#24
SENSITIVE FLOW CYTOMETRIC ANALYSIS OF ANTI-HLA ANTIBODY PRODUCED BY PERIPHERAL BLOOD B LYMPHOCYTES.
Denis P. Snider, PhD, Jason Fernandes, BSc, Azim Gangji, M.D. and Dianne Arlen, M.D. Hamilton ON, Canada, McMaster University, L8N-3Z5, Pathol.Mol.Med.; Hamilton ON, Canada, St. Joseph
s Hospital, L8N-4A6, Nephrology 
Renal Transplantation and Hamilton ON, Canada, Hamilton Health Sciences, L8N-4A6, Hamilton Regional Lab Med Program.
RATIONALE: Pre-exisiting anti-HLA antibody remains a significant barrier to renal transplantation. Recent crossmatch and antibody detection methods based on flow cytometry allow sensitive detection of anti-HLA antibodies, but rely on serum antibody. Frequently these serum antibodies are transient, but latent B and T cell memory is presumed to continue, despite the decline of anti-HLA antibody in serum. Techniques to probe latent anti-HLA memory would greatly enhance assessment of various patient groups and could be used to predict and thereby avoid antibody-mediated graft rejection. METHODS: An in vitro microculture technigue has been developed for enhanced Ig production by blood B lymphocytes, using PWM(poke weed mitogen), IL-2 and IL-6. Anti-HLA antibodies elicited from these cultures are detected using sensitive bead-based flow cytometry, in which beads are coated with HLA molecules. RESULTS: Microcultures produce Ig in sufficient quantities to measure specific anti-HLA antibodies. Stimulated micro-ultures of blood B cells from patients with known anti-HLA antibodies in their serum were found to produce detectable anti-HLA antibodies. DISCUSSION: We will present data on anti-HLA antibody from blood B cells from patients with historic anti-HLA antibody, but no current anti-HLA antibodies in serum. This work in principle provides a means to detect an evaluate latent anti-HLA antibody in patients.