29-P

Abstract

Aim Studies have revealed that dilution of human sera can release blocking mechanisms for HLA antibodies (Ab) when testing with Single Antigen Beads (SAB). These blocking mechanisms are not clearly defined, but they can inhibit binding of HLA Ab to purified antigen on the beads, giving a false negative result. Normal Human Serum (NHS) is commonly added to patient sera as a diluent. Use of NHS maintains the serum protein level of the diluted serum. However, the NHS can contain HLA Ab that may interfere with HLA Ab analysis. The purpose of this study was to determine if Fetal Bovine Serum (FBS) could be substituted for NHS in the SAB assay. FBS was chosen as it’s a common supplement in other HLA assays with no adverse effects. Methods Sera were selected containing a wide range of HLA Ab specificities. 11 sera were tested in SAB HLA Ab assay (10 pos, 1 neg). All sera were pretreated with DTT. The sera were tested: neat, diluted 1:4 in NHS, and diluted 1:4 in FBS. Results The prescreened neg serum also tested negative both neat and diluted 1:4 in FBS. Low levels of HLA Class I (HLA Ab of moderate to high reactivity >5000 MFI), as detected in neat sera, were also detected in sera diluted in FBS or NHS. However, the HLA Ab activity in the diluted sera was not as strong due to dilution effect. The FBS was also tested for blocking properties by adding FBS to SAB and incubating for 30 minutes before adding patient serum to the assay. HLA Ab detected in patient sera were no different when using FBS-pretreated SAB or by routine protocol. Conclusions We have found, in parallel testing, that FBS is a superior diluent over NHS in the SAB HLA Ab assay. FBS maintains an adequate protein level without adding unwanted background, it does not inhibit binding of HLA antibodies to the beads, and allows for easily interpretable test results.