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Coagulation testing

Part 1: Current methods and challenges

David G.M. Carville and Kirk E. Guyer

Surgical procedures can put patients at risk of developing blood clots that may result in life-threatening clinical conditions, including deep vein thrombosis (DVT) and pulmonary embolism (PE). Procedures in which such risks are especially high include cardio-pulmonary bypass surgery (CPB), coronary artery bypass grafting (CABG), gynecological procedures, neurosurgery, orthopedic surgery, percutaneous transluminal coronary angioplasty (PTCA), and posttrauma reconstructive surgery (see Table I).^1–4 The risk of clotting is higher for these operations than for other procedures because they are inherently more invasive and expose the patient's blood to foreign surfaces that can initiate coagulation.^5,6

Table I. Incidence of deep vein thrombosis (DVT) and pulmonary embolism (PE) during surgical procedures.

To minimize the risks associated with such surgical procedures, patients are administered anticoagulants. Without appropriate prophylaxis, it is estimated that thrombotic complications would occur in as many as 50% of patients undergoing orthopedic surgery and as many as 25% of patients undergoing other surgeries. But while anticoagulant prophylaxis may reduce the risk of thrombosis, it may also increase the potential for serious hemorrhagic complications.⁷ Such a heightened risk can be especially dangerous for patients with previously silent lesions (e.g., carcinoma, gastrointestinal ulcers, trauma) that may be revealed for the first time during anticoagulant therapy. For these patients, the physician must adjust the dose of anticoagulant, reducing it to a level sufficient to prevent thrombosis without inducing hemorrhage.⁸

Reaction tray used in optical clot detection on the Coag-a-mate analyzer by Organon Teknika Corp. (Durham, NC). Photo Courtesy Reeves/Conlon/DUMC PhotoPath

For all these reasons, it is extremely important that health professionals be able to obtain an accurate assessment of patients' hemostatic status. Significant social and economic costs are incurred when patients develop thrombotic or hemorrhagic complications as a result of under- or overdosing. To ensure that surgical patients receive the correct dose of anticoagulant and antithrombotic agents, clinicians must evaluate each patient's hemostatic ranking before, during, and after the surgical procedure. For patients who are placed on long-term oral anticoagulant therapy, additional postdischarge monitoring is also critical.⁹

Phenotype coagulation testing plays a central role in providing the physician with important information to aid in minimizing patient risks. Whether the tests are performed in the central coagulation laboratory or in a near-patient setting (at the patient's bedside or in the patient's home after hospital discharge), they all aim to characterize the functioning of the patient's coagulation system so that the clinician can make appropriate decisions for therapeutic intervention. Following is a brief review of the tests currently used for coagulation testing and their utility in clinical settings.

Screening Tests

The traditional coagulation tests performed in clinical laboratories have been available for decades. The majority of such tests are functional end-point assays, in which a patient sample (plasma or whole blood) is incubated with exogenous reagents that activate the coagulation cascade, and the time until clot formation is measured. The clotting time of the patient sample is then compared to the clotting time of pooled normal plasma or whole blood to provide a standard measurement of the patient's hemostatic status. As described below, such clotting assays are commonly used as screening tests that evaluate the functioning of both the patient's intrinsic and extrinsic coagulation systems (see Figure 1).

Figure 1. The intrinsic and extrinsic coagulation pathways.

Activated Clotting Time Test. The ACT is a screening test that resembles the activated partial thromboplastin time (APTT) test, but is performed using fresh whole blood samples. ACT is used primarily to monitor a patient's coagulation status in connection with clinical procedures that involve the administration of high doses of heparin (e.g., CPB and PTCA). It is important to monitor a patient's response to heparin during such procedures because underdosing can result in pathological thrombus formation, whereas overdosing can lead to serious hemorrhagic complications.¹⁰ Because the ACT test is commonly performed outside the central coagulation laboratory, it is considered a point-of-care assay.

Activated Partial Thromboplastin Time Test. A common central laboratory test, APTT is used to evaluate the intrinsic coagulation pathway, which includes factors I, II, V, VIII, IX, X, XI, and XII. The test is performed using a plasma sample, in which the intrinsic pathway is activated by the addition of phospholipid, an activator (ellagic acid, kaolin, or micronized silica), and Ca²⁺. Formation of the Xase and prothrombinase complexes on the surface of the phospholipid enables prothrombin to be converted into thrombin, with subsequent clot formation. The result of the APTT test is the time (in seconds) required for this reaction. APTT is used to assess the overall competence of a patient's coagulation system, as a preoperative screening test for bleeding tendencies, and as a routine test for monitoring heparin therapy.¹¹

Bleeding Time Test. The bleeding time test is widely used for the diagnosis of hemostatic dysfunction, von Willebrand's disease, and vascular disorders. It is also used to screen for platelet abnormalities prior to surgery. The test is performed by making a small incision on the forearm and wicking away the blood from the wound site. The time it takes for bleeding to stop is recorded and in control subjects is approximately 3.5 minutes. Prolongation of the bleeding time is indicative of qualitative or quantitative platelet defects.

Prothrombin Time Test. First described by Quick in 1935, the PT test measures the tissue factor–induced coagulation time of blood or plasma.¹² It is used as a screening test to evaluate the integrity of the extrinsic coagulation pathway, and is sensitive to coagulation factors I, II, V, VII, and X. The test is performed by adding thromboplastin and Ca²⁺ to a patient sample and measuring the time for clot formation. A prolonged clotting time suggests the presence of an inhibitor to, or a deficiency in, one or more of the coagulation factors of the extrinsic pathway. But PT clotting time can also be prolonged for patients on warfarin therapy, or for those with vitamin K deficiency or liver dysfunction. The PT test can provide an assessment of the extrinsic coagulation pathway, and is widely used to monitor oral anticoagulation therapy.¹³ Again, this test is traditionally performed in the central laboratory.

Thrombin Clotting Time Test. The TCT test measures the rate of a patient's clot formation compared to that of a normal plasma control. The test is performed by adding a standard amount of thrombin to a patient's plasma that has been depleted of platelets, and measuring the time required for a clot to form. This test has been used as an aid in the diagnosis of disseminated intravascular coagulation (DIC) and liver disease.¹¹ The thrombin time test is generally performed in the central laboratory.

Diagnostic Tests

Beyond the tests commonly used for screening, there are a number of tests that may be used in the diagnosis of a patient's coagulative status. These fall into two categories: complex tests, some of which are based on the screening tests outlined above, and immunoassays.

Complex Tests. This category includes specific factor assays based on laboratory tests, such as the APTT, PT, and TCT tests. Such assays enable clinicians to reduce the number of possible explanations for a prolonged clotting time found in screening assays.

One such test is a clotting assay for factor VIIa, which has found utility in monitoring patients with severe factor IX deficiency. The level of factor VIIa in these patients has been reported to be less than 10% of the level found in healthy control subjects.

Another example is the assay for factor VIII, which constitutes a diagnostic test for classical hemophilia.

Another assay measures the level of the activation peptide factor IXa or the factor IXa–antithrombin III complex. These measurements are used to determine the levels of factor IXa or factor VII–tissue mediated complex. Patients with congenital deficiencies of factor VII may be monitored with this test.

Assays for activated protein C resistance, antithrombin, protein C deficiency, and protein S deficiency are also part of this group. Asymptomatic individuals who have heterogeneous deficiencies of proteins C and S, and resistance to activated protein C, have significantly elevated levels of the prothrombin fragment F1.2 compared to controls.

Immunoassays. In recent years, researchers have developed immunochemical assays that characterize a patient's hemostatic status by determining the concentration of peptides, proteins, and factors of the coagulation cascade found in the patient's sample. The most common immunoassays for coagulation factors and their by-products, together with their clinical utility, are listed in Table II.

Table II. Current immunochemical tests for measuring coagulation factors (an index of thrombin formation) and thrombosis (an index of thrombin activity).

As tests for thrombin formation, some of the commonly used immunoassays are considered more reflective of a hypercoagulable state rather than indicative of thrombosis. Such assays include tests for activated protein C; factors VIIa, IX, and X; prothrombin fragment F1.2; and thrombin-antithrombin (TAT) complexes. Another test for the measurement of factor XIIa (but not factor XII), has been proposed as a reliable indicator for activation of the contact coagulation system.

Other widely used immunoassays to evaluate thrombin activity include tests for fibrin D-dimer (a degradation product), fibrin monomers, fibrinopeptide A and B (activation products of fibrinogen), and soluble fibrin polymers. It is hoped that these tests will provide clinicians with a better overall assessment of the coagulation and thrombotic processes. Until the clinical utility of such immunoassays has attained widespread acceptance, however, most coagulation testing is likely to involve the traditional, laboratory-based clotting assays.

Challenges to Advanced Technologies

It is estimated that the annual number of at-risk surgeries in the United States is approximately 12 million (and 36 million worldwide). Since prophylactic anticoagulation is indicated for the majority of these patients, the market for laboratory coagulation assays and associated instrumentation is expected to exceed $600 million in 1998.

The coagulation testing market is not immune from the cost-reduction pressures being felt throughout the world's health-care systems. To capitalize on this market, therefore, manufacturers have had to develop alternative coagulation testing technologies with potential to reduce overall health-care costs. During the past few years, diagnostics firms have made considerable progress in this area, resulting in the appearance of near-patient tests using novel analyzers that boast microvolume test capabilities. Such tests have been promoted for use in both acute areas of the hospital and by outpatients on warfarin therapy who do not have ready access to a central coagulation laboratory. In the latter instance, patients must be specifically trained to perform prothrombin time self-testing at home.

Although the emergence of near-patient, microvolume coagulation testing has met with a generally positive reception in the marketplace, researchers continue to study the suitability and accuracy of such advanced technologies for assessing the adequacy of a patient's hemostatic system.¹⁴ Concerns arise because of the potential for surface-to-volume effects when small samples are employed, the extreme complexity of the medium being tested (blood), and the effects that sample processing can have on both the coagulation and thrombotic pathways.

Sample Size. The use of smaller sample volumes has been touted as a major advantage of next-generation coagulation analyzers. Near-patient analyzers now in use include models that use standard milliliter volumes of whole blood, and others designed to use microliter volumes of whole blood or citrated plasma.^15,16 However, the benefit of using microvolumes of blood may have overstated clinical appeal. Because of the difficulties involved in handling and processing microliter-volume samples, clinicians tend to draw much larger volumes of blood than required by a microanalyzer, discarding any portion of the sample that is not needed. With the possible exception of pediatric patients, blood loss from routine volume coagulation testing poses no known disadvantage.

The use of reduced sample volumes is attractive, in part, because it implies reduced reagent use—and therefore lower testing costs. But as sample and reagent volumes become very small, other factors such as the effects of surface-to-volume ratios may become greater sources of bias. For this reason, the most important performance issue for next-generation microanalyzers is whether a coagulation test using microliter volumes of sample can deliver results comparable to those of traditional laboratory tests using larger sample volumes. To be certain that such results are comparable, the results of assays run on microanalyzers should be compared to laboratory tests using the NCCLS method comparison protocol.¹⁷

In addition to these limitations that affect all coagulation testing, it is possible (if not probable) that microvolume coagulation analyzers may also encounter significant operational limitations resulting from the use of such small sample volumes. One such limitation could be caused by the chemistry of blood and its components.

Blood Component Chemistry. Like all physiological fluids, blood has an extremely complex makeup. It is composed of an aqueous portion (plasma) and cellular components (red cells, white cells, and platelets), each of which has a role in coagulation.¹⁸ But the importance of such hemostasis components does not imply that they are present in high concentrations. Of the more than 700 proteins contained in plasma, for instance, fewer than 20 are factors of hemostasis (Figure 1). It has also been estimated that factor II (prothrombin), the precursor to thrombin, represents less than 1 x 10^–4 of 1% of blood.

Working with such small amounts of analyte would add to the potential for preanalytical error by any microvolume analyzer, but is an even more important source of error for those instruments used to evaluate coagulation. This is especially true when the sample under analysis is whole, nonanticoagulated blood. Meticulous attention to sample processing is an important factor in ensuring that such tests provide valid results.

Processing Requirements. Conventional coagulation tests are laborious and difficult to perform. For this reason, the majority of coagulation testing is performed in a central laboratory, where the technicians are specifically trained to execute the exacting procedures necessary to run each test. Such procedures include correct sample collection into the appropriate anticoagulant, transportation of sample to the laboratory, precise pipetting and sample processing, and exact timing.¹⁹

Mechanical problems can begin with the sample-drawing procedure. Most laboratory-based clotting tests make use of venous blood that has been anticoagulated, and this is the type of sample most commonly recommended.¹⁸ But fingerstick assays use capillary blood, which may not be comparable to the blood in larger vessels.²⁰ Moreover, the traumatic nature of the draw for a fingerstick assay can initiate platelet and hemostatic activation prior to introduction of the sample into the analyzer.²¹ Before the results of a microvolume test using capillary blood can be accepted, it is therefore necessary to establish a correlation with standard-volume tests using venous anticoagulated blood.

Herein lies another problem for the laboratorian. To achieve accurate anticoagulation, the blood must be mixed exactly at a ratio of nine volumes of blood to one volume of sodium citrate. This ratio must be used even if only 40–50 µl of sample are to be used. Any deviations from this ratio can cause additional analytical error.

All whole blood and plasma microanalyzers use ultralow sample volumes in which approximately 50% of the content is cellular and 50% is plasma. The surface area of the sample vial or test strip is lubricated as the specimen is pulled along by capillary action. Such contact increases the likelihood of nonspecific adherence of plasma proteins to test surfaces, with consequent loss of analyte. In addition, exposure of blood to a foreign surface can initiate the coagulation cascade.²¹ Collectively, these factors have the potential to introduce significant analytical error.

An Evolving Dilemma

Coagulation testing continues to gain importance as a means of identifying and assessing bleeding tendencies in patients' complex coagulopathies. But while conventional, laboratory-based tests may provide an accurate assessment of a patient's hemostatic status, they are difficult to perform. In addition, clinicians and laboratorians are challenged to provide coagulation test results in a timely manner and at very low cost.

These challenges have led to the development of both central laboratory and near-patient analyzers for which little or no sample handling is required. Near-patient analyzers have the advantage that they can be used in critical-care settings such as operating rooms and coronary and intensive care units, as well as in patients' homes.^15,22,23

The manufacturers of such next-generation coagulation analyzers are striving to achieve a set of operational and economic characteristics that have become internationally recognized as the optimal elements for a coagulation analyzer.²² An analyzer that satisfied all of these requirements would have all of the following characteristics.

• Portability (small size and light weight).

• Achieves physiological temperatures (37°C).

• Easy to use (CLIA waivable, requiring no specialized training).

• Provides rapid results.

• Provides quantitative data (preferably with a hard copy for inclusion in patient files).

• Uses small volumes of whole blood.

• Only disposable parts contact patient sample.

• Capable of performing all coagulation tests.

• Uses reagents that have been standardized to provide international normalized ratios.

• Low equipment and operating costs to patient and institution.

The challenge of attaining all of these characteristics with a single analyzer is a difficult one. To accomplish this goal, manufacturers must confront not only the technological requirements of such systems, but also an evolving dilemma over which elements must be included (and which omitted) to create a commercially successful device.

Despite these difficulties, the introduction of such instruments is at hand. In the September issue of IVD Technology, part 2 of this article will examine the progress that manufacturers have made toward achieving the optimal analyzer for coagulation testing, and the benefits that such devices have for overall patient care.

References

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2. Poller L, Hirsh J, "Optimal Therapeutic Ranges for Oral Anticoagulation," in Thrombosis in Cardiovascular Disorders, Fuster V, Verstraete M (eds), Philadelphia, Saunders, pp 161–173, 1992.

3. Ogilby JD, Kopelman HA, Klein LW, et al., "Adequate Heparinization during PTCA:Assessment Using Activated Clotting Times," Cath Cardio Diag, 18:206–209, 1989.

4. Narins CR, Hillegass WB, Nelson CL, et al., "Relation between Activated Clotting Time during Angioplasty and Abrupt Closure," Circulation, 93:667–671, 1996.

5. Bick RL, "Disseminated Intravascular Coagulation: A Clinical/Laboratory Study of 48 Patients," Ann NY Acad Sci, 370:843, 1981.

6. Vogel G, Spanuth E, "Predictive Value of Fibrin Monomers in Postoperative Deep Vein Thrombosis," Klin Wochenscher, 68:1020–1026, 1990.

7. Levine MN, Hirsch J, "Hemorrhagic Complications of Long-Term Antithrombotic Treatment," in Thrombosis in Cardiovascular Disorders, Fuster V, Verstraete M (eds), Philadelphia, Saunders, pp 515–522, 1992.

8. Hull RD, Kakkar VV, Raskob GE, "Prevention of Venous Thrombosis and Pulmonary Embolism," in Thrombosis in Cardiovascular Disorders, Fuster V, Verstraete M (eds), Philadelphia, Saunders, pp 451–464, 1992.

9. Hirsh J, Salzman EW, Marder VJ, "Treatment of Thromboembolism," in Hemostasis and Thrombosis: Basic Principles and Clinical Practices (3rd ed), Colman RC, Hirsh J, Marder VJ, et al. (eds), Philadelphia, Lippincott, pp 1346–1366, 1994.

10. Esposito RA, Culliford AT, Colvin SB, et al., "The Role of the Activated Clotting Time in Heparin Administration and Neutralization for Cardiopulmonary Bypass," J Thorac Cardiovasc Surg, 85:174–185, 1983.

11. The Illustrated Guide to Diagnostic Tests, Springhouse, PA: Springhouse, pp 52–65, 1993.

12. Quick AJ, Stanley-Brown M, Bancroft FW, "A Study of the Coagulation Defects in Hemophilia and in Jaundice," Am J Med Sci, 190:501, 1935.

13. Verstraete M, Wessler S, "Heparins and Oral Anticoagulants," in Thrombosis in Cardiovascular Disorders, Fuster V, Verstraete M (eds), Philadelphia, Saunders, pp 121–140, 1992.

14. Fareed J, Iqbal O, Hoppenstadt JM, et al., "Point-of-Care Testing of Different Drugs and Hemostatic Formations," presented at the American Association for Clinical Chemistry Oak Ridge Conference, St. Louis, 1997.

15. Rose VL, Dermott SC, Murray BF, et al., "Decentralized Testing for Prothrombin Time and Activated Partial Thromboplastin Time Using a Dry Chemistry Portable Analyzer," Arch Pathol Lab Med, 117:611–617, 1993.

16. Chapman B, Coagulation Testing at the Bedside, Northfield, IL, College of American Pathologists, 1995.

17. National Committee on Clinical Laboratory Standards, Method Comparison and Bias Estimation Using Patient Samples, Approved Guideline, Wayne, PA, NCCLS, 1995.

18. Harker LA, Mann KG, "Thrombosis and Fibrinolysis," in Thrombosis in Cardiovascular Disorders, Fuster V, Verstraete M (eds), Philadelphia, Saunders, pp 1–16, 1992.

19. Wilson SG, Leumas JB, "Multicenter Evaluation of a Near-Patient Coagulation Test System," Am Clin Lab, 1995.

20. Peters RHM, Van den Besselar AMPH, Olthuis FMFG, "A Multicenter Study to Evaluate Method Dependency of the International Sensitivity Index of Bovine Thromboplastin," Thromb Hemost, 66:442–445, 1991.

21. Mann KG, Nesheim ME, Church WR, et al., "Surface-Dependent Reactions of the Vitamin K–Dependent Enzyme Complexes," Blood, 76:1–16, 1990.

22. Reich DL, "Monitoring Hemostasis in the Perioperative Period: Anticoagulation Control," J Card Vasc Anest, 5(6):4–7, 1991.

23. Andrew M, Adams M, "Prothrombin Time Testing at Home," Clin Hemost Rev, 10(11):22, 1996.

David G.M. Carville, PhD, is vice president for new product development, and Kirk E. Guyer, BS, is manager of the research and development laboratory, at Array Medical Laboratories (South Bend, IN).

Continue to Part II of this article.