Dimeric FcγR ectodomains detect pathogenic anti-platelet factor 4-heparin antibodies in heparin-induced thromobocytopenia

Abstract

Essentials FcγRIIa mediates life-threatening heparin-induced thrombocytopenia (HIT). Most anti-platelet factor (PF)4-heparin IgGs are not pathogenic so diagnosis of HIT is challenging. Dimeric rsFcγRIIa was used to quantify receptor-binding activity of anti-PF4-heparin antibodies. Dimeric rsFcγRIIa binding specifically correlated with occurrence of HIT. SUMMARY: Background Heparin-induced thrombocytopenia (HIT) is a major and potentially fatal consequence of antibodies produced against platelet factor 4 (PF4)-heparin complexes following heparin exposure. Not all anti-PF4-heparin antibodies are pathogenic, so overdiagnosis can occur, with resulting inappropriate use of alternative anticoagulation therapies that have associated risks of bleeding. However, definitive platelet functional assays are not widely available for routine analysis. Objectives To assess the utility of dimeric recombinant soluble FcγRIIa (rsFcγRIIa) ectodomains for detecting HIT antibodies. Patients/Methods Plasma from 27 suspected HIT patients were tested for pathogenic anti-PF4-heparin antibodies by binding of a novel dimeric FcγRIIa ectodomain probe. Plasmas were also tested by the use of PF4-heparin IgG ELISA, the HemosIL AcuStar HIT IgG-specific assay, and a serotonin release assay (SRA). Results The dimeric rsFcγRIIa test produced no false positives and excluded four samples that were positive by IgG ELISA. In this small patient cohort, the novel assay correctly assigned 93% of the suspected HIT patients, with two of the HIT patients being scored as false negatives. The improved discrimination of the novel assay over the IgG ELISA, which scored four false positives, supports the mechanistic interpretation that binding of dimeric rsFcγRIIa detects pairs of closely spaced IgG antibodies in PF4-heparin immune complexes. Conclusions This study found the cell-free, function-based dimeric rsFcγRIIa assay to be convenient, simple, and potentially predictive of HIT. The assay had improved specificity over the IgG ELISA, and correlated strongly with the AcuStar HIT IgG-specific assay, warranting further evaluation of its potential to identify HIT in larger patient cohorts.

Keywords: enzyme immunoassay; heparin; platelet factor 4; thrombocytopenia; thrombosis.

Figure 1

Comparison of the novel dimeric recombinant soluble FcγRIIa (rsFcγRIIa) assay with existing serological assays for the detection of heparin‐induced thrombocytopenia (HIT) antibodies. (A) Suspected HIT patients were segregated on the basis of a positive serotonin release assay (SRA) result and a 4Ts score of ≥ 4. The three dashed lines indicate the cut‐off values for a positive result with each assay. These thresholds were 3% of the normalized signal for the dimeric rsFcγRIIa assay: 1 U mL ⁻¹ for the AcuStar HIT IgG‐specific assay, and an OD of 0.4 (right y‐axis) for the platelet factor 4 (PF4)–heparin IgG ELISA. Two sets of parentheses mark the false negatives from the assays: two from the dimeric rsFcγRIIa assay, and one from the AcuStar HIT IgG‐specific assay. The third set of parentheses marks the four false positives from the PF4–heparin IgG ELISA. (B) The dimeric rsFcγRIIa and AcuStar HIT IgG‐specific assays were correlated for the HIT‐positive (i.e. 4T score of ≥ 4 and SRA‐positive) samples. Cut‐off values for the two assays are shown as dotted and dashed lines, respectively. HIT samples with low antibody levels are more sensitively identified by combining both the dimeric rsFcγRIIa and AcuStar HIT IgG‐specific assays. When HIT positivity is defined as rsFcγRII a positivity and/or AcuStar HIT IgG‐specific assay positivity, sensitivity is improved over that with either assay used alone.

Figure 2

Correlation of the novel dimeric recombinant soluble FcγRIIa (rsFcγRIIa) assay and the existing serological assays with serotonin release. Results from all the serological assays of samples positive in the IgG ELISA, including four HIT false positives (filled blue star and black circles), were compared against the SRA results. The SRA (release at 0.1 U mL⁻¹ heparin) was compared with (A) the PF4–heparin IgG ELISA, (B) the AcuStar HIT IgG‐specific assay, and (C) the novel dimeric rsFcγRIIa assay. The dashed lines indicate the cut‐off values for a positive result with each assay. Open symbols represent HIT‐diagnosed patients (positive SRA result and a 4Ts score of ≥ 4). Filled symbols represent PF4–heparin IgG ELISA‐positive HIT‐negative patients, with the blue star symbol indicating one SRA‐negative patient with equivalent levels of serotonin release with both high‐dose and low‐dose heparin stimulation. The open triangle indicates an HIT patient plasma sample that was weakly AcuStar HIT IgG‐specific assay‐positive and dimeric rsFcγRIIa assay‐negative, and the open square indicates an HIT patient plasma sample that was weakly AcuStar HIT IgG‐specific assay‐positive and strongly dimeric rsFcγRIIa assay‐positive. [Color figure can be viewed at wileyonlinelibrary.com]