P024 : POTENTIAL FOR A FALSE POSITIVE B CELL FLOW CYTOMETRY CROSSMATCH WITH THE USE OF FLUORESCEIN ISOTHYOCYANATE-GOAT-ANTI-HUMAN IMMUNOGLOBULIN-G ANTIBODY

Abstract

Background In flow cytometry crossmatches (FCXM), sera from AB blood group male donors with none or minimal anti-HLA IgG antibodies are used as the negative control serum (NCS). As NCS can give higher median channel fluorescence (MCF) on certain donor cells, it is advised to use cells treated with phosphate buffer saline (PBS) as an additional negative control for comparison. Aim We examined whether using PBS or NCS as the negative control and subsequent staining with FITC-goat-anti-human IgG polyclonal antibody (g α IgG pAb; Jackson Immunoresearch Laboratory; Cat# 109-096-098) or FITC-mouse-anti-human IgG monoclonal antibody (m α IgG mAb; BD biosciences; Cat# 555786; Clone: G18-145) impacts FCXM results. Methodology Peripheral blood ( N = 5) and serum ( N = 5) were collected from human subjects. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density gradient separation. In 96 well V-bottom plate, 300,000 PBMC were incubated with 30 μl of serum for 20 min at 22 °C, washed and stained with 10 μl each of anti-CD3 PerCP and of anti-CD19 PE, and with either 10 μl (1:100 diluted) of FITC-gαIgG pAb or 60 μl of FITC-mαIgG mAb for 30 min at 4 °C. The cells were washed and flow cytometry was performed using BD FACS Canto II instrument and the data was analyzed using the DiVa software. A channel shift of >40 from the NCS for T cell and a shift of >50 for B cells is considered positive here. Results PBMCs treated with PBS gave positive B cell channel shift when stained with g α IgG pAb, but not when stained with m α IgG mAb ( Fig. 1 ). Unlike m α IgG mAb, the g α IgG pAb seems to bind non-specifically to B cells in the absence of human serum. Conclusion This observation cautions that in a clinical FCXM testing, the non use of NCS can lead to a false positive FCXM result. It is also anticipated that a technical error of not adding patient’s serum to the donor cells can lead to positive B cell crossmatch reporting. Using m α IgG mAb or any other α IgG antibody that does not give non-specific binding to donor cells is recommended for FCXM testing.