A 25-PERCENT ERROR RATE IN SEROLOGIC TYPING OF HLA-B HOMOZYGOTES

The microlymphocytotoxicity technique has been the accepted method for HLA class I typing since the early 1960s. However, it is often diffic ult to distinguish two related alleles expressed in an individual due to the crossreactive nature of the alloantibodies used in this techniq ue. This is especially evident at the HLA-B locus, whose more than 180 alleles fall into only 4 major interrelated cross-reactive antigen gr oups. To estimate the error rate in serologic typing due to the cross- reactive nature of sera, we used polymerase chain reaction with sequen ce-specific primers (PCR-SSP) amplification to retype 40 individuals w ho were previously typed as serologic HLA-B locus homozygotes. PCR-SSP revealed that 10 of these 40 individuals (25%) were actually heterozy gous at their HLA-B loci. The HLA-B locus alleles of 9 of these 10 dis crepant individuals were further analyzed by denaturing gradient gel e lectrophoresis followed by direct sequencing. The sequence analysis co nfirmed that all nine individuals were indeed HLA-B locus heterozygote s. This surprisingly high error rate in serologic definition of HLA-B molecules argues for the use of rapid DNA-based techniques in HLA clas s I typing, even in the setting of solid organ transplantation.