Functional Assays in the Diagnosis of Heparin-Induced Thrombocytopenia: A Review

Abstract

A rapid and accurate diagnosis in patients with suspected heparin-induced thrombocytopenia (HIT) is essential for patient management but remains challenging. Current HIT diagnosis ideally relies on a combination of clinical information, immunoassay and functional assay results. Platelet activation assays or functional assays detect HIT antibodies that are more clinically significant. Several functional assays have been developed and evaluated in the literature. They differ in the activation endpoint studied; the technique or technology used; the platelet donor selection; the platelet suspension (washed platelets, platelet rich plasma or whole blood); the patient sample (serum or plasma); and the heparin used (type and concentrations). Inconsistencies in controls performed and associated results interpretation are common. Thresholds and performances are determined differently among papers. Functional assays suffer from interlaboratory variability. This lack of standardization limits the evaluation and the accessibility of functional assays in laboratories. In the present article, we review all the current activation endpoints, techniques and methodologies of functional assays developed for HIT diagnosis.

Evaluation of functional assays for the diagnosis of heparin induced thrombocytopenia using 5B9, a monoclonal IgG that mimics human antibodies

“…In this context, platelet functional assays are mandatory, useful tools, and SRA is considered today the gold standard for confirming the diagnosis of HIT . However, this assay is technically challenging, requires the use of radio‐elements, and other functional tests have therefore been proposed . Recently, we developed 5B9, a monoclonal IgG specific to PF4/heparin complexes, which has a human Fc fragment, and behaves in vitro and in vivo like human HIT antibodies .…”

“…Indeed, the sensitivity of SRA to high, intermediate, and low concentrations of 5B9 was ≥95%, and SRA was also positive with 9 of the 10 samples from patients with “likely HIT.” The use of washed platelets is likely essential for explaining the excellent performance of SRA, because the removal of plasma abolishes any competition between HIT antibodies and donors' IgG for binding to platelet FcγRIIA receptors . This benefit of washing platelets probably explains why heparin‐induced activation assay (HIPA), performed with WP collected from four independent donors and under stirring in microtiter plates, is also considered a gold standard for HIT diagnosis . However, other parameters clearly contribute to the sensitivity of each assay, such as the measured parameter and the detection method used.…”

“…All functional assays used for HIT confirmation share the same principle, as they assess the ability of serum/plasma from suspected HIT patients to activate fresh platelets from healthy donors in the presence of variable concentrations of unfractionated heparin (UFH) . Depending on the assay, donors' platelets are stimulated either in whole blood (WB), platelet‐rich plasma (PRP), or after washings (WP).…”

Development and validation of a liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of serotonin and thromboxane B2 from activated platelets

“…Different methodologies to assess platelet function are currently available, to screen different platelet pathologies such as platelet secretion disorders or heparin‐induced thrombocytopenia (HIT), a severe immune adverse drug effect of heparin . The available assays allow either the study of global platelet function or the evaluation of particular aspects of platelet activation.…”

“…The available assays allow either the study of global platelet function or the evaluation of particular aspects of platelet activation. Current diagnosis techniques present limitations (use of radioactivity, light interference, time‐consuming…) . There is a major need to develop and evaluate new specific, standardized, more rapid, and easy methodologies that overcome these limitations …”