Sensitive and Specific Functional Flow Cytometry Assay for Rapid Diagnosis of Heparin Induced Thrombocytopenia and Thrombosis (HIT)

Background: Reliable diagnosis of Heparin Induced Thrombocytopenia and Thrombosis (HIT) is crucial for patient management. A test that detects the presence of functional antibodies, capable of inducing platelet activation, is far superior to the commonly performed immunoassays having poor specificity. Unfortunately, the currently applied functional assays are technically difficult and require a high level of expertise not readily available in clinical laboratories, thus limiting their performance feasibility.

Aims: We aimed to assess the sensitivity and the specificity of a simple, easily performed functional flow cytometric assay (FCA) to detect heparin stimulated platelet activating antibodies.

Methods: Samples from patients clinically suspected of HIT were routinely tested by the PF4/H-PaGIA immunoassay (DiaMed, Switzerland), followed by the functional FCA. The capacity of the patient's serum to induce platelet activation in the presence of heparin was tested using two color flow cytometry. The FCA results, were calculated as the ratio of % activated normal donor platelets stimulated by the patient sample /% the same donor platelets activated by TRAP. Results were compared to those of the gold-standard, the radioactive serotonin-release assay (SRA). The assays results were also correlated with the HIT presentation based on the 4Ts score.

Results:

Samples were consecutively accrued from patients (n=650) suspected of HIT by hematologists, 99 (15.3%) were positive by H/PF4-PaGIA immunoassay and 31 (4.8%) were positive by FCA. Sample dilution of 1:32, decreased the H/PF4-PaGIA-positive results to 29 (4.5%), while the FCA-positive results (4.8%) remained consistent, with 93.1% and 94.3% sensitivity and specificity respectively. SRA was performed on 63 samples. Of the 21 SRA positive samples, 19 were positive by FCA (relative sensitivity 90.5%), and of 42 SRA negative samples, 40 were negative by FCA (relative specificity 95.2%).

Patients with PaGIA+/ FCA+ showed average platelet activation ratio of 2.2+2.9 SD (n=31), compared to the average of patients negative by both tests 0.1 + 0.1 SD(n=30). The high resolution of this test is demonstrated even at the 2SD level with the average negative + 2SD (0.3) being smaller by 7.3fold than the average (2.2) of the positive patient thus facilitating easy discrimination between positive and negative values. Patients with PaGIA+/ FCA-negative were also easily resolved from the PaGIA+/FCA+. These patients with mean of 0.2 +0.2 SD, and +2SD of 0.6, had significantly smaller values than the mean of the positive patients (ratio 3.7). Overall, the FCA showed significantly higher correlation with the clinical presentation of HIT (4Ts score) performed on 182 patients, compared to the PF4/H-PaGIA (ROC-plot analysis, AUC 0.93 vs. 0.63, p<0.001). At a cut-off level of 92% sensitivity, the respective specificity of the FCA was 96% .

Conclusions: The present study demonstrates that this flow cytometric functional assay is easily performed, practical for routine daily use and provides reliable, well resolved and rapid confirmation of heparin stimulated platelet activating antibodies in the patients blood.