Photo-optical: This can be evaluated by the change in optical density (spectrophotometric) or turbidity (nephelometric) in the sample as a clot is formed.This is performed on platelet-poor citrate anticoagulated plasma. Mechanical: There are different mechanical methods, but these detect fibrin formation because the fibrin restricts the movement of a metal ball (moved between two magnets) or probes inserted within the patient sample.a poorly formed clot indicates problems with clot formation even if the time to clot formation is not abnormal. This is the least sensitive of all methods (small fibrin strands may be missed as the eye can only pick up large clots), but is useful for ascertaining the nature of the clot, e.g. Visual tilt-tube: The platelet-poor citrate anticoagulated plasma is placed in a tube, along with activators and the time to clot formation is recorded.These not only differ in how they detect the clot but also on the provided results. There are different methods to detect the formation of fibrin in these assays, including the visual tilt tube method, mechanical,photo-optical and viscoelastographic techniques. Thrombin can be used to measure the thrombin clot time (fibrinogen conversion to fibrin). ![]() Russell’s viper venom, can activate factor X. Common pathway activator: Viper venoms, e.g.Commonly used activators are kaolin, ellagic acid, glass (which is why blood clots in a red top tube) and siliceous earth (e.g. Intrinsic pathway activator: There are various substances that activate factor XII.Nowadays human recombinant tissue factor can be added instead. Extrinsic pathway activator: This is tissue factor which is usually provided as an extract of brain (tissue thromboplastin) from rabbits or other species.Different activators will trigger different pathways. ![]() After addition of these reagents, the time for fibrin clot formation is recorded. Since citrate anticoagulated plasma is used for most of these assays, calcium needs to be added, along with a clotting activator. Most screening coagulation assays are based on how rapidly fibrin clots form in patient samples. There are also differences in how people interpret the results (with respect to reference intervals, percentage change compared to controls etc). ![]() It is critically important to collect samples properly for hemostasis testing. Normal clotting assays in a bleeding animal do not rule out a defect in secondary hemostasis (because the used assay may not be sensitive) and prolonged clotting times do not mean the animal is bleeding due to a defect in secondary hemostasis (particularly if there were sample collection issues). Interpretation of all assays should only be done with respect to clinical signs. Indeed, short clotting times are often a laboratory or sampling error. Short clotting times do not necessarily mean a hypercoagulable state and should not be interpreted as such. only prolonged times are of diagnostic relevance. ![]() Coagulation screening assays are only sensitive to factor deficiencies and not a hyperactive or excessively activated clotting system (hypercoagulability), i.e. This may involve dilution of a specific reagent, evaluating which method of clot detection is optimal for a given species, or using different clot activators or reagents to measure clotting times. Some laboratories, such as the Comparative Coagulation Laboratory at Cornell University, optimize testing to be sensitive to coagulation factor deficiencies. It is important to note that not all laboratories are created equal with respect to coagulation testing. The combination of results from coagulation screening tests (PT, APTT and fibrinogen) can be used to determine the defect is in coagulation factors, as shown in the image and table below. Laboratories offer different screening assays, but most hemostasis or coagulation profiles consist of the PT and APTT at the minimum, which are interpreted together based on the classic cascade model of secondary hemostasis. Also the dilute Russell’s viper venom time (dRVVT) can be used to evaluate clot formation with the common pathway but is not done routinely. Additionally, a clotting assay that is sensitive to deficiencies of vitamin-K dependent factors (PIVKA assay) can also be performed, but has largely fallen out of favor. The activated coagulation time (ACT) is an in-house point-of-care test that provides some information on secondary hemostasis and is useful as a screening tool for severe coagulation factor deficiencies. The thrombin clot time can be modified to measure fibrinogen concentration. Screening coagulation assays are the “bread and butter” of secondary hemostasis testing and consist of the prothrombin time (PT), activated partial thromboplastin time (APTT) and the thrombin clot time. Screening coagulation assays and the cascade model of secondary hemostasis
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