Download PDF
Semin Thromb Hemost 2004; 30(3): 359-368
DOI: 10.1055/s-2004-831049
Copyright © 2004 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA.

A Critical Evaluation of Assays for Detecting Antibodies to the Heparin-PF4 Complex

John L. Francis1
  • 1Director, Florida Hospital Center for Hemostasis and Thrombosis, Orlando, Florida.
Further Information

Publication History

Publication Date:
29 July 2004 (online)

    Permissions and Reprints

Heparin‐induced thrombocytopenia (HIT) is a potentially catastrophic complication of heparin therapy. The syndrome is the result of the production of an antibody to the complex that forms between heparin and platelet factor 4 (H‐PF4) released from activated platelets. IgG antibodies bind to platelet Fc receptors and cause platelet activation, resulting in thrombocytopenia and greatly increased risk of thrombosis. Tests for the H‐PF4 antibody can be classified into functional assays (which rely on the demonstration of platelet activation) and immunoassays (which detect the presence of an antibody without regard for its functional ability). The functional assays have a greater specificity for clinical HIT, but require normal donor platelets and are relatively unstandardized. The immunoassays have the advantage of better standardization and do not require normal platelets, but may give positive results in the absence of clinical HIT. The choice of test is usually dictated by what is possible for a given laboratory in terms of instrumentation, expertise, and interest. For most institutions this will be a commercially available enzyme‐linked immunosorbent assay. Although HIT is a true clinicopathological syndrome, there are several reasons why its diagnosis still rests primarily on clinical grounds. First, laboratory tests may not be available locally. Second, they may not be available in a sufficiently timely manner. Finally, available tests are not completely sensitive or specific for the condition. There is, therefore, a continuing need to develop more rapid testing strategies with greater specificity for clinical HIT.

KEYWORDS

Heparin‐induced thrombocytopenia - HIT - platelet counts - heparin‐platelet factor 4 antibodies - laboratory diagnosis

REFERENCES

  • 1 Kelton J G, Smith J W, Warkentin T E, Hayward C P, Denomme G A, Horsewood P. Immunoglobulin G from patients with heparin‐induced thrombocytopenia binds to a complex of heparin and platelet factor 4.  Blood. 1994;  83 3232-3239
    PubMedGoogle Scholar
  • 2 Chong B H, Fawaz I, Chesterman C N, Berndt M C. Heparin‐induced thrombocytopenia: mechanism of interaction of the heparin‐dependent antibody with platelets.  Br J Haematol. 1989;  73 235-240
    PubMedGoogle Scholar
  • 3 Chong B H. Heparin‐induced thrombocytopenia.  J Thromb Haemost. 2003;  1 1471-1478
    CrossrefPubMedGoogle Scholar
  • 4 Warkentin T E. Clinical presentation of heparin‐induced thrombocytopenia.  Semin Hematol. 1998;  35 9-16
    PubMedGoogle Scholar
  • 5 Warkentin T E, Kelton J G. Temporal aspects of heparin‐induced thrombocytopenia.  N Engl J Med. 2001;  344 1286-1292
    CrossrefPubMedGoogle Scholar
  • 6 Warkentin T E, Kelton J G. Delayed‐onset heparin‐induced thrombocytopenia and thrombosis.  Ann Intern Med. 2001;  135 502-506
    CrossrefPubMedGoogle Scholar
  • 7 Rice L, Attisha W K, Drexler A, Francis J L. Delayed‐onset heparin‐induced thrombocytopenia.  Ann Intern Med. 2002;  136 210-215
    PubMedGoogle Scholar
  • 8 Warkentin T E. Platelet count monitoring and laboratory testing for heparin‐induced thrombocytopenia.  Arch Pathol Lab Med. 2002;  126 1415-1423
    PubMedGoogle Scholar
  • 9 Warkentin T E, Hayward C P, Smith C A, Kelly P M, Kelton J G. Determinants of donor platelet variability when testing for heparin‐induced thrombocytopenia.  J Lab Clin Med. 1992;  120 371-379
    PubMedGoogle Scholar
  • 10 Brandt J T, Isenhart C E, Osborne J M, Ahmed A, Anderson C L. On the role of platelet FcgammaRIIa phenotype in heparin‐induced thrombocytopenia.  Thromb Haemost. 1995;  74 1564-1572
    PubMedGoogle Scholar
  • 11 Greinacher A, Amiral J, Dummel V, Vissac A, Kiefel V, Mueller-Eckhardt C. 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
    CrossrefPubMedGoogle Scholar
  • 12 Sheridan D, Carter C, Kelton J G. Clinical usefulness of testing for a heparin‐dependent platelet‐aggregating factor in patients with suspected heparin‐associated thrombocytopenia.  J Lab Clin Med. 1984;  103 606-612
    PubMedGoogle Scholar
  • 13 Warkentin T E, Greinacher A. Laboratory testing for HIT. In: Warkentin TE, Greinacher A Heparin-Induced Thrombocytopenia New York; Marcel Dekker 2001: 231-269
    Google Scholar
  • 14 Harenberg J, Huhle G, Giese C et al.. Determination of serotonin release from platelets by enzyme immunoassay in the diagnosis of heparin‐induced thrombocytopenia.  Br J Haematol. 2000;  109 182-186
    CrossrefPubMedGoogle Scholar
  • 15 Koch S, Harenberg J, Odel M, Schmidt-Gayk H, Walch S, Budde U. Development of a high‐pressure liquid chromatography method for diagnosis of heparin‐induced thrombocytopenia.  Am J Clin Pathol. 2002;  117 900-904
    CrossrefPubMedGoogle Scholar
  • 16 Gobbi G, Mirandola P, Tazzari P L et al.. Flow cytometry detection of serotonin content and release in resting and activated platelets.  Br J Haematol. 2003;  121 892-896
    CrossrefPubMedGoogle Scholar
  • 17 Stewart M W, Etches W S, Boshkov L K, Gordon P A. Heparin‐induced thrombocytopenia: an improved method of detection based on lumi‐aggregometry.  Br J Haematol. 1995;  91 173-177
    CrossrefPubMedGoogle Scholar
  • 18 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
    Article in Thieme ConnectPubMedGoogle Scholar
  • 19 Warkentin T E, Hayward C P, Boshkov L K 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
    CrossrefPubMedGoogle Scholar
  • 20 Lee D H, Warkentin T E, Denomme G A, Hayward C PM, Kelton J G. A diagnostic test for heparin‐induced thrombocytopenia: detection of platelet microparticles using flow cytometry.  Br J Haematol. 1996;  95 724-731
    CrossrefPubMedGoogle Scholar
  • 21 Tomer A. A sensitive and specific functional flow cytometric assay for the diagnosis of heparin‐induced thrombocytopenia.  Br J Haematol. 1997;  98 648-656
    CrossrefPubMedGoogle Scholar
  • 22 Vitale M, Tazzari P, Ricci F et al.. Comparison between different laboratory tests for the detection and prevention of heparin‐induced thrombocytopenia.  Cytometry. 2001;  46 290-295
    CrossrefPubMedGoogle Scholar
  • 23 Jy W, Mao W W, Horstman L L, Valant P A, Ahn Y S. 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
    PubMedGoogle Scholar
  • 24 Chong B H, Grace C S, Rozenberg M C. Heparin‐induced thrombocytopenia: effect of heparin platelet antibody on platelets.  Br J Haematol. 1981;  49 531-540
    PubMedGoogle Scholar
  • 25 Chong B H, Burgess J, Ismail F. The clinical usefulness of the platelet aggregation test for the diagnosis of heparin‐induced thrombocytopenia.  Thromb Haemost. 1993;  69 344-350
    Article in Thieme ConnectPubMedGoogle Scholar
  • 26 Trossaert M, Gaillard A, Commin P L, Amiral J, Vissac A M, Fressinaud E. High incidence of anti‐heparin/platelet factor 4 antibodies after cardiopulmonary bypass surgery.  Br J Haematol. 1998;  101 653-655
    CrossrefPubMedGoogle Scholar
  • 27 Francis J L, Palmer III G J, Moroose R, Drexler A. Comparison of bovine and porcine heparin in heparin antibody formation after cardiac surgery.  Ann Thorac Surg. 2003;  75 17-22
    CrossrefPubMedGoogle Scholar
  • 28 Mattioli A V, Bonetti L, Sternieri S, Mattioli G. Heparin‐induced thrombocytopenia in patients treated with unfractionated heparin: prevalence of thrombosis in a 1-year follow‐up.  Ital Heart J. 2000;  1 39-42
    PubMedGoogle Scholar
  • 29 Williams R T, Damaraju L V, Mascelli M A et al.. Anti‐platelet factor 4/heparin antibodies: an independent predictor of 30‐day myocardial infarction after acute coronary ischemic syndromes.  Circulation. 2003;  107 2307-2312
    CrossrefPubMedGoogle Scholar
  • 30 Look K A, Sahud M, Flaherty S, Zehnder J L. Heparin‐induced platelet aggregation vs platelet factor 4 enzyme‐linked immunosorbent assay in the diagnosis of heparin‐induced thrombocytopenia‐thrombosis.  Am J Clin Pathol. 1997;  108 78-82
    PubMedGoogle Scholar
  • 31 Walenga J M, Jeske W P, Fasanella A R, Wood J J, Bakhos M. Laboratory tests for the diagnosis of heparin‐induced thrombocytopenia.  Semin Thromb Hemost. 1999;  25(suppl 1) 43-49
    PubMedGoogle Scholar
  • 32 Vun C M, Evans S, Chesterman C N. Anti-PF4-heparin immunoglobulin G is the major class of heparin‐induced thrombocytopenia antibody: findings of an enzyme‐linked immunofiltration assay using membrane‐bound hPF4-heparin.  Br J Haematol. 2001;  112 69-75
    CrossrefPubMedGoogle Scholar
  • 33 Untch B, Ahmad S, Jeske W P et al.. Prevalence, isotype, and functionality of antiheparin‐platelet factor 4 antibodies in patients treated with heparin and clinically suspected for heparin‐induced thrombocytopenia. The pathogenic role of IgG.  Thromb Res. 2002;  105 117-123
    CrossrefPubMedGoogle Scholar
  • 34 Lindhoff-Last E, Gerdsen F, Ackermann H, Bauersachs R. Determination of heparin‐platelet factor 4-IgG antibodies improves diagnosis of heparin‐induced thrombocytopenia.  Br J Haematol. 2001;  113 886-890
    CrossrefPubMedGoogle Scholar
  • 35 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
    Article in Thieme ConnectPubMedGoogle Scholar
  • 36 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
    PubMedGoogle Scholar
  • 37 Izban K F, Lietz H W, Hoppensteadt D A et al.. Comparison of two PF4/heparin ELISA assays for the laboratory diagnosis of heparin‐induced thrombocytopenia.  Semin Thromb Hemost. 1999;  25(suppl 1) 51-56
    PubMedGoogle Scholar
  • 38 Arepally G, Reynolds C, Tomaski A et al.. Comparison of PF4/heparin ELISA assay with the 14C-serotonin release assay in the diagnosis of heparin‐induced thrombocytopenia.  Am J Clin Pathol. 1995;  104 648-654
    PubMedGoogle Scholar
  • 39 Newman P M, Swanson R L, Chong B H. Heparin‐induced thrombocytopenia: IgG binding to PF4-heparin complexes in the fluid phase and cross‐reactivity with low molecular weight heparin and heparinoid.  Thromb Haemost. 1998;  80 292-297
    PubMedGoogle Scholar
  • 40 Meyer O, Salama A, Pittet N, Schwind P. Rapid detection of heparin‐induced platelet antibodies with particle gel immunoassay (ID‐HPF4).  Lancet. 1999;  354 1525-1526
    CrossrefPubMedGoogle Scholar
  • 41 Eichler P, Raschke R, Lubenow N, Meyer O, Schwind P, Greinacher A. The new ID-heparin/PF4 antibody test for rapid detection of heparin‐induced antibodies in comparison with functional and antigenic assays.  Br J Haematol. 2002;  116 887-891
    CrossrefPubMedGoogle Scholar
  • 42 Alberio L, Kimmerle S, Baumann A, Taleghani B M, Biasiutti F D, Lämmle B. Rapid determination of anti‐heparin/platelet factor 4 antibody titers in the diagnosis of heparin‐induced thrombocytopenia.  Am J Med. 2003;  114 528-536
    CrossrefPubMedGoogle Scholar
  • 43 Tazzari P L, Ricci F, Vitale M et al.. Heparin‐induced thrombocytopenia: detection of antiheparin/PF4 antibodies by means of heparin/PF4-coated beads and flow cytometry.  Transfus Med. 2002;  12 193-198
    CrossrefPubMedGoogle Scholar
  • 44 Wang L, Huhle G, Malsch R, Hoffmann U, Song X, Harenberg J. Determination of heparin‐induced IgG antibody by fluorescence‐linked immunofiltration assay (FLIFA).  J Immunol Methods. 1999;  222 93-99
    CrossrefPubMedGoogle Scholar
  • 45 Warkentin T E. Heparin‐induced thrombocytopenia: pathogenesis and management.  Br J Haematol. 2003;  121 535-555
    CrossrefPubMedGoogle Scholar
  • 46 Refaai M A, Laposata M, Van Cott E M. Clinical significance of a borderline titer in a negative ELISA test for heparin‐induced thrombocytopenia.  Am J Clin Pathol. 2003;  119 61-65
    CrossrefPubMedGoogle Scholar
  • 47 Rugeri L, Bauters A, Trillot N et al.. Hemostasis and thrombosis: clinical usefulness of combined use of platelet aggregation test and anti PF4-H antibodies ELISA test for the diagnosis of heparin‐induced thrombocytopenia.  Hematology. 1999;  4 367-372
    PubMedGoogle Scholar
  • 48 Walenga J M, Jeske W P, Fasanella A R, Wood J J, Ahmad S, Bakhos M. Laboratory diagnosis of heparin‐induced thrombocytopenia.  Clin Appl Thromb Hemost. 1999;  5(suppl 1) S21-S27
    CrossrefPubMedGoogle Scholar
  • 49 Pouplard C, Amiral J, Borg J Y, Laporte-Simitsidis S, Delahousse B, Gruel Y. Decision analysis for use of platelet aggregation test, carbon 14-serotonin release assay, and heparin‐platelet factor 4 enzyme‐linked immunosorbent assay for diagnosis of heparin‐induced thrombocytopenia.  Am J Clin Pathol. 1999;  111 700-706
    PubMedGoogle Scholar

 Dr.
John L FrancisPh.D. 

Florida Hospital Center for Hemostasis and Thrombosis

2501 North Orange Avenue, Suite 786, Orlando, FL 32804

Email: john.francis@flhosp.org