Lorentzen D F, Iwanaga K K, Meuer K J, Moritz T L, Watkins D I
Histocompatibility and Molecular Diagnostics Laboratory, University of Wisconsin Hospital and Clinics, Madison, USA.
Tissue Antigens. 1997 Oct;50(4):359-65. doi: 10.1111/j.1399-0039.1997.tb02888.x.
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. PCR-SSP revealed that 10 of these 40 individuals (25%) were actually heterozygous at their HLA-B loci. The HLA-B locus alleles of 9 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. 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.
自20世纪60年代初以来，微量淋巴细胞毒性技术一直是HLA I类分型的公认方法。然而，由于该技术中使用的同种抗体具有交叉反应性，通常很难区分个体中表达的两个相关等位基因。这在HLA - B位点尤为明显，其180多个等位基因仅分为4个主要的相互关联的交叉反应抗原组。为了评估由于血清的交叉反应性导致的血清学分型错误率，我们使用序列特异性引物聚合酶链反应（PCR - SSP）扩增对40名先前被血清学鉴定为HLA - B位点纯合子的个体进行重新分型。PCR - SSP显示，这40名个体中有10名（25%）在其HLA - B位点实际上是杂合子。对这10名有差异个体中的9名的HLA - B位点等位基因通过变性梯度凝胶电泳随后直接测序进行了进一步分析。序列分析证实所有9名个体确实是HLA - B位点杂合子。HLA - B分子血清学定义中这种惊人的高错误率表明，即使在实体器官移植的情况下，在HLA I类分型中也应使用基于DNA的快速技术。
