Coagulation Tests and Their Use
Tests of coagulation cascade
Most coagulation tests measure the time required for fibrino-gen from plasma to form fibrin strands, which can be detected by either optical or electrical devices. Prolongation may represent a low factor concentration, inactive factor or factors, or the presence of inhibitors.
Partial Thromboplastin Time
The partial thromboplastin time (PTT), sometimes termed the activated PTT (aPTT), tests the intrinsic coagulation system. A negatively charged surface (e.g., kaolin or silica), followed by cephalin, is added to whole plasma to activate factors XII and XI. The PTT is most sensitive to abnormalities and deficiencies in the sequence of the coagulation cascade before factor X activation. The PTT is also quite sensitive to the action of heparin. It is used to monitor and adjust anticoagulant therapy with regular hep-arin but not with low-molecular-weight heparins.
Prothrombin Time
The prothrombin time (PT) is a test of the extrinsic system. It detects deficiencies in fibrinogen, factor II (prothrombin), factor V, factor VII, and factor X. Tissue factor is added to whole plasma, leading to fibrin formation, normally in 9 to 12 seconds. Results are usually reported using the international normalized ratio (INR). The INR is calculated by using the following equation:
INR = (Log patient PT / Log control PT)C
where C represents the international sensitivity index (ISI). In this way, the thromboplastin used in an individual laboratory, with its specific ISI, is calibrated against a standard reference thromboplastin, and the PT is reported as an INR.28 The presence of a lupus anticoagulant may also interfere with the PT.29
Dilute Russell Viper Venom Time
Russell viper venom contains an enzyme that activates factor X; therefore, the dilute Russell viper venom time (DRVVT) measures the common pathway of the clotting cascade. It is sensitive to the presence of a lupuslike anticoagulant that inhibits the phospholipid-dependent prothrombinase complex.
Thrombin Time
The thrombin time (TT) is used to test abnormalities of the conversion of fibrinogen to fibrin. It can be prolonged because of hypofibrinogenemia, abnormal fibrinogen (dysfibrinogen), or the presence of inhibitors (e.g., fibrin degradation products) that interfere with fibrin polymerization. The clinical factors commonly associated with prolonged TT are severe liver disease, disseminated intravascular coagulation, and heparin therapy.
Reptilase Time
Reptilase is a thrombinlike enzyme that converts fibrinogen to fibrin. The reptilase time (RT) is prolonged under conditions similar to those for prolonged TT, with one significant difference: reptilase is not inhibited by the AT-III-heparin complex. Therefore, RT is not prolonged by heparin. A long thrombin time and normal RT suggest a heparin effect.
Fibrinopeptide A
Thrombin activates fibrinogen by splitting off two peptides, fibrinopeptide A (FPA) and FPB, from the Aa and Bp chain of fibrinogen and converting fibrinogen to fibrin monomer. Measurement of FPA in the blood can be used as an index of throm-bin activity in vivo. Because the clotting cascade can be activated during the blood-sample collection, however, precautions are required in the measurement and interpretation of FPA levels.
Fibrinogen
The fibrinogen level in plasma can be measured either anti-genically or more commonly by clotting assays. The results are reported in mg/dl.
D-Dimer and Fibrin-Fibrinogen Degradation Products
Fibrinogen degradation products (FDP) and fibrin-fibrinogen split products (FSP) result from plasmin degradation of fibrino-gen and fibrin clot [see Figure 9]. D-dimer is released by the plas-min-mediated degradation of fully polymerized fibrin. Plasmin cleavage of fibrinogen or soluble fibrin monomer does not yield the D-dimer. Thus, elevated D-dimer is a specific measure of in-travascular fibrin deposition and plasmin degradation characteristic of disseminated intravascular coagulation. The D-dimer test has largely replaced the FSP test.
Factor XIII
Factor XIII is the only clotting factor whose activity is not assessed in PT or PTT because the end point for both tests is the formation of fibrin polymers, irrespective of whether these polymers are cross-linked covalently by activated factor XIII. Factor XIII deficiency may be suspected in an infant who has significant bleeding after circumcision or, more rarely, in an adult patient who has unexplained bleeding.
Plasminogen and a2-Antiplasmin
The activation of the plasminogen-plasmin system can be inferred from the findings of a long TT, a low plasma fibrinogen level, and an elevated D-dimer level. Another crude test used to measure plasminogen-plasmin activation is the euglobulin lysis time. The sensitivity and specificity of this test is not well defined, however. During extensive thrombosis and fibrinolysis, both plasminogen and a2-antiplasmin (the physiologic inhibitor of plasmin) are consumed. The direct measurement of plasma levels of plasminogen and a2-antiplasmin is sometimes useful to assess the extent of fibrinolysis and the requirement for replenishment of these plasma proteins using fresh frozen plasma.
Tests of platelets and of platelet function
Peripheral Blood Smear Evaluation
This examination provides quick, definitive information to confirm or question a platelet count. Normally, there are eight to 12 platelets per high-power field (1,000 x magnification), corresponding to a normal platelet count of 150,000 to 300,000/ml. The smear also shows platelet granularity and whether mega-thrombocytes are present.
Bleeding Time
This test primarily measures platelet function. A spring-loaded device is used to make a standard skin incision on the forearm. A prolonged bleeding time with platelets greater than 100,000/ml suggests impaired function. The bleeding time is difficult to standardize, and a normal bleeding time does not predict the safety of surgical procedures or accurately predict hem-orrhage.30 It should not be used as a general screening test in a preoperative setting. Although once used in the screening of patients for von Willebrand disease or certain platelet function disorders, for these purposes bleeding time has been largely replaced by the platelet function-100 assay (PFA-100).
Platelet Function Assay-100
PFA-100 is a newly developed automated test for platelet function. Citrated whole blood is aspirated through a capillary tube under high shear onto a membrane coated with collagen and ep-inephrine or collagen and ADP in which a central aperture is made. The time it takes for blood flow through the membrane to stop is denoted as closure time and is a measure of platelet function. The closure time is prolonged in patients with von Wille-brand disease or other platelet functional defects.31 PFA-100 should be considered the first-line test for platelet function disorders.
Platelet Aggregometry
Platelet aggregometers are photometric devices for recording the transmission of light through a suspension of platelets. When platelets aggregate, light passes through the suspension more readily. To test aggregation, dilute concentrations of platelet agonists (e.g., ADP, epinephrine, collagen, and ristocetin) are added to citrated platelet-rich plasma. With the weak agonists, such as ADP and epinephrine, the initial primary wave of aggregation is followed by a secondary wave. The secondary wave reflects the induction of the platelet release reaction, in which platelet granule contents are released to augment further platelet aggregation. A suboptimal secondary wave is seen with platelet storage pool defects in which either platelet granule content is diminished or its release activity is impaired. The latter is commonly associated with aspirin intake or uremia-related thrombo-cytopathy. Patients with von Willebrand disease will have a sub-optimal platelet aggregation response to ristocetin but a normal response to the other agonists. Platelet aggregation testing is labor intensive and expensive and should be performed only in clinical coagulation laboratories that do this test regularly.
Tests of inhibitors of hemostasis
Mixing Studies
A prolonged clotting time (e.g., PTT of 60 seconds [normal, 28 to 30 seconds]) can be caused by either a clotting factor deficiency or an inhibitor. An inhibitor is generally an antibody directed against a specific clotting factor or against a phospholipid-pro-tein complex, the so-called lupus anticoagulant [see 5:XIV Thrombotic Disorders]. In a mixing study, one volume of a patient’s plasma is mixed with an equal volume of normal plasma. The resulting mixture will provide at least 50% of a deficient factor and correct the abnormality. If the problem is caused by an inhibitor, the resulting plasma mixture still has a prolonged clotting time. A mixing study should always be done when a prolonged clotting time is noted.
Antithrombin III
Bioassays and immunoassays are available for assessing AT-III activity. A functional assay is preferable to an antigenic assay.
Protein C and Protein S
Functional and immunologic methods are available. Because protein C and protein S are vitamin K dependent, their measurement can be problematic in patients taking warfarin. It is best to measure protein C or protein S when the patient has been off warfarin for 3 to 4 weeks.
