The identification of irregular antibodies and autoantibody by the single molecule sequencing technology and serological method
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摘要: 目的 探讨单分子测序技术联合血清学方法对于不规则抗体及自身抗体的鉴定。方法 将收集的1例不规则抗体筛查阳性患者样本利用单分子测序技术对Rh血型系统和Kidd血型系统红细胞抗原表型进行测序，利用血清学方法进行不规则抗体的筛查和鉴定及血型系统红细胞抗原表型鉴定。结果 采用血清学方法进行不规则抗体鉴定结果为抗E、抗JKb、类抗E抗体和自身抗体; 应用单分子测序方法检测Rh血型系统红细胞抗原表型，检测结果为Ccee，Kidd血型系统红细胞抗原表型，检测结果为JK(a+wb-)。结论 血清学方法不规则抗体鉴定结果与单分子测序技术红细胞抗原表型结果相符，不规则抗体鉴定结果为抗E、抗JKb、类抗E抗体和自身抗体。血清学方法与单分子测序技术相互配合，规避和补充目前的血清学分析的局限性，保证检测结果的准确性，更为患者筛选合适的血液提供指导，确保输血安全。Abstract: Objective To explore the identification of irregular antibodies and autoantibody by the single molecule sequencing technology combined with serological methods.Methods One patient with positive irregular antibody screening was collected, and the phenotypes of red blood cells in Rh blood group system and Kidd blood group system were sequenced by the single molecule sequencing technology, the irregular antibody was screened and identified by serological method and phenotype identification of red blood cells in blood group system were carried out by serological method.Results The results of irregular antibody identification by serological method were anti-E, anti-JKb, anti-E-like antibody and autoantibody. The single molecule sequencing method was used to detect the erythrocyte antigen phenotype of Rh blood group system, and the detection result was Ccee, and the detection result of erythrocyte antigen phenotype of Kidd blood group system was JK(a+wb-) .Conclusion The results of irregular antibody identification by serological method are consistent with the results of erythrocyte antigen phenotype by the single molecule sequencing technology, the results of irregular antibody identification were anti-E, anti-JKb, anti-E-like antibody and autoantibody, and the serological method and the single molecule sequencing technology cooperate with each other to avoid and supplement the limitations of current serological analysis, ensure the accuracy of test results, and provide guidance for patients to screen suitable blood and ensure the safety of blood transfusion.
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表 1 抗筛红细胞试剂盒抗原格局表及不规则抗体筛查结果
序号 Rh-hr Kidd MNS Duffy Lewis P 盐水试管法 微柱凝集法 D C E c e JKa JKb M N S s FYa FYb Lea Leb P1 1 + 0 + + 0 + + + + 0 + + 0 + + 0 0 3+ 2 + + 0 0 + 0 + + + + + + 0 0 + 0 0 2+ 3 + + + + + + + 0 + 0 + + 0 0 + + 0 3+ 自身 0 2+ 
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表 2 十六系抗筛谱红细胞试剂盒抗原格局表及不规则抗体筛查结果
序号 RH-Hr Kell Duffy RH-Hr C D E c e Cw f V K k Kpa Kpb Jsa Jsb Fya Fyb 1 R1wR1 + + 0 0 + + / / 0 + 0 + / + + 0 2 R1R1 + + 0 0 + 0 / / + + 0 + 0 + 0 + 3 R2R2 0 + + + 0 0 / / + + 0 + 0 + + + 4 R0 0 + 0 + + 0 / / 0 + 0 + / + + 0 5 r′r′ + 0 0 0 + 0 / / 0 + 0 + / + + 0 6 r″r″ 0 0 + + 0 0 / / 0 + 0 + / + 0 + 7 rr 0 0 0 + + 0 / / 0 + 0 + 0 + + 0 8 rr 0 0 0 + + 0 / / 0 + + + 0 + + 0 9 rr 0 0 0 + + 0 / / + 0 0 + / + 0 + 10 rr 0 0 0 + + 0 / / 0 + 0 + 0 + + + 11 RzR1w + + + 0 + + / / 0 + 0 + 0 + 0 + 12 RzR2 + + + + 0 0 / / 0 + 0 + / + + 0 13 rr 0 0 0 + + 0 / / 0 + 0 + / + 0 + 14 r′wr + 0 0 + + + / / 0 + 0 + / + 0 + 15 R2r 0 + + + + 0 / / 0 + 0 + 0 + + + 16 r′r + 0 0 + + 0 / / 0 + 0 + / + + + 自身 序号 Kidd Lewis P MNS Luther Xg a1 a2 a3 a4 Jka Jkb Lea Leb P1 M N S s Lua Lub Xga 1 + 0 0 + + + 0 0 + 0 + + 0 0 0 0 2 0 + 0 + + + + + 0 0 + / 2+ 2+ 1+ 0 3 0 + + 0 + 0 + + + 0 w / 4+ 4+ 1+ 0.5+ 4 + 0 0 + + + 0 + 0 + + + 0 0 0 0 5 + + 0 + + 0 + + + 0 + + 1+ 1+ 0.5+ 0 6 + + 0 + + + 0 + 0 0 + + 4+ 4+ 1+ 0.5+ 7 + 0 0 0 0 + + 0 + + + / 0 0 0 0 8 0 + 0 + + + + 0 + + 0 + 2+ 2+ 1+ 0 9 0 + + 0 + + + 0 + 0 + + 2+ 2+ 1+ 0 10 + 0 0 + + 0 + + 0 0 + / 0 0 0 0 11 + + + 0 + + + 0 + 0 + / 4+ 4+ 0.5+ 0.5+ 12 + + 0 + + + + 0 + 0 + / 4+ 4+ 0.5+ 0.5+ 13 0 + 0 + + 0 + 0 + + + + 2+ 2+ 1+ 0 14 0 + 0 + + + + 0 + 0 + 0 2+ 2+ 1+ 0 15 + + 0 + 0 + + + + + 0 + 4+ 4+ 1+ 0.5+ 16 + + 0 + + + + 0 + 0 + / 2+ 2+ 1+ 0 自身 2+ / / / 注：a1：微柱玻璃珠法检测患者血清抗体; a2：微柱玻璃珠法检测患者红细胞放散液抗体; a3：微柱玻璃珠法检测用CceeJKb+红细胞吸收患者血清后放散液抗体; a4：微柱玻璃珠法检测用CceeJKb-红细胞吸收患者血清后放散液抗体。
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表 3 患者红细胞抗原血清学分型
反应条件 Rh MN Kidd Lewis 抗-D 抗-C 抗-c 抗-E 抗-e 抗-M 抗-N 抗-JKa 抗-JKb 抗-Lea 抗-Leb 盐水试管法 4+ 3+ 3+ 0 3+ 0 3+ 1+ 0 0 0
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表 4 抗体效价测定
筛检红细胞(检测方法) 1∶1 1∶2 1∶4 1∶8 1∶16 1∶32 1∶64 1∶128 eeJKb+型红细胞(微柱玻璃珠法) 2+ 2+ 1+ 1+w 0 0 0 0 E+JKb+型红细胞(微柱玻璃珠法) 4+ 4+ 3+ 3+ 2+ 1+s 1+w 0
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表 5 红细胞抗原基因鉴定结果
表型1 等位基因名称1 突变1 表型2 等位基因名称2 突变2 最终预测表型 JK(a+w) JK*01W.06 c.130G>A; c.588A>G JK(a+w) JK*01W.06 c.130G>A; c.588A>G JK(a+wb-) RH：2 or C
RH：5 or e
RH：7 or CeRHCE*02 or RHCE*Ce c.48G>C; c.150C>T;
c.178C>A; c.201A>G;
c.203A>G; c.307C>TRH：4 or c RH：5 or e RH：6 or f(ce) RHCE*01 or RHCE*ce Ccee
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