USE OF FLOW PRA TO IDENTIFY UNACCEPTABLE ANTIGENS AFTER in vitro IVIG TREATMENT.
D Crowe, S Wood, R Eddings, and W Won. DCI Laboratory and Vanderbilt Transplant Center. Nashville, TN.
Previous studies (Tyan, Czer, and Jordan) have shown that intravenous gamma globulin (IVIG) can inhibit HLA-specific alloantibodies both in vitro and in vivo. This treatment has been used in highly sensitized patients to reduce antibody levels and allow subsequent transplantation. IVIG treatment may not be equally effective against different HLA antigens in patients with multiple allo-specific antibodies. In this study, we have used Flow PRA analysis to examine the effect of IVIG treatment on a high PRA sample. After treatment, the titer of the antibodies in serum treated with IVIG was compared with titers in a sample incubated with glycine (the carrier molecule in the IVIG preparation). Flow specific beads were used to determine antibody specificity and to see if unacceptable antigens could be identified after IVIG treatment. Two-fold serial dilutions were made on the serum using a negative AB serum as the diluent (neat, 1:2, 1:4, and 1:8). Each dilution was then mixed with an equal volume of either IVIG or Glycine (200mM). The samples were then analyzed using Flow Bead Analysis (One Lambda). With the FlowPRA screen, a marked inhibition was seen with the serum diluted 1:4 and treated with IVIG. This sample was used with the Flow Specific beads to determine antibody specificity. The Glycine sample was 91% positive with the flow specific beads and no specificity could be determined. The IVIG-treated sample was 50% positive with the Flow specific beads and the specificity was found to be against A2, 68, 23,24, and a weak response to the some 7 CREG antigens. Cytotoxic assays demonstrated complete inhibition in the IVIG treated samples against the cells with 7CREG or A2 only, but not when the cells had both A2 and 7 CREG antigens. This study shows that the FlowPRA may be useful in pretesting high PRA samples with IVIG in vitro to determine which specificities will respond to IVIG treatment.