8-P

Abstract

Aim Both bead and cell-based assays for HLA antibody detection can be impacted by non-specific background staining. In some (but not all) instances, adsorption of serum with control beads prior to testing reduces background. Herein we demonstrate that the addition of small amounts of FBS to patient sera can reduce or eliminate non-specific background activity. Methods Briefly, 5ul of heat-inactivated FBS was added to 50ul of patient or control serum. Samples were incubated from 0-30 minutes and then centrifuged before testing in bead or cell-based assays. Results Informative patient’s sera were treated with FBS and tested by FlowPRA, LABScreen Single Antigen Beads and flow crossmatch. Following treatment, 3 general patterns were observed:1) No effect on sera exhibiting well-defined HLA antibody specificities; 2) Improved resolution of true HLA antibodies in bead and cell based assays among sera with questionable (but real) antibodies; 3) Elimination of spurious activity among sera devoid of HLA antibody activity. FBS Treatment of beads alone had no effect.[figure1] Conclusions Many variables can contribute to high background staining in HLA antibody testing for which there are few remedies. These high backgrounds can confound/preclude the detection of true HLA antibody. Here we describe a simple procedure that can significantly reduce non-specific binding. FBS treatment allows easy resolution of true positive results from spurious background staining without any apparent loss in sensitivity and specificity. While its mechanism is unknown, we speculate that FBS treatment provides target antigens that block the binding of naturally occurring heterophile antibodies to beads and cells.