25% ERROR RATE IN SEROLOGIC TYPING OF HLA B HOMOZYGOTES

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The microlymphocytotoxicity technique has been the accepted method for HLA class I typing since the early 1960s. However, it is often difficult to distinguish two related alleles expressed in an individual due to the cross-reactive nature of the alloantibodies used in this technique. This is especially evident at the HLA-B locus, whose more than 180 alleles fall into only 4 major interrelated cross-reactive antigen groups. To estimate the error rate in serologic typing due to the cross-reactive nature of sera, we used polymerase chain reaction with sequence-specific primers (PCR-SSP) amplification to retype 40 individuals who were previously typed as serologic HLA-B locus homozygotes. OBJECTIVE: To estimate the error rate in serologic typing due to the cross-reactive nature of sera. RESULTS PCR-SSP revealed that 10 of these 40 individuals (25%) were actually heterozygous at their HLA-B loci. The HLA-B locus alleles of these 10 discrepant individuals were further analyzed by denaturing gradient gel electrophoresis followed by direct sequencing. The sequence analysis confirmed that all nine individuals were indeed HLA-B locus heterozygotes. FUTURE DIRECTIONS This surprisingly high error rate in serologic definition of HLA-B molecules argues for the use of rapid DNA-based techniques in HLA class I typing, even in the setting of solid organ transplantation. KEY WORDS cross-reaction, HLA-B, DGGE, homozygote, microlymphocytotoxicity, PCR-SSP, serology