1 TOXICOLOGY 707 EVALUATION OF COAGULATION IN TOXICOLOGY STUDIES Dr. Gregory Travlos Fall 2006 Introduction: Hemostasis is a complex series of physiological and biochemical processes (see attached figure) which result in the formation of a stable plug (clot) that seals an injured blood vessel. The process involves the following sequence of events: the interaction between the blood vessel wall and platelets, blood coagulation, and fibrinolysis. For most toxicity and safety assessment studies, platelet counts and coagulation screening tests are the most frequently used methods for evaluating the potential of a specific compound for causing a hemostatic disorder. Proper sample collection for coagulation studies cannot be overemphasized. Cleanliness is a must and you must avoid rough handling of the specimen. Smooth surfaces are important to avoid activation of factor XII and to inhibit spontaneous platelet clumping. Thus, plastic or siliconized glass syringes and tubes and glassware should be used for sample collection and storage. Careful venipuncture is necessary to avoid sample contamination with tissue juices (containing factor III) which will activate the coagulation system within seconds. Samples collected via an indwelling catheter are generally not acceptable. A small clot in a blood sample will activate enough of the coagulation system to invalidate the results and any subsequent interpretation. Since most animal blood clots faster than human blood, it is advisable to add anticoagulant to the needle before collecting samples for coagulation studies. Trisodium citrate is the anticoagulant of choice for coagulation studies. Oxalate anticoagulants are acceptable but are used less frequently. Heparin and EDTA are unacceptable anticoagulants. General Screening Tests for Coagulation: A. Evaluation of the Extrinsic System The prothrombin time (one-stage prothrombin time, Quick time; PT, OSPT) is used to evaluate the extrinsic system of coagulation. It measures clotting factors I, II, V, VII, and X in plasma after activation with tissue thromboplastin (factor III) and recalcification. Many of the commercially available thromboplastins are crude extracts of rabbit brain. Since animal plasmas typically clot in less than 10 seconds, a minor deficiency will not be detected. Thus, it is essential that a homologous normal plasma be used as an assay control and dilute the commercial thromboplastin until the clotting time for the control is between 10-15 seconds. Similar to the PT, the Russel’s viper venom time (stypven time; RVVT) is another measure of the extrinsic system. Except that that the RVVT is insensitive to factor VII deficiency. It also requires phospholipid (supplied by platelet factor 3) for activation. The RVVT gives essentially the same results for all species (approximately 7-10 seconds) eliminating the problems of species specificity for the brain thromboplastin preparations. It is an expensive test and not used routinely. An increased PT and a normal RVVT are diagnostic for a factor VII deficiency. 2 B. Evaluation of the Intrinsic System The activated partial thromboplastin time (APTT) is used to evaluate the intrinsic system of coagulation. The APTT is performed on kaolin- or ellagic acid-activated citrated plasma. Since APTT varies in different species with the source of the partial thromboplastin, the use of a pooled homologous normal control plasma is essential. The APTT for rats is approximately 13-18 seconds; dogs, 14-18 seconds; humans, 25-45 seconds. The activated coagulation time (ACT) is a simplified version of the APTT and is measure of the intrinsic system performed on whole blood. Its advantages are that it can be done at the animals side, and requires a whole blood sample, activator, calcium, and depends on activation of the patients platelets. Thus, the ACT is sensitive to marked alterations in the platelet count. A disadvantage is that the ACT is not adaptable for use in small laboratory animals. C. Evaluation of Fibrinogen The fibrinogen concentration can be measured by several methods and is a quantitative measure of fibrinogen (factor I). For most animal species the fibrinogen concentration is between 100-400 mg/dL. The thrombin clotting time (TCT) provides information on fibrinogen clottability. It is performed by recording the time (seconds) it takes a standardized thrombin solution to clot a patient plasma and is a measure of the rate of conversion of fibrinogen to fibrin. The TCT will give abnormal results if there is a decrease in fibrinogen concentration, dysfibrinogenemia (nonfunctional fibrinogen), increased fibrinolysis (for example, with DIC), and with heparin or heparin-like anticoagulants. Thus the TCT is an indicator of both quantitative and qualitative fibrinogen disorders. The presence of fibrinogen and/or fibrin degradation products prolongs the TCT. Dysfibrinogenemias, caused by an abnormal fibrinogen molecule, are detectable by the TCT but the fibrinogen concentration may be normal. D. Evaluation of Fibrinogen-Fibrin Degradation Products Fibrinogen-fibrin degradation products (FDP) is an evaluation that is useful for identifying the presence of disseminated intravascular coagulation (DIC). It is performed by using a commercially available latex agglutination method that has been designed for human testing but also is applicable to animal testing. For most animal species the FDP is <10 g/mL.
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