Interference of Direct Bilirubin on Lymphocyte Subset Determination in Human Peripheral Blood by Flow Cytometry and Mitigation Strategies

Context.— Flow cytometry assay is a common tool used to determine the different clusters of lymphocyte cells in peripheral blood. However, the test is commonly influenced by interference factors such as specimen conditions. Objective.— To define the specific interference factors that affect the classification of lymphocyte subsets. Design.— In our study, we investigated the effect of direct bilirubin (DBIL) in peripheral blood in daily routine tests, using an interference kit, and attempted to determine the best reverse measurement to manage icterus samples. Results.— We found that DBIL affects lymphocyte subsets including CD3 + T cells, CD4 + T cells, and CD16 + CD56 + natural killer cells. Stain-lyse-wash (SLW) solution is an ideal protocol to deal with such interference samples. By comparing the wavelength of bilirubin and different fluorescence channels, we found superposition in the fluorescein isothiocyanate (FITC), P-phycoerythrin (PE), peridinin-chlorophyll-protein complex (PerCP), PE-cyanine7 (PE-Cy7), and Brilliant Violet 510 (BV510) channels, indicating that a potential reason is disturbance in some of the fluorescence channels. Conclusions.— The interference effects of DBIL are usual causes of unsuccessful flow cytometry assay in determining the presence of lymphocyte subsets. SLW should be conducted to reverse these interference effects.