Heparin-induced thrombocytopenia: effect of heat inactivation of patient's serum on functional flow cytometric assay results

Abstract

Background: Heat inactivation of a patient's sample is not systematically performed in the diagnostics of heparin-induced thrombocytopenia (HIT). Some authors recommend that the patient's sample is heat-inactivated to avoid the effect of thrombin on platelet activation in a functional assay. Others do not find this additional step essential or even advise against it.

Material and methods: An enzyme-linked immunosorbent assay (ELISA) and flow cytometry-based functional assay with CD62P as a marker of platelet activation were performed. Forty-seven patients with suspected HIT and three healthy controls were included in the study. Each serum sample was divided into two aliquots: one was heat-inactivated and the other was not. Both aliquots were tested in parallel using the same donor platelets from four randomly selected individuals. We designed an index of platelet activation for both protocols to assess platelet activation in the assay and to compare the results.

Results: We observed a higher percentage of platelet activation in heat-inactivated compared to non-heat-inactivated sera. This phenomenon was seen in low and high heparin steps, although it did not occur for all samples. There were discrepant results in seven samples, which tested negative in the non-heat-inactivated protocol and positive in the heat-inactivated protocol. There was no case in which the result of a non-heat-inactivated aliquot was positive and the corresponding heat-inactivated aliquot was negative.

Discussion: Due to the higher percentages of donor platelet activation, all seven non-compliant cases met our current criteria for a positive result. However, those results were probably false-positive based on ELISA optical density value and 4T score. Therefore, in the current settings, heat inactivation of serum is not suitable for our flow cytometric functional assay since it can cause an elevated risk of creating false-positive results.

Figure 1

Indices of platelet activation depending on the optical density values of the enzyme-linked immunosorbent assay The scatter correlation diagram represents the correlation between enzyme-linked immunosorbent assay (ELISA) optical density (OD) values and the Index_non-HI (black circles) or Index_HI (white circles). There is a statistically significant positive correlation between ELISA OD and Index_(non-HI) (r=0.761) as well as between ELISA OD and Index_(HI) (r=0.763). The dark grey and the light grey dotted lines represent the regression lines for the non-heat-inactivated protocol with a slope of 1.66 and the heat-inactivated protocol with a slope of 0.86, respectively.