Phlebotomy Preanalytic Variables

COURSE DESCRIPTION
Errors in laboratory testing can result from issues occurring before the testing process
(preanalytic), during the testing process (analytic), and after the testing process
(postanalytic). All healthcare professionals responsible for blood specimen collection must
be knowledgeable about preanalytic causes of error; i.e., variables, as many of them can
occur as a result of patient identification, specimen collection, and specimen transport. If a
quality patient specimen is not collected, the test results on that specimen may be useless,
even if the analytic and postanalytic processes are 100% free of error. This CE course
identifies and discusses the most common causes of preanalytic error.
*Valid for P.A.C.E.® credit through 04/30/2024

* ASCLS P.A.C.E.® is an approved continuing education agency by the California
Department of Health Laboratory Field Services, Accrediting Agency #0001.
*NCCT is approved as a provider of continuing education programs in the clinical

laboratory sciences by the ASCLS P.A.C.E.® Program, provider #122.
Rev 4
Updated February 2022
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COURSE TITLE: Phlebotomy Preanalytic Variables
Author: Sheryl Whitlock, MA, MT(ASCP)BB
Laboratory Coordinator, Student Health Service
University of Delaware
Newark, DE
Editors: Lucia Johnson, MEd, MT(ASCP)SBB
Tami Maffitt, MLS(ASCP)CM
National Center for Competency Testing
Number of Clock Hours Credit: 2.0
Course # 1220622
Level of instruction: Intermediate
P.A.C.E.® Approved: _X_ Yes __ _ No
Objectives
Upon completion of the continuing education course, the healthcare professional will be able to:
1. Identify sources of preanalytic, analytic, and postanalytic error.

2. List preanalytic causes of error outside the control of phlebotomists.
3. Identify methods to identify inpatient, hospital outpatients, and physician office patients.
4. Identify unique patient identifiers acceptable for use in patient identification.
5. Identify locations of veins in the antecubital fossa in both the H and M arrangements.
6. Describe the veins recommended for venipuncture use in the antecubital fossa, and the
vein(s) that should be used only after no other sites are available or with physician
permission.
7. Describe patient safety issues that may arise when the basilic vein is used for venipuncture.
8. Identify sites for skin puncture collection and describe why these sites are used.
9. Identify consequences of performing venipuncture on an arm ipsilateral to lymph node
removal or dissection.
10. Describe the process for venipuncture on a site distal and proximal to IV fluid infusion.
11. Identify errors that may occur due to underfilling and overfilling evacuated tubes.
12. List the order of draw/collection for venipuncture and skin puncture, and describe the
rationale for the order.
13. Describe methods to prevent the formation of micro-clots in blood specimens.
14. Identify the correct way to position labels on evacuated tubes.
15. List tests that require chilling, maintenance at 37° C, and protection from light after
collection and during transportation.
16. Identify consequences of not allowing skin cleansing preparations to dry prior to blood
collection.
17. List causes of hemolysis and identify issues hemolysis presents with laboratory analysis.
18. Describe the importance of patient identification for collection of blood for blood bank
testing.
19. Describe the importance of collecting the correct quantity of blood for PT and aPTT testing.
20. Identify the purposes of employee training, performance approval, and competency
assessment.
Disclaimer
The writers for NCCT continuing education courses attempt to provide factual information based on literature review and
current professional practice. However, NCCT does not guarantee that the information contained in the continuing
education courses is free from all errors and omissions.
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INTRODUCTION TO THE LABORATORY PROCESS
Note: The use of the term ‘phlebotomist’ in this course refers to all qualified healthcare professionals who
collect blood specimens including but not limited to medical assistants, patient care technicians, and
phlebotomy technicians.
Laboratory science is a complex specialty and a medical laboratory has multiple

departments or sections. Each department has a test menu and each test has specific
specimen requirements. The quality of the specimen collected for laboratory testing is
directly proportional to the quality of the tests results provided for diagnosis and treatment
of patient illnesses. To be useful, patient tests results must be accurate and reliable,
reflecting the actual status of the patient at the time the specimen was collected.
The phlebotomist is the first step in the production of an accurate and quality test result.
The physician depends on accurate test results for correct management of all patients. In
each instance, the absence of a quality specimen may alter treatment steps and change
the possibility of a positive outcome for the patient.
Correct patient and specimen identification are included in patient safety goals. The safety
of a patient is not only physical safety (such as fall prevention), but also the safe and timely
delivery of proper patient care. If a patient or a specimen is not correctly identified, this
impacts safe patient treatment and a safe patient outcome.
As implied in this process, the phlebotomist is the strong base on which the testing
process rests. Accurate and reliable laboratory test results are dependent on the
collection of the blood specimen.
PROCESS APPROACH
In any industry, there is a process that produces an endpoint. All healthcare procedures
are composed of a series of processes and therefore fall under this umbrella. Phlebotomy
and specimen collection are procedures composed of a series of steps. Each procedural
step is a process with an endpoint. A successful endpoint to each step is required to
proceed to the next procedural step. The entire testing process may also be labeled as
the "total testing process" or TTP.
The process approach may be visualized below:
INPUT  PROCESS  OUTPUT
Laboratory testing utilizes the process approach with different labels. The process
approach for a laboratory test is visualized below:
PREANALYTIC  ANALYTIC  POSTANALYTIC
The preanalytic phase consists of all processes that occur before the specimen is
analyzed; i.e., tested. Phlebotomy is a part of the preanalytic phase of laboratory testing.
Therefore, phlebotomists have a very important role in the preanalytic process of
laboratory testing.
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The analytic phase is the actual testing phase. Some laboratory tests are performed
manually, others are performed by automated instruments. Quality control of reagents,
calibration of instruments, and competency assessment of testing personnel are performed
to minimize analytic causes of error.
The postanalytic phase consists of the steps that occur after the testing is completed. This
includes the steps involved with taking test results from worksheets, instrument printouts,
and computer interfaces and placing them in the medical record of the patient. This
process can be electronic or manual.
Healthcare management and accreditation organizations have examined the process

approach and developed corresponding alternative terminology for healthcare processes.
The terminology provided by International Organization for Standardization (ISO) is
outlined below the standard laboratory test process notations.
Standard Laboratory Process Terminology

ISO Process Terminology
PRE-EXAMINATION  EXAMINATION  POST-EXAMINATION
While the standardization of terminology has not occurred, the implied concepts are
identical. Many medical laboratories are adopting ISO standards and phlebotomy staff
should be aware of variation in terminology.
With the exception of some point of care testing (POCT), the phlebotomist is not involved
in the analytic or postanalytic processes. It is, however, important for a phlebotomist to
understand the analytic and postanalytic steps of the testing process. Factors and issues
that may result in errors in all phases of laboratory testing are called variables. Therefore,
there are preanalytic variables, analytic variables, and postanalytic variables. To assure
quality patient test results, policies and procedures must be followed to minimize variables
in all phases of testing.
A brief summary of the three phases of laboratory testing and the variables associated with
each is shown below.

 Test order  Testing variables are  Test reporting variables
variables minimized by o Recording of results
 Patient variables o Performance and o Reporting of results
 Specimen variables evaluation of quality o Interpretation of
o Collection control results
o Handling o Calibration of
o Transport instruments
o Processing o Competency
assessment
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THE IMPACT OF PREANALYTIC ERROR
There is no good data identifying how many laboratory tests are inaccurate due to
preanalytic, analytic, or postanalytic error. There are 7-10 billion laboratory tests
performed in the United States each year. Assuming each test represents one person,
even if the error rate is as little as 0.1% that means 7-10 million people are affected by
laboratory error of some sort. Therefore, a ‘zero tolerance’ approach must be applied to all
phases of laboratory testing in an effort to minimize errors.
Patients can be seriously harmed as a result of preanalytic error. All it takes is the
incorrect patient label being placed on a tube of blood or a patient identity not being
correctly confirmed to result in one of the following scenarios: patient death due to
incompatible blood transfusion, a diagnosis of cancer where no cancer is present, patient
hemorrhage from inaccurate laboratory coagulation test results, delay in treatment of
serious medical conditions, and more.
The preanalytic phase is labor intensive. The steps in each process are detailed. The
specific training for phlebotomy technicians is usually thorough. However, individuals who
are not phlebotomy technicians frequently perform specimen collection. Nurses,
physician's assistants, medical assistants, patient care technicians, and other
professionals involved in direct patient care collect specimens and forward them to the
laboratory for analysis. This fact contributes to the difficulty of orienting, training, and
maintaining competency of all staff that collect specimens.
PREANALYTIC VARIABLES PRIOR TO PHLEBOTOMIST

INVOLVEMENT
There are some preanalytic steps that occur prior to the phlebotomist’s involvement that
are usually outside of their control. However, they can impact the production of reliable
test results. Following are some of the preanalytic variables outside the control of
phlebotomists.
 Patient registration: The first step of proper patient identification takes place during
registration. Errors at this step can include incorrectly identifying the patient as another
patient within the database, incorrectly transcribing personal identifiers from the
patient’s ID card, or placement of an ID band onto the wrong patient.
 Specimen requirements: Specific collection requirements for all orderable tests should
be available to all personnel performing phlebotomy. The collection requirements for
each test may be in a written manual, electronic system, and/or may print with the
labels or written order. The specimen requirements should be revised and updated on
an ongoing basis. If proper updating does not occur, the phlebotomist may collect an
incorrect specimen.
 Laboratory test orders: Placing a test order begins a series of steps that end with a test
result for use in diagnosis and treatment. A physician (or other licensed practitioner)
orders the test in written or electronic form. In some healthcare facilities, test order
entry may be performed by phlebotomy technicians.
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Written orders have several potentials for error. Written orders may require
transcription by a clerk, administrative assistant, or nursing personnel. The
transcription may be onto a paper form or into an electronic system. Errors that may
occur at the ordering phase are summarized below.
o Physician orders incorrect test

o Order illegible
o Incorrect test transcribed into medical record
o Test omitted in the transcription
o Incorrect test chosen from a list (e.g. screening test vs. specific test)
o Requested test not included in the test dictionary
o Duplication of a test that is included in an ordered panel of tests
 Patient preparation: Patient preparation is important for collection of some specimens.

While this is not the responsibility of the phlebotomist, the phlebotomist can assist in
obtaining a quality test result by asking the patient certain questions that relate to the
accuracy of the test results, such as for glucose, lipids, peak/trough drug levels, and
other testing. Some preparation factors that may be important to specimen collection
include:
o Fasting or no ingestion of food or drink (except for water) for a specific period of
time
o Postprandial collection or collection following food ingestion with a specific time
of collection
o Prior to a medication dosage
o After a medication dosage and at a specific time frame
The constituents of blood are affected by diet, exercise, and other factors. Therefore,
the ideal time for collecting blood specimens is early in the morning while the body is
still at rest and fasting, or approximately 12 hours after the last intake of food. This
condition is known as the basal state. Physiologic factors affecting basal state follow.
o Age: Values for numerous blood components vary considerably with the age of
the patient. For example, red blood cell and white blood cell values are higher in
newborns than in adults. Some physiologic functions decrease with age in
adults. For example, creatinine clearance, a kidney function test, naturally
declines as a patient ages.
o Altitude: Decreased oxygen content of the air at higher altitudes causes the
body to produce more red blood cells to fulfill the body’s oxygen requirements.
The higher the altitude, the greater the increase in red cell production. Red
blood cell counts and related determinations such as hemoglobin and hematocrit
have higher reference ranges at higher elevations.
o Dehydration: Dehydration that occurs with persistent vomiting or diarrhea, for

example, causes hemoconcentration, which can falsely increase some blood
components such as red blood cell counts, enzymes, iron, calcium, and sodium.
o Diurnal variations: Many blood constituents show diurnal variation (normal

fluctuations) throughout the day. White blood cell counts, eosinophil counts, and
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iron levels are lower in the morning than in the afternoon. Cortisol levels are
highest in the morning.
o Diet: Blood composition is significantly altered by ingestion of food. Glucose

levels increase dramatically with the ingestion of food substances but should
return to normal within two hours. Ingestion of fatty substances increases lipid
content in the blood for anywhere from one to 10 hours or more. Some
chemistry determinations should not be performed on non-fasting specimens, as
the results will be inaccurate. Excessive fluid intake may cause decreased
hemoglobin levels and later, electrolyte imbalance. Consumption of caffeine has
been demonstrated to affect cortisol levels.
o Medications: Many prescription medications alter physiologic functions. For an

example, thiazide diuretics often cause increased calcium levels and may cause
low potassium levels. Other examples of drug alterations include the following.
 Chemotherapy drugs often lower white blood cell and platelet levels.
 Acetaminophen (Tylenol®) may affect liver and kidney tests.
 Steroids can cause pancreatitis and an increase in amylase and lipase.
Per recommendations from the College of American Pathologists, medications

known to interfere with blood tests should be stopped or avoided 4 to 24 hours
prior to obtaining the blood sample for testing.
o Environment: Temperature and humidity are known to affect test values.

Environmental factors associated with geographic location are accounted for
when the laboratory establishes reference values. The laboratory monitors
temperature and humidity to maintain specimen integrity.
o Exercise: Muscular activity, however moderate, will elevate the blood levels of a
number of blood components such as lactic acid, creatinine, protein, and certain
enzymes. Levels of these substances return to normal soon after the activity is
stopped with the exception of the enzymes creatine phosphokinase (CK) and
lactic dehydrogenase (LD), which may remain elevated 24 hours or more.
o Position: The position of a patient both before and during venipuncture

influences blood composition. Going from a supine to standing position causes
the water or plasma portion of the blood to filter into the tissues. This causes a
decrease in plasma volume and increase in nonfilterable elements or
substances such as proteins, iron, calcium, and blood cells.
o Pregnancy: Pregnancy causes physiologic changes in many body systems.

Because of these changes, a number of laboratory test results are affected. For
example, body fluid increases, which are normal during pregnancy, have a
diluting effect on the red blood cells, leading to lower red blood cell counts.
o Gender: A patient’s gender has an effect on the concentration of numerous

blood components. Most differences are apparent only after sexual maturity.
These differences are reflected in separate reference range values for male and
female patients. For example, red blood cells, hemoglobin, and hematocrit
reference ranges are higher in males.
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o Stress: Emotional stress in the form of fear or anxiety has been shown to cause
short-lived elevations in white blood cell counts, decreases in serum iron, and
increases in adrenal hormone values.
 Storage and shipping of phlebotomy items from the manufacturer to a facility:

Collection tubes should be stored and shipped within the temperature and
environmental requirements specified by the manufacturer. If the requirements are
exceeded, the stability of the additives and the vacuum in the tubes may be
compromised.
PREANALYTIC VARIABLES UNDER CONTROL OF PHLEBOTOMISTS
There are important specimen collection steps that are the responsibility of the
phlebotomist. Every healthcare professional that performs phlebotomy is accountable for
the quality of the specimen he/she obtains. It helps to understand the potential errors in
order to avoid the consequences. Some specific errors that may occur as a part of
phlebotomy are summarized below.
 Failure to confirm patient identification

 Screening for patient preparation
 Incorrect collection site chosen
 Extensive tourniquet application
 Improper skin cleansing
 Tissue damage from probing
 Improper collection containers used
 Damaged/outdated/nonsterile equipment used
 Incorrect order of draw/collection
 Incomplete filling of collection tubes
 Insufficient mixing of collection tubes
 Improper labeling of specimens
 Incorrect handling of specimens
 Delay in delivering specimens to the test site
Consequences of incorrectly obtained specimens include specimen rejection, the

necessity of recollection of specimens, unusable specimens, and specimens of insufficient
volume and quality that when tested provide inaccurate results. These consequences
impact on the correct diagnosis and treatment of the patient, impeding the goal of patient
safety.
PATIENT IDENTIFICATION
The phlebotomist is responsible for confirming the identity of each patient prior to
specimen collection. Positively identifying the patient is crucial to accurate specimen
collection and patient safety.
The phlebotomist should identify patients following the facility’s protocol. The procedures
may vary depending on the patient status – hospitalized, outpatient, and physician office
patient. Phlebotomists will have laboratory test orders and/or specimen collection labels
8
that contain information to link the specimens to the patient. At a minimum, these records
have a name (first name, last name, middle initial or name), and a unique number such as
a medical record number, or a person-specific number such as the date of birth, address,
or telephone number. This information is considered to be unique identifiers. Two unique
identifiers should be used to confirm the identity of each patient.
The following are not considered unique identifiers: patient room/bed number,
billing/encounter number, physician name, patient age, date of collection, city, state, last 4
digits of the Social Security number, and procedure/test.
Patients in hospitals (inpatients): Identification of inpatients should always include

checking an identification band (wristband) attached to the patient and comparing the
unique identifiers on it to the unique identifiers on the laboratory test request/specimen
labels. The use of a medical record chart, bed label, sign on the wall, or other "loose"
document should not be used to identify a patient.
The use of two patient identifiers is required, which is usually the full patient name (first,
last, middle name/initial), and a unique identifier such as a medical record number to
confirm the identity of hospitalized patients. If the patient is lucid, CLSI GP33 requires that
phlebotomists verbally confirm patient identification by asking the patient to state their full
name, spell their first and last names, and state their date of birth. Identification of a
patient who is unable to participate in the identification process must be verbally confirmed
the same way except by the patient’s nurse or family member.
Specimen collections should not occur if.

 discrepancies exist between the patient identification band and the patient’s verbal
identity confirmation,
 discrepancies exist between the patient identification band and laboratory test
request/specimen label, or
 the patient does not have an identification band.
In the above situations, the patient’s charge nurse or laboratory supervisor should be
notified. Specimens can be collected upon resolution of identification discrepancies and
placement of an identification band on the patient.
In emergency situations, healthcare facilities generally have a system to uniquely identify a

patient for whom no identification has been confirmed.
Outpatients/Physician Office Patients: Outpatients and patients in physician offices

may not have identification bands. In these situations, the patient must state their full
name and spell their first and last names, state their date of birth, and provide proof of
identification. In the event the patient has no form of personal identification, follow facility
policy. The phlebotomist must then compare this information with the information on the
test order/specimen labels.
Patient Identification Technology
Many healthcare facilities use barcode systems as an additional method of patient

identification. These systems include many of the same features. Each patient is given a
unique barcode in the hospital electronic health record system. This barcode is included
9
on the identification band. Using a handheld device interfaced with the hospital electronic
health record, the phlebotomist accesses the barcode on the identification band and
confirms the identity with the identification number in the electronic health record.
Following verbal confirmation of matching identifiers, specimen labels are printed at the
bedside. The labels may include a variety of information such as specimen requirements,
location, time of collection, phlebotomist identity, and other items as specified by the
institution. The photograph below shows a patient identification band being scanned prior
to labels being printed for specimen containers/evacuated tubes.
Scanning barcodes on identification band before specimen collection

Clinical Center News, April 2009
http://clinicalcenter.nih.gov
The Clinical Center located the National Institutes of Health in Bethesda, Md.,
is the nation's largest hospital entirely dedicated to clinical research.
Patient Safety Considerations

Failure to properly identify a patient and/or mislabel patient specimens has very serious
implications. Patients could die from transfusion of ABO incompatible blood products or from
overdoses/underdoses of medications.
SITE SELECTION, TOURNIQUET APPLICATION, & PREVENTION OF TISSUE

TRAUMA
Site Selection
Selection of an appropriate site to collect a blood specimen is important to produce quality

test results and to ensure patient safety.
Venipuncture: Veins available for blood collection include the principal veins of the arms
and hands. The veins of the antecubital fossa provide the ideal venipuncture location in
most adults, older toddlers, and children. These veins include the median cubital,
cephalic, and basilic veins.
10
Following are graphics demonstrating the two most common anatomic arrangements of
these veins in the antecubital fossa – H and M arrangements. The H arrangement is most
prevalent.
Vein Prioritization
Median basilic vein 1. Median (center)
2. Lateral (outer)
3. Medial (inner)
NCCT Graphic
The median cubital vein (H arrangement) or the median cephalic/median basilic vein
(M arrangement), all located in the center, are the recommended veins of choice for
venipuncture in adults. Advantages of these veins follow.
 Typically the closest vein to the skin's surface, making it readily accessible
 Typically the best anchored of the antecubital veins making it less likely to ‘roll’
 Safest vein to puncture as its location is such that nerves and tendons are least
likely to be damaged
 Puncture generally causes the least amount of pain
The second choice for venipuncture are veins in the lateral (outer) aspect, i.e. the cephalic
vein and accessory cephalic vein (not pictured). The third choice for venipuncture are
veins in the medial (inner) aspect such as the basilic vein and medial aspects of the
median cubital or median basilic veins.
Additional venipuncture sites include veins on the back of the hand, and possibly the
medial ankle or dorsum of the foot. The basilic and cephalic veins of the back side of the
hand are acceptable veins for blood collection. These veins are not well anchored, and
may roll when then the needle is inserted. Veins on the palm side of the wrist, thumb side
of the wrist, and forearm should never be considered as an alternate site. Veins of the foot
and ankle should be used for venipuncture only if the physician has granted permission.
To select a vein for venipuncture, a tourniquet is applied and the patient is requested to
make a fist (but should not “pump” the fist). These actions allow the veins to become more
prominent. However, the tourniquet should not be applied for more than one minute as
this may result in hemoconcentration which can alter laboratory tests. If it takes longer
than one minute to locate a vein and perform a venipuncture, the tourniquet should be
released and not reapplied for at least two minutes to allow the blood flow to normalize.
The phlebotomist can assemble the needed phlebotomy equipment and reapply the
tourniquet immediately before needle insertion.
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NCCT Graphic
Capillary Blood Collection: Capillary blood collection requires the use of a dermal site.
The two dermal sites most frequently utilized in pediatric patients are the heel and the
finger. Per CLSI GP42 (2017), the heel is the only site for all infants under 6 months of
age and infants less than 12 months of age who weigh 10kg or less. A finger can be used
for infants between 6 and 12 months of age who weigh more than 10kg as long as the
lancet depth does not exceed 1.5mm.
The site selected for heel puncture must avoid the midline of the heel surface. When
performing a capillary puncture of the heel, care must be taken to avoid puncture of the
calcaneus bone located at the posterior curve of the heel as this can cause an infection of
the bone. The acceptable surface of the heel for puncture has been established as lateral
or medial plantar surface of the foot as shown in the following graphic.
The shaded areas indicated

by the arrows are the
appropriate sites for a heel
puncture.
The skin puncture on an

infant’s heel must be no
deeper than 2.0 mm!
Punctures deeper than this
are associated with bone
injury and infection.
As an infant ages and begins to walk, the skin of the heel becomes thick and calloused
making the heel an unacceptable choice for blood collection. This is generally at 12
months (one year) of age. At one year, capillary collection from the dorsal surface of the
finger should be considered. This site can safely be performed in children older than one
year through adulthood.
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The ideal site for skin puncture in adults and children is the palmar surface of the distal
phalanx (fingertip) of the middle or ring finger of the non-dominant hand. The puncture
should be slightly off-center in the fleshy area of the thickest part of palmar side of the
fingertip. The sides (near the nail’s edge) and extreme tip must be avoided. The little
finger is avoided due to thin skin, and the thumb and index finger are avoided due to
calluses on these fingers from use. Age-appropriate sites are selected based on the
judgment of an experienced, well-trained phlebotomist.
X
X
Dermal sites that are bruised, edematous, or have a hematoma should not be used for
collection of a capillary specimen. The quality of the specimen may be unacceptable in
addition to increasing the pain level of the patient. Alternate sites should be investigated
when the usual site is unacceptable.
1. The basilic vein should be used for a venipuncture only after both arms have been
evaluated and no other prominent antecubital vein is present. The basilic vein is close to both
the brachial artery and median nerve, and venipuncture can lead to nerve damage or accidental
arterial puncture. Nerve injuries can be debilitating and permanent. Accidental arterial
puncture can result in prolonged bleeding, blood clot formation, arterial spasms, compression
neuropathy, and more.
2. Venipuncture/capillary blood collection should never be performed using the arm on a side
where lymph node removal or dissection has occurred unless permission has been granted by
the attending physician. This is often seen in women who have had mastectomy and lymph
node removal. However, it is also seen in patients with lymphoma, head and neck cancer,
melanoma, and many other types of malignancies.
When lymph nodes have been removed from the axillary and chest areas, lymph fluid can
accumulate in the arm, causing lymphedema, a serious painful chronic condition. Patients with
lymphedema are at risk for recurrent infections and gross disfigurement. Their quality of life is
decreased.
Blood collection, as well as blood pressure measurement and injections, can trigger the
development of lymphedema, make existing lymphedema worse, and result in the development
of an infection. If performed in error, these procedures are also very painful for the patient.
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Patient Safety Considerations - continued
3. Venipuncture/capillary blood collection should not be performed in areas that are edematous,
bruised, scarred, burned, or have hematomas. Not only is specimen collection in these areas
painful for the patient, tissue substances present can contaminate the blood specimen affecting
test results.
Other Collection Site Factors
After choosing an appropriate site, the collection of a specimen without traumatizing the
collection site is vital for providing a quality specimen. Some specific steps to be avoided
during collection include the following.
 Application of tourniquet for more than one minute

 Excessive probing or repositioning of the needle
 Excessive squeezing or milking during capillary collection
 Using a small gauge needle with a collection of multiple tubes
 Excessive warming of tissue prior to capillary collection
 Drawing blood proximal (above) to an IV line in use
Patient diagnosis and treatment may be affected as a result of erroneous test results obtained from
the following actions.
1. Extended application of the tourniquet causes a rise in the capillary pressure. This results in an
increase in the levels of potassium, total protein, lactic acid, glucose, and some enzymes.
2. Hemoconcentration, a false increase in red cell concentration, results from extended tourniquet
application. Hemoconcentration falsely increases red cell counts, white cell counts, platelet
counts, and many other laboratory test values.
3. Extended needle probing, excessive needle repositioning, and squeezing/milking capillary blood
collection extremities results in tissue damage and dilution of the blood with tissue fluids.
Thromboplastin, a coagulation factor, is present in tissue fluid and can cause decreased
coagulation test results. Dilution of specimens with tissue fluids will cause results to be invalid.
4. While patients may be asked to make a fist as preparation for venipuncture, they should not
clench their fist or rapidly open and close (pump) their fist. These actions induce anaerobic
metabolism in the forearm environment, and increase lactic dehydrogenase and potassium
levels.
5. If blood is collected from a site proximal to (above) an IV infusion, the blood is diluted with the
fluid being infused. The dilution causes falsely decreased test results. If the fluid being infused
contains the substance the blood for which the blood is to be tested; i.e., glucose, potassium,
etc., these test results will be greatly increased.
Before collecting blood distal to an IV line, it is recommended to have the patient’s nurse stop
the IV infusion for a minimum of two minutes before collection.
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Patient Safety Considerations - continued
If the only site available for a venipuncture is proximal to the IV line, the patient’s nurse must
stop the IV infusion for a minimum of two minutes before collection. The first 5 mL of blood
collected must be discarded.
A comment may be placed in the laboratory report that the specimen was collected above an
IV line and the IV solution must be identified in the comment.
Other Patient Variables
While outside the control of the phlebotomist there are other patient factors that can result
in erroneous test results. Two of the more common variables follow.
 A change in a patients’ position can alter some laboratory test results. A change in
patient posture from supine to upright leads to an increase in capillary pressure in the
legs. An intravascular concentration of large proteins occurs as fluids and small
molecules leave the intravascular space. These changes may result in elevated
protein, triglycerides, cholesterol, iron, calcium, alanine aminotransferase (ALT) test
results.
 Strenuous exercise is also known to affect test results. Potassium values can be
decreased following strenuous exercise, and the following test results can be
increased: creatinine, ALT, lactic acid, growth hormone, antidiuretic hormone, uric
acid, WBC count, lactic dehydrogenase (LD), creatine kinase (CK), and aspartate
aminotransferase (AST).
NEEDLE SIZE, TUBES, ORDER OF DRAW, TUBE VOLUME, & SPECIMEN HANDLING
Needle selection: There are two criteria used to select the proper needle size for
venipuncture.
 The needle should be an appropriate size for the vein selected so to cause minimal
patient discomfort and little or no tissue damage.
 The needle must be of sufficient size to allow all sufficient blood to be collected with no
visible hemolysis.
Typical needles used for venipuncture are 21 to 23 gauge (the larger the gauge number
the smaller the needle, and the smaller the gauge number the larger the needle). In
venipuncture scenarios on young children and the elderly where a small needle is needed
(25-gauage), the newly designed red-cell-friendly 25-gauge needles are recommended.
Conventional 25-gauge needles cause red cell damage and hemolyzed specimens.
Evacuated tubes: Choosing the proper tube size and collecting the minimum amount of
blood needed for analysis is important for patient safety. Guidelines for minimum
specimen volume needed for analysis should be included in the specimen collection
manual.
Expiration dates on tubes should always be checked prior to using. Expired tubes should
not be used for blood collection as the vacuum may be compromised and the
anticoagulants and additives may not be viable.
15
Each evacuated tube has a maximum fill volume. This is the volume of blood that the
vacuum will pull into the tube during specimen collection. Evacuated tubes can be
overfilled if the plunger of a syringe is forcibly pushed when transferring blood into an
evacuated tube. This practice should be avoided as it may result in a hemolyzed
specimen, and if the tubes contain an anticoagulant, there may be too much blood for the
quantity of anticoagulant and result in the presence of micro-clots.
Filling the tube with less than the maximum volume of blood is generally acceptable.
However, there are instances when underfilling a tube has negative impact on test results.
1. Repeated venipunctures in neonates and those with serious illnesses can result in anemia from
the removal of too much blood. Maximum amounts of blood to be drawn in any one given time
period from special patient populations should identified in laboratory procedures.
2. Underfilling tubes containing liquid anticoagulants results in dilution of the blood specimen,
altering the laboratory test results.
Tubes anticoagulated with sodium citrate (light blue closure) are most affected by
underfilling, resulting in falsely lengthened PT test results.
Underfilling tubes anticoagulated with EDTA (lavender closure) results in excessive

anticoagulation. This falsely decreases hematocrit, MCV and MCHC.
Underfilling tubes anticoagulated with heparin (green closure) falsely lowers sodium, and
alters ALT, AST, lipase, and potassium test results.
Underfilling blood culture bottles can result in an insufficient amount of blood to detect
fungal or bacterial microorganisms, causing a false negative blood culture result.
3. Hemolysis can result from use of small needles, using smaller needles with larger
size evacuated tubes, or exerting excessive force when pulling back on a syringe plunger.
Order of draw/collection: Once the proper tubes have been selected for the lab tests,
the order in which the tubes are filled with blood is important to assure additives or
anticoagulants in one tube type do not carry over and contaminate the blood collected in
the following tube. This is called the order of draw. The order of draw is the same for
blood collected by evacuated systems and by syringe. However, the order of collection for
capillary specimens differs from venipuncture and is discussed separately.
Guidelines for the venipuncture order of collection of the most commonly used evacuated
tubes follow.
16
CLSI Recommend Order of Draw for Venipuncture
Order Tube Closure* Color

1 Blood culture and other specimens requiring  Blood culture bottles
sterility  Yellow closure SPS tubes
 Any other test that must be sterile
2 Coagulation tube  Light blue closure

(sodium citrate anticoagulant)
3 Serum tube (with or without clot activator,  Red & black closure
with or without gel)  Gold closure
 Red closure
 Orange closure
4 Heparin tube (lithium heparin anticoagulant  Green & gray closure

with or without gel plasma separator)  Light green closure
 Green closure
5 EDTA tube  Lavender closure

(ethylenediaminetetraacetic acid  Pink closure
anticoagulant)  White (pearl) closure
6 Glycolytic inhibitor tube  Gray closure

(EDTA anticoagulant, sodium fluoride
glycolytic inhibitor)
*Glass tubes typically have a color rubber stopper; plastic tubes typically have a color shield that covers a rubber
stopper that may be a different color than the shield.

The rationale for the order of draw follows.
1. EDTA is rich in potassium and can falsely elevate potassium test results. Thus, tubes used
to test for potassium must be collected before tubes containing EDTA. Therefore, all tubes that
may be used for potassium testing (red/black, gold, green/gray, green, and red closure) are
collected before lavender, pink, or white closure tubes.
2. The additives in gray stopper tubes distort the microscopic appearance of blood cells on a
WBC differential test. Therefore, tubes for WBC tests/CBC tests (lavender closure) are
collected before gray closure tubes.
3. Clot activators can interfere with coagulation tests such as prothrombin (PT) and activated
partial thromboplastin time (aPTT), resulting in erroneous test results. Therefore, tubes for
coagulation testing (light blue closure) are collected before red/gray, gold, red, gray/yellow, and
orange closure tubes.
4. Bacteria from non-sterile tube closures can contaminate blood collected into
bottles/tubes used for blood cultures, resulting in the growth of bacteria erroneously leading a
physician to think his/her patient has a blood infection. Therefore, blood culture bottles and
yellow closure SPS tubes (or other tests requiring sterile blood) are collected first and before
any other tubes.
17
Guidelines for the capillary collection order of collection of the most commonly used
microcontainers follow.
CLSI Recommend Order of Draw for Capillary Collection
Order Microcontainer Closure Color

1 EDTA Lavender closure
2 Heparin tube with gel Mint green

 Clear plastic
 Amber plastic (light protection)
3 Glycolytic inhibitor tube Gray
4 Serum with clot activator and gel Gold

 Clear plastic
 Amber plastic (light protection)
5 Serum with no additive Red
The primary factor determining the capillary order of collection is filling the microcontainers
that have anticoagulants before the specimen can begin to clot. Therefore, EDTA and
heparin microcontainers are collected before serum containers.
Specimen handling: A few guidelines for specimen handling are listed below.
 Immediately upon filling with blood, evacuated tubes and collection containers must be
gently inverted to mix the blood with the anticoagulant or additive. The inversions of
tubes/collection containers containing anticoagulants allow the blood to mix with the
anticoagulant to prevent clotting of blood specimens. The inversion of tubes/collection
containers with clot activators has the opposite effect – the blood comes into contact
with the clot activator and forms a clot more quickly. The number of inversions differs
with the substance in the container.
 Venipuncture evacuated tubes

 EDTA, sodium fluoride, heparin: 8-10 inversions
 Citrate: 3-4 inversions
 Serum with clot activator and gel: 5 inversions
 Capillary collection containers

 EDTA, sodium fluoride, heparin: 10 inversions
 Serum with clot activator and gel: 5 inversions
 Evacuated tubes and collection containers should not be shaken or tapped against a
surface to mix the blood and anticoagulants/additives. These actions may result in a
hemolyzed specimen.
 Failure to thoroughly mix blood with anticoagulant leads to the presence of micro-clots.
Micro-clots can also form due to a prolonged collection procedure as they form in the
18
needle, syringe, or winged infusion set tubing. As the blood contains the micro-clots
before coming into contact with an anticoagulant, the anticoagulant will not ‘unclot’
them. To prevent this, select a vein and equipment to fill tubes as quickly as possible,
and transfer blood from a syringe into collection tubes without delay.
Anticoagulated specimens that contain micro-clots are not acceptable for analysis, and the patient
must undergo another collection procedure to obtain accurate test results.
1. The presence of micro-clots in whole blood specimens for whole blood analysis (such as CBC,
hemoglobin & hematocrit, etc.) will invalidate all blood counts. The micro-clots can also clog the
mechanisms of the hematology analyzer causing it to malfunction.
2. Testing of partially clotted blood can lead to either prolonged or shortened coagulation testing
results depending on the extent of the clotting components in the specimen that are used up in
the clots. Affected tests include PT, aPTT, and others.
3. When micro-clots are formed in an evacuated tube/container containing heparin, upon
centrifugation, the liquid portion of the specimen is now serum, not plasma. The serum often
contains very small clots of micro-fibrin particles that provide inaccurate troponin and some
immunoassay test results. The micro-fibrin particles can also clog the mechanisms of chemistry
analyzers causing them to malfunction.
Micro-fibrin particles can also be present in the serum of specimens collected in evacuated
tubes with no additives (or clot activators) that are centrifuged before a well-defined blood clot
has formed.
SPECIMEN LABELING
 Specimens must be labeled immediately following collection at the patient’s bedside

(inpatients) or in the specimen collection area before the patient leaves (outpatients,
physician office patients). Failure to label the specimens at this time greatly increases
the chance that another patient’s labels will be placed on the collection containers.
 Computer generated specimen labels or handwritten labels can be used. The writing
on handwritten labels must be legible. At a minimum specimen labels should contain
the two unique patient identifiers, the time, date, and a method by which to identify the
individual who collected the specimen; i.e., initials or unique identification number.
When computer generated labels lack this information it should be handwritten on the
label.
 Labels must be placed on evacuated tubes in a manner that easily allows the specimen
to be identified, the specimen to be evaluated for hemolysis, volume, etc., and the tube
to be placed directly on an analyzer if applicable. Labels should be attached by placing
the collection label on the tube directly over the existing label on the tube so both labels
are completely aligned. If the label has a barcode it must be straightly aligned and in
the correct location (this may vary by facility) to enable an analyzer to identify the
specimen and perform the requested tests.
 When placing labels on evacuated tubes, the following should be avoided.
o Wrapping the label around the tube and making a tab
o Placing it opposite the existing label on the tube
19
o Wrapping a single label around multiple tubes
o Attaching in a crooked manner or upside down (this may vary by facility)
o Wrinkling the label so the patient identification information and barcode
cannot be read
o Wrapping the label around the tube, covering up information by adhering a
portion of the label over another portion of the label
o Covering a portion of the tube closure
Examples of correctly aligned and misaligned labels follow.
Courtesy and © Becton, Dickinson and Company
Placing another patient’s label on a specimen collection tube/container has serious consequences.
The physician receives and acts on test results from the wrong patient, leading to serious medical
errors that involve significant adverse events for the patient.
Specimens arriving in the lab that lack all needed information on the label may be rejected for
testing. This requires another specimen collection procedure for the patient.
Improperly placed labels on evacuated tubes require laboratory personnel to attempt to reposition
the label without destroying the label. Repositioning the label may damage the barcoded
specimen label leading to the need to relabel and the potential for labeling errors. In addition,
repositioning labels disrupts the efficiency of the analysis of specimens and prolongs turnaround
times if it must be done with as many 200-300 tubes of blood a day.
20
SPECIMEN TRANSPORT
 Specimens should be transported to the laboratory without delay. This transport may
be provided by the phlebotomist, transport staff, or pneumatic tube systems. Prompt
transportation of specimens to the testing site not only assures the quality of the
specimens will not be altered, but also provides for a rapid turnaround time.
 Specimens transported via pneumatic tube may be hemolyzed while in transit due to
the length, speed, and angles or turns in the system. If pneumatic tube systems are
used they should be checked for acceptability at installation and periodically thereafter.
 Some laboratory tests have special handling requirements that must be followed from
the time the specimen is collected until the specimen is analyzed. The most common
of these are chilling the specimen, protecting the specimen from light, and maintaining
the specimen at 37° C (body temperature). Test specific transportation requirements
should be stated in the collection manual and they generally appear on computer
generated collection labels.
The following chart identifies the most common tests that require special handling in
most laboratories.
Tests That Require Chilling Tests That Require Tests That Require
Maintenance at 37ºC Protection From Light
 Gastrin  Cold agglutinin  Bilirubin
 Ammonia  Cryofibrinogen  Vitamin A
 Lactic acid  Cryoglobulin  Vitamin B6
 Catecholamine  Beta carotene
 Pyruvate  Porphyrins
 Parathyroid hormone
o Chilling specimens is accomplished by placing the filled labeled evacuated

tubes in a slurry (mixture) of ice and water and immediately transported to
the laboratory. Filled tubes should never be placed directly in containers of
ice only.
o To maintain blood specimens at body temperature, heat blocks are used.

Heat blocks are kept in the laboratory and maintained at a temperature of
37°C. They have wells designed to hold tubes of blood. When it is time to
collect a specimen that must be maintained at body temperature, the heat
block is unplugged, taken to the patient room, and plugged in. The filled
labeled evacuated tubes are placed in the heat block immediately after
collection, the heat block unplugged, and immediately transported to the
laboratory. In some instances, infant heel warmers may be wrapped around
the tube as a means to keep the blood specimen warm during transportation.
o To protect specimens from light, the filled labeled evacuated tubes can be
wrapped in aluminum foil or paper towels. Capillary collection containers are
available in an amber color which will protect the specimen from light.
21
OTHER VARIABLES IN SPECIMEN COLLECTION
Skin Preparation
Prior to performing a venipuncture or skin puncture the skin is cleansed with an antiseptic
solution. Alcohol is the most frequently used product for antisepsis. After cleansing, the
antiseptic solution must be allowed to dry naturally. It should not be wiped off, fanned,
blown on, etc.
Following are issues that may occur if the alcohol or other skin preparation solution is not
allowed to dry.
 Solution can mix with the blood in the collection container, interfering with test
results
 Solutions may cause hemolysis of the specimen
 Causes a stinging sensation in the patient when the needle is inserted
 Fails to provide asepsis; i.e., the patient could develop an infection because the
venipuncture site is not aseptic
The skin preparation prior to blood culture specimen collection is more rigorous than that
for routine specimen collection. The preparation usually includes using a mixture of
iodine/alcohol or chlorhexidine for skin asepsis. Preparation is performed using back and
forth friction over the collection area. The solutions must dry before collection and the site
cannot be repalpated before needle insertion. Improper skin preparation may result in
contamination of the cultures with bacteria from the skin causing false positive test results.
Central Venous Catheters
Blood collection from central venous catheters (CVCs) is common in hospitals and
oncology clinics. Examples of CVCs include peripherally inserted central catheter (PICC)
line, tunneled catheter, and implanted ports. Protocols for accessing CVCs are facility
specific and they are also not within the scope of practice for phlebotomy technicians.
However, phlebotomy technicians and other healthcare professionals should understand
correct procedures for CVC access and the effects of incorrectly collected specimens.
The two most commonly encountered problems with blood specimens collected from
CVCs are hemolysis and contamination with IV fluids or heparin.
 Hemolysis occurs due to mechanical trauma. Some causes of mechanical trauma

follow.
o Frothing of blood from a loose connection in the blood collection assembly
o Excessively high flow rate of the blood from the catheter into the syringe or
evacuated tube
 CVCs may be used to infuse IV fluids or they may contain heparin to assure the
patient’s blood does not clot in the line. Therefore, before blood is collected for
laboratory tests from CVCs, the line must be flushed with normal saline to assure all
fluids are sufficiently removed from the line so as not to contaminate the blood
specimen. After flushing, the first blood collected, generally 3-6 mL, is discarded to
further assure fluid from the line does not contaminate the blood used for testing.
22
Contamination can give falsely elevated levels of analytes such as electrolytes and
glucose. Fluids can also dilute the blood and cause falsely decreased laboratory test
results.

Delayed transport of specimens to the laboratory can increase turnaround time, and in some
instances, delay life-saving patient treatment.
Failure to follow special handling requirements seriously affects the results of the ordered
laboratory tests.
Failure to allow skin preparations to dry before collecting a specimen can result in inaccurate test
results, unneeded treatment of the patient for an infection due to a false positive blood culture, or
actually result in the patient developing an infection as the collection site was not aseptic.
Specimens collected incorrectly from a CVC may be hemolyzed, resulting in inaccurate test results.
OTHER VARIABLES RELATED TO SPECIMEN QUALITY
There are many special considerations for phlebotomists regarding specimen collection
and quality. Some considerations are general issues that apply to many specimen types.
Other considerations are specific to a particular type of analysis or laboratory department.
A few of the more common issues are discussed below.
CENTRIFUGATION AND SPECIMEN SEPARATION
Specimen processing may be part of a phlebotomist's duties. When serum or plasma is

needed for laboratory tests, the evacuated tubes are centrifuged to separate the serum
from the blood clot or the plasma from the whole blood.
Evacuated tubes should be centrifuged without excessive delay. Extended contact of the
serum or plasma with cells may result in increases in some analytes (e.g. potassium) and
a decrease in other analytes (e.g. glucose).
There are two general types of centrifuges: swing bucket and fixed angle. The type of
centrifuge used depends on the type of specimen collected, the need for serum or plasma
for the laboratory test, the equipment available in the laboratory, and the number of
specimens to be processed. Serum and plasma must be free of red cells before analysis
to assure accurate test results. If a collection tube contains a gel, the centrifugation time
must assure the gel has time to move up and form a solid barrier between the red
cells/blood clot and the plasma/serum. Exposure of plasma/serum to the red cells/blood
clot results in substances leaking from the red cells into the plasma/serum which can
cause unreliable laboratory test results.
The centrifugation time is based on the relative centrifugal force (RCF) needed to separate
the serum/plasma from the blood clot/red cells. The RCF is determined by the diameter of
the centrifuge rotor and the spin speed. Manufacturers suggest recommended times,
usually 5-10 minutes. Laboratories must initially evaluate the effectiveness of the
centrifuge and adjust the recommended times as needed. Periodic evaluations must also
be performed.
23
Tubes for which serum will be used for testing must be allowed to clot for at least 30
minutes before centrifugation. A firm clot must form before centrifugation to assure the
serum does not contain fibrin micro-clots which will interfere with the analytic process.
Therefore, serum specimens must be collected, transported to the laboratory, allowed to
firmly clot, and centrifuged for generally 10 minutes. It may take up to 60 minutes before
the tests can actually be performed on the serum. In many instances, physicians expect
test results before this length of time. For this reason, many laboratories request
heparinized specimens instead of clotted specimens for testing whenever possible.
Heparinized specimens can be centrifuged immediately upon arrival and plasma can be
available for analysis much more quickly.
HEMOLYSIS
Hemolysis of blood specimens is a common occurrence. There are many technical

causes of hemolysis and all attempts must be made to minimize its development.
Hemolysis occurs when red blood cells are ruptured and the contents inside the cells are
released into the liquid portion of the blood. The chemical substances formerly inside the
cell result in falsely elevated laboratory test results, especially potassium, lactic
dehydrogenase, sodium, calcium, bilirubin, total protein, iron, folate, and more.
Hemolysis gives a reddish color to the normally clear/yellow plasma and serum. This
reddish color can interfere with those analytes quantitated by spectrophotometric methods.
These methods identify a change in color intensity that is proportional to the amount of
analyte in a specimen. When the plasma or serum is red, this interferes with the analysis.
Hemolysis can occur at various times during the specimen collection and processing.
Some of the causes of hemolysis follow; some have previously been discussed.
 Prolonged tourniquet application

 Failure to allow skin preparation solutions to completely dry before collecting
specimen
 Using a small needle
 Tissue trauma from probing
 Slow blood flow
 Needle occlusion due to lumen too close to vein wall
 Pulling syringe plunger back too far/too forcibly
 Shaking/tapping evacuated tubes to mix anticoagulants/additives with blood
 Frothing of blood from loose connections of the blood collection equipment
 Freezing of blood during storage or transportation
 Exposure of cells to excessive heat during transportation
 Prolonged contact of serum/plasma with red cells/blood clot
Hemolyzed specimens are often rejected for testing and require a new specimen
collection. Factors affecting hemolysis should then be considered in the recollection and
processing of the repeat specimen.
Unfortunately, there are many conditions that lead to the development of hemolysis inside
the vascular system of patients (in vivo hemolysis). Some prescription medications, blood
24
transfusion reactions, abnormal hemoglobin types, mechanical heart valves, and
autoimmune disorders are a few causes of in vivo hemolysis. When hemolysis is seen in a
patient’s specimen, all technical causes of hemolysis must be ruled out to assure the
hemolysis seen is actually the result of what is occurring in the patient’s body.
BLOOD BANK SPECIMENS
Specimens for blood bank testing (ABO and Rh type, type and screen, type and
crossmatch, antibody identification, and more) generally require special labeling to doubly
assure patient safety. Transfusion of ABO blood group incompatible blood can result in
death of the patient.
Extra attention in the identification of patients and labeling of patient specimens is required
for blood bank testing. Many facilities use an additional identification system to provide an
additional method of patient identification. Most identification systems include a wrist band
with a unique alpha-numeric identification and evacuated tube labels with the same alpha-
numeric identification. The band is attached to the patient and the labels attached to all
specimens and blood components. The patient then is identified for blood collection and
transfusion using both the hospital identification band and the blood bank identification
band.
Some blood bank identification systems use barcode technology to assist with patient
identification. Following are examples of two systems for this process.
Securline® Blood Band by Precision Dynamics Corporation
Reprinted with permission of Precision Dynamics Corporation
25
Hematrax-ID System by Digi-Trax
Reprinted with permission by Digi-Trax Corporation
Attention to detail with the identification of the patient, the patient’s specimens used for
testing, and the blood products for transfusion provides a safeguard against transfusion
reactions due to incompatible blood.
COAGULATION TESTING
There are multiple factors that can impact the accuracy of coagulation tests. These tests
include protime/International Normalized Ratio (PT/INR), activated partial thromboplastin
time (aPTT), and others. A few variables that impact the results of these tests follow.
 Release of tissue thromboplastin into the blood specimen from a traumatic

venipuncture
 Extended tourniquet application
 Ratio of blood to anticoagulant in the evacuated tube
26
The majority of coagulation tests are performed to monitor the amount of anticoagulant
patients are taking. Warfarin (Coumadin® is a common brand name) is monitored with the
PT/INR tests; heparin is monitored with the aPTT test. Heparin is generally used as an
anticoagulant only on hospitalized patients.
Dosage amounts of anticoagulants vary from patient to patient, and are dependent of
height, weight, age, or sex. Assuring the patient is receiving a therapeutic dose is
important. If patients are taking too small a quantity of anticoagulant, they may develop
blood clots. If patients are taking too much anticoagulant, they may have spontaneous
bleeding.
Evacuated tubes for coagulation testing contain the anticoagulant sodium citrate (light blue
closure/stopper). The correct ratio of anticoagulant to blood is 1:9 and this is extremely
important to assure accurate test results. The tubes have minimum and maximum draw
lines on the label to assure the correct ratio is collected. Collecting an amount of blood
within these two lines assures accurate test results as the proper anticoagulant to blood
ratio is achieved. The chart below shows the filling requirements for sodium citrate tubes.
Courtesy and © Becton, Dickinson and Company
If a tube in underfilled, the excess anticoagulant will lengthen the test result and this may
negatively impact patient treatment. A tube forcibly overfilled will develop micro-clots and
cause erroneous test results.
27
All attempts to avoid hemolysis of blood specimens must be taken. The presence of hemolysis adversely
affects patient safety as it falsely increases many lab test results, interferes with the methods used to test for
certain analytes, and masks the presence of an in vivo hemolytic process occurring in the patient.
Failure to follow policies for identification of blood specimens for blood bank testing can result in transfusion
of incompatible blood. This may result in patient death from massive in vivo hemolysis.
Patients taking anticoagulant drugs must be carefully monitored to assure they are receiving therapeutic
doses. If they take too little of the drug, they may develop blood clots which can cause strokes and heart
attacks. If they take too much of the drug, they risk spontaneous hemorrhage. Patient doses of
anticoagulant drugs are determined by the results of the laboratory PT and aPTT tests. Therefore, blue
stoppered anticoagulant tubes must be filled with the correct amount of blood to assure the PT and aPTT
test results are accurate.
PERSONNEL VARIABLES
Another important factor in minimizing preanalytic errors in phlebotomy is the evaluation of

the knowledge, skills, and behaviors of those who perform phlebotomy.
 All phlebotomists should receive initial training on their assigned duties upon hire. The
training should be comprehensive, covering all relevant policies and technical
procedures for which employees are responsible. Training must be documented.
Implementation of new policies and procedures requires additional training for all
employees, again with documentation of the training.
 Training should include information about the legal responsibility phlebotomists have to
their employer and their patients. Phlebotomists must know they can be held legally
responsible for not following facility/laboratory policies and procedures if patient safety
is compromised due to an action on their part.
 Assessment of the phlebotomist’s ability to perform his/her job-related duties, i.e.,

competency, should be assessed upon hire after initial training, six months after hire,
and annually thereafter. Assessment of competency is also recommended upon
implementation of new policies, procedures, or equipment. If at any time competency
assessment identifies employees in need of additional training in a particular skill(s),
those employees should not perform the identified skill(s) until remedial training is given
and competency is once again evaluated and shown to be acceptable. This training,
assessment, remedial training, and reassessment cycle must continue until employees
have satisfactory performance.
 Employee training, performance appraisal/evaluation, and competency assessment are

often confused and thought to be the same. This is not true; however, they are
interrelated. Each is important to assure employees are successful at their jobs.
o Employee training is a process used to confirm the employee has the

knowledge, skills, and behaviors needed to meet the job requirements.
o Performance appraisals/evaluations assess behaviors and qualities that

contribute to an employee’s performance on the job. While they do ask about
the employee’s ability to perform job-related tasks, other questions are asked
28
such as: Is the employee prompt? Is he/she safety-oriented? Does the
employee exhibit quality internal and external customer service skills? Does the
employee follow policies?
o Competency assessment, as stated above, identifies the employee’s ability to

apply his/her skill, knowledge, and experience in the performance of job-related
duties.
 An employee
o can be properly trained, assessed as competent in the performance of a
particular skill, but choose not to follow procedures/policies or
o can be assessed as not competent in a particular skill because the training was
inadequate, or for other reasons.
When evaluations or assessments identify issues such as these, documentation is

required, and follow up training is needed to bring the employee’s performance to an
acceptable level.
 Employers must maintain records of all training, education, and competency

assessment for the duration of employment as well as post-employment for the period
of time required by state, local, and accreditation agencies. These records must be
made available to regulatory and accrediting agencies upon request.
Most regulatory organizations and accrediting agencies recommend or require

employers to provide professional development opportunities for their employees.
Documentation of employee attendance at continuing education offerings should be
maintained in the employee file. Studies have shown that continuing education
increases the professional behavior and improves the knowledge of healthcare
professionals, thus improving patient care.
CONCLUSION
The phlebotomists’ role in the prevention of preanalytic error in laboratory testing cannot
be minimized. With their attention to detail and the proper performance of all processes
involved with specimen collection, laboratory testing can be performed on quality
specimens.
Healthcare professionals responsible for medication administration are familiar with

mediation ‘rights’ (right individual, right medication, right dose, right time, right route, right
documentation). There are also rights for specimen collection and phlebotomists should
always remember their ‘rights’.
Equals
Right Right Right Right Right
Right Right quality
Tech- Handl-
Patient Site Tube Order Label test
nique ing
results!
29
Assuring all the phlebotomy ‘rights’ are followed provides the very important first step in
the production of an accurate and reliable patient test result.
REFERENCES
Arzoumanian, Lena. "What is Hemolysis." BD Tech Talk. Vol 2, No. 2, October 2003.
Brecher, Mark E. (editor). Technical Manual of the American Association of Blood Banks 15th Edition. 2005,
pp. 4-5.
Central Venous Catheters – Topic Overview. WebMd. www.webmd.com. Accessed 5 October 2015.
Chuang, Juan, et al. "Impact of Evacuated Collection Tube Fill Volume and Mixing on Routine Coagulation
Testing Using 2.5 ml (Pediatric) Tubes." Chest. Vol 126, Issue 4, 2004 pp. 1262-6.
CLSI Proposed Guideline. Accuracy in Patient and Specimen Identification. 2009.
CLSI. Accuracy in Patient and Specimen Identification. 2nd ed. CLSI Standard GP33. Wayne, PA: Clinical
and Laboratory Standards Institute; 2019.
CLSI. Collection of Diagnostic Venous Blood Specimens. 7th ed. CLSI Standard GP41. Wayne, PA: Clinical
and Laboratory Standards Institute; 2017.
CLSI. Collection of Capillary Blood Specimens. 7th ed. CLSI Standard GP42. Wayne, PA: Clinical and
Laboratory Standards Institute; 2020
Epner, Paul L., Gans, Janet E., and Grabner, Mark L. “When diagnostic testing leads to harm: a new
outcomes-based approach for laboratory medicine. BMJ Quality and Safety. 2013:22-ii6-ii10.1136/bmjqs-
2012-001621.
Ernst, Dennis J. and Ernst, Catherine. Phlebotomy for Nurses and Nursing Personnel. Health Star Press,
Ramsey, Indiana, 2001.
Favalaro E., Funk (Adcock)D., and Lippi G. ‘Pre-analytical Variables in Coagulation Testing Associated with
Diagnostic Errors in Hemostasis’. Laboratory Medicine.2012;43(2):1-10.
Garon, Jack E. "Patient Safety and the Preanalytic Phase of Testing." Clinical Leadership and Management
Review. November/December 2004, pp. 322-327.
Hayden, Randall T., et al. "Computer-Assisted Bar-Coding System Significantly Reduces Clinical Laboratory
Specimen Identification Errors in a Pediatric Oncology Hospital." The Journal of Pediatrics. Vol. 152, Issue 2,
2008, pp. 219-224.
Hemoconcentration. Center for Phlebotomy Education. www.phlebotomy.com. 2008 May. Accessed 28

October 2015.
Iancu, Dan M. and Handy, Beverly C. "The Value of Laboratory Medicine." Advance for Laboratory
Administrators. March 2009, pp. 60-66.
Laboratory Errors Can Cause Patient Harm. ThinkReliability. www.thinkreliability.com. Accessed 5 October
2015.
Lippi, Giuseppe. "Influence of Hemolysis on Routine Clinical Chemistry Testing." Clinical Chemistry and
Laboratory Medicine. Vol. 44, Issue 3, 2006, pp. 311-6.
Lippi, Giuseppe, et al. "Preanalytic Variability: The Dark Side of the Moon in Laboratory Testing." Clinical
Chemistry and Laboratory Medicine. Vol. 44, Issue 4, 2006, pp. 358-365.
30
Lippi, Giuseppe, et al. "Quality and Reliability of Routine Coagulation Testing: Can We Trust That
Specimen?" Blood Coagulation and Fibrinolysis. Vol. 17, Issue 7, 2006, pp 513-519.
Magee, Leslie S. "Preanalytic Variables in the Chemistry Laboratory." BD Lab Notes. Vol, 15, No. 1, 2005.
Plumhoff, E. A., Masoner, D and Dale, J. D. "Preanalytic Laboratory Errors: Identification and Prevention."
Communique. December 2008.
Pretlow, Lester. "A Quality Improvement Cycle: Hemolyzed Specimens in the Emergency Department."
Clinical Laboratory Science: Journal of the American Soceity for Medical Technology. Vol. 21, Issue 4, 2008,
pp 219-224.
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Journal of the Royal Society of Medicine. Vol. 92, July 1999, pp. 339-341.
Smith, Brian. "Preanalytic Errors in the Emergency Department." BD Lab Notes. Vol. 17, No. 1, 2007.
Smith, Todd J. "Strategies for Error Reduction." Advance for Laboratory Administrators. April 2009, pp. 25-
30.
Stankovic, Ana K. "Elevated Serum Potassium Values, The Role of Preanalytic Variables." American Journal
of Clinical Pathology. June 2004, pp. S105-112.
Wager, Elizabeth, et al. "Patient Safety in the Clinical Laboratory: A Longitudinal Analysis of Specimen
Identification Errors." Archives of Pathology and Laboratory Medicine. Vol 130, Issue 11, 2006, pp. 1662-
1668.
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Journal of the Americal College of Surgery. 2011 Oct; 213(4):543-551.
QUESTIONS
Phlebotomy Preanalytic Variables #1220622
Directions:
 Answer sheets: Read the instructions to assure you correctly complete the answer
sheets.
 Online: Log in to your User Account on the NCCT website www.ncctinc.com.
o NOTE: If the online test questions differ from the course test that follows the
reading material, the CE course you are using is outdated or the question has been
revised since you downloaded it. The online question is the most current and it
should be answered accordingly.
 Select the response that best completes each sentence or answers each question from
the information presented in the course.
 If you are having difficulty answering a question, go to www.ncctinc.com and select
Forms/Documents. Then select CE Updates and Revisions to see if course content
and/or a test questions have been revised. If you do not have access to the internet,
call Customer Service at 800-875-4404.
1. Which of the following is part of the preanalytic phase of testing?
a. Recording test results

b. Equipment calibration
c. Result interpretation
d. Specimen transport
31
2. Which of the following can result from a preanalytic error?
a. Death from incompatible blood transfusion

b. Delay in patient treatment
c. Patient hemorrhage
d. All of the above can occur as a result of a preanalytic error
3. Which of the following preanalytic variables is outside the control of the phlebotomist?
a. Confirming patient identification

b. Ordering correct test
c. Screening of patient preparation
d. Using expired evacuated tubes
4. Which of the following physiologic factors causes iron levels to be lower in the morning
than in the afternoon?
a. Altitude
b. Diet
c. Diurnal variation
d. Gender
5. Which of the following physiologic factors can falsely elevate red blood cell counts?
a. Dehydration
b. Diurnal variation
c. Exercise
d. Medication
6. You just collected blood from a young adult who just completed a 5k run. Which of the
following blood components may remain elevated for 24 or more hours?
a. Calcium
b. Creatinine
c. Hemoglobin
d. Lactic dehydrogenase
7. When the patient’s name (first, last, and middle initial/name) is used as a unique
identifier for patient identification, which of the following is acceptable for use as a
second identifier?
a. Age
b. Bed/room number
c. Date of birth
d. Date of collection
32
8. The phlebotomist finds a discrepancy between the patient’s first name on the
identification band (John) and the laboratory test order (Jonathan). What should the
phlebotomist do at this point?
a. Ask the patient to state his correct name and then change either the
identification band or the laboratory test order to match that name.
b. Change the first name on the laboratory test order to John and collect the blood
specimen.
c. Collect the blood specimen as the name on the identification band is a
recognized shortened version of the name on the laboratory test order.
d. Notify the charge nurse or laboratory supervisor of the discrepancy and inform
him/her the patient will not have blood collected until the discrepancy is
resolved.
9. Identify the 1st recommended vein of choice for an adult venipuncture in the graphic
below.
a. Vein A
b. Vein B
c. Vein C
d. Both B and C
10. What is the name of the vein labeled “A” in question 9?
a. Basilic
b. Cephalic
c. Median cubital
d. Median cephalic
11. Venipuncture of the basilic vein should be avoided due to its proximity to the brachial
artery and the __________.
a. Biceps brachii tendon

b. Median nerve
c. Radial artery
d. Superficial fascia
33
12. Which of the following veins can acceptable for venipuncture as long as physician
permission is first obtained?
a. Veins on the underside of the wrist

b. Basilic vein in antecubital area
c. Veins of the top of the foot
d. Veins on the back of the hand
13. At what age does a heel generally become an unacceptable location for capillary blood
collection?
a. 10 months
b. 12 months
c. 16 months
d. 24 months
14. Why is blood not collected from the arm on the same side where the patient has had
axillary lymph node removal (unless physician permission is granted)?
a. It can make existing lymphedema worse.

b. It can trigger the development of lymphedema.
c. It is very painful for the patient.
d. All of the above are reasons.
15. The phlebotomist is preparing to perform a skin puncture on a finger of an adult. The
patient’s left arm is broken and in a cast. The fingers on the left hand are somewhat
edematous and bruised. On the right hand, the middle and index finger are splinted
together and the little finger has sutures. What is the best finger to use for the capillary
blood collection?
a. Left hand middle finger

b. Left hand thumb
c. Right hand ring finger
d. Right hand thumb
16. Which of the following can result from extended application of a tourniquet?
a. Decreased potassium
b. Decreased red cell counts
c. Increased glucose
d. Increased iron
34
17. Which of the following statements is TRUE?
a. The order of collection for capillary blood is the same as the order of draw for
venous blood.
b. Venipuncture tubes with citrate anticoagulants are drawn before serum tubes,
and venipuncture tubes with heparin anticoagulants are drawn after serum
tubes.
c. When collecting blood using a syringe, the order of draw is reversed.
d. When doing a venipuncture, lavender stopper/shield tubes should be collected
first to make sure the blood does not clot.
18. A phlebotomist deviates from the proper order of draw for venipuncture and draws a
lavender EDTA tube directly before a green heparin tube. What affect might this error
have on the patient’s lab test results?
a. Falsely elevated potassium results

b. Distortion of the microscopic appearance of the white blood cells (WBCs)
c. Erroneous coagulation test results
d. Bacterial contamination interference
19. What is the primary factor that determines the order of draw for capillary (skin puncture)
specimens?
a. Carryover of additive contents from one microtainer to the next can cause
erroneous test results.
b. Filling the microtainer for the most important test first before the specimen
begins to clot.
c. Filling the microtainers that have no additives first before the specimen begins to
clot.
d. Filling the microtainers that have anticoagulants first before the specimen begins
to clot.
20. A blood specimen for a CBC is rejected due to the presence of micro-clots. Which one
of the following will help the phlebotomist prevent this problem when recollecting the
specimen?
a. Collect less blood in the tube so there is sufficient anticoagulant to mix with the
blood.
b. Do a fingerstick capillary collection as capillary blood is less likely to clot than
venous blood.
c. Mix the blood and anticoagulant by inverting the tube no more than 4 times.
d. Select a vein and venipuncture equipment that will allow for a quick collection.
21. An evacuated tube containing a clot activator is inverted a minimum of __________

times.
a. 2
b. 3
c. 5
d. 8
35
22. When placing labels on evacuated tubes, which of the following is an acceptable
practice?
a. Wrapping the label around the tube and making a tab.

b. Wrapping a single label around multiple tubes.
c. Covering a portion of the tube closure with the label.
d. None of the above are acceptable practices.
23. Specimens for which of the following lab tests should be transported in a slurry of ice
and water?
a. Ammonia
b. Beta carotene
c. Cold agglutinin
d. Vitamin A
24. Specimens for which of the following lab tests should be transported in a method that
protects the container from light?
a. Catecholamine
b. Cryofibrinogen
c. Gastrin
d. Bilirubin
25. Specimens for which of the following lab tests should be transported in at 37°C?
a. Vitamin B6
b. Parathyroid hormone
c. Gastrin
d. Cryoglobulin
26. What could happen if a blood specimen is collected while the skin antiseptic solution is
still wet?
a. For a skin puncture, it makes the ‘stick’ hurt less painful.

b. Nothing because the solution should be wiped off before the specimen is
collected.
c. The patient could develop an infection because the collection site is not aseptic.
d. For a venipuncture, the needle enters the vein with less resistance.
27. Which of the following is TRUE regarding central venous catheters?
a. Accessing central venous catheters is within the phlebotomists’ scope of

practice.
b. Blood from a central venous catheter flows very slowly into the syringe or
evacuated tube.
c. Hemolysis of the blood specimen can occur if the specimen is collected
incorrectly.
d. The first blood collected from a central venous catheter is placed in a lavender
stopper tube for hematology testing.
36
28. Delayed or inadequate centrifugation of a specimen can result in some erroneous lab
test results.
a. True
b. False
29. Why is hemolysis of a blood specimen such an issue with lab testing?
a. It can mask the presence of hemolysis occurring in the patient’s circulation.

b. It falsely increases some laboratory tests results.
c. It interferes with some laboratory testing methods.
d. All of the above are issues with hemolysis.
30. Phlebotomists can be held legally responsible for failure to follow facility policies and
procedures.
a. True
b. False
*end of test*
37
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38
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Course Title: Phlebotomy Preanalytic Variables Course #: 1220622
OBJECTIVES
____Yes ____No 1. Did you meet the objectives while reading this CE course?
____Yes ____No 2. Did the test measure what you learned?
COURSE CONTENT
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COURSE DESCRIPTION

*Valid for P.A.C.E.® credit through 04/30/2024

*NCCT is approved as a provider of continuing education programs in the clinical

1. Identify sources of preanalytic, analytic, and postanalytic error.

Laboratory science is a complex specialty and a medical laboratory has multiple

The process approach may be visualized below:

INPUT  PROCESS  OUTPUT

PREANALYTIC  ANALYTIC  POSTANALYTIC

Healthcare management and accreditation organizations have examined the process

Standard Laboratory Process Terminology

PREANALYTIC  ANALYTIC  POSTANALYTIC

PREANALYTIC VARIABLES PRIOR TO PHLEBOTOMIST

o Physician orders incorrect test

 Patient preparation: Patient preparation is important for collection of some specimens.

o Dehydration: Dehydration that occurs with persistent vomiting or diarrhea, for

o Diurnal variations: Many blood constituents show diurnal variation (normal

o Diet: Blood composition is significantly altered by ingestion of food. Glucose

o Medications: Many prescription medications alter physiologic functions. For an

Per recommendations from the College of American Pathologists, medications

o Environment: Temperature and humidity are known to affect test values.

o Position: The position of a patient both before and during venipuncture

o Pregnancy: Pregnancy causes physiologic changes in many body systems.

o Gender: A patient’s gender has an effect on the concentration of numerous

 Storage and shipping of phlebotomy items from the manufacturer to a facility:

PREANALYTIC VARIABLES UNDER CONTROL OF PHLEBOTOMISTS

 Failure to confirm patient identification

Consequences of incorrectly obtained specimens include specimen rejection, the

Patients in hospitals (inpatients): Identification of inpatients should always include

Specimen collections should not occur if.

In emergency situations, healthcare facilities generally have a system to uniquely identify a

Outpatients/Physician Office Patients: Outpatients and patients in physician offices

Patient Identification Technology

Many healthcare facilities use barcode systems as an additional method of patient

Scanning barcodes on identification band before specimen collection

Patient Safety Considerations

SITE SELECTION, TOURNIQUET APPLICATION, & PREVENTION OF TISSUE

Selection of an appropriate site to collect a blood specimen is important to produce quality

Median basilic vein 1. Median (center)

The shaded areas indicated

The skin puncture on an

Patient Safety Considerations

Other Collection Site Factors

 Application of tourniquet for more than one minute

Patient Safety Considerations

Other Patient Variables

Patient Safety Considerations

Underfilling tubes anticoagulated with EDTA (lavender closure) results in excessive

Order Tube Closure* Color

2 Coagulation tube  Light blue closure

4 Heparin tube (lithium heparin anticoagulant  Green & gray closure

5 EDTA tube  Lavender closure

6 Glycolytic inhibitor tube  Gray closure

Patient Safety Considerations

CLSI Recommend Order of Draw for Capillary Collection

Order Microcontainer Closure Color

2 Heparin tube with gel Mint green

3 Glycolytic inhibitor tube Gray

4 Serum with clot activator and gel Gold

5 Serum with no additive Red

Yes No 2. Did the test measure what you learned?

Yes No 3. Were you satisfied with this course?

Yes No 6. Did you learn anything new?

Please include this evaluation with your answer sheet.