Pathogenic Heparin Induced Thrombocytopenia and Thrombosis (HIT) Antibodies Determined By Simple Rapid Functional Flow Cytometry
Authors: Deutsch Varda R , Cipok Michal , Kirgner Ilya , Kay Sigi , Elalamy Ismail , Tomer Aaron 
Published: October 2019

Abstract
Background: HIT diagnosis is mandatory for patient management, yet prompt determination of pathogenic antibodies remains an unmet clinical challenge. Commonly used immunoassays carry inherent limitations and functional assays which determine antibody-mediated platelet activation are not readily available being technically demanding and require high level expertise. Additionally, they are time consuming and not rapid enough to provide quick detection of the pathologic antibodies required at time of diagnosis.
Aims: To overcome these limitations, we aimed to develop an easily performed functional flow cytometric assay (FCA) to rapidly detect platelet activating antibodies for the initial diagnosis and/or confirmation of HIT.
Methods: Serum samples from patients clinically suspected of HIT (n=650) were tested by the PF4/H-PaGIA immunoassay (DiaMed, Switzerland). The capacity of the patient's serum to induce platelet activation in the presence of heparin was assessed by a rapid 2-hour functional FCA. Platelets were gated by CD41 expression and a positive cut-off gate of the high 2% CD62p positive events was set on normal control sample. Each assay assessed the fraction of positive events above the 2% cut-off gate of the control sample. Background fluorescence was normalized in each sample. TRAP stimulation provided the positive control. A positive test result was defined as a value ≥2 fold greater than that of the control. Results were compared to those of the radioactive serotonin-release assay (SRA) and to the clinical 4Ts score HIT presentation
Results: Consecutive HIT-suspected patient samples were tested, 15.3% were positive by the PaGIA-Heparin/PF4 immunoassay and only 4.8% by FCA. When compared to TRAP, PaGIA+/FCA+ samples were 11-fold higher in fluorescence vs. PaGIA-/FCA- samples +2SD. SRA was performed on 63 samples. Of 21 SRA positive samples, 19 were positive by FCA (relative sensitivity 90.5%), and of 42 SRA negative samples, 40 were negative by FCA (specificity 95.2%). The robustness of the FCA was confirmed by an alternative calculation, using the ratio of the % platelet activation induced by patient samples/the % activated by thrombin-receptor-activating-peptide (TRAP). Platelet activation, generated by the patient samples was 11-fold higher in PaGIA+/FCA+ vs. PaGIA-/FCA- samples, confirming the high resolution of this assay to facilitate discrimination between positive and negative values. The FCA showed significantly higher correlation with the clinical presentation of HIT (4Ts score) performed on 182 patients, compared to PaGIA-Heparin/PF4 (ROC-plot analysis, AUC 0.93 vs. 0.63, p<0.001). At 92% sensitivity, the assay specificity was 96%.
Conclusions: We demonstrate that pathologic HIT antibodies can be detected rapidly by routine clinical laboratory methodology. Thus, the present assay provides a feasible answer to the unmet clinical need of prompt and reliable diagnosis of true HIT. Hence, validation of this assay by other laboratories, might offer a practical replacement for the non-readily available radioactive SRA, or other complex tests, thus contributing to improved clinical decision making and patient care.

Disclosures
Tomer: Azapharm: Other: patent pending and consultation fees from Azapharm outside of submitted work..