Coagulation Tests | myADLM.org

When damage to small blood vessels and capillaries occurs, the body controls blood loss via physiological processes referred to as hemostasis. In vivo, hemostasis depends on an interaction between the plasma–based coagulation cascade, platelets, and the endothelium of blood vessels. In the clinical laboratory, in vitro analytical assays are capable of measuring only the first two components of this system. Consequently, laboratory measurements of blood coagulation represent only a close approximation of the body's hemostatic system.

Clinicians frequently order coagulation tests, such as the prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT), to assess blood clotting function in patients. While these laboratory tests may be helpful in elucidating the cause of unexplained bleeding, they are not helpful in predicting if bleeding will occur. In fact, no single test can predict bleeding in the perioperative or post-operative period. Furthermore, these common laboratory tests are of little help in predicting blood clotting or thrombosis in the absence of vessel injury. Well-described assays are available to test for hereditary predisposition to thrombosis, but the majority of thrombophilic states cannot be quantified by any current laboratory tests.Clearly, laboratory assessment of hemostasis presents many challenges for laboratorians and the clinicians who interpret the results. This review briefly explains the common tests used to assess hemostasis, as well as their clinical context, and provides a guide for clinical chemists to assess unexplained bleeding.

Clearly, laboratory assessment of hemostasis presents many challenges for laboratorians and the clinicians who interpret the results. This review briefly explains the common tests used to assess hemostasis, as well as their clinical context, and provides a guide for clinical chemists to assess unexplained bleeding.

The ABCs of Coagulation Tests

Laboratory tests for hemostasis typically require citrated plasma derived from whole blood. Specimens should be collected into tubes containing 3.2% sodium citrate (109 mM) at a ratio of 9 parts blood and 1 part anticoagulant. The purpose of the citrate is to remove calcium ions that are essential for blood coagulation; however, failure to fill the draw tube adequately causes the final citrate concentration of the patient sample to be too high. This is important because PT and aPTT tests require the addition of calcium. If the specimen contains excess citrate, addition of calcium may be inadequate, and the low plasma calcium will lead to an artificial prolongation of PT or aPTT.

A similar but more subtle problem might arise if the patient's hematocrit is unusually high, typically ≥55%. Normally, 10 mL of blood with a hematocrit of 40% contains 6 mL of plasma. If the hematocrit is abnormally elevated, for example 65%, the specimen will contain only 3.5 mL of plasma, effectively under-filling the draw tube with plasma, leading to overcitration of the plasma.

For the PT test, adding a thromboplastin reagent containing a tissue factor, calcium, and phospholipids initiates coagulation of the pre-warmed specimen via the extrinsic coagulation pathway (Figure 1). Similarly, the aPTT test is initiated by adding a negatively charged surface such as silica to the plasma, as well as a phospholipid extract that is free of tissue factor. The coagulation pathway that occurs in the aPTT test represents the intrinsic coagulation pathway (Figure 1).

Figure 1
Cogualtion Cascade

The coagulation cascade is a series of enzymatic reactions that turn inactive precursors into active factors. The end result of the cascade is the production of fibrin, a protein that binds platelets and other materials in a stable clot. The cascade has two initial pathways: the extrinsic (tissue factor-mediated) and the intrinsic (contact system-initiated). These two pathways converge to become the common pathway with the activation of factor X. The steps in the cascade that are measured by the three common coagulation assays, PT, aPTT, and TT, are indicated.

coagulation cascade info graphic

When a patient has an abnormally prolonged PT or aPTT, laboratories should perform a mixing study of the specimen (Table 1). To perform the test, the technologist mixes an equal volume of the patient's citrated plasma with normal pooled plasma (NPP) and repeats the PT and/or aPTT. If the clotting assay time now falls within the PT and/or aPTT reference intervals, the initial abnormal result was due to one or more clotting factor deficiencies. In contrast, the presence of inhibitors in patient plasma interferes with the clotting factors in the NPP, but the mixing study results will not produce normal clotting times. Another common assay used to assess hemostasis is TT (Figure 1). This test measures the ability of fibrinogen to form fibrin strands in vitro. To perform the test, the technologist adds exogenous thrombin to pre-warmed plasma. This step ensures that the result is independent of endogenous thrombin or any of the other clotting factors. TT is particularly sensitive to heparin.

Table 1 Assessment of Prolonged aPTT This table shows how coagulation assays can be combined to elucidate the possible causes of a prolonged aPTT.
Clinical Features	Increased Bleeding	Increased Bleeding	Increased Bleeding	No Hemostatic Problems	Thrombophilia, DVT, PE
1:1 Mixing Study	Corrects	Corrects	No correction	Corrects	No correction
PT	Normal	Prolonged	Normal	Normal	Normal
Pathology	Deficiency of factors VIII, IX and some cases of factor XI deficiency	Deficiency of factors II, V, X, fibrinogen Warfarin Rx	Autoantibodies to factor VIII (acquired hemophilia)	Deficiency of factor XII Deficiency of other contact factors such as prekallikrein Some cases of factor XI deficiency	Antiphospholipid syndrome
Abbreviations: DVT, deep vein thrombosis; PE, pulmonary embolism; Rx, treatment.

Antiphospholipid syndrome. An autoimmune prothrombotic acquired condition, antiphospholipid syndrome (APLS) is frequently associated with a markedly prolonged aPTT, leading to a concern that the affected individual might be at risk for a major hemorrhage. Not only is this highly unlikely, but as a prothrombotic state, APLS is typically associated with venous thromboembolism and/or arterial thrombosis. The condition may also present with fetal loss or stillbirth, which likely occurs as a result of placental inflammation or thrombosis.

Individuals with APLS have antibodies known as lupus anticoagulants (LA). These antibodies are directed to complexes of beta-2-glycoprotein I/phospholipid or prothrombin/phospholipid, and they interfere with and prolong in vitro clotting assays. In the body's vascular system, however, the presence of endothelial cells and leukocytes, as well as many other components that are absent from the simplified in vitro clotting assay, increase the likelihood of clotting.

The classic laboratory findings in APLS patients are prolonged aPTT, normal PT, and no correction of the aPTT 1:1 mixing study. Adding excess phospholipid to the aPTT assay, however, reduces the clotting time. This is the basis for the so-called LA assay. One version of the LA assay is the dilute Russell's viper venom time (dRVVT). The assay components activate only the common coagulation pathway via factor X, and they are independent of factor VIII or antibodies to factor VIII. Laboratories also can confirm APLS by detecting IgG or IgM antibodies to cardiolipin or to beta-2-glycoprotein I in an ELISA-type assay.

Factor V Leiden. A variant of factor V, factor V Leiden causes a hereditary hypercoagulability disorder. Individuals with the disorder have a point mutation in the factor V gene that produces a single amino acid switch (arginine to glutamine, R506Q) that makes the protein resistant to inactivation by activated protein C. Heterozygosity for factor V Leiden increases the risk for venous thromboembolism about two- or three-fold.

Laboratory results for this genetic condition include PT and aPTT within the normal range. In addition, aPTT will be resistant to activated protein C, and in normal individuals, adding activated protein C to a fresh plasma specimen will cause prolongation of aPTT. This effect is blunted for individuals with factor V Leiden. DNA studies definitively confirm the G1691A nucleotide switch.

Prothrombin G20210A. Another hereditary thrombophilia, the G20210A polymorphism in the prothrombin gene elevates the plasma concentrations of prothrombin (FII) without changing the amino acid sequence of the protein.

Patients with this mutation have PT and aPTT results that fall within the normal range, as well as normal functional clot-based studies. DNA studies will show a G-to-A substitution in the 3'-untranslated region of prothrombin gene at nucleotide 20,210.

Protein C and S deficiency. These two vitamin K-dependent factors interrupt the activity of clotting factors V and VIII. Activated protein C is a proteolytic enzyme, while protein S is an essential co-factor.

Antithrombin deficiency. AT, formerly called AT III, is a vitamin K-independent glycoprotein that is a major inhibitor of thrombin and other coagulation serine proteases, including factors Xa and IXa. AT forms a competitive 1:1 complex with its target but only in the presence of a negatively charged glycosaminoglycan, such as heparin or heparin sulfate.

Patients with AT deficiency will have little-to-no AT III activity as measured in a chromogenic assay.

Interpretation of Coagulation Tests

As with any laboratory test, our goal as laboratorians is to assist clinicians with utilization and interpretation of tests that assess hemostasis. Unlike an elevated serum creatinine or a high thyroid stimulating hormone that indicate impaired renal function and hypothyroidism respectively, tests of hemostasis have to be interpreted in the context of clinical findings as well as other laboratory findings. Given the dire consequences of unexplained bleeding, clinical laboratorians should actively advise clinicians on use and interpretation of coagulation tests. A shotgun approach to hemostatic disorders is rarely successful and can result in delayed diagnosis and treatment for patients.