Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

XII. Blood Coagulation
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Coagulation in birds is thought to be mainly initiated
through the extrinsic pathway by the release of tissue
thromboplastin (factor III) after vascular injury. Five proteases
(factors VII, IX, X, protein C, and prothrombin) act
with two cofactors (factors V and VII), Ca 2 , and phospholipids
to form thrombin (factor IIa) from prothrombin
(factor II), followed by the formation of fibrin (factor Ia)
from fibrinogen (factor I).
The existence of an intrinsic pathway in birds (involving
factors XII, XI, IX), which is initiated by the exposure
to vascular subendothelium, is controversial. Although the
intrinsic pathway is generally considered to be unimportant,
Doerr and Hamilton (1981) did provide some evidence
for the existence of such a pathway in chickens.
C . Diagnostic Tests
1 . Sample Collection
Blood samples for coagulation studies in birds should be
collected in plastic or siliconized tubes containing 3.8%
sodium citrate. Samples should be fresh as freezing and
thawing may interfere with the results. The principle of
various tests is that clotting in the blood sample is inhibited
by the binding of calcium by sodium citrate, and the
presence of sufficient coagulation factors is examined by
establishing the clotting time after the addition of calcium
chloride and the missing factor. In the prothrombin test,
where the extrinsic and common pathways are evaluated,
tissue thromboplastin is added to initiate the clotting cascade
(discussed later).
2 . Whole Blood Clotting Time
The whole blood clotting time (WBCT), which evaluates the
intrinsic and common coagulation pathways, should be performed
in samples collected in nonsiliconized glass tubes or
capillary tubes (discussed later), whereby contact with tissue
thromboplastin should be avoided. Excessive contamination
with tissue juices will reduce the WBCT considerably.
3 . Blood Smears for Thrombocyte Counts
In birds in which there is a clinical suspicion of a coagulation
disorder, a thrombocyte count should be estimated from
a peripheral blood smear. The best-quality blood smears for
an estimated thrombocyte count can be obtained from whole
fresh blood without an anticoagulant, using the two-slide
wedge technique with bevel-edged microscope slides.
Avian thrombocytes are oval nucleated cells that
are smaller and more rounded than avian erythrocytes.
Because thrombocytes tend to clump in a peripheral blood
smear, an actual thrombocyte count is difficult. In normal
birds, one or two thrombocytes are expected to be seen in
an average monolayer oil immersion field. The estimated
number of thrombocytes in a bird with a normal hematocrit
is equal to the average number of thrombocytes in five
monolayer fields multiplied by 3500 and should normally
range between 20,000 and 30,000/ul.
4 . Prothrombine Time
The single most useful coagulation test in birds is establishment
of the prothrombine time (PT) or tissue thromboplastin
time. PT is a measure of the extrinsic and common coagulation
pathways. It should be stressed that PT in birds should be
performed with homologous brain thromboplastin, as PT significantly
increases when heterologous avian or even mammalian
thromboplastin is used. The use of the PT in birds has
been considered inconvenient because of the unavailability of
species-specific brain thromboplastin. Reportedly, commercially
available Russels’s viper venom (RVV) may be used
instead of homologous brain thromboplastin ( Powers, 2000 ).
PT times using RVV are considerably shorter compared to
those using mammalian brain thromboplastin, but they are
longer than those using a homologous brain thromboplastin
( Timms, 1977 ). Details of performing a prothrombin test in
chickens have been reported by Doerr et al. (1975) .
5 . Modifi ed Russels’s Viper Venom Test (MRVVT)
Russels’s viper venom, in the presence of Ca 2 and phospholipids,
is a powerful coagulant of normal plasma and plasma
from humans deficient in factors VII, VIII (antihemophilic
factor A), and IX (antihemophilic factor B, Christmas factor).
It is an activator of the common coagulation pathway
by activation of factor X. In humans with factor V or X deficiency,
the RVVT is prolonged. The conventional RVVT
cannot be used in birds because the phospholipids in the rabbit-brain
cephalin inhibits coagulation in the presence of raw
RVV. However, use of purified factor X activating enzyme
(RVV-X) eliminates this interference. It has been shown that
experimental infection of turkeys with Pasteurella multocida
increases MRVVT, indicative of a consumptive coagulopathy,
possibly caused by increased consumption of factors X,
V, II, or I (Friedlander and Olson, 1995) .
6 . Fibrinogen Estimation
Fibrinogen is formed and stored in the liver and is important
for the final stage of blood coagulation where it is
transformed into fibrin. Plasma fibrinogen concentrations
decrease when there is severe liver damage. A variety of
inflammatory, suppurating, traumatic, and neoplastic diseases
can increase fibrinogen concentrations in humans
and domestic animals ( Schalm et al. , 1975 ). Hawkey and
Hart (1988) concluded that fibrinogen estimation in conjunction
with a heterophil count was a useful screening test
for birds to detect infections.
Fibrinogen can be measured by the micro heat-precipitation
test at 56°C. This test is based on the principle that fibrinogen