TOX 207 Hemo Outline by HC120314114829

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                                        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.
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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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