Standardization of HIT Diagnostic Assays, with K070, a chimeric human-mouse antibody, mimicking heparin dependent pathogenic antibodies
Main Article Content
Abstract
This study reports the K070 monoclonal antibody, a human-mouse recombinant chimeric monoclonal antibody, IgG1 isotype, targeted to Heparin and PF4 complexes, which mimics Heparin Induced Thrombocytopenia. This antibody has a strong reactivity to Heparin-PF4 complexes, and a much weaker one to PF4 alone. When plasmas spiked with this antibody at concentrations ≥ 0.5 µg/ml are tested at the standard immunoassay dilution, a dose dependent reactivity is obtained. This opens the perspective to use this material to calibrate immunoassays for a quantitative measurement of heparin dependent antibodies, when the disease is suspected. Furthermore, when plasmas are spiked at a higher concentration, from 20 to 200 µg/ml, they test positive in functional assays, like Platelet Aggregation/Activation Tests or Flow Cytometry methods, in presence of a low (0.1-1.0 IU/ml) but not a high (10-100 IU/ml) heparin concentration. In functional assays K070 reactivity level depends on the platelet rich plasmas used. In Platelet Aggregometry Tests some platelet rich plasmas are already positive when tested with plasmas supplemented with 20 µg/ml, whilst others require 100 to 200 µg/ml. At this latter concentration, all tested plasmas generate a strong positive responses with functional assays. K070 is then a useful antibody for calibration of immunoassays and standardization of functional methods used to diagnose Heparin Induced Thrombocytopenia.
Keywords:
K070 HIT mimicking antibody; Functional assays; Immunoassays; Calibration; Heparin Induced Thrombocytopenia; HPF4 complexes
Article Details
How to Cite
AMIRAL, Jean et al.
Standardization of HIT Diagnostic Assays, with K070, a chimeric human-mouse antibody, mimicking heparin dependent pathogenic antibodies.
Medical Research Archives, [S.l.], v. 11, n. 11, nov. 2023.
ISSN 2375-1924.
Available at: <https://esmed.org/MRA/mra/article/view/4760>. Date accessed: 16 may 2024.
doi: https://doi.org/10.18103/mra.v11i11.4760.
Section
Research Articles
The Medical Research Archives grants authors the right to publish and reproduce the unrevised contribution in whole or in part at any time and in any form for any scholarly non-commercial purpose with the condition that all publications of the contribution include a full citation to the journal as published by the Medical Research Archives.
References
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21. Price EA, Hayward CPM, Moffat KA, Moore JC, Warkentin TE, Zehnder JL. Laboratory testing for heparin-induced thrombocytopenia is inconsistent in North America: a survey of North American specialized coagulation laboratories. Thromb Haemost. 2007;98(6):1357-1361. doi:10.1160/th07-06-0401
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(1):21-28.
23. Luna E, Agrawal P, Mehta R, et al. Evaluation of Immunostimulatory Potential of Branded and US-Generic Enoxaparins in an In Vitro Human Immune System Model. Clin Appl Thromb Off J Int Acad Clin Appl Thromb. 2015;21(3):211-222. doi:10.1177/1076029614562037
24. Amiral J, Seghatchian J. An update on evidence based diagnostic and confirmatory testing strategies for heparin induced thrombocytopenia using combined immunological and functional assays. Transfus Apher Sci Off J World Apher Assoc Off J Eur Soc Haemapheresis. 2018;57(6):804-811. doi:10.1016/j.transci.2018.10.019
25. I E, C L, Mh H, J C, Mm S. Heparin-induced thrombocytopenia: laboratory diagnosis and management. Ann Med. 2000;32 Suppl 1. Accessed October 31, 2023. https://pubmed.ncbi.nlm.nih.gov/11209984/
26. Runser A, Schaning C, Allemand F, Amiral J. An Optimized and Standardized Rapid Flow Cytometry Functional Method for Heparin-Induced Thrombocytopenia. Biomedicines. 2021;9(3):296. doi:10.3390/biomedicines9030296
27. Tardy-Poncet B, Montmartin A, Piot M, et al. Functional Flow Cytometric Assay for Reliable and Convenient Heparin-Induced Thrombocytopenia Diagnosis in Daily Practice. Biomedicines. 2021;9(4):332. doi:10.3390/biomedicines9040332
28. Minet V, Dogné JM, Mullier F. Functional Assays in the Diagnosis of Heparin-Induced Thrombocytopenia: A Review. Mol Basel Switz. 2017;22(4):617.
doi:10.3390/molecules22040617
29. Greinacher A, Michels I, Kiefel V, Mueller-Eckhardt C. A rapid and sensitive test for diagnosing heparin-associated thrombocytopenia. Thromb Haemost. 1991;66(6):734-736.
30. Nagler M, Bakchoul T. Clinical and laboratory tests for the diagnosis of heparin-induced thrombocytopenia. Thromb Haemost. 2016;116(5):823-834. doi:10.1160/TH16-03-0240
31. Gonthier MC, Gendron N, Eloy P, et al. Heparin-induced Thrombocytopenia Diagnosis: A Retrospective Study Comparing Heparin-induced Platelet Activation Test to 14 C-serotonin Release Assay. TH Open Companion J Thromb Haemost. 2021;5(4):e507-e512. doi:10.1055/a-1653-5065
32. Arman M, Krauel K. Human platelet IgG Fc receptor FcγRIIA in immunity and thrombosis. J Thromb Haemost JTH. 2015;13(6):893-908. doi:10.1111/jth.12905
33. Althaus K, Strobel U, Warkentin TE, Greinacher A. Combined use of the high heparin step and optical density to optimize diagnostic sensitivity and specificity of an anti-PF4/heparin enzyme-immunoassay. Thromb Res. 2011;128(3):256-260. doi:10.1016/j.thromres.2011.05.003
34. Amiral J, Pouplard C, Vissac AM, Walenga JM, Jeske W, Gruel Y. Affinity purification of heparin-dependent antibodies to platelet factor 4 developed in heparin-induced thrombocytopenia: biological characteristics and effects on platelet activation. Br J Haematol. 2000;109(2):336-341. doi:10.1046/j.1365-2141.2000.02034.x
35. Leroux D, Hezard N, Lebreton A, et al. Prospective evaluation of a rapid nanoparticle-based lateral flow immunoassay (STic Expert(®) HIT) for the diagnosis of heparin-induced thrombocytopenia. Br J Haematol. 2014;166(5):774-782. doi:10.1111/bjh.12939
2. Arepally GM, Cines DB. Pathogenesis of heparin-induced thrombocytopenia. Transl Res J Lab Clin Med. 2020;225:131-140. doi:10.1016/j.trsl.2020.04.014
3. Cuker A, Gimotty PA, Crowther MA, Warkentin TE. Predictive value of the 4Ts scoring system for heparin-induced thrombocytopenia: a systematic review and meta-analysis. Blood. 2012;120(20):4160-4167. doi:10.1182/blood-2012-07-443051
4. Greinacher A. Heparin-induced thrombocytopenia. J Thromb Haemost JTH. 2009;7 Suppl 1:9-12. doi:10.1111/j.1538-7836.2009.03385.x
5. Lindhoff-Last E, Nakov R, Misselwitz F, Breddin HK, Bauersachs R. Incidence and clinical relevance of heparin-induced antibodies in patients with deep vein thrombosis treated with unfractionated or low-molecular-weight heparin. Br J Haematol. 2002;118(4):1137-1142. doi:10.1046/j.1365-2141.2002.03687.x
6. Amiral J, Marfaing-Koka A, Poncz M, Meyer D. The biological basis of immune heparin-induced thrombocytopenia. Platelets. 1998;9(2):77-91. doi:10.1080/09537109876843
7. Castelli R, Cassinerio E, Cappellini MD, Porro F, Graziadei G, Fabris F. Heparin induced thrombocytopenia: pathogenetic, clinical, diagnostic and therapeutic aspects. Cardiovasc Hematol Disord Drug Targets. 2007;7(3):153-162. doi:10.2174/187152907781745251
8. Amiral J, Peynaud-Debayle E, Wolf M, Bridey F, Vissac AM, Meyer D. Generation of antibodies to heparin-PF4 complexes without thrombocytopenia in patients treated with unfractionated or low-molecular-weight heparin. Am J Hematol. 1996;52(2):90-95. doi:10.1002/(SICI)1096-8652(199606)52:2<90::AID-AJH4>3.0.CO;2-0
9. Arepally GM, Ortel TL. Heparin-induced thrombocytopenia. Annu Rev Med. 2010;61:77-90. doi:10.1146/annurev.med.042808.171814
10. Onishi A, St Ange K, Dordick JS, Linhardt RJ. Heparin and anticoagulation. Front Biosci Landmark Ed. 2016;21(7):1372-1392. doi:10.2741/4462
11. Rubino JG, Arnold DM, Warkentin TE, Smith JW, Kelton JG, Nazy I. A comparative study of platelet factor 4-enhanced platelet activation assays for the diagnosis of heparin-induced thrombocytopenia. J Thromb Haemost JTH. 2021;19(4):1096-1102. doi:10.1111/jth.15233
12. 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(4):625-629.
13. Eekels JJM, Althaus K, Bakchoul T, et al. An international external quality assessment for laboratory diagnosis of heparin-induced thrombocytopenia. J Thromb Haemost JTH. 2019;17(3):525-531. doi:10.1111/jth.14383
14. Arepally GM, Kamei S, Park KS, et al. Characterization of a murine monoclonal antibody that mimics heparin-induced thrombocytopenia antibodies. Blood. 2000;95(5):1533-1540. doi:10.1182/blood.V95.5.1533.005k01_1533_1540
15. Kizlik-Masson C, Vayne C, McKenzie SE, et al. 5B9, a monoclonal antiplatelet factor 4/heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies. J Thromb Haemost. 2017;15(10):2065-2075. doi:10.1111/jth.13786
16. Pouplard C, Rollin J, Vayne C, et al. Multicentre evaluation of 5B9, a monoclonal anti-PF4/heparin IgG mimicking human HIT antibodies, as an internal quality control in HIT functional assays: Communication from the ISTH SSC Subcommittee on Platelet Immunology. J Thromb Haemost JTH. 2022;20(1):252-259. doi:10.1111/jth.15560
17. Tardy B, Lecompte T, Mullier F, Vayne C, Pouplard C. Detection of Platelet-Activating Antibodies Associated with Heparin-Induced Thrombocytopenia. J Clin Med. 2020;9(4):1226. doi:10.3390/jcm9041226
18. Griffiths E, Dzik WH. Assays for heparin-induced thrombocytopenia. Transfus Med Oxf Engl. 1997;7(1):1-11. doi:10.1046/j.1365-3148.1997.d01-77.x
19. Zwicker JI, Uhl L, Huang WY, Shaz BH, Bauer KA. Thrombosis and ELISA optical density values in hospitalized patients with heparin-induced thrombocytopenia. J Thromb Haemost JTH. 2004;2(12):2133-2137. doi:10.1111/j.1538-7836.2004.01039.x
20. Pearson MA, Nadeau C, Blais N. Correlation of ELISA optical density with clinical diagnosis of heparin-induced thrombocytopenia: a retrospective study of 104 patients with positive anti-PF4/heparin ELISA. Clin Appl Thromb Off J Int Acad Clin Appl Thromb. 2014;20(4):349-354. doi:10.1177/1076029613513319
21. Price EA, Hayward CPM, Moffat KA, Moore JC, Warkentin TE, Zehnder JL. Laboratory testing for heparin-induced thrombocytopenia is inconsistent in North America: a survey of North American specialized coagulation laboratories. Thromb Haemost. 2007;98(6):1357-1361. doi:10.1160/th07-06-0401
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(1):21-28.
23. Luna E, Agrawal P, Mehta R, et al. Evaluation of Immunostimulatory Potential of Branded and US-Generic Enoxaparins in an In Vitro Human Immune System Model. Clin Appl Thromb Off J Int Acad Clin Appl Thromb. 2015;21(3):211-222. doi:10.1177/1076029614562037
24. Amiral J, Seghatchian J. An update on evidence based diagnostic and confirmatory testing strategies for heparin induced thrombocytopenia using combined immunological and functional assays. Transfus Apher Sci Off J World Apher Assoc Off J Eur Soc Haemapheresis. 2018;57(6):804-811. doi:10.1016/j.transci.2018.10.019
25. I E, C L, Mh H, J C, Mm S. Heparin-induced thrombocytopenia: laboratory diagnosis and management. Ann Med. 2000;32 Suppl 1. Accessed October 31, 2023. https://pubmed.ncbi.nlm.nih.gov/11209984/
26. Runser A, Schaning C, Allemand F, Amiral J. An Optimized and Standardized Rapid Flow Cytometry Functional Method for Heparin-Induced Thrombocytopenia. Biomedicines. 2021;9(3):296. doi:10.3390/biomedicines9030296
27. Tardy-Poncet B, Montmartin A, Piot M, et al. Functional Flow Cytometric Assay for Reliable and Convenient Heparin-Induced Thrombocytopenia Diagnosis in Daily Practice. Biomedicines. 2021;9(4):332. doi:10.3390/biomedicines9040332
28. Minet V, Dogné JM, Mullier F. Functional Assays in the Diagnosis of Heparin-Induced Thrombocytopenia: A Review. Mol Basel Switz. 2017;22(4):617.
doi:10.3390/molecules22040617
29. Greinacher A, Michels I, Kiefel V, Mueller-Eckhardt C. A rapid and sensitive test for diagnosing heparin-associated thrombocytopenia. Thromb Haemost. 1991;66(6):734-736.
30. Nagler M, Bakchoul T. Clinical and laboratory tests for the diagnosis of heparin-induced thrombocytopenia. Thromb Haemost. 2016;116(5):823-834. doi:10.1160/TH16-03-0240
31. Gonthier MC, Gendron N, Eloy P, et al. Heparin-induced Thrombocytopenia Diagnosis: A Retrospective Study Comparing Heparin-induced Platelet Activation Test to 14 C-serotonin Release Assay. TH Open Companion J Thromb Haemost. 2021;5(4):e507-e512. doi:10.1055/a-1653-5065
32. Arman M, Krauel K. Human platelet IgG Fc receptor FcγRIIA in immunity and thrombosis. J Thromb Haemost JTH. 2015;13(6):893-908. doi:10.1111/jth.12905
33. Althaus K, Strobel U, Warkentin TE, Greinacher A. Combined use of the high heparin step and optical density to optimize diagnostic sensitivity and specificity of an anti-PF4/heparin enzyme-immunoassay. Thromb Res. 2011;128(3):256-260. doi:10.1016/j.thromres.2011.05.003
34. Amiral J, Pouplard C, Vissac AM, Walenga JM, Jeske W, Gruel Y. Affinity purification of heparin-dependent antibodies to platelet factor 4 developed in heparin-induced thrombocytopenia: biological characteristics and effects on platelet activation. Br J Haematol. 2000;109(2):336-341. doi:10.1046/j.1365-2141.2000.02034.x
35. Leroux D, Hezard N, Lebreton A, et al. Prospective evaluation of a rapid nanoparticle-based lateral flow immunoassay (STic Expert(®) HIT) for the diagnosis of heparin-induced thrombocytopenia. Br J Haematol. 2014;166(5):774-782. doi:10.1111/bjh.12939