Subrat Kumar Dash1 and Gurpreet Singh2
1Assistant Professor-cum-Junior Scientist, Department of Veterinary Biochemistry, College of Veterinary Science and Animal Husbandry, Birsa Agricultural University, Ranchi-834 006, Jharkhand, India.
2Associate Professor, Department of Veterinary Physiology and Biochemistry, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Rampura Phul, 151 103, Bathinda, Punjab, India
Introduction
Following are brief description of some known coagulation disorders and tests used for their diagnosis in recent times.
Haemostatic or bleeding disorders in birds are clinically important from health and production point of view. Timely identification of the causes and diagnosis of bleeding disorders are helpful for poultry farmers for economic farming. Common causes of bleeding disorders in poultry are vitamin K deficiency, bacterial and viral infections, aflatoxicosis, fatty liver hemorrhagic syndrome etc.
Laboratory Diagnosis:
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.
Different Tests:
The principle of various tests is that clotting in the blood sample is inhibited by the binding of calcium by sodium citrate. The presence of sufficient coagulation factors is examined by establishing the clotting time after the addition of calcium chloride and the missing factor.
a. 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. 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/µl.
b. Estimation of Fibrinogen
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. Fibrinogen can be measured by the micro heat-precipitation test at 56°C. This test is based on the principle that fibrinogen will precipitate at 56°C, while the other plasma proteins remain in solution. EDTA rather than heparin should be used as an anticoagulant when this test is performed. Protein concentrations can be estimated in the plasma column of two hematocrit tubes, one of which has been placed in a water bath at 56°C to 58°C for 3 min. Fibrinogen concentration is the difference between the protein concentrations of the two plasma columns. Because the difference between plasma and serum is the absence of fibrinogen in the latter, it is possible to locate the fibrinogen fraction in the electrophoretic gel of a particular species by performing a comparative protein electrophoresis in serum and plasma from the same sample of this particular avian species.
c. Determination of blood Clotting Time
The whole blood clotting time (WBCT), which evaluates the intrinsic and common coagulation pathways, should be performed in samples collected in non-siliconized glass tubes or capillary tubes, whereby contact with tissue thromboplastin should be avoided. Excessive contamination with tissue juices will reduce the WBCT considerably.
d. Determination of tissue thromboplastin 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. 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.