Determination Of RHD Zygosity In Chinese Population And Function Research Of Rh Protein Complex Components CD47

Rh blood group system is very complicated blood group system,the clinic significance of Rh blood group system is just after the ABO blood group system.D is the most immunocompetent of all Rh antigens.Hemolytic disease of the newborn and blood transfusion reaction which are caused by D antigens are very common.D antigen encoded by the RHD gene,determination of RHD zygosity is a new technology,it has important clinical value in diagnosing,guardianship and control of Rh same species immunity and hemolytic disease of newborn.Such as in 2000, Wagner established restriction fragment length polymorphism normality(RFLP) method to test RHD gene zygosity,but in the majority of Chinese people,only use RFLP method to detect RHD zygosity,and to speculate RhD serological phenotype might be miscarriage of justice,we tested RHD zygosity based on Rhesus box and RHD gene exon.In this study,265 cases(93 cases of RhD positive and RhD-negative 72 cases,84 cases of Del,16 cases other variant D)were taken from healthy blood donors of Shanghai and Xinjiang Blood Center during September 2006 to December 2007,in addition to 5 Uighur and 1 Hui donors,the majority Han nationality.After using IgM, IgG monoclonal anti-D to determinate RhD antigen and weak D,ues D epitope determination kit type to test D variant and absorb emission to test Del.TIANamp Genomic DNA kit extraction extracted genomic DNA of all samples.Through three methods on detection of RHD zygosity,including PCR-RFLP, duplex PCR which amplified RHD exon 1 and hybrid Rhesus box,amplification of real-time fluorescence quantitative PCR which amplified RHD 5 and exon 7,the result showed that PCR-RFLP and duplex PCR has same results,and there were 9 discrepancies(3 RhD-negative,2 Del and 4 variant D)between RFLP and real-time fluorescence quantitative PCR.Used multiplex PCR and sequencing for further research,the genotype of these samples were D^{ⅥⅢ}/RHD^-,RHD-CE(2-9)-D/RHD^-, RHD 1227A /RHD 711 del C.This study introduced real-time quantitative PCR, through detecting RHD exons directly to determine RHD zygosity,which is not only a partial solution to the"invalid RHD genes"miscarriage of justice,but also to solve the general PCR can only detect if RHD exons exist unable to determine zygosity problems.Both methods for testing Rhesus box and RHD gene must be used to determine RHD zygosity,in this way inaccurate results can be avoided.Another 20 cases were randomly selected from different RhD phenotype(7 Rh-positive,7 Del,3 Rh negative,3 variant D)for rhesus box sequencing,results showed that hybrid Rhesus box does not appear polymorphism,which is consistent with the preliminary studies.7 variants s were found in Downstream rhesus box, 4951G→A mutation,5161T→C mutation,5222G→A mutation,5442G→A mutation,5467 G→C mutation,7403 G→C mutation,7443A→T mutation. However,due to limited number of sequenced samples,and the variants are not distribution ruly,there is no evidence showed any relevant between RhD phenotype and these variants.As Rh protein complex components,research of CD47 function in recent years become a new hot topic.CD47 is self-marker of erythrocyte membrane,it can prevent macrophages phagocytosising normal erythrocyte through interacting with SIRPa which is on the membrane of macrophage.Therefore Researchers infer that CD47 play an important role in regulating erythrocyte life during blood circulation.This study randomly selected fresh blood samples of 18 samples from Shanghai Blood Center,taken through the capillary hematocrit centrifugation divided erythrocyte into young,middle and old,which were detected by flow cytometry. Divided erythrocyte were stored in erythrocyte maintenance fluid at 4℃,and a follow testing,continuing five-week,gathering 5 groups effectively data in total.Results showed that although there is difference among individuals CD47 expression,but both in vivo and in vitro,CD47 showed downward trend during the aging process, while the differences of downward percentage among individuals were also exist, after the preservation for 28 days,CD47 of total erythrocytes decreased 51%,44% decline in young erythrocytes,46%decline in old erythrocytes.In same test CD47 of old erythrocytes decline 40%than young erythrocytes.This phenomenon indicates that both in vivo and in vitro,CD47 showed large downward trend during erythrocytes aging process,CD47 may play an important role in inhibiting phagocytosis.