it is the “patient” that experience an adverse reaction. However, the two terms
must be distinguished from “adverse event”, that is an averse outcome that occurs while a
patient is taking a drug, but is not necessarily attributable to it (Edwards, 2000).
The interest in ADRs was stimulated by the thalidomide tragedy in the 1960s and, over the
past several years, ADRs have gained worldwide attention: the Food and Drug
Administration (FDA) has planned a system of pharmacovigilance to be followed by
regulatory agencies, pharmaceutical companies, and individual health care providers (U.S.
Department of Health and Human Services Food and Drug Administration, 2005).
The occurrence of ADRs is associated with morbidity and mortality and substantial costs of
medical care. Numerous studies provide a wide range of epidemiological data regarding
adverse drug reactions. ADRs are one of the top ten causes of death in the United States,
causing over 100 000 deaths annually; approximately 2–5% of all hospital admissions can be
attributed to adverse drug reactions (Lazarou, 1998). In a study performed in 1999 in
emergency departments of French public hospitals, out of a total of 1937 patients consulting,
328 (21%) of these patients consulted an physician because of an ADR (Queneau, 2007).
During the year 2000, a prospective Italian study was performed in two observational
periods of 10 days each in 22 Italian emergency departments: on 18 854 enrolled patients,
629 (3.3%) were affected by ADR and among these, 244 (38.8% of ADR patients) reported a
serious event (Trifirò G, 2005). In a prospective Scandinavian study with 13.992 patients of
internal medicine, the incidence of lethal ADR was estimated to be 0.95% (Ebbesen, 2001).
Another prospective study conducted in the UK demostrated that about 6.5% of hospital
admissions were ADR related in 18.820 patients (Pirmohamed, 2004). In a nationwide study
in Spain, during a six-year period (2001-2006), the total number of hospitalized patients with
ADR diagnosis was 350 835, 1.69% of all acute hospital admissions (Carrasco-Garrido, 2010);
in The Netherlands, in 2001, 12 249 hospitalisations were coded as ADR related, 1.83% of all
acute hospital admissions (van der Hooft, 2010).
Unfortunately, many physicians still consider adverse drug reactions to be an exception,
rather than a primary diagnosis and adverse drug reactions have become cases of medical
professional liability, with great increase of lawsuits (Wooten, 2010).
The classification of ADRs distinguished dose-related and non-dose related reactions,
named type A and type B, respectively; type A reactions are common, predictable and
therefore potentially preventable, based on the drug’s pharmacological action, while type B
reactions are more troublesome, uncommon and unpredctable. More recently, additional
types were added, such as chronic (type C) and delayed (type D) effects, as well as
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Pharmacogenetics Role in Forensic Sciences 253
withdrawal or end of use syndromes (type E) and therapeutic failures (type F) (Edwards,
2000).
Different subjects with the same diagnosis could respond differently to the same drug
administered at the same dose, with a diminished, absent or excessive response or
interaction with other drugs (Mancinelli, 2000; Meyer, 2004). Potential risk factors for ADRs
include patients’ age, sex, race, nutritional status, organ function, especially of liver and
kidneys, co-morbidities, co-medication, as well as some lifestyle variables (smoking habits,
concomitant use of alcohols and drugs) and, of course, genetics. Some ADRs caused by
genetic variation, previously considered unpredictable, may now be preventable. In general,
genetic factors are estimated to account for 15-30% of interindividual differences in drug
metabolism and response, but for certain drugs this can be as high as 95% (Evans, 2004;
Weinshilboum, 2003)
ADRs may be reduced by means of the introduction of “personalized medicine”, which
anticipates the screening of patients for polymorphisms associated with a drug response,
usually performed prior to the initiation of therapy. Despite significant progress in this field,
only few drugs, such as cetuximab, dasatinib, maraviroc and trastuzumab, require a
pharmacogenetic test before being prescribed: there are several gaps that limit the
application of pharmacogenetics based upon the complex nature of the drug response itself
(Gervasini, 2010).
This kind of policy foresees the introduction of new sophisticated tests, especially in the
field of genetics, like DNA microarrays or DNA chips.
3. Drug addiction
Drug addiction is a chronic, relapsing disorder in which compulsive drug-seeking and drug-
taking behavior persists despite serious negative consequences; continued use induces
adaptive changes in the central nervous system that lead to tolerance, physical dependence,
sensitization, craving, and relapse (Goodman, 2008). This mental health disorder imposes a
significant burden on those directly affected, health care systems, and society in general,
since it is associated with considerable morbidity and mortality, violence, and legal issues.
According to World Health Organization (WHO) figures, about 2 billion people worldwide
consume alcoholic beverages, 1.3 billion nicotine and 185 million illegal drugs. In Europe the
use of alcohol, nicotine and illicit substances is responsible for respectively 10%, 12% and 2%
of the total cost of illness. (WHO, 2002).
Polydrug use of psychoactive substances, legal and illegal, characterizes and defines the
style of consumption prevailing more and more common among younger subjects. There is
another emerging market worldwide for an increasing number of psychoactive substances
whose compositions are not well known and whose effects have not yet been recorded by
physicians and they are difficult to recognize, delaying the diagnosis and treatment of
patients themselves.
In addition there is another phenomenon in recent years: it is a marked shift in the
marketing of licit and illicit drugs through online pharmacies, without requiring a
prescription. The new generations are particularly vulnerable to this risk because they are
very prone to use new technologies.
The nonmedical use of a prescription or over-the-counter (OTC) medication is another
significant international emerging problem. OTC medications are pharmaceuticals that do
not require a prescription and are sold on the shelves of markets, stores and pharmacies.
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The several classes of medications that are commonly abused include: analgesics opioids,
which are most often prescribed to treat severe pain (morphine, oxycodone, hydrocodone,
hydromorphone, codeine); central nervous system depressants, commonly prescribed to
treat anxiety and sleep disorders (barbiturates and benzodiazepines); stimulants, which are
used primarily to treat attention defict disorder, attention defict hyperactivity disorder
(ADHD) and narcolepsy (dextroamphetamine and methylphenidate). The OTC medicines,
such as certain cough suppressant (dextromethorphan), sleep aids (doxylamine),
antihistamines (diphenhydramine), decongestants and others can be abused for their
psychoactive effects (Lessenger, 2008).
According to figures reported by NIDA, in 2009 approximately 7 million (M) reported past
month non-medical use of psychotherapeutic drugs (2.8 percent of the U.S. population). The
medications most commonly abused are: pain relievers (5.3 M), tranquilizers (2.0 M),
stimulants (1.3 M), sedatives (0.4 M). The abuse of drugs is particularly problematic in
adolescents, shows that in boys aged 12 to 17 years 8,3% reported abuse of Vicodin
(hydrocodone) and 5% of Oxicontin (oxycodone hydrochloride) (NIDA, 2010).
All substances that are abused have ability to induce dependence and withdrawal.
The current challenge is to transfer the important increase of the knowledges of addiction's
neurobiology in patients with addiction problems and to identify specific genes responsible
for the particular vulnerability or resistance to addiction. Some schools of thought contend
that addiction is entirely preventable through proper legislative action and individual
choice, and claim that genetic research in this field is to assume a role as a low priority
(Merikangas, 2003). Genetic research, however, plays a very important role, since the origins
of addiction susceptibility are complex and wide-ranging; the underlying genetic factors
need to be identified to solve the puzzle of what causes these pervasive and relatively
intractable disorders (Goldman, 2005).
Both genetic and environmental variables contribute to the initiation of use of addictive
agents and to the transition from use to addiction (Bevilacqua, 2009). Evidence from twin
and adoption studies suggest that 40-60% of the risk of developing substance abuse
disorders is due to genetic factors, with the percentage depending on the substance (Nestler,
2000). The addiction are complex disorders involving multiple genes and environment
interaction (G x E). The genetic influences are more prominent in the later phases of
individuals’ progression toward substance dependence; this variation could add to allelic
variations that could produce effects on addiction susceptability phenotypes by other routes
that could include: differences in pharmacokinetic characteristics of the substance such as
metabolism and biodistribution; differences in drug’s rewarding properties; differences in
traits manifest by the addict, including personality differences; differences in addict’s
psychiatric comorbidities (Uhl, 2004). This suggests two broad types of genetic
predisposition to addiction: genetic profiles that make people more likely to find the acute
effect of drugs rewarding and genetic profiles that make people more or less likely to
developing addiction if they use drugs (EMCDDA, 2009). Finally, evidence indicates that
there is a genetic predisposition that is shared between the different substance use disorders;
nearly 25-36% of the genetic influences of alcohol, nicotine and cannabis problem use is
attributable to overlapping factors (Young, 2006).
The inheritance of addictions has been evaluated in many ways, including studies on
families and adoptees, but the main reference of our knowledge comes from the patterns of
correlations in monozygotic (MZ) and dizygotic (DZ) twins. The overall genetic influence
for substance use disorders has proved to be consistent and heritabilities for most substance
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use disorders are estimated to be moderate to high (Wong, 2008). This moderate to high
inheritance may seem paradoxical: addiction depends initially on individual choice to use
an additive agent. Cocaine and opiates, among the most addictive of substances, are among
the most heritable; in contrast, hallucinogens, are among the least addictive, and are also the
least heritable (Bevilacqua, 2009).
The genes involved in the of the condition are very numerous (Kreek, 2005).
The phenotype for addiction to drugs is not well defined, and the heritability of addiction to
drugs of abuse is far from clear. Knowledge of genetic factors in etiology and treatment
response may enable the individualization of prevention and treatment, as well as the
identification of new therapeutic targets (Buckland, 2008).
4. Variability in the human genome

Individuals are all different from each other and much of this difference has a genetic basis.
Two unrelated human beings also share 99.9% of their genomic sequence, and could be
considered genetically almost identical: the difference has been estimated to be of 0.1%
overall, but still, this means that there are at least several million nucleotide differences per
individual. There are, on average, three million genetic differences between any two people;
the human genome contains approximately 3 billion base pairs of DNA and the variability
of genetic material between any two individuals averages approximately one variation for
every 1,000.
This genetic diversity in most cases have no functional significance, but in some cases have
important consequences (Marchant, 2003). The most dramatic examples are seen with
inherited disorders, where small alterations in gene sequence can result in premature death
or severe disability (Alberts, 2002; Habener and Williams, 2002; King, 2002). It is also
responsible of the phenotypic diversity, which results in the heterogeneous capacity of each
individual to respond to exogenous substances, such as drugs and xenobiotics, and in the
different susceptibility to induce adverse health effects.
The types of genetic variations used in these studies have changed in the past 25 years and
can be classified into five major classes: RFLP (restriction fragment length polymorphism),
VNTR (variable number of tandem repeat), STR (short tandem repeat), SNP (single-
nucleotide polymorphism) and CNV (copy-number variation); furthermore, construction of
the international SNP database and recent development of high-throughput SNP typing
platforms enabled us to perform genome-wide association studies, which have identified
genes or genetic variations susceptible to common diseases or those associated with drug
responses (Nakamura, 2009).
SNPs are found at a frequency of about 1:1000 bases in humans and they are changes in a
single base at a specific position in the genome, in most cases with two alleles. By definition,
the more rare allele should be more abundant than 1% in the general population; if the
variant is rare, with a frequency below 0.1%, it is referred to as a mutation. More than 99%
of these genetic variations are biologically silent, while some polymorphisms can affect
biological function according to their position within the genome.
Following the scheme of Orphanides and Kimber (2003), it is possible to distinguish:
1. SNPs that fall within the coding region of a gene can give rise to a protein that has an
amino acid substitution, or is truncated, causing a change in activity, localization, or
stability;
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2. SNPs that induce shifts in translational reading frames will lead to the synthesis of
proteins with altered aminoacid sequence and, perhaps, activity;
3. nucleotide alterations in the regulatory regions of a gene can also have a significant
impact on the integrity of protein function;
4. polymorphisms in promoter regions may change the regulation and level of expression
of a protein, whereas those that fall near intron-exon junctions may cause alterations in
mRNA splicing;
5. more dramatic polymorphisms involving larger segments of the genome include gene
deletions, gene conversions, and gene duplications (Orphanides and Kimber, 2003).
5. Pharmacogenetic variability in drug response

Gene polymorphisms account for the polymodal distribution of the frequency of response to
a drug in a non-homogeneous population, i.e. one encompassing multiple genetic profiles
capable of affecting response.
Current pharmacogenetic studies are exploring individual responses to drugs in relation to
the genetic variations in the proteins involved in pharmacokinetics (absorption, distribution,
metabolism and excretion) and pharmacodynamics (receptors, ion channels and other
enzymes) (Roses A, 2000; Nebert, 2008) (figure 1).
Fig. 1. Pharmacogenetic variations affecting the individual response to a drug

Drug-metabolizing enzymes (DMEs) play a key pharmacokinetic role (Meyer, 1997). Drugs
are turned to metabolites in the liver, by transformation of functional groups (phase I
reactions) and subsequent conjugation with endogenous lipophilic substances to form
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inactive compounds (phase II reactions) for ready excretion in urine or bile. Oxidative drug
metabolism is mainly catalyzed by the enzymes of the large CYP450 gene family, that
contains 57 functional genes and 58 pseudogenes playing an important role in the
metabolism of therapeutic drugs and other xenobiotics. CYP450 are so named because they
are bound to membranes within a cell (cyto) and contain a heme pigment (chrome and P)
that absorbs light at a wavelength of 450 nm when exposed to carbon monoxide. The
corresponding genes are highly polymorphic and genetic variability underlies
interindividual differences in drug response. CYP2D6, 2C19, and 2C9 polymorphisms
account for the most frequent variations, since almost 80% of drugs in use today are
metabolized by these enzymes (Danielson, 2002; Ingelman-Sundberg, 1999; Nebert, 2002;
Nelson, 1999; Zhou, 2009). The polymorphisms of CYP2D6 significantly affect the
pharmacokinetics of about 50% of the drugs in clinical use (De Gregori M, 2010).
The CYP450 enzimes convert the substances into electrophilic intermediates, which are then
conjugated by phase II enzymes, of which the most important are the highly polymorphic
UDP-glucuronil transferase, sulfotransferase, catechol-O-methyltransferase (COMT) and
glutathione transferase, to facilitate substrate excretion by turning them into more water-
soluble forms.
All these factors determine the trend of concentration over time and therefore the
effectiveness of drugs and duration of effect. DME genetic variations result in marked
phenotypic consequences; these range from poor metabolizers (where toxic drug effects may
arise due to the absence of the gene product) to ultrarapid metabolizers (where therapeutic
failure may be induced by the indicated dosage, with the risk of achieving high plasma
concentrations and concentration-dependent side effects due to gene overexpression). The
resulting phenotypes are poor (PMs), intermediate (IMs), extensive (EMs) and ultrarapid
(UMs) metabolizers.
To leave the cell, some drugs are actively transported by membrane transporter proteins.
The major transporter enzymes are MDR1 (multidrug resistance proteins), MRP (multidrug
resistance-associated proteins) and OATP (organic anion-transporting polypeptides), where
several genetic polymorphisms have been demonstrated. MDRs are transmembrane
transporters of the large ABC protein family: P-glycoprotein (P-gp, or MDR1/ABCB1), the
best known, is highly polymorphic. It can influence substrate absorption at the level of the
blood brain barrier; high P-gp concentrations can limit entry of the required amount of
drug, whereas low levels may result in abnormal accumulation. Recently, allele frequencies
and findings regarding functional variants in drug transporter systems were reported in an
interesting review (Kroetz, 2010).
Pharmacodynamic processes mediate the biochemical and physical effects of drugs on the
organism. Variations in the sequence of the genes encoding the primary therapeutic target,
such as receptors and ion channels, are capable of inducing protein forms with different
functional characteristics. This can account for abnormal drug responses, which may also
underpin some adverse reactions.
Recently, researchers are focusing on most important genetic variations that could
contribute to the initiation of use of addictive agents and to the transition from use to
addiction. The complex genetic constitution is partly accounted for by heterogeneity and
polygenicity: the first assumes that a single or a few genetic variation(s) determine
vulnerability and resiliency, but different alleles would lead to the same clinical presentation
in different individuals; the second, on the other hand, assumes that a phenotype is a result
of simultaneous function of multiple genetic variants (Goldman, 2005; Wong, 2008).
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Pharmacogenetic studies can assess the effects of genetic variation on the risk for particular
phenotypes for addiction, for example being an alcoholic (Onori, 2010; Buscemi, 2011). In
recent years abundant evidence has accumulated demonstrating that alcoholism, a major
health and social issue, being one of the most frequent disease and cause of premature
death, is a multifaceted disease of the brain, caused by numerous genetic, neurobiological,
environmental factors that are still not yet fully understood. Numerous genes are up-
and/or down-regulated by alcohol exposure: the ethanol-responsive genes mainly encode
functional proteins such as proteins involved in nucleic acid binding, transcription factors,
selected regulatory molecules, and receptors. Currently there are only three medications
approved by the U.S. Food and Drug Administration (FDA) for use in the treatment of
alcohol abuse and alcohol dependence: the aldehyde dehydrogenase inhibitor disulfiram,
the micro-opioid receptor antagonist naltrexone, and the N-methyl-D-aspartate (NMDA)
receptor inhibitor acamprosate (Wang, 2010).
6. Pharmacogenetic approaches
Pharmacogenetic studies can be categorized into two methodologic approaches: genome-
wide linkage analysis and candidate gene approach.
Linkage analysis is applied to families with several affected individuals, to establish
whether specific alleles of marker genes are found more often in individuals with the
disease than in healthy subjects. The whole genome is analysed using markers that are
uniformly distributed on all chromosomes, seeking chromosome regions that could contain
genes involved in complex disorder susceptibility. The linkage is sought only in recent
ancestors. Since only a small number of recombination events are involved, the gene regions
detected by linkage analysis are likely to be large and to encompass hundreds or even
thousands of genes. Genetic association studies assess correlations between genetic variants
and trait differences on a population scale and they have been used widely to identify
regions of the genome and candidate genes that contribute to complex disease.
A disease-associated SNP that falls within a gene can provide information on the
mechanistic basis for disease, while a SNP that is in linkage disequilibrium with a genetic
allele that confers disease predisposition may be used to identify susceptible individuals,
and naturally this can include those genetic variations that influence relative susceptibility
or resistance to toxicants (Roses, 2000). The common errors encountered in association
studies of complex diseases are the small sample size, subgroup analysis and multiple
testing, random error, poorly matched control group, failure to attempt study replication
and to detect linkage disequilibrium with adjacent loci, overinterpreting results and positive
publication bias, unwarranted candidate gene declaration after identifying association in
arbitrary genetic region (Cardon, 2001). Despite these known limitations, the power of
association analysis to detect genetic contributions to complex disease can be much greater
than that of linkage studies (Risch, 2000).
Association studies can be distinguished into family-based, which use the transmission
disequilibrium test, and population-based, which use case-control testing. Case-control
studies compare genes from two groups of individuals, healthy and diseased. Ideally, the
two groups should be homogeneous, with subjects matching for measures like age,
ethnicity, years of education, and marital status, and differing only in terms of the disease
studied. The allele frequency of the gene markers (e.g. SNPs) in or close to the genes are
analysed and frequency differences between the groups taken to indicate that the gene
contributes to the disease.
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Association studies draw from historic recombination so disease-associated regions are

extremely small in outbred random mating populations, encompassing only one gene or
gene fragment. As the disease mutation is transmitted from one generation to the next,
recombination will separate it from the alleles of its original haplotype.
A specific genetic profile, or haplotype, i.e. the combination of allelic states in a set of
polymorphic markers found on the same chromosome, could be identified by association
studies by analyzing a number of markers of a given chromosome region in a group of
affected subjects and in a control group (case-control study). Different haplotypes can be
found in a population as a result of mutation or genetic recombination. The recombination is
principally determined by the genetic distance between markers and by the propierties of
the locus where they are found (recombination hotspots). Markers that do not undergo
recombination are characterized by linkage disequilibrium (LD). The tendency of some
alleles at distinct loci to be co-inherited, due to reduced rates of, or absent, genetic
recombination may lead to their association in a population, i.e. to LD. Recent LD studies by
analysis of SNP haplotypes have suggested a block structure, at least in some portions of the
genome. Haplotype blocks appear as regions made up of consecutive alleles that are co-
inherited. Given the limited haplotype diversity within blocks, several SNPs will be
redundant, enabling a minimum number of informative markers to be used to identify the
common haplotypes in each block: these markers are called tag-SNPs. Different block
structures can be found in different populations, with significant implications for association
studies, since the tag-SNPs identified in a population will be useless in another if they are
found in different blocks.
7. Ethical issues
It is necessary to make a reflection on how informations from the human genome will be
used. The collection of genetic data has attracted much public attention for the possible
ethical, moral and political issues relating to the use of these informations.
Genome-wide association studies trying to identify genes that contribute a small risk to
common diseases can only be performed on an international scale; meanwhile, it is
becoming more and more clear that genomic information is hard to hide. Thus the
traditional promise in research that privacy will be protected appears to be less realistic. The
deciphering of the genetic code may pose a threat to the protection of one’s privacy; some
variants that predict drug response are also markers for disease predisposition. This may
subsequently lead to medico-legal implications, such as the issue of data confidentially:
whether employers and insurance companies should given rights to assess the genetic data
(Koo, 2006). Access to genetic might lead to discrimination of individuals with an
unfavourable genetic constitution; for example, individuals who have a genetic
predisposition for a certain condition, or who would only tolerate expensive drugs, might be
charged higher insurance premiums (Vijverberg, 2010).
Most European countries have adopted genetic anti- discrimination legislation; Belgium was
the first in 1990, and many countries followed. After a 13-year battle in Congress-longer
than it took to map the human genome-the Genetic Information Nondiscrimination Act
(GINA) was passed into law on 21 May 2008. Francis Collins, the director of the National
Human Genome Research Institute, said that the success of personalized medicine hinged
on the passing of the legislation.
Van Hoyweghen and Horstman state that many European genetic non-discrimination laws
only provide the illusion of protection and the protection against potential risks of
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discrimination based on predictive medical information is still so far. Some insurance

companies may still use genetic test results or genetic information derived from physician
records or insurance questionnaires (Van Hoyweghen, 2008). This practice is mainly caused
by ignorance, confusion and misunderstanding, but also due to the lack of clear legal
definitions of ‘genetic data’ and ‘genetic tests’(Vijverberg, 2010). The definitions of genetic
testing used by 65 organisations and entities, including genetics professional organisations,
insurance organisations, pharmaceutical companies, and legal organisations, was reviewed;
it was found that the definitions used were extremely variable; ranging from DNA testing
solely, to any source that can provide unambiguous genetic information, including family
history (Sequeiros, 2005).
It has been suggested that potential problems with the ethical use of this kind of genetic data
can be minimized by selecting SNPs that are of pharmacogenetic and toxicogenetic value,
while avoiding those that predict genetic disease (Roses, 2002).
Toxicogenetics can learn from the forensic sciences: the widely used technique of “genetic
fingerprinting” uses a small number of highly polymorphic, unlinked genetic markers that
have no known implications to the health of an individual (Orphanides and Kimber, 2003).
8. Conclusion
The potential of pharmacogenetics to improve the clinical practice is only at the very
beginning but will present an important biomedical tool in the post-genomic era. The aim is
to aid physicians in the prescription of the right medicine to a person in an attempt to obtain
maximum efficacy and minum toxicity based on a genetic test, according the new strategy
named “personalized medicine”: prescribing the right drug in the right dose to the right
patient according to specific health needs and individual characteristics.
Advanced diagnostic analyses, genetic counselling, and interdisciplinary and
multidisciplinary approach, involving neurobiological, genetical, toxicological,
psychological, and social sciences, should be integral parts of forensic practice.
Although a relatively novel concept in the forensic context, pharmacogenetics has the
capability to assist in the interpretation of drug related deaths, particularly in unintentional
drug poisonings where the cause of death remains unclear (Pilgrim, 2010). The
recommendation number eleven of the report from the National Academy of Sciences (NAS)
titled “Strengthening Forensic Science in the United States: A Path Forward” is concerned
with improving medicolegal death investigations: “Best practices should include the
utilization of new technologies such as laboratory testing for the molecular basis of
diseases”.
The forensic science community, however, has not yet fully received this directive and only
few studies to date have been able to ascertain a correlation between genotype and
phenotype for a limited number of drugs and to establish a link with the death (Koski, 2006;
Koski, 2007; Launiainem, 2010; Levo, 2003).
The correlation between genotype and phenotype still remains a limitation in a molecular
autopsy ant it is complicate for a number of reason. Only individuals completely lacking the
enzyme activity (PMs) are highly correlated with the expected phenotype. There is
substantial overlap in activity within and between the other phenotypic classes: subjects
with identical genotypes may also exhibit different phenotypic activities which may be
explained by population-specific factors, such as unidentified genetic, such as other enzyme
and proteins, and non-genetic factors, such as diet. In addition, the funtional consequence of
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the genetic variation may be substrate (e.g. drug or its metabolite) specific (Gaedigk, 2008;
Sajantila, 2010).
The new opportunities, offered by pharmacogenetics, to analyse the genetic variations
related to the risk of ADRs or to susceptibility to drug addiction are of considerable interest
to forensic scientists, for their role in the evaluation of drug addiction in its various phases
of development, from beginning to end stage. A better understanding of genetic
susceptibility to addiction may be also useful for ascertaining the causes and circumstances
of death. Some gene variants may, in fact, determine in some individuals more sensitive to
the substance, with an increased risk of toxic effects, even death.
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12
Forensic Pharmacogenetics
Susi Pelotti and Carla Bini
University of Bologna, Section of Legal Medicine
Italy
1. Introduction
Pharmacogenetics and pharmacogenomics are gaining importance both in the clinical setting
and in forensic pathology to investigate causes of death where no findings emerge from
autopsy, and in the medical liability arena where scientific issues meet the justice system
(Pilgrim et al., 2011). Generally speaking, Pharmacogenetics is the study of how genetic
variations give rise to differences in drug response, while pharmacogenomics (PGx) is the
application of genomic technologies to the discovery of new therapeutic targets (Evans &
Relling, 1999). Nevertheless, there is a diversity of opinion regarding the definitions and
benefits of pharmacogenetics and pharmacogenomics. Depending on the purpose,
pharmacogenetics can be used to define applications of single gene sequences or a limited
set of multiple gene sequences, but not gene expression or genome-wide scans, to study
variations in DNA sequences related to drug action and disposition. Pharmacogenomics can
be used to define applications of genome-wide single-nucleotide polymorphism (SNP) scans
and genome-wide gene expression analyses to study variations influencing drug action
(Lesko et al., 2003). Some authors use a very broad definition of pharmacogenomics
including the study of inter-individual variations in whole-genome or candidate gene single
nucleotide polymorphisms (SNP maps), haplotype markers and alterations in gene
expression or inactivation that might be correlated with pharmacological function and
therapeutic response. Pharmacogenetics is narrower in definition and refers to the study of
inter-individual variations in DNA sequence related to drug absorption and disposition
(pharmacokinetics) or drug action (pharmacodynamics), including polymorphic variation in
genes encoding transporters, drug-metabolizing enzymes, receptors and other proteins
(Lesko & Woodcock, 2004). With the accumulating knowledge of human genomic variation,
the Human Genome Project offers the opportunity to develop personalized medicine,
decreasing adverse drug reactions and increasing the efficacy of drug treatment
(Weinshilboum & Wang, 2004). Historically, Johansson & Ingelman-Sundberg (Johansson &
Ingelman-Sundberg, 2011) consider that inter-individual variation in response to a
xenobiotic was probably described first by Pythagoras in 510 BC when he noted that some
individuals developed hemolytic anemia after ingestion of fava beans. Then, they record
that at the beginning of the last century, Garrod and Oxon suggested the involvement of a
genetic component in biochemical processes where the cause of inter-individual differences
in adverse reactions was because of enzyme deficiencies (Garrod, 1902, as cited in Johansson
& Ingelman-Sundberg, 2011). Observations implying that genetic variation was responsible
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for the diversity in some drug responses was established nearly 50 years ago, with the
discovery that deficiency in glucose-6-phosphate dehydrogenase (G6DP) results in
hemolytic anemia following ingestion of the anti-malarial primaquine (Beutler, 1959).
Another example is succinylcholine, which is administered as an adjunct to anesthesia and
can induce prolonged apnea due to altered kinetics of a pseudocholinesterase (Lehmann &
Ryan, 1956). In 1959, Vogel coined the term of pharmacogenetics to describe inherited
differences in drug response (Vogel, 1959). The best-known example of a genetic defect in
drug biotransformation is the acetylation polymorphism in tuberculosis therapy with
Isoniazid characterized by mutations in N-acetyltransferase-2 (NAT2) on chromosome 8
(Evans et al., 1960). Alvan et al. (Alvan et al., 2001) remember the case of debrisoquine, an
antihypertensive agent inducing orthostatic hypotension in a small percentage of
individuals. The reason for the exaggerated effect was found to be the lack of an enzyme
almost exclusively responsible for the metabolic elimination of debrisoquine and the
affected subjects were classified as poor metabolizers of debrisoquine (Mahgoub, 1977, as
cited in Alvan et al, 2001). The enzyme named “debrisoquine hydroxylase” is now known as
CYP2D6. The oxidation of sparteine was found to be catalyzed by the same enzyme
(Eichelbaum, 1975, 1979 as cited in Alvan et al, 2001). Now it is well-established that the
therapeutic failure of drugs, and adverse side-effects in individuals may also have a genetic
component due to genetic variations in the receptors, ion channels, transporter, enzymes
and regulatory proteins involved in drug metabolism that may influence
pharmacodynamics (e.g. the binding and functional capacity of the receptor or regulatory
proteins) and pharmacokinetics, consisting in drug bio-availability at the level of metabolic
enzymes and transporters. Most studies have focused on single nucleotide polymorphisms
(SNPs) in genes encoding important metabolizing enzymes, like the cytochrome P450
enzyme superfamily, revealing an association with clinical phenotypes of drug
efficacy/toxicity (Bishop & Ellingrod, 2004; Korkina et al., 2009; Mellen & Herrington, 2005;
Wilkinson, 2005; Yang et al., 2010). One of the goals of pharmacogenetics is to develop
predictive genetic tests to reduce the risks associated with drug administration (de Leon et
al., 2006, 2009). According to the World Health Organization (WHO), adverse drug reactions
(ADRs) are any harmful, unintended reactions to medicines that occurs at doses normally
prescribed for prophylaxis, diagnosis and therapy and in some cases can lead to death
(Edwards & Aronson, 2000). ADRs represent a significant clinical and economic problem: a
prospective study conducted in the United Kingdom showed that 6.5% of hospital
admissions are related to ADRs (Pirmohamed et al., 2004). According to the Food and Drug
Administration (FDA), the frequency of reported serious and fatal adverse drug events
increased 2.6 fold from 1998 through 2005 (Moore et al., 2007). Moreover, it has been
estimated that ADRs were between the fourth and sixth leading causes of death in the world
due to treatment with drugs like anti-inflammatories, analgesics, antidepressants, sedatives,
anticoagulants and antibiotics (Carleton et al., 2009; Lazarou et al., 1998). Given the
association between response to treatment and genetic variability on the basis of clinical
trials, the European Medicines Agency (EMEA) and the FDA currently recommend the use
of biomarkers in informing prescribing decisions for certain drugs (Frueh et al., 2008).
Moreover, growing information is available on biomarkers indicating whether a therapy
could work on a particular individual. In 2004, a “Personalized Medicine Coalition” was
launched in the USA, giving strong input to the US Senate bill on the Patient-Centered
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Forensic Pharmacogenetics 269
Outcome Research Act. But questions arise, including: Will pharmacogenetics in general be
accepted by physicians and patients? “Safe and effective medicines for all” is a vision that
will not come true in general. Furthermore, even if its outcome and cost-effectiveness have
to be proven, personalized medicine can currently contribute to solving the problems of lack
of efficacy, drug resistance and adverse effects in some indications, and this opportunity
should be used (Cascorbi, 2010). The scientific literature highlights the magnitude of this
public health problem at different levels and point of views and illustrates the need for
improved systems to select the appropriate drug dosage to achieve the optimal therapeutic
response, avoid therapeutic failure and minimize side-effects and toxicity (Davies et al.,
2009). Recently, Sim et al. (2011) emphasize the need of updated databases for providing
guidance to both scientists, physicians, regulatory agencies, and industries to cope with this
major problem in human health. The medico-legal implications are evident both for medical
liability issues and in forensic death investigation.
2. Genetic polymorphism of Cytochrome P450 (P450 or CYP)

The primary site of drug metabolism is the liver, where enzymes chemically change drug
components into substances known as metabolites that are then bound to other substances
for excretion mainly through the kidneys, lungs or bodily fluids or by intestinal re-
absorption. Some drugs do not change chemical structure and are removed from the body
as such. Drug pharmacokinetics and pharmacodynamics are regulated by complex chemical
reactions with the participation of numerous proteins encoded by different genes, deputies
for the transport and metabolism of drugs, or involved in their mechanism of action
(Weinshilboum, 2003). Two different types of metabolic reactions are involved: in phase 1
molecules are characterized by oxidation, reduction and hydrolysis reactions, in phase 2
drugs are conjugated with other compounds and then discarded (Johansson & Ingelman-
Sundberg, 2011; Zhou et al., 2008). If two or more polymorphic genes regulate drug
metabolism and transport inside a cell, the variability in the response to treatment depends
on the interaction of these gene variants. The cytochrome P450 enzyme system plays a
central role in phase I oxidative metabolism of the vast majority of prescribed drugs and
also of endogenous substances (Bertz & Granneman, 1997). The genes coding for these
enzymes are called CYP. The Human Genome Project identified 57 human CYPs divided
into families and subfamilies based on structural similarity in amino acid sequence of the
enzymes. Enzymes in families 1 to 3 are involved in the detoxification of exogenous
chemicals, whereas the remaining groups are mainly active in the metabolism of
endogenous compound like steroids, fatty acids and bile acids (Ingelman-Sundberg & Sim,
2010). Many of the genes involved in drug metabolism are highly polymorphic and all
researchers specify the different CYP variants as reported at the Human CYP allele
nomenclature web site www.cypallele.ki.se (Oscarson & Ingelman-Sundberg, 2002). Sim et
al. (Sim et al, 2011) describe that the main purpose of the CYP-allele website is the
management of new allele designations based on recognized nomenclature guidelines,
facilitation of rapid publication, as well as providing a readily available summary of alleles
and their associated effects. In addition they summarize the inclusion criteria of the new
alleles in the website: submission of new alleles is achieved by contacting the Webmaster of
the CYP-allele Website, whereby the data characterizing the allele is reviewed for potential
allele name designation. All information is kept confidential and a manuscript in
preparation can often serve as a good basis for review and discussion between the author
and the Webmaster. Designation of allele names outside the CYP-allele nomenclature
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committee is not advised, due to the apparent risk of confusion in the literature.
Submissions with respect to additional functional in vivo and in vitro characterization of
alleles listed are also highly relevant, and aids in keeping the CYP-allele Website up to date.
2.1 P 450 genes family: From genotype to phenotype

Of all the isoforms of the P450 gene family, CYP2D6, CYP2C19, CYP3As, CYP2C9, CYP2B6,
CYP2C8, CYP2A6 and CYP1A2 are the most important and polymorphic enzymes and are
responsible for several phase I metabolism xenobiotics (Anzenbacher & Anzenbacherová,
2001; Daly, 2003; Ingelman-Sundberg, 2004). In particular, CYP2D6, characterized by a high
inter-individual variability in catalytic activity mainly caused by genetic polymorphisms,
will be described in depth. The genetic bases of the polymorphism are single nucleotide
polymorphisms, insertions/deletions and gene copy number variations (Ingelman-
Sundberg et al., 1999). Because of such variability, individuals could be classified into four
different phenotypes: ultra-rapid metabolizers (UM) with more than two active gene copies
on the same allele or increased expression of a single gene, extensive metabolizers (EM)
carrying two functional alleles, intermediate metabolizers (IM) with one defective allele or
two partially defective alleles, and poor metabolizers (PM) lacking functional enzymes due
to defective or deleted genes. PM and UM are the most clinically important phenotypes: PM
individuals are at risk of having a higher than expected serum concentration in relation to
the drug dose and hence more side-effects, especially in the case of drugs with a narrow
therapeutic index (White, 2010; Prandota, 2010). Instead, UMs may require higher doses and
more frequent administration of a drug in an active form to achieve optimal therapeutic
concentrations. However, when an inactive “pro-drug” must be converted to the active
metabolite (e.g. codeine and tamoxifen), the therapy will be ineffective in PM subjects and
UMs will metabolize it quickly with accumulation of the metabolite and consequent toxicity.
As a result, drug toxicity is related to metabolizer status (Ingelman-Sundberg et al., 1999;
van der Weide & Steijns, 1999). Moreover, the phenomenon of phenocopying must be taken
into account where EM individuals turn into apparent PM or IM phenotypes because of
drug-drug interactions (Owen et al., 2009). Many drugs also inhibit or induce the activity of
CYPs and knowledge of CYP–drug relations is therefore essential to recognize incompatible
drug combinations and allows individualized therapies (Mishra et al., 2010). For this
purpose, a user-friendly platform for researchers and health professionals was developed
where each drug was attributed to those CYPs involved in drug metabolism as substrate,
inhibitor or inducer. The SuperCYP database contains 1170 drugs with more than 3800
interactions including references (Preissner et al., 2010). Nevertheless, epigenetics, defined
as heritable phenotypic changes not involving alteration in nuclear DNA, promises answer
to interindividual variability in drug response not associated to genetic polymorphism
(Ingelman-Sundberg & Gomez, 2010). Indeed, the CYPs expression can be influenced by
diet, lifestyle and environmental pollutants. Update of P450 epigenetics knowledge and its
relevance for cancer risk and treatment is reported in a recent review: CYP1A1, CYP1A2,
CYP1B1, CYP2E1, CYP2W1, CYP2A13 have been shown to have epigenetics component in
their expression regulation (Rodriguez-Antona et al., 2010).
2.2 CYP2D6
CYP2D6 is the most extensively studied drug metabolizing enzyme in humans and its
polymorphism was the first among polymorphic P450s to be characterized at the molecular
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level. About 20-25% of clinically used drugs are metabolized by this enzyme including beta-
blockers, antiarrhythmics, antidepressants, neuroleptics, analgesics and anti-cancer drugs.
Most of them are metabolized to the inactive form; others like codeine, tramadol and
tamoxifen are bio-activated. CYP2D6 is the only drug metabolizing CYP which is not
inducible and therefore genetic variation plays a major role in the inter-individual variation
in enzyme activity (Ingelman-Sundberg et al., 2007). The gene, located near two cytochrome
P450 pseudogenes on chromosome 22q13.1, is highly polymorphic and more than 80 allelic
variants related to the gene activity have been described (Zhou, 2009). The wild type allele is
CYP2D6*1 and major variants associated with decreased and abolished enzyme catalytic
activity are CYP2D6*2, CYP2D6*4, CYP2D6*5, CYP2D6*10, CYP2D6*17 and CYP2D6*41.
Multiple active gene copies are responsible for ultra-rapid metabolizer individuals. CYP2D6
phenotypying is characterized in vivo by the ratio of urinary amounts of parent compound
relative to oxidative metabolite. The most commonly used probe substrates have been
debrisoquine, sparteine and dextromethorphan. On the basis of the urinary metabolic ratio
(MR), PM, UM, EM and IM phenotypes have been classified, but CYP2D6 genotyping to
predict metabolic status is considered a valid alternative to traditional phenotyping
methods because genetic characteristics remain unchanged throughout life and are not
influenced by environmental and physiologic factors (Gaedigk et al., 2003; Zanger et al.,
2004). One of the most commonly used methods for CYP2D6 genotyping consists in a
combination of a first long-PCR (polymerase chain reaction) step designed to amplify the
entire CYP2D6 gene in a single fragment of about 5 kb followed by minisequencing, a
multiplex PCR by SNP genotyping method (Fig. 1), screening the 11 most important
polymorphic positions of the gene (Sistonen et al., 2005). The inferring phenotype from
CYP2D6 genotype information is based on different approaches including the conventional
classification in PM, IM, EM and UM, established on the assumption of dominance, in which
the phenotype is determined by the most efficient allele (Sistonen et al., 2007). However, this
method does not consider inter-individual variability in urinary metabolic ratio-based
phenotypes of subjects with identical genotype and the complexity of allele combinations.
For this reason, the “activity score” (AS) system has been evaluated to translate genotype
into a qualitative measure of phenotype and to overcome the difficulties of interpretation
and comparison of different studies on CYP2D6 activity. For each variant allele a value is
assigned based on CYP2D6 activity: “1” to the fully functional alleles and “0” to non-
functional alleles, “0.5” or “0.75” to reduced activity alleles and double the value to
duplicated genes. The AS is the sum of the individual allele values (Gaedigk et al., 2008).
2.2.1 Distribution of CYP2D6 polymorphism

Inter-ethnic differences in the distribution of CYP2D6 genotypes have been described
(Bernard et al., 2006; Gaedigk et al., 1999, 2002; Gaedigk & Coetsee, 2008; Griese et al., 2001;
Kitada, 2003; Leathart et al., 1998; Luo et al., 2004; Wan et al., 2001; Zhou et al., 2009). In a
worldwide survey (Sistonen et al., 2007) 5-10% of Europeans are PMs with the highest
frequency of CYP2D6*4 variant, while the UMs are most represented in North Africa and
Oceania (40% and 26% respectively) due to the high percentage of gene duplication. In
Asian populations alleles with absent CYP2D6 activity are very rare, but the *10 allele,
causing a decreased enzyme function, occurs quite frequently leading to a high percentage
of IMs. The *1 and *2 variants are the most represented in all population groups and their
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CYP2D6 *1/*1
a)
CYP2D6 *4/*4
b)
Fig. 1. Electropherograms of the multiplex PCR by SNP genotyping method

(minisequencing) of two different individuals. Peaks correspond to 11 polymorphic
positions (100C>T, 1023C>T, 1661G>C, 1707delT, 1846G>A, 2549delA, 2613-15delAGA,
2850C>T, 4180G>C, 2988G>A, 3183G>A) of the CYP2D6 gene. a) Electropherogram of a
homozygous CYP2D6*1/*1 wild type genotype and b) Electropherogram of a homozygous
CYP2D6*4/*4 genotype
homogeneous geographic distribution could be regarded as the result of a long-term
selective pressure maintaining the high frequency of alleles coding for a full-function
enzyme. However, few rarely region-specific alleles associated with an altered enzymatic
activity are observed and seem to be geographically dispersed over all four continents
(Gaedigk et al., 2006, 2007, 2009, 2010; Luo et al., 2005). Ethnic specificity has become an
integral part of pharmacogenetics research but caution is required against the use of
continental labels to lump together heterogeneous populations. The Asian category, for
example, is applied to individuals of distinct ethnicity and/or living in different countries or
regions of the vast continent of Asia. Not surprisingly, significant variation in the
distribution of pharmacogenetics polymorphism is detected among Asians (Suarez-Kurtz,
2008). Nevertheless, with increasing global migration, admixture gains relevance as an
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additional challenge to the successful worldwide implementation of pharmacogenetics in

clinical practice. The Brazilian population, with tri-hybrid ancestral roots in Amerindian,
European and African groups and five centuries of extensive inter-ethnic mating, provides a
valuable model for studying the impact of admixture on the conceptual development and
clinical implementation of pharmacogenetics-informed prescription. Recognition of this fact
is important in the design and interpretation of pharmacogenetics clinical trials in
Brazilians, but does not imply that pharmacogenetics-informed drug prescription requires
investigation of individual ancestry. Rather, individual genotyping should be directed to
polymorphisms of proven clinical utility, irrespective of biogeographical ancestry (Suarez-
Kurtz, 2010).
2.3 CYP2C9
CYP2C9 accounts for approximately 20% of total hepatic CYP content and metabolizes
approximately 15% of clinically used drugs including S-warfarin, tolbutamide, phenytoin,
losartan, diclofenac and celecoxib (Goldstein, 2001). To date, at least 33 variants of CYP2C9
(*1B through to *34) have been identified (Yasar et al., 1999, 2002). CYP2C9*2 and CYP2C9*3
differ from the wild-type CYP2C9*1 by a single point mutation: CYP2C9*2 is characterised
by a 430C>T exchange in exon 3 resulting in an Arg144Cys amino acid substitution, whereas
CYP2C9*3 shows an exchange of 1075A>C in exon 7 causing an Ile359Leu substitution in the
catalytic site of the enzyme (Wang et al., 2009). The CYP2C9 polymorphism is clinically
highly significant and also substrate-dependent (Rosemary & Adithan, 2007; Xie et al., 2001).
Marked inter-racial differences have been reported: CYP2C9*2 and CYP2C9*3 were both
found with highest frequencies in Northern African and European populations. The
frequency of CYP2C9*2 decreases rapidly when moving from Europe toward the east, and it
is practically zero in Eastern Asian populations. CYP2C9*3 occurs more evenly in different
geographic regions. (Sistonen, et al., 2009). The polymorphism in admixed populations was
studied in the context of warfarin dose requirements. Variant alleles CYP2C9*5, CYP2C9*6,
CYP2C9*8 and CYP2C9*11 occur in Africans but are rare or absent in Europeans.
Genotyping of the six polymorphisms could be justified in Brazilians and, most likely
African-Americans, but not Europeans, in whom only CYP2C9*2, CYP2C9*3 might be
adequate for predicting the CYP2C9 polymorphism (Suarez-Kurtz, 2010). Candidate-gene
association studies for warfarin response have identified CYP2C9 and VKORC1, which
codes for warfarin's target, vitamin K epoxide reductase, responsible for most of the genetic
effect. VKORC1 is a key enzyme of the vitamin K cycle and molecular target of coumarin
anticoagulants. Among whites and Asians, VKORC1 polymorphisms have shown a
consistently significant influence on warfarin response, accounting for 11% to 32% of dose
variability. Among North American blacks, VKORC1 polymorphisms account for 4% to 10%
of the variability in dose. Given that genetic diversity is known to be greater in persons of
African descent, investigators have hypothesized that other VKORC1 polymorphisms, or
combinations of multiple polymorphisms (haplotypes), may better explain the variation in
dose in this group (Limdi et al., 2010).
2.4 CYP2C19
The metabolism of tricyclic antidepressants, benzodiazepines and proton pump inhibitors is
catalyzed mainly by CYP2C19. The most common genetic variation, designated CYP2C19*2
(c.G681A), leads to a splicing defect that functionally affects the enzyme. Other alterations
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have also been reported such as loss-of-function: CYP2C19*3 (c.G636A; stop codon),
CYP2C19*4 (c.A1G; transition in the initiation codon), and CYP2C19*5 (c.C1297T; amino acid
substitution) (Santos et al., 2011). A variant defining an ultra-rapid metabolizer has been
identified (Sim et al., 2006). Pronounced ethnic differences exist in the frequencies of the
non-functional alleles: a low frequency of up to 5% in the Caucasian and African
populations, higher in Oriental populations (23%). CYP2C19*2 and CYP2C19*3 are together
responsible for the majority of PM alleles, of which CYP2C19*3 is mainly found in Asians
(Chen et al., 2008; Xie et al., 2001).
2.5 CYP1A2 and CYP2A6

CYP2A6 is an inducible enzyme primarily expressed in the liver and was first recognized for
its involvement in the metabolism of coumarin. The CYP2A6 gene locus spans a region of
6kbp and has been physically mapped to the long arm of chromosome 19. Thirteen allelic
variants have been discovered due to point mutation, deletion, and gene conversion, and
several of these result in altered enzyme activity. CYP2A6 is involved in the metabolism of
nicotine, some procarcinogens and several toxins. Variants may affect smoking, cancer and
the treatment of cigarette smoking. (Xu et al., 2002). CYP1A2 metabolizes clozapine, tacrine,
tizanidine and theophylline, a number of procarcinogens like benzo[a]pyrene and aromatic
amines, and several important endogenous compounds (e.g., steroids). CYP1A2 is subject to
reversible and/or irreversible inhibition by a number of drugs, natural substances and other
compounds. The CYP1A gene cluster has been mapped on chromosome 15q24.1, with a
close link between CYP1A1 and 1A2 sharing a common 5‘-flanking region. More than 15
variant alleles and a series of subvariants of the CYP1A2 gene have been identified and
some of them have been associated with altered drug clearance and response and disease
susceptibility (Zhou et al., 2010).
2.6 CYP2B6 and CYP2C8

CYP2B6, mapped to the CYP2 gene cluster on chromosome 19, plays a major role in the
biotransformation of several therapeutically important drugs including cyclophosphamide,
ifosfamide, tamoxifen, ketamine, artemisinin, nevirapine, efavirenz, bupropion, sibutramine
and propofol. This enzyme also metabolizes arachidonic acid, lauric acid, 17-estradiol,
estrone, ethinylestradiol and testosterone (Mo et al., 2009). Genetic polymorphisms in
CYP2B6 are defined in terms of 29 allelic variants many of which are associated with
increased, decreased or abolished enzyme activity (Watanabe et al., 2010). Overall, there is a
marked inter-individual variability in CYP2B6 activity, but current pharmacogenetic
knowledge is not sufficient to provide efficient tools to predict the specific capacity for
metabolism of CYP2B6 substrates (Ingelman-Sundberg et al., 2007). CYP2C8 is a
polymorphic phase I drug-metabolizing enzyme involved in the metabolism of several
therapeutic drugs including paclitaxel, amodiaquine, troglitazone, amiodarone and
verapamil, and has also been implicated in the activation of procarcinogenic compounds
(Totah & Rettie, 2005). The gene is located on chromosome 10q24 in a cluster with CYP2C19
and CYP2C18 and 14 different allelic variants have been reported (http://www.
cypalleles.ki.se/cyp2c8.htm). The main CYP2C8 polymorphisms code for the amino acid
changes I269F, R139K, K399R and I264M. These single nucleotide polymorphisms define
three main non-wild-type alleles, CYP2C8*2 (I269F), CYP2C8*3 (R139K and K399R) and
CYP2C8*4 (I264M). The CYP2C8*2 allele has been found in black populations with an allele
frequency of 18% but is very rare in white subjects (Dorado et al., 2008).
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2.7 CYP3A
The CYP3A drug-metabolizing enzymes facilitate the metabolism and elimination of a wide
range of structurally different xenobiotics and of 50% of all clinically used therapeutic
drugs. In addition, they participate in the metabolism of key endogenous substrates, such as
retinoic acid, steroid hormones and bile acids (Domanski et al., 2001; Ingelman-Sundberg et
al., 2007; Thummel & Wilkinson, 1998). The four CYP3A genes lie within a 218 kb region of
chromosome 7q22.1 in the following order: CYP3A5, CYP3A7, CYP3A4, and CYP3A43
(Lamba et al., 2002; Westlind et al., 2001). More than 30 SNPs have been identified in the
CYP3A4 gene (Du et al., 2006; Garsa et al., 2005). Generally speaking, variants in the coding
regions of CYP3A4 occur at allele frequencies <5% and appear heterozygous with the wild-
type allele. These coding variants may contribute, but are unlikely to be the major cause of
inter-individual differences in CYP3A-dependent clearance, because of the low allele
frequencies and limited alterations in enzyme expression or catalytic function (Eiselt et al.,
2001). The most common variant, CYP3A4*1B, is an A-392G transition in the 5'-flanking
region with an allele frequency ranging from 0% in Chinese and Japanese to 45% in African-
Americans. Studies have not linked CYP3A4*1B with alterations in CYP3A substrate
metabolism (Garcia-Martin et al., 2002; Lamba et al., 2002). CYP3A5 is polymorphically
expressed in adults with readily detectable expression in about 10–20% in Caucasians, 33%
in Japanese and 55% in African-Americans. The primary causal mutation for its
polymorphic expression (CYP3A5*3) confers low CYP3A5 protein expression and its allele
frequency varies from approximately 50% in African-Americans to 90% in Caucasians
(Adler et al., 2009; Lamba et al., 2002). CYP3A7 is considered to be the major fetal liver
CYP3A enzyme. Three of the mutations represent SNPs (CYP3A7*1B, *1D and *1E) and
occurs in regions outside those associated with the regulation of CYP3A transcription. The
fourth mutation (CYP3A7*1C) consists of the replacement of 60 bp from the CYP3A4 gene
with the corresponding sequence from the CYP3A7 gene (Lamba et al., 2002).
3. Personalized therapy: ethical and legal issues

Current prescribing practice involves administration of a standard “one size fits all” starting
dose and is often a process of trial and error, varying the prescription until the most suitable
treatment is found (Sadee, 1998). However, such a procedure exposes the patient to possible
side-effects. In fact, around 21% of all outpatients suffer some kind of adverse reaction to
drugs prescribed by their physician (Queneau et al., 2007). By investigating drug
metabolism related to individual genetic polymorphism, pharmacogenetics has a significant
impact on the clinical setting so that forensic implications may arise throughout the public
health sector. The concept of “therapy with the right drug at the right dose in the right
patient” was highlighted just about ten years ago (Mancinelli et al., 2000) and
pharmacogenetic tests are now available for a number of drugs by biotechnology
companies, some with FDA approval, as reported in Wong et al., (2010): Luminex xTag®
(Luminex Corporation, TK, USA), Roche AmpliChip® (Roche, Basel, Switzerland),
Affymetrix DMET® chip (Affymetrix, CA, USA), Autogenomics INFINITI™ Analyzer
(Autogenomics, CA, USA), Osmetech eSensors® (WA, USA), ParagonDx (NC, USA), and
ABI PRISM® SNaPshot™ (Applied Biosystems, CA, USA) and TaqMan® assays (Applied
Biosystems). In addition, the Authors underlined the limitations: existing evidence to
demonstrate significant and medically relevant correlations for many disease-causing genes
and variants, limited detection of genetic variants within the context of each testing
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platform, clinical interpretation of genotype results including environmental factors, and

transplanted organs interfering with testing. Nevertheless, SNP arrays covering 5 million
SNPs will soon become a reality and the cost for whole-genome sequencing is rapidly
decreasing (Sim & Ingelman-Sundberg, 2011). The influence of genetic polymorphism on
drug failure or toxicity can be illustrated by some significant examples.
3.1 Moving to clinical practice: significant examples

An increasing number of studies on psychiatric patients have shown that genetic variation
of CYP2D6 and CYP2C19 affects the metabolism of antidepressants and antipsychotics,
thereby explaining the different therapeutic effects in different patients (Kirchheiner &
Seeringer, 2007). Of the patients treated with antidepressants 10-20% react adversely and 25-
35% do not respond to the medication. This can lead to treatment for patients being selected
in what has been described as a “trial and error” fashion, as physicians try different
pharmacological agents with their patients in order to select the best treatment (Morley &
Hall, 2004). Given their selective mechanism of action, selective serotonin reuptake
inhibitors (SSRIs) replaced tryciclic antidepressants and monoamino oxidase inhibitors, but
because of their inhibitory effects on various CYP enzymes, SSRIs may be associated with
clinically relevant pharmacokinetic interactions with other medications (Spina et al., 2008).
In dementia, approximately 10-15% of direct costs are attributed to pharmacological
treatment and only 10-20% of the patients are moderate responders to conventional anti-
dementia drugs, with questionable cost-effectiveness (Cacabelos, 2008). Pharmacogenetic
and pharmacogenomic factors are reported to be responsible for 75-85% of the therapeutic
response in Alzheimer’s disease (AD) patients treated with conventional drugs.
Cholinesterase inhibitors of current use in AD, such as donepezil, tacrine and galantamine,
are metabolized via CYP-related enzymes. EMs and IMs are the best responders, and PMs
and UMs are the worst responders to pharmacologic treatments in AD. At this early stage of
the development of pharmacogenomic/pharmacogenetic procedures in AD therapeutics, it
seems very plausible that the pharmacogenetic response in Alzheimer's disease depends on
the interaction of genes involved in drug metabolism and genes associated with Alzheimer's
disease pathogenesis (Cacabelos, 2007). Paracetamol and tramadol were the most popular
analgesics among individuals taking warfarin. A recent study supports clinical evidence of
the significance of the adverse warfarin-paracetamol interaction, although the mechanism
responsible for the interaction is not clear. Some case reports of warfarin tramadol
interactions have been published and a CYP2D6-related mechanism has been proposed
(Launiainen et al., 2010). Warfarin (4-hydroxy coumarin), the most frequently prescribed
oral anticoagulant, has a narrow therapeutic index and a wide inter-individual variability in
dose requirement: an increase or decrease in anticoagulant activity are associated with the
risk of hemorrhagic or thrombotic events (Kamali & Wynne, 2010). Variants in the
cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase (VKOR) genes have been
shown to have a significant effect on warfarin dose requirement. Warfarin
pharmacogenetics has become a case study for personalized medicine. Algorithms
incorporating selected SNPs in two genes, CYP2C9 and VKORC1, show improved dose
prediction compared with algorithms based solely on clinical and demographic factors.
However, the performance of these algorithms differs among racial groups, with a higher
proportion of variability in dose explained in whites than in Asians or blacks. The first
comprehensive assessment of the influence of six common VKORC1 SNPS and haplotypes
on warfarin dose among Asians, blacks and whites with the use of the largest racially
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diverse cohort captured the influence of common genetic variation in VKORC1 on

variability in warfarin dose by a single polymorphism (either -1639G>A or 1173C>T) across
all racial groups. Incorporation of additional VKORC1 SNPs or haplotypes did not improve
dose prediction. Therefore, current evidence supports the use of -1639G>A (or 1173C>T) to
capture dose variability related to VKORC1. Both VKORC1 and CYP2C9 influenced warfarin
dose among individual patients in all three racial groups studied (Limdi et al., 2010). The
case of tamoxifen is well-documented in the management of women with hormone receptor
(HR) positive breast cancer (Hoskins et al., 2009). Tamoxifen is a pro-drug that is
metabolized to its main active metabolite endoxifen by the cytochrome P450 2D6. Null or
reduced enzyme activity in women treated with tamoxifen results in worse outcomes in
terms of cancer relapse and lower event-free survival rates compared to extensive
metabolizers. Addition data are required for a mandatory use of a CYP2D6 genetic test,
considering the demonstrated potential effect of CYP2C19 polymorphism on relapse risk
during tamoxifen treatment (Sim & Ingelman-Sundberg, 2011). In addition, selective
serotonin reuptake inhibitor antidepressant drugs such as paroxetine and fluoxetine have
also been used to manage tamoxifen-induced hot flushes. These drugs potently inhibit the
metabolism of tamoxifen by CYP2D6 and thus potentially may lessen the efficacy of
tamoxifen (Singh et al., 2011). Recently, it has been pointed out that there is sufficient reason
to be aware of the real possibility of herbal products inhibiting cytochrome P450 enzymes.
Considering that self-administration of complementary products is an increasingly common
trend worldwide, the interactions may significantly increase the risk of ADR (Sevior et al.,
2010). The picture that emerges highlights the crucial role of the physician to understand
when to order the pharmacogenetics test and how to use the results in clinical decision-
making (Robertson et al., 2002). The authors emphasized that if the risk of side-effects from
a drug is great, a physician who failed to order the test indicative of toxicity could be found
to have been negligent and future lawsuits for failure to give a test or properly interpret it
will ensue, both reflecting and reinforcing acceptance of pharmacogenetic testing as an
ordinary part of medical practice.
3.2 Legal and ethical issues

The development of pharmacogenetics has important implications in the medico-legal and
forensic field because the classic topics of informed consent, shared genetic information,
privacy and data base collection arise (Vaszar et al., 2002). Nevertheless the problem of
orphan patients, non-responders for all available drug options, might have unforeseen
consequences to avoid the label “hard to treat” (Robertson, 2001). Some patients might not
want to be tested for pharmacogenetics profiles and this leads to the question of what a
physician should do if a patient refuses to be genotyped. The responsibility of health care
professionals will need to be defined as to who (doctors, pharmacist, clinical chemists) is
responsible for the application of new technologies (genotyping) and what kind of patient
counseling is needed (van Delden et al., 2004). A recent paper (Hamburg & Collins, 2010)
highlighted that today about 10% of labels for FDA-approved drugs contain
pharmacogenomic information — a substantial increase since the 1990s. Furthermore, there
has been an explosion in the number of validated markers but relatively little independent
analysis of the validity of the tests used to identify them in biologic specimens. The National
Institute of Health (NIH) and the FDA will invest in advancing translational and regulatory
science, and will better define regulatory pathways for coordinated approval of co-
developed diagnostics and therapeutics, develop risk-based approaches for appropriate
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review of diagnostics to assess their validity and clinical utility more accurately, and make
information on tests readily available. As the field advances, they expect to see more
efficient clinical trials based on a more thorough understanding of the genetic basis of
disease. The impact on the medico-legal field is evident. But, there are two schools of
thought on how tort liability may affect personalized medicine, i.e., whether fear of lawsuits
will tend to accelerate progress or slow it down. Tort suits include product liability suits
against manufacturers and negligence suits against physicians and other providers of
health-related services (Evans, 2007). Recently, Wong et al. (2010) stressed that personalized
medicine by means of pharmacogenomics may have a dramatic impact on the justice system
in ways we are only beginning to understand. They stated that if personalized medicine has
already entered the curricula of well-regarded medical schools such as that of Johns
Hopkins University (MD, USA), law schools offer no analogue. For example, “The FDA
relabelled some drugs such as warfarin with CYP2C9 and vitamin K epoxide reductase
complex 1 to reduce bleeding. If pharmacogenetics retrospectively reveals that the warfarin
patient was at high risk and testing was not initially performed, litigation may follow.
Indeed, some lawyers advertise on the Internet for cases involving warfarin-related errors.
Consequently, pharmacogenomics may become part of defensive medicine”. An important
issue for legal and ethical use of pharmacogenetic tests arises from the evidence of genetic
polymorphism distribution in different ethnic groups. CYP2D6 gene polymorphism shows
that the distribution of PMs, IMs and UMs varies in different ethnic groups so inter-ethnic
differences in drug response highlight the need to evaluate the genetic makeup of
individuals before prescribing drugs, also considering that the recent demographic
movements and back migration of populations are reshaping the picture of genetic diversity
within the native population (Suarez-Kurtz, 2010). A recent review by McNamara
(McNamara, 2008) discusses investigations of pharmacogenomics in heart failure and the
challenge of converting genomic heterogeneity into a usable clinical tool, concluding that
investigators are beginning to delineate the genomic basis for differences in drug efficacy
between black and white heart failure cohorts. The influence of CYP2C9 and VKORC1
genotypes on warfarin dose requirements has been demonstrated in diverse racial and
ethnic patient groups and to choose a warfarin starting dose, dosing algorithms have been
developed that incorporate clinical, demographic and genetic information. Because of these
significant issues, the Journal of Health & Life Sciences Law explored personalized or
patient-tailored medicine addressing the relevance of genetic information, and how race and
genetics have affected and may impact on the development of medicines,
pharmacogenomics and personalized medicine in the United States (Braff et al., 2008). A
second part discussed current and proposed federal and state laws and regulations intended
to protect individuals from the misuse of genetic information, including uses that
discriminate based on genetic predispositions (Braff et al., 2009). It was recently pointed out
(Nebert et al., 2008) that numerous reasons exist to show that an "unequivocal genotype" or
even an "unequivocal phenotype" is virtually impossible to achieve in current limited-size
studies of human populations. The problem of insufficiently stringent criteria leads to a
decrease in statistical power and consequently an equivocal interpretation of most
genotype-phenotype association studies. It remains unclear whether personalized medicine
or individualized drug therapy will ever be achievable by DNA testing alone. The authors
ask “Where are we, today, in our understanding of the role of human genetics and genomics
in drug toxicity, efficacy and therapeutic failure? Few high-prevalence predominantly
monogenic genes that do make a difference in metabolism of various drug substrates (e.g.,
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CYP2D6, NAT2, TPMT, CYP2C19) might contribute perhaps 15% to 20% to the (EM or PM)
phenotype, whereas a contribution of 90% or more is expected for the gene responsible for a
monogenic human disease. If we know that dozens or hundreds of additional downstream
genes might affect the ultimate outcome of a particular drug, how can we integrate and
assemble this knowledge into a diagram or equation?” A recent paper discussed the
possible application of genotyping for depression, cardiovascular diseases and
thromboembolic disorders, gastric ulcer, malignant diseases and tuberculosis (Tomalik-
Scharte et al., 2008). The authors noted that thousands of manuscripts addressing
pharmacogenetic questions in in vitro studies and clinical trials have been published, but it
seems that the way to a broader use of pharmacogenetics approaches at the patients’
bedside is quite laborious. The unknown exact relationship between the genotype and
phenotype, although in many cases the genotype explains most of the inter-individual
variability, the lack of prospective clinical studies in large patient cohorts and no reliable
data on the cost effectiveness of screening procedures explain the slow progress in clinical
pharmacogenetics/pharmacogenomics. In this respect, Zhou (Zhou, 2009) wrote that the
functional impact of most CYP2D6 alleles has not been systematically assessed for most
clinically important drugs that are mainly metabolized by CYP2D6, though some initial
evidence has been identified for a very limited number of drugs. The majority of reported in
vivo pharmacogenetic data on CYP2D6 are from single-dose and steady-state
pharmacokinetic studies of a small number of drugs. Pharmacodynamic data on CYP2D6
polymorphisms are scanty for most drug studies. Given that genotype testing for CYP2D6 is
not routinely performed in clinical practice and there is uncertainty regarding genotype-
phenotype, gene-concentration and gene-dose relationships, further prospective studies on
the clinical impact of CYP2D6-dependent metabolism of drugs are warranted in large
cohorts. The expectation is that this research field will provide both the industry and
clinicians with useful pharmacogenomic biomarkers that can aid in procedures for drug
development and specific drug treatment in order to optimize the results and improve
human health. The process is slow, and for a solid basis for decisions on mandatory
biomarkers to be used further large prospective clinical studies are required. One fruitful
manner in which this can be achieved is a closer collaboration between industry and
academics (Johansson & Ingelman-Sundberg, 2011).
4. Forensic investigation
In the forensic context, pharmacogenetics can assist in the interpretation of drug-related
deaths, especially accidental drug poisonings or cases of sudden death with “nearly normal
autopsy "(Karch, 2007), called “white autopsy” in Italy. The author claims that the ability to
identify "invisible diseases" with post-mortem genetic testing has become a reality far more
quickly than anyone had ever imagined and this development is not without irony: “at the
same time that many clinicians are expressing frustration about the lack of tangible gains
provided by the Human Genome Project and pathologists are wondering about the viability
of their field, DNA technology is about to reshape the field of forensic pathology”. The role
of pharmacogenetic analysis in forensic investigation has already been emphasized as the
holistic approach of molecular analysis connected to macroscopic, microscopic and
toxicological observations, constituting an integral part of modern medico-legal study of
death (Koski et al., 2007). Nevertheless, the area of medico-legal investigation also involves
occupational medicine due to the consequences on toxic-exposed workers. In this field the
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role of CYP2D6 genotype in determining parkinsonism resulting from pesticide exposure

may play an important role (Elbaz et al., 2004). Pesticide exposure significantly increases the
risk for Parkinson’s disease even when the poor metabolizer allele is in the heterozygote
state. Interestingly, poor metabolizers are less common in the Parkinson’s disease group in
rarely exposed subjects (Deng et al., 2004). Pharmacogenetics also plays an important role in
drug-addiction studies: methadone is metabolized through the liver by cytochrome P450
enzymes CYP3A4, CYP2D6 and CYP1A2 and buprenorphine is mainly metabolized by
CYP3A4 enzyme. A recent review reported that Caucasians who lack CYP2D6 function
appear to be protected from oral opioid dependence since this genotype is under-
represented in the opiate-addicted population and these poor metabolizers are satisfied with
the withdrawal and anticraving relief provided by methadone treatment. Ultra-rapid
metabolizer heroin-dependent patients have felt dissatisfied with methadone therapy and
can do well using buprenorphine because it is not significantly metabolized by CYP2D6
(Haile et al., 2008). In a limited number of cases of methadone toxicity, Wong et al. (2003)
showed that the prevalence of poor metabolizers was higher but not significantly different
from that of a control group (n=23). They concluded that CYP2D6 mutations may not yet be
directly associated with methadone toxicity, and pharmacogenomics, complementing other
case findings in molecular autopsy, is considered an adjunct in interpreting the methadone
toxicity of poor and intermediate metabolizers.
4.1 Post mortem analysis

Genetic variation and its effects on metabolism can be applied to post-mortem analysis to
help resolve cases initially believed to be suicide or classified as sudden unexplained deaths
especially in cases where poisoning, incapacitation, inebriation or certain diseases where
pharmacotherapy is an essential treatment (such as epilepsy, depression, cardiac diseases or
diabetes) are factors in the cause of death. An additional benefit is that pharmacogenetics
analysis may provide health information (certainly only via proper ethical disclosure
practices) to at-risk relatives (Budowle & van Daal, 2009). As reported recently, the medico-
legal community has yet to fully exploit genetic variation as a parameter in determining the
causes of death as done by the National Academy of Science in recognizing the
underutilization of molecular autopsies (Sajantila et al., 2010). The authors of this valuable
review stressed that an individual’s pathophysiological phenotype affecting drug efficacy
depends on genetic constitution and several other factors such as developmental stage,
physiological and environmental factors, association with disease or specific conditions.
Hence, some of these studies may be ethically unacceptable or practically impossible to
perform in the clinical setting, but may be more readily performed post-mortem as part of
the cause of death investigation or retrospectively with proper authorization. Therefore they
recommend that serious consideration and support be given to studies of medico-legal
genetics not just because of the impact on death investigation but because of the tremendous
value such information can have for personalized medicine. From this point of view and
due to the increasing attention paid to sudden cardiac death, the role of pharmacogenetics is
now studied in more depth considering that cytochrome P450 enzymes in acquired Q-T
prolongation are more prevalent than the congenital form. Several risk factors have been
identified with use of Q-T prolonging drugs as the most frequent cause (van Noord et al.,
2010). The CYP2D6 hydroxylation capacity has already been implicated in causing
elongation of the Q-T interval: patients treated with thioridazine that inhibits CYP2D6
activity itself, may be prone to an increased risk of death due to sudden arrhythmia such as
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“torsades de pointes” (Llerena et al., 2002). In general, fatal drug toxicity has been associated
with either slow or ultra-rapid CYP2D6 metabolism depending on the substrate activation
or inactivation. Sallee et al., (2000) described the clinical course of a nine-year-old boy
diagnosed with attention-deficit hyperactivity disorder, obsessive-compulsive disorder and
Tourette's disorder and treated with a combination of methylphenidate, clonidine and
fluoxetine. After experiencing signs and symptoms suggestive of metabolic toxicity marked
by bouts of gastrointestinal distress, low-grade fever, incoordination and disorientation for
more than ten months, the patient presented generalized seizures, lapsed into status
epilepticus followed by cardiac arrest and subsequently expired. At autopsy, blood, brain
and other tissue concentrations of fluoxetine and norfluoxetine were several-fold higher
than expected based on literature reports for overdose situations. The medical examiner's
report indicated death caused by fluoxetine toxicity. As the child's adoptive parents
controlled medication access, they were investigated by social welfare agencies. Further
genetic testing of autopsy tissue revealed a gene defect at the cytochrome P450 CYP2D6
locus, resulting in poor metabolism of fluoxetine. As a result of this and other evidence, the
investigation of the adoptive parents was terminated. One of the first demonstrations that
genetic variation in drug metabolizing enzyme can be analyzed in post-mortem blood was
performed studying the CYP2D6 gene variations correlated to the tramadol metabolite ratio
in blood in 33 Finnish autopsy cases where tramadol was found (Levo et al., 2003). A series
of fatal poisonings due to amitriptyline (AT) abuse not all due to suicides was reported in
Finland in 2005 (Prahlow & Landrum, 2005). In view of this, Koski et al. (2006) investigated
the genetic variation at CYP2D6 and CYP2C19 genes with the metabolic ratio of the drug in
post-mortem samples. No cases of fatal poisoning due to a combination of AT treatment and
poor metabolizer phenotypes was found, with the exception of one case of female suicide
with a very high concentration of AT and homozygous null alleles at CYP2D6. The authors
emphasized the role of confounding factors in the interpretation of pharmacogenetics
results such as age and enzyme inhibition by drugs. The pharmacogenetics analysis in a
post-mortem forensic setting to reveal the cause and manner of death demonstrated doxepin
poisoning associated with a completely non functional CYP2D6 genotype, considering that
CYP2D6 is a major factor involved in the large inter-individual variation in doxepin
metabolism (Koski et al., 2007). However, ultrarapid metabolizer for duplication in CYP2D6
may also be responsible for fatal toxicities. The risk of opioid poisoning to breast-fed
neonates whose mothers had been prescribed codeine was studied after a fatal case.
Neonatal morphine plasma concentrations were simulated for various combinations of
CYP2D6 genotype and morphine clearance. Neonates of mothers with the ultrarapid
CYP2D6 genotype and neonates of mothers who are extensive metabolizers have
comparable risks of opioid poisoning (Willmann et al., 2009). A tragic case was reported in
2006 when a 13-day-old baby died from morphine poisoning. Review of the medical records
revealed that the mother had been prescribed Tylenol® 3 (codeine 30 mg and acetaminophen
500 mg) in the immediate post-partum period. Initially she took two tablets twice daily, but
she halved the dose on post-partum day two owing to somnolence and constipation.
Following the development of poor neonatal feeding, the mother expressed milk and stored
it in a freezer. Analysis of the milk for morphine using a specific enzyme-linked
immunosorbent assay method for morphine revealed a concentration of 87 ng/mL. The
mother was later classified as a UM of CYP2D6 substrates, carrying one extra copy of a
functional CYP2D6 gene (Madadi et al., 2007). Since 2007, the FDA has required the
manufacturers of prescription codeine products to include information on the label to
inform prescribing doctors about these risks and to help prevent morphine overdose in
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breast-fed infants. Recently, the death of a ten-month-old boy found dead in the bed after
exposure to-ethylmorphine was reported. The morphine-to ethylmorphine ratio in blood
from the deceased was higher than expected for the exclusive ingestion of ethylmorphine.
The explanations considered that the metabolism of ethylmorphine involves both the de-
ethylation to morphine by CYP2D6 and the conjugation to glucuronide by the
glucuronosyltransferase UGT2B7. The activity of the enzymes varies for genetic reasons and
is influenced by age. Infants have lower glucuronidation capabilities than adults and
CYP2D6 activity may exceed adult levels in infancy. The CYP2D6 genotyping excluded the
hypothesis of an ultra-rapid phenotype (Helland et al., 2010). Nevertheless, the genetic
variability of CYP2D6 and possibly in UGT2B7 was studied in a large number of women
receiving codeine for obstetric pain while breast-feeding. Breast-fed infants of mother who
were CYP2D6 UM combined with UGT2B7 *2/*2 are at increased risk of potentially life-
threatening central nervous system depression (Madadi et al., 2009). Molecular autopsy
research was also performed on fentanyl that is clinically used as an adjunct to surgical
anaesthesia or for chronic pain management and its toxicity may be partially due to CYP
3A4*1B and 3A5*3 variant alleles, resulting in variable fentanyl metabolism. The study of 25
fentanyl-related deaths by the analysis of fentanyl and norfentanyl in post-mortem blood
samples showed the first scientific evidence of CYP3A5 involvement in fentanyl metabolism:
homozygous CYP3A5*3 causes impaired metabolism of fentanyl, and CYP3A4*1B and 3A5*3
variants may help to certify the fentanyl toxicity (Jin et al., 2005). Furthermore,
pharmacogenetic analysis has gained burgeoning interest in suicidal cases both for the
correlation with antidepressant therapy and in researching endogenous serotonin
metabolism. A study of the genetic profile of individuals in relation to the presence of
CYP2D6 and CYP2C19 genes in 242 fatal intoxications, 262 suicides and 212 natural deaths
showed that those dying from suicide (including hanging, shooting, sticking/cutting or
jumping from a height) included a higher number carrying more than two active CYP2D6
genes. A possible explanation was the lower concentration level of prescribed medication in
these ultra-rapid metabolizers resulting in ineffective treatment, but no information was
available on the medical history of the suicide cases (Zackrisson et al., 2010). Discussing the
results of Isacsson et al. (Isacsson et al., 2009) and the treatment of depression with
antidepressants, mainly SSRI, in preventing suicide, Bertilsson (2010) recently reported that
the data seem to indicate that serotonergic mechanisms are involved in the etiology of
suicidal behaviour. An alternative explanation to the overrepresentation of CYP2D6 gene
duplication among suicide cases was based on the presence of the CYP2D6 enzyme in the
human brain where its distribution follows that of dopamine nerve terminals,
demonstrating several endogenous substrates of CYP2D6 among which of special interest is
the O-demethylation of 5-methoxytryptamine to serotonin. The author reported that
serotonin might then be synthesized in dopaminergic neurons by CYP2D6 and act as a false
transmitter in wrong (inhibitory?) neurons.
5. Conclusion
Current pharmacogenetics research in the clinical and medico-legal settings provides new
options for disease treatment and prevention of ADR avoiding correlated death, and for
screening interactions with the polymorphic P450 enzymes early on in drug development.
The ensuing information will be translated into routine clinical practice in the years to come
benefitting millions of patients worldwide (Ingelman-Sundberg & Sim, 2010). In the future,
the research in relatively new fields such as epigenetics and small nuclear RNA mediated
www.intechopen.com
mechanisms will increase the number of useful biomarkers for personalized therapy.
Indeed, epigenetics providing answers to interindividual variability in drug response not
associated to genetic polymorphism, could represent the bridge that connects the
environment to the genome (Gomez & Ingelman-Sundberg, 2009). In this respect, the area of
pharmacoepigenomics has a promising future (Ingelman-Sundberg & Gomez, 2010). In
medico-legal setting, molecular autopsy is becoming a reality also considering that robust
techniques suitable for implementation in forensic laboratories are broadening the genetic
analysis of P450 gene polymorphism. But we agree with Sajantila et al. (2010) that “in some
ways this situation now confronting medico-legal geneticists is similar to the early years of
the DNA level human identification era. The societal and judicial systems sought the
technology and scientists had serious challenges to cope with demands. Like DNA-based
identification at that time, fundamental pharmacogenetic research needs to be performed so
that our knowledge is sufficient to render valid and reliable interpretations related to
medico-legal genetic findings”. Significantly, Wong et al. (2010) state that personalized
justice complements personalized medicine, but “personalized justice” in a firm foundation
should be based on sound legal principles as well as reliable and valid evidence-based
studies, not on ‘junk’ science and unsubstantiated case reports. Furthermore, the American
Academy of Forensic Sciences supports the National Academy of Science’s 13
recommendations (National Academy of Sciences, 2009, as cited in Wong et al., 2010) and
the following principles: the need for strong scientific foundations; laboratory accreditation;
certification of technicians; the standardization of terminology; ethical protocols;
governmental oversight; and the education of legal professionals, including judges, in
forensic scientific methods and principles.
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13
Forensic Microbiology
Herbert Tomaso and Heinrich Neubauer
Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health,
Institute of Bacterial Infections and Zoonoses, Jena,
Germany
1. Introduction
Biocrime or bioterrorism is the threat or use of microorganisms, toxins, pests, prions, or their
associated ancillary products to commit acts of crime or terror. Microorganisms can
malevolently be used as biological warfare agents, in bioterrorist acts, and in crimes without
political intentions. Such actions can be directed against humans and animals and can lead
to outbreaks of infections with high morbidity and mortality. In recent years microbial
forensics has been established as a new scientific discipline to strengthen the law
enforcement response especially in a bioterrorism event (3). These tools can also be applied
to investigate the transmission of pathogenic microorganism caused by sexual abuse and
other physical offenses (1).
The aim of this review is to describe the corner stones of microbial forensics as a novel type
of forensic analysis defined as “the detection of reliably measured molecular variations
between microbial strains and their use to infer the origin, relationships, or transmission
route of a particular isolate” (10).
Several microorganisms are a severe threat to human and/or animal health and a country's
agricultural economy. Their malevolent use can have a major socio-economic impact. A
number of these pathogens can affect both humans and animals (zoonoses), can contaminate
the environment for decades, or may establish new enzootic foci. The World Organisation
for Animal Health (OIE) lists several of these agents as diseases of importance to
international trade with serious export restrictions for countries where the diseases are
endemic.
Biological warfare agents to be used against humans and animals were developed and
weaponized in the fifties of the last century in several countries including the USA, the
former Soviet Union and the United Kingdom. An international arms control and
disarmament treaty, the Biological Weapons Convention (BWC), banned the use of
biological weapons in 1972 (http://www.opbw.org/convention/documents/btwctext.pdf).
Today only few states are under suspicion of having biological warfare programs.
Politically-binding confidence building measures provide a permanent transparency tool for
building confidence in compliance with the BWC.
In the aftermath of the anthrax letters attacks in October 2001 that killed five people it has
become evident that biocrimes can only be solved when genomic information can be used to
identify the source of an organism. Evidence in a criminal investigation must be collected
within the constraints of legal rules to ensure that any prosecution based upon that evidence
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can withstand judicial review in a court. Therefore, first responders must learn how to
secure evidence and preserve the chain of custody (34). Quality-assurance and –control
procedures have to assure that reliable evidence can be presented in court (3). Laboratories
that have been officially accredited will be able to provide all relevant documents regarding
quality control and assurance, proficiency test results, qualification of laboratory personnel
etc. Also case specific material like photographs of gels, benchnotes, validation studies, and
controls will usually be adequately documented. Evidence collection, transport, and storage
need more attention than is usually needed for clinical routine samples. Test procedure will
be according to standard operating procedures (SOPs) and any deviation of protocols will
have to be documented. The final report should contain information about the specificity
and accuracy of the applied tests and provide an interpretation of the result and its
limitations.
Nucleic amplification and molecular-epidemiological techniques are essential tools in
clinical microbiology for identifying pathogens and in outbreak investigations. Various
typing tools have been developed for phylogenetic and phylogeographic studies. In forensic
microbiology these methodologies can be used to detect and trace back the spread of
microorganisms in the context of a crime. Whole-genome sequencing provides the most
comprehensive, reliable and reproducible information about a strain, but until recently this
technique was expensive and time consuming. The subsequent annotation of sequences was
also a major endeavor. Nowadays this technique has become affordable and reference
genomes for all select agents have been sequenced. They can be used to clarify the
relationship of suspicious isolates with reference genomes.
Centralized reporting and surveillance systems on the national and international level are
essential as single cases may be regarded as sporadic although they are part of a larger
transboundary outbreak. Surveillance systems have already been established that store and
provide DNA fingerprints of microbes being major causes of hospital-acquired or food
borne infections.
Descriptive epidemiological data have to be analyzed with caution. Natural outbreaks can
be difficult to discriminate from intentional use of microorganisms, especially if the
organisms are endemic. Only molecular-epidemiological tools can corroborate the chain of
infection.
This review will discuss the value of diagnostic and molecular-epidemiological tools
developed for select agents and will provide examples of investigations focused on for
example Bacillus anthracis, Francisella tularensis, and Yersinia pestis. The critical role of sample
collection, packaging, transport, and storage will be highlighted.
2. Sample collection
Laboratories involved in forensic microbiology analysis must be prepared to deal with
chain-of-custody documentation, secure storage of evidence, tracking of individual items of
evidence and their derivatives and all the legal requirements for handling evidence. Chain-
of-custody protocols document the unbroken chain of records showing who had handled
the evidence, where and under which conditions (temperature, time etc.) the material had
been stored and whether access to the samples was restricted (27). The NATO document
AEP-10 “Handbook for Sampling and Identification of Biological and Chemical Agents
(SIBCA)”, 2007, 5th Edition, Procedures and Techniques, Volume 1 (STANAG 4329) provides
practical guidelines how to sample select agents in the field even in a contaminated
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Forensic Microbiology 295
environment. These guidelines are used by NATO and Partnership for Peace (PFP)
countries. Countries may have different national requirements, but general principles can be
a guideline for Civilian-Military Cooperation (CIMIC) or purely civilian operational and
forensic investigation teams. The European Guideline on Principles of Field Investigation
“Biological Incident Response and Environmental Sampling” was published by the EU
Commission, DG Health and Consumer Protection, Health Threats Unit in October 2006 and
“describes the principles of response in the initial phase of a biological incident where the
goal is to identify what has happened in order to initiate appropriate countermeasures”.
These documents underline the necessity of planning and pre-mission briefings as the
environment may be life-threatening. Moreover, the quality of primary samples is critical for
subsequent analyses. The personal protective equipment is also affecting personnel by
limiting mobility, flexibility, and time available to work at the scene (2).
3. Sample matrix analysis

In clinical microbiology the sample matrix is important to decide, whether the analyses
requested by the clinician are appropriate and which tests should be performed.
Unfortunately, requests are not always justified by the clinical presentation and the sample
matrix is sometimes conflicting. For example a microscopical inspection of sputum samples
will indicate, if the quality of the specimens is adequate. In forensic microbiology the same
rules apply, but more detailed investigations may be necessary to obtain relevant
information about the history of a specimen, environmental conditions, chemical and
physical constitution of the matrix, presence of pollen etc. (2).
This can be achieved by particle sizing, electron microscopy, analytical chemistry, isotope
analysis, and other techniques. However, several analyses will have to be performed outside
appropriate laboratory safety containment (e.g. BSL-3 for Y. pestis) and therefore, specimens
will have to be inactivated. Especially when anthrax spores have to be killed the inactivation
with chemical or physical techniques is quite aggressive. This treatment does not only
denature the pathogens but will also cause changes of the matrix. It has to be demonstrated
for each method that the inactivation process does not interfere with the subsequent tests.
4. Biological agents
The Centers for Disease Control (CDC) in Atlanta have evaluated the priority of agents
according to their relevance for national security due to ease of dissemination and
transmission from person to person, high mortality rates, the potential for major public
health impact, risk of public panic and social disruption, and the requirement of special
action for public health preparedness. Category A includes the most dangerous agents:
Variola major virus (smallpox), Bacillus anthracis (anthrax), Yersinia pestis (plague),
Clostridium botulinum toxin (botulism), Francisella tularensis (tularemia), and viral
hemorrhagic fever viruses (33).
4.1 Microbe identification by classical microbiology

The identification of microbial agents – as defined by the SIBCA handbook - can be
provisional (presumptive), when immunological methods, nucleic acid detection or
cultivation and metabolic assays have been tested positive. Identification is confirmed by the
combination of at least two of the above mentioned criteria. Unambiguous identification
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requires cultivation and in vivo studies (animal models) that prove the pathogenicity of the
agent. However, animal models should be avoided for ethical reasons whenever possible.
Biological agents can be difficult to cultivate due to sample contamination, low number of
bacteria or pretreatment of patients with antibiotics. Some bacteria are fastidious (F.
tularensis, Brucella spp.) and require special nutrient media, and some need prolonged
cultivation times (Brucella spp.). Phenotypical characteristics such as antibiotic susceptibility
and biochemical reaction profiles, susceptibility to specific phages, colony morphology and
others are not always reliable. Mutations of agents can be induced or engineered, but
naturally occurring atypical strains have also been found e.g. among Bacillus anthracis and
Yersinia pestis isolates which can result in misidentification and treatment failure (52).
Commercial biochemical identification systems are not optimized for these agents and can
result in misidentification. Multiple antimicrobial resistances can occur through natural
horizontal gene transfer or by genetic manipulation. Natural resistance to a multitude of
antimicrobials is typical for Burkholderia pseudomallei. Francisella tularensis is naturally
resistant to penicillins and cephalosporines. A very dangerous multidrug resistant strain of
Yersinia pestis has been isolated from a patient with bubonic plague in Madagascar. This
strain carries a self-transmissible plasmid with a genetic backbone also prevalent among
Escherichia coli, Klebsiella spp. and Salmonella spp. conferring high-level resistance to
streptomycin, tetracyclin, chloramphenicol, and sulfonamides (50). These facts underline the
importance of cultivation and the assessment of antimicrobial susceptibility in addition to
more rapid diagnostic tools. A polyphasic approach for identification and typing will help
to avoid problems due to atypical genotype and phenotype, inhibition, or lack of specificity
or sensitivity of assays.
Handling of select agents is highly dangerous and cumbersome and restricted to
laboratories with biosafety-level 3 containment. Biosafety-level 3 laboratories have to be
operated according to special regulations that require e.g. a sophisticated ventilation system
and personal protective equipment (e.g. FFP3 masks, overalls, face shields, gloves etc.).
4.2 Nucleic acid amplification techniques

Many real-time PCR assays are highly specific and sensitive and shorten the time required
to establish a diagnosis in comparison with conventional PCR protocols, cultivation, and
biochemical identification methods. Therefore, real-time PCR assays have been developed
for the identification of Bacillus anthracis, Brucella spp., Burkholderia mallei and Burkholderia
pseudomallei, Francisella tularensis and Yersinia pestis (21). PCR results can be false negative
due to inadequate quality of clinical samples, low number of bacteria in samples, DNA
degradation, inhibitory substances and inappropriate DNA preparation.
4.3 Serology
Seroconversion may prove the exposure to a certain agent in the past. However,
seroconversion can be expected only after several days or weeks and is of little use for
rapidly diagnosing infections caused by highly pathogenic agents. It will be difficult to
organize serological investigations (including follow-up tests) when a terrorist attack causes
mass casualties that need medical treatment or when the situation is complicated by civil
unrest, war or natural catastrophes at the same time.
Various immunological assays have also been used to identify pathogens in samples of
patients and environmental samples. Hand-held test kits can be used as bed-side tests and
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are useful under field conditions, but clinical validations hardly exist and most tests are “for
scientific use only”. Immunochromatographic lateral-flow assays have been developed e.g.
for brucellosis, tularemia, and plague (4; 29; 37; 44). Limitations of these immunological
assays are that they are frequently not available commercially, not specific enough, or have
not been validated and licensed for use in humans or animals. Moreover, cross-reactions
may cause false positives and modified or missing antigenic structures can cause false
negatives.
5. Typing and strain identification

Differences among microbes have to be assessed to determine whether strains are from the
same source or lineage or from a different origin. The accuracy and precision will depend on
the typing method, expected mutation rates, and other characteristics of the organism. In
court scientists may need to quantify the reliability of a relationship among strains
determined using molecular phylogenetic analyses. This will establish the probability of
association to a certain source of infection (16).
Techniques for forensic microbiology can be very similar to those being used for
phylogenetic and epidemiological investigations e.g. for food-borne outbreaks.
Molecular-epidemiological tools used for genotyping are most promising and have been
applied in the past to elucidate the origin of biological agents. Especially whole genome
sequencing and bioinformatic tools for comparison of genomes are potent tools, but
technical complexity and costs are still prohibitive for routine application.
In several chapters of the highly recommendable book “Microbial Forensics” by Bruce
Budowle and many other “founders” of this new scientific discipline it was demonstrated
that only highly specialized knowledge of microbial genetics will allow an assessment of the
relevance of typing results obtained by Multi-locus Sequence Typing (MLST), Variable
Number of Tandem Repeats (VNTR), Single Nucleotide Polymorphisms (SNPs) analysis or
other typing tools (“Microbial Forensics” B. Budowle. ISBN 978-0-12-382006-8). Validation of
typing assays and data of large collections of strains from all over the world are crucial for
microbial forensic investigations. Typing methods should be reproducible, stable during the
study period, applicable to every isolate, discriminating among isolates, and discrimination
should be concordant with the epidemiological picture (46). DNA sequence-based data are
robust, portable, easy to compare, and amenable to computerised analysis for
phylogeographical and epidemiological studies. However, the quality of open access
sequence databases depends on the accuracy of submitted sequences and is consequently
sometimes not reliable.
6. Select agents
6.1 Anthrax
Bacillus (B.) anthracis is the causative agent of anthrax and a member of the Bacillus cereus
group. This group includes B. anthracis, B. cereus, B. thuringiensis, B. weihenstephanensis, and
B. mycoides. These closely related bacteria can be discriminated by using phenotypic
characteristics. B. anthracis is typically non-motile, susceptible to penicillin, lysed by the
gamma phage, and colonies are non-hemolytic with a typical morphology. However, more
than 24 hours are required to assess these characteristics and misidentification can
occasionally occur due to variations of the phenotype. Natural infections result mostly in
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cutaneous anthrax. Anthrax caused by inhalation is rare, but the spores of B. anthracis can
easily be disseminated in aerosols. The spores are very stable and persist in the environment
especially in soil for many years or even decades (35).
In the fall of 2001 an attack with “anthrax letters” resulted in 22 cases including five deaths
in the USA. This incident was investigated using VNTR analysis as described by Keim (22)
and sequencing of pag A coding for the protective antigen which is one of the toxin genes of
the bacterium. The obtained B. anthracis strains were all identical and could be identified as
the Ames strain (18; 31). The Ames strain was originally isolated from cattle in Jim Hogg
County, Texas, in 1981 (47). Whole genome sequencing revealed Ames specific SNPs and
real-time PCR assays using TaqMan MGB probes were designed to rapidly identify the
strain that was used in the bioterrorist attacks (47). This was a novel microbial forensic tool
for differentiating natural outbreaks from an attack. However, in 2001 the CDC used MLVA
for subtyping isolates and in a future attack other strains might be used which would
require fast adaptation of this methodology. Massively parallel sequencing (MPS)
technology will allow rapid whole-genome characterization (9).
6.2 Plague
Plague is caused by the gram-negative bacterium Yersinia pestis and is still endemic in
natural foci of Asia, Africa, and America in rural areas (19). The affected population is
mostly poor and is living under deplorable hygienic conditions. Due to the high lethality of
plague a rapid and reliable identification of the organism is crucial, but medical services and
laboratory facilities are very scarce in the endemic regions of Africa and Central Asia.
The validation of diagnostic assays for infectious diseases like plague can be demanding
because of very limited access to clinical samples and isolates. None of the previously
published real-time PCR assays for diagnosing plague had been clinically validated so far.
In Madagascar a relevant number of cases is reported each year and a good surveillance
system based on the well equipped laboratory facility at the Institut Pasteur in the capital
Antananarivo is in place. In a retrospective clinical study we evaluated real-time PCR assays
by testing lymph node aspirates from 149 patients with a clinical diagnosis of bubonic
plague. In this study results of real-time PCR assays targeting the virulence plasmids pPCP1
(pla), and pMT1 (caf1 and Ymt) were compared with an F1-antigen immunochromatographic
test (ICT) and cultivation of the organism. Out of the 149 samples infection with Y. pestis
was confirmed by culture in 47 patients while ICT was positive in 88 patients including all
culture proven cases. The most efficient real-time PCR assay was the 5´-nuclease assay
targeting pla being positive in 120 cases. It can therefore be recommended as diagnostic tool
for establishing a presumptive diagnosis when bubonic plague is clinically suspected (32).
Assays for targets on the chromosome and on the second Y. pestis specific plasmid were
included because strains lacking one of the specific plasmids occur naturally and can also be
highly pathogenic (13; 51).
The evolution and phylogenetic analysis of Y. pestis has been studied with MLVA and
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) (8; 24). These
genotyping tools can also be used to trace a particular isolate and to determine its
geographic origin. VNTRs will facilitate to distinguish between infections caused by attacks
and naturally occurring plague infections. Isolates from human cases can be linked now
effectively with isolates from environmental sources (25). A genetic match with
environmental isolates was found in four out of nine human cases.
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Whole-genome characterization of Y. pestis strains revealed dozens of SNPs per strain that
differ relative to a reference genome and can be used for forensic microbiological
investigations in the future (9).
6.3 Glanders and melioidosis

Burkholderia (B.) mallei is a gram-negative bacterium causing glanders and farcy in horses,
donkeys, and mules (solipeds) and has been classified by the CDC as a priority category B
biological agent.
Glanders in horses presents with pneumonia, purulent nasal discharge, and poor general
condition, whereas farcy is a chronic cutaneous disease with massively enlarged lymph
vessels (“farcy-pipes”) and nodules developing into ulcers. Equines are the only known
reservoir for sporadic infections in humans. Humans develop a clinical picture resembling
melioidosis which is caused by the closely related bacterium B. pseudomallei.
In 2004, an outbreak of glanders in horses was reported to the Office International des
Epizooties by the United Arab Emirates. In addition to cultivation and phenotypical
identification a new real-time PCR assay was developed for the specific identification of B.
mallei, which detected the bacteria in tissues of two horses (42).
B. pseudomallei is the etiologic agent of melioidosis, a tropical disease that is highly endemic
in Southeast Asia and Northern Australia. B. pseudomallei is a facultative intracellular,
opportunistic pathogen that can be acquired by inhalation or by contact of skin lesions with
contaminated soil or water. The clinical presentation of melioidosis is variable including
subclinical infection, cutaneous lesions, fulminate septicaemia and rapidly progressing
pneumonia. Identification of the pathogen and specific antimicrobial therapy are critical,
because B. pseudomallei is resistant to ampicillin and broad- and expanded-spectrum
cephalosporines due to the production of a beta-lactamase.
Real-time PCR assays have been developed as rapid identification tools, but several assays
were evaluated with strain collections and spiked samples only (30; 40; 41; 43). In fatal
septicaemia the amount of bacterial DNA that can be extracted from blood is high enough to
be detectable using real-time PCR (38), but a study in Thailand has shown that this
diagnostic tool may be of little clinical value when compared with conventional diagnostic
approaches (5). MLST analyses have shown that strains from Thailand and Australia can be
discriminated and that some sequence types found in environmental samples are
underrepresented among clinical isolates thus indicating that they may be less pathogenic
for humans (12; 48). However, VNTR typing did not reproduce this geographic
discrimination, but proved to have a higher resolving power that can be used to analyse
outbreaks for microbial forensic purposes (11; 45).
6.4 Tularemia
Francisella (F.) tularensis is a biological agent of category A and the causative agent of
tularemia. The subspecies F. tularensis subsp. holarctica can be found in many regions of the
northern hemisphere, but the subspecies F. tularensis subsp. tularensis occurs only in North
America. Surprisingly, isolates of F. tularensis subsp. tularensis were recovered repeatedly
from fleas and mites captured in the region of the Danube river basin, close to Bratislava in
Slovakia (17). This was extremely unusual and warranted further investigations. Multiple-
locus variable-number tandem repeat analysis has been developed to elucidate the
worldwide genetic relationships among F. tularensis isolates and to distinguish natural
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outbreaks from intentional (terrorist) dissemination (20). The two Slovakian isolates
clustered with the highly pathogenic laboratory strain Schu4. The isolate FSC198 was finally
sequenced completely and found to be almost identical to the laboratory strain Schu4 (6). A
comparison of mutation patterns of an isolate propagated from Schu4 in vitro (FSC043) and
FSC198 indicated that FSC198 diverged from its progenitor Schu4 and has subsequently
passed life-cycles in a natural environment (36). This is a remarkable example for a
microbial forensic investigation that also demonstrates how much effort is needed to
elucidate the potential origin of an isolate and under which conditions it may have
propagated.
In the above mentioned study it was not possible to clarify the phylogeographic expansion
of F. tularensis completely (20). This can be explained by the low genetic diversity of F.
tularensis subsp. holarctica and the limited number of Francisella strains available from certain
geographic regions. Local inconsistencies in the genetic relationship were found and
attributed to homoplasy effects. Recently, the phylogeography of F. tularensis was further
investigated using SNP analysis and insertion/deletion events (INDELS) (39; 49). This is a
promising straight forward approach that can be used to analyze the relationship of closely
related strains. Outbreaks on a local scale can be investigated and the work load of
sequencing of relevant gene loci is acceptable and affordable.
7. Animal pathogens and agroterrorism

“Agroterrorism is the deliberate tampering with and/or contamination of the food supply
with the intent of adversely affecting the social, economic, physical and psychological well-
being of society” (23). Agroterrorism carries less risk for the terrorist, could be carried out
more covertly, and does not require sophisticated methodology for weaponization (53).
Important vulnerabilities are intensive production practices, increased susceptibility of
immunologically naïve animal populations, and rapid and fast movement of animals and
their products over long distances (7). Attacks can result in disastrous economic losses due
to eradication measures (mass culling), international trade embargos, loss of jobs, increased
consumer costs, and may even cause difficulties in sustaining the food supply (26).
8. Quality assurance
Quality assurance is required to verify whether practices and test results are providing
reliable and relevant information and quality control can verify whether test conditions are
functioning appropriately to yield reproducible results. The Scientific Working Group on
Microbial Genetics and Forensics has developed Quality Assurance Guidelines for Laboratories
Performing Microbial Forensic Work to provide a framework for laboratories that carry out
microbial forensic analysis (3).
9. Reporting and surveillance systems

The Global Early Warning and Response System for Major Animal Diseases, including
Zoonoses (GLEWS) is the combined effort of WHO, FAO, and OIE. For zoonotic events,
alerts of animal outbreaks can provide direct early warning so that human surveillance
could be enhanced and preventive action taken.
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10. Limitations
Crops, rangeland and forests can also be targets of biological attacks. However, the field of
plant pathogen forensics is beyond the scope of this chapter. The interested reader can be
referred to a comprehensive review written by Fletcher et al. (14).
Profiling of forensic soil samples by determining the bacterial content may provide valuable
information, but depends on several factors such as heterogeneity within a habitat, distance
of collection sites, and time (15; 28).
11. Conclusion
Microbial forensics is a young scientific discipline and probably only few scientists and
institutions are aware of the methodological and quality assurance requirements.
Epidemiological tools can be used to trace strains and to clarify the chain of infection, but
typing systems have to be especially evaluated for forensic purposes. Classical
microbiological techniques are indispensable, but most recent developments including very
rapid whole genome sequencing complement the polyphasic approach needed for
diagnostics and typing. Only large collections of strains from all over the world and high
quality sequence data will provide the basis for meaningful results in microbial forensic
investigations. International and interdisciplinary cooperation will improve our capabilities
to rapidly identify the agents, elucidate the source, and provide these results as evidence in
court.
12. Acknowledgment
The authors thank Lisa Sprague, Helmut Hotzel and Sascha Rommeiss for reading the
manuscript and valuable discussions.
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14
Advanced Medical Imaging and

Reverse Engineering Technologies
in Craniometric Study
Supakit Rooppakhun1, Nattapon Chantarapanich2
and Kriskrai Sitthiseripratip3
1School of Mechanical Engineering, Suranaree University of Technology
2Institute ofBiomedical Engineering, Prince of Songkla University
3National Metal and Materials Technology Center (MTEC)
Thailand
1. Introduction
In crime scenes and accidents, the standard operating protocols for personal identification is
not difficult when the entire body is found. As a result, the investigators can directly collect
the sample of the decrease such as facial photograph, DNA, fingerprint and dental record in
order to compare to possible relatives or with ante-mortem profile (De Valck, 2006).
However, the investigators are not always lucky. In severe accidents such aircraft crashes,
only little information of the decrease is available, the skin and soft tissue may be
completely burnt out and the skeleton can be broken into small pieces due to the impact.
The skeleton examination in historical sites by archaeologists presents even more
complication. The archaeologists need not only to identify general aspects of the skeleton,
for instant age, sex, cause of death, stature and race, but also to estimate the period of death
and the possibility to discover the particular person that might be significant in the history.
Several techniques have been applied to assist the identification of the decrease ranging
from the simplest technique which may acquire the evidence from the personal belonging
until using the advance scientific techniques. These techniques can be generally categorized
into two methods, invasive and non-invasive. The invasive method includes biochemical
analysis, microscopy, accelerator mass spectrometry radiocarbon dating (with standard C-
14), ancient DNA analysis, histology and endoscope whereas the non-invasive technique
involved the aid of engineering technologies such as radiographic analysis and computed
tomography (CT) examination.
From 1975 – 2005, the archaeological researches had been increasingly conducted by means
of non-invasive techniques which 112 of 245 researches have applied the non-invasive
technique and the trend of investigation gradually moved from invasive to non-invasive
examinations (Zweifel, et al., 2009). The first non-invasive technique has been presented to
the public in 1896 which the radiographic analysis was used to examine the ibis mummy in
Belgium (Van Tiggelen, 2004). Soon after that, archaeologist realized the importance of
radiological technique and applied broadly to examine great numbers of mummy (Dedouit,
et al., 2010; Friedrich, et al., 2010; Recheis, et al., 1999; Zweifel, et al., 2009). One main
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advantage of radiography includes the ability to access general aspects of mummy without
releasing the bandages which may be destroyed important features or contaminated. In the
early 1900s, another non-invasive medical examination device, computed tomography
scanner, has been invented by Alessandro Vallebona, but the technology has remained
unpopular until the 1970s which modern era of computed tomography scanner began (Hill,
2009). Not too long, computed tomography scanner became an effective tool in examination
the autopsy. The computed tomography generally relies on medical imaging processing
combined with reverse engineering principles which the object is captured the profile and
presented the virtual three-dimensional models in computer. With these advanced features,
the perspective of archaeology and forensic medicine has changed into three-dimensional
aspect. Consequently, The two-dimensional radiographic image occlusion and the problem
such uncertainty from bias of investigator in direct measurements are eliminated.
Apart from the advantages of computed tomography in forensic medicine, it also becomes
an effective clinical diagnosis device in many hospitals. The use of three-dimensional model
allows the surgeon to examine the abnormality of organs in any configurations which the
two-dimensional technique may be inaccessible. Moreover, the three-dimensional models
can be used to simulate the surgical operation prior surgery (Fürnstahl, et al., 2010),
subsequently reduce operating time and increase safety of patients. Alternative uses of
computed tomography include morphometric study (Chantarapanich, et al., 2008;
Mahaisavariya, et al., 2002) and the evaluation the risk of implant usage (Mahaisavariya, et
al., 2004; Sitthiseripratip, et al., 2003)
The purpose of this chapter is to present and discuss the medical imaging and reverse
engineering techniques by means of demonstrating the application in craniometric study.
Obviously, recent craniometric studies have been employed by two-dimensional techniques
or direct measurement (Steyn & Işcan, 1997; Deshmukh & Devershi, 2006; Dayal, et al, 2008;
Sangvichien, et al., 2008; Matamala, et al, 2009) such as the use of spreading caliper, sliding
vernier calliper, mandibulometer and standard flexible steel tape. The diverse mentioned
measurement techniques reflect the lack of engineering aids which the current trend in
forensic medicine need the advanced technologies to provide the accurate measurements.
Therefore, the scope of demonstration includes the brief detail of reverse engineering as to
provide for who may not familiar with, data acquisition technique using computed
tomography scanner, measurement of skull anatomical parameters and sex determination
method based on logistic function. By this way of demonstration, the overview of forensic
medicine by aid of advance medical imaging and reverse engineering is obtained.
2. Reverse engineering
Reverse engineering has been widely applied several years in clinical fields and forensic
medicine (Aamodt, et al., 1999; Aghayev, et al., 2008; Fürnstahl, et al., 2010). Initially, reverse
engineering was first used in free-form product designs which the conventional “Forward
Engineering” has limited drawing functions and time consumption. Product design based
on “Forward Engineering”, the process involves turning the conceptual product design to
physical product whereas reverse engineering is inversed (Zhou & Xi, 2002).
The process of reverse engineering involves turning the physical product back to the virtual
models (normally three-dimension) (Li, et al., 2002; Várady, et al., 1997). From the three-
dimensional virtual models, the conceptual design can be obtained. Reverse engineering can
be described as two phases which are digitization and reconstruction phase, as summarized
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Advanced Medical Imaging and Reverse Engineering Technologies in Craniometric Study 309
in Fig. 1. The digitization phase involves the data acquisition of the physical model using
various types of scanner. The initial geometry from the scanner is then obtained in three
forms, point clouds, polygon model and series of image depending on the acquisition
technique of each scanner. In the reconstruction phase, the obtained data is processed in
order to reconstruct the three-dimensional model. with “At this step, the elimination of
noise data and filtering of unnecessary data may also be performed. For the production
phases, it may be added as a final step in reverse engineering. This phase employs various
manufacturing processes to fabricate the three-dimensional virtual model. However, this
phase may not be mandatory, because sometime the geometry is only stored in database
without any further processing.
Fig. 1. Diagram of reverse engineering process.

The digitization methods can be categorized into two approaches which are tactile approach
(contact method) and non-contact approach (Li, et al., 2002; Várady, et al., 1997). For tactile
approach, the device contacts to the physical object directly whereas the non-contact
approach uses the medium in digitization instead without contact of device.
The characteristic of tactile approach is relatively simple. Touch probe is used in
conjunction with robotic mechanism such as coordinate measurement machine (CMM),
articulated arm or computer numerical control (CNC) devices to determine the position of
the object (Cartesian coordinate). The accuracy is considered to be a main advantage of
tactile approach, nevertheless the digitization process is quite slow and difficult to
digitize complex geometry. A wide range of object can be applied with this approach
regardless of color, shininess and transparency, this approach is not appropriate for
deformable materials.
In non-contact approach, the medium is used to measure the physical object using the
principle of reflection or penetration. Laser beam and white light are medium sources
commonly found in many three-dimensional scanners and they rely on the principle of
reflection. The medium travels from the generator to the object before reflects and transmits
to the receiver unit. The determination of geometry can be processed using at least one two-
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Fig. 2. Digitization Methods.
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dimensional images combined with some optical parameters such as reflection angle,
distance and time of flight. The initial geometry is presented in form of cloud point or of
polygon model. Use of laser beam and white light has advantage in fast digitization and
continuous data, but too shiny and transparent object present complication.
Even the laser beam and white light systems are applied in many forensic studies (Park, et
al., 2006; Thali, et al., 2003; Vanezis, et al., 2000), but probably, the most efficient system for
forensic studies is the use of non-contact device based on principle of penetration. This
system uses the medium that can go though the object to capture both internal and external
geometries. The most popular device is computed tomography scanner which involves the
use of X-ray.
The digitization process initializes the transmission of the X-ray through the object. A set of
data acquisitions is performed with the constant interval throughout entire object which
subsequently give a series of slice image (Hounsfield, 1980). Each slice contains the
information of object’s position and the value of Hounsfield unit (HU). The density of object
is proportional to the Hounsfield value. The higher Hounsfield value indicates high-density
object such as enameled and cortical bone whereas the lower Hounsfield value indicates
low-density object such as cancellous bone, fat and soft tissue. Various Hounsfield values of
various substances are given in Table 1. In order to reconstruct the three-dimensional model,
the optimal Hounsfield values must be selected (threshold). After that the threshold regions
of each slice are combined to construct the volumetric model.
For the computed tomography device, the speed of digitization and ability to examine the
internal topology are considered to be superior, but the artifact (noise data) caused by
metallic structure is drawback.
Substances Hounsfield values

Air -1000
Fat -70 to -90
Water 0
Tissue +20 to +35
Blood Volume Approx. +40
Bone +900
Table 1. Hounsfield values of various substances. (Hounsfield, 1980)
3. Data acquisition of skull

Specimen of 104 dry cadaveric skulls donated by the Department of Anatomy, Faculty of
Medicine, Khon Kaen University, Thailand is used to demonstrate the application of
advanced medical imaging and reverse engineering technologies in craniometric study. The
cadaver includes 63 males with average age of 55.16 years (standard deviation 18.38 years)
and 41 females with average age of 49.00 years (standard deviation 17.94 years). The age
rages from 17–81 years at the time of death. The skulls are placed in acrylic box with a set of
four. The reverse engineering technique by means of spiral computed tomography scanner
(Siemens AG, Germany) is used to capture the profile of each skull as shown in Fig. 3. The
data acquisition protocol is axial scan with tube voltage of 120 kV and tube current of 100
mA. The digitization is performed with 1.5-mm slice thickness and the reconstruction is
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done at 1-mm thickness. Each slice contains the volumetric data that represent the density
and position (contour) of the skull. The computed tomography images are then processed
with medical image processing software (Mimics, Materialise NV, Belgium). To begin the
reconstruction of three-dimensional model, a proper Hounsfield value (normally +900 for
bone structure) is selected. After that, the threshold regions are used to calculate the
complete topology of three-dimensional skull. The reconstruction process of skull model is
illustrated in Fig. 4.
Fig. 3. A set of skull during data acquisition using computed tomography scanner.
Fig. 4. Three-dimensional model reconstruction.
4. Three-dimensional computerized craniometric study

The anatomical landmarks in craniometric study are categorized in to median and bilateral
landmarks (Rooppakhun, et al., 2010). The median landmarks are approximately located on
sagittal plane. Each of them has only one location. There are 13 median landmarks which the
specific definitions can be described as follows:
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 Glabella (GL) - the most anterior point of frontal bone between supraorbital in the
sagittal plane.
 Bregma (BR) - the crossing of the coronal and sagittal sutures on the top of the skull.
 Opisthocranium (OPC) - the most posterior point in midline of inion bone which length
of the skull is maximum when measure from Galbella point.
 Nasion (NA) - the intersection point of the internasal and frontonasal sutures in the
sagittal plane.
 Opistion (OPS) - the most posterior midsagittal point on the posterior margin of the
foramen magnum.
 Basion (BA) - the most anterior point of the great foramen magnum in the sagittal plane.
 Orale (OR) - the midpoint on the intersection of posterior alveolar sockets rim of the
cavities of two upper central incisors.
 Prosthion (PR) - the lowest, most anterior point on the alveolar portion of the premaxilla,
in the median plane, between the upper central incisors.
 Staphylion (STA) - point in the medial line (interpalatal suture) of the posterior part of
the hard palate where it is crossed by a line drawn tangent to the curves of the posterior
margins of the palate.
 Nasospinale (NAS) - the lowest point of lower anterior nasal aperture in mid-sagittal
plane.
 Gnathion (GN) - The midpoint on the lower border of the mandible in the sagittal plane.
 Pogonion (PG) - The most projecting point of the chin in the standard sagittal plane.
 Infradentale (ID) - The anterior superior point on the mandible at its labial contact
between mandibular central incisors.
For the bilateral landmarks, each of them is located on both sides of skull. There are 17
bilateral landmarks which the specific definitions can be described as follows:
 Euryon (EU) - the lateral point on either side of the greatest transverse diameter of the
skull.
 Staphanion (ST) - the intersection of the superior temporal line and the coronal suture.
 Frontotemporale (FT) - the most anterior point on either side of temporal crest of the
minimum transverse breadth of frontal bone.
 Bolton (BO) - The superior point of the curvature between occipital condyle and
posterior margin of foramen magnum.
 Orbitale (ORB) - the most inferior point of each infraorbital rim .
 Ectoconchion (EC) - the most lateral point on each orbital's margin where a line running
parallel to upper orbital border cut the lateral orbital margin.
 Maxillo-frontale (MF) - the intersection point on anterior lacrimal crest and fronto-
maxillary sutures.
 Supraorbitale (SOR) - the most superior point of each superior orbital rim.
 Zygonion (ZG) - The most lateral point on the outline of each zygomatic arch.
 Zygomaxillare (ZM) - The most interior point on each zygomatico-maxillary sutures.
 Nasal (NS) - The most lateral point on each nasal's margin where maximum nasal
breadth.
 Endomolare (ENM) - the most medial point of internal curvature surface of alveolar
ridge corresponding to second molar tooth.
 Coronion (CO) - The most superior point on each coroniod process.
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 Condylion superior (CS) - the most superior point on each mandibular condyle.
 Conlylion laterale (CDL) - the most lateral point on each mandibular condyle.
 Gonion (GO) - the point at each mandibular angle that is defined by dropping a
perpendicular from the intersection point of the tangent lines to the posterior margin of
the mandibular vertical ramus and inferior margin of the mandibular body or
horizontal ramus.
 Laterla infradentale (LID) - the midpoint of a line tangent to the outer margins of the
cavities of the lateral incisor of each lower canine teeth.
In order to better understand the previous described definitions, every landmark is also
illustrated in Fig. 5.
Each of the three-dimensional models of skull is used to determine the anatomical landmark
according to previous description. Only one investigator locate the entire landmarks in
every skull to avoid uncertainty of intra-observer. The anatomical landmarks are then used
to obtain 40 craniometric parameters as shown in Table 2. The measurements are interpreted
using statistical analysis and reported in form of average values and standard deviation
regarding to gender. In order to distinguish craniometric parameters of each gender, an
unpaired t-test is utilized for analysis. A p-value < 0.05 is a significant level that used to
determine the difference. In addition, the linear regression and the correlation coefficient are
also used for the pair-wise tests.
As also shown in Table 2, the craniometric parameters of male are larger than female.
Thirty-one of forty parameters show the statistical significant differences among both
genders, especially, Maximum cranial breadth, Facial length, Orbital height-left, Orbital
height-right, Palatal breadth, Bicoronion breadth, Bizygometic breadth, Maxillary breadth,
Upper facial height, Orbital breadth-left, Orbital breadth-Right, Nasal height, Bicondylar
breath, Bi-gonion breadth, Coronion height-left, Coronion height-right, Mandibular body
length-left, Mandibular body length-right, Maximum mandibular length-left and Maximum
mandibular length-right which present the p-value << 0.001. For Maximum cranial breadth,
Facial length, Orbital height-left, Orbital height-right, Palatal breadth, Bicoronion breadth
are considered to be relatively different as the p-value < 0.01. The parameters such Ramus
height-left, Ramus height-right, Symphysic height shows some degrees of significance. In
addition, nine parameters do not present the statistical differences which are Maximum
frontal breadth, Anterior inter-orbital breadth, Nasal breadth, Palatal length, Mandibular
angle-left, Mandibular angle-right, Notch length-left, Notch length-right, and Symphysic
breadth.
From the pair-wise tests, the correlations of craniometric parameters are different among
male and female population. Table 3 and Table 4 show the sample of correlations of
craniometric parameters which correlation coefficient (r) are above 0.500. In both
populations, the bilateral anatomy presents some degrees of correlation which can be
signified the facial symmetry. The linear regression equations are considered to be useful to
predict the craniometric parameters in forensic medicine. For example, the defected skull
usually miss some landmarks, the use of known craniometric parameters can be used to
determine the missing parameters. However, it should be noticed that the obtained missing
parameters may not be always precise. The possibility of being can only be determined, but
the accuracy strongly depends on the correlation coefficient. The regression scatters plot and
95% interval bands of some pair-wise tests are plotted in Fig. 6 and Fig. 7.
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Fig. 5. The anatomical landmarks of skull.
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Average (S.D.)
Craniometric Parameter Landmark p-value
Male Female
Maximum cranial length GL-OPC 173.6 (5.2) 165.4 (6.4) << 0.001
Maximum cranial breadth EUL-EUR 144.9 (5.6) 141.2 (5.5) 0.001361
Maximum frontal breadth STL -STR 115.8 (6.7) 113.3 (6.7) 0.070663
Minimum frontal breadth FTL-FTR 94.9 (5.1) 91.4 (4.9) 0.000584
Basion-brema height BA-BR 138.6 (4.8) 132.4 (5.2) << 0.001
Nasion-basion length NA-BA 101.8 (4.0) 96.0 (3.4) << 0.001
Foramen magnum length BA-OPC 36.7 (2.1) 34.5 (2.4) 0.000006
Foramen magnum breadth BOL-BOR 30.5 (2.1) 28.9 (1.8) 0.000051
Nasion-bregma length NA-BR 112.9 (4.2) 107.3 (6.0) 0.000002
Facial length BA-PR 96.1 (5.4) 92.9 (5.5) 0.004704
Bi-orbital breadth ECL-ECR 97.4 (3.8) 94.0 (3.8) 0.000024
Bi-zygometic breadth ZGL-ZGR 133.7 (5.1) 127.7 (5.2) << 0.001
Maxillary breadth ZML–ZMR 104.5 (5.3) 99.1 (4.9) 0.000001
Upper facial height NA-PR 70.3 (4.2) 66.2 (4.6) 0.000019
Orbital breadth-left ECL-MFL 41.2 (2.2) 39.6 (2.4) 0.000743
Orbital breadth-right ECR-MFR 41.5 (2.0) 39.8 (2.0) 0.000044
Orbital height-left ORBL-SORL 36.2 (2.3) 34.7 (2.5) 0.002714
Orbital height-right ORBR-SORR 36.3 (2.5) 34.9 (2.1) 0.004341
Anterior inter-orbital breadth MFL-MFR 21.0 (2.2) 20.7 (2.4) 0.497642
Nasal breadth NSL-NSR 27.0 (2.2) 26.8 (2.2) 0.632101
Nasal height NA-NAS 52.7 (3.0) 49.6 (3.1) 0.000003
Palatal length OR-STA 42.6 (4.2) 42.6 (4.4) 0.952028
Palatal breadth ENML-ENMR 39.1 (3.1) 37.6 (2.4) 0.006430
Bi-coronion breadth COL-COR 98.6 (5.2) 94.3 (5.8) 0.002652
Bi-condylar breadth CDLL-CDLR 123.1 (5.2) 118.7 (5.1) 0.000870
Bi-gonion breadth GOL-GOR 99.1 (6.3) 92.8 (6.2) 0.000113
Coronion height-left COL-GOL 62.3 (4.9) 57.2 (4.8) 0.000065
Coronion height-right COR-GOR 62.83 (5.2) 57.5 (4.8) 0.000043
Mandibular angle-left* CSL-GOL-GN 112.4 (5.1) 113.9 (6.4) 0.298443
Mandibular angle-right* CSR-GOR-GN 112.4 (5.5) 112.8 (5.6) 0.759332
Mandibular body length-left GOL-PG 92.0 (4.8) 87.0 (4.9) 0.000083
Mandibular body length-right GOR-PG 92.5 (4.9) 87.6 (5.0) 0.000128
Maximum mandibular length-left CSL-PG 119.8 (6.1) 114.6 (5.1) 0.000383
Maximum mandibular length- CSR-PG 120.6 (6.1) 115.0 (5.0) 0.000079
right
Notch length-left COL-CSL 35.3 (2.7) 34.1 (4.2) 0.202213
Notch length-right COR-CSR 35.1 (2.7) 33.7 (4.0) 0.105663
Ramus height-left CSL-GOL 58.2 (5.0) 55.2 (4.5) 0.010815
Ramus height-right CSR-GOR 58.3 (4.6) 55.3 (4.7) 0.010342
Symphysic breadth LIDL-LIDR 23.1 (6.0) 23.7 (6.0) 0.689146
Symphysic height GN-ID 31.6 (3.5) 26.5 (3.1) 0.011128
Table 2. Average values of craniometric parameters regarding to gender derived from 104
skulls (63-males and 41-females), (Unit: millimeter, *degree).
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Craniometric Parameter (x vs. y) Regression (Correlation, r)

Maximum mandibular length-left vs. -right y = 0.916x + 10.470 (0.920)
Mandibular angle-left vs. -right y = 0.976x + 2.630 (0.904)
Mandibular body length-left vs. -right y = 0.883x + 11.227 (0.860)
Coronion height-left vs. -right y = 0.908x + 6.233 (0.858)
Orbital height-left vs. -right y = 0.875x + 4.561 (0.835)
Orbital breadth-left vs. -right y = 0.741x + 10.969 (0.835)
Ramus height-left vs. -right y = 0.724x + 16.146 (0.835)
Notch length-left vs. -right y = 0.761x + 8.239 (0.770)
Bi-orbital breadth vs. Orbital breadth-left y = 0.400x + 2.283 (0.685)
Bi-zygometic breadth vs. Bi-orbital breadth y = 0.473x + 34.210 (0.643)
Upper facial height vs. Symphysic height y = 0.485x – 2.733 (0.636)
Maximun cranial breadth vs. Bi-zygometic breadth y = 0.109x + 17.063 (0.626)
Basion-brema heigth vs. Nasion-bregma length y = 0.516x + 41.339 (0.594)
Nasal height vs. Upper facial height y = 0.840x + 25.980 (0.592)
Palatal length vs. Facial length y = 0.734x + 64.806 (0.570)
Nasion-basion length vs. Bi-coronion breadth y = 0.730x + 24.429 (0.533)
Bi-condylar breadth vs. Bi-zygometic breadth y = 0.528x + 67.980 (0.570)
Table 3. The correlation of craniometric parameters of Thai male population.
Fig. 6. The linear regression scatters plot and 95% interval bands of Maximum mandibular
length-left (x) vs. Maximum mandibular length –right (y) (Unit: Millimeter).
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Craniometric Parameter (x vs. y) Regression (Correlation, r)

Mandibular body length-left vs. -right y = 0.930x + 6.626 (0.922)
Maximun mandibular length-left vs. -right y = 0.883x + 13.821 (0.904)
Coronion height-left vs. -right y = 0.889x + 6.599 (0.895)
Notch length-left vs. -right y = 0.831x + 5.335 (0.880)
Ramus height-left vs. -right y = 0.912x + 4.958 (0.871)
Orbital breadth-left vs. -right y = 0.693x + 12.370 (0.849)
Orbital height-left vs. -right y = 0.728x + 9.680 (0.839)
Mandibular angle-left vs. -right y = 0.737x + 28.837 (0.832)
Upper facial height vs. Nasal height y = 0.522x + 15.065 (0.765)
Bi-orbital breadth vs. Orbital breadth-left y = 0.478x - 5.357 (0.759)
Minimun frontal breadth vs. Maximum frontal breadth y = 0.997x + 22.127 (0.723)
Maximun cranial breadth vs. Bi-zygometic breadth y = 0.624x + 39.615 (0.662)
Symphysic height vs. Upper facial height y = 0.784x + 42.987 (0.641)
Facial length vs. Palatal length y = 0.507x - 4.481 (0.637)
Maxillary breadth vs. Bi-orbital breadth y = 0.474x + 46.949 (0.609)
Anterior interorbital breadth vs. Bi-coronion breadth y = 1.638x + 60.166 (0.590)
Maximum cranial length vs. Nasion-basion length y = 0.314x + 43.994 (0.584)
Table 4. The correlation of craniometric parameters of Thai female population.
Fig. 7. The linear regression scatters plot and 95% interval bands of Mandibular body length
-left (x) vs. Mandibular body length -right (y) (Unit: Millimeter).
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5. Sex determination using logistic regression analysis

The determination of sex from skeleton is one of the critical components in forensic
medicine as well as archaeology. The determination can be interpreted based on the
average data from craniometric parameters of certain population. The accuracy of sex
determination depends on several factors which one of them is osteological elements.
Various osteological elements have been used to predict sex of skeleton which includes
femur (Yaşar Işcan & Shihai, 1995), tibia (Steyn & Işcan, 1997), pelvis (Durić, et al., 2005),
skull (King, et al., 1998) , humerus (Işcan, et al., 1998; Robinson & Bidmos, 2009), hyoid
(Mukhopadhyay, 2010), talus (Murphy, 2002a) and calcaneus (Murphy, 2002b). However,
among aforementioned elements, the best for prediction is pelvis, follow by skull and
long bone (Durić, et al., 2005, Işcan, 2005). Rather than osteological element, population
variation and method of data processing (data acquisition and statistical analysis) are also
among important factors.
Logistic regression analysis is a method to analysis multi-variate analysis which aims to
predict the probability of occurrence of an event under consideration by fitting the raw data
using to a logit function logistic curve. The advantage of logistic regression is less of
parameter restrictions than discriminant analysis and regression analysis. The output from
the logistic regression analysis can only be “Yes” or “No” which normally designated as “0”
and “1”, respectively.
Basically, logistic regression function (Z) is written in form of
Z = β0 + β1x1 + β2x2 + β3x3 + β4x4 + … +βnxn + βn+1xn+1 (1)

where β0 is called intercept and β1, β2, β3 ,β4, …,βn and βn+1 are regression coefficients of x1,
x2, x3, x4, …,xn and xn+1 variable, respectively. From the following logistic model of Z, the
probability of event (P.E.) under consideration can be calculate through
P.E. = eZ / (1 + eZ) (2)
In equation (2) e is a natural logarithm, its value is approximately 2.71828.

The probability of event varies from “0” to “1”. The near “1” value means the independent
variables influences the probability of event whereas the value close to “0” means the
independent variables have little effect to probability of event.
In this study, a binary logistic regression using forward stepwise is applied to determine sex
based on average data of craniometric parameters in previous section. The fitting process
excludes the measurement with high co-linearity. The probability from logistic regression
function, “0” indicates male whereas “1” indicates female, respectively. All statistical
analysis is preformed using statistic commercial software (SPSS, SPSSTM Inc, United States
of America).
There are three logistic regression models used in sex prediction. Model A uses only cranial
parameters whereas Model B uses only mandibular parameters. Model C uses combination of
cranial and mandibular parameters. From the analysis, the coefficient (β), standard error of
means and significant value are reported as shown in Table 5 to Table 7.
For Model A, there are four significant predictors (p < 0.05) in mandible parameters which
are Nasion-basion length, Palatal length, Upper facial height and Maxillary breadth. The
function is as follows:
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Coefficient Standard error of mean Significant

Craniometric Parameter
(β) (S.E.) level
Intercept 76.340 15.9577 0.00000
Nasion-basion length -0.387 0.0984 0.00008
Palatal length 0.362 0.1238 0.00349
Upper facial height -0.497 0.1608 0.00199
Maxillary breadth -0.197 0.0708 0.00530
Table 5. Logistic response model using logistic regression analysis of Model A.

(β) (S.E.) level
Intercept 70.589 18.7774 0.00017
Bi-gonion breadth -0.184 0.0631 0.00347
Coronion height-left -0.310 0.1042 0.00289
Mandibular angle-right -0.208 0.0812 0.01033
Mandibular body length-right -0.125 0.0888 0.15810
Table 6. Logistic response model using logistic regression analysis of Model B.

(β) (S.E.) level
Intercept 88.202 24.4335 0.00031
Coronion height-left -0.276 0.1242 0.02614
Nasion-basion length -0.596 0.1741 0.00062
Palatal length 0.581 0.2032 0.00422
Upper facial height -0.567 0.2186 0.00956
Table 7. Logistic response model using logistic regression analysis of Model C.
Z = 76.340 - 0.387(NA-BA) + 0.362(OR-STA) - 0.497(NA-PR) - 0.197(ZML–ZMR) (3)

where
NA-BA = Nasion-basion length (unit: millimeter)
OR-STA = Palatal length (unit: millimeter)
NA-PR = Upper facial height (unit: millimeter)
ZML–ZMR = Maxillary breadth (unit: millimeter)
For Model B, there are four significant predictors (p < 0.05) in mandibular parameters which
are Bigonion breadth, Coronion height-left, Mandibular angle-right and Mandibular body
length-right. The function is as follows:
Z = 70.589 - 0.184(GOL-GOR) - 0.310(COL-GOL) - 0.208(CSR-GOR-GN) - 0.125(GOR-PG) (4)

where
GOL-GOR = Bi-gonion breadth (unit: millimeter)
COL-GOL = Coronion height-left (unit: millimeter)
CSR-GOR-GN = Mandibular angle-right (unit: degree)
GOR-PG = Mandibular body length-right (unit: millimeter)
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For Model C, there are four significant predictors in cranial and mandibular parameters (p <
0.05) which are Coronion height-left, Nasion-basion length, Palatal length, and Upper facial
height. The function is as follows:
Z = 88.202 - 0.276(COL-GOL) - 0.596(NA-BA) + 0.581 (OR-STA) - 0.567 (NA-PR) (5)

where
COL-GOL = Coronion height-left (unit: millimeter)
NA-BA = Nasion-basion length (unit: millimeter)
OR-STA = Palatal length (unit: millimeter)
NA-PR = Upper facial height (unit: millimeter)
The set of skulls are tested according to logistic regression models (Model A, Model B and
Model C) to evaluate the accuracy as reported in Table 8.
Model Male Female Overall

Model A 92.1 90.2 91.3
Model B 85.0 86.2 85.5
Model C 95.0 93.1 94.2
Table 8. Accuracy of each logistic model (percentage).

The logistic regression analysis revealed that 4 of 21 cranial parameters and 4 of 11
mandibular parameters are significant predictors (p < 0.05). The Basion-nasion length and
Bigonion breadth are the most dimorphic of the cranial and mandibular measurement,
respectively. For the best prediction model, Model C which includes both cranial and
mandibular parameters is recommended.
In order to predict the gender by the parameters in Model C, the probability of being female
can be calculated using equation (2) whereas the probability of being male is then inversed
(1-P).
The accuracies from Model C of the present study are compared to previous studies on
sexual dimorphism using the skull of some previous studies based on South African white,
South African black, Indian and Thai population. Table 9 shows a comparison of
measurement techniques and average accuracies obtained from the present study and
previous studies which derived from many populations.
To the best of author’s knowledge, there is only one study (Sangvichien, et al., 2007) that
determine the sex of skull based on logistic function using on four parameters which as
Nasion-basion length, Maximum breadth of the cranium, Facial height, and Bi-zygometic
breadth. This presented the accuracy 88.8% for overall sex classification and 82.9% and
92.1% among females and males, respectively.
From the table, it reveals that the present study yields higher accuracies than the many
previous studies. Therefore, the Model C presents in this study can be used to determine
the gender of skeleton for intact skull in forensics and archaeology. However, in order to
ensure the effectiveness of logistic regression, three skulls with known gender from
Thammasat University Hospital, Thammasat University, Thailand are used to test the
concept. The skull No.1 and skull No. 3 are adult males whereas the skull No. 2 is adult
female.
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Based on logistic regression Model C which is consider to be the best model for sex
prediction in Thais’ skull , Coronion height-left, nasion-basion length, palatal length, upper
facial height are measured and predicted the gender. As shown in Table 10, all cases are
predicted the gender correctly (3/3). Hence, the determination of sex based on the Model C
of logistic regression is considered to be accurate.
Population Measurement Statistical Analysis Average

Technique Method Accuracies
South African White Direct Discriminant function 80% - 86%

(Steyn & Işcan, 1997) measurement
 44 males
 47 females
South African black 3D tactile Discriminant function 75% - 80%

(Franklin, et al, 2005) digitizer
 182 males
 150 females
Indian Direct Discriminant function 85% - 90%

(Deshmukh & Devershi, 2006) measurement
 40 males
 34 females
Thai Direct Logistic function 83% - 92%

(Sangvichien, et al., 2007) measurement
 66 males
 35 males
South African black Direct Discriminant function 80% - 85%

(Dayal, et al, 2008) measurement
 60 males
 60 males
Combination Direct Discriminant function 82%

(Matamala, et al, 2009) measurement
 149 males
 77 males
Thai Three- Logistic function 93% - 95%

(Present study) dimensional
 63 males CAD Model
 41 males
Table 9. Comparison on methods of data assessment and statistical analysis among different
studies.
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Fig. 8. The reconstruction of skull fragment (A) intact (B) intact with reconstruction unit.
Craniometric Parameter Skull No.1 Skull No.2 Skull No.3

Intercept 88.202 88.202 88.202
Coronion height-left 69.63 53.12 72.09
Nasion-basion length 105.07 95.24 101.06
Palatal length 58.50 45.82 55.17
Upper facial height 76.91 72.26 71.81
Regression Analysis (Z) -3.26 2.43 -0.59

Probability of event (P.E.) 0.037 0.919 0.357
Prediction Gender male female male
Exact Gender male female male
Result correct correct correct

Table 10. Craniometric measurement and sex prediction.
6. Outlook
Since there is possibility to find skull as fragment bone in forensics and archaeology, then
the assessment of necessary craniometric parameters becomes complex. Although the
missing craniometric parameters can be predicted using correlation as shown in Table 3 and
Table 4, but some correlation coefficients among these craniometric parameters are mostly
inferior. With low correlation coefficient, the equation may not be an appropriate solution to
determine those missing craniometric parameters.
As a result, a purposed alternative is to reconstruct defected skull surface based on the
mirror surface topology of normal side with aids of Computer Aided Design technique as
shown in Fig. 8. In fact, this technique is a standard protocol for implant design in cranial
reconstruction (Müller, et al., 2003). The purposed alternative relies on the discovery that the
symmetric between left and right of bilateral craniometric parameters as presented in Table
2. However, this concept should be further investigated to ensure the usability.
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7. Conclusion
This study presents advantages of using advance medical imaging and reverse engineering
through computed tomography scanner to reconstruct the three-dimensional model of
skulls. This is very useful to analyze and measure craniometric parameters based on virtual
model. Generally, in forensic medicine and archaeological researches, the study relies on
direct measurement and other two-dimensional techniques which may not be accurate. The
measurement errors can be influenced by human error, instrumental error, image
magnification and image occlusion. The advantage of three-dimensional computed
tomography technique includes the analysis of specimen without destruction or damage of
specimens as well as the ability to analyze the specimens in configuration which the
conventional technique cannot provide. Comparing to the other reverse engineering
technologies, computed tomography presents the superior ability in accessing the internal
geometry which the other tools find the difficulty in data capturing.
In the craniometric analysis, the medians and bilateral landmarks are accessed. From the
analysis, the result reveals that Thai male presents the craniometric parameters greater than
Thai female, especially, Maximum cranial breadth, Facial length, Orbital height-left, Orbital
height-right, Palatal breadth, Bicoronion breadth, Bizygometic breadth, Maxillary breadth,
Upper facial height, Orbital breadth-left, Orbital breadth-Right, Nasal height, Bicondylar
breath, Bi-gonion breadth, Coronion height-left, Coronion height-right, Mandibular body
length-left, Mandibular body length-right, Maximum mandibular length-left and Maximum
mandibular length-right. In both populations, the bilateral anatomy presents some degrees
of correlation which may be concluded the facial symmetry.
Logistic regression is used to derive functions for sex determination based on average
numerical values which obtained from craniometric parameters. Three models are
purposed, Model A is based on cranial parameters, Model B is based on mandible parameters
and Model C is based on both cranial and mandibular parameters. From the result, Model C
provides the best accuracy among other models which is 94.2%. The prediction equation
relies on four parameters which are Coronion height-left, Nasion-basion length, Palatal
length, and Upper facial height which subsequently produce logistic regression as in
equation (5).
As seen in Table 9, our study yields higher accuracies than other previous studies. This is
due to two main factors, the accurate landmark identification by the three-dimensional
technique and developing logistic regression function from difference sample.
In addition, the authors suggest that the techniques described in this chapter which includes
data acquisition, three-dimensional computerized craniometric study and sex determination
based on logistic regression function can effectively be applied to the other osteological
elements in specific race.
8. Acknowledgment
The research is supported the funding and facilities in part by the National Metal and
Material Technology Center (MTEC), National Science and Technology Development
Agency (NSTDA) of Thailand and Suranaree University of Technology, Thailand. In
addition, the authors are grateful to Department of Anatomy, Faculty of Medicine, Khon
Kaen University, Thailand for providing cadaveric skull specimens.
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15
House Dust Mites, Other Domestic

Mites and Forensic Medicine
Solarz Krzysztof
Medical University of Silesia in Katowice
Poland
1. Introduction
Many species of mites are the sources of potent allergens that sensitize and induce IgE-
mediated allergic reactions in humans. Most of the mite allergens are proteins, and the
allergic response mechanism to these allergens is the same as it is for allergens from other
sources such as plant pollens, molds and foods (Arlian, 2002). Mites occurring in house dust,
besides the ticks (Acari: Ixodida), are one of the most medically important group of mites. In
total at least 150 species of mites have been found in dwellings, including plant parasites,
animal parasites, predatory mites, oribatid mites and storage mites. But the most abundant
mites in house dust are members of the family Pyroglyphidae (Astigmatina) (Wharton, 1976;
Arlian, 2001; Arlian et al., 2002; Solarz, 2010). The three mite species, most often and most
abundant found in house dust throughout the world, are Dermatophagoides pteronyssinus, D.
farinae and Euroglyphus maynei (Arlian, 2001; Colloff, 2009; Pope et al., 1993). The house dust
mites now constitute the most dangerous pests of temperate climate countries, causing both
significant loss of human life and immense waste of resources. These mites are the major
sources of indoor inhalant allergens facilitating both the sensitization of atopic subjects and
asthmatic attacks in patients (Pope et al., 1993; Arlian, 2002; Colloff, 2009). For the first time,
Voorhorst et al. reported that house dust contained mites of the genus Dermatophagoides and
suggested that these were the source of the house dust allergen (Voorhorst et al., 1969).
Therefore they are studied recently as causing atopic diseases in humans, known in
medicine as house-dust-mite allergy or house-dust-mite atopy (Wharton, 1976; Van
Bronswijk, 1981; Platts-Mills et al., 1992; Pope, 1993; Arlian, 2001; Colloff, 2009). These
diseases are atopic asthma, atopic dermatitis (eczema) and allergic rhinitis,
keratoconjunctivitis or oculorhinitis (Arlian & Platts-Mills, 2001; Arlian et al., 2008; Colloff,
2009). Many-faceted studies of house dust mites of the Pyroglyphidae family have been
continued since 1964 in many countries of the world, including the surveys on their
taxonomy and fauna, biology and ecology, epidemiology, allergenicity and control. To date,
only 15 species were found in house-dust samples: Dermatophagoides pteronyssinus, D. farinae,
D. evansi, D. microceras, D. siboney, D. neotropicalis, Hirstia domicola, H. chelidonis,
Malayoglyphus intermedius, M. carmelitus, Sturnophagoides brasiliensis, Hughesiella africana, Hu.
valerioi, Euroglyphus maynei and Gymnoglyphus longior. The house dust mites have been
reported from human dwellings and a wide variety of other habitats associated with man
and his environment, both indoor and outdoor (Van Bronswijk 1981; Fain et al., 1990; Solarz
& Solarz, 1996; Arlian, 2002; Solarz, 2003; Solarz et al., 2007; Colloff, 2009; Perotti et al., 2009).
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Most often they are found in habitats intimately associated with man, such as beds, bed
linen, couches, sofas, other upholstery furnitures, clothing, curtains, window stills, floors
and carpets (Van Bronswijk, 1981; Fain et al., 1990; Mehl, 1998; Colloff, 2009; Arlian & Platts-
Mills, 2001; Arlian, 2002; Solarz, 2001a, b, 2004a, 2010). These domestic environments are
very important locations for forensic investigations, but this richness of mite diversity has
not been exploited by forensic investigators (Perotti et al., 2009). Next chapters are focused
on the mites in a variety of indoor habitats. The natural sources of allergenic mites in
dwellings or stores are still not quiet known (Solarz et al, 2007). The possible sources of
these mites in house dust are nests of synanthropic birds and stored products (Hughes,
1976; van Bronswijk, 1981; Fain et al., 1990). In Poland, the knowledge of their occurrence in
house dust is still poor and the number of faunistic surveys on dust acarofauna is anywhere
from ten to twenty. So, exists indispensability of these surveys in Poland, especially in
Upper Silesian region, where air vitiation may to have stimulating influence to sensitization
of human beings with house dust allergens. It is also commonly known that certain
industrial dusts may cause chronic lung diseases in occupational populations, including also
coal workers (Solarz & Solarz, 1996). It cannot be excluded that apart from coal dust itself,
certain constituents of biological origin may also contribute to the pathogenic effect. Our
previous results reveal the occurrence of allergenic mites in samples of dust and debris from
coal-mines of the Upper Silesian region (Solarz & Solarz, 1996; Solarz, 2003). Thus they
should all be regarded as a potential source of mite allergens in this environment. Our
studies suggest also that the allergenic mites belonging to Acaridae, Glycyphagidae,
Pyroglyphidae and Tetranychidae should be considered as occupational risk factors
contributing to the occurrence of respiratory and dermal diseases among workers of ZOO
gardens (Solarz et al., 2004a, b). As the occurrence and concentration of mites in samples
from different places may vary to a considerable extent, further studies are highly desirable.
An understanding of the seasonal dynamics, as well as environmental factors influencing
mite populations, can be exploited in mite control. Most studies on house dust mites within
dwellings have traditionally sampled beds, carpets and upholstered furniture as the 3 main
types of the indoor microhabitats of these mites. House-dust-mite species have been
recovered from animal and human carcasses and only recently they are being studied for
their potential as forensic indicators (Baker, 2009; Braig and Perotti, 2009; OConnor, 2009;
Solarz, 2009). The usability of domestic mites in forensic investigations is dependent on a
thorough knowledge of their diversity and abundance in a variety of indoor environments.
The ubiquity of mites means that there are many situations in which human beings and
different objects associated with crime are exposed to these arachnids (Perotti et al., 2009).
This chapter gives a faunistic review of the pyroglyphid mites that have actually been
recorded in Southern Poland, in dwellings (beds, carpeted and non-carpeted floors,
upholstery furniture, desks, walls), hospitals, libraries, and other workplaces and/or public
places, in heaps of litter soiled with communal wastes near fences, houses and public
buildings. There are no detailed differential diagnoses and identification keys to the
pyroglyphid dust mites and other domestic mites, especially for the juvenile stages.
Therefore, an acarological diagnostics in forensic studies may be difficult. The taxonomic
relationships and number of valid species within the family Pyroglyphidae are not
established to date. The measurements and analysis of variation between individuals,
populations, species and genera is fundamental to the study of systematic, ecology and
evolution, and has numerous applications in the medical, veterinary and agricultural
sciences, including the forensic medicine. Therefore, this chapter presents also results of
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House Dust Mites, Other Domestic Mites and Forensic Medicine 329
morphological studies, including most medically important taxa of mites, especially genera
from the families Pyroglyphidae (Dermatophagoides, Euroglyphus, Gymnoglyphus,
Sturnophagoides), Acaridae (Acarus, Tyrophagus), Glycyphagidae (Glycyphagus, Lepidoglyphus,
Gohieria) and Chortoglyphidae (Chortoglyphus). I obtained most mite specimens from house
dust samples, bird nests, farming environments, and from research collections in the United
States, UK and Belgium. Own detailed descriptions of all species examined, differential
diagnoses and identification keys are presented. Results suggest that the current division of
the subfamilies of the family Pyroglyphidae should be revised. These results are compared
with published studies with the aim of outlining and examining the weaknesses of the
approach. It is commonly known, that more detailed descriptions of mites in different
indoor situations are very important for forensic investigations (Perotti et al., 2009).
2. Recent studies on the house-dust-mite acarofauna in Poland

The study was carried out from July 2007 to December 2009. During this period 1875 house
dust samples were collected from flats and one-family homes at different localities in Poland
(Silesian Province, Malopolskie Province, Swietokrzyskie Province); 1053 (56.16%) samples
were positive for mites. A total of 15,698 mites were isolated and 18 species were identified,
including 5 species from the family Pyroglyphidae (house dust mites). Among them
Dermatophagoides pteronyssinus was predominant (6,870 specimens; 43.8% of the total count),
followed by D. farinae (6,553 ones; 41.7%). The second species was predominant in
Czestochowa (88.3%), Katowice (91.9%), Sosnowiec (89.4%), Chorzow (94.8%), Tychy (59.4-
92.8%), Bytom (50.9%) and generally in Upper Silesia (55.8-87.61%), whereas D. pteronyssinus
dominated in old buildings in Chorzow and Sosnowiec (60.31%), Bytom (52.7%) and vicinity
(68.4%), in old buildings in Upper Silesia (49.6%), in Upper Silesian dwellings of allergic
patients (70.8%), in Cracow (centre of the city; 50.83%), one-family homes in Zywiec and
vicinity (90.3%), Miechow and vicinity (43.8%), Swietokrzyskie Province (mainly Staszow,
Sedziszow and vicinity, Skarzysko-Kamienna; 41.9%) and Bielsko-Biala vicinity (76.51%).
Another pyroglyphid mites, E. maynei, G. longior and H. chelidonis, occurred in very small
numbers (190, 1 and 6 specimens, respectively). Highest mite densities per gram of dust
were noted in one-family homes. D. pteronyssinus was more abundant per gram of dust
mainly in the one-family houses on agricultural or subagricultural settlements, especially in
bed mattresses, whereas D. farinae in samples from the remaining indoor places examined,
and especially from dwellings in urban regions. Generally, the highest dust mite
concentrations were usually found in dust from beds, upholstery furniture and carpeted
floors. An influence of some abiotic indoor factors on the mite prevalence in the examined
dwellings were analysed separately in relation to samples of bed dust, floor dust and dust
from upholstery furniture. The density of mites was influenced mainly by the type of
heating, temperature, relative humidity, type of sleeping accommodation, type of floor or
furniture, age of building, type of building, number of inhabitants and weight of samples.
Mean relative humidities were 59.7%, 60.4% and 72.0% for samplings of dust from the
Upper Silesian dwellings, from other urban localities and from agricultural or
subagricultural settlements, alternatively. In hospitals a total of 80 samples were examined,
always from two sites – floor and patient’s mattresses. Mites (209 specimens) were isolated
from 44 samples (55%). The most abundant mites were members of the family
Pyroglyphidae, which formed 99.01%of the total count. D. farinae was predominant (66.5%
of all mites collected), followed by D. pteronyssinus (32.1%). The former species was more
frequent in samples from floors than from patients’ beds, whereas D. pteronyssinus was
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collected more frequently from beds than from floors. D. farinae was distinctly more
abundant per 1 gram of dust (arithmetic mean 19.8) than D. pteronyssinus (7.7), whereas
alive mites were slightly more numerous in populations of the second species (5.4 vs. 5.3).
Populations of both species were dominated by adult mites. The density of mites was
influenced mainly by the type of mattress, number of patients and relative humidity. The
research has revealed differences in the occurrence and abundance of both species of house
dust mites between hospitals examined and between particular places within the same
hospital. Moreover, the study suggests that the house dust mites and other mites, including
also some allergenic taxa, should be considered as occupational risk factors contributing to
the occurrence of respiratory and dermal diseases among patients and different workers of
hospitals. Most probably, these mites are introduced into hospitals by humans from their
houses and/or flats. House dust mite prevalence was also studied in the rooms of a tertiary
care hospital in Knurow (Upper Silesia). A total of 60 samples were examined, always from
two sites - from floors and patient’s beds. Only 10 samples were positive for mites (16,67%)
and only 19 mites were isolated; among them the most abundant ones were pyroglyphids
(16 specimens; 84.21% of the total count), two species of the family – D. farinae (12
specimens) and D. pteronyssinus (4 ones). The first species was more abundant per gram of
dust (mean number 34.9) than D. pteronyssinus (27.0). Main sources of mites in libraries and
drug-stores are shelves, upholstery chairs and carpeted floors, whereas in offices carpeted
floors and upholstery furniture. Although beds are commonly known as the main indoor
places of mite occurrence, they were however – besides the dwellings – more abundant in
libraries than in hospitals. The highest numbers of D. farinae per gram of dust were found in
samples of dust from book-shelves and carpets. D. farinae was the dominant constituting
approximately 575 of mites collected. It is possible that older books, and also book-shelves
and carpets contain significant quantities of skin scales or dander from the readers or library
workers (librarians) which skin scales serve as suitable food for pyroglyphid mites. These
data confirm the results of several acarologists of small numbers of mites in hospitals,
offices, hotel rooms and other social buildings or public places, suggesting that the
environment in these places is unsuitable for the mite growth. Repeated routine cleaning
practices as well as maintenance of low relative humidity could in part explain the small
abundance of mites in public places/buildings. This research, as well as some other studies,
has revealed differences in the occurrence and prevalence of different species of domestic
mites between geographical areas and between dwellings within the same geographical
area, between particular places within the same dwelling, and between the seasons of the
year. This knowledge may be very useful in the forensic medicine. Moreover, the study
suggests that the house dust mites and other domestic mites, including also some allergenic
or parasitic taxa, should be considered as occupational risk factors contributing to the
occurrence of respiratory and dermal diseases among librarians, barkers, cleaners, different
workers of hospitals, drug-stores, airports, offices, and many other occupational categories.
Exposure to indoor allergens, especially dust mites has been recognized as a risk factor for
sensitization and allergy symptoms that in extreme conditions could develop into asthma
(Spiewak et al., 1995).
3. Species composition of the house-dust-mite acarofauna

Pyroglyphid mites usually make up 60-90% of the house dust acarofauna in temperate
climate regions throughout the world (Van Bronwijk 1981; Fain et al., 1990; Colloff, 2009),
also in Poland (Solarz, 2010). In the case of mite density, the number of mites per gram of
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dust may range from a few to 16,000 or more, although the results of the surveys are
difficult to compare and evaluate because of the lack of standardization of both dust
collecting methods and reporting procedures. It has been shown, however, that occurrence
and abundance of house dust mites may vary in particular topographical regions and are
associated to a large degree with the climate of a region, and especially with outdoor and
indoor humidity (Korsgaard, 1998; Mumcuoglu, 1976; Mumcuoglu et al., 1999; Colloff,
2009). Ratios of numbers of the particular pyroglyphid dust mite species, especially between
D. pteronyssinus and D. farinae, are different in separate regions of the world (Fain et al.,
1990; Mumcuoglu, et al. 1999). Decisive factors influencing their occurrence and abundance
are mainly relative humidity and temperature of both outdoor and indoor air (Solarz, 2001
a, b). It is commonly known that the optimal temperature is higher (25-30 oC) and optimal
humidity lower (50-75 %RH) for D. farinae than for D. pteronyssinus. The former species
appear to survive better in dryer habitats than the latter, whereas lower temperature (15-20
oC) and higher humidity (75-80 %RH) favours D. pteronyssinus in mixed laboratory cultures
(Arlian et al., 1998). Within the wide zone of temperate climate D. pteronyssinus is the most
common and dominant species in more damp areas, at the seaside or in lowlands, which
have a more humid climate. D. farinae, however, is more common and abundant in areas
with a dry continental climate (intercontinental and alpine regions) (Voorhorst et al., 1969;
Fain et al., 1990; Colloff, 2009; Solarz, 2001a, b, 2010). In Europe, most abundant mite
populations were usually collected from bed mattresses (Van Bronswijk, 1981; Fain et al.,
1990; Hallas & Korsgaard, 1997; Horak et al., 1996; Solarz, 2006). In previous surveys
(Solarz, 1998) of house-dust-mite fauna in dust samples from dwellings and hospitals
located in Sosnowiec and Katowice (Upper Silesia), the dominance of the Pyroglyphidae was
demonstrated (Solarz, 2001 a, b). Approximately 63.5% of the total mite population belonged
to this family. Most abundant were the following members of Pyroglyphidae – D.
pteronyssinus (29% of the total mite population), D. farinae (25.5%) and E. maynei (6.0%).
Unidentified Dermatophagoides spp. formed 3% of mites isolated from the samples examined.
As indicated by the results of other previous surveys in Poland, D. pteronyssinus was found
to be the dominant species in Warsaw, Poznan, and two Upper Silesian cities-towns
Katowice and Sosnowiec (Horak et al., 1996; Solarz, 1998). As demonstrated by the more
direct investigations of the house dust samples from dwellings (in Katowice, Sosnowiec,
Myslowice, Chorzow, Tarnowskie Gory, Bytom, Zabrze, Gliwice, Dabrowa Gornicza and
Ogrodzieniec), libraries (in Sosnowiec), institutes (in Katowice) and hospitals (in Katowice
and Sosnowiec), the dominance of Pyroglyphidae was more significant than previously
(Solarz, 1998, 2001a, b). About 89.2% of the total mite population from dwellings constituted
the following members of this family – D. pteronyssinus (45.1%), D. farinae (40.2%), E. maynei
(2.6%), G. longior (0.05%) and unidentified Dermatophagoides (1.24%). A total of 31 species of
mites from 15 families were identified, of which 18 species belonged to Astigmatina, 3 to
Prostigmata, 3 to Oribatida sensu lato and 7 to Mesostigmata. The fauna of house dust mites
was therefore rather differentiated in this region. This was particularly apparent in
dwellings, where 49 combinations of species composition in collected mite populations were
observed. This study has clearly established that mite species in beds and other sleeping
accommodations are different from the mite species on bedroom floors. The pyroglyphid
mites were most abundant in samples from beds and upholstery furniture whereas floors
were dominated with the non-pyroglyphid mites. A total of 402 samples were analysed: 238
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samples from dwellings, 122 samples from hospitals, 14 from libraries and 28 from
institutes. Mites were present in 51.3%, 50.0%, 21.3% and 17.9% of dust samples from
dwellings, libraries, hospitals and institutes, respectively. Generally, they were found in 160
samples (39.8% of the total count). The majority of mites (96.0 %) were found in samples
from the dwellings, especially in dust from upholstery furniture, couches, sofas and beds.
Altogether, the pyroglyphid mites constituted 89.2%, 78.9% and 57.5% of a total population
of mites collected from dwellings, libraries and hospitals, respectively, but were not found
in institutes. In total, D. pteronyssinus was the most dominant, especially in libraries and
hospitals, however in dwellings D. farinae was more abundant per 1 gram of dust than the
former species. Another pyroglyphid mite, E. maynei, occurred in very small numbers
(Solarz, 1998, 2001a, b). Mites of families Glycyphagidae, Chortoglyphidae and Acaridae are
considered to be much more sensitive to desiccation than pyroglyphids (Van Bronswijk,
1981; Fain et al., 1990; Hallas & Korsgaard, 1997). It has also been suggested that some
domestic mite species thrive in very damp conditions; this group include domestic acarids,
glycyphagids (L. destructor, G. domesticus) and cheyletids (Cheyletus spp). Therefore, the
presence and abundance of these mite species can be used as an indicator of humid
environments (Fain et al., 1990; Solarz, 1998, 2003). The relatively low frequency of mites in a
total of samples from dwellings examined and relatively lower abundance of glycyphagids,
acarids, cheyletids and E. maynei mites is clear, taking into account the aforementioned
values of indoor relative humidity observed in these dwellings (Fain et al., 1990; Solarz,
1998, 2003). In general, these mites are not as abundant or frequent in Europe as in the
Tropics (Fain et al., 1990; Puerta et al., 1993; Mehl, 1998; Mumcuoglu et al., 1999; da Silva et
al., 2001; Colloff, 2009). The mean concentration of mites in examined samples and mite
frequency was at the lower end of the published range for more humid regions (Van
Bronswijk, 1981; Fain et al., 1990; Mumcuoglu, et al. 1999; Colloff, 2009) and was comparable
with some European results from France, Denmark and Holland (Van Bronswijk, 1981; Fain
et al., 1990; Harving et al., 1993) and with other results from Poland (Horak et al., 1996;
Racewicz, 2001).
4. Acarofauna of the synanthropic outdoor sites

The occurrence of allergenic mites (pyroglyphid house-dust mites, acarid and glycyphagid
storage mites and others) in synanthropic outdoor sites in a densely populated urban area
was investigated. Litter soiled with communal wastes was sampled. 80.5% of the total
population was formed by allergenic mites. These mites (11 species) belong to Acaridae and
Winterschmidtiidae. Among the astigmatic mites two acarids were dominant: Tyrophagus
silvester and T. longior (28.7% and 25.1% of all mites respectively), with the latter being the
most frequent (44.2% of all samples). The age structures of the two species versus relative
humidity were investigated. The correlations between the age structures of T. silvester, T.
longior, T. molitor and T. similis were statistically analysed. The most important allergenic
mites from Pyroglyphidae (house dust mites) or Glycyphagidae (stored food mites) were
not found. However, allergenic mites from Tarsonemidae – important for house dust, and
Acaridae – reported from food stores, were presented numerously in the samples, which
bring us to the conclusion that litter can be an important source of invasion of the mites into
dwellings or food stores.
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5. Allergenic mites as the occupational and/or environmental risk factors

Many species of mites that humans come in contact with, besides the house dust mites or
storage mites, induce allergic reactions. These include some species of spider mites (e.g., the
2-spotted spider mite Tetranychus urticae and Panonychus ulmi), which are common pests in
orchards, greenhouses, and gardens. These mites were recently proved to induce IgE-
mediated reactions (Arlian, 2001, 2002; Solarz, 2004a, 2006). It should come as no surprise to
learn that chigger mites (larvae of Trombiculidae), ticks (Ixodida) and other species of
ectoparasitic mites (Mesostigmata) of fowl, pigeons, other birds, mice, guinea pigs, and
other mammals and some predaceous mites sensitize and induce allergic reactions in
human (Arlian and Platts-Mills, 2001). Many species of stored-product mites occur in both
residential and occupational environments and in processed foods and can cause allergic
disease. In addition, humans may contact or be exposed to predaceous mites and parasitic
mites of plants and animals that are also the sources of allergens that induce allergic disease.
5.1 Storage mites

The stored-product mites, especially several species from the families Acaridae,
Glycyphagidae and Chortoglyphidae (Acari: Astigmatina), are commonly found in different
stored food products, hay, straw, granaries, barns and other farming and occupational
environments, as well as in samples of house dust. The most abundant and most often
reported are Acarus siro, A. farris and Tyrophagus putrescentiae from Acaridae, Lepidoglyphus
destructor, Glycyphagus domesticus and Gohieria fusca from Glycyphagidae and Chortoglyphus
arcuatus from Chortoglyphidae (Hughes, 1976; Franz et al., 1997; Mehl, 1998; Baker, 1999;
Müsken et al., 2000; Solarz, 2011; Solarz et al., 1997, 2004b). These mites are also the source
of clinically important allergens and the cause of occupational allergies (known as an allergy
to storage mites) among farmers, grain-storage workers and other agricultural workers (van
Hage-Hamsten & Johansson, 1998; Morgan & Arlian, 2006; Fernández-Caldas et al., 2007).
The greates exposure to storage mites usually occurs in an occupational and/or rural setting
where allergies to these mites are of major importance (van Hage-Hamsten et al., 1992; van
Hage-Hamsten & Johansson, 1998; Arlian, 2001, 2002; Stejskal & Hubert, 2008). Many
species of storage mites are found in processed foods (flour, boxed baking mixes such as
cakes, pancakes, and beignets); in stored hay, grain and straw; in dust in grain and hay
storage and livestock feeding facilities. Exposure to storage mite allergens can be by ingestion
or by inhalation. Several studies report anaphylactic reactions after patients consumed
beignets, cakes, pancakes, pizza, pasta, cornmeal cakes, and bread made from ingredients
contaminated with mites. The importance of storage mites as ingested or aeroallergens in
the urban population has not been studied extensively. The greatest exposure to storage
mites usually occurs in an occupational setting where allergies to storage mites are of major
importance. Kronqvist et al. (2000) reported that 12% of 440 farmers tested were sensitive to
the storage mites Acarus siro, L. destructor, T. putrescentiae, and Glycyphagus domesticus.
Similar sensitivities to storage mites have been reported for farmers in Scotland and USA
(Arlian et al., 1997; Arlian, 2002). Important storage mite species in occupational settings are
L. destructor, G. domesticus, A. siro, T. putrescentiae, Tyrophagus longior, Aleuroglyphus ovatus,
Suidasia medanensis, Chortoglyphus arcuatus, and Carpoglyphus sp. Sensitization to the storage
mite T. putrescentiae is present in the urban population of Upper Silesia in similar
proportions as to pyroglyphid house-dust mites (Dermatophagoides spp.) (Szilman et al.,
2004). Testing with storage mites should be considered routine allergological diagnostic
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procedure. In other words it is necessary to establish methods for identification and

quantification of mites in the Upper Silesia (Szilman et al., 2004).
5.2 Other mites inducing allergic reactions

Many species of mites that humans come in contact with, besides those found in house dust,
induce allergic reactions. These include the citrus red mite (Panonychus citri) and the two-
spotted spider mite (Tetranychus urticae), which are common pests of apple orchards. Mites
are ubiquitous and thrive in many diverse environments. Thus humans contact many
species of mites and their products in their daily lives. The role of these less-known mites in
causing allergic disease is not yet known. Phytophagous mites of fruit trees, vegetable crops,
and yard, greenhouse, and house plants can cause allergic disease in at-risk populations. A
study of 725 Korean farmers working in apple orchards found that 23.2% and 21.2% were
sensitive based on skin tests to the two-spotted spider mite (T. urticae) and the European red
mite (Panonychus ulmi), respectively. In this study population, the prevalence of sensitivity
to these mites was greater than it was to the house dust mites, D. farinae and D. pteronyssinus
(Kim et al., 1999; Arlian, 2002; Solarz 2003, 2004a, 2006). Another study by this group found
that skin tests of 14.2% of 1055 children living around citrus orchards were positive to P. citri
(Lee et al., 2000). Multiple erythematous papules accompanied by severe pruritus were
observed in humans bitten by the mites Pyemotes tritici (Pyemotidae), Dermanyssus gallinae
(Dermanysidae), Ornithonyssus bacoti (Macronyssidae) and Andrlaelaps casalis (Laelapidae) in
Israel (Rosen et al., 2002). The mite Hemisarcoptes cooremani, a parasite of scale insects that is
commonly found in orchards, yards, and gardens, was recently discovered to induce IgE-
mediated reactions (Arlian et al., 1999). Hemisarcoptes cooremani that feeds on scale insects
that parasitize trees and shrubs in orchards, yards, and gardens can induce allergic diseases.
Immunoblotting of extracts of this mite showed that they contain 16- and 19-kDa proteins
that bound IgE in the serum of an exposed and symptomatic individual (Arlian et al., 1999).
It should come as no surprise to learn that chigger mites (Trombiculidae), ticks (Ixodida)
and other species of ectoparasitic mites (Mesostigmata) of fowl, pigeons, other birds, mice,
guinea pigs, and other mammals and some predaceous mites either sensitize or induce
allergic reactions in human as well (Arlian & Platts-Mills, 2001). The saliva of ticks (Ixodida)
contains immunogenic proteins that can induce IgE-mediated reactions while they feed;
cases of anaphylactic reactions from a tick bite have been reported (Arlian, 2002).
Predaceous mites such as Phytoseiulus persimilis and Amblyseius cucumeris, that feed on
spider mites and larvae of thrips, respectively, can sensitize greenhouse workers (Kronqvist
et al., 2000). This raises the possibility that predaceous mites, used for biological control of
pest species in fields and orchards, may be important sources of allergens for gardeners,
farmers, and people living around fields and orchards (Arlian, 2002). Molecules of from the
human itch mite (Sarcoptes scabiei var. hominis), which burrow in the stratum corneum of the
skin, induce IgE production and an IgE-mediated reaction in some parasitized human hosts
(Arlian, 2002; Arlian & Platts-Mills, 2001). Many of these immunogens are cross-reactive
with antigens of the house dust mites D. farinae and D. pteronyssinus. Capability of other
parasitic mites, such as follicle mites (Demodex folliculorum, Demodex brevis), red chicken
mites (Dermanyssus gallinae), rat mites (Ornithonyssus bacoti) and sheep scab mites (Psoroptes
ovis), inducing IgE reactions in humans needs to be investigated (Arlian, 2002).
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6. Identification diagnoses to mites of the families Acaridae, Glycyphagidae,

Chortoglyphidae and Pyroglyphidae in taxonomic order
The measurements and analysis of variation between individuals, populations, species and
genera (or subfamilies), is fundamental to the study of systematics, ecology and evolution, and
has numerous applications in the agricultural, veterinary and medical sciences (Klimov &
OConnor, 2009; Dabert et al., 2010), including the forensic medicine (Perotti, 2009a). In forensic
acarology it is necessary to produce the data that clearly demonstrate how mites can contribute
to investigations (Perotti et al., 2009). Therefore, a following priority is to to provide more user
and friendly identification aids, such as differential diagnoses and/or keys for different groups
of synanthropic or semisynanthropic mites, including the domestic and storage mites (Baker,
1999; Colloff, 1998; 2009; Desch, 2009; Perotti et al., 2009; Solarz, 2011).
6.1 Family: Acaridae

6.1.1 Genus: Acarus L.
Differential diagnosis: In both sexes: solenidion sigma 1 on genu I at least 3 times as long as
sigma 2 (Fig. 1A); setae v e less than half the length of setae v i ; setae v e arising near the
anterior angles of the dorsal propodosomal or slightly posterior; ventral apex of tarsus I
with proral (p, q) and unguinal (u, v) setae as thin, not short, stout spines; setae c 1 and d 2
short; setae sc e about equal in length to sc i (Fig. 1B); seta sc x generally expanded basally
and thickly pectinate (Figs 1B, 2). In males: legs I enlarged; femur I with a stout ventral
apophysis or process (seta vF) (Fig. 1); ventral side of genu I with small thickenings of
cuticle; lateral arms of the penis support diverge posteriorly. In females: characteristic
structure of reproductive system, with chitinised depression in body wall, external opening
of expansible tube of bursa copulatrix, chitinised bell-shaped structure leading into
receptaculum seminis and two armed flame-like chitinised opening to thin-walled oviduct;
claws of female never bifid.
6.1.1.1 Acarus siro L.
Differential diagnosis: In both sexes: setae sc e slightly shorter than sc i; setae d 1
intermediate in length between c 1 and e 1, never more than 3 times longer than c 1; seta sc x
expanded basally and thickly pectinate; solenidion omega 1 on tarsi I and II long, recumbent
and the angle between the dorsal surface of the tarsus and the anterior face of the solenidion
rarely exceeds 45 degrees (generally about 30). In males: tarsus II of male with seta s large,
about equal in length to length of empodial claw; ventro-posterior margin of this seta
concave, claw tip directed backwards; seta sc x expanded basally and thickly pectinate (Fig.
2); lateral arms of the penis support diverge posteriorly; penis (aedeagus) as an arc-shaped
tube with a blunt end. In females: tarsi I-II with seta s large, about equal in length to length
of empodial claw (Fig. 3); ventro-posterior margin of the seta concave, claw tip directed
backwards; setae more sparsely pectinate as that of the male; bursa copulatrix opens into a
narrow expansible tube which joins a sclerotized bell-shaped structure; setae ps 3 are twice
and ps 2 almost four times the lengths of ad 3, ad 2 and ad 1; vulva characteristic, positioned
between coxae III and IV. Hypopi: mobile form; gnathosomal remnant with well developed
solenidia; posterior ventral attachment organ (sucker plate) well developed, with 8 distinct
suckers; legs well developed, with normally formed setae and solenidia; scapular setae
relatively long; length of sc i greater than distance between alveoli; hysterosomal setae c 1, d
1, d 2 and e 1 long, extending beyond base of next most posterior seta; sc i about 1.5 times
length of c 1 and 1.2 times length of d 1; setae c 1 and d 1 about 3 times longer than h 1; bases
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of genital setae and flanking pair of coxal suckers almost in line, distance between base of
sucker and base of seta is less than width of setal base.
6.1.1.2 Acarus farris (Oudemans)
Differential diagnosis: In both sexes: setae sc e about equal in length to sc i; setae d 1
intermediate in length between c 1 and e 1, never more than 3 times longer than c 1; most
dorsal setae short, setae d 1 and e 1 not extending to base of next posterior seta; solenidion
omega 1 of tarsus II short, compact, with sides expanding gradually from the base, then
narrowing to an indistinct neck before expanding into a terminal head, width of widest part of
head equal to width of widest part of shaft; the angle between anterior margin of the
solenidion and the dorsal surface of the tarsus II is nearer to 90 degrees than 45 degrees. In
males: tarsus II of male with seta s slender, about 1/2-2/3 the length of empodial claw and
points anteriorly; ventro-posterior margin of the seta convex, seta tip directed forward. In
females: tarsi I-II with seta s slender, about half to two-thirds the length of empodial claw;
ventro-posterior margin of seta convex, seta tip directed forward; setae ad 3, ad 2 and ad 1
almost equal in length, ps 3 about 1/3 and ps 2 about twice as long; vulva as in A. siro. Hypopi:
hysterosomal setae c 1, d 1, d 2 and e 1 appreciably shorter than scapulars, and not extending to
bases of next most posterior setae; setae sc i at least 2 times length of c 1 and d 1; setae c 1 and d
1 about equal in length to h 1; bases of genital setae just forward of coxal sucker bases;
distance between base of sucker and base of seta about equal to width of setal base.
Fig. 1. Acarus sp. – A. Acarus immobilis, male: legs I and II; B. Acarus nidicolous, male in dorsal
aspect: seta vF on femur I, shape of supracoxal seta (sc x) and propodosomal chaetotaxy;
key: solenidia omega 1 on tarsi I and II; solenidia sigma 1 and 2 on genu I; vF = seta vF on
femur I; sc x = supracoxal seta; external (sc e) and internal (sc i) scapular setae; dorsal setae c
1 and c 2.
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6.1.1.3 Acarus immobilis Griffiths

Differential diagnosis: In both sexes: setae sc e about equal in length to sc i; setae d 1
intermediate in length between c 1 and e 1, never more than three times longer than c 1; most
dorsal setae short, setae d 1 and e 1 not extending to base of next posterior seta; tarsus II with
solenidion omega1 with sides almost parallel, expanding into a distinct egg-shaped terminal
head which is wider than widest part of shaft (Fig. 1A); the angle between the dorsal surface
of the tarsus and the anterior face of the solenidion generally about 45-50 degrees. In males:
tarsus II with seta s slender, about half the length of empodial claw; ventro-posterior margin
of the seta convex, seta tip directed forward. In females: tarsi I-II with seta s slender; ventro-
posterior margin of the seta convex, seta tip directed forward. Hypopi: inert forms; legs
short (in dorso-ventral mounts tarsi of legs I and II are the only segments completely
visible); setae very reduced or absent; gnathosoma rudimentary, flagelliform bristles
(solenidia) not present; posterior ventral attachment organ rudimentary, with at most one
pair of well-developed suckers on sucker plate, the central pair of suckers vestigial, anterior
peripheral pair moderately well developed; all setae on tarsus III and IV shorter than length
of tarsus, spine-like, never leaf-shaped; solenidion omega 1 on tarsus I long, at least twice
the length of empodial claw.
Fig. 2. Acarus siro – male, dorsal aspect: supracoxal seta; key: sc x = supracoxal seta.
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Fig. 3. Acarus siro – female, legs I and II; key: s = seta (spine) s.
6.1.2 Genus: Tyrophagus Oudemans

Differential diagnosis: In both sexes: setae v e distinctly barbed, relatively long, longer than
the length of the genu I and usually positioned near anterior lateral corners of
propodosomal shield; setae v e at almost the same level as v i and curve downwards; setae sc
i longer than sc e; setae c 1 and d 2 usually almost equal in length, and shorter than e 1 and h
1; the dorsal terminal seta e on tarsi I needle-shaped; presence of 5 ventral setae on tarsi I, of
which the three central ones are thickened; solenidion sigma 1 on tibia I always less than
three times as long as sigma 2; ventral apex of tarsi with proral and unguinal setae usually
in the form of short, stout spines, occasionally one or both pairs strongly reduced or absent;
tarsi I-II more than twice as long as basal width; proral setae thinner than unguinal setae
but similar in length; Grandjean's organ finger-like. In males: without modifications of leg I;
legs I not enlarged and the femur does not bear a ventral apophysis.
6.1.2.1 Tyrophagus putrescentiae (Schrank) (Fig. 4)
Differential diagnosis: In both sexes: anterior margins of propodosomal shield with
pigmented spots (corneae); coxal plate II with a sinuous posterior margin so that the plate
narrows sharply along the distal 1/3; tarsi I and II with solenidion omega 1 terminating in a
distinctly expanded tip; distal 2/3 of solenidion omega 1 on tarsi I widened. In females:
genital seta g longer than vulva; paraproctal setae ad 1 much shorter than ad 2; setae ad 2
distinctly longer than anal slit; internal spermathecal apparatus large, base of spermatheca
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expanded, flat, spermathecal duct with distinct constriction about halfway along length;
proximal part of spermathecal duct gradually widened. In males: aedeagus shorter (<20µm),
S-shaped with two deep curves, one at base, the other in apical third; distal 1/3 of aedeagus
curved at an angle of about 75-100° to its median part; tarsal copulatory suckers on tarsus IV
equidistant from the base and apex of the segment; setae ps 2 more than 1.8-2 times longer
than the anal slit.
6.1.2.2 Tyrophagus longior (Gervais)
Differential diagnosis: In both sexes: anterior margins of propodosomal sclerite without
pigmented spots (corneae); supracoxal seta (sc x) thin, not expanded basally, pectination
variable; setae d 2 short, at most only slightly longer than c 1; setae d 1 about 1.3-2 times as
long as c 1 and d 2; tarsi I-II with solenidion omega 1 long, slender, tapering toward tip; tip
pointed and not expanded apically. In males: supracoxal seta (sc x) thin, with short lateral
barbs of about equal lengt; the lateral sclerites supporting the penis (aedeagus) turned
inwards; aedeagus straight only slightly curved distally and shaped like the spout of a
teapot; aedeagus long, slender and tapering towards its free end; tarsus IV longer than the
combined length of the genu and tibia; both tarsal copulatory suckers positioned nearer to
the base of tarsus than the apex. In females: anterior margins of propodosomal sclerite
without pigmented spots (corneae); internal spermathecal apparatus large, spermathecal
duct moderate or large, base of spermatheca moderately long or very wide, distinctly wider
than spermathecal duct; setae ad 2 considerably longer than ad 3, ps 3, ps 2 and ad 1.
6.2 Family: Glycyphagidae

6.2.1 Genus: Lepidoglyphus Zachvatkin
Differential diagnosis: In both sexes: setae v e absent; setae v i long and barbed, extend well
beyond the tips of chelicerae; scapular setae arranged in a trapezoid or rectangle;
gnathosoma at anterior apex of the body; prodorsal aclerite crista metopica absent; seta sc x
slender, much branched; trochanteres I-II not surrounded basally by large, thickened
apodemes (as in the genus Xenocaster); with pectinate subtarsal scales on all tarsi; on genu I
solenidion sigma 2 more than 3 times longer than solenidion sigma 1; setae la, ra, ba on
tarsus I arise in the distal third of the tarsus. In males: penis (aedeagus) posterior to coxal
epimera I and positioned between coxae II and III and with anterior end marked by a
triangular plate. In females: epigynium not fused to coxal apodemes I; vulva positioned
between coxae II and III.
6.2.1.1 Lepidoglyphus destructor (Schrank)
Differential diagnosis: In both sexes: ventral seta nG of genua III not widened to form a
pectinate scale; solenidion sigma on genu II not thickened. In males: without modifications
of leg I; on genu I sigma 2 more than 4 times longer than sigma 1; ventral setae kT on tibiae
III and IV does not arise from the edge of the arthrodial membrane as in L. michaeli. In
females: setae 4 a arises behind the posterior edge of the genital opening; anus terminal; 2
pairs of setae (ad 3, ps 3) inserted on either side of its anterior end. Hypopi: inactive, oval
colourless, with reduced legs, rudiment of the genital slit and apodemes I and II feebly
sclerotized; enclosed within the protonymphal cuticle.
6.2.2 Genus: Glycyphagus Hering

Differential diagnosis: In both sexes: without a subtarsal pectinate scale on all tarsi; usually
with a prodorsal sclerite crista metopica; seta sc x more robust, forked and branched;
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solenidion sigma 2 on genu I is more than twice as long as solenidia sigma 1 and omega 1; 2
ventral setae present on tibiae I and II; genital opening lies between coxae II and III.
6.2.2.1 Glycyphagus domesticus (De Geer)
Differential diagnosis: In both sexes: crista metopica extends from the base of the
chelicerae to level of the anterior scapular setae (= sc i) (Fig. 5A); setae v i inserted
almost in the middle of the crista metopica; setae d 1 arising almost at the same
Fig. 4. Tyrophagus putrescentiae – legs I and II and dorsal propodosomal chaetotaxy; key:
solenidia omega 1 on tarsi I and II; sc x = supracoxal seta; external (v e) and internal (v i)
vertical setae; external (sc e) and internal (sc i) scapular setae.
level as e 1; the subtarsal seta is replaced on all the legs by a pectinate seta wa arising from
the middle of the tarsus; setae la, ba and ra arise between the base of wa and the apex of the
tarsus; solenidion omega 1 on tarsus I as a slender rod longer than omega 1 on tarsus II;
solenidion omega 2 is relatively long and about half the length of solenidion omega 1 and
much longer than the famulus epsilon; on genu I solenidion sigma 1 is less than half the
length of sigma 2 and about the same length as omega 1. In males: with normal setae on
tibiae I and II; on tibiae III and IV seta kT is well removed from the distal edge of the
segment. In females: genital opening extends to the posterior edge of of acetabula III and it
is shorter than the distance separating it from the anterior end of the anus; setae 4a inserted
just behind the hind end of the genital opening; 2 pairs of setae (ad 3, ps 3) lie on either side
of the anterior end of the anal slit; a tubular bursa copulatrix projects from the hind margin
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of the opisthosoma; setae d 1 arising almost at the same level as e 1; distal setae (la, ra, ba, wa)
on tarsus I more widely spaced. Hypopi: oval body, white in colour, with small bud-like
gnathosoma, enclosed in protonymphal cuticle without reticulations.
cm
vi
cm
sc x
sc i
sc i
sc e
sc e
vi
cm
B
Fig. 5. Glycyphagus spp., females, dorsal aspects – shape of crista metopica: A. G. domesticus; B.
G. privatus; key: cm = crista metopica; v i = internal vertical setae sc i = internal scapular setae;
sc e = external scapular setae; sc x = supracoxal seta.
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6.2.2.2 Glycyphagus privatus Oudemans (Syn.: G. cadaverum Schrank)

Differential diagnosis: In both sexes: setae v i inserted almost at the anterior end of the crista
metopica (Fig. 5B); setae d 1 arising anterior to e 1 and on the same level as c 1; solenidion
omega 2 on tarsus I very short, the same length as the famulus; solenidion sigma 1 on genu I
shorter than in G. domesticus, much shorter than omega 1; seta sc x forked and branched. In
females: genital opening (vulva) longer than in G. domesticus, extending as far back as the
posterior edge of acetabula IV and longer than the distance between it and the anus. In
males: with normal setae on tibiae I and II.
6.2.3 Genus: Gohieria Oudemans; Gohieria fusca (Oudemans)

Differential diagnosis: In both sexes: propodosoma without a division into 2 planes and
extended forward and overhangs the gnathosoma; chelicerae with 1 seta; cuticle evenly
sclerotized, pitted deeply tanned, pinkish-brown and ornamented with fine setae;
ornamentation not in the form of small triangular microtrichae; no distinct propodonotal
sclerite (or crista metopica) present; lateral body setae of both sexes not arising from
tubercles; lamellae absent; setae v i and sc x pectinate, the remaining setae of the idiosoma
only slightly serrated; setae v i situated almost on the apex of the propodosoma (only
slightly more posterior); setae v e much more posterior, positioned almost in the same
transverse line as setae sc x; seate sc i, sc e and c 2 on a level with one another; legs short and
massive, distinctly ridged; apodemes of all legs slender and unite to surround the genital
opening; empodial claws present; tibiae and genua distinctly ridged especially in females;
genua and femora freely articulated on all legs, distal edges of genua and femora expanded,
femora without large ventral keel; tibiae I-II with 2 ventral setae; genu III with solenidion
sigma present; solenidion phi on tibia I unusually long. In males: pretarsi expanded basally
and arising ventrally on tarsal apex; legs III and IV with long pretarsi and noticeably bent;
genitalia relatively simple, aedeagus anteriorly directed, positioned between coxal fields IV;
body form of males similar to females, sexual dimorphism slight. In females: legs more
slender than in males, the longitudinal ridges better developed; setae on tarsus I spaced out
along the segment instead of being concentrated at the distal end; genital papillae everting
in posterior region of oviporus (vulva); female oviporus region elongate, parallel sided;
coxal apodemes I fusing separately with epigynium; ventral seta of femur I nude, without
barbs.
6.3 Family: Chortoglyphidae

6.3.1 Genus: Chortoglyphus Berlese; Chortoglyphus arcuatus (Troupeau)
Differential diagnosis: In both sexes: chelicerae chelate, conspicuous; idiosoma oval and not
divided into propodosoma and hysterosoma; body cuticle smooth and shining, without
striations and microtrichae; discrete propodosomal sclerite absent; body setae short and
nearly all smooth; genital papillae normal in form, strongly enlarged and reduced;
solenidion omega 1 on tarsus I formed as a long curved rod arising close to a small
solenidion omega 2; tarsus I with seta wa enlarged to form a stout spine and ba as a thin seta;
only 1 solenidion sigma on genu I; tibiae I-II with dorsal solenidion and 0-2 ventral setae;
legs ventrally positioned; praetarsi fleshy with inserted claws; empodial claws simple; anus
positioned near posterior margin of body; both sexes with empodial claws simple or absent;
ventral subcapitulum without external ridges; discrete coxal apodemes III and sometimes IV
absent. In females: vulva (oviporus) longitudinal with genital valves fused to body anteriorly,
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free posteriorly, and situated between coxae III and IV; genital papillae always associated
with genital opening; 5 pairs of setae in anal region. In males: aedeagus long, situated
between coxae I and II; adanal suckers conspicuous; 2 pairs of setae in anal region; tarsal
suckers present on tarsi IV; legs III similar to legs IV.
6.4 Family: Pyroglyphidae

6.4.1 Subfamily Pyroglyphinae
Differential diagnosis: In both sexes: setae v i absent; tegmen well developed; cuticle from
slightly to strongly sclerotized; striations either relatively thick, irregular and well spaced, or
completely lacking the median area separating the epimera I punctate; idiosomal setae
variable, either all very thin and very short, or with some setae (sc e, h 2 and h 3) very long
and strong. In females: cuticle lacking projections as in the genus Glycyphagus; setae sc i
never very strong and long; ventral surface of tarsi I and II without projections; cuticle
variable; epigynium distinctly separated from epimera I; tarsi III and IV lacking apical
spines (except in Weelawadjia which presents these spines); posterior lip of the vulva always
punctate; posterior vulvar lip generally fairly long and in some species incised in its anterior
angle. In males: cuticle variable - slightly or strongly sclerotized; striations either present but
irregular, thick and well spaced, or absent; setae sc i never very strong and very long; tarsi
IV normal (not very short); legs III variable; femora III lacking a spur; tarsi IV always
without a bifid or trifid subapicoventral spine; tarsi III with or without the subapicoventral
spine; adanal copulatory suckers either present or lacking; tarsal copulatory suckers (on
tarsi IV) either present or (generally) replaced by thin and short setae; setae sc e either long
and strong (genera Weelawadjia and Campephilocoptes) or short and thin (other genera); tarsi
III lacking an apical forked spine.
6.4.1.1 Genus: Euroglyphus Fain; Euroglyphus maynei (Cooreman)
Differential diagnosis: In both sexes: tegmen well developed, triangular with rounded apex
(not bifid in the male as figured in the typical description); cuticle slightly sclerotized with
rather well formed striations or folds; hysteronotum with in a median shield with poorly
distinct margins; anterior legs lacking chitinous membranes; chaetotaxy reduced:
trochanterals I-III, tibials IV, genital anterior setae (3 b) and anal external (ps 2) setae are
lacking; tarsi III with 5 setae; tarsi IV with 3 setae; dorsal setae very thin and short; setae h 3
very thin and short (maximum length 50 µm); setae h 2 short (not exceeding 30 µm) and very
thin; genu I with one solenidion. In males: tegmen with rounded, unforked apex; dorsal
setae variable; opisthosoma slightly but regularly narrowed backwards; anus more posterior
(anal suckers situated at 25 µm from the posterior margin of the body); posterior margin of
the body straight and wide with 2 very small paramedian lobes; adanal suckers well
developed; tarsi IV lacking suckers. In females: setae sc e thin and short (maximum 50 µm);
no chitinous pouches at the bases of legs II; tegmen either triangular and prominent but
with apex rounded and not forked or poorly developed, rounded with a small median
notch; posterior lip of vulva punctate and short, not covering the vulvar slit; anterior angle
of posterior vulvar lip not incised; tegmen triangular with rounded, not incised apex;
hysteronotum striated with a median shield; copulatory vestibule ovoid, strongly
sclerotized and opaque; tarsi I-IV without apical processes nor spines.
6.4.1.2 Genus: Gymnoglyphus Fain
Differential diagnosis: In both sexes: tegmen is triangular with bifid apex and the chaetotaxy
is not reduced: the setae trochanterals I-III, tibials IV, setae ps 2 and 3 b are present; the tarsi
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III and IV bear 6 and 5 setae, respectively. In males: adanal suckers well developed; dorsal
setae short and thin; setae h 3 and h 2 very thin and short (maximum length 50 µm); tegmen
deeply incised at apex; opisthosoma strongly narrowed backwards; anus more anterior
(adanal suckers at 40 µm from posterior margin of body); posterior margin of body
(idiosoma, opisthosoma) narrower, concave in the middle and with 2 small but well distinct
paramedian lobes; chaetotaxy normal (trochanteral setae I-III, tibial IV setae, ps 2 and 3 b
setae present). In females: setae sc e thin and short (maximum 50 µm); setae h 2 and h 3 short
or very short (less than 50 µm) and thin; no chitinous pouches at bases of legs II; tegmen
triangular, prominent with apex forked; posterior vulvar lip very long; anteriorly and
completely covering the vulvar slit, its anterior angle not incised; opening of bursa
copulatrix situated near the posterior extremity of anus and followed by a small ovoid
strongly sclerotized pouch (vestibule); dorsum with thick striations and a small median
opisthosomal (opisthonotal) shield; epimera I free.
6.4.1.2.1 Gymnoglyphus longior (Trouessart)
Differential diagnosis: posterior region of opisthonotum not punctate; setae ps 3 situated at
the junction of the anterior third and the posterior two thirds of the anus; setae h 2 20-25 µm
long; idiosoma 280-290 µm long.
6.4.1.2.2 Gymnoglyphus osu Fain et Johnston
Differential diagnosis: only the female of this species is known; posterior region of dorsum
(opisthonotum) punctate; setae a i situated close to the anterior angle of the anus; setae h 2
40-50 µm long; larger species; idiosoma 328-345 µm long; smaller length of the solenidia of
tibiae I and II.
6.4.2 Subfamily: Dermatophagoidinae

Differential diagnosis: In both sexes: setae v i absent; tegmen absent; cuticle lacking
projections as in the genus Glycyphagus; cuticle soft, with well-developed striations;
striations generally very thin and set close together, only rarely of the striated-punctate type
(Sturnophagoides); the area separating epimera I exceptionally punctate; setae sc e strong and
long (except in the genus Malayoglyphus where they are short or very short); setae h 2 and h 3
long. In females: setae sc i never very strong and long; ventral surface of tarsi I and II
without projections; epigynium distinctly separated from epimera I; tarsi III and IV lacking
apical spines; cuticle soft and finely striated; hysteronotal shield present only in
Sturnophagoides; posterior lip of vulva (vulvar lip) soft and striated, not punctate and not
incised anteriorly (except in Sturnophagoides where this lip is punctate, either partly or
completely, and incised anteriorly. In males: cuticle soft and striated; setae sc i never very
strong and very long; tarsi IV normal (not very short); legs III variable; femora III lacking a
spur; tarsi IV always without a bifid or trifid subapicoventral spine; tarsi III with or without
the subapicoventral spine; adanal copulatory suckers present; tarsal copulatory suckers (on
tarsi IV) present (except in Malayoglyphus); tarsi III with an apico-ventral forkate spine
(except in Malayoglyphus and Sturnophagoides, where this spine is lacking).
6.4.2.1 Genus: Sturnophagoides Fain
Differential diagnosis: In both sexes: striations of the cuticle punctate and more or less
sclerotized over a part or all of the body; the region between epimera I completely punctate;
setae sc e, h 2 and h 3 long or very long; setae c p very short.
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In females: dorsum with a median hysteronotal shield (absent in the other genera of
Dermatophagoidinae); cuticle striated-punctate over a large part or all of the body; posterior
lip of vulva long, punctate (either partially or completely) and incised in its anterior angle
(as in some Pyroglyphinae). In males: perianal chitinous frame not denticulate; legs III and
IV less unequal; the legs III a maximum of 1.6 times as long as IV; tarsi III either with a
subapical conical unforked spine and 5 thin setae or with all setae simple; tarsi IV with 2
small tarsal copulatory suckers; adanal copulatory suckers well developed; legs III distinctly
stronger and longer than legs IV; at least tarsus I with an apical process; setae sc e strong and
long (minimum 110 µm).
6.4.2.1.1 Sturnophagoides brasiliensis Fain
In females: small species; idiosoma 246-262 µm long; posterior lip of vulva punctate only in
its lateral parts; hysteronotal shield situated inside setae e 1; striations behind the
hysteronotal shield distinctly thickened, more punctate and more spaced than on other parts
of the body; solenidia of genua I very short (10 and 4 µm). In males: idiosoma 175-185 µm
long; perianal chitinous frame narrow, oval in shape; tarsi III with an apical curved process,
but lacking a subapical spine.
6.4.2.1.2 Sturnophagoides bakeri Fain
Differential diagnosis: In females: larger species (idiosoma 390-420 µm long); posterior lip of
vulva completely punctate; striations behind the hysteronotal shield not modified; setae d 1
and e 1 situated outside the hysteronotal shield; solenidia of genua I 30-35 and 6 µm long,
respectively. In males: idiosoma 270-290 µm long; perianal chitinous frame wide, piriform
(pear-shaped); tarsi III ending in a conical spine and a curved apical process; hysteronotal
shield piriform passing hardly beyond setae d 1 (heteromorphic males).
6.4.2.1.3 Sturnophagoides petrochelidonis Cuervo et Dusbabek
Differential diagnosis: In females: idiosoma 310-379 µm long; posterior lip of vulva
completely punctate; striations behind the hysteronotal shield not modified; setae d 2 and d
3 situated on the margins of the hysteronotal shield. In males: idiosoma 245-272 µm long;
perianal chitinous frame wide, piriform (pear-shaped); tarsi III ending in a conical spine and
a curved apical process; hysteronotal shield rectangular, reaching setae c 1 (heteromorphic
males).
6.4.2.2 Genus: Hirstia Hull
Differential diagnosis: In both sexes: striations of the cuticle are finer and set closer together;
striations are separated by less than 1 µm (at the level of setae c 1); more distinct reduction
of legs IV compared to legs III. In females: hysteronotum striated, lacking a median shield;
cuticle with non-punctate striations; cuticle between epimera I not punctate; posterior lip of
vulva smaller and shorter and with anterior angle not incised; legs III distinctly longer
(length of the four apical segments - tarsus-femur) and thicker than legs IV; the ratio of the
lengths of legs IV / lengths of legs III = 1 : 1.4 (to 1.56); cuticle with very thin striations
separated by less than 1 µm (at the level of setae d 1). In males: perianal chitinous frame
finely denticulate inside; legs III much thicker than legs IV and from 1.8 to 1.9 times longer
than the latter (length of the 4 apical segments); tarsi III bearing in their middle 2 strong
conical spines (setae w and r).
6.4.2.2.1. Hirstia chelidonis Hull
Differential diagnosis: In both sexes: larger species. In females: idiosoma 395-426 µm long;
posterior region of dorsum not punctate and not sclerotized; length of legs III 174 µm, legs
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IV 118 µm (= length of 4 apical segments); length of tarsi I-IV = 40-43-66-48 µm. In males:
idiosoma 321 to 345 µm long; tarsi I-IV = 33-39-51-24 µm long.
6.4.2.2.2. Hirstia domicola Fain, Oshima et van Bronswijk
Differential diagnosis: In females: idiosoma 298-310 µm long; posterior region of dorsum
sclerotized and punctate mainly around the bases of setae d 5 and l 5; legs III and IV – 123-
129µm and 85-90 µm long, respectively (= length of 4 apical segments); tarsi I-IV = 40-43-66-
48 µm long, respectively. In males: idiosoma 240 to 248 µm long; tarsi I-IV shorter, 22-27-32-
18 µm long, respectively.
6.4.2.3 Genus: Malayoglyphus Fain, Cunnington et Spieksma
Differential diagnosis: In both sexes: short, thin shape of setae of the setae sc e ; the normal
development of legs IV which are as long as legs III; the presence of only one solenidion on
genu I. In females: hysteronotum striated, lacking a median shield; cuticle with non-
punctate striations; cuticle between epimera I not punctate; posterior lip of vulva smaller
and shorter and with anterior angle not incised; legs III and IV equal or subequal in length
and in width; cuticle with dorsal striations more spaced (separated by 1.2 to 2.3 μm at level
of setae d 2); setae sc i and sc e thin and short, either equal or suequal, or slightly unequal (sc
e less than 35 µm long); epigynium poorly developed and slightly sclerotized; genu I with
only one very short solenidion (5-6 µm). In males: perianal chitinous frame not denticulate;
legs III and legs IV less unequal; the legs III a maximum of 1.6 times as long as legs IV; tarsi
III with only thin setae (there is no forked spine on the apex of tarsus III as in
Dermatophagoides) setae sc e thin and short (maximum 30 µm long); adanal copulatory
suckers poorly developed (reduced); tarsi I and II without apical processes; legs III and IV
subequal; tarsi IV without suckers; tarsus IV bears 3 thin setae and one rounded papilla
which is a remnant of a sucker.
6.4.2.3.1 Malayoglyphus intermedius Fain, Cunnington et Spieksma
Differential diagnosis: In both sexes: smaller species. In females: idiosoma 218-243 µm long;
setae sc i and sc e equal or subequal (about 12-15 µm long); posterior half of opisthonotum
distinctly punctate and with thicker and more spaced striations than on other parts of the
dorsum. In males: idiosoma 168 to 175 µm long; setae sc e and sc i equal or subequal (12-15
µm long); striations of the posterior half of hysteronotum thick, punctate and sclerotized.
6.4.2.3.2. Malayoglyphus carmelitus Spieksma
Differential diagnosis: In both sexes: larger species. In females: idiosoma 320-348 µm long;
setae sc e distinctly longer (30-35 µm) than sc i (15 µm); punctation of posterior half of
opisthonotum indistinct. In males: idiosoma 240 to 283 µm long; setae sc e distinctly longer
(30 µm long) than setae sc i (15 µm); posterior half of hysteronotum with a large punctate
and not striated shield.
6.4.2.4 Genus: Dermatophagoides Bogdanov
Differential diagnosis: In both sexes: cuticle with dorsal striations more spaced (separated by
1.2 to 2.3 µm at level of setae d 1); setae sc i and sc e very unequal; the sc e long and strong;
genu I with 2 very unequal solenidia. In females: hysteronotum striated, lacking a median
shield; cuticle with non-punctate striations; cuticle between epimera I not punctate;
posterior lip of vulva smaller and shorter and with anterior angle not incised; legs III and IV
equal or subequal in length and in width; setae sc i and sc e very unequal; the sc e long and
strong; epigynium well developed and sclerotized. In males: perianal chitinous frame not
denticulate; legs III distinctly stronger and longer than legs IV; but the legs III a maximum of
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1.6 times as long as legs IV; tarsi III with a strong subapical forked (bifid) spine (seta f); setae
sc e minimum 110 µm long; tarsi IV with two small copulatory tarsal suckers; adanal suckers
well developed; at least tarsus I with an apical process.
6.4.2.4.1 The Dermatophagoides pteronyssinus group
Differential diagnosis: In females: median area comprized between setae d 1 and e 1 [= M
area] completely striated longitudinally; opening of the bursa copulatrix situated on
posterior margin of the body. In males: hysteronotal shield long, reaching forwards to setae
d 1 and/or further in front (anteriorly).
epigyniumm
b.c.
Fig. 6. Dermatophagoides pteronyssinus, female – ventral aspect; key: b.c. = sclerite

surrounding an internal opening of bursa copulatrix; epigynium = anterior genital apodeme,
pregenital sclerite.
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6.4.2.4.1.1. Dermatophagoides pteronyssinus (Trouessart)

Differential diagnosis: In females (Fig. 6): bursa copulatrix very narrow, of uniform calibre
and ending inside (proximally) in a daisy-like sclerite. In males: hysteronotal shield distinctly
extending forward to beyond the bases of setae d 1; coxae II closed; adanal suckers 12 µm in
diameter; males homeomorphic: epimera I free; tarsi I with 2 unequal apical processes
(ongles); tarsus II with a small apical process; legs III 1.3 times thicker (at level of femur) and
1.46 times longer (length of 4 distal segments) tthan legs IV; setae h 2 and h 3 with bases poorly
sclerotized; setae cp 80-90 µm long; setae d 2 situated at 40 µm from the opening of the fat
gland; the male differs from that of D. evansi mainly by: the dorsal shield is shorter and
broader than in D. evansi; ratio width (at level of d 1) : length = 1 : 1.8-1.9 [whilst in D. evansi
this ratio is 1 : 2.5]; legs III and IV are much less unequal than in D. evansi.
epigynium
b.c.
Fig. 7. Dermatophagoides evansi, female – ventral aspect; key: b.c. = sclerite surrounding an
internal opening of bursa copulatrix; epigynium = anterior genital apodeme, pregenital
sclerite.
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6.4.2.4.1.2 Dermatophagoides evansi Fain, Hughes et Johnston

Differential diagnosis: In females (Fig. 7): bursa copulatrix strongly enlarged in its distal
third and very narrow in its proximal two thirds (internal); spermatheca sclerotized and
tulip-like. In males: hysteronotal shield distinctly extending forward to beyond the bases
of setae d 1; coxae II closed; adanal suckers 12 µm in diameter; males homeomorphic,
epimera I free; tarsi I with 2 unequal apical processes (ongles); tarsus II with a small
Fig. 8. Dermatophagoides farinae, female – ventral aspect, vulva and epigynium (anterior genital
apodeme, pregenital sclerite); genital chaetotaxy: setae 3 b (or genital anterior setae g a),
genital setae g (or genital median setae g m), setae 4 a (or genital posterior setae g p); g. p. =
genital papillae.
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apical process; legs III 1.8 times thicker (at level of femur) and 1.6 times longer (length of 4
distal segments) than legs IV; setae h 2 and h 3 with bases strongly sclerotized; setae cp 110 µm
long; setae d 2 situated at 55-65 µm from the opening of the fat gland; the male differs from
that of D. pteronyssinus mainly by: the dorsal hysterosomal shield is longer and narrower; ratio
width (at level of setae d 1) : length = 1 : 2.5 [whereas in D. pteronyssinus this ratio is 1.8 to 1.9];
legs III and IV are much more unequal than in D. pteronyssinus.
aedeagus
anal plate
Fig. 9. Dermatophagoides farinae, heteromorphic male – ventral aspect.

6.4.2.4.2 The Dermatophagoides farinae group
Differential diagnosis: In females: striations of the posterior half of the area M only slightly
convex; opening of the bursa situated ventrally on the side of the posterior third of the anus;
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first part of the bursa forming (sometimes not) a sclerotized pocket (vestibule). In males:
hysteronotal shield short, extending forwards to a point situated at equal distance from
setae d 1 and e 1; hysteronotal shield wider than long (in its middle); seta r on tarsus III is
thin and situated basally epimera I free in the homeomorphic male.
6.4.2.4.2.1 Dermatophagoides siboney Dusbabek, Cuervo et Cruz
Differential diagnosis: In both sexes: small species. In females: idiosoma 258-311 µm long;
propodonotal shield approximately twice as long as wide. In males: small species; idiosoma
199-245 µm long; all known males are homeomorphic.
6.4.2.4.2.2 Dermatophagoides farinae Hughes
Differential diagnosis: In both sexes: larger species. In females: idiosoma 395 to 435 µm long;
propodonotal shield approximately 1.4 times as long as wide; vestibule of bursa well
sclerotized and shaped like a calabash pipe; beyond this vestibule the bursa is not expanded;
tarsus I generally with well developed curved process (ongle); epigynium crescent-shaped
(Fig. 8). In males: idiosoma 285-345 µm long; males either homeomorphic with epimera I
free (and normal first legs) or heteromorphic with epimera I fused to form a V or Y (and
enlarged I legs) (Fig. 9).
6.4.2.4.2.3. Dermatophagoides microceras Griffiths et Cunnington
Differential diagnosis: In females: idiosoma 395 to 435 µm long; propodonotal shield
approximately 1.4 times as long as wide; vestibule of bursa lacking, the bursa opens at the
bottom of a non-sclerotized depression of the tegumen; the first part of the bursa proper is
slightly dilated and distinctly sclerotized; apical process of tarsus I generally very small or
lacking. In males: idiosoma 285-345 µm long; males either homeomorphic with epimera I
free (and normal first legs) or heteromorphic with epimera I fused to form a V or Y (and
enlarged I legs).
7. Phylogenetic reconstructions of the Pyroglyphidae

The family Pyroglyphidae presently consists of 47 species and 20 genera, whose species are
associated with birds, mammals or house dust and stored products (Mumcuoglu et al., 1976;
Fain et al., 1990; Evans, 1992; Colloff, 2009; Krantz and Walter, 2009; Solarz, 2004b, 2011).
The majority of these species are nidicolous, with bird associates outnumbering mammal
associates. They are associated with many avian groups, but most of them are nidicoles of
Passeriformes. These mites feed on animal detritus and/or dander in the nests and have
given rise to several more typically parasitic taxa (Fain et al., 1990). The Pyroglyphidae are
small mites, whitish in colour; the length of idiosoma of adults ranges from 168 μm
(Malayoglyphus intermedius) to 585 μm (Onychalges longitarsus). The idiosoma is generally
oval in shape with parallel sides, and broadly rounded anterior and posterior margins. The
degree of the cuticle sclerotization is variable; in some subfamilies or genera it is almost
completely sclerotized, without true striations, whereas in others the cuticle is soft and
striated, in which case the dorsal shields are more poorly developed. Morphology and
biology of the pyroglyphids seems to indicate that the free-living forms have evolved from
obligate parasitic ancestors (OConnor, 1982; Woolley, 1988). These mites morphologically
show some characters of parasitic Psoroptidia, particularly a regression of legs IV (especially
in males), dorsal shields (mainly the hysterosomal), copulatory suckers (vestigial or
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reduced, in the shape of small sclerotized rings), tarsal claws (only in the form of a small
median axis). On the other hand, it has been suggested by Fain (Fain et al. 1990) that in the
Pyroglyphidae the regression of the organs has preceeded the invasion of the host as if there
were a preadaptation. This regression involves also an idiosomal and leg chetotaxy.
Pyroglyphids have 8 setae on tarsi I and on II, and 1 seta on each of tibiae I and II, whereas
in the Acaridae there are 13 and 12 tarsal setae on tarsi I and II, respectively, and 2 tibial
setae on tibiae I and II. Vertical setae are generally absent; thus, the scapular setae (sc e and
sc i) are the first pair, most anteriorly located. The absence of setae v e is the typical feature of
the Pyroglyphidae; whereas setae v i occur only in the members of the genus Paralgopsis
(subfamily Paralgopsinae). The external scapulars (sc e) are longer, more or less, as the
internal setae (sc i). Female have only 2 pairs of adanal setae (ps 2 and ps 3), whereas females
of the family Acaridae have 5-6 pairs of anal/adanal setae. Phylogeny basically means, the
history of the tribe. Reconstructing a phylogeny is similar to compiling a genealogic tree in
that both indicate the degree of relatedness between the members of the tribe, or other
taxon. However, the family tree is based on known fact and documentary evidence, whereas
a phylogeny is only ever, at best, an hypothesis of the most likely evolutionary history of the
taxon (Colloff, 1998). There has been no detailed phylogenetic revision of the family
Pyroglyphidae to date. In opinion of Colloff (1998), the host relationships of pyroglyphid
mites with birds indicate that the two subfamilies which contain species found in house
dust, the Pyroglyphinae and Dermatophagoidinae, are the most widespread geographically
and the most species-rich. They are associated with a higher diversity of avian taxa than the
other subfamilies, that do not contain the house-dust-mite species (Colloff, 1998). Indeed,
the Pyroglyphidae probably form a link between the free living and parasitic Astigmatina. It
was also shown that some pyroglyphids are able to adopt a parasitic mode of life and
feeding (Fain et al., 1990; Proctor, 2003; Dabert et al., 2010). But mites of this family are
perhaps best known as the house dust mites, because of their occurrence in human
dwellings. Main sources of the mite allergens in dwellings usually are beds, couches or sofas
in bedrooms and couches or sofas in living-rooms (family rooms). New dwellings might be
colonized via mite-infested furniture, by humans, on skin, clothing, or by their pets (Hewitt
et al., 1973; Hoeven et al., 1992; Perotti et al., 2009) The natural sources of allergenic mites in
stores are still not quiet known (Hallas & Iversen, 1996; Solarz et al., 2007; Hallas, 2010).
Possible sources of these mites in farming environments are also the nests of synanthropic
birds (Hughes, 1976; Wharton, 1976; van Bronswijk, 1981; Fain et al., 1990; Solarz et al.,
1999). On the other hand, it has been suggested that the majority of the mite population is
brought from the cultivated field into the stores, and that the open field is the main source of
storage-mite populations (Hallas & Iversen, 1996), whereas the bird nests are less important
(Solarz, 2003).
Indoor acarofauna depends on the people living and working in these buildings. Different
indoor environments are very important places for forensic investigations, but the richness
of mite biodiversity, phylogenetic relationships between different groups of domestic mites
and their associations with potential hosts has not been exploited by forensic investigators.
Summarizing it should be stressed that knowledge about occurrence of particular species of
parasitic and/or synanthropic on man or in human environment as well as the correct
identification of mites colonising dead body are very important factors in forensic
investigations of past human activity (Perotti, 2009b; Braig & Perotti, 2009; Perotti & Braig,
2009; OConnor, 2009; Turner, 2009; Solarz, 2009; Desch, 2009; Baker, 2009, Proctor, 2009).
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8. Domestic mites and forensic medicine. Final remarks

More than 100 species of mites from over 60 families were collected from animal carcases,
and apprioximately 75 mite species from over 20 families from human corpses (Braig &
Perotti, 2009), including also the astigmatid mite taxa. Within the Astigmatina were
involved some domestic mite species such as Acarus siro, A. farris, A. immobilis, Tyrophagus
longior, T. putrescentiae, Tyrolichus casei, Caloglyphus berlesei, Rhizoglyphus echinopus,
Lardoglyphus spp. and Lepidoglyphus destructor. Among them from human carcases were
noted Myianoetus diadematus (mass population), C. berlesei (many), A. siro (common), A.
immobilis (common), T. longior (abundant), T. putrescentiae (common and abundant) and L.
destructor (very rare) (Braig & Perotti, 2009). Tyrophagus longior is particularly active at lower
temperatures. This mite is almost always associated with dry cheese and/or meat. It was
known to feed on the fatty acids and soapy substances containing ammonia that form on
carcasses during dry decomposition (Perotti et al., 2009). In 1878 these mites were found in
the mummified body of a newborn baby girl in Paris and they were studied by Mégnin. He
estimated and identified approximately 2.4 million specimens of T. longior in this material
(Perotti, 2009). In a more recent case reported from Germany a child corpse found in a
basement was also associated with a mass populations of Myianoetus diadematus, A. immobilis
and T. putrescentiae (OConnor, 2009; Braig & Perotti, 2009). Mites originating from outdoor
environments can also be useful trace evidence and give accurate evidence about
movements or transportation of a body (Perotti et al., 2009). Many mite species arrive at a
carcase via phoresy on insects. The phoresy is often taxon specific (Braig and Perotti, 2009).
Phoretic hypopi (heteromorphic deutonymphs) are known in Acarus farris, A. siro,
Caloglyphus berlesei, Rhizoglyphus echinopus and Myianoetus diadematus. Summarizing, it
should be stressed that the astigmatid mites have played little role in the field of forensic
medicine to date (OConnor, 2009). Domestic mites have not yet been explored in forensic
investigations, but there is every reason for doing this. Dust mites and other domestic mites
are globally present, yet species composition may vary between seasons, between dwellings
and even places within a single indoor environments (e.g., beds vs. floors, floors vs.
upholstery furniture, or dust from a book shelve vs. a librarian’s desk) (Solarz, 2009). Subtle
differences in the house-dust-mite acarofauna between sites may yield valuable information,
for instance as indicator of time and circumtances of death (Solarz 2009; Perotti, 2009).
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Forensic Medicine - From Old Problems to New Challenges
Edited by Prof. Duarte Nuno Vieira
ISBN 978-953-307-262-3
Hard cover, 382 pages
Publisher InTech
Published online 12, September, 2011
Published in print edition September, 2011
Forensic medicine is a continuously evolving science that is constantly being updated and improved, not only
as a result of technological and scientific advances (which bring almost immediate repercussions) but also
because of developments in the social and legal spheres. This book contains innovative perspectives and
approaches to classic topics and problems in forensic medicine, offering reflections about the potential and
limits of emerging areas in forensic expert research; it transmits the experience of some countries in the
domain of cutting-edge expert intervention, and shows how research in other fields of knowledge may have
very relevant implications for this practice.
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From Old Problems to New Challenges, Prof. Duarte Nuno Vieira (Ed.), ISBN: 978-953-307-262-3, InTech,
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16
Types and Subtypes of the Posterior Part

of the Cerebral arterial Circle in
Human Adult Cadavers
Ljiljana Vasović1, Milena Trandafilović2, Ivan Jovanović1,
Slađana Ugrenović1, Slobodan Vlajković1 and Jovan Stojanović3
University of Niš,
1-3Facultyof Medicine,
1Department of Anatomy,
3Institute of Forensic Medicine
Serbia
1. Introduction
The main cerebral distribution center of 15-20% blood flow from the cardiac output is the
cerebral arterial circle or circle of Willis, a nonagon of collateral vessels on the human brain
base.
At about 4 mm stage of the embryo, the primitive internal carotid arteries, which develop as
cranial extensions of the paired dorsal aorta, are formed. Paired longitudinal neural arteries
appear along the hindbrain and coalesce to form the basilar trunk at the 7- to 12-mm stage.
The caudal division of the primitive internal carotid artery anastomoses with ipsilateral
neural artery and becomes the posterior communicating artery. At the 40-mm stage the
posterior cerebral arteries are as extensions of the posterior communicating arteries. The
vertebrobasilar system develops and thus participates in the supply of the posterior cerebral
artery through the segment between the basilar artery and the post-communicating part of
the posterior cerebral artery. In that phase, the component vessels of the circle of Willis all
have the same caliber (Silver & Wilkins, 1991).
The posterior cerebral artery originates from the basilar bifurcation within the
interpeduncular cistern. From its origin the artery curves superior to the corresponding
oculomotor nerve in relation to the antero-medial portion of the cerebral peduncle and joins
ipsilateral posterior communicating artery (Yasargil, 1984).
The posterior communicating artery takes origin from the infero-lateral wall of the cerebral
part of the internal carotid artery. It is encased in a sleeve of arachnoid along the course
from the carotid cistern to the piercing of the interpeduncular cistern and junction with
posterior cerebral artery (Yasargil, 1984).
Topographically, the circle of Willis is divided on anterior and posterior parts. The anterior
part composes five vascular components – bilateral cerebral parts of internal carotid arteries
(communicating and choroid subparts) and the pre-communicating part of anterior cerebral
arteries interconnected by the anterior communicating artery; the posterior part composes
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four vascular components. As a rule, the posterior part of the circle of Willis is normal as it is
formed with posterior communicating artery and the pre-communicating part of the
posterior cerebral artery on both sides, where the left and right pre-communicating parts of
the posterior cerebral arteries have normal calibres which are larger in relation to posterior
communicating arteries, as well as that posterior communicating arteries are not hypoplastic
(Saeki & Rhoton, 1977). In the fetal period, the posterior circle part develops into one of
three variants: an adult configuration, a transitional configuration or a fetal configuration
(Silver & Wilkins, 1991).
It has been assumed that the arrangement of the arteries at the base of the brain is
symmetrical and that the architecture of the circle of Willis provides a structural basis for
free anastomotic flow (Riggs & Rupp, 1963). The collateral potential of the circle of Willis is
believed to be dependent with the presence and size of its component vessels, which vary
among normal individuals. The anterior communicating artery and posterior
communicating arteries are designated as primary collateral pathways (Hartkamp et al.,
1999). The integrity of the circle of Willis may be critical because anomalies and hypoplasia
of the circle of Willis are frequent (Merkkola et al., 2006). Anomalies found in a pattern of
the posterior part of the circle of Willis result due to persistence of vessels that normally
disappear or disappearance of normal vessels (Vasović, 2004; Kapoor et al., 2008; Vasović et
al., 2010). Although the association of variations and aneurysms had been used as an
argument in favor of a congenital theory of aneurismal development, it should be
interpreted in terms of the hemodynamic stress caused by variations (Kayembe et al., 1984).
However, a significantly higher percentage of complete posterior circle configurations were
demonstrated on magnetic resonance angiograms in some patients rather than in the
controlled subjects (Hartkamp et al., 1999). The size and patency of primary collateral
pathways may be risk factors for cerebral infarction in patients with severe stenosis or
occlusion of a carotid artery (Schomer et al., 1994).
The purpose of the present morphologic study is to obtain wider, more precise and more
detailed information about the relationship between caliber variations in the posterior part
of the circle of Willis forming different (sub) types of its posterior part, as well as to compare
those results with previous literature data.
2. Material and methods

Examination was carried out on the brains of human adult cadavers, during the period
between 2006 and 2010. Approval was granted by the Research Ethics Committee (No. 01-
9068-1) of the Faculty of Medicine of Niš. All subjects had died from natural or violent death
and were candidates for autopsy for medico-legal reasons in Institute of Forensic Medicine
of Niš, Serbia. The brain base with blood vessels was photographed on each cadaver; the
ventral side of the brainstem and diencephalon was specially zoomed. Each case was
recorded as the schematic drawing in the workbook. External morphology (calibre of the
arteries, possible abnormalities) was inspected using magnifying glass. We used only cases
with complete Willis’ circle (Figure 1). These circles originated from 110 cadavers of both
sexes (62 male and 48 female) and of different ages (from 20 to 95). The numbers of cadavers
in each age deciles were as follows: 2nd decade, two cadavers; 3rd decade, three cadavers;
4th decade, seven cadavers; 5th decade, 16 cadavers; 6th decade, six cadavers; 7th decade, 23
cadavers; 8th decade, 35 cadavers, 9th decade, 17 cadavers; and 10th decade, one cadaver.
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Types and Subtypes of the Posterior Part of the
Cerebral arterial Circle in Human Adult Cadavers 361
Only one cadaver had cerebral infarction, while the others were on autopsy because of
cardiac, respiratory, incidental and other causes of no cerebral pathology.
Measurement of the outer diameter of the pre-communicating part of the posterior cerebral
artery and the posterior communicating artery of both sides, as well basilar bifurcation
angle was performed using ImageJ (http://rsb.info.nih.gov/ij/index.html). Statistical
analysis of quantitative data is performed with PASW statistical software SPSS version 15.
According to Kamath (1981), values of ≤ 0.5 and 1.0 mm for the posterior communicating
and pre-communicating part of the posterior cerebral arteries are respectively used as
hypoplastic calibers.
Fig. 1. Diagram of vessels of the carotid and vertebrobasilar systems. Vascular components
of the circle of Willis (choroids and communicating subparts of the cerebral part of the
internal carotid artery, ICA-C4*; pre-communicating part of the anterior cerebral artery, A1;
anterior communicating artery, ACoA; posterior communicating artery, PCoA; pre-
communicating part of the posterior cerebral artery, P1), and some of other arteries of the
vertebrobasilar system (post-communicating part of the posterior cerebral artery, P2; basilar
artery, BA) are marked. Vessels (PCoAs and P1s) of the circle posterior part are white
colored.
Form of types and subtypes of the posterior part of 110 circles of Willis follows their
previous description in human fetuses (Vasović et al., 2008a). Bilateral fetal or FF-type is
characterized with the larger diameter of the posterior communicating arteries in relation to
the pre-communicating part of the posterior cerebral arteries (normal or hypoplastic);
bilateral adult or AA-type is characterized with bilaterally larger diameter of the pre-
communicating part of the posterior cerebral arteries in relation to the posterior
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communicating arteries (normal or hypoplastic); bilateral transitory or TT-type is

characterized with the same diameter (normal or hypoplastic) of the pre-communicating
part of the posterior cerebral artery and the posterior communicating artery on both sides;
the fetal-transitory or transitory-fetal type (FT/TF) is characterized with the same size of the
posterior communicating and pre-communicating part of the posterior cerebral artery on
one side and the larger posterior communicating artery in relation to the pre-
communicating part of the posterior cerebral artery on the right or left side; the adult-
transitory or transitory-adult type (AT/TA) is characterized with the same size of the
posterior communicating artery and pre-communicating part of the posterior cerebral artery
on one side and the larger pre-communicating part of the posterior cerebral artery in
relation to the posterior communicating artery on the right or left side; the adult-fetal or
fetal-adult type (AF/FA) is characterized with the larger diameter of the pre-communicating
part of the posterior cerebral artery in relation to the posterior communicating artery and
the larger diameter of the posterior communicating artery in relation to the pre-
communicating part of the posterior cerebral artery on the right or left side. Subtypes with
the ideal caliber relations are marked with zero in the subscript. The mark of every next
subtype retains the first letters of the basic type but in the subscript contains Arabic
numerals starting from number 1. To differentiate the side of the TT, FF, and AA subtypes in
schemes, we emphasize that the numeral in the subscript designates right side and the
numeral with the apostrophe designates left side (as the images in the mirror). To
differentiate the side on FT/TF, AT/TA, and AF/FA schemes, we emphasize that the first
letter in the designation marks right-sided origin, and the second marks left-sided origin of
arteries. The lines of different thicknesses and broken lines on the schemes of the circle
posterior part show the difference in the caliber size, independently from whether the
caliber of an artery was normal and/or (slightly) hypoplastic or hyperplastic.
Macroscopic visualization of atheromatous plaques in vessels’ wall of the circle of Willis is
graduated according to personal proposes (grade 0 – without atheromatous changes; grade
1 - atheromatous plaques at vessels bifurcations; grade 2 - atheromatous plaques at
bifurcations and up to half vessels length; grade 3 – islands or diffuse atheromatous
plaques).
3. Results
Basic statistic analysis of calibres of the pre-communicating part of the posterior cerebral
artery and posterior communicating artery on both sides in the circle of Willis of human
cadavers of different ages and genders is presented in Table 1.
The least caliber of the pre-communicating parts of the right and left posterior cerebral
arteries (0.89 and 0.80 mm) is found in a 75-year-old female and a 42-year-old male, while
their largest caliber (3.69 and 3.60 mm) is found in 47-year-old and 80-year-old males. The
least caliber of the right and left posterior communicating arteries (0.30 and 0.45 mm) is
found in a 44-year-old and in a 64-year-old woman, while the largest caliber (2.86 and 3.31
mm) is found in 80-year old woman. Average values of the caliber and the standard
deviations for right and left pre-communicating part of the posterior cerebral artery are 2.17
(±0.58) mm and 2.29 (±0.48) mm, as well 1.19 (±0.54) mm and 1.22 (±0.57) mm for the right
and left posterior communicating artery, respectively (Table 1).
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Table 1. Statistical parameters of caliber values of the pre-communicating part (P1) of the
posterior cerebral artery (PCA) and posterior communicating artery (PCoA) on both sides in
the posterior circle part of human cadavers
We established four (two symmetric and two asymmetric) types. The incidences of types in
adult specimens are as follow: 8 cases (7.27%) of the FF-type (four subtypes), 75 cases
(68.18%) of the AA-type (six subtypes), 15 cases (13.63%) of the AF-type (four subtypes) and
12 cases (10.91%) of the AT-type (three subtypes). Sequences of same or special subtypes in
fetal and adult specimens are compared and showed in Table 2.
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Table 2. Sequences of subtypes of the posterior part of fetal and adult circles of Willis.
Special subtypes are in gray rows;
*Vasović et al. (2008a);
**recent findings.
It is obvious that an incidence of bilateral adult type dominates (68.18%) and that the cases
of bilateral transitory and fetal-transitory types as well some subtypes of bilateral fetal and
adult types are missing; an unilateral adult configuration is associated with fetal or
transitory configuration in about 25% of these adult cases; there is the least incidence (7.27)
of bilateral fetal configuration in relation to other three patterns of posterior circle part
(Table 2).
Distribution of cases of corresponding subtypes according to causes of death in female and
male adults cadavers is showed in Table 3. A presence of atheromatous plaques in carotid
and vertebrobasilar systems on the brain base is noted according to age and graduated
independently from cause of death (Table 4).
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Singular
cases of
Myoca- Cerebral
Cardiorespira- Polytrau- Intoxica- no
Types / rdial patho-
N tory arrest ma tion cerebral
Subtypes infarction logy
patho-
logy
(females + males)
AA0 29 3+0 5+2 2+5 2+2 3+4 0+1
AA1/AA1, 7 0+1 2+2 0+1 0+1
AA2/AA2, 27 1+4 3+3 1+6 0+1 5+3
AA
AA3/AA3, 2 0+1 0+1
AA4/AA4, 7 0+3 1+2 1+0
AA5/AA5, 3 1+0 1+1
FF0 3 0+1 1+0 1+0
FF1/FF1, 3 1+1 0+1
FF
FF2, 1 0+1
FF4, 1 1+0
AF0/FA0 8 1+3 0+2 2+0
FA4 1 0+1
AF/FA
AF5/FA5 5 1+0 1+1 2+0
AF7 1 0+1
AT0/TA0 8 0+1 1+2 0+1 2+1
AT/TA AT1 1 0+1
AT3/TA3 3 1+0 1+0 1+0
Table 3. Sex distribution of number of cases of the posterior circle subtypes according to
causes of death
Atheromatous changes (grade 1) were first visualized in a 34-years old male (AA2 subtype),
as well as, in five cases (one in FF2' , two in AAo and two in AA2 subtype) of the fifth
decade of life. Atheromatous changes of grades 2 and 3 were first visualized in 42-years old
and 47-years old males, respectively. Incidences of atheromatous changes are as follow:
Grade 1: Five (2 females and 3 males) or 62.5% of the FF-type cases; 24 (8 females and 16
males) or 32% of the AA-type cases; four (2 females and 2 males) or 26.6% of the AF/FA-
type cases, and two (males) or 16.6% of the AT/TA-type cases. Grade 2: Two (female and
male) or 25% of the FF-type cases; 26 (12 females and 14 males) or 34.6% of the AA-type
cases; three (two females and one male) or 20% of the AF/FA-type cases, and two (males) or
16.6% of the AT/TA-type cases. Grade 3: One (male) or 12.5% of the FF-type cases; four (two
females and two males) or 5.3% of the AA-type cases; four (two females and two males) or
26.6% of the AF/FA-type cases, and one (female) or 8.3% of the AT/TA-type cases. There
are 21 (9 females and 12 males) or 28% of the AA-type cases from the 20th to 78th year, then
four (two females and two males) or 26.6% of the AF/FA-type cases from the 28th to 54th
year, and seven (two females and five males) or 58.3% of the AF/FA-type cases from the
35th to 82nd year without atheromatous changes.
Diagrams and appropriate pictures are showed at Figures 2-5. General incidences of types
and/or subtypes are compared with other literature data in Table 5.
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Types Subtypes Grade 0 Grade 1 Grade 2 Grade 3 N

f 78 80 2
FF0
m 84 1
f 75 1
FF1/FF1,
m 79 70 2
FF
f 0
FF2,
m 41 1
f 79 1
FF4,
m 0
f 27,34,58,67 46,72,85 68,74,75,80,95 71,87 15
AA0
m 20, 37,45,47,50,60,75 45,52,81,83 70,86 70 14
f 34,43 2
AA1/AA1,
m 70 69 78,80,90 5
f 44,44,61 45,71,82 65,74,76,80 10
AA2/AA2,
m 23,51,78 34,43,54,61,70,79,81 68,69,72,77,78,79,82 17
AA
f 0
AA3/AA3,
m 63,65 2
f 79 64 2
AA4/AA4,
m 20 80,84 70 85 5
f 64 85 2
AA5/AA5,
m 42 1
f 45,54 81 3
AF0/FA0
m 28,42 80 74 82 5
f 0
FA4
m 89 1
AF/FA
f 68,70 78 78 4
AF5/FA5
m 47 1
f 0
AF7
m 74 1
f 82 78 72 3
AT0/TA0
m 39,50,76,80 61 5
f 0
AT/TA AT1
m 35 1
f 40 73 67 3
AT3/TA3
m 0
Table 4. Graduation of atheromatosis according to gender (female - f and male - m) and age
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Fig. 2. Diagrams and corresponding photos of subtypes of the bilateral fetal (FF) type.
Right or left posterior communicating artery, rPCoA or lPCoA; right or left pre-communicating part of
the posterior cerebral artery, rP1 or lP1.
FFo (rPCoA > rP1 = lP1 < lPCoA ^ lPCoA = rPCoA): 3 cases (37.5%).
FF1/FF1’ (lP1 < rP1 < rPCoA = lPCoA) or (rP1 < lP1 < lPCoA = rPCoA): 3 cases (37.5%).
FF2’ (lP1 = rP1 < rPCoA < lPCoA): 1 case.
FF4’ (rP1 < lP1 < lPCoA < rPCoA): 1 case. Note: PCoAs are accidentally broken.
In FF-type, more frequent are the cases where calibers of paired posterior communicating
arteries and pre-communicating parts of the posterior cerebral arteries are equaled,
respectively with larger caliber of posterior communicating arteries (FFo-subtype), as well
as the cases with same relationship of posterior communicating arteries and caliber
difference of pre-communicating parts of the posterior cerebral arteries (FF1/FF1’subtype).
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Fig. 3. Diagrams and corresponding photos of subtypes of the bilateral adult (AA) type.
Right or left posterior communicating artery, rPCoA or lPCoA; right or left pre communicating part of
AAo (rPCoA < rP1 = lP1 > lPCoA ^ lPCoA = rPCoA): 29 cases (38.6%).
AA1/AA1’ (lPCoA = rPCoA < rP1 < lP1) or (rPCoA = lPCoA < lP1 < rP1): 7 cases (9.3%).
AA2/AA2’ (lPCoA < rPCoA < rP1 = lP1) or (rPCoA < lPCoA < lP1 = rP1): 27 cases (36%).
AA3/AA3’ (rPCoA < rP1 = lPCoA < lP1) or (lPCoA < rPCoA = lP1 < rP1): 2 cases.
Note: left PCoA is accidentally broken in AA3 subtype.
AA4/AA4’ (lPCoA < rPCoA < lP1 < rP1) or (rPCoA < lPCoA < lP1 < rP1): 7 cases (9.3%).
AA5/AA5’ (lPCoA < rPCoA < rP1 < lP1) or (rPCoA < lPCoA < lP1 < rP1): 3 cases.
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In AA-type, more frequent are the cases where calibers of paired pre-communicating parts of
posterior cerebral arteries are equaled and larger in relation to equaled caliber of posterior
communicating arteries (AAo-subtype), as well as the cases with same relationship of
posterior cerebral arteries and caliber difference of posterior communicating arteries
(AA2/AA2’ subtype). Cases of two subtypes (AA1/AA1’ and AA4/AA4’) have same
frequency (9.3%), while cases of AA3/AA3’ (2/75) and AA5/AA5’ (3/75) have the least
frequency.
Fig. 4. Diagrams and corresponding photos of subtypes of the adult-fetal or fetal-adult

(AF/FA) type.
Right or left posterior communicating artery, rPCoA or lPCoA; right or left pre communicating part of
AFo/FAo (rP1 > rPCoA = lP1 < lPCoA ^ lPCoA = rP1) or (rPCoA > rP1 = lPCoA < lP1 ^ lP1 = rPCoA): 8
cases (53.3%).
Note: rPCoA and lP1 are accidentally broken in AFo.
FA4 (lPCoA < lP1 = rP1 < rPCoA ^ lPCoA < rPCoA): 1 case.
AF5/FA5 (lPCoA = rP1 > lP1 > rPCoA) or (rPCoA = lP1 > rP1 > lPCoA): 5 cases (33.3%).
AF7 (rP1 > lP1 > lPCoA > rPCoA): 1 case.
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Fig. 5. Diagrams and corresponding photos of subtypes of the adult-transitory or transitory-

adult (AT/TA) type.
Right or left posterior communicating artery, rPCoA or lPCoA; right or left pre-communicating part of
ATo/TAo (rPCoA < rP1 = lP1 = lPCoA) or (rP1 = rPCoA = lPCoA < lP1): 8 cases (66.6%).
Note: rPCoA and lP1 are accidentally broken.
AT1 (rP1 > lP1 = lPCoA = rPCoA): 1 case.
AT3/TA3 (rP1 > lP1 = lPCoA > rPCoA) or (lP1 > rP1 = rPCoA > lPCoA): 3 cases (25%).
Note: Left PCoA origin is infundibular.
In AF/FA-type, more frequent are the cases where calibers of the posterior communicating
artery and pre-communicating part of the posterior cerebral artery are equaled on opposite
sides (AFo/FAo subtype), as well as cases with same caliber of the left or right posterior
communicating artery and pre-communicating part of the posterior cerebral artery on
corresponding opposite side simultaneously with larger caliber of the left or right pre-
communicating part of the posterior cerebral artery in relation to opposite posterior
communicating artery (AF5/FA5 subtype). Cases of two subtypes (FA4 and AF7) are singular.
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In AT/TA-type, more frequent are the cases where calibers of the left or right posterior
communicating artery and pre-communicating part of both posterior cerebral arteries are
equaled and larger in relation to opposite posterior communicating artery (ATo/TAo
subtype), as well cases where the left or right pre-communicating part of posterior cerebral
artery is larger than corresponding opposite pre-communicating part of posterior cerebral
artery and posterior communicating artery, simultaneously with their larger caliber than
ipsilateral posterior communicating artery (AT3/TA3 subtype).
TYPES OF POSTERIOR PART OF THE CIRCLE OF WILLIS (%)
AUTHORS N COUNTRY AGE* METHOD BILAT BILAT BILAT ADULT- ADULT- UNILAT UNILAT UNILAT UNKNOWNED UNILATERAL OR
ADULT FETAL TRANSIT FETAL TRANSIT ADULT FETAL TRANSIT BILATERAL
(AA) (FF) (TT) (AF) (AT) Adult Fetal Transitory
Riggs and Rupp
994 USA ADULTS AUTOPSY 72.8 5.6 5.4 ≈16
(1963)
Saeki and Rhoton
50 USA ADULTS AUTOPSY 60 2 8 20
(1977)
Zeal and Rhoton
25 USA ADULTS AUTOPSY 58 40 20
(1978)
RETROSPECTIVE
Zada et al. (2007) 271 USA ANEURYSMATIC SERIE 10 11
REVIEW
Kayembe et al. 44 ADULTS 9.1 6.8

Japan ANEURYSMATIC AND AUTOPSY
(1984) 148 6 8.3
CONTROL SERIES
Tanaka et al. (2006) 117 Japan 20-29 MRI ≈95
111 <40 27.9 5.4 ≈9

Chen et al. (2004) China MRA
396 >40 10.6 6 ≈4
45 CHILDREN 6.6
Sahni et al. (2007) India AUTOPSY
280 ADULTS 0.3 6.8
Krishnamurthy et al.
89 India ADULTS AUTOPSY 2.2
(2008)
Pai et al. (2007) 25 India 40-84 AUTOPSY 78 10 12
Poudel and Bhattarai
35 Nepal ADULTS AUTOPSY 91.4 8.6
(2010)
225 Sri Lanka ADULTS AUTOPSY 88 1.3 0.8 8.8 6.1 2.6
De Silva et al. (2010)
26 (right)
102 Iran 15-75 (male) AUTOPSY
Efekhtar et al. (2006) 28 (left)
Songur et al. (2008) 110 Turkey 17-58 AUTOPSY 35.4
Al-Hussain et al.
50 Jordan 20-55 AUTOPSY 77 15 8
(2001)
Alawad et al. (2009) 143 Sudan 40-65 MRA 97.5
Malamateniou et al. INFANTS (PRETERM-AT-

94 UK MRA 25.5 ≈8
(2009) TERM AND TERM-BORN)
Van Overbeeke et al.

100 Netherlands ADULTS AUTOPSY 84 14 2
(1991)
Hartkamp et al. PATIENTS 54 1 8
175 Netherlands MRA
(1999) CONTROLS 25 8 14
Yasargil (1984) 200 Switzerland ADULTS AUTOPSY 67.5 24.5 8
Gabrovsky (2002) 35 Bulgaria AUTOPSY 41.4 11.4
Papantchev et al.
99 Bulgaria ADULTS AUTOPSY 30.4 9.1
(2007)
RECENT STUDY 110 Serbia 20-95 AUTOPSY 68.18 7.27 13.63 10.91
Table 5. Incidences of (sub) types of the circle posterior part in human specimens in same
and/or different countries on four continents.
Thicker black lines separate corresponding continents;
N (number of investigated specimens);
*population group or years.
There are about 65% of hypoplastic posterior communicating arteries (≤0.5 mm + 1SD) in
AA type; about one half of the cases are bilateral. There is about 44% of unilaterally
hypoplastic posterior communicating artery in AF/FA and AT/TA types with relatively
more frequency on the left side. Generally, about 60% of hypoplastic posterior
communicating arteries are in adults after 65. There are 6.6% of bilaterally and about 14% of
unilaterally hypoplasia of the pre-communicating part of the posterior cerebral artery (≤1
mm + 1SD) in FF, AF/FA and AT/TA types, respectively.
There is significant correlation (R=0.51, p<0.01) between right and left pre-communicating
parts of the posterior cerebral arteries calibers, between the right pre-communicating part of
the posterior cerebral artery and left posterior communicating artery caliber (R=0.39, p<0.01)
and between right and left posterior communicating arteries caliber (R=0.44, p<0.01) in AA
type. In AF type, significant correlation is present between right pre-communicating part of
the posterior cerebral artery and left posterior communicating artery caliber (R=0.76,
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p<0.01). Significant correlation is present between right pre-communicating part of the

posterior cerebral artery and right posterior communicating artery (R=-0.87, p=0.012) and
left posterior communicating artery (R=0.87, p=0.011) caliber, as well as, right and left
posterior communicating artery caliber (R=0.86, p=0.013) in AT type of cases. Correlations
between calibers of right and left pre-communicating parts of the posterior cerebral arteries,
as well as, right and left posterior communicating arteries of AA and corresponding arteries
of both AF and AT types are not statistically significant (Fig. 6).
Fig. 6. Correlation of calibers of the left and right pre-communicating parts of the posterior
cerebral arteries (PCAs-P1) and posterior communicating arteries (PCoAs) in corresponding
type (group) of the circle posterior part
In our specimens, basilar bifurcation angle ranges from 63.4-168.1° (111.9°) in AA type and
from 37.8-154.6° (96.6°) in FF type, as well from 67.3-142.2° (111.6°) and from 71.4-161.1°
(128.4°) in AT/TA and AF/FA types, respectively; in average, it is 109.68° (Fig. 7). We found
only one case of the basilar aneurysm originating from an obtuse basilar bifurcation angle in
AAo subtype.
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Fig. 7. The basilar bifurcation angle in corresponding type of the posterior circle part. The
least and the greatest angle are encircled by white and black lines.
4. Discussion
We selected 110 circles of Willis according to the presence of four vascular components
(posterior communicating and pre-communicating parts of the posterior cerebral arteries) in
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the circle posterior part. The "typical" circle of Willis was defined with respect to its
components as a closed circuit in which fluid may circulate from any entrance point back to
the same point with all vessels more than 1 mm in external diameter and with no excess
vessels (Kayembe et al., 1984). Variable five vascular components in the circle posterior part
are previous described in fetuses and adults (Vasović, 2004; Vasović et al., 2010). Many
authors wide world have studied about the length, diameter and anomalies in the origin of
vascular components of the circle of Willis and found that the parameters in different
geographical limits were different. However Efekhtar et al. (2006) concluded that there is no
evidence that the distributions of the variations of the circle of Willis varied in different
populations. Our selection of authors in Table 5 had the aim to check previous data.
Different incidences of types of the circle posterior part are obvious as from continent to
continent, as from the city to city in same country. Many years ago, Kayembe et al. (1984)
found that the incidence of variations in the circle of Willis was significantly higher in the
aneurysm series than in the control. Hartcamp et al. (1999) noted that several factors may
explain a significantly higher percentage of entirely complete circle anterior or posterior
configurations in different patients, including population selection, adaptation of circle
morphology and improved detection of vessels with increased flow on magnetic resonance
angiograms. Recently, in a study of volume flow rates of the basilar and bilateral internal
carotid arteries in 125 healthy volunteers, Tanaka et al. (2006) found that the relative
contribution of each of the proximal arteries correlated significantly with variations in the
circle of Willis.
Average diameter of the posterior cerebral artery was seen to be 1.7 mm (Krishnamurthy et
al., 2008) or 1.8 mm (Chen et al., 2004) on both sides, or 2.76 mm on the right and 2.5 mm on
the left side (Pai et al., 2007), as well 1.83 mm on the right and 1.88 mm on the left side
(Songur et al., 2008). Many years ago, Kamath (1981) noted that average diameter of the
posterior cerebral artery was 2.1 mm on the right and 2.2 mm on the left side. Similar to
these data, average diameter of the pre-communicating part of the posterior cerebral artery
were 2.17 mm on the right and 2.20 mm on the left in our specimens. We found the least
caliber of the pre-communicating parts of the right and left posterior cerebral arteries in a
75-year-old female and a 42-year-old male, as well the largest caliber in 47-year-old and 80-
year-old males, respectively. Average diameter of the posterior communicating artery was
seen to be 1.5 mm on the right and 1.4 mm on the left side (Kamath, 1981). The diameter of
the posterior communicating artery varied from hypoplastic (less than 0.5 mm) to 4 mm
(fetal circulation) on the right side (average 1.25 mm) and 0.5 mm to 3 mm (average 1.12
mm) on the left side (Pai et al., 2007). In our specimens, average diameter of this artery was
similar on both sides (1.22 mm on the right and 1.23 mm on the left). We found the least
caliber of posterior communicating arteries in 44-year-old and in a 64-year-old woman,
respectively, while the largest caliber in 80-year old woman, too. However, values of
calibers of these vascular components in our study were in the function of forming basic
types and their subtypes of the posterior circle part.
Al-Hussain et al. (2001) noted a possibility that the circle of Willis may have different
configurations of the posterior bifurcation of the posterior communicating artery on both
sides. In that case, two primary visual areas of the same individual may receive their blood
from different sources; one from the basilar artery through the pre-communicating segment
(adult configuration) and the other from the internal carotid artery through the posterior
communicating artery (fetal configuration).
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Because of many anatomical variations of the circle of Willis, its classification into clearly
arranged groups is hardly possible. Urbanski et al. (2008) divided arterial variations into
three groups in regard to the possible impact on the cerebral cross-perfusion. In the first
group, there was only one location of hypoplasia or aplasia within the circle of Willis. In the
second group, the abnormalities existed within the posterior pre-communicating or
communicating arteries. In the third group, the pathologies were found in both anterior and
posterior vascular components of the circle of Willis. According to Saeki & Rhoton (1977) the
posterior part of the circle of Willis is normal if the posterior communicating arteries are not
hypoplastic. However, we include those arteries during formation different subtypes
because of no cerebral cause of death, oldest of cadavers and the presence of posterior
communicating side branches. In continuation of the study on fetuses (Vasović et al., 2008a),
we accepted a classification of the circle posterior part into bilateral adult (AA-), fetal (FF-)
and transitory (TT-) types and their three combinations. We presented incidences of some
patterns of the circle part in Table 5. According to the fact that many authors have noted
incidence of one of three configurations (adult or fetal or transitory) without data about
unilateral or bilateral pattern (Zeal & Rhoton, 1978; Al-Houssain et al., 2001; Gabrovsky,
2002; Pai et al., 2007; Papantchev et al., 2007; Songur et al., 2008), complete comparison is
difficult. We calculated the percentages of different configurations according to diameters of
the vessels, and presented in Table 5. We found four (two symmetric and two asymmetric)
types and labeled with initial letters of their marks, opposite to the authors listing in Table 5.
As early as seven weeks after conception, a preliminary form of the circle of Willis is present
with approximately equal diameters of all vessels. As fetal growth occurs, the relative sizes
of the various components of the circle of Willis change significantly to assume the eventual
adult morphology (Cuchiara & Detre, 2007). Adult configurations occur in the majority of
circles of Willis through further fetal and newborn life (Van Overbeeke et al., 1991), as well
in majority of other and our study of adults, except in the studies of Chen et al. (2004) and
Songur et al. (2008). No sexual prevalence or prevalence for the right or left side of the adult
or fetal configuration was noted in a study of complete circles of Willis of 53 brains taken
from fetuses and neonates aged 12 to 60 weeks after conception (Van Overbeeke et al., 1991).
The preterm infants born after 30 weeks’ gestation age have more significantly increased
prevalence in the bilateral fetal-type origin of the posterior cerebral artery (83.3%) than those
infants born before 30 weeks (Malamateniou et al., 2009). We found bilateral adult and fetal
(AA and FF) types in 68.18% and 7.27%, respectively, while De Silva et al. (2010) noted in
88.8% and 1.3%, respectively in Sri Lankan population. Hendrikse et al. (2005) cited
literature data that in the circle posterior part, the variant type with a unilateral fetal-type
posterior cerebral artery is present on up to 25% of angiograms, and a bilateral fetal-type
posterior cerebral artery present on up to 10% of angiograms. Zada et al. (2008) noted that
the overall incidence of the fetal-type posterior circulation has been reported to occur in 4 to
29% of patients, whereas bilaterally occurring fetal posterior cerebral arteries have been
reported to occur in 1 to 9% of patients. We also found unilateral adult configuration
associated with opposite fetal (AF/FA) or transitory pattern (AT/TA) in 13.63% and 10.91%,
respectively. A central hypothesis is that the circle of Willis anomalies correlates with
alterations in cerebral hemodynamic. Songur et al. (2008) described that cerebral blood flow
was in direct proportion to vessel diameter and thus there is more blood flow in larger
vessels. In addition, a large posterior communicating artery may protect against watershed
infarction in patients with ipsilateral occlusion of the internal carotid artery, while a very
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small or absent ipsilateral posterior communicating artery increases the risk of a watershed
infarction in these patients (Schomer et al., 1994). The patients with fetal type could be more
prone to develop vascular insufficiency (Van Raamt et al., 2006) or internal carotid-posterior
communicating aneurysms (Zada et al., 2008). According to De Silva et al. (2010), fetal
configuration has been reported in adults at 4% to 46% on the basis of anatomical and
angiographic studies, and a higher percentage of on the basis of anatomical studies has been
reported in older fetuses and newborns: 35% and 56%, while only a minority of circles
showed a transitional configuration, 14% and 20%, respectively. In our adult cases there was
not bilateral transitory configuration as in other studies of infants or adults (Riggs & Rupp,
1963; Hartkamp et al., 1999; Malamateniou et al., 2009, De Silva et al., 2010) or fetuses (Van
Overbeeke et al., 1991; Vasović et al., 2008a; De Silva et al., 2010).
Anastomotic flow between carotid and basilar arteries is limited when one or both posterior
communicating arteries are hypoplastic, while effective circulation across the circle and
between its anterior and posterior components is restricted when both anterior and
posterior anastomotic stems are hypoplastic, as well similar limitation of collateral flow
results when all component stems of the circle are hypoplastic (Riggs and Rupp, 1963). The
resistance to flow across the posterior communicating artery is greater than across the
anterior communicating artery, because the posterior communicating artery is usually a
longer vessel (Hartkamp et al., 1999). Arteries of less than 1 mm diameter were considered
abnormal, barring the communicating arteries, where less than 0.5 mm diameter was
considered abnormal (Kamath, 1981; Pai et al., 2007). The relative importance of the caliber
values of the brain arteries have thus been assessed, although no clear consensus is found
among reports. One of limitations is also potential minor changes in the diameter of the
vessels during time (Efekhtar et al., 2006). Hypoplastic vessels were defined to be those with
external diameters less than 0.8 mm (Hartkamp et al., 1999; Chen et al., 2004) or 1.0 mm
(Schomer et al., 1994; Gabrovsky, 2002; Efekhtar et al., 2006; Tanaka et al., 2006). When
taking into account only static anatomic observations, Merkkola et al. (2006) hypothesized
that the circulation to the left hemisphere would be sufficient in the majority of patients
when using a threshold of 0.5 mm. The results of Hoksbergen et al. (2000) indicate that the
threshold diameter for collateral function of the posterior communicating artery lies
between 0.4 and 0.6 mm. This threshold diameter might be used in future studies evaluating
the influence of its collateral ability in symmetric or asymmetric circle posterior part. The
most common anatomic variation in the group without vascular-related abnormalities was
the absence or hypoplasia of the posterior communicating artery (Malamateniou et al.,
2009). Dysregulation of cerebral blood flow may allow relative ischemia to develop in the
setting of increased metabolic demand related to neuronal hyper excitability, may trigger
cortical spreading depression, and may predispose individuals to ischemic lesions and
stroke (Cucchiara and Detre, 2008).
The study of Sahni et al. (2007) was conducted on brains of 280 adults and 45 children. The
hypoplastic posterior communicating artery on the right side was present in 2 children and
17 adults, whereas on left side, it was seen in 1 child and 6 adults; bilateral hypoplastic
posterior communicating artery was observed in 4 children and 17 adults. Fetal type on the
right side was observed in 2 children and 12 adults, whereas on the left side, it was in 1 child
and in 7 adults; bilaterally, it was found to be present only in 1 adult male. In the literature it
was also noted that absence/hypoplasia of the posterior communicating artery was more
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common in preterm boys than in girls, while absence/hypoplasia of the pre-communicating

part of the posterior cerebral artery occurred twice as often in term-born girls than in boys
(Malamateniou et al., 2009). In a study of 1000 brains, the pre-communicating part of the
posterior cerebral artery was hypoplastic in 10.6% and the posterior communicating artery
in 16.4% of cases (Kapoor et al., 2008). There are about 65% of hypoplastic posterior
communicating arteries in bilateral adult (AA) group, and about 44% of unilaterally
hypoplastic posterior communicating artery in asymmetric groups with relative more
frequency on the left side in our study. Generally, about 60% of hypoplastic posterior
communicating arteries are in adults aging after 65. There are 6.6% of bilaterally and about
14% of unilaterally hypoplasia of the pre-communicating part of the posterior cerebral
artery in bilateral fetal (FF), adult-fetal (AF/FA) and adult-transitory groups (AT/TA),
respectively. We evidenced percentile of hypoplastic pre-communicating part of the
posterior cerebral and posterior communicating arteries in corresponding type and subtype,
while other authors presented it as general data, except of Gabrowsky (2002). He noted that
the mean diameter of the adult and fetal types of the posterior communicating artery was
1.28 and 2.33 mm, respectively. He also found that a reduction of the diameter from the
anterior to the posterior third of this artery in both configurations and more frequently for
the hypoplastic (51%), than for the adult type (37%). A hypoplasia of the right posterior
communicating artery in 16% and left posterior communicating artery in 11%, as well of
both arteries in 33% of males were seen (Efekhtar et al., 2006). Incidence of a hypoplastic
pre-communicating segment of the posterior cerebral artery, according to Tanaka et al.
(2006), ranges from 4% to 14% of subjects. However, a hypoplasia of the pre-communicating
segment of the posterior cerebral artery and posterior communicating artery was 1% and
33%, respectively (Al-Houssain et al., 2001). A hypoplasia of the posterior cerebral artery
also was noted in 2.1% on the right and in 1.4% on the left side (Alawad et al., 2009).
Since a significant inverse relationship existed between the diameters of ipsilateral posterior
cerebral and posterior communicating arteries, as well as a smaller posterior communicating
artery on the left would be associated with a larger posterior cerebral artery on that side
(Kamath, 1981). In our study there was significant correlation between right and left pre-
communicating parts of the posterior cerebral arteries calibers, between the right pre-
communicating part of the posterior cerebral artery and left posterior communicating artery
caliber, and between right and left posterior communicating arteries caliber in AA group. In
AF group, significant correlation is present between right pre-communicating part of the
posterior cerebral artery and left posterior communicating artery caliber. It was also present
between right pre-communicating part of the posterior cerebral artery and right posterior
communicating artery and left posterior communicating artery caliber, as well as, right and
left posterior communicating artery caliber in AT group of cases.
Atheromatous changes (grade 1) were first visualized in a 34-years old male (AA2 subtype)
and in five cases (one in FF2' , two in AAo and two in AA2 subtype) of the fifth decade of
life. We did not visualize atheromatous changes in 21 adults (9 female and 12 male) of the
fifth decade and older. Atheromatous changes of grades 2 and 3 were first visualized in 42
and 47-years old males, respectively, as well as in females of the seventh decade of life
(Table 4). Ravensbergen et al. (1998) noted that a strong argument for the (causal)
relationship between hemodynamics and atherosclerosis can be found if variations of
the geometry result in changes of the location of the atheromatous lesions, which
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correspond to the changes of the flow force distribution. Songur et al. (2008) established
atheromatous plaques in the basilar artery in 30% of cases. We visualized these plaques
particularly for every type. Very interesting findings were that atheromatous changes of
Grade 1 and 3 were the most frequent in FF (62.5) and AF/FA (26.6%) types, while the
least frequency was observed both in AT/TA (16.6%) and AA- (5.3%) types. Atheromatous
changes of grade1 for male gender were more frequent in AA type; a relation was 2:1.
In our adult specimens, basilar bifurcation angle ranged from 37.8-168.1°, in average, it was
109.68°. The average basilar angle in adults was 109 degrees and ranged between 30 and 180
degrees in a study of Caruso et al. (1990). The average basilar bifurcation angle of 98 patients
aging from 12 to 78 years was determined to be 117.7° (30.93°–172.2°) (Żurada et al., 2008).
During a study of fetal specimens, this angle ranged from 35 to 175 degrees (Vasović et al.,
2008b). Żurada et al. (2008) described three types of the basilar bifurcation angle in which a
type "T" is with an angle greater than 145°, a type "Y" with an angle being equal or less than
145° but greater than 100° and a type "V" for angles less than 100°. Ingebrigsten et al. (2004)
hypothesized that normal bifurcations of cerebral arteries beyond the circle of Willis would
follow optimality principles of minimum work and that the presence of aneurysms would
be associated with deviations from optimum bifurcation geometry. We found only one case
of basilar tip aneurysm arising from an obtuse basilar bifurcation angle. However Valleé et
al. (2003) showed two basilar apex aneurysms originating from bifurcation angles of about
90 degrees and 180 degrees.
We presented six AA-subtypes of the AA-type, four subtypes of the FF-type, three subtypes
of the AT/TA-type and four subtypes of the AF/FA-type of the posterior part of the circle of
Willis. Van Raamt et al. (2006) proposed to define a partial fetal-type posterior circle of
Willis, in which a small pre-communicating part between the basilar artery and the post-
communicating part of the posterior cerebral artery is present, and a full fetal-type posterior
circle of Willis, in which the pre-communicating part is absent. Besides our study confirms a
domination of bilateral adult configuration of the circle posterior part as other authors, we
are the first who classified and marked posterior circle subtypes, as well as new subtypes,
which missing in fetal status (Vasović, 2004). We found some similar schemes of (sub) types
in Riggs’ paper (Riggs & Rupp, 1963) and Yasargil’s book (Yasargil, 1984); however, we can
not do precise comparison with them. It is difficult confirm that some subtypes of the
posterior circle part is a feature of Serbian population. This study offers a possibility to
evaluate that angioarchitecture in different health population groups or in patients with or
without cerebral pathology wide our or other countries, especially if proposed (sub) types of
the circle posterior part many authors could be accept.
5. Acknowledgment
Contract grant sponsor: Ministry of Science and Technological Development of Republic of
Serbia (No: 41018 and 175092).
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Forensic Medicine - From Old Problems to New Challenges

Edited by Prof. Duarte Nuno Vieira

InTech Europe InTech China

Avoiding Errors and Pitfalls in Evidence

2. Biological sample collection

2.2 Collecting methodologies

3.2 DNA quantitation

3.3 DNA amplification

3.4 Detection of amplified product

3.5 Difficulties of the typing of trace DNA

3.6 Pitfall at the interpretation step

3.7 Mixture interpretation

Recommendations were published by the International Society of Forensic Genetics on

Sewell J, Quinones I, Ames C, Multaney B, Curtis S, Seeboruth H, Moore S, Daniel B:

Death Scene Investigation from

2. Crime scene & death scene

3. Investigative tools and equipment

4. Steps of death scene investigation

4.1 Pre-planning the death scene investigation

4.2 Cooperation among investigators

4.3 Documentation of the scene

4.3.1 Taking notes at the death scene

4.3.2 Videotaping the death scene

4.3.3 Photographing the death scene

4.3.4 Sketching the death scene

4.4 Identification of the deceased

4.5 Examination of the body

performed on a decedent's body at a scene, such as gunshot residue or fingerprinting,

4.6 Other scene information collection

4.7 Determining what information has already been developed

4.8 Collecting evidences which may be found at the death scene

4.9 Interviewing persons regarding the death

4.10 Estimating the post-mortem interval at the scene

4.11 Ending the death scene investigation

Fig. 7. A buried homicide victim in a desolate land.

5.1 Homicide by poisoning

Fig. 18. A soft piece of cloth against the ligature.

In some cases, it is important to distinguish accidental manner from suicidal or homicidal

Fig. 30. Fatality involving power take-off (water pump) entanglement.

9. Sudden and unexplained infant death

9.1 SUID scene investigation

Di Nunno, N., Costantinides, F., Vacca, M. & Di Nunno, C. (2006). Dismemberment: a

4.2 Biological tests

Suto M, Abe S, Nakamura H, Suzuki T, Itoh T, Kocbi H, Hoshiai G, Hiraiwa

Forensic Investigation in Anaphylactic Deaths

release of secondary mediators of ‘de novo’ synthesis such as leucothrienes,

lachrymation), upper airway (nasal congestion, sneezing, hoarseness, stridor, pharyngeal or

3. Proposal of a methodological protocol

3.1 Medical history and investigations on the spot

3.2 Complete necroscopic exam

3.3 Laboratory tests

3.3 Auxiliary techniques: histochemical and immuno-histochemical

Cytotypes EMBP Chymase Tryptase CD117 Pagoda red

Greenberger, P.A.; Rotskoff B.D.; Lifschultz, B. (2007) Fatal anaphylaxis: postmortem

Forensic Age Estimation in Unaccompanied

In accordance to the guidelines proposed by Ritz-Timme et al for an age assessment method

4. Bone age in carpus

5.1 Dental eruption

5.2 Dental maturation

5.3 The third molar in age assesment.