Molecular biological analysis of serological preliminary screening RhD negative population and D variant population
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摘要: 目的 探讨血清学结合分子生物学在鉴定RhD阴性及D变异血型中的重要意义。方法 采用盐水法对医院2021年5月—2023年12月的54 380例标本进行RhD初筛；采用间接抗人球蛋白试验对初筛阴性的标本进行阴性确认和抗体筛选；采用抗C、抗E标准试剂进行C、E抗原检测；采用吸收放散试验筛选DEL型；采用聚合酶链式反应-序列特异性引物(PCR-SSP)方法进行RHD基因分型，并对其10个外显子序列进行扩增；采用Sanger法对血清学与PCR-SSP结果不一致或基因扩增格局特殊的标本进行测序。结果 在54 380例标本中检出251例RhD阴性标本及3例弱D(IgM抗-D < 3+)标本，经阴性确认IgG抗-D阳性12例，占初筛阴性样本的4.78%；抗筛阳性4例，经鉴定均为抗-D。254例标本可划分为8种表型，以cde表型最多，占比59.84%(152/254)；其次是Cde，占比27.95%(71/254)；cDe最少见，仅有1例。吸收放散试验共筛选出39例DEL变异型，占初筛阴性样本的15.35%；其中38例为CDe表型，1例为cDe表型。分子生物学试验结果显示，254例标本中，仅178例完全缺失RHD基因，占比70.08%；剩余76例中共检测出9种RHD变异类型。其中最常见的为RHD*DEL1 (c. 1227G>A)，发生频率约为0.07%(40/54 380)；其次为RHD-CE(2-9)-D，发生频率约为0.03%(18/54 380)。DVI type3和weak D type15为该人群第三常见的基因型，发生频率约为0.01%。另外还发现2例RHD*711DELC基因型和3种罕见RHD基因类型：RHD-CE(2-7)-D、weak D type 1 (c. 809T>G)和RHD*970del3, 976del16，均只有1例。本研究还发现了2例RHD*01/RHD*01N.01杂合型，其中1例血清学为弱D，经测序在其第8内含子上的1154-31位点发生了T>C的突变，该突变并未被国际输血学会数据库收录，其血清学意义不明。而另1例的血清学为阴性，经RHD外显子测序并未发现相应的突变，具体机制有待进一步研究。结论 本研究通过血清学与分子生物学联用的方法，检测出研究人群独特的RHD基因类型，进一步丰富了沈阳地区的RHD等位基因库，在准确鉴定血型、保障患者输血安全方面具有重要意义，有利于提高临床精准输血管理水平。Abstract: Objective To explore the significance of serology combined with molecular biology in the identification of RhD negative and D-variant blood groups.Methods RhD screening was performed on 54 380 samples from May 2021 to December 2023 by saline method. Indirect antiglobulin test(IAT) was used to confirm the negative and screen the antibody of the negative specimens. C and E antigen were detected by anti-C and anti-E standard reagents. DEL type was screened by absorption and release test. RHD was genotyped by polymerase chain reaction-sequence-specific primer(PCR-SSP), and its 10 exon sequences were amplified. Sanger method was used to sequence the samples with different serological and PCR-SSP results or special gene amplification pattern.Results Among the 54 380 samples, 251 RhD-negative specimens and 3 weak D(IgM anti-D < 3+) specimens were detected, and 12 cases were confirmed positive by IgG anti-D, accounting for 4.78% of the negative samples. There were 4 anti-ethmoid positive cases, all of which were identified as anti-D. The 254 samples were classified into 8 phenotypes, and cde phenotype was the most common, accounting for 59.84%(152/254). The second was Cde, accounting for 27.95%(71/254). cDe is the rarest, with only 1 case. A total of 39 cases of DEL variant were screened by absorption and release test, accounting for 15.35% of the negative samples. Of these, 38 were CDe phenotypes and 1 was cDe phenotype. The results of molecular biology experiments showed that only 178 of 254 samples were real RHD deletion, accounting for 70.08%. A total of 9 RHD variants were detected in the remaining 76 subjects. The most common was RHD*DEL1 (c. 1227G>A), with a frequency of about 0.07%(40/54 380). It was followed by RHD-CE(2-9)-D, with a frequency of about 0.03%(18/54 380). DVI type3 and weak D type15 were the third most common genotypes in this population, with a frequency of about 0.01%. In addition, 2 cases of RHD*711DELC genotype and 3 rare RHD genotypes were found: RHD-CE(2-7)-D, weak D type1 (c. 809T>G), and RHD*970del3, 976del16, with only 1 case each. It is worth noting that this study also found 2 cases of RHD*01/RHD*01N. 01 heterozygous type, of which 1 case was serologically weak D and had a T>C mutation at sites 1154-31 on intron 8 after sequencing. This mutation was not included in the ISBT database and its serological significance was unknown. The serology of the other case was negative, and no corresponding mutation was found by RHD exon sequencing. The specific mechanism needs to be further studied.Conclusion In this study, the unique RHD genotype of the study population was detected through the combination of serology and molecular biology, which further enriched the RHD allele pool in Shenyang area, which is of great significance in accurately identifying blood types and ensuring the transfusion safety of patients, and is conducive to improving the level of clinical accurate transfusion management.
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Key words:
- serology /
- molecular biology /
- RhD negative /
- D variation /
- RHD gene /
- safe blood transfusion
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表 1 254例RhD阴性及弱D受检者的血清学统计结果
例 分型 RhD(-) IgM抗-D(＜3+) IgG抗-D 抗筛阳性 吸收放散阳性 CDE 2 2 CDe 7 7 38 cDE 2 3 2 cDe 1 1 1 CdE 4 Cde 71 cDE 12 cde 152 4 合计 251 3 12 4 39 
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表 2 254例血清学RhD阴性及弱D受检者的RHD基因型、表现型和分布频率统计结果
例(%) 编号 基因型 表现型 发生频率 核苷酸变化 氨基酸变化 杂合型 ISBT命名 1 RHD-CE(2-9)-D D negative 18(0.03) Hybrid RHCE(2-9) / RHD+/RHD- RHD*01N.03 2 RHD-CE(2-7)-D D negative 1(0.002) Hybrid RHCE(2-7) / RHD+/RHD- RHD*01N.05 3 DVI type3 Partial D 6(0.01) Hybrid RHCE(3-6) / RHD+/RHD+
RHD+/RHD-RHD*06.03 4 RHD*DEL1 DEL 40(0.07) c.1227G>A p.Lys409Lys RHD+/RHD+
RHD+/RHD-RHD*01EL.01 5 weak D type15 weak D 5(0.01) c.845G>A p.Gly282Asp RHD+/RHD-
RHD+/RHD+RHD*15 6 weak D type1 weak D 1(0.002) c.809T>G p.Val270Gly RHD+/RHD- RHD*01W.1 7 RHD*711delC D negative 2(0.004) c.711delC p.Val238Cysfs*8 RHD-/RHD- RHD*01N.16 8 970del3, 976del16 D negative 1(0.002) c.970_972delCAC；c.976_991delTCCATCATGGGCTACA p.His324
Serfs*29RHD-/RHD- RHD*01N.28 9 RHD*01/RHD*01N.01 D negative 1(0.002) / / RHD+/RHD- RHD*01/RHD*01N.01 weak D 1(0.002) c.1154-31T>C / RHD+/RHD- 未命名 10 RHD deletion D negative 178(0.33) RHD deletion / RHD-/RHD- RHD*01N.01
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