May 2000
Coagulation Case Study

John T. Brandt, MD

Table 1 | Figure 1 | Figure 2

This is the sixth in a periodic series of articles written by members of the CAP Coagulation Resource Committee and focusing on laboratory evaluation of coagulation disorders.

Case history. A 67-year-old woman with a 20-year history of type 2 diabetes mellitus was admitted for therapy of acute exacerbation of her peripheral vascular disease. Several years ago she underwent a revascularization procedure of the right leg that failed in the immediate postoperative period, necessitating a below-the-knee amputation. The amputation was subsequently revised to an above-the-knee amputation due to tissue necrosis. Two months prior to the current admission the patient developed nonhealing ulcers of the left lower extremity. An arteriogram at the time of admission revealed diffuse arterial disease. A left femoral-anterior tibial artery graft was placed.

On the first postoperative day she became cyanotic and short of breath, with a pO₂ of 40 mm Hg and an arterial pH of 7.17. She was resuscitated and did well until the 10th postoperative day, when progressive cyanosis of both hands was noted. The lab-oratory was requested to eval-uate the patient for acute disseminated intra-vas-cular coagulation (DIC). Laboratory studies at that time showed the values listed in Table 1.

Consider the following questions:

• Do the results of the laboratory studies indicate a diagnosis of acute DIC?
• What other diagnoses should be considered?
• What additional testing would you recommend to establish your alternate diagnoses?

A platelet aggregation assay for the diagnosis of HIT was performed and was strongly positive. Intermittent subcutaneous heparin was discontinued without improvement of the platelet count over two days. A heparin-coated catheter was then removed and the platelet count increased over the next two days. Unfortunately, the patient developed multiple organ failure and died on the 15th postoperative day.

Overview of heparin-induced thrombocytopenia. Heparin-induced thrombocytopenia is a serious clinicopathologic syndrome characterized by immune-mediated activation and clearance of platelets. Immune-mediated platelet activation may lead to significant arterial and/or venous thromboembolism while enhanced platelet clearance may contribute to moderate to severe thrombocytopenia.¹ Typically the question of whether a patient has HIT arises when either a decrease in the platelet count or onset of thrombosis is noted in the setting of heparin therapy.

The basic pathogenesis of HIT is now reasonably well characterized.^2-4 Upon exposure to exogenous heparin, multimolecular complexes composed of platelet factor 4 (PF4) and heparin form (Figure 1). The binding of PF4 to heparin in these complexes is associated with a conformational change in PF4, exposing a neoepitope. This heparin-dependent neoepitope elicits an immune response in some patients; the antibody response is usually an IgG type of antibody. This IgG binds to the PF4/heparin complex, creating an immune complex that binds to the receptor for IgG on the platelet surface (FcyRIIa). Aggregation of platelet FcyRIIa receptors by the immune complex triggers platelet activation and aggregation through transmembrane signaling. In addition, during the process of platelet activation, platelets release phospholipid microparticles derived from the cell membrane.⁵ These microparticles support the enzymatic reactions of the coagulation cascade, leading to thrombin formation; consequently, the release of these microparticles into the circulation is thrombogenic. In addition, the PF4/heparin immune complexes can bind to the endothelium, leading to endothelial injury.⁶ The endothelial damage may also participate in the “thrombotic storm” that can be associated with this syndrome.

As the PF4/heparin complex is necessary for the pathogenesis of this disorder, cessation of all sources of exogenous heparin is an essential component of the therapeutic approach to these patients. However, it should be remembered that increased thrombin generation is a component of this syndrome and simple cessation of heparin may not immediately reverse this process. Thus, it may also be critical to introduce an alternate immediate--acting anticoagulant, such as lepirudin or danaparoid, to control the risk or propagation of thrombosis.^1,7,8 Recent clinical data demonstrating a high rate of venous thromboembolic disease in the period following cessation of heparin without immediate alternate anticoagulation support this hypothesis.⁹ Warfarin should be avoided in patients with newly diagnosed HIT as it may be associated with venous limb gangrene.¹⁰ The pathogenesis of venous limb gangrene appears to be related to the combination of a rapid decrease in protein C due to warfarin and the continued generation of thrombin related to HIT.

Laboratory methods for diagnosing HIT. The laboratory is often called on to confirm the diagnosis of HIT to justify the continued use of alternate anticoagulants and guide future exposure to heparin. Two basic types of assays are used for diagnosing HIT. One group of assays, the functional assays, depends on detecting platelet activation following exposure of normal plate-lets to patient serum or plasma in the presence of heparin. The second group of assays, the antigenic assays, depends on detection of antibody binding to PF4/heparin (or heparin substitute). Recent clinical experience suggests these two different assay approaches are complementary, as neither alone is 100 percent sensitive.

The functional assays are based on the ability of patient immunoglobulin to activate normal platelets in the presence of heparin. The source of the patient immunoglobulin may be either serum or plasma. Detection of platelet activation may be based on platelet aggregation, release of granular contents (for example, serotonin), or change in membrane properties (flow cytometry). There are advantages and disadvantages to each of the major functional methods described.

The platelet aggregation method is the most widely used functional assay.¹¹ It is based on the observation that adding patient platelet-poor plasma (as a source of immunoglobulin) to normal platelet-rich plasma in the presence of heparin can induce platelet aggregation. Attention to technical details is important for the assay to be used successfully.¹² Donor platelets vary in their response to HIT-positive plasmas. Therefore, it is important to use platelets that are known to respond to HIT plasma and to use platelets from more than one donor. The final concentration of heparin in the assay is also important. If a single concentration of heparin is used, it should be about 0.4-0.5 U/mL. A common weakness of studies comparing the platelet aggregation assay to other functional assays is that insufficient heparin (for example, 0.1 U/mL) is used in the aggregation assay. We have found it useful to use two concentrations (0.2 and 0.5 U/mL) to increase the assay sensitivity. The platelet aggregation assay should be allowed to run for a sufficient period—a minimum of 10 to 15 minutes—before it is called negative. An aggregation response >20 percent greater than a buffer control (no heparin) is usually regarded as a positive result. A positive result is specific for HIT, but a negative result does not exclude HIT.

The serotonin release assay is a well-characterized assay that is technically demanding for the routine hospital laboratory. Donor platelets must be isolated from platelet-rich plasma by differential centrifugation and incubated with radiolabeled serotonin. The platelets must then be washed to remove free serotonin. The labeled platelets are then incubated at room temperature with patient serum (heat-treated) in the presence of low (0.1 U/mL) and high (100 U/mL) concentrations of heparin. A positive assay is defined as greater than 20 percent release in the presence of the low concentration of heparin and less than 20 percent release in the presence of the high concentration of heparin.¹³

As with the platelet aggregation assay, selection of donor platelets is critical for success with this assay. It may also be important to run controls (positive HIT samples) to ensure the platelets are still functional after the isolation and serotonin labeling procedure. The serotonin release assay is thought to be more sensitive than the platelet aggregation assay.¹⁴ Again, a positive result is specific for HIT, but a negative result does not exclude the syndrome.

The heparin-induced platelet agglutination (HIPA) assay is similar to the serotonin release assay in that it uses washed donor platelets and heat-treated patient serum.^15,16 In contrast to the serotonin release assay, the endpoint for the HIPA assay is macroscopic agglutination of platelets in a microtiter well. The results of the HIPA assay appear to compare well with functional and antigenic assays for HIT.¹⁶ The sensitivity and specificity of this assay appear to be similar to those of the serotonin release assay.

Flow cytometric assays for the diagnosis of HIT were developed recently.^17-20 The assays are based on incubating patient plasma with normal donor platelets in the presence or absence of heparin and then measuring a change in the platelet surface, such as binding of annexin to negatively charged phospholipids or expression of an activation marker such as P-selectin (CD62P). These assays appear to have a similar sensitivity and specificity for the diagnosis of HIT as does the serotonin release assay. Of particular interest, the assay described by Jy et al suggests it may be possible to distinguish patients at high risk for thrombosis from other patients with HIT.¹⁸ These assays do not require use of radioactive material and can be performed fairly rapidly in an efficient mode.¹⁹ This approach, however, requires a skilled flow cytometry laboratory.

The second group of assays measures the presence of antibodies capable of binding to PF4 bound to heparin or a heparin-like molecule.^21-23 The detection system can be modified to detect IgG, IgM, and/or IgA antibodies.²⁴ These assays are usually performed in an enzyme-linked immunosorbent assay (ELISA) format and thus are less amenable to single patient, rapid turnaround testing.

The ELISA assays appear to be more sensitive for the detection of PF4/heparin antibodies than the functional assays. However, it is now apparent that not all patients with antibodies detectable by the ELISA method have clinically evident HIT. For example, 40 to 60 percent of patients undergoing cardiopulmonary bypass procedures develop antibodies to PF4/heparin detectable by the ELISA method, yet few of these patients develop HIT.^25-27 In addition, it is apparent that other proteins can substitute for PF4 in the immunogenic complex.²⁸ These antibodies are not detected by the PF4/heparin ELISA method but may cause clinically significant HIT. Thus the ELISA method may be negative in some patients with HIT or give false positive results in patients without HIT.

Strategy for diagnosing HIT. The laboratory needs to provide access to appropriate testing to establish the diagnosis of HIT. There are two components to this issue: identifying the appropriate patients to evaluate and selecting the appropriate assays to perform. Laboratory testing should be performed whenever there is clinical evidence of HIT. This may include any of the following (Figure 2):

1. A greater than 50 percent decrease in the platelet count from baseline (pre-heparin) in association with exposure to heparin. The platelet count should be monitored at least every other day (q. 48 hours) in patients receiving heparin.
2. A decrease in the platelet count to <100,000/µl associated with exposure to heparin.
3. Evidence of a new throm-bo-embolic event (venous or arterial) while the patient is exposed to heparin.

Once the possibility of HIT is suspected on clinical grounds, the most important next step is to discontinue heparin, including removing heparin-coated catheters. The diagnosis of HIT should be confirmed using appropriate laboratory studies. As no method is 100 percent sensitive for the diagnosis of HIT, it is now recommended that a functional assay and an ELISA-type of method be performed before excluding the diagnosis.² A positive result with a functional assay is specific for the diagnosis of HIT, so it makes sense to perform the functional assay first. If a positive result is obtained, then the diagnosis is established and no further testing is required. If the functional assay is negative, then an ELISA may provide useful information. If both tests are negative, then HIT is unlikely. If the ELISA is positive, the patient should be treated as positive for HIT.

Occasionally patients with HIT may be negative using both functional and antigenic assays. These patients may develop recurrent thrombocytopenia or thromboembolism when re-exposed to heparin. The recurrence of either of these manifestations of HIT upon re-exposure to heparin should be taken as positive evidence for HIT, and the patient should be treated accordingly. Among patients with HIT, the platelet count usually starts to rise toward normal within a couple of days of stopping the heparin. Failure of the platelet count to rise following cessation of heparin should prompt a search for alternate sources of heparin, including heparin-coated catheters. If present, such catheters should be exchanged for nonheparin-coated catheters. Occasionally, the return of the platelet count may be delayed for weeks, even in the absence of heparin exposure.

Patients identified as having HIT should be educated about their condition, and the condition should be documented in their medical records. Patients with HIT should also consider carrying some indicator of their heparin “allergy” for use in emergency situations. Finally, patients should be strongly counseled against future exposure to heparin.

References

1. Warkentin TE. Heparin-induced thrombocytopenia: a clinicopathologic syndrome. Thromb Haemost. 1999;82: 439-447.
2. Warkentin TE, Chong BH, Greinacher A. Heparin-induced thrombocytopenia: towards consensus. Thromb Haemost. 1998;79:1-7.
3. Chong BH, Eisbacher M. Pathophysiology and laboratory testing of heparin-induced thrombocytopenia. Semin Hematol. 1998;35:3-8; discussion 35-36.
4. Visentin GP. Heparin-induced thrombocytopenia: molecular pathogenesis. Thromb Haemost. 1999;82:448-456.
5. Warkentin TE, Hayward CP, Boshkov LK, et al. Sera from patients with -heparin-induced thrombocytopenia generate platelet-derived microparticles with procoagulant activity: an explanation for the thrombotic complications of heparin-induced thrombocytopenia. Blood. 1994; 84:3691-3699.
6. Kwaan HC, Sakurai S. Endothelial cell hyperplasia contributes to thrombosis in heparin-induced thrombocytopenia. Semin Thromb Hemost. 1999;25:23-27.
7. Greinacher A, Janssens U, Berg G, et al. Lepirudin (recombinant hirudin) for parenteral anticoagulation in patients with heparin-induced thrombocytopenia. Heparin-Associated Thrombocytopenia Study (HAT) investigators. Circulation. 1999;100:587-593.
8. Greinacher A. Treatment of heparin--induced thrombocytopenia. Thromb Haemost. 1999;82:457-467.
9. Greinacher A, Volpel H, Janssens U, et al. Recombinant hirudin (lepirudin) provides safe and effective anticoagulation in patients with heparin-induced thrombocytopenia: a prospective study. Circulation. 1999;99:73-80.
10. Warkentin TE, Elavathil LJ, Hayward CP, Johnston MA, et al. The pathogenesis of venous limb gangrene associated with heparin-induced thrombocytopenia. Ann Intern Med. 1997;127:804-812.
11. Nguyen P, Lecompte T. Heparin--induced thrombocytopenia: a survey of tests employed and attitudes in haematology laboratories. Groupe d'Étude sur l'Hemostase et la Thrombose (GEHT) de la Societe Française d'Hematologie. Nouv Rev Fr Hematol. 1994; 36:353-357.
12. Isenhart CE, Brandt JT. Platelet aggregation studies for the diagnosis of heparin-induced thrombocytopenia. Am J Clin Pathol. 1993;99:324-330.
13. Sheridan D, Carter C, Kelton JG. A diagnostic test for heparin-induced thrombocytopenia. Blood. 1986;67:27-30.
14. Favaloro EJ, Bernal-Hoyos E, Exner T, Koutts J. Heparin-induced thrombocytopenia: laboratory investigation and confirmation of diagnosis. Pathology. 1992;24:177-183.
15. Greinacher A, Michels I, Kiefel V, Mueller-Eckhardt C. A rapid and sensitive test for diagnosing heparin--associated thrombocytopenia. Thromb Haemost. 1991;66:734-736.
16. Greinacher A, Amiral J, Dummel V, Vissac A, et al. Laboratory diagnosis of heparin-associated thrombocytopenia and comparison of platelet aggregation test, heparin-induced platelet activation test, and platelet factor 4/heparin enzyme-linked immunosorbent assay. Transfusion. 1994;34:381-385.
17. Tomer A. A sensitive and specific functional flow cytometric assay for the diagnosis of heparin-induced thrombocytopenia. Br J Haematol. 1997;98:648-656.
18. Jy W, Mao WW, Horstman LL, Valant PA, et al. A flow cytometric assay of platelet activation marker P-selectin (CD62P) distinguishes heparin-induced thrombocytopenia (HIT) from HIT with thrombosis (HITT). Thromb Haemost. 1999;82:1255-1259.
19. Tomer A, Masalunga C, Abshire TC. Determination of heparin-induced thrombocytopenia: a rapid flow cytometric assay for direct demonstration of antibody-mediated platelet activation. Am J Hematol. 1999;61:53-61.
20. Walenga JM, Jeske WP, Fasanella AR, Wood JJ, et al. Laboratory tests for the diagnosis of heparin-induced thrombocytopenia. Semin Thromb Hemost. 1999;25:43-49.
21. Amiral J, Bridey F, Dreyfus M, et al. Platelet factor 4 complexed to heparin is the target for antibodies generated in heparin-induced thrombocytopenia. Thromb Haemost. 1992;68:95-96.
22. Amiral J, Bridey F, Wolf M, et al. Antibodies to macromolecular platelet factor 4-heparin complexes in heparin-induced thrombocytopenia: a study of 44 cases. Thromb Haemost. 1995;73:21-28.
23. Visentin GP, Ford SE, Scott JP, Aster RH. Antibodies from patients with -heparin-induced thrombo-cyto-pen-ia/ throm-bosis are specific for platelet factor 4 complexed with heparin or bound to endothelial cells. J Clin Invest. 1994;93:81-88.
24. Amiral J, Wolf M, Fischer A, Boyer-Neumann C, et al. Pathogenicity of IgA and/or IgM antibodies to heparin-PF4 complexes in patients with heparin--induced thrombocytopenia. Br J Haematol. 1996;92:954-959.
25. Visentin GP, Malik M, Cyganiak KA, Aster RH. Patients treated with unfractionated heparin during open heart surgery are at high risk to form antibodies reactive with heparin:platelet factor 4 complexes. J Lab Clin Med. 1996;128: 376-383.
26. Bauer TL, Arepally G, Konkle BA, et al. Prevalence of heparin-associated antibodies without thrombosis in patients undergoing cardiopulmonary bypass surgery. Circulation. 1997;95:1242-1246.
27. Trossaert M, Gaillard A, Commin PL, Amiral J, et al. High incidence of anti--heparin/platelet factor 4 antibodies after cardiopulmonary bypass surgery. Br J Haematol. 1998;101:653-655.
28. Amiral J. Antigens involved in heparin-induced thrombocytopenia. Semin Hematol. 1999;36:7-11.