179-P: A BLIND STUDY OF HLA-A TYPING BY REFERENCE STRAND MEDIATED CONFORMATION ANALYSIS (RSCA)

Abstract

The aim of this work was to test in a blind study the application of RSCA for HLA-A typing in a panel of 244 DNA samples of known HLA type from different ethnic groups and evaluate the power of resolving alleles that differ by a single nucleotide in a DNA fragment of almost 1000bp. We also test the capacity of RSCA in resolving ambiguous combinations of HLA-A alleles. DNA was extracted using the salting-out method from a panel of 244 cell lines and from cells available locally. The samples were coded and the HLA type unknown to all involved in the study. On completion of the study, the code was broken and the HLA type of the samples were revealed and compared with that obtained by RSCA. RSCA was performed using two fluorescent labeled references generated by PCR from the cell lines STEINLIN (IHW9087) and from the cell line AMALA (IHW9064). They were labeled with Cy5 at the 5’ end. We found 99% concordance between RSCA results and those obtained by conventional methods such as serology, sequencing, SSOP or SSP typing. Nine samples did not match the original type. Retyping of these nine samples by either SSP or SBT demonstrated that seven gave typing in agreement with the RSCA results and two matched the original typing. In this study two new HLA-A alleles differing from other alleles by a single nucleotide were discovered and six know alleles not previously tested by RSCA were identified. We demonstrate that RSCA is capable of resolving ambiguous combinations without the necessity of extra PCR amplifications or DNA manipulations and without previous knowledge of broad HLA type. RSCA for HLA typing is reliable, accurate and simple to perform, and should prove valuable for donor selection and patient matching, for population genetics and disease susceptibility studies.