Determination of heparin-induced thrombocytopenia: A rapid flow cytometric assay for direct demonstration of antibody-mediated platelet activation

Abstract

Heparin-induced thrombocytopenia (HIT) and thrombosis are serious complications of heparin therapy. Recently, we have reported a practical and rapid functional flow cytometric assay (FCA) for the diagnosis of HIT with high specificity and sensitivity compared with the radioactive serotonin-release assay (SRA). In the present study, we added an immune-neutralization assay to directly demonstrate the antibody-mediated process, and tested the immune compatibility of low-molecular-weight heparin (LMWH) Lovenox and the heparinoid Orgaran (danaproid) using plasma from 18 patients with HIT confirmed by both FCA and SRA. The clinical utility of this modified method is demonstrated by a pediatric patient with a complex clinical presentation who developed thrombocytopenia with multiple thromboses while on heparin therapy. ELISA and SRA (performed in three independent laboratories) for diagnosis of HIT were both negative. In contrast, the FCA for detecting activated platelets expressing anionic phospholipids, was highly and reproducibly positive with both unfractionated and LMWH. Another FCA also demonstrated the surface expression of the alpha-granule membrane p-selectin (CD62p). Compatibility testing with the heparinoid Orgaran was also positive (and with plasma from 4 of the 18 patients with HIT). Heparin was discontinued, along with full recovery of the platelet count. The capacity of the patient's plasma to activate platelets in the presence of heparin gradually decreased over 4 weeks consistent with antibody clearance. The responsible mechanism was clarified using an immune-neutralization assay, which showed a dose response neutralization of the plasma activity by antibodies against human Immunoglobulin G (IgG) and IgM. This assay was also reproducible in the 18 patients with HIT. We conclude that the functional FCA with its modification is practical, sensitive, and specific for reliable diagnosis of HIT. It can simultaneously assess the compatibility of alternative therapies and directly confirm the antibody-mediated process. Further, it is particularly useful to clarify mechanisms of thrombocytopenia and thrombosis and to direct therapy in patients with a complex presentation and confounding laboratory results who often need prompt diagnosis and treatment.

Diagnosis and Management of Heparin-Induced Thrombocytopenia

“…Whereas functional assays, in particular the SRA, have high specificity (> 95%) 44 and are associated with a high positive predictive values (89-100%), 21 the sensitivity of functional assays are less robust (62-100%) 44,65,94,95 due to a number of technical variables affecting platelet reactivity. 96 Other important drawbacks to these assays include lack of standardization, 97 complexity of the assays and the use of radioactive isotopes with the SRA.…”

Heparin‐induced thrombocytopenia and thrombosis

“…Other end points used to detect heparin-dependent activation of platelets by HIT antibodies include expression of the alpha granule protein P-selectin (CD62) on the platelet surface [53], binding of annexin V [54], and formation of platelet-derived microparticles [55]. These assays are technically demanding and difficult to standardize, and their use is largely restricted to a research setting.…”

Heparin-Induced Thrombocytopenia-Associated Thrombosis in Pediatric Intensive Care Patients

“…Most data on HIT in children are confined to case reports. [13][14][15][16][17] To date, only 1 prospective study has evaluated the incidence of HIT in newborns: Spadone et al 18 found an inci-dence of 1%, and in this group, 85% developed umbilical or aortal thrombosis.…”