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January 2012 Clinical Laboratory News: Coagulation Tests

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January 2012: Volume 38, Number 1


Coagulation Tests
A Primer on Hemostasis for Clinical Chemists

By Neil S. Harris, MBChB, MD, Lindsay A. L. Bazydlo, PhD, and William E. Winter, MD

Coagulation Test

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.

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
Coagulation 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

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.

The quantitative fibrinogen assay is a modification of the TT assay. The test requires addition of exogenous thrombin, except that in this case, the technologist dilutes the plasma several-fold and adds greater amounts of thrombin compared to the TT assay. The results, which are reported in mg/dL rather than seconds, are determined from a standard curve generated from a calibrator plasma.

Another test frequently ordered to assess hemostatic function is D-dimer. These degradation products of mature cross-linked fibrin are formed by the action of the enzyme plasmin on fibrin, and elevated levels are an indicator of thrombosis. Two types of laboratory assays measure D-dimers: noncompetitive, sandwich immunoassays, and immunoturbidimetric assays. D-dimer determination is used for its high negative-predictive value of thrombosis. When the concentration is within the reference range, clinicians can exclude a suspected deep venous thrombosis (or pulmonary embolism). Patients who suffer trauma, undergo surgery, or are pregnant commonly have elevated levels of D-dimers.

The activated clotting time (ACT) test uses whole blood, which is mixed with a clot activator, usually diatomaceous earth or kaolin. Clotting typically takes 70–180 seconds, and can be measured mechanically or by an electrochemical procedure. Hospitals use the test at the bedside to monitor high-dose heparin anticoagulation during cardiopulmonary bypass surgery as well as during cardiac catheterization.

The coagulation factor activity assay determines the level of various coagulation factors. An essential component of the assay is a factor-deficient plasma that lacks the specific factor being tested. To perform the assay, the technologist dilutes the patient’s citrated plasma in a buffer and mixes the diluted specimen one-to-one with the factor-deficient plasma. The patient’s specimen supplies the missing factor to the assay, which is completed by performing a standard PT or aPTT, depending on the factor being tested. As an example for factors VIII and IX, which participate in the intrinsic coagulation pathway, the lab should run the aPTT test. In contrast, a PT assay would be used to determine factor VII, which is involved in the extrinsic coagulation pathway. Calibration of the assays involves a standard reference plasma with a known concentration of the factor being tested.

Platelet Function

Another component of laboratory assessment of hemostasis is platelet function testing. Platelet aggregometry is considered the classic test of platelet function. This test includes a platelet-activating agonist such as ADP, epinephrine, or collagen. The technologist adds the agonist to platelet-rich plasma and monitors platelet aggregation in a photometer cuvette. Optical density changes are plotted to view the aggregation curve.

Other devices measure platelet adhesion under high shear conditions. In this method, the instrument first draws an aliquot of the specimen through a capillary under conditions of high shear stress and then through a very small aperture in a disposable cartridge. The latter is coated with collagen and epinephrine or collagen and ADP. In normal individuals, this produces platelet adhesion and activation that eventually plugs the aperture and stops the flow. The assay end-point is called the closure-time.

The Anti-Xa Assay

Clinicians occasionally order the anti-Xa test to monitor and adjust patients’ levels of unfractionated heparin, a widely used anticoagulant. This chromogenic method generally contains exogenous factor Xa and anti-thrombin (AT), both in excess, as well as a chromogenic substrate for factor Xa. Some versions of the assay use the patient’s own AT, a serine protease inhibitor that is the major inhibitor of coagulation proteases, instead of adding exogenous AT. In either approach, heparin present in the specimen complexes with AT and this complex inhibits factor Xa. Any residual factor Xa cleaves the chromogenic substrate, thereby releasing a yellow-colored chromophore. Labs report these results as units per mL of anti-Xa activity.

Clinical Problems Associated with Bleeding

Many clinical conditions can lead to unexplained bleeding. Proper testing can help clinicians discern the cause. This section presents some of the more common causes and which tests should be used (Table 2).

Table 2
Sequential Use of Coagulation Assays to Assess Bleeding

This table shows how coagulation assays can be combined to elucidate the possible causes of unexplained bleeding.

Step
Test
Indication for Performing Test
Purpose of Test
Go to Step…
1
PT, aPTT, fibrinogen, platelet count unexplained bleeding assess extrinsic, intrinsic and common pathways and adequacy of platelet numbers
2
2
1:1 mixing study prolonged PT, aPTT assess deficiency/dysfunction vs inhibitor
3 or 4 or 5
3
specific factor activity assays mixing studies show correction assess factor deficiency or warfarin Rx
if factor VIII is low, proceed to 7 and 8
4
TT mixing study does not correct rule-out heparin contamination
if heparin is excluded, proceed to 6
 
5
anti-Xa assay mixing study does not correct
6
assay for specific factor inhibitors mixing study does not correct and heparin is excluded rule-out acquired hemophilia and other factor inhibitors
 
7
von Willebrand antigen and activity reduced factor VIII activity rule-out vWD
 
8
platelet function screen reduced factor VIII activity or normal plasma-based coagulation tests with normal platelet counts rule-out vWD and platelet dysfunction
 
9
factor XIII screen, tests for disorders of fibrinolysis (e.g., TEG) all previous testing is within normal limits rule-out fibrinolytic problems
 

Congenital hemophilia. The classic bleeding disorder, congenital hemophilia, is an X-linked, recessive genetic abnormality. Males with one copy of the defect are affected; however, females are asymptomatic carriers. The disease originates from one of two altered proteins in the coagulation cascade, factor VIII (hemophilia A) or factor IX (hemophilia B), which are indistinguishable clinically. The incidence of hemophilia A is 1 in 5,000 male live births, and that of hemophilia B is 1 in 30,000.

Patients with hemophilia have reduced factor VIII or factor IX activity in fresh, citrated plasma, and the disease is classified as severe (<1%), moderate (1–5%), or mild (6–30%) depending on the amount of factor activity. In the severe form, patients have spontaneous, deep bleeding into muscles and joints, as well as severe bleeding after injury. These individuals present in infancy. In contrast, the mild form may be diagnosed in adulthood and even as late as middle age. Affected individuals bleed after surgery or trauma but not spontaneously.

The following lab results are consistent findings for hemophilia: 1) significantly prolonged aPTT that corrects in a 1:1 mixing study; 2) PT within the reference range; 3) fibrinogen and TT within the reference range; 3) normal platelet function; and 4) normal von Willebrand test results (see below).

von Willebrand Disease. Defective synthesis or release of functional von Willebrand factor (vWF) causes defective platelet adhesion and leads to a spectrum of conditions, such as epistaxis, heavy menstrual bleeding, and easy bruising.

Labs use an immunoturbidimetric technique to measure the plasma concentration of vWF, and they measure vWF activity using the patient’s plasma in combination with formalin fixed, normal donor platelets and ristocetin, which serves as a cofactor in the assay. Another informative test examines the structure of vWF multimers. An ancillary test is factor VIII activity, since low vWF is frequently associated with low factor VIII activity. If the factor VIII activity is sufficiently decreased, aPTT may be prolonged. In most forms of von Willebrand disease, bleeding is a consequence of platelet dysfunction that results from the insufficiency of active vWF and not from reduced factor VIII activity.

Acquired hemophilia. Although rare, acquired hemophilia results from spontaneous formation of autoantibodies to factor VIII in a previously healthy individual, either male or female. As the name suggests, there is no overt genetic component. Factor VIII activity is significantly decreased as a result of the autoantibodies, and severe and uncontrolled bleeding results.

Laboratory studies reveal a markedly prolonged aPTT, a normal PT, and a low FVIII activity. For these patients, a 1:1 mixing study does not correct the abnormal aPTT.

Acquired von Willebrand Syndrome. Also uncommon, acquired von Willebrand syndrome (AVWS) refers to the development of von Willebrand deficiency relatively late in life in a previously healthy individual. AVWS may be seen in a variety of lymphoproliferative disorders, monoclonal plasma cell disorders, autoimmune disease, and cardiac valvular disease or septal defects. vWF activity in these individuals may be reduced by inhibitory antibodies, adsorption to cell surfaces, or areas of elevated shear stress that occur via a stenotic heart valve or septal defect.

Lab studies show decreased factor VIII activity, prolonged aPTT if factor VIII is <40%, impaired platelet response to ristocetin, and prolonged closure times.

None of the standard laboratory tests described above provide diagnostic information for identifying thrombophilic states. In the majority of cases, hypercoaguable disorders are acquired, and, except for antiphospholipid syndrome and the hereditary thrombophilias, there are no informative laboratory tests (Table 3).

Table 3
Laboratory Assessment of Hypercoaguable States
Hypercoaguable State
Clinical Scenario
Mechanisms
Laboratory Tests

Inflammation

Trauma

Surgery

Immobilization

Common in almost every postoperative surgical and trauma patient Elevated factor VIII, vWF, fibrinogen, inflammatory cytokines combined with venous stasis
None
Malignancy
(Trousseau
Syndrome)
Apparent or occult neoplasm Inflammation, cytokines, proteases. Production of tissue factor by tumor
None
Hormonal

Oral contraceptives

Pregnancy

Estrogen is associated with elevated factor II, VII, VIII, X and fibrinogen, decreased antithrombin, decreased protein S
None
Antiphospholipid Syndrome Acquired autoimmune disorder Antibodies to phospholipid and phospholipid binding proteins. Inflammation and endothelial damage. glycoprotein I Lupus anticoagulant testing antibodies to cardiolipin and beta2
Heparin-induced thrombocytopenia (HIT) Acquired antibody to heparin/platelet factor 4 complex Platelet activation and release of microparticles Test for antibodies to heparin/PF4 by ELISA
Hereditary Thrombophilia Unexplained thrombosis at an early age or thrombosis at unusual sites or recurrent thrombosis Multiple (see text)

PCR-based DNA analysis

Functional activity assays

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.

SUGGESTED READING

Eby CS. Warfarin Dosing. Should Labs Offer Pharmacogenetic Testing? CLN 2009;35(6).

Harris NS, Winter WE. The International Normalized Ratio A Tool for Monitoring Warfarin Therapy. CLN 2010;36(11).

Harris NS, Winter WE, Ledford-Kraemer MR. Hemostasis: A Review & Methods of Assessment in the Clinical Laboratory. Chapter 21, pp 265-283. IN: Contemporary Practice in Clinical Chemistry 2nd Edition, Clarke, W. 2011 (AACC Press, Washington DC).

Hoffbrand AV, Moss PAH. Essential Haematology. 6th Edition. Chapters 26 (Coagulation Disorders) and 27 (Thrombosis and Antithrombotic Therapy). Wiley-Blackwell 2011.

Hoffman M, Monroe DM. Coagulation 2006: A modern view of hemostasis. Hematol Oncol Clin N Am 2007;21:1–11.

Kroll MH. ThromboelastographyTheory and Practice in Measuring Hemostasis. CLN 2010;36(12). 

Neil Harris
Neil S. Harris, MBChB, MD, is clinical associate professor and co-director of the Core Diagnostic Laboratory in the Department of Pathology, Immunology, and Laboratory Medicine at the University of Florida College of Medicine in Gainesville. Email

Lindsay Bazydlo
Lindsay A.L. Bazydlo, PhD, is clinical assistant professor and director of clinical chemistry in the Department of Pathology, Immunology and Laboratory Medicine at the University of Florida College of Medicine in Gainesville. Email

 

William Winter
William E. Winter, MD, is professor of pathology and pediatrics at the University of Florida in Gainesville. Email

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