3. Prolonged coagulation time will be noted in afibrinogenemia and marked hyperheparinemia.
1. Quality of venipuncture: The venipuncture must be
carefully done because either tissue thromboplastin
obtained as a contaminant when the venipuncture is
done, or hemolyzed red blood cells suctioned when the
blood is drawn, can cause a marked shortening of the
a normal value when a poor venipuncture is done.
Clinical Hematology: Bleeding Disorders 277
2. Type of test tube: The coagulation time will be
lengthened to 20 to 40 minutes if plastic or silicone
3. Drugs: Increased coagulation time may be seen with:
Decreased coagulation time may be seen with:
When whole blood is allowed to clot spontaneously, the
initial coagulum is composed of all elements of the blood.
With time the coagulum reduces in mass and fluid serum is
expressed from the clot. This is due to an action of platelets
¾ Equipment for collecting blood
¾ Clean, dry plain glass graduated centrifuge tube
1. 5 mL blood is obtained with a standard two-syringe
technique and transferred to the centrifuge tube.
2. Incubate it at 37°C in vertical position.
3. Record degree of retraction after 1, 2, and 4 hours. It
may be necessary to loosen the clot gently from the
wall of the test tube if contraction is not apparent at
the end of 1 hour. The degree and rate of retraction
should be noted. Note also any digestion of clot or
Clot retraction is directly related to platelet count,
hence, it is impaired in thrombocytopenia, but is normal in
hemophilia. In the method just described, one can remove
the clot by using a hooked long needle and the volume of
serum left behind can be measured. The percentage of clot
can be calculated from the initial 5 mL of blood taken. In
normal individuals, the clot percentage is about 50% at the
end of one hour of the original blood volume taken.
plump, poorly demarcated clot.
2. The clot is small and serum voluminous if the patient
3. Patients with polycythemia have poor clot retraction
because the large numbers of captured red cells
separate fibrin stands and interfere with platelet
4. If fibrinogen levels are low, the initial clot is so fragile
that the delicate strands rupture and red cells spill out
into the serum when retraction begins.
5. Serum contamination by red cells is especially striking
if fibrinolysis is abnormally brisk, as often happens
with reduced fibrinogen levels. Sometimes in these
cases, the incubated tube contains only cells and
plasma with no fibrin clot at all.
1. When fibrinogen is reduced in amount, the clot may
be very small and retraction may be interpreted as
normal even though it is inadequate.
2. In the presence of active fibrinolytic activity, the clot
3. In normal blood the exuded serum will be clear and
free of RBC’s. The presence of significant number of
RBC’s in the serum suggests fibrinolytic activity.
4. With a low hematocrit value, the mass of the clot will
be proportionately small and may give enormously
Protocols and Blood Coagulation Tests
1. Heparin combines in the blood with an alpha globulin
(heparin cofactor) for a potent antithrombin.
2. The intravenous injection of heparin will give an
immediate anticoagulant effect, so it is used when
3. Because of heparin not remaining in the blood very
long, the clotting time is measured before each
4. The coagulation time is ordinarily maintained at two
to two and one half times the normal limit.
5. To evaluate the effect of heparin, the blood is tested
• Before therapy is started for baseline
• One hour before the next dose is administered
• Dependent upon the status of patient during heparin
6. Protamine sulfate is the antidote for heparin overdose
Coagulation tests for the routine assay parameters in
laboratories are fairly simple to perform and master.
The performance of basic tests require simple apparatus
such as water bath, test tubes, pipettes and stop watch
and/or automatic clot timer. Moreover, It is precisely for
this reason that these techniques appear deceptively easy.
There are a number of pretest variables that affect the
accuracy and precision of coagulation results. These may
relate to collection techniques, processing of samples,
selection and preparation of reagents. In order to achieve
optimum and reproducible results the impact of variables
Various variables that affect the results are discussed, along
with the basis that leads to such recommendation.
Although no special preparation of patients is required
prior to approve techniques, it is preferable that patients
are not heavily exercised before blood collection. Fasting
patients or patients on a light non-fatty meal are preferable.
• Patients who are on fasting or on a light non-fatty
meal prior to blood collection provide samples with
desirable lower opacity this improves the sensitivity
of clot detection especially when photo-optical
• Turbid, icteric, lipemic or grossly hemolyzed samples
generate erroneous results due to varying opacity.
Sample Collection Techniques (Phlebotomy)
1. Blood should be withdrawn without undue venous
stasis and without frothing into a plastic syringe with
a short needle of 19 to 20 SWG.
2. The venipuncture must be a ‘clean’ one and incase of
difficulty with a new syringe and needle another vein
should be tried. The tourniquet should not be placed
too tightly or for extended lengths of time. Patting the
venipuncture site should also be avoided.
3. Distribute blood into test tubes (preferably plastic)
after detaching the needle from the syringe. Do not
delay mixing blood with anticoagulant by gentle
• ‘Clean’ venipuncture is essential to avoid formation
of microclots at the site of venipuncture and
consumption of factors, which will lead to artificially
• Usage of short bigger bore needle allows free flow of
blood within the syringe and reducing blood contact
with metal surface. With smaller bore longer needles,
blood will remain in contact with metal surface for
longer time. This will lead to initiation of clotting or
partial consumption of factors being assayed leading
to erroneous results during test procedures.
• Frothing when distributing the blood into anticoagulant tube should be avoided because frothing
1. The anticoagulant of choice for most coagulation
procedures is sodium citrate or preferably buffered
2. Sodium citrate is an ideal anticoagulant since Factor V
and Factor VII are more stable in citrate. These factors
are more labile in sodium oxalate. Heparin neutralizes
action of thrombin on fibrinogen.
3. The recommended molarity of sodium citrate for
coagulation studies is 0.109M, which equates to 3.2%
4. Use of buffered sodium citrate is preferred over plain
• After collection of blood in citrate during centrifugation for preparation of platelet poor plasma
(PPP) or platelet free plasma (PFP), the pH of
the solution shifts releasing of carbon dioxide
(CO2). This shift in pH affects the labile factor V
leading to erroneous results during test. Use of
appropriately formulated buffered citrate overcomes
• When samples are collected in 3.8% citrate (129 mm)
the prothrombin time of samples especially with
patients receiving oral anticoagulants give prolonged
results. Also the ISI of thromboplastins is lowered.
It is for this reason 3.2% citrate is recommended
universally instead of 3.8% for increasing accuracy
5. The optimum ratio of citrate to blood is 1 part of
anticoagulant to 9 parts of blood.
• When the molarity of citrate is accurate the anticoagulant supplied in this amount and ratio is
sufficient to bind all the available calcium in the
collected sample so as to prevent clotting. A shift in
this ratio leads to erroneous results as follows:
• More blood less citrate: The chelating activity of
citrate will not be sufficient to bind the calcium
present in the sample. This will lead to formation
Clinical Hematology: Bleeding Disorders 279
of clots, consumption of factors and subsequent
prolongation of results during test
• More citrate less blood: Excess citrate remaining in
the blood sample would consume the calcium from
the reagents thereby giving prolonged results.
6. The optimum concentration of calcium chloride to be
used for APTT test should be 0.02 M:
• The concentration of 0.02 M CaCl2 replaces the
calcium necessary to activate the intrinsic coagulation cascade. This ultimately generates thrombin
from prothrombin via the coagulation cascade.’
• Appropriate volumes of CaCl2 should be aspirated
for the day's work. Prewarmed CaCl2 should always
be discarded at the end of the working day.
7. The standard ratio of blood to anticoagulant of 9:1 is
• For occasional patients with PCV less than 20% (e.g.
microcytic hypochromic anemia) and greater than
50% (e.g. polycythemia vera) the anticoagulant to
blood ratio must be readjusted using the following
C = Volume of sodium citrate in mL
V = Volume of whole blood–sodium citrate in mL
• When the PCV is higher than 55% the patient blood
contains so little plasma that excess unutilized
anticoagulant remains and is available to bind
reagent calcium to prolongation of test results.
• On the other hand, if the PCV is less than 20 percent
the patient blood contains excess of plasma but less
of anticoagulant and the chelating activity of citrate
will not be sufficient to bind the calcium present in
sample. This will lead to formation of clots in vitro,
consumption of factors and prolongation of results.
¾ Containers for collection and processing of plasma
should be ideally made out of plastic or siliconized
glass tubes. They should be scrupulously clean and dry.
• The containers should be ideally made out of plastic
and not from glass as scratched glass surfaces can
activate in vitro the coagulation mechanism within
the sample due to contact with silica. While plastic
tubes overcome this problem they should be free
from leavening chemicals used by the plastic
industry during molding. These chemicals usually
have an inhibitory effect. Scrupulous washing and
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