Phenotypic Distribution And D Variant Genotypes Of Rhnegative Voluntary Blood Donors In Xinxiang Area

BackgroundRh blood group is a more complex system of human red blood cells,and its clinical importance is second only to ABO blood group system.55 antigens are included in the Rh blood group system,among which D antigen is the strongest immunogenicity.If one unit of Rh-positive red blood cell is injected into Rh-negative individuals in clinical,about 27%to56%of individuals can produce anti-D antibody.The RH gene is located in human chromosome 1p34.3-36.1,and the deletion,recombination and mutation of the RHD gene can lead to the generation of D variant.Therefore,accurate identification of blood type is the primary work to avoid severe hemolytic transfusion reaction and neonatal hemolytic disease and other adverse consequences.However,due to the limitations of conventional serological methods,it is difficult to accurately detect specimens of the D variant in clinical.Therefore,we propose to implement Sanger sequencing to genotype individuals with D variant in blood donors in Xinxiang area,in order to accurately identify blood types,lay the foundation for genotype-matched transfusion therapy,and ensure the scientific and efficient blood transfusion.ObjectiveTo observe the distribution of ABO and Rh phenotypes of Rh-negative voluntary blood donors in Xinxiang area,and to genotype D variant individuals in order to understand the genetic characteristics and polymorphisms of D variants in Rh-negative blood donors,and to build a database of D variants in the region,so as to provide an important basis for clinical formulation of individualized and precise blood transfusion protocols.Methods1.Blood group serology tests for identification of ABO and Rh blood groups:According to the National Clinical Laboratory Practice,all voluntary blood donors underwent ABO positive and negative typing and RhD blood group identification by standard blood group serological methods.After initial screening of 269 RhD negative specimens,RhD negative confirmation was performed by indirect anti-human globulin test（IAT）using anti-D reagents from three different manufacturers,while self-control was done to exclude autoantibodies and other interference.13 D variant specimens were finally detected and Rh typing was performed to confirm C,c,E and e phenotypes.2.Genotyping of D variant specimens:For individuals suspected of RhD variants by serological detection,2 ml of EDTA-K₂anticoagulant whole blood was collected,and genomic DNA was extracted according to the reagent operation instructions,PCR amplification and Sanger gene sequencing were performed on the exons of RHD gene,and the types of RhD variants were determined according to their single nucleotide polymorphisms（SNPs）sites.Results1.Distribution of ABO blood groups among Rh-negative voluntary blood donors in Xinxiang:Among 110,667 unpaid donors,269 cases were initially screened RhD-negative,accounting for 0.24%of all unpaid blood donors.Further confirmation of Rh negativity was256 cases（95.17%,256/269）.Among the 256 Rh-negative individuals,the gene frequency of ABO blood group antigen was r>q>p,and the phenotypic characteristics were B>A>O>AB.The distribution of ABO blood groups among the RhD-negative blood donors in Xinxiang area was consistent with the Hardy-Weinberg population genetic pattern.2.Distribution of Rh phenotypes among Rh-negative unpaid blood donors in Xinxiang:Among the 256 confirmed Rh-negative donors,the Rh antigen phenotypes were ccdee and Ccdee,which accounted for the largest proportion,respectively,155 cases of ccdee（60.55%,155/256）,75 cases of Ccdee（29.30%,75/256）,14 cases of ccd Ee（5.47%,14/256）,CCdee 9 cases（3.51%,9/256）,and ccd EE 3 cases（1.17%,3/256）.3.Rh antigen phenotype distribution and genotype analysis of D variant individuals in Xinxiang area:13 D variant specimens were found by Rh negative confirmation test,and their Rh CE phenotypes were CCee（2/13）,Ccee（6/13）and cc Ee（5/13）.After Sanger gene sequencing analysis,7 specimens were found to be weak D15 type with 845G>A heterozygous mutation in exon E6;4 DEL type specimens had 1227G>A heterozygous mutation in exon E9,and one of them also had C>A mutation in the 152nd base of the 7th intron;the other 2 specimens had exons E6～E9 replaced by RHCE gene,which showed RHD-CE（6-9）-RHD.Conclusions1.The ABO blood types of Rh-negative voluntary blood donors in Xinxiang area were B>A>O>AB,the distribution of Rh phenotypes was mostly ccdee and Ccdee,and the Rh antigens of RhD variant individuals were mostly C and E positive.2.The allele types of RhD variants in the Xinxiang area were mostly RHD*15 and RHD*DEL1,and the less common partial D type RHD*DIV.3 was also found.