VERIFICATION OF HOMOZYGOSITY BY QUANTITATIVE PCR-SSP BASED FLUOROTYPING.
          K Slateva, M Albis Camps and R. Blasczyk. Department of Hematology and Onkology, Blood Bank, Charité, Campus Virchow-Klinikum, Humboldt-University Berlin, Germany.
          In PCR-SSP, the way to determine homozygosity is based on the absence of an amplification product. This bears the risk to oversee unknown alleles not amplified with the current primers. The aim of our study was to examine homozygosity in HLA class I and DRB using the quantitative features of fluorotyping. In homozygotes the individual allele is present in a double amount. However, the agarose gel-based read out is not capable of recognizing this double allele content. In contrast, fluorotyping is capable of delivering quantitative information. In this study we investigated 30 homozygous samples. In HLA class I, the results after 33 cycles indicated clearly higher fluorescence intensities as usually observed in heterozygotes. This quantitative relationship was not found in HLA-DRB. For that reason we explored the PCR kinetics and determined the increase of the fluorescence every 2nd cycle. The signals obtained in the homozygotes were essentially higher than those of the heterozygotes during the monitored period from cycle 23 to 43. The signal intensities for HLA class I increased from cycle 23 to cycle 39, and revealed the maximum differences between homozygotes and heterozygotes at cycle 37. The PCRs led in individual plateaus at cycle 39. In contrast to HLA class I, in HLA-DRB the fluorescence intensities of the homozygotes and heterozygotes increased more rapidly and converged after 25 cycles in the same PCR plateau. This is probably due to the extremely high efficiency of the amplification of the short HLA class II exon 2 sequences. Based on these PCR profiles it should be possible to design nomograms for each individual specificity which might be very helpful to provide evidence for homozygosity. This method can not replace family studies. Nevertheless, the quantitative typing information essentially increases the reliability of fluorotyping compared to conventional agarose gel based PCR-SSP.