A multiplexed reverse-SSO hybridization assay will be developed to achieve high resolution DNA typing for improved matching of donor and recipient for bone marrow transplantation (BMT). The system has already been applied to low-resolution typing (e.g. for preliminary screening of bone marrow donors), and will be further developed to provide allele-level typing information that is important to improve the clinical outcome of BMT, particularly for the HLA-DRB1 locus (which affects the incidence of Graft vs. Host Disease (GvHD). Over the 3-year project period, HLA-A and -B group-specific typing assays will be added for resolution of serological ambiguities such as Bw4, Bw6, and high resolution typing of HLA-C following National Marrow Donor Program (NMDP) priorities. To complete product line capabilities for HLA typing, generic kits will also be developed for DPB1, DQA1, and DPA1 (DQB1 and DRB3,4,5 were already produced). Additional work will be aimed at rSSO typing of the KIR region genes, since recent evidence has shown a beneficial effect of mismatching for these genes in acute myeloid leukemia (AML) patients, where a Graft vs Leukemia (GvL) effect was noted to reduce the recurrence of leukemia. The application of a high throughput multiplexed assay to this clinical testing offers the advantage of both cost and labor savings, as well as enabling the resolution of many typing ambiguities observed with current methods such as sequence based typing. In particular, the use of proprietary gap-primers and gap-probes allows for fewer rounds of PCR amplification to achieve the desired rSSO typing. In addition, primers for KIR will be designed to allow for multiplex PCR of the various loci. Finally, analytical software will be developed, which will require new algorithms for assignment of KIR typing results, due to the multiple and variable number of alleles expressed for each individual. Following validation testing and regulatory clearance, a complete line of superior HLA and KIR typing products will be available to the NMDP and HLA laboratories.