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

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.

An evaluation of a volumetric method for the flow cytometric determination of residual leukocytes in blood transfusion units.

The removal of leukocytes from blood components helps to prevent or reduce some adverse reactions that occur after blood transfusions. The implementation of the leukodepletion process in the preparation of blood units requires quality control, consisting of a reliable cell counting method to determine residual leukocytes in blood components. The most widely used methodology is a flow cytometric bead-based counting method. To avoid the need for commercial counting beads, we evaluated a volumetric counting method of leukocyte enumeration. A total of 160 specimens of leukodepleted plasma, red cell and platelet units, as well as 58 samples of commercially available controls containing different concentration levels of leukocytes, were included in the study. The conventional quality control method using the bead-based counting method performed with the FACSCalibur flow cytometer was compared to the bead-based counting method and the volumetric counting method performed with the MACSQuant 10 flow cytometer. Our results show that the MACSQuant bead-based method, as well as the volumetric MACSQuant method, meet the sensitivity requirements of residual leukocyte enumeration when compared to the gold standard, bead-based FACSCalibur method. We conclude that the volumetric method can be a substitute for the bead-based counting of residual leukocytes in a variety of blood components.

Heparin-induced thrombocytopenia: ELISA optical density value and 4T score in correlation with panel donor platelets activation in functional flow cytometric assay.

BACKGROUND: Serological assays for the diagnosis of heparin-induced thrombocytopenia (HIT) detect both platelet-activating and platelet non-activating anti-heparin/platelet factor 4 (PF4) antibodies and have therefore a limited positive predictive value. Functional assays confirm the presence of platelet-activating antibodies but require platelets from healthy donors, whose response to patient serum can differ. Our aim was to investigate the correlation between the level of anti-heparin/PF4 antibodies, 4T score, and the extent of panel donor platelet activation in the functional assay. MATERIALS AND METHODS: In total, 38 sera from enzyme immunoassays (ELISA) positive patients were tested against panel platelets obtained from 10 healthy, randomly selected donors, using our routine flow cytometry functional test for CD62P expression. Levels of anti-heparin/PF4 antibodies from medical and surgical patients and 4T pretest probability scores (where available) were correlated with the number of activated panel platelets. RESULTS: Sera with low ELISA optical density (OD) values (0.4-1) activated on average 5.6, sera with intermediate ELISA OD values (>1-2.5) activated on average 7.3, and sera with high ELISA OD values (>2.5) activated on average 8.6 out of 10 panel platelets. One serum with low 4T score did not activate donor platelets, 12 sera with intermediate 4T score activated on average 6.3 donors, 8 sera with high 4T score activated on average 8.5 panel platelets. DISCUSSION: Sera with higher ELISA OD values activated platelets from a higher number of platelet donors, independently of patient type (medical or surgical). The average number of activated panel platelets increased with rising 4T score. Results indicate that both donor platelet reactivity and quantity of anti-heparin/PF4 antibodies affect the result of the functional assay, meaning special attention is needed in platelet donor selection when testing sera with low levels of antibodies.