SIMULTANEOUS HLA-A AND DRB TYPING BY MULTIPLEXED PCR-SSP.
          Marta Albis Camps and Rainer Blasczyk. Deptartment of Hematology and Oncology, Blood Bank, Charité, Campus Virchow-Klinikum, Humboldt-University Berlin, Germany. 
          Sequence-specific primed PCR (PCR-SSP) has been proven to be a very reliable typing technique but suffers from the drawback that a large amount of primer mixes is necessary for HLA-A, B, C, DRB and DQB1 typing. Therefore, PCR-SSP turns out to be very labour-intensive. In order to sort out these limitations, we took advantage of the possibility to amplify simultaneously different fragments of a certain DNA sample in the same reaction tube (MULTIPLEX-PCR). The main requirement to be fulfilled is that the simultaneously amplified fragments have to differ significantly in their size, so that they can be clearly resolved by electrophoresis. The aim of the present study was to develop a multiplex-PCR protocol for two different HLA genes. Due to the high degree of sequence homology among HLA class I genes, co-amplification of different HLA class I loci in the set up of a complex typing system is infeasible. In contrast, a multiplex-PCR approach for HLA class I and class II genes is possible since they are of distinct evolutionary origin and are, thus, significantly differing in their structure and sequence. From a practical point of view, the HLA-A and DRB PCR-SSP sets applied previously did both consist of 24 primer mixes, suggesting themselves suitable for combination. Therefore, we developed a multiplex-PCR typing system consisting of 24 primer mixes for the simultaneous detection of HLA-A and DRB alleles. So far 120 samples were analysed using this method and the results are completely concordant with those obtained by conventional PCR-SSP. In conclusion, the application of this multiplexed HLA-A/DRB PCR-SSP typing system can effectively increase the laboratory's capacity and will bring the advantage of a complete PCR-based typing of all patients and donors closer to reality.