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目的 RhD阴性存在多种变异体,通过研究成都地区RhD阴性献血者,了解其遗传背景。方法 2009年6—11月采集的74947人份全血中获得盐水法初筛阴性标本318例,采用间接抗球蛋白试验(IAT)筛选部分D和弱D,对IAT阴性的标本采用吸收放散试验,筛查Del型,采用序列特异性引物PCR(PCR-SSP)检测各种变异D基因型。检测的Del等位基因包括RHD(M285)、RHD(IVS3+1)、RHD(IVS1+1)、RHD(1227A)、RHD(DelEX9)、RHD(M1I)、RHD(R10W)、RHD(L84P)。吸收放散试验阳性标本使用DelDNA分型试剂盒筛查,未分出型别的标本检测RHD基因1—7和9—10外显子,并对检测到的各种变异D以及疑难标本进行测序分析。结果 318份盐水法初筛阴性标本中检出IAT阳性标本3例,弱D152例,部分D(RhDⅥⅢ)1例。成都地区献血人群中RhD阴性比例为0.42%,表型dccee(56.19%)和dCcee(32.06%)比例高。对可获得的303例IAT阴性标本进行微量吸收放散试验,发现阳性71例。通过DelDNA分型试剂盒检测,RHD(1227A)型57例、RHD(M1I)型1例、无法确定为已知的Del型13例。无法确定的13例标本通过检测RHD基因1—7和9—10外显子,发现11例部分或全部缺失上述外显子,2例含有完整的RHD基因。34例吸收放散阴性含有C或E抗原标本,经检测RHD基因1—7和9—10外显子及1227A,发现RHD(1227A)型9例、部分或全部缺失型25例。血清学试验和分子生物学试验确定的Del型共69例,占IAT筛查RhD阴性的22.77%(69/303)。结论成都地区常规血清学筛查RhD阴性献血人群中RHD基因存在多态性,RHD1227A等位基因是Del型的主要基因类型。吸收放散试验检测Del准确率偏低。
Purpose There are many variants of RhD negative. Through studying RhD-negative blood donors in Chengdu, we can understand the genetic background. Methods Seventy-four (74) and nine-nine (749) samples of whole blood collected from June to November in 2009 were screened by indirect immunoglobulin test (IAT) for partial D and weak D, respectively. , Screening Del type, using sequence specific primer PCR (PCR-SSP) to detect various variant D genotypes. The detected Del alleles include RHD (M285), RHD (IVS3 + 1), RHD (IVS1 + 1), RHD (1227A), RHD (DelEX9), RHD (M1I), RHD (R10W), RHD . Absorption and desorption test positive specimens using DelDNA typing kit screening, undifferentiated specimens detected RHD gene 1-7 and 9-10 exons, and the detected variety of variation D and difficult specimens sequencing analysis . Results Among the 318 samples with negative IAP, 3 were positive for IAT, 152 were weak and 1 was D (RhD Ⅵ). The RhD-negative rate was 0.42% in blood donors in Chengdu. The phenotypes of dccee (56.19%) and dCcee (32.06%) were high. A total of 303 IAT negative specimens were obtained for microprecipitation, and 71 patients were positive. According to the DelDNA typing kit, 57 cases of RHD (1227A) and 1 case of RHD (M1I) could not be identified as 13 cases of Del type. Of the 13 indeterminate samples, 11 and 9-10 exons of RHD gene were detected, and 11 of them were found to have partial or complete deletion of the above exon and 2 of them contained the complete RHD gene. Of the 34 patients with negative or negative samples containing C or E antigen, 9 and 13 RHD (1227A) and 25 partially or completely deleted RHD were detected by detecting exon 1-7 and 9-10 and 1227A of RHD gene. Serotypes and molecular biology tests confirmed a total of 69 cases of Del type, accounting for 22.77% (69/303) of Rh-negative IAT screening. Conclusion There are polymorphisms of RHD gene in routine serological screening of RhD-negative blood donors in Chengdu. The RHD1227A allele is the major type of Del-type. Absorption test to detect Del low accuracy.