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Thromb Haemost 2017; 117(01): 127-138
DOI: 10.1160/TH16-06-0482
New Technologies, Diagnostic Tools and Drugs
Schattauer GmbH

Identifying patients at high risk of heparin-induced thrombocytopenia-associated thrombosis with a platelet activation assay using flow cytometry

Takuma Maeda
1   Division of Transfusion Medicine, National Cerebral and Cardiovascular Center, Osaka, Japan
,
Katsura Nakagawa
2   Department of Anesthesiology, National Cerebral and Cardiovascular Center, Osaka, Japan
,
Kuniko Murata
2   Department of Anesthesiology, National Cerebral and Cardiovascular Center, Osaka, Japan
,
Yoshiaki Kanaumi
1   Division of Transfusion Medicine, National Cerebral and Cardiovascular Center, Osaka, Japan
,
Shu Seguchi
1   Division of Transfusion Medicine, National Cerebral and Cardiovascular Center, Osaka, Japan
,
Shiori Kawamura
1   Division of Transfusion Medicine, National Cerebral and Cardiovascular Center, Osaka, Japan
,
Mayumi Kodama
1   Division of Transfusion Medicine, National Cerebral and Cardiovascular Center, Osaka, Japan
,
Takeshi Kawai
1   Division of Transfusion Medicine, National Cerebral and Cardiovascular Center, Osaka, Japan
,
Isami Kakutani
1   Division of Transfusion Medicine, National Cerebral and Cardiovascular Center, Osaka, Japan
,
Yoshihiko Ohnishi
2   Department of Anesthesiology, National Cerebral and Cardiovascular Center, Osaka, Japan
,
Koichi Kokame
3   Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Osaka, Japan
,
Hitoshi Okazaki
4   Department of Transfusion Medicine, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan
,
Shigeki Miyata
1   Division of Transfusion Medicine, National Cerebral and Cardiovascular Center, Osaka, Japan
› Author Affiliations Financial support: This research was supported in part by a Health and Labour Sciences Research Grant from the Ministry of Health, Labour and Welfare of Japan; the Intramural Research Fund (27–1–2) for Cardiovascular Disease of the National Cerebral and Cardiovascular Center; and a grant-in-aid from the Takeda Science Foundation.
Further Information

Publication History

Received: 29 June 2016

Accepted after major revision: 17 September 2016

Publication Date:
01 December 2017 (online)

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Summary

To diagnose heparin-induced thrombocytopenia (HIT), detection of platelet-activating antibodies (HIT antibodies) is crucial. However, serum platelet activation profiles vary across patients and depend on test conditions. We evaluated the association between clinical outcomes and platelet-activating profiles assessed by a platelet microparticle assay (PMA), which detects activation of washed platelets induced by HIT antibodies, in 401 consecutive patients clinically suspected of having HIT. We made modifications to the assay, such as donor selection for washed platelets that increased sensitivity. Serum that activated platelets at a therapeutic (but not high) heparin concentration was defined as positive. Of these, serum that activated platelets within 30 minutes or in the absence of heparin was defined as strongly positive. The remaining samples were considered weakly positive. As a result, 97 % and 93 % of patients who tested strongly and weakly positive had clinical profiles consistent with HIT, respectively. The incidence of thromboembolic events (TEEs) after heparin exposure in patients who tested strongly positive, weakly positive, and negative was 61 %, 40 %, and 29 %, respectively. Among patients who did not experience a TEE on the day HIT was suspected, there was no significant difference in the cumulative incidence of subsequent TEEs between patients who tested strongly and weakly positive when argatroban was initiated on the same day (19.0 % vs 7.1 %, p=0.313), but there was a significant difference when argatroban therapy was delayed by one or more days (61.1 % vs 17.6 %, p=0.007). The modified PMA is effective in diagnosing HIT and identifying patients at high risk for HIT-associated TEEs.

Supplementary Material to this article is available online at www.thrombosis-online.com.

Keywords

Platelet-activating antibodies - heparin - platelet activation - thrombocytopenia - thromboembolism

 

  • References

  • 1 Greinacher A. CLINICALPRACTICE. Heparin-Induced Thrombocytopenia. N Engl J Med 2015; 373: 252-261.
    CrossrefPubMedGoogle Scholar
  • 2 Greinacher A, Gopinadhan M, Gunther JU. et al. Close approximation of two platelet factor 4 tetramers by charge neutralization forms the antigens recognized by HIT antibodies. Arterioscl Thromb Vasc Biol 2006; 26: 2386-2393.
    CrossrefPubMedGoogle Scholar
  • 3 Greinacher A, Alban S, Omer-Adam MA. et al. Heparin-induced thrombocy-topenia: a stoichiometry-based model to explain the differing immunogenicities of unfractionated heparin, low-molecular-weight heparin, and fondaparinux in different clinical settings. Thromb Res 2008; 122: 211-220.
    CrossrefPubMedGoogle Scholar
  • 4 Hughes M, Hayward CP, Warkentin TE. et al. Morphological analysis of micro-particle generation in heparin-induced thrombocytopenia. Blood 2000; 96: 188-194.
    PubMedGoogle Scholar
  • 5 Padmanabhan A, Jones CG, Bougie DW. et al. Heparin-independent, PF4-dependent binding of HIT antibodies to platelets: implications for HIT pathogenesis. Blood 2015; 125: 155-161.
    CrossrefPubMedGoogle Scholar
  • 6 Kasthuri RS, Glover SL, Jonas W. et al. PF4/heparin-antibody complex induces monocyte tissue factor expression and release of tissue factor positive microparticles by activation of FcgammaRI. Blood 2012; 119: 5285-5293.
    CrossrefPubMedGoogle Scholar
  • 7 Visentin GP, Ford SE, Scott JP. et al. Antibodies from patients with heparin-induced thrombocytopenia/thrombosis are specific for platelet factor 4 complexed with heparin or bound to endothelial cells. J Clin Invest 1994; 93: 81-88.
    CrossrefPubMedGoogle Scholar
  • 8 Blank M, Shoenfeld Y, Tavor S. et al. Anti-platelet factor 4/heparin antibodies from patients with heparin-induced thrombocytopenia provoke direct activation of microvascular endothelial cells. Int Immunol 2002; 14: 121-129.
    CrossrefPubMedGoogle Scholar
  • 9 Warkentin TE, Sheppard JA, Heels-Ansdell D. et al. Heparin-induced throm-bocytopenia in medical surgical critical illness. Chest 2013; 144: 848-858.
    CrossrefPubMedGoogle Scholar
  • 10 Lo GK, Sigouin CS, Warkentin TE. What is the potential for overdiagnosis of heparin-induced thrombocytopenia?. Am J Hematol 2007; 82: 1037-1043.
    CrossrefPubMedGoogle Scholar
  • 11 Lubenow N, Eichler P, Lietz T. et al. Lepirudin in patients with heparin-induced thrombocytopenia - results of the third prospective study (HAT-3) and a combined analysis of HAT-1, HAT-2, and HAT-3. J Thromb Haemost 2005; 03: 2428-2436.
    CrossrefPubMedGoogle Scholar
  • 12 Warkentin TE, Kelton JG. A 14-year study of heparin-induced thrombocytopenia. Am J Med 1996; 101: 502-507.
    CrossrefPubMedGoogle Scholar
  • 13 Linkins LA, Dans AL, Moores LK. et al. Treatment and prevention of heparin-induced thrombocytopenia: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest 2012; 141 (02) Suppl e495S-530S.
    CrossrefPubMedGoogle Scholar
  • 14 Cuker A, Gimotty PA, Crowther MA. et al. Predictive value of the 4Ts scoring system for heparin-induced thrombocytopenia: a systematic review and meta-analysis. Blood 2012; 120: 4160-4167.
    CrossrefPubMedGoogle Scholar
  • 15 Warkentin TE, Sheppard JA, Horsewood P. et al. Impact of the patient population on the risk for heparin-induced thrombocytopenia. Blood 2000; 96: 1703-1708.
    PubMedGoogle Scholar
  • 16 Warkentin TE, Sheppard JI, Moore JC. et al. Quantitative interpretation of optical density measurements using PF4-dependent enzyme-immunoassays. J Thromb Haemost 2008; 06: 1304-1312.
    CrossrefPubMedGoogle Scholar
  • 17 Kawano H, Yamamoto H, Miyata S. et al. Prospective multicentre cohort study of heparin-induced thrombocytopenia in acute ischaemic stroke patients. Br J Haematol 2011; 154: 378-386.
    CrossrefPubMedGoogle Scholar
  • 18 Warkentin TE, Arnold DM, Nazi I. et al. The platelet serotonin-release assay. Am J Hematol 2015; 90: 564-572.
    CrossrefPubMedGoogle Scholar
  • 19 Warkentin TE. How I diagnose and manage HIT. Hematology / the Education Program of the American Society of Hematology American Society of Hematol-ogy Education Program 2011; 2011: 143-149.
    CrossrefPubMedGoogle Scholar
  • 20 Warkentin TE, Levine MN, Hirsh J. et al. Heparin-induced thrombocytopenia in patients treated with low-molecular-weight heparin or unfractionated heparin. N Engl J Med 1995; 332: 1330-1335.
    CrossrefPubMedGoogle Scholar
  • 21 Nazi I, Arnold DM, Warkentin TE. et al. Distinguishing between anti-platelet factor 4/heparin antibodies that can and cannot cause heparin-induced throm-bocytopenia. J Thromb Haemost 2015; 13: 1900-1907.
    CrossrefPubMedGoogle Scholar
  • 22 Padmanabhan A, Jones CG, Curtis BR. et al. A novel PF4-dependent platelet activation assay identifies patients likely to have heparin-induced thrombocytopenia/thrombosis (HIT). Chest 2016; 150: 506-515.
    CrossrefPubMedGoogle Scholar
  • 23 Lee DH, Warkentin TE, Denomme GA. et al. A diagnostic test for heparin-induced thrombocytopenia: detection of platelet microparticles using flow cytometry. Br J Haematol 1996; 95: 724-731.
    CrossrefPubMedGoogle Scholar
  • 24 Maeda T, Noguchi T, Saito S. et al. Impact of heparin-induced thrombocytopenia on acute coronary artery thrombosis in patients undergoing PCI. Thromb Haemost 2014; 112: 624-626.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 25 Warkentin TE, Linkins LA. Non-necrotizing heparin-induced skin lesions and the 4T’s score. J Thromb Haemost 2010; 08: 1483-1485.
    CrossrefPubMedGoogle Scholar
  • 26 Asada R, Wanaka K, Walenga J. et al. Murine monoclonal antibody to platelet factor 4/heparin complexes as a potential reference standard for platelet activation assays in heparin-induced thrombocytopenia. Clin Appl Thromb Hemost 2013; 19: 37-41.
    CrossrefPubMedGoogle Scholar
  • 27 Kanda Y. Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transplant 2013; 48: 452-458.
    CrossrefPubMedGoogle Scholar
  • 28 Greinacher A, Farner B, Kroll H. et al. Clinical features of heparin-induced thrombocytopenia including risk factors for thrombosis. A retrospective analysis of 408 patients. Thromb Haemost 2005; 94: 132-135.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 29 Nagler M, Bachmann LM, ten Cate H, ten Cate-Hoek A. Diagnostic value of im-munoassays for heparin-induced thrombocytopenia: a systematic review and meta-analysis. Blood 2016; 127: 546-557.
    CrossrefPubMedGoogle Scholar
  • 30 Greinacher A, Ittermann T, Bagemuhl J. et al. Heparin-induced thrombocytopenia: towards standardization of platelet factor 4/heparin antigen tests. J Thromb Haemost 2010; 08: 2025-2031.
    CrossrefPubMedGoogle Scholar
  • 31 Greinacher A, Juhl D, Strobel U. et al. Heparin-induced thrombocytopenia: a prospective study on the incidence, platelet-activating capacity and clinical significance of antiplatelet factor 4/heparin antibodies of the IgG, IgM, and IgA classes. J Thromb Haemost 2007; 05: 1666-1673.
    CrossrefPubMedGoogle Scholar
  • 32 Amiral J, Marfaing-Koka A, Wolf M. et al. Presence of autoantibodies to inter-leukin-8 or neutrophil-activating peptide-2 in patients with heparin-associated thrombocytopenia. Blood 1996; 88: 410-416.
    PubMedGoogle Scholar
  • 33 Selleng S, Selleng K, Friesecke S. et al. Prevalence and clinical implications of anti-PF4/heparin antibodies in intensive care patients: a prospective observational study. J Thromb Thrombolysis 2015; 39: 60-67.
    CrossrefPubMedGoogle Scholar
  • 34 Greinacher A, Michels I, Kiefel V. et al. A rapid and sensitive test for diagnosing heparin-associated thrombocytopenia. Thromb Haemost 1991; 66: 734-736.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 35 Eichler P, Budde U, Haas S. et al. First workshop for detection of heparin-induced antibodies: validation of the heparin-induced platelet-activation test (HIPA) in comparison with a PF4/heparin ELISA. Thromb Haemost 1999; 81: 625-629.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 36 Greinacher A, Feigl M, Mueller-Eckhardt C. Crossreactivity studies between sera of patients with heparin associated thrombocytopenia and a new low molecular weight heparin, reviparin. Thromb Haemost 1994; 72: 644-645.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 37 Nazi I, Arnold DM, Moore JC. et al. Pitfalls in the diagnosis of heparin-Induced thrombocytopenia: A 6-year experience from a reference laboratory. Am J He-matol 2015; 90: 629-633.
    CrossrefPubMedGoogle Scholar
  • 38 Greinacher A, Eichler P, Lubenow N. et al. Heparin-induced thrombocytopenia with thromboembolic complications: meta-analysis of 2 prospective trials to assess the value of parenteral treatment with lepirudin and its therapeutic aPTT range. Blood 2000; 96: 846-851.
    PubMedGoogle Scholar
  • 39 Warkentin TE, Kelton JG. Delayed-onset heparin-induced thrombocytopenia and thrombosis. Ann Intern Med 2001; 135: 502-506.
    CrossrefPubMedGoogle Scholar
  • 40 Rice L, Attisha WK, Drexler A. et al. Delayed-onset heparin-induced throm-bocytopenia. Ann Intern Med 2002; 136: 210-215.
    CrossrefPubMedGoogle Scholar
  • 41 Crowther MA, Cook DJ, Albert M. et al. The 4Ts scoring system for heparin-induced thrombocytopenia in medical-surgical intensive care unit patients. J Crit Care 2010; 25: 287-293.
    CrossrefPubMedGoogle Scholar