It is well recognized that the anti-HLA antibodies in recipients are associated with prognosis of graft rejection in transplantation including both solid organ transplantation and bone marrow stem cell transplantation. Recently, a detection system for anti-HLA antibodies using the HLA-antigen-conjugated fluorescence beads has been widely used in the laboratory testing. We have experienced three cases in which a discrepancy of results between the cross-match tests and bead-based detection system for anti-HLA-antibodies. Dilution of serum with the discrepant results showed that the bead-based detection system failed to detect several specific anti-HLA-antibodies, suggesting that the discrepancy was representing the prozone-like phenomenon. Treatment of the serum with discrepant results by freeze-thaw or heat inactivation for 30 min at 56℃ suggested the involvement of complement C1q in the prozone-like phenomena. Further experiments using rabbit complement or human serum also supported the involvement of C1q in the phenomena. In addition, analysis of sequential 20 cases with anti-HLA-antibodies revealed that 10 of them showed discrepancy results and anti-HLA-antibodies were failed to be detected by using the bead-based detection system in three of them. These observations indicated the importance of considering a possibility for the prozone-like phenomena in detection of anti-HLA-antibodies.

Current HLA-DNA typing methods are mainly PCR-SBT and PCR-Luminex used as routine testing. However, these methods generally yield ambiguous typing results because of lacking of oligonucleotide probes and phase ambiguity for HLA allele determination. In this paper we described the development and first application of 8-digit level super high resolution single molecule-sequence based typing (SS-SBT) method for HLA-A, -B and -C loci using next generation sequencer, Roche GS Junior Bench Top System aimed at elimination of ambiguities. HLA-A, -B and -C specific PCR primers were designed to amplify the entire gene regions from the enhancer-promoter to the 3' untranslated region. The PCR condition was set to amplify both of the HLA alleles of the HLA-A, -B and -C gene with 1:1 ratio. By this SS-SBT HLA-DNA typing of the HLA class I loci using DNA samples that were observed with ambiguities for HLA alleles when defined by conventional HLA-DNA typing method, all of them were unequivocally defined to single HLA alleles at the 8-digit level without ambiguity by next generation sequencing. Therefore, our SS-SBT method is a superior, complete and ultimate HLA-DNA typing method to efficiently detect new HLA alleles and null alleles along with effective 8-digit level DNA typing for the HLA class I genes without ambiguity.