Practical Clinical Hematology
Sampling:
General considerations prior to or during blood sample collection:
1- Blood sample should be collected from animals in rest condition,
to avoid physiological variation in blood picture due to fear or
stress.
2- It is favorable to collect blood from fasting animals to avoid the
effect of postprandial lipemia on hematological parameters.
3- Prior to blood collection make disinfection of the site with ethyl
alcohol.
4- Blood sample should be collected slowly to avoid vein collapse
and should be with adequate volume for routine hematological
examination about 5ml.
5- Concerning to laboratory utensils by and in which blood will be
collected should be sterile, clean and dry. Such as, needle and
syringe or vacutainer tubes
Needle and syringe:
gauge of needle (the size or internal diameter of opening or the
pore of the needle) should be suitable according to age to avoid
disruption of blood cells.
Remove the needle from the syringe before emptying of the
blood sample into the vial or the tube contain the anticoagulant
to avoid disruption of blood cells.
Push the blood slowly on the wall not directly towards the
bottom of the tube containing the anticoagulant to minimize cell
damage.
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Practical Clinical Hematology
Vacutainer tubes:
They are tubes contain vacuum which make automatic suction of
blood just when introduced into the suitable vein from which blood
collected.
6- Minimizes tissue injury during blood collection to avoid
contamination of blood sample with tissue thromboplastin which
causes partial clot of the sample making it unsuitable for
quantative tests.
7- Suitable type and dose of anticoagulant for hematological
parameter required to be measured to avoid laboratory artifacts.
8- Thorough gentle mixing between blood sample and
anticoagulant by inverting the tube contains blood and
anticoagulant several times in eight figure manner to provide
homogenous distribution of anticoagulant within blood sample.
9- Blood sample should be kept in refrigerator (at 4 oc) if blood
analysis will be done later.
Site of blood collection:
Animal species Site of blood collection
Ruminants Jugular vein
(cattle, sheep & goats)
Equines Jugular vein
Canine & feline Cephalic vein or femoral vein
Pigs Anterior vena cava
Birds Wing vein or heart
Rats and mice Medial canthus of eye
(retro-orbital venous plexus) or
heart
Camel Jugular vein
Rabbit Ear vein or heart
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Handling of the blood sample for CBC in the laboratory:
For hematological measurement, after the blood is collected it is
transferred immediately to clean, dry, sterile tubes containing
suitable dose and type anticoagulant and make thorough mixing
between blood and anticoagulant by gentle tilting the tubes several
times.
ANTICOAGULANTS
Characteristics
An anticoagulant selected for use in hematological examination
must have the following qualities:
1- It must not alter the size of the red cells.
2- It must not cause hemolysis.
3- It must minimize platelet aggregation.
4- It must minimize disruption of the staining and cells morphology.
5- It must be readily soluble in blood.
Types of Anticoagulants
EDTA (Ethylene diamine tetra acetic acid) Salts.
It is commonly anticoagulant used in hematological studies, mostly
salts of EDTA best meets the above requirements.
Two forms are used: The tripotassium salt (K3EDTA), and the
disodium salt (Na2EDTA). The potassium salts (liquid or dry
powder) are used in commercial tubes because they are more
soluble.
Mode of action: It forms insoluble calcium salts by chelation .
Amount: 1 mg EDTA per1 ml of blood will preserve blood
excellently for at least 6 hours. Refrigeration will extend the
preservation to 24 hours Or can be used in fluid form at
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concentration 15% (the dose used from this form 15 µl per 1 ml
blood).
Uses & advantages
EDTA preserves the staining and morphological characteristics of
leukocytes and other cells.
Used for making a blood smear for cell morphology studies, test for
microfilaria, perform cell counts, BUN, plasma protein, fibrinogen,
and glucose, determination of Coomb's Test.
Disadvantages:
1- Excessive concentrations of EDTA will cause shrinkage of RBC's
and erroneous PCV, MCV and MCHC results. Thus, it is important
to fill vacutainer tubes with the required quantity of blood.
2- EDTA interferes with blood chemistry tests as follows:
a) Falsely decreases alkaline phosphatase by binding magnesium.
b) Decreases the C02 combining power of blood.
c) Interferes with Jaffe reaction for creatinine test.
d) Alters Na+, K+ and Ca++ concentrations in plasma.
Oxalate salts mixture (Heller & Paul Formula)
A mixture of dry ammonium oxalate and potassium oxalate in the
ratio of 3:2 is used together as anticoagulant in one formula known
as Heller and Paul oxalate mixture because potassium oxalate
alone causes red cells to shrink meanwhile ammonium oxalate
alone causes red cells to swell.
Uses: The oxalate mixture may be used for immediate some
hematological studies, such as manually counting RBCs & WBCs.
Disadvantages:
1- It does not prevent platelet aggregation in vitro as effectively as
EDTA.
2- It is poisonous and should not be used for blood transfusion.
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3-This oxalate mixture cannot be used in measurements of the
levels of plasma potassium, alkaline phosphatase, amylase, LDH,
uric acid, urea or nitrogen by a method which involves converting
the urea to ammonia. Potassium oxalate alone, in a concentration
of 2 mg/ml of blood, may be used for the estimation of plasma urea
(BUN). Lithium oxalate, but not ammonium oxalate may also be
used for BUN determination.
4- Blood examination is preferred within one hour of collection, as
some cellular distortion becomes evident thereafter.
Heparin
It is a natural anticoagulant in the body, found in the liver (from the
Greek "hepar" meaning liver), and may also be within basophils
and mast cells. Heparin is also called antithromboplastin or
antithrombin.
Heparin is available in a liquid or dry form as sodium, calcium,
ammonium and lithium salts. Each of these will interfere with
determinations of their respective ions in the plasma.
Mode of action: It interferes with conversion of prothrombin to
thrombin.
The optimum concentration: is 0.1 to 0.2 mg/ml of blood or a rinse
of syringe /5 ml blood.
Uses: It is the anticoagulant of choice for blood pH and blood gas
analysis for acid-base balance. It may be used for special trace
element studies and some cytology. Excessive heparin does not
alter RBC volume.
Disadvantages:
1- It causes clumping of platelets.
2- It interferes with the staining of leukocytes.
3- It is the most expensive of the anticoagulants.
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4- Blood will clot in 8-12 hours because clotting is only delayed and
not prevented. So not used for agglutination tests, coagulation
studies (prothrombin time tests) or plasma fibrinogen
determination.
6- It may interfere with some automated biochemical analyses of
plasma.
Citrates:
The common citrate salt used as anticoagulant is sodium citrate
Solution (3.8%):
Uses:
It is s the anticoagulant of choice for blood transfusion and
studies of platelet function and morphology (1 part of it to 9 parts
of blood).
It is used for ESR estimating (1part of it to 4 parts of blood).
Disadvantages :
Na-citrate is generally not used for CBC because it makes a 10%
dilution of blood.
It interferes with many chemical tests.
Sodium Fluoride
Mode of action: Na fluoride inhibits the glycolytic enzymes
responsible for the breakdown of glucose in the blood. (At room
temperature, about 10% of the glucose is lost per hour from an
untreated sample.)
N.B. Na fluoride has a poor anticoagulant effect so used in
combination with oxalate salts where the oxalate is the primary
anticoagulant.
Optimum concentration: 1 mg of the mixture per 1 ml of blood.
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Uses: The anticoagulant of choice used for glucose determination.
Disadvantages:
It is poisonous.
Alkaline phosphatase, amylase and uric acid cannot be
determined in blood containing sodium fluoride.
Collection tubes used in routine clinical pathology testing
Stopper Additive Sample Usual Tests
Color
Lavender EDTA Whole blood Hematology (CBC)
(Na or K salt)
Red None Serum Chemistry
parameters
Black Citrate (Na) Whole blood ESR
Green Heparin Whole blood or CBC or blood gas
(Na, Li or NH4) plasma analyses & pH
Blue Citrate (Na) Plasma or whole Coagulation tests
blood
Gray Na fluoride Plasma or whole Glucose
blood
Yellow Acid citrate dextrose whole blood blood culture &
(ACD) or DNA analyses
physiological saline
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Time Limits for proper estimation of CBC parameters:
1 hr for Blood film preparation and platelet count.
Three hours for PCV, fragility test and ESR.
24 hrs for Hb concentration, WBCs and RBCs count.
N.B. The better of all, blood samples are doing as quick as
possible, and always keep the blood sample in refrigerator
Sample interferences:
Hemolysis
Lipemia
Clotting of blood sample
Bacterial decomposition
Hemolysis:
# Intravascular hemolysis (within the body) as in case of extensive
hemolysis.
# Extravascular hemolysis (outside the body) during and after
collection of blood sample due to:
1- Direct Trauma, the trauma represented by drawing of the blood
under excessive pressure, excessive forces to expel the blood
from the syringe, excessive shaking of sample container to mix
blood and anticoagulant, freezing the blood sample and centrifuge
of blood sample at high speed or for long period.
2- Contact with hypotonic solution.
3- Contact with chemicals; alcohol, ether and acids.
4- Excessive heat.
5- Lipemia increases the mechanical fragility of RBCs.
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Practical Clinical Hematology
Mechanisms by which, hemolysis interferes with laboratory tests:
Increased absorbance: Released hemoglobin increases
absorbance in the hemoglobin spectral range.
Inhibition of reactions: Released hemoglobin can directly inhibit
chemical reactions (lipase enzyme).
Analytes release: Release of analytes found in high
concentrations in red blood cells will falsely elevate the values of
these analytes (e.g., potassium).
Enzyme release: Release of enzymes which participate in
chemical reactions, (e.g., creatine kinase).
Water release: Release of intraerythrocyte water which dilutes
analytes and decrease analyte values in serum.
Effect of hemolysis on laboratory tests:
Variables increase:
Hb, MCHC
Phosphorus
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k
Total protein, albumin
CK, AST, ALT, LDH
Arginase
Variables decrease:
Decrease PCV and RBCs count.
Decrease in plasma insulin level due to release of insulinase
enzyme.
Effect of lipemia on laboratory tests:
Falsely increase glucose, protein and hemoglobin value with
spectrophotometric estimation method.
Falsely decrease sodium and potassium measurement.
Mechanisms by which, lipemia interferes with laboratory tests:
Light scattering: Results in falsely increased absorbance
readings of some analytes, e.g. total bilirubin.
Volume displacement: This falsely decreases values of some
analytes, e.g. electrolytes.
Hemolysis: Hemolysis of erythrocytes is enhanced in the
presence of lipemia.
Management of Lipemic blood sample:
Fasting for 24 hrs can eliminate lipemia.
N.B. If lipemia persists after fasting, intravenous injection of
heparin at a dose 100 IU/Kg in dogs and blood sample collected
after 15 minutes.
Refrigeration of lipemic sample and collection of sample under
accumulated lipid layer which formed by the effect of refrigeration
if the cause of lipemia is chylomicrons.
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Centrifuge the sample, then after a creamy layer seen at the top
this indicates postprandial hyperlipemia whereas, a turbidity
remains after centrifugation this indicates metabolic diseases.
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