12-P

Abstract

Use Real Time PCR (RT-PCR) to verify the identity of donor cells used in flow crossmatch. Following lymphocyte isolation for flow crossmatch, an aliquot of cells is used for DNA isolation using the Qiagen EZ1™ extractor. The aliquot is taken from the SAME tube used to count cells for the crossmatch. The DNA is plated on LinkSeq™ABDR typing trays from Linkage Biosciences™. The trays are amplified and melt curve analysis is performed on the Viia 7™ RT-PCR instrument from Life Technologies™. This method of HLA typing requires no hybridization or gels, so it is possible to perform a typing concurrently with the flow crossmatch. The size of the cell aliquot used for DNA extraction is dependent on the cell count. As part of validation, a range of cell counts was outlined.[ Table 1 ] All cells yielded enough DNA to get a complete ABDR typing. There was concern this would increase crossmatch turn-around-times to an unacceptable level, but because this is a semi-automated method, it did not increase the TAT significantly. The DNA is extracted while the crossmatch is being set up; the RT-PCR reactions are set up during the 30 minute cell/serum incubation. After the crossmatch has been completed, the HLA typing is analyzed to the level that donor cell identity can be confirmed. The cells we used for verification typing had been previously HLA typed. Verification HLA typing can be less “perfect” than when doing an HLA typing for deceased donor organ allocation. This is a semi-automated HLA typing platform that has proven to be robust, reproducible, and well suited to HLA typing to verify the identity of donor cells used in flow crossmatching. It does not prevent a sample mix-up, but it provides a strong barrier to reporting crossmatches in which the wrong donor cells may have been used.