Visual scoring versus histogram subtraction of in vivo binding of immunoglobulins against platelets after transfusion

Background: We developed a technique, in vivo binding of immunoglobulins in the platelet immunofluorescence test (IVBI-PIFT), that detects immunoglobulins bound in vivo to transfused platelets. The visually scored results of this technique, however, are susceptible to interobserver variation. We describe a more objective method to generate results in IVBI-PIFT.

Methods: We studied 201 samples in 120 patients with hematologic malignancies in the IVBI-PIFT. Histogram subtraction, i.e., fluorescence (anti-immunoglobulin G and fluorescein isothiocyanate) histogram before platelet transfusion subtracted from the histogram after platelet transfusion, was compared with visual scoring (pattern 1: no enhanced fluorescence before and after transfusion; pattern 2: enhanced fluorescence before and after platelet transfusion; pattern 3: enhanced fluorescence before transfusion; pattern 4: enhanced fluorescence after transfusion, interpreted as alloimmunization). After histogram subtraction, the number of remaining events (events post substraction, EPS) and the mean amount of fluorescence of these remaining events (mean channel post substraction, MCPS) were used and compared with the visual scoring and with platelet survival after transfusion.

Results: In 26 (13%) of the 201 samples studied in the IVBI-PIFT, fewer than three of five observers agreed on the visually scored pattern. In the 175 (87%) remaining samples, histogram subtraction showed a significant differentiation between pattern 4 and patterns 1 and 2 by using EPS, whereas patterns 4 and 3 were distinguished by using MCPS. The combination of EPS and MCPS differentiated best between pattern 4 and patterns 1, 2, and 3 (73% sensitivity, 96% specificity, 79% positive predictive value, and 95% negative predictive value). In contrast, the predictive value for platelet recovery after 1 and 16 h of pattern 4 from the visual scoring method and the results of histogram subtraction were poor.

Conclusion: This objective method of histogram subtraction correlated well with the visual scoring method of IVBI-PIFT.