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

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Primate Research Center Grants (P51)
Project #
5P51RR000167-38
Application #
6277725
Study Section
Project Start
1998-05-01
Project End
1999-04-30
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
38
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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