An Optimized and Standardized Rapid Flow Cytometry Functional Method for Heparin-Induced Thrombocytopenia

Abstract

Heparin-induced thrombocytopenia (HIT) is a thrombocytopenia caused by heparin and mediated by an atypical immune mechanism leading to a paradoxical high thrombotic risk, associated with severe morbidity or death. The diagnosis of HIT combines a clinical scoring of pretest probability and laboratory testing. First-line routine tests are antigen binding assays detecting specific antibodies. The most sensitive of these tests have a high HIT-negative predictive value enabling HIT diagnosis to be ruled out when negative. However, HIT-positive predictive value is low, and a functional assay evaluating the pathogenicity of the antibodies should be performed to exclude false-positive results. In contrast to screening assays, functional assays are highly specific but technically challenging, and are thus performed in referral laboratories, where platelet activation is detected using radioactive serotonin (serotonin release assay, SRA) or visually (heparin-induced platelet activation, HIPA). Flow cytometry is a possible alternative. It is, however, currently not widely used, mostly because of the lack of standardization of the published assays. This article describes and discusses the standardization of a HIT flow cytometry assay (HIT-FCA) method, which subsequently led to the development and commercialization of a CE-marked assay (HIT Confirm^®, Emosis, France) as a suitable rapid HIT functional test.

Keywords: HIT functional assay; flow cytometry; heparin-induced thrombocytopenia diagnosis.

Figure 1

Principle of HIT flow cytometry assay (HIT-FCA), a flow-cytometry-based HIT functional assay, using donor platelets and dye-conjugated monoclonal antibodies. This assay uses four tubes: a negative control (NEG), a positive control (POS), and two tubes with patient sample containing different heparin concentrations (H0.3 and H100). The PE-conjugated anti-CD41 antibody (red) specifically labels platelets. In the presence of anti-PF4/H antibodies, donor platelets are activated, and they express CD62 or P-selectin in their plasma membrane. The latter is then recognized by the second FITC-conjugated anti-CD62 antibody (green). The tubes are read by flow cytometry after the incubation times.

Figure 2

Optimization of assay reagents. The median fluorescence intensities (MFIs) of (a) two anti-CD41 and (b) three anti-CD62 clones were measured. These monoclonal antibodies (MoAbs) were labeled with either FITC or PE fluorophores. Three different suppliers for (c) thrombin-receptor-activating peptides (TRAPs) and (d) heparin were also tested.

Figure 3

Donor platelet stability. (a) Negative and (b) positive reference values for HIT Confirm were set according to 25 batch samples of donor platelets. The solid lines correspond to the mean, and dashed lines show the limits of mean ± 2 standard deviations (SDs). The change of activation percentage of (c) negative and (d) positive controls as a function of time overlayed with the mean and limits of mean ± 2 SD as defined in the previous plots. Seven batch samples were tested.

Figure 4

Gating strategy for HIT-FCA assay. Left: PE profile in the four tubes used to identify platelet population. Middle: negative (NEG) and positive (POS) FITC profiles in solid and dashed lines, respectively, overlayed to define the analytical cutoff of activated platelets. Right: examples of HIT-FCA-positive and HIT-FCA-negative with the calculated %HEPLA.

Figure 5

HEPLA interpretative algorithm. If HEPLA is above 13%, the HIT-FCA result is considered positive [41]. In case of HEPLA below 9.6%, %H100 is to be checked; if %H100 is high (↑)—that is, above 23% (see text)—the result is considered indeterminate, and the assay must be repeated with new PRPs. If %H100 is low (↓), the HIT-FCA result is considered negative. If HEPLA is in the gray zone from 9% to 13%, the HIT-FCA result is considered indeterminate. If indeterminacy of HEPLA remains after repeats, the HIT-FCA result is deemed inconclusive.