AMBIGUOUS SEROLOGICAL AND PCR-SSP HLA-B LOCUS TYPING RESOLVED BY DNA SEQUENCING. MS Kiamar, D J Mancuso, TM Vyvial, CM Kegelman, JD Williams and JA Danilovs, Immunogenetics Laboratory, Donor Network of Arizona, Phoenix, AZ

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Routine tissue typing of a Black/Hispanic/American Indian patient, typed elsewhere as
HLA-A23,A24,B60,B62,Bw4,Bw6, failed to confirm the presence of B60 or Bw4. Three of 7
B60 alloantisera were positive with no extra reactions observed. PCR-SSP testing was
performed using 2 different commercial kits. With both kits B*40 and B*07 reactions were
observed, but were inconsistent with known allele patterns, thus suggesting a rare, or possibly
new, B60 related allele. Additional PCR-SSP testing was performed by an independent
laboratory using a third commercial kit. Surprisingly, B*48 was assigned. However, the
laboratory was not provided with previous typing data. None of the 3 PCR-SSP kits used
detected Bw4. This difficult B-locus allele was identified by DNA sequencing at 2 different laboratories as HLA-B*4016. Subsequently, we obtained updated primers and were able to confirm the B*4016 allele by PCR-SSP testing. Notably, the DNA sequence of the B*4016 allele is identical to B*40012 for exon 2 and B*0705/B*0706 for exon 3 which may account for the ambiguous typing results observed. Other rare alleles consisting of sequences from 2 or more known alleles could also lead to ambiguous typing results for both serology and PCR-SSP.
In conclusion, this case study demonstrates the importance of staying current on updated
allele sequences and primers.