摘要：
Objective:To investigate the effect of loss of heterozygosity (LOH) in HLA region at initial diagnosis and remission of leukemia patient before transplantation on HLA typing.Methods:The HLA typing was performed in DNA extracted from peripheral blood obtained at diagnosis (Sample 1 and Sample 2) and remission (Sample 3) in one pretransplant male patient with mixedphenotype acute leukemia (MPAL).HLA typing for HLA-A,B,C,DQB1,DRB1 was performed by Sequence-based typing (SBT),Sequence-specific oligonucleotide probe hybridization (SSO) and Sequence-specific primers (SSP).To define more precisely a cutoff limit for the detection of a heterozygous DNA present in a fraction of the cells by the SBT technology,DNA mixing experiments were performed.Results:SBT results showed that Sample 1 and Sample 2 were both homozygous HLA results at five loci (lost one haplotype) although the sequencing background of Sample 1 was a little high.Except HLA-C locus was homozygous,Sample 3 was heterozygous HLA results at four loci.Based on DNA mixing experiments,a cutoff limit for the detection of heterozygous DNA was 20％ by SBT technology,and a detection threshold for HLA-A,B,C,DQB1,DRB1 heterozygosity in blood sampies was ＜ 75％ blasts.Conclusion:Because LOH may be partial,any homozygous HLA result obtained during a blast crisis,especially ≥75％ blasts,would have to be confirmed by a second typing on a buccal swab or on peripheral blood from the patient in complete remission.%目的:研究移植前白血病患者初诊时期和缓解期HLA区域杂合性缺失(LOH)对HLA分型的影响.方法:用3种HLA基因分型常用的方法(SBT、SSO和SSP方法)分别对1例急性混合细胞白血病(MPAL)患者初诊静脉血样本1(骨髓原始细胞为81.7％)、样本2(骨髓原始细胞为93.07％)和缓解期静脉血样本3(骨髓原始细胞为＜0.01％)进行HLA-A、B、C、DQB1、DRB1基因分型;通过2种DNA混合实验进一步确定SBT方法检测HLA杂合峰的检出阈值和检测HLA分型时对静脉血原始细胞比例的要求.结果:样本1和样本2的HLA-A、B、C、DQB1、DRB1均为纯合位点,其中样本1有些HLA位点的测序背景峰偏高,无法确定位点的具体结果;样本3的5个HLA座位除了C座位外均为杂合位点.DNA混合实验结果表明,用SBT方法检测HLA双等位基因杂合峰的最低检出限为20％;移植前白血病患者采用SBT方法检测HLA分型的要求是静脉血原始细胞＜75％,若≥75％,则可能因为原始细胞HLA区域出现LOH,从而导致HLA位点漏检.结论:对于白血病初诊患者,特别是原始细胞≥75％,如果HLA某一位点分型结果为纯合位点,应该再次采集患者缓解期静脉血或者正常体细胞,如口腔黏膜细胞,重新对该位点进行复核,排除HLA区域发生LOH造成假性纯合的可能,以免HLA配型结果发生错误,影响移植供者的选择和最终的移植效果.
