Specimen Considerations in Hematology 7.
Gender: differences of reference values also exist
Specimen collection is a preanalytic variable that may
have serious implications in laboratory testing. According
to studies done in recent years, 32-75% of testing errors
occur during the preanalytic phase.
Pre-Collection variables.
Physiologic factors influence laboratory determinations.
These include:
1. Diurnal variation: most commonly encountered
when testing for hormones, ACP, and urinary
excretion of electrolytes.
2. Exercise: Physical activity may have both long-
term and transient effects on the patient. Exercise
may activate coagulation, fibrinolysis and
platelets, but such activation is due to increased
metabolic activity and returns to normal (pre-
exercise) levels soon after cessation of exercise.
3. Diet: An individual’s diet may greatly affect
laboratory test results, notably for blood
chemistry determinations.
4. Stress: mental and physical stress has been
implicated in production of ACTH, cortisol, and
catecholamines. Total cholesterol has been
reported to increase with stress while HDL-chole
has been observed to decrease. Hyperventilation
increases WBC count.
5. Posture: supine or upright position of the patient
during specimen collection should be taken note
of as this could affect laboratory tests. Upright
position of the patient decreases plasma volume,
thus there is an increase in plasma concentrations
of proteins. Hemoglobin concentrations of
patients may decrease after bed rest in the
hospital.
6. Age: Generally, four age groups have been
defined: Newborn, Childhood to Puberty, Adult,
and Elderly Adults. Most tests have different
reference values with regard to each age group.
With some tests, the differences are more
pronounced with each age group, compared to
others.
between men and women.
Non-physiologic interferences.
In vivo: Tobacco Smoking.
Tobacco smokers generally have high blood
concentrations of carboxyhemoglobin, plasma
catecholamines, and cortisol. These hormonal changes
also result in lower eosinophil counts and higher
neutrophil and monocyte counts. Chronic smoking leads
to increase Hgb, RBC count, MCV, and WBC count.
In vitro
Collection-Associated Variables:
1. Hemolysis: mechanical hemolysis may occur as a
result of:
a. difficult extraction,
b. needle bore size too small,
c. pulling a plunger too fast,
d. improper transfer of blood to the tubes,
e. vigorous shaking or mixing of tubes, or
f. performing venipuncture without allowing
the alcohol to dry.
2. Hemoconcentration/Hemodilution: can occur
due to positional changes. Such problems can be
avoided by allowing the patient to sit in a supine
position 15-20 minutes prior to drawing blood.
Tourniquet application must also not exceed one
(1) minute to avoid concentrating the blood.
When using anticoagulants, the order of draw,
appropriate blood to anticoagulant ratio, and
adequate mixing should be observed.
Non-Collection-Associated Variables:
1. In vivo hemolysis: may be due to hemolytic
anemias or hemolytic transfusion reactions,
among others. In vivo hemolysis may be
differentiated from collection-associated
hemolysis by observing the serum or plasma from
subsequent repeat collections.
2. Lipemia: may be observed shortly after a high-fat
meal or due to high-fat diet.
3. Icterus: may be observed in patients with high
concentrations of bilirubin (≥2.5 mg/dL)
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SPECIMEN COLLECTION
Anticoagulants used in the hematology laboratory:
1. EDTA/versene/sequestrene: chelates calcium
K2EDTA is the preferred form due to its solubility,
followed by K3EDTA (or Li2EDTA), and Na2EDTA.
Na3EDTA is not recommended due to its high pH.
1.5-2.2 mg EDTA per mL of blood is the
recommended ratio.
Uses:
a. Cell counts (RBC, WBC, Platelets)
b. RBC parameters (Hgb, Hct)
c. RBC indices (MCV, MCH, MCHC)
d. ESR determination (mod. Westergren)
e. Peripheral blood smear*
f. Differential count*
Disadvantages:
a. Not recommended for coagulation testing
due to interference of the anticoagulant with
Fibrin formation and instability of FV and
FVIII:c in EDTA.
b. Using old (>2 hrs) blood will cause
morphological changes:
i. Crenation of RBCs
ii. Vacuolization of WBCs
iii. Formation of artifacts and crystals and
phagocytosis of these crystals
iv. Nuclear changes in WBCs (eg. separated
PMN nuclei)
v. Disintegration of platelets
c. May cause platelet satellitism occasionally
2. Citrate: binds with calcium in an unionized form
May be 0.105M (3.13%) or 0.109M (3.2%)
trisodium citrate (Na3C6H5O7•2H2O) in a 9:1
blood-to-anticoagulant ratio. 0.129M (3.8%)
concentrations are no longer recommended
because it affects coagulation test results.
Uses:
a. Standard Westergren method of ESR: blood
to anticoagulant ration is 4:1
b. Coagulation testing (using platelet poor
plasma+)
c. Platelet aggregometry tests (using platelet
rich plasma++)
d. Platelet counts when platelet satellitism or
clumping is encountered
3. Heparin: accelerates antithrombin action to
inhibit thrombin formation; inhibits
thromboplastin
Use 15-30 units per mL of blood.
Uses:
a. RBC count
b. RBC parameters
c. Osmotic fragility
Disadvantages:
a. Causes agglutination of WBC
b. Causes aggregation of platelets
c. Not recommended for coagulation tests
because it affects all stages of coagulation
d. Produces a blue background with Wright’s
stain
e. Expensive
4. Oxalate: precipitates calcium
May be used as Na2C2O4, Li2C2O4, or a mixture of
K2C2O4 and (NH4) 2C2O4.
K2C2O4-(NH4) 2C2O4 mixture (in a 2:3 ratio) is also
known as double oxalate, balanced oxalate, Paul-
Heller’s fluid, or wintrobe fluid. It is prepared as a
mixture because K2C2O4 causes shrinkage of RBCs
while (NH4) 2C2O4 causes swelling of RBCs.
Uses:
a. Coagulation Testing
b. Lithium oxalate is used to prevent clotting of
bloody body fluids
c. Double oxalate can be used for:
 RBC count
 RBC parameters
 ESR determination
*smear has to be prepared within 2 hours of collection
to prevent morphological changes in the blood cells.
+
Platelet-poor plasma (PPP) contains <10,000
platelets/μL and is prepared by heavy spin:
centrifugation at 1500-2000g for 10 minutes.
++
Platelet-rich plasma (PRP) contains 200,000-300,000
platelets/µL and is prepared by light spin: centrifugation
at 50-100g for 10 minutes.
Page 2 of 4
 Disadvantages:
a. Causes agglutination of WBC
b. Causes aggregation of platelets
c. Produces the same morphologic changes seen
when using old EDTA-anticoagulated blood.
Order of draw:
 blood culture tubes (yellow)
 coagulation tubes ctg. citrate (blue)
 serum tubes with or without gel separator
 heparin tubes with or without gel separator
(green)
 EDTA tubes (lavender)
 Fluoride tubes (gray)
Sources of blood for Hematology testing:
1. Skin / Peripheral puncture – source of peripheral
blood (contains a mixture of blood from venules,
arterioles, capillaries, as well as interstitial and
intercellular fluids)
- Finger (ring or middle finger): least used
- Ear lobe (free margin): less nerve endings
: less tissue juice
: no longer recommended due to less capillary
access
- Heel: for newborns and infants
- Big toe: for infants
 depth of puncture should be 2-3 mm
 do not milk the site to avoid contamination
with tissue juice
 do not puncture cyanotic, swollen, inflamed,
and edematous sites.
 Warm the site by massage, warm compress,
or warm water bath
2. Venipuncture – source of venous blood
- Median cubital vein
- Cephalic cubital vein
- Basilic cubital veins
Indications of skin puncture:
1. Infants/neonates/pediatric patients
2. Geriatric patients
3. Adults who are/have:
 Extremely obese
 History of thromboembolism, stroke, or DIC
 Extensive burns
4. Examination requested necessitates skin puncture
(bleeding time, micro methods of clotting time)
Indications of venipuncture: all examinations except
those that are indicated by skin puncture, especially when
a large amount of blood is needed for testing
Patient considerations during venipuncture:
1. Identification
- Ask the patient to state his/her COMPLETE
name. (Do not volunteer the patient’s name)
- Verify the name of the patient with the wrist
tag
- If the patient only speaks a foreign language
or cannot speak, verify the patient’s name
with the companion, nurse or the doctor.
2. Isolation restrictions
a. Isolation – for patients with contagious
disease.
- Everything is left in the patient’s room
after obtaining the specimen (gloves,
gown, mask, etc.)
b. Reverse Isolation – for patients who are
immunocompromised
- Nothing should be left in the patient’s
room after obtaining the specimen
3. Note the time of extraction. WBC count is noted
to be increased in the morning while RBC count,
Hemoglobin, and Hematocrit are decreased.
Page 3 of 4
 4. If both arms are receiving intravenous (IV) - confidentiality of identity
infusion, c. Right to be informed
a. ask the doctor or nurse to stop infusion for at - as to purpose of testing
least 2 minutes. Discard the first tube of - as to results of tests
blood collected.
b. collect below the IV line. Discard the first tube Complications of venipuncture:
collected. 1. Hematoma
c. collect from the lower extremities, if and only 2. Hemoconcentation
if, the patient is not diabetic. 3. Hemolysis
5. Respect the patient’s rights: 4. Syncope
a. Right to refuse extraction
b. Right to confidentiality
- confidentiality of results

References:
[1] Aceron, Z. B. (unpublished). Lecture notes
[2] Benett, S.T., et. al. (Eds.). (2007.) Laboratory hemostasis: A practical guide for pathologists. New York, NY: Springer
Science+Business Media, LLC
[3] Fritsma, G. A. (2012). Laboratory evaluation of hemostasis. In Rodak’s Hematology: Clinical principles and
correlations (pp 734-764) Singapore: Elsevier Pte. Ltd.
[4] Hillyer, C.D., et. al. (2009). Transfusion medicine and hemostasis: Clinical and laboratory aspects. New York, NY:
Elsevier
[5] Jury, C. (2011). Collection and handling of blood, in B. J. Bains (Ed) Dacie and Lewis’ Practical haematology (pp 1-9).
China: Churchill Livingstone
[6] Sanford, K. W. & McPherson, R. A. (2012). Preanalysis. In R. A. McPherson & M. R. Pincus (Eds.) Clinical diagnosis and
management by laboratory methods (23rd Ed.). pp 24-36. Singapore: Elsevier Pte. Ltd.
[7] Tricore Reference Laboratories (2011). Preparation of platelet poor plasma for coagulation testing. Retrieved from
http://www.tricore.org/Healthcare-Professionals/Test-Information/Testing-Protocols/Preparation-of-Platelet-Poor-
Plasma-for-Coagulatio
[8] Turgeon, M.L. (2012). Clinical hematology: Theory and procedures. Baltimore, MD: Lippincott Williams and Wilkins
[9] Zhou, L. & Schmaier, A.H. (2005). Description of procedures with the aim to develop standards in the field. American
Journal of Clinical Pathology, 123, 172-183. DOI: 10.1309/Y9EC63RW3XG1V313

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