#9-OR
FLOW CYTOMETRIC DETECTION OF HLA ANTIBODY: SENSITIVITY OF SINGLE ANTIGEN COATED BEADS VS LYMPHOCYTE CROSSMATCHING.
Wayne Shumway BS 1, Jennifer McKie MT 1, Mayra Cepero-Lopez PhD 1 and William M. LeFor PhD 1. 1 LifeLink Foundation, Inc., Tampa, FL .
Although microscopic beads coated with purified HLA antigens are a powerful tool, the meaning of single antigen flow bead results is not always clear. Sera tested against single antigen beads and in flow XMs with T and B cells, showed that while beads are usually more sensitive than cells, how much more varies tremendously. Nine Class-I and 6 Class-II alloantibodies were tested against both cell and bead targets. Quantitative fluorescence (MESF) showed the binding of anti-Class-I to beads was 1.8
32.8 times that seen on T cells. Anti-Class-II bead binding was 0.52.9 times that seen on B cells. The wide ranges can be explained by the marked difference in antigen density among the beads. Using the pan-HLA Class-I MoAb W6/32, the antigen density on beads from two different lots of 
One Lambda Flow PRA Class-I Single Antigen Beads
was compared. The binding of W6/32 with different beads ranged from 16,000 MESF units to 113,000 MESF units in bead group#1 from Lot#4, and from 70,000 to 155,000 MESF units in group#1 from Lot#7. Between lots, W6/32 signal on beads bearing the same antigen differed by anywhere from 17,000 to 134,000 MESF units. The increased sensitivity of beads relative to cells is due to increased bead antigen density as well as a much lower 
Antigen carrier/Antibody ratio (56 target beads per 1ul of serum vs 12,500 cells per 1 ul of serum). Ultimately, single antigen assays should accurately predict (replace?) the crossmatch, and provide a more reliable gauge of clinical risk. To reach these goals requires not only greater uniformity or normalization among the targets used, but also the application of objective and quantitative methods of analysis (for example, using MESF data instead of channel data for flow based assays).