RhD双群患者的血清学及分子生物学分析--附1例报道

Molecular genetic analysis of a patient with double population of red blood cells for RhD antigen

目的对1名配血困难的RhD双群(double population,DP)患者进行血清学及分子生物学分析,解决其RhD血型鉴定及临床用血问题。方法用毛细管离心法分离患者红细胞,对近心端、远心端红细胞分别进行ABO和Rh血型鉴定,以及直接抗人球蛋白试验;对患者血浆进行不规则抗体筛选及鉴定以及交叉配血试验;采用限制性片段长度多态性聚合酶链反应(PCR-RFLP)检测RHD基因合子型,采用序列特异性聚合酶链反应(PCR-SSP)进行RHD和RHCE基因型检测。结果血型鉴定的结果显示该患者血型为B型,RhD及RhCE的c和E血型抗原均为DP,直抗阳性,抗体筛查及鉴定结果显示血浆中含有抗-D;PCR-RFLP结果显示该患者RHD基因盒子型为D-/D-纯合子,即为RHD编码基因缺失型所致的RhD阴性;PCR-SSP的结果也显示该患者RHD编码基因外显子均缺失,RHD基因型为RHD*01N.01/01N.01,RHCE基因型为Ccee。结论本例患者在急救情况下输注了RhD阳性血液,之后被误判为RhD阳性血型,实则为RhD阴性血型。本中心在血型鉴定为DP时,利用分子生物学方法对患者进行了Rh血型准确鉴定,为该患者的精准临床用血提供了依据。

Objective To solve the difficulty of RhD blood group typing in a patient with double population(DP)of red blood cells for RhD antigen by serological and genotyping analysis.Methods Separation of the two populations of red blood cells of the patient was performed using capillary centrifugation method.ABO,RhD and RhCE typing,direct anti-human globulin test(DAT),irregular antibody screening,antibody identification and blood crossmatching of the patient were conducted using the standard serological methods.The hybrid Rhesus zygosity analysis of the RHD gene was performed by PCR-RFLP method.RHD and RHCE genotype of the patients were identified by PCR-SSP method.Results The patient was B type but with DP of red blood cells for RhD,Rhc and RhE antigens.DAT of the patient was positive and the alloanti-D was detected in serum.The RHD zygosity was D-/D-homozygote.PCR-SSP testing showed the RHD gene deletion(RHD~*01N.01/01N.01 genotype)and Ccee of RHCE genotype in the patient,which was consistent with RHD zygosity analysis.Conclusion This is a special case with D-negative phenotype which was wrongly detected as D-positive type after D-positive red blood cells transfusion in emergency.When the DP of red cells for D antigen encountered like this case,the RhD typing can be accurately determined by using RHD genotyping analysis to provide strong evidence to the clinical blood transfusion.