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 ␣-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. Am. J. Hematol. 61:53-61, 1999.

High sensitivity and specificity of a new functional flow cytometry assay for clinically significant heparin‐induced thrombocytopenia antibodies

“…Patient sera that showed ≥7.6% platelet activation in the presence of therapeutic heparin were only considered positive if platelet activation was subsequently reduced ≥50% [15,16] in the presence of supratherapeutic heparin.…”

“…Subsequently, PRP was added to patient serum in the presence or absence of therapeutic concentration of unfractionated heparin [15,16] in a total volume of 50 lL. To determine heparin dependence of platelet activation, a supratherapeutic dosage of heparin [15,16] was added. After 1-h incubation at room temperature (+20 to +24°C) on a horizontal orbital shaker, 5 lL of the suspension was transferred to 45 lL staining solution containing fluorescent-labeled antibodies to a platelet activation marker annexin V [15,16] and a platelet marker, followed by 15-min incubation in the dark.…”

“…To determine heparin dependence of platelet activation, a supratherapeutic dosage of heparin [15,16] was added. After 1-h incubation at room temperature (+20 to +24°C) on a horizontal orbital shaker, 5 lL of the suspension was transferred to 45 lL staining solution containing fluorescent-labeled antibodies to a platelet activation marker annexin V [15,16] and a platelet marker, followed by 15-min incubation in the dark. Samples were measured and analyzed on a standard flow cytometer, capable of detecting FITC and R-PE fluorescence (Braunschweig: FACS Calibur, BD, Franklin Lakes, NJ, USA, London: Beckman Coulter UK Ltd, High Wycombe, UK, Stanford: BD FACS Canto II).…”

“…In this study, the HITAlert was used, which is an IVD-registered, rapid, commercialized version of the highly sensitive and specific flow cytometry (FCM) assay originally described by Tomer [15,16]. Sensitivity and specificity of this FCM assay, as compared to the SRA, were found to be 95% and 100%, respectively, in 19 patients and 10 healthy controls [15].…”

Heparin‐induced thrombocytopenia in children

“…5 Severe HIT is defined as a reduction of >50% in the platelet count occurring ≥ 5 days after heparin exposure. 2,5,13,14 This reduction is a response to antibody development against the heparin-platelet antigen complex and can be detected by several laboratory techniques. 4 Mild HIT presents as a reduction in the platelet count and can be asymptomatic.…”