Chapter six

The next chapter will be on other

body fluids
 Chapter outline

Introduction to body fluids

Types of body fluids
Cerebrospinal fluid analysis
Synovial fluid analysis
Serous (pleural, pericardial and peritoneal) fluid analysis
Semen analysis
Amniotic fluid analysis
Nasal smear analysis
Chapter outline

Routine laboratory assays

Collection of sample

Gross appearance

RBC &WBC counts

Chemical analysis

Morphological Examination

Microbiological Examination

Serological Examination

Quality control in body fluid analysis

Cerebral Spinal Fluids
Learning Objectives

Upon completion of this chapter the student will

be able to:
1. Describe the formation of CSF from blood.
2. Describe cellular neurochemistry and the function of
the choroids plexus.
3. Discuss diffusion mechanism across the blood brain
barrier: simple diffusion, carrier mediated diffusion,
and active transport. Relate which types of
molecules are transported with each diffusion
mechanism.
4. Explain the mechanism of glucose uptake into the
brain.
Learning Objectives (continued)

5. Describe the role of Hexokinase and Enolase as

enzymes in the glycolytic pathway of glucose
utilization.
6. List the function of amino acids in CSF formation.
7. Identify cells observed in the microscopic evalulation
of CSF: Ependymal cells, neutrophils, bands,
immature granulocytes, lymphocytes, plasmacytoid
cells, motts cells, macrophages, basophils, nucleated
red blood cells, Erythrophages, Leukophages,
Siderophages, Hematoidin crystals, and malignant
cells.
Learning Objectives (continued)

8. Given the laboratory observations of a bloody CSF,

evaluate the supernatant and propose the type of
pathological condition associated with a clear
supernatant versus a xanthochromic supernatant.
9. Compare the difference of pathological conditions
associated with the types of cells observed in a CSF.
10. List the normal range of glucose, protein, and cell
count for a CSF.
11. Evaluate abnormal laboratory results with a
pathological condition related to CSF.
12. Discuss appropriate collection requirements for CSF
following a lumbar puncture.
Cerebrospinal fluid analysis

Collectionof CSF sample

Routine Laboratory assays of CSF
Gross appearance

RBC &WBC counts

Morphological Examination

Microbiological Examination

Serological Examination
Cerebrospinal fluid (CSF)
Fluid in the space called sub-arachnoid space between
the arachnoid mater and pia mater
Protects the underlying tissues of the central nervous
system (CNS)
Serve as mechanical buffer to
prevent trauma,

regulate the volume of intracranial pressure

circulate nutrients

remove metabolic waste products from the CNS

Act as lubricant

Has composition similar to plasma except that it has

less protein, less glucose and more chloride ion
 CSF contd
Maximum volume of CSF
Adults 150 mL
Neonates 60 mL
Rate of formation in adult is 450-750 mL per day or 20 ml
per hour
reabsorbed at the same rate to maintain constant
volume
Collection by lumbar puncture done by experienced
medical personnel
About 1-2ml of CSF is collected for examination
lumbar puncture is made from the space between the 4th
and 5th lumbar vertebrae under sterile conditions.
 Fig. Collecting a CSF specimen
Collected in three
sequentially labeled tubes
Tube 1 Chemical and
immunologic tests
Tube 2 Microbiology
Tube 3 Hematology
(gross examination, Location of CSF
total WBC & Diff)
This is the list likely
to contain cells
introduced by the
puncture procedure
 Lab analysis
Clinical Significance
Diagnosis of meningitis of bacterial, fungal,
mycobacterial and amoebic origin or differential
diagnosis of other infectious diseases
subarachnoid hemorrhage or intracerebral hemorrhage
Principle of the test
CSF specimen examined visually and microscopically
and total number of cells can be counted and identified
Specimen: the third tube in the sequentially collected
tubes*
must be counted within 1 hour of collection (cells
disintegrate rapidly). If delay is unavoidable store 2-8oC.
All specimens should be handled as biologically
hazardous
Cellular Neurochemistry

Concept: Brain cells exist in an almost invariant

chemical environment, which is different from
that surrounding cells in other organs.
Barrier mechanisms help to maintain proper
environment
Cellular Neurochemistry

Permeability barriers
Capillary endothelial cell tight junctions: limit
movement between blood and brain extracellular
space.
Choroid plexus epithelial cells: limits movement
between blood and Cerebral Spinal Fluid (CSF).
Glial cells
Diffusion Mechanisms

Active Transport
Movement against a concentration gradient
Requires energy
Highly selective
Usually moves substances out of CNS
Uptake and Utilization of
Glucose

Glucose is major energy substrate for brain as

well as a major carbon source for many
molecules.
Brain uses 20-25% of total oxygen and 15% of
cardiac output is directed to CNS.
Glucose Utilization

When body glucose supply is decreased, other

organs decrease glucose utilization to maintain
adequate supply of glucose to brain.
Other organs can readily switch to oxidation of
another substrate for energy production.
Under certain conditions, such as chronic
starvation, the brain can oxidize other
substances but maintains a minimal obligatory
requirement for glucose.
Brain Utilization of Glucose

Glycolysis--conversion to lactic acid

Hexokinase has high activity in brain
Serves to trap glucose and maintain concentration
gradient for diffusion
Brain Utilization of Glucose

2-deoxyglucose is also taken up by brain and

phosphorylated by hexokinase, but then
becomes trapped
Markerto correlate changes in neural activity with
changes in glucose utilization.
Enolase, an enzyme in glycolytic pathway, exists
in nerve cells in unique isoform (neuron specific
enolase, NSE)
Used as a specific marker for neurons.
Brain Utilization of Glucose

Pentose shunt
Provides source of D-ribose for synthesis of
DNA and RNA
Produces NADPH required for lipid syntheses
Most active during development
Utilization of Amino Acids

Concept: Amino acids serve many functions in

CNS
Peptide and Protein synthesis
Precursors for transmitters
Neurotransmitters
Proteins in the CNS

Concept: Neurons must produce those proteins

essential for their special functions:
conduction of action potentials
synaptic transmission
axoplasmic transport
establishment of specific connections
Specific Neural/Glial
Peptides/Proteins

Structual-cytoskeletal
Cell Surface proteins play a role during
development in directing neural connections
Contractile proteins
function in axoplasmic movement
Neurotubular protein
Glial proteins (glial fibrillary protein)
Specific Neural/Glial
Peptides/Proteins

Enzymes for transmitter synthesis and

degradation
Transmitter receptors
Membrane transporters
Ion Channels
Growth Factors
Synaptic Vesicle
Microscopic
Observations of Cerebral
Spinal Fluid
Ependymal Cells

Cells lining the ventricles (ependymal) or choroid plexus

may be shed into the CSF
Single or in clumps
Nuclei are round to oval with a definitive smooth nuclear
membrane
Chromatin is distributed evenly and is finely granular or
hyperchromatic
Nucleoli are not present
The cytoplasm can be basophilic or pink. Microvilli may
be present.
Neutrophils and Bands

Morphologically identical to neutrophils and

bands in blood
Occasionally granulation disappears and
pseudo-hypersegmentation is observed.
Lymphocytes

Almost identical morphology to lymphocytes in

the blood
Due to "flattening-out" of the lymphs during
cytocentrifugation, nucleoli may be visible.
Found in all fluid
Macrophages

Leukophages:Macrophagescontaining
phagocytized WBC. WBCs are often pyknotic
and easily confused with NRBC's. Found in all
fluids.
Erythrophages: Macrophages containing
phagocytized RBC or RBC fragments. May
contain several RBC. Found in all fluids.
Siderophages: Macrophages containing
phagocytized particles of hemosiderin, which
stain a blue-black color.
Hematoidin Crystals

These are bright-yellow diamond-shaped

crystals of hemosiderin
intracellular or extracellular on the slide.
They are iron-negative on the Prussian blue
stain and therefore
Can be noted on the patient report without
performing an iron stain.
Immature Granulocytes

Metamyelocytes, myelocytes, and

promyelocytes may be found in fluids, though
they are rarely seen.
They are morphologically identical to those in
the blood
May be due to bone marrow contamination in
CSF
Blasts

Morphologically similar to blasts found in the

blood
There may be some clover-leaf shaped nuclei
due to cytocentrifugal distortion.
May be found in all fluids
Seen in association with leukemias, lymphomas
Bone marrow contamination of CSF
Nucleated Red Blood Cells

NRBC are rarely seen body fluids. If observed,

they should be reported as the number of NRBC
per number of WBC counted
They must be differentiated from pyknotic WBCs
NRBCs are commonly due to peripheral blood
or bone marrow contamination of CSF
Abnormal Lymphocytes

Plasmacytoid lymphs: Identical in morphology to

plasmacytoid lymphs in blood
Found in all fluids.
Mott cells: Plasma cells with numerous clear
cytoplasmic vacuoles containing
immunoglobulins
Reactive Macrophages

These are most common in CSF from small

children with subarachnoid hemorrhage but may
be found in all body fluids
May be very difficult to distinguish
morphologically from large atypical lymphocytes
Malignant Cells

Malignant cells may be shed from solid tissue

(non-hematopoietic) neoplasms into CSF or
body cavity fluid submitted for cell counts
Fluid will be turbid or bloody
Malignant cells are usually seen in clusters of 3-
5 or more, but may occur singly
Microorganisms

Intracellular bacteria or yeast can be observed in

acute bacterial or fungal infections
It is important to coordinate your findings with
those of the Microbiology Section of the
laboratory
Bloody CSF

When the CSF is pinkish red, this usually

indicates the presence of blood, which may have
resulted from:
Sub arachnoid hemorrhage
Intra cerebral hemorrhage
Infarct
traumatic tap
Order of Draw of Lumbar Puncture

1st - Chemistry
2nd - Microbiology
3rd - Hematology
Physical Examination

Color Xanthochromia
Hyperbilirubinemia
Increased Protein
Turbidity
Increased White Blood Cells (Pleocytosis)
CSF Supernatant

A traumatic tap shows progressively decreasing

RBC in serial samples
Generally, in subarachnoid hemorrhage, the
RBC would be consistent from one tube to the
next
CSF Supernatant

After the CSF is centrifuged, the supernatant

fluid is clear in a traumatic tap, but it is
xanthochromic in a subarachnoid hemorrhage
Xanthochromia of the CSF refers to a pink,
orange, or yellow color of the supernatant after
the CSF has been centrifuged
Cell Count

The white cell count is increased when there is

inflammation of the central nervous system,
particularly the meninges
Bacterial infections are usually associated with
the presence of neutrophils in the CSF
Cell Count

Viral infections are associated with an increase

in mononuclear cells
An increase in mononuclear cells may also be
seen with:
cerebral abscess
acute leukemia
Lymphoma
intracranial vein thrombosis
cerebral tumor
multiple sclerosis
CSF Normal Adult Lab Ranges

Normal CSF Levels:

Protein (10 - 45 mg/dL)
Glucose (40 - 70 mg/dL)
Physical Appearance
Clear/colorless
RBC <5/mL
WBC <5/mL
Increased CSF Protein
>80mg/dL
Diabetes Mellitus
Brain tumor
Meningioma
Acoustic neuroma
Ependymoma
Encapsulated brain abscess
Spinal cord tumor
Multiple Sclerosis
Acute purulent Meningitis
Increased CSF Protein
>80mg/dL
Granulomatous Meningitis
Carcinomatous Meningitis
Syphilis (protein may be normal if longstanding)
Guillain-Barre Syndrome (Infectious polyneuritis)
Cushing's Disease
Connective tissue disease
Uremia
Myxedema
Cerebral hemorrhage
Glucose

Low glucose levels, as compared to plasma

levels, are seen in:
bacterialmeningitis
cryptococcal meningitis
malignant involvement of the meninges and
sarcoidosis
Glucose levels are usually normal in viral
infections of the CNS
Glucose

Increased CSF Glucose

Reflects serum hyperglycemia
CSF glucose lags Serum Glucose by 1 hour
CSF glucose is two thirds of Serum Glucose
Lactate

In bacterial and cryptococcal infection, an

increased CSF lactate is found earlier than a
reduced glucose
In viral meningitis, lactate levels remain normal,
even when neutrophils are present in the CSF
Raised levels may also occur with severe
cerebral hypoxia or genetic lactic acidosis
Summary

You should now be able to discuss the formation

and collection procedure for cerebral spinal fluid,
normal and abnormal findings in CSF and
methods of analysis and evaluation of CSF
Semen analysis
 Semen analysis

Used in the evaluation of reproductive dysfunction

(infertility) in the male
Used to select donors for therapeutic insemination
Is a cost-effective and relatively simple procedure.
Consists of microscopic and macroscopic components
Collection and transport of semen

1. Give the person a clean, dry, leak-proof container,

and request him to collect a specimen of semen at
home following 3 days of sexual abstinence
condom is used to collect the fluid, this must be well-
washed to remove the powder which coats the rubber.
It must be dried completely before being used.
Collection and transport, contd

2. Lable the container (name ,date and time of

collection, period of abstinence
Deliver the specimen to the laboratory within 1 hour
Fluid should be kept as near as possible to body
temperature.
This is best achieved by placing the container inside a
plastic bag and transporting it in the person's armpit . .
Tests for semen

Macroscopic
-Physical (volume, viscosity, liquefaction)
-chemical l(eg. ph)
Microscopic
-stained preparation
- wet-mount
 Semen analysis

When investigating infertility, the basic analysis of

semen (seminal fluid) usually includes:
Measurement of volume
Measurement of pH
Examination of a wet preparation to estimate the
percentage of motile spermatozoa and viable forms and
to look for cells and bacteria.
Sperm count
Examination of a stained preparation to estimate the
percentage of spermatozoa with normal morphology.
 EXAMINATION OF SEMEN

Caution: Handle semen with care because it may

contain infectious pathogens, e.g. HIV, hepatitis
viruses, herpes viruses.
 Macroscopic Examination

Measure the volume

Normal semen is thick and viscous when ejaculated.
It becomes liquefied usually within 60 minutes due to a
fibrinolysin in the fluid.
Failure to liquefy may indicate inadequate prostate
secretion.
When liquefied, measure the volume of fluid in millilitres
using a small graduated cylinder.
Normal specimens: Usually 2 ml or more
Macroscopic Examination, cont.

Measure the pH
Using a narrow range pH paper, e.g. pH 6.48.0, spread
a drop of liquefied semen on the paper.
After 30 seconds, record the pH.
pH of normal semen: Should be pH 7.2 7.8
When the pH is over 7.8 this may be due to infection.
When the pH is below 7.0 and the semen is found to
contain no sperm, this may indicate dysgenesis (failure
to develop) of the vas deferens, seminal vesicles or
epididymis.
Microscopic Examination

be performed to obtain estimates of sperm

concentration, motility, and agglutination.
polygonal cells of the urethral tract and round cells' such
as spermatogenic cells and leukocytes can also be
observed when sperm are counted in a hemocytometer.
Motility (normal range 50% or above) is expressed as
the percentage of sperm that move.
Microscopic Examination

Estimate the percentage of motile and viable

spermatozoa
Motility
Place 1 drop of well-mixed liquefied semen on a slide
and cover with cover glass.
Focus the specimen using the 10_ objective.
Ensure the spermatozoa are evenly distributed
if not, re-mix the semen and examine anew preparation.
Using the 40_ objective, examine several fields
Microscopic Examination,contd

to assess motility, i.e. whether excellent (rapid

and progressive) or weak (slow and non
progressive).
Count a Normal motility: Over 50% of
spermatozoa are motile within 60 minutes of
ejaculation.
 Reporting of results

Motility (normal range 50% or above) is expressed as

the percentage of sperm that move.
Sperm moving rapidly in a straight line with little yaw and
lateral movement are Grade 4
if they move more slowly, Grade 3.
Grade 2 sperm move even more slowly and with
substantial yaw.
Grade 1 sperm have no forward progression.
Zero progression denotes absence of any motility
contd

If motility is less than 50%, a viability stain of

eosin Y with nigrosin as a counterstain is done.
dead sperm will stain red, whereas live sperm
will exclude the dye and appear unstained.
In samples with no visible sperm, such as post-
vasectomy semen, the entire sample should be
centrifuged, and the pellet examined for intact or
damaged sperm fragments.
Cont

The spermatozoa remain motile for several

hours.
Perform gram stain smear:
When more than 60% of spermatozoa are non motile,
when more than a few leucocytes and
> 6 red blood cell/ HPF
Look for the type of bacteria that exist in the semen
Viability

procedure
Mix one drop of semen with 1 drop of 0.5% eosin
solution on a slide.
After 2 minutes examine microscopically.
Use the 40X objective to count the percentage of viable
and non-viable spermatozoa.
Viable spermatozoa remain unstained,
non-viable spermatozoa stain red.
Normal viability: 75% or more of spermatozoa should be
viable (unstained).
 Staining procedure Contd

sperm count
Using a graduated tube or small cylinder, dilute the
semen 1 in 20 as follows:
Fill the tube or Thoma pipette to the 1 ml mark with
well-mixed liquefied semen.
Add sodium bicarbonate-formalin diluting fluid to the
20 ml mark, and mix well.
Using a Pasteur pipette or Thoma pipette, fill an
Improved Neubauer ruled chamber .
Staining procedure Contd
Wait 35 minutes for the spermatozoa to settle.
Count the number of spermatozoa in an area of 2 sq
mm, (i.e. any 2 large squares within the 9 squares).
Calculate the number of spermatozoa in 1 ml of fluid by
multiplying the number counted by100, 000.
Normal count: 20 106X106 spermatozoa/ml or more.
Counts less than 20 - 106 X106/ml are associated
with male sterility. total of 100 spermatozoa, and note
out of the hundred how many are motile.
Record the percentage that are motile and non motile.
Staining procedure Contd

Make a thin smear of the liquefied well-mixed semen on

a slide.
While still wet,fix the smear with 95% v/v ethanol for 5
10 minutes
allow to air-dry.
Wash the smear with sodium bicarbonate formalin
solution to remove any mucus which may be present.
Rinse the smear with several changes of water.
Cover the smear with dilute (1 in 20) carbol fuchsin and
allow to stain for 3 minutes.
Wash off the stain with water.
 Staining contd

Counter stain, by covering the smear with dilute (1 in 20)

Loefflers methylene blue for 2 minutes.
Wash off the stain with water.
Allow the smear to air-dry.
Staining results
Nucleus of head . . . . . . . . . . . . . . . . . . . . Dark blue
Cytoplasm of head . . . . . . . . . . . . . . . . . . . Pale blue
Middle piece and tail . . . . . . . . . . . . . . . . . Pink-red
Alternative stains: Other staining techniques used to
stain spermatozoa include Giemsa and Papanicolaou.
Reporting morphology of spermatozoa

Examine the preparation for normal and abnormal

spermatozoa using the 40X objective
Use the100X objective to confirm abnormalities.
Count 100 spermatozoa and estimate the percentage
showing normal morphology and the percentage that
appear abnormal.
Normal spermatozoa

Measure 5070 m in length.

Each consists of an oval-shaped head (with acrosomal
cap) which measures 35 X 23 m
a short middle piece
a long thin tail (at least 45 m in length).
In normal semen, at least 50% of spermatozoa should
show normal morphology.
Most specimens contain no more than 20% abnormal
forms.
Abnormal spermatozoa

The following abnormalities may be seen:

Head
Greatly increased or decreased in size.
Abnormal shape and tapering head (pyriform)
Acrosomal cap absent or abnormally large.
Nucleus contains vacuoles or chromatin is
unevenly distributed.
Two heads.
Additional residual body, i.e. cytoplasmic droplet.
Middle piece
Two heads Contd

Absent or markedly increased in size.

Appears divided (bifurcated).
Angled where it meets tail.
Morphology contd

Tail
Absent or markedly reduced in length.
Double tail.
Bent or coiled tail.
Synovial Fluid
Synovial Fluid
Definition:
Synovium refers to the tissue lining synovial
tendon sheaths, bursae, and diarthrodial joints
except for the articular surface.
Synovial fluid (synovia, SF) is an imperfect
ultrafiltrate of blood plasma combined with
hyaluronic acid produced by the synovial cells.
Synovial Fluid contd

Small ions and molecules (e.g., Na+, K+, glucose,

urea, etc.) readily pass into the joint space and are,
therefore, similar in concentration to plasma, while
large molecules are absent or present in trace
amounts
Classification of Synovial Fluid
*Examination of the synovial fluid is essential to
distinguish infectious from noninfectious arthritis

Non inflammatory effusions (Group I)

Typically have leukocyte counts less than
3000/L, with a minority of neutrophils..
Non inflammatory response*
Non inflammatory effusions**
Inflammatory effusions (Group II)

have leukocyte counts 3000 - 75 000, with neutrophils

accounting for over 50% .
Examples of this reaction group:
Rheumatoid arthritis
systemic lupus erythematosus (SLE)
Reiter's syndrome
rheumatic fever
acute crystal-induced arthritis
arthritis associated with inflammatory bowel disease
psoriatic arthritis
fat droplet synovitis
Purulent (infectious) effusions (Group III)

typically have leukocyte counts greater than 50,000, of

which 90% or more are neutrophils.

Bacterial, fungal, and tuberculous joint infections

constitute this group.
Hemorrhagic effusions (Group IV)

WBC count between 5010,000WBC/ mL, with < 50%

neutrophils
RBCs may be present
may be seen in association with:
traumatic arthritis
pigmented villonodular synovitis
synovial hemangioma
neuropathic osteoarthropathy
joint prostheses
hematologic disorders (hemophilia,
thrombocytopenia, anticoagulant therapy, sickle cell
disease or trait, myeloproliferative syndrome).
 Synovial Fluid Findings by Disease Category
Category

Finding Normal Group I Non- Group II Group III Group IV

inflammatory Inflammatory Infectious Hemorrhagic

Clarity Transparent Transparent Transparent/ Opaque Opaque

opaque
Color Clear to Xanthochromic Xanthochromic White Red-brown or
pale yellow to white/bloody xanthochromic
WBCs/mL 0150 < 3000 300075 000 50 000 5010 000
200 000
PMNs (%) < 25 < 30 > 50 > 90 < 50

RBCs No No No Yes Yes

Glucose 010 (0 010 (00.56 040 (02.2 20100 020 (01.11

(blood/SF 0.56 mmol/L) mmol/L) (1.115.5 mmol/L)
difference mmol/L) mmol/L)
mg/dL)
Specimen Collection
Joint fluid aspiration (arthrocentesis) should be confined
to patients with an undiagnosed effusion

It should be performed by an experienced operator

using good sterile technique.

Large joints (knee) normally contain< 4.0 mL of synovia

small sample size is common unless effusion is
present.
Synovial Fluid Analysis
Routine tests
Gross examination (color, clarity)
Total and differential leukocyte counts
Gram's stain and bacterial culture (aerobic and anaerobic)
Crystal examination with polarizing microscope

Useful tests in certain circumstances

Fungal and acid-fast stains and cultures
PCR for bacterial and mycobacterial DNA
Serumsynovial fluid glucose differential
Lactate and other organic acids
Complement
Enzymes
Uric acid
Recommended Tests
Major importance to differentiate crystal-induced joint
disease from infectious arthritis.
When either disease is suspected perform:
Arthrocentesis
systematic examination of the synovial fluid
Examination diagnostic if performed correctly
In other joint diseases a specific diagnosis may not be
possible

Note: Fluid examination is important if only to rule out

infectious arthritis, which is a critical diagnosis to make
as a joint may be irreversibly damaged within 48 hours if
not properly treated
Gross Examination of Synovial Fluid

1. Total volume
Recorded at bedside by professional collecting fluid

At least 5 mL of specimen needed for testing

Volumes generally > than CSF and ascitic fluid

collections
Gross Examination of Synovial Fluid
2. Color
Should be evaluated in a clear glass tube against a
white background.

Interpretation:
Normal SF is colorless but is often pale yellow.
Non inflammatory and inflammatory disorders are
usually straw- to yellow-colored (xanthochromia).
Septic fluid may be yellow, brown, or green
Gross Examination of Synovial Fluid

3. Traumatic tap
produces an uneven distribution of blood during
arthrocentesis or streaking in the syringe
pale yellow xanthochromia is difficult to
distinguish from normal, a red-brown color
following centrifugation is good evidence of
pathologic hemarthrosis.
Gross Examination of Synovial Fluid
4. Clarity
relates to number and type of particles within the
synovia.

Normal SF is transparent (newsprint is easily read

through the tube)
Translucent fluid obscures details,
black and white areas can be distinguished
opaque fluid completely obscures the background.
Increased turbidity is less often due to concentrations of
fibrin, free-floating rice bodies
Microscopic Examination

Total Cell Count

Should be done within 1 hour following
arthrocentesis to avoid degenerative cell loss.
Cell counts are usually performed in a standard
hemocytometer and Automated cell counters**
Microscopic Examination, contd
A wet-prep slide count of 0-2 WBCs /HPF (averaged
over 10 fields) predicts less than 1300 WBCs by cell
count
Leukocyte counts > 10 000/L, and often > 50 000/L,
are characteristic of:
o crystal-induced arthritis(e.g., gout, pseudogout)
o chronic inflammatory arthritis (e.g., rheumatoid arthritis
o systemic lupus erythematosus
o ankylosing spondylitis, and others)
o septic arthritis
Microscopic Examination, contd

Leukocyte counts over 50 000/L require dilution, which

should be done with saline, not acetic acid, to avoid
mucin clot formation and cell clumping.
Highly viscous synovial fluid should be incubated with
hyaluronidase before counting, especially if automated
counters are used.
Osteoarthritis, osteochondritis dissicans, trauma, and
synovioma usually have total WBC counts less than 10
000/L.
Microscopic Examination, contd

The upper reference level for SF leukocytes is 150-

200/L .
Elevated cell counts are used to help divide findings into
different disease categories, but are nonspecific for any
particular disease because of extensive overlap
Microscopic Examination, contd

Notes:
Erythrocytes should be routinely counted unless
it is an obvious traumatic tap.
If a large number of red cells interferes with the
leukocyte count, they may be lysed by dilution
with 0.3 normal saline or 1% saponin in saline
If there is the appearance of the fluid as bloody,
count it because it indicates some infectious
diseases.
Differential Leukocyte Count.

Differential Leukocyte Count.

Cytospin preparations are preferred over smears from
centrifuged SF because the cell morphology is
significantly better.
Treatment with hyaluronidase may be necessary to
produce thin smears in viscous specimens.
Neutrophils normally account for about 20% of SF
leukocytes.
Neutrophils generally exceed 50% in urate gout,
pseudogout, and rheumatoid arthritis (RA); they most
often exceed 75% in acute bacterial arthritis.
Differential Leukocyte Count.

These cells frequently exhibit degenerative changes and

may contain bacteria, crystals, lipid droplets, vacuoles,
or dark blue to black granular inclusions (ragocytes, RA
cells) which are similar to toxic granulation occasionally
seen in peripheral blood smears.
The presence of ragocytes in patients with RA may
indicate a poorer outcome.
Differential Leukocyte Count.

LE (lupus erythematosus) cells

commonly present in patients with lupus arthritis,
are most often neutrophils which have
phagocytosed the nuclei of degenerating cells (
Fig. 28-10 ). (see next slide)
However, LE cells are not pathognomonic for
systemic lupus erythrematosus since they have
also been identified in the synovial fluid of RA
patients
Figure 28-10 LE cell in the synovial fluid from a patient with
systemic lupus erythematosus
Differential Leukocyte Count.

Lymphocytes
normally constituting about 15% of the SF cells, are
prominent in early RA and other collagen disorders, and
chronic infections.
Reactive forms, including immunoblasts, are also
occasionally present.

Monocytes and macrophages

most common cells present in normal SF, accounting
for approximately 65% of the cell count.
Reiter's cells, originally believed to be specific for
Reiter's syndrome, are macrophages containing
degenerating neutrophils
Differential Leukocyte Count.

Eosinophilia
over 2% of the leukocyte count, has been reported in
rheumatoid arthritis, rheumatic fever, metastatic
carcinoma, Lyme disease, parasitic infections, chronic
urticaria, angioedema, following arthrography (allergic
reaction to dye), and irradiation
 Crystal Examination.

Crystals formed from crystallization of solutes like

gout due to accumulation of urate crystals
Crystals in synovial fluid lead to acute inflammation
with increased WBC counts and a neutrophils-
predominant infiltrate. Crystal identification, especially
if intracellularly in neutrophils or macrophages, is
pathognomonic for a crystal-induced arthritis
Crystal Examination contd

Gout : the process of crystal deposition in articular

tissue.
most common types of endogenous crystals responsible
for gouty arthritis are monosodium urate monohydrate
(urate gout), calcium pyrophosphate dihydrate
(pyrophosphate gout, chondrocalcinosis, or
pseudogout), apatite and other basic calcium
phosphates (BCP; apatite gout), calcium oxalate (oxalate
gout), and lipids (lipid gout).
Crystal Examination contd

Initial examination should be performed on a wet

preparation using polarized light.
Phase-contrast microscopy enhances crystal detection.
Most crystals are scanned with a 10 objective
evaluated with at least a 40 objective, concentrating
especially on cellular areas.
Complete examination requires 100 oil immersion,
however, because apparently negative fluids on
scanning may contain a large population of small
crystals.
Crystal Examination contd?

The *Diff Quik staining method may be a reliable

alternative to polarized microscopy.
The overall specificity, sensitivity, and accuracy were
respectively 87.5%, 94.4%, and 91.9%.
Crystal Examination contd
Monosodium urate (MSU) crystals appear as needle-
shaped rods 5-20 m long, but may be only 12 m in
length or, rarely, appear as rounded spherolites.
They are strongly birefringent ( Fig. 28-12 ): yellow
when oriented parallel to the compensator, blue with
perpendicular orientation (negative birefringence or
elongation) ( Fig. 28-13 ).
Quality Control:
A control slide of MSU crystals should always be used
for comparison.
Alternatively, betamethasone, a steroid that appears
as a strongly negative birefringent rod, can be used to
prepare a reference slide for the polarizing microscope
Figure 28-12 Monosodium urate crystals under
polarized light from a patient with urate gout.
 Figure 28-13 Monosodium urate crystals in synovial fluid.
Compensated polarized light.
Crystal Examination contd

Calcium pyrophosphate dihydrate (CPPD) crystals are

found in a group of conditions collectively known as
CPPD crystal deposition disease.
 Figure 28-15 Cholesterol crystals in synovial fluid.
Polarized light
limitations
Interfering factors in crystal identification:
Glove powder
introduced during joint surgery appears as round,
strongly birefringent particles 5-30 m in diameter
with a Maltese cross appearance when polarized

Other crystals or particulates

monoclonal immunoglobulin crystals or cryoglobulins,
CharcotLeyden crystals
amyloid, cartilage, and prosthetic fragments
collagen fibrils and fibrin strands
hematoidin crystals from prior hemorrhage
crystals from certain anticoagulants
nail polish
dust particles
Chemical Analysis

offers only supportive information to the routine

tests .
High viscosity may be remedied by dilution with
normal saline, sonication, or hyaluronidase
treatment
 Reference Ranges for SF Constituents
Total protein 13 g/dL 68 g/dL
Albumin 5570% 5065%
Alpha-1-globulin 68% 35%
Alpha-2-globulin 57% 713%
Beta-globulin 810% 814%
Gamma-globulin 1014% 1222%
Hyaluronic acid 0.30.4 g/dL
Glucose 70110 mg/dL 70110 mg/dL
Uric acid 28 mg/dL 28 mg/dL
Lactate 929 mg/dL 929 mg/dL
Chemical analysis

Mucin Clot Test.

the mucin clot test has minimal clinical utility
Glucose.
Proper interpretation of SF glucose values requires
comparison with serum levels,
ideally preceded by a fast of 8 hours to allow glucose to
equilibrate across the synovial membrane.
The serumsynovia differential is less than 10 mg/dL in
normal and many non inflammatory conditions.
In septic arthritis, this difference ranges from 20-60
mg/dL, but overlaps significantly with other inflammatory
conditions, thereby limiting its clinical usefulness.
Chemical Analysis contd

Protein.
Reference interval is 1.0-3.0 g/dL.
With increasing inflammation, larger proteins (e.g.,
fibrinogen) enter the synovial space.
Spontaneous clot formation may be detected in non-
anticoagulated specimen tubes (fibrin clot test
Measurement of SF protein is very nonspecific
The total protein level is not generally useful in patient
diagnosis, treatment, or outcome.
Chemical Analysis contd

Enzymes.
enzymes have been studied in SF,
lactate dehydrogenase,
aspartate aminotransferase,
adenosine deaminase,
acid and alkaline phosphatase,
and lysozyme among others
currently not clinically relevant, the measurement of
various hydrolases may have significant predictive
value in joint prognosis, especially RA.
Chemical Analysis contd

Organic Acids.
SF lactic acid levels are usually increased in patients
with septic arthritis .
Levels >30 mg/dL are commonly associated with
septic arthritis due to Gram-positive cocci and Gram-
negative bacilli.
Using gasliquid chromatography, the presence of
other organic acids not normally present in SF may
be very helpful in differentiating septic from non septic
arthritis
Chemical Analysis contd
Uric Acid.
SF uric acid levels generally parallel serum levels in
gout and noninflammatory arthropathies.
Exception is inflammatory joint disorders other than
gout, where SF urate levels may be significantly lower
than in the paired serum.
Lipids
normal synovial fluid contains extremely low
concentrations of lipids
quantification of lipids currently has no clinical value
in joint fluid analysis except in cases where
cholesterol crystals may resemble MSU or CPPD
Immunologic studies
Immunologic Studies
Rheumatoid factor (RF) is found in synovia of about 60%
of RA patients, usually at a titer equal to or slightly lower
than the serum titer.
Antinuclear antibodies (ANA) are found in the SF of
about 70% of patients with SLE and 20% of patients with
RA.
Neither is specific enough for practical use. SF
complement levels, normally about 10% of serum levels,
increase to 40-70% of serum activity with inflammation,
proportional to the increase of protein exudation.
 Microbiological Examination
Immediate transportation of joint fluid are extremely important
in the rapid identification of an infectious agent
Septic arthritis may be acute or chronic
Gram stain and culture should be performed as part of the
routine synovial fluid evaluation.
Gram stain sensitivity varies from about 75% for staphylococcal
infections, 50% for most Gram-negative organisms, to < 25%
for gonococcal (GC) infections .
Microbiological Examination contd

Culture sensitivity :
75-95% for non gonococcal joint infections in patients
who have not received antibiotics.
For patients with gonorrhea, the sensitivity is only 10-
50%.
PCR with universal primers to detect bacterial DNA for
the more fastidious, uncultivable pathogens like:
Borrelia burgdorferi
Chlamydia s
Mycoplasma sp.
 Microbiological Examination contd

Arthritis develops in approximately 60% of patients with Lyme

disease resulting from exposure to ticks infected with Borrelia
burgdorferi.
In patients with outdoor occupations, synovial fluid/tissue
should be examined for fungal pathogens by KOH/calcofluor
white stain
and cultured on selective fungal media.
For example
a patient with a recent travel history to Arizona may present
with a monoarticular arthritis secondary to Coccidioides
immitis.
Patients with a chronic arthritis and risk factors for
Mycobacterium tuberculosis or nontuberculous infections
should undergo a synovial biopsy.
Microbiological Examination contd

ZiehlNeelsen or Kinyoun stains for acid-fast organisms

have a sensitivity of about 20%.
Cultures for M. tuberculosis are positive in about 80% of
proven cases..
Synovial biopsy is recommended for suspected
tuberculous arthritis to provide a more rapid diagnosis.
Synovial Testing Limitations
It is critical that tests be performed accurately to make
specific diagnostic information.

Major problem in the laboratory examination of

synovial fluid is there is no consensus as to what
constitutes a routine analysis

Quality performance is not consistent, due to the fact

that the average laboratory examines only one to two
synovial fluids each month
 Pleural Fluid
The pleural cavity normally contains a small amount of fluid that
facilitates movement of the visceral and parietal pleura each
other.
A plasma filtrate derived from capillaries of the parietal pleura.
Produced continuously at a rate dependent on capillary
hydrostatic pressure, plasma oncotic pressure, and capillary
permeability.
Reabsorbed through the lymphatic and venules of the visceral
pleura.
Pleural Fluid contd

Effusion: an accumulation of fluid, which results from an

imbalance of fluid production and reabsorption.
Serous effusion: fluid accumulation in the pleural,
pericardial, and peritoneal cavities
Pleural Fluid contd
Specimen Collection
The physician is expected to draw approprate
amount of sample from the patient
Should be labeled with all information like above
one
Should be handled appropriately during delivery to
the laboratory
Avoid in appropriate testing
Except for an EDTA tube for total and differential
cells counts, the specimen should be collected in
heparinized tubes to avoid clotting
Specimen Collection contd
Aliquots for aerobic and anaerobic bacterial cultures are best
inoculated into blood culture media at the bedside.
If malignancy, fungal infection, or mycobacterial infection is
suspected, all remaining fluid (100 mL or more) should be
submitted to maximize yield of stains and culture.
fresh specimens for cytology may be stored up to 48 hours
in the refrigerator with satisfactory results
initial classification of a pleural fluid as a transudate or an
exudate greatly simplifies the process of arriving at a correct
final diagnosis
Transudates and Exudates contd.

Transudates:
are usually bilateral owing to systemic conditions
leading to increased capillary hydrostatic pressure or
decreased plasma oncotic pressure
Non-inflammatory condition
No need further investigation
Transudates:cont

increased hydrostatic pressure or decreased

plasma oncotic pressure
Congestive heart failure
Hepatic cirrhosis
Hypoproteinemia (e.g., nephritic syndrome)
Exudates:

Exudates are more often unilateral, associated with

localized disorders that increase vascular
permeability or interfere with lymphatic resorption
Because of any inflammatory condition
 Exudates: cont
Exudates: increased capillary permeability or decreased
lymphatic resorption
Infections
Bacterial pneumonia
Tuberculosis
granulomatous diseases (e.g., sarcoidosis,
histoplasmosis, etc.)
Viral or mycoplasma pneumonia
Neoplasms
Bronchogenic carcinoma
Lymphoma
Pulmonary infarct (may be associated with hemorrhagic
effusion)
Pleural fluid/serum protein 0.50
ratio
Pleural fluid/serum LD ratio 0.60

Pleural fluid LD upper limit of normal

serum LD
Pleural fluid cholesterol > 45 mg/dL

Pleural fluid/serum 0.30

cholesterol ratio
Serumpleural fluid albumin 1.2 g/dL
gradient
Pleural fluid/serum bilirubin 0.60
ratio
Transudates and Exudates contd

Classical teaching stressed that exudates and

transudates can be distinguished on the basis of total
protein concentrations above (exudates) or below
(transudates) 3.0 g/dL.
However, using total protein alone misclassifies both
exudates and transudates by about 30%
Recommended Tests

The evaluation of serous body fluids (pleural,

pericardial, peritoneal) is directed first toward
differentiating transudative from exudative
effusions.
Gross Examination

Transudates
are typically clear, pale yellow to straw-colored,
odorless, and do not clot.
Approximately 15% of transudates are blood tinged.
A bloody pleural effusion (hematocrit > 1%) suggests
trauma, malignancy, or pulmonary infarction
A traumatic tap is suggested by uneven blood
distribution, fluid clearing with continued aspiration, or
formation of small blood clots.
 Exudates
may grossly resemble transudates, but most show
variable degrees of cloudiness or turbidity, and often clot
if not heparinized.
A fecalent odor may be detected in anaerobic infections.
Turbid, milky, and/or bloody specimens should be
centrifuged and the supernatant examined.
If the supernatant is clear, the turbidity is most likely due to
cellular elements or debris.
If the turbidity persists after centrifugation, a chylous or
pseudochylous effusion is likely
Characteristic Features of Chylous and
Pseudochylous Effusions
Feature Chylous Pseudochylous

Onset Sudden Gradual

Appearance Milky-white, or Milky or greenish,

yellow to bloody metallic sheen
Microscopic Lymphocytosis Mixed cellular reaction,
examination cholesterol crystals
Triglycerides[*][ 110 mg/dL ( < 50 mg/dL (< 0.56
] 1.24 mmol/L) mol/L)
Lipoprotein Chylomicrons Chylomicrons absent
electrophoresis present
Microscopic Examination
Cell Counts.
Leukocyte counts are unreliable in separating
transudates (< 1000/L) from exudates (> 1000/L).
Although red cell counts above 100 000/L are highly
suggestive of malignancy, trauma, or pulmonary
infarction, they have little practical value.
Differential Leukocyte Count and Cytology.
Examination on a stained smear, prepared by
cytocentrifugation and an air-dried Romanowski's
stain.
Indeed, examination by the hematology laboratory can
be highly effective in the detection of malignant cells
 Mesothelial cells
are common in
pleural fluids from
inflammatory
processes
well -differentiated breast carcinoma cells in pleural fluid
 Undifferentiated oat cell carcinoma of lung
showing typical molding of nuclei
 Neutrophilia
(> 50%)Bacterial pneumonia (Para pneumonic
effusion)
Pulmonary infarction
Pancreatitis
Subphrenic abscess
Early tuberculosis
Transudates (over 10%)
 Lymphocytosis
(> 50%)Tuberculosis (mesothelial cells are rare)
Viral infection
Malignancy
True chylothorax
Rheumatoid pleuritis
Systemic lupus erythematosus
Uremic effusions
Transudates (approximately 30%)
 Eosinophilia (> 10%)Pneumothorax (air in pleural space)

Trauma
Pulmonary infarction
Congestive heart failure
Infection (especially parasitic, fungal)
Hypersensitivity syndromes
Drug reaction
Rheumatologic diseases
Hodgkin's disease
Idiopathic
Chemical Analysis

Protein.
The measurement of pleural fluid total protein or
albumin has little clinical value except when combined
with other parameters to differentiate exudates from
transudates.
Protein electrophoresis shows a pattern similar to
serum except for a higher proportion of albumin; it
has little value for differential diagnosis
 Glucose.
The glucose level of normal pleural fluid, transudates,
and most exudates is similar to serum levels.
Decreased pleural fluid glucose, accepted as a level
below 60 mg/dL (3.33 mmol/L) or a pleural
fluid/serum glucose ratio less than 0.5, is most
consistent and dramatic in rheumatoid pleuritis and
grossly purulent parapneumonic exudates ( Sahn,
1982 ). Low pleural fluid glucose may also be present
in malignancy, tuberculosis, nonpurulent bacterial
infections, lupus pleuritis, and esophageal rupture.
 Lactate.
Pleural fluid lactate levels can be a useful adjunct in
the rapid diagnosis of infectious pleuritis. Levels are
significantly higher in bacterial and tuberculous
pleural infections than in other pleural effusions.
Moderate elevations are generally observed in
malignant effusions ( Brook, 1980 ). Values greater
than 90 mg/dL (10 mmol/L) have a positive predictive
value for infectious pleuritis of 94% and a negative
predictive value of 100% ( Gastrin,
Enzymatic tests

Amylase elevations
above the serum level (usually 1.5-2.0 or more times greater)
indicate the presence of pancreatitis, esophageal rupture, or
malignant effusion
Elevated amylase derived from esophageal rupture or malignancy
is the salivary isoform, which differentiates it from pancreatic
amylase
Pleural fluid lactate dehydrogenase (LD)
levels rise in proportion to the degree of inflammation.
In addition to its use in separating exudates from transudates,
declining LD levels during the course of an effusion indicate that the
inflammatory process is resolving.
 Interferon-gamma (INF-gamma).
Pleural fluid INF-gamma levels are significantly
increased in pleural fluid of patients with tuberculous
pleuritis.
The sensitivity of levels 3.7 IU/L or greater is 99% and
the specificity is 98%.
The test sensitivity does not differ in HIV-positive and
HIV-negative patients. Only about 20% of patients with
effusions due to hematologic malignancies have INF-
gamma levels slightly above 3.7 IU/L
 pH.
Pleural fluid pH measurement has the highest diagnostic
accuracy in assessing the prognosis of parapneumonic
(pneumonia-related) effusions.
A parapneumonic exudate with a pH greater than 7.30
generally resolves with medical therapy alone.
A pH less than 7.20 indicates a complicated
parapneumonic effusion (loculated or associated with
empyema) requiring surgical drainage.
Immunologic Studies

Approximately 5% of patients with rheumatoid arthritis

(RA) and 50% with systemic lupus erythematosus (SLE)
develop pleural effusions sometime during the course of
their disease.
Rheumatoid factor (RF) is commonly present in pleural
effusions associated with seropositive RA.
Although a pleural fluid titer of 1:320 or greater in a
patient with known RA is reasonable evidence of
rheumatic pleuritis
Antinuclear antibody (ANA) titers may be useful in the
diagnosis of effusions due to lupus pleuritis
Microbiological Examination

Bacteria most commonly associated with parapneumonic

effusions are
Staphylococcus aureus,
Streptococcus pneumoniae,
beta-hemolytic group A streptococci
gamma-streptococci
some Gram-negative bacilli.
patients with suspected M. tuberculosis, direct
staining of tuberculous effusions for acid-fast bacteria
has a sensitivity of 20-30 %
Pericardial Fluid
From 10-50 mL of fluid is normally present in the
pericardial space
produced by a transudative process similar to pleural
fluid.
Pericardial effusion caused by :
viral infection
enterovirus being the most common.
bacterial, tuberculous or fungal infections
autoimmune disorders, renal failure, myocardial
infarction.
Many of the recommended laboratory tests described for
pleural fluid also pertain to pericardial effusions
Pericardial Fluid contd

Specimen Collection
Performed by experienced professional
should be delivered within 1 hours if delay is mandatory the
approprate preservative for the desired test should be added
as soon as it reached the lab gross examination should be
performed
Pericardial Fluid contd

Gross Examination
Normal pericardial fluid : pale yellow and clear.
Large effusions (> 350 mL) are most often caused by
malignancy or uremia, or are idiopathic.
Turbid effusions :Infection or malignancy clear and
straw-colored: effusions due to uremia
 Microscopic Examination
The hematocrit and red cell count document the
presence of a hemorrhagic effusion
Total leukocyte counts over 10 000/L suggest
bacterial, tuberculous, or malignant pericarditis.
Although formal leukocyte differentials add little
diagnostic information, a stained smear should
always be examined.
Chemical Analysis

Protein.
A value greater than 3.0 g/dL has a sensitivity of 97%
for exudative effusions
Glucose.
Pericardial glucose levels less than 60 mg/dL have a
diagnostic accuracy of only 36% in identifying
pericardial exudates
pH.
Pericardial fluid pH may be markedly decreased
(< 7.10) in rheumatic or purulent pericarditis
Microbiological Examination

The sensitivity of the Gram stain and culture for bacterial

pericarditis is similar to other serous body fluids
Diagnosis of a specific etiologic agent in viral pericarditis
is difficult because the viruses (e.g., Coxsackieviruses,
influenza virus, mumps) are rarely isolated from
pericardial fluid.
Obtaining acute and convalescent sera for antibody
response to suspected viral pathogens may help support
the diagnosis.
The sensitivity of acid-fast stains and culture for
tuberculous pericarditis is about 50%
PCR is a sensitive technique
Peritoneal Fluid
Ascites is the pathologic accumulation of excess fluid in the
peritoneal cavity.
Up to 50 mL of fluid is normally present in this mesothelial-
lined space.
As with pleural and pericardial fluids, it is produced as an
ultrafiltrate of plasma dependent on vascular permeability,
and hydrostatic and oncotic Starling forces.
Peritoneal Fluid contd

Specimen Collection
Done by clilinicians
Collected in in sterile test tube
Delivered to the lab within 1 hour
The test performed in appropriate technique
Sufficient sample is needed( A minimum of 30 m)
Sample of cell count should be placed in an EDTA-
anticoagulated venipuncture tube.
Culture specimens should include blood culture bottles
that have been inoculated at the bedside with ascetic fluid
(10 mL per culture bottle).
Recommended Tests in Peritoneal Effusion
Useful in most patients
Gross examination
Cytology
Stains and culture for microorganisms
Serumascites albumin concentration gradient
Useful in selected disorders
Total leukocyte and differential cell counts
RBC count (lavage)
Bilirubin
Creatinine/urea nitrogen
Enzymes (ADA, ALP, amylase, LD, telomerase)
Lactate
Cholesterol (malignant ascites)
 Peritoneal Fluid contd

Gross Examination
Milkyfluid that does not clear with centrifugation suggests a
chylous or pseudochylous effusion.
True chylous peritoneal effusions are significantly less
common than chylous pleural fluids.
Caused by disruption or blockage of lymphatic flow by
trauma, lymphoma, carcinoma, tuberculosis or other
granulomatous diseases , hepatic cirrhosis, adhesions, or
parasitic infestation.
Peritoneal Fluid contd

Microscopic Examination
The total leukocyte count is useful in distinguishing
ascites due to uncomplicated cirrhosis from spontaneous
bacterial peritonitis (SBP), which is caused by migration
of bacteria from the intestine into the ascetic fluid.
Approximately 90% of patients with SBP will have
leukocyte counts greater than 500/L, over 50% of which
are neutrophils
Neutrophils in a patient with bacterial peritonitis
Peritoneal Fluid contd

Eosinophilia
most commonly > 10% associated with the chronic
inflammatory process with chronic peritoneal dialysis.
it is also reported in congestive heart failure,
vasculitis, lymphoma, and ruptured hydatid cyst.
Peritoneal Fluid contd

Chemical Analysis
Protein.
Spontaneous bacterial peritonitis is commonly associated with
low total protein (< 3.0 g/dL) and a high serumascites albumin
gradient (> 1.1 g/dL), making total protein measurements of little
value in this disorder.
Glucose.
peritoneal fluid glucose levels of 50 mg/dL or less are present in
30-60% of cases of tuberculous peritonitis and about 50% of
patients with abdominal carcinomatosis
Peritoneal Fluid contd
Microbiological Examination
The bacteria in SBP are most often normal intestinal
flora and over 92% are monomicrobial.
The Gram stain has a sensitivity of 25% in SBP and
routine cultures are positive in only about 50% of cases
Ascetic fluid total neutrophils count is the preferred
method for the diagnosis of SBP
Amniotic Fluid

Amniotic fluid: liquid that surrounds the fetus in the

amniotic cavity
The primary function is to provide protective cushion for the
fetus, allow for movement, and regulate temperature.
Amniocentesis is the fluid collection procedure in
which a sample of the amniotic fluid surrounding a fetus
is removed by means of a fine needle inserted through
the abdomen and into the uterus of the pregnant woman.
 Amniotic Fluid contd
Sample collection
Collected by experienced professional
A maximum of 30mL of fluid can be collected.
The first 2 or 3 mL should be discarded because they
may contain maternal blood, tissue fluid, and maternal
cells.
Sterile plastic syringes and conical centrifuge tubes
should be used for the collection and transportation of
the amniotic fluid.
Specimens for cytogenetic studies are maintained at
25 - 37 0C incubation prior to analysis to prolong the
life of the cells needed for analysis.
Do not freeze, refrigerate, or centrifuge.
Amniotic Fluid specimen
Specimens for fetal lung maturity tests should be
placed in ice for delivery to the laboratory and
refrigerated prior to testing.
The specimen will be centrifuged or filtered before
analysis.
Specimens for bilirubin analysis in cases of Hemolytic
Disease of the Newborn, must be protected from light.
The sample should be collected in an amber-colored
tube.
The appearance of the amniotic fluid can indicate
presence of certain chemicals. For example, degree
of redness indicates presence of hemoglobin.
Gross examination of amniotic fluids color

COLOR SIGNIFICANCE
Colorless Normal
Blood-streaked Traumatic tap, abdominal
trauma, intra-amniotic
hemorrhage
Yellow Hemolytic Disease of the
Newborn (HDN), Bilirubin
Dark green Meconium (first bowel
movement)
Dark red-brown Fetal Death
Amniotic Fluid testing

Laboratory testing of amniotic fluid involves analysis of

bilirubin, alpha-fetoprotein and a variety of tests for fetal
lung maturity (FLM).
During the third trimester of pregnancy but less than 35 to
36 weeks gestation, fluid collected from the
amniocentesis procedure is analyzed to evaluate fetal
lung maturity.
Limitations:
Contamination of the amniotic fluid specimen by blood
or meconium invalidates the FLM results.
Amniotic Fluid testing

Bilirubin Scan in Amniotic Fluid

Hemolyis and bilirubin assessed by optical density of
amniotic fluid at 450 nm
Change in Absorbance due to bilirubin
Clinical significance
evaluate fetal hemolysis in hemolytic disease of
newborn
Summary for Amniotic Fluid

Mother and unborn child testing

Heath care provide makes decisions about care and
treatment
Assess chemical changes in mother and fetus
Understanding the stage of the fetus
Nasal smear analysis
Exercises

1.What may cause yellow color in amniotic fluids

2. Mention the limitation of chemical tests in amniotic fluid
for specimen transport
3. Describe the principle of bilirubin test in amniotic fluid
4. Describe the clinical significance of amniotic fluid tests