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An HLA-B null allele was identified in a Japanese family during histocompatibility testing for bone marrow transplantation. The propositus was a healthy Japanese woman with three children, and her parents were cousins. Serological HLA typing of the family members indicated that the propositus was homozygous for the A24-Cw4-B blank (B null)-DR4.2-DQ3 haplotype. Total RNA was extracted from peripheral blood of the propositus was converted to first-strand cDNA using reverse transcriptase. The cDNA was amplified by the polymerase chain reaction (PCR) using HLA-B locus-specific primers. The PCR product showed no change in size upon polyacrylamide gel electrophoresis (PAGE) compared to that of normal controls, suggesting that HLA-B gene mRNA was normally expressed. The nucleotide sequence of the cDNA was the same as that of B*1501 except at nucleotide 369 or codon 123, where C was replaced with A; TAC encodes Tyr whereas TAA is a stop codon. This point mutation may have truncated the HLA-B molecule of the propositus, resulting in the negative results we obtained with anti-HLA-B sera.

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Abstract:The first nationwide nucleic acid amplification testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-l) of voluntarily donated blood after serological pre-screening and before release of cellular components and plasma for fractionation was implemented by the Japanese Red Cross Blood Transfusion Services. From February 1, 2000 to April 30, 2001, specimens from 6,805,010 units of serologically negative donation were screened in minipools of 50 samples within 24 hr after blood donation by NAT using multiplex HBV/HCV/HIV-l reagent for blood transfusion including short shelf-life platelets. Among them, 112 HBV DNA-positives, 25 HCV RNA positives and 4 HIV-l RNA positives were screened out and we could prevent transfusion of these NAT positive units. Subtypes/genotypes of HBV DNA, adr/C, adw/A, adwlB, adw/C, ayr/C and aywlD were found and adr/C was predominant. A total of 61.6 % of them (69/112) were negative by overnight EIA. Sixth three of HBV NATpositive samples carried virus loads less than 10 4 copies ImL and 92.1 % of them (58/63) were negative by overnight EIA. The virus growth curves of HBV in 6 cases obtained by retrospective and prospective follOW-Up study showed exponential straight lines in the early stage of serological window periods and the log times ofHBV growth (10 fold increase) in serological window period were between 4.6 and 7.6 days. NAT screening with highly sensitive reagents in pool of specimens is useful to exclude blood units with low level of HBV and HBV mutants from blood transfusion.

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