The Research About The Correlation And The Mechanism Between DEL Phenotype And Anti-D Alloimmunization

In the Rh blood group system,the D antigen(RH1)carried by the Rh D polypeptide is the most important and highly immunogenic antigen.Anti-D isoantibody is the commonest cause of haemolytic disease of the fetus and newborns.D variants are usually divided into two categories: partial D and weak D.They show substantial ethnic variability.Partial Rh D variants result from RHD/RHCE gene recombination events or RHD nucleotide polymorphisms affecting generally exofacial Rh D sections and leading to loss of one or more Rh D epitopes.The Rh phenotype DEL(D-elute)can only be detected by adsorbing and eluting anti-D from the cell surface,which exhibits trace amounts of the D antigen on the red blood cell and are routinely typed Rh D-negative.To date,Rh DEL phenotypes have been traced to more than seventeen distinct alleles: RHD1227 A,RHD(IVS3+1A),RHD885 T,RHD(IVS5-38del4),RHD1252 T,RHD(del Ex8),RHD1222 C,RHD(IVS1+1A),RHD3 A,RHD(L18P),RHD(L84P),RHD(R10W),RHD(A137E),RHD-CE(4-9)-D,RHD-CE(2-5)-D,RHD(1-9)-CE.DEL phenotypes can both exhibit a qualitative variation and a quantitative decrease in D antigen expression.A new concept has been reported distinguishing between DEL phenotypes expressing complete repertoire of D antigen epitopes versus a partial DEL with detectable D epitopes loss.The DEL varianthas not been reported in Africans,and is rare in Caucasians.Approximately 30% of apparently Rh-negative peoples of East Asia actually were DEL.So studies on the DEL phenotypes in Chinese Han population not only have the advantage of resources,but also contribute to increase data on the Rh blood group.In this study the anti-D alloimmunization,molecular properties and D epitopes of the pregnant women with DEL phenotyp were described.We tracked 142 DEL pregnant women who delivered Rh D-positive infant,with a history of gestations or parturitions for the evidence of alloanti-D.It is an imitate model to evaluate if the DEL phenotype can receive Rh D-positive blood,because transfusion DEL patients with Rh D-positive red blood cells is forbidden according to current blood transfusion policy.In the retrospective study,alloanti-D antibody was found in 6 of 142 DEL pregnant women(4.2%).All the 6 cases showed loss of some Rh D epitopes.HDFN occurred in two of six cases and one case had severe HDFN.We also retrospectively evaluated the alloimmunisation status of 21 true D-negative recipients that transfused with DEL red blood cells.Data from our study suggested that partial DEL could be immunized by D antigen and not all the DEL variants can safely receive transfusions from Rh D-positive donors.Objective:To determinate the genetic structure of DEL phenotype in Anhui Han population.To discover the new alleles except for the RHD 1227 A allele and to determinate the D antigen epitopes and intensity for DEL phenotype and analyse anti-D alloimmunization due to pregnancy or transfusion by DEL red blood cells in Anhui.In order to evaluate whether DEL patients could receive the RHD positive blood cell,we will raise the transfusion strategy of DEL patients in a preliminary way and save the Rh negative blood resources to a certain extent,and enhancing the security of blood transfusion.Methods:Blood samplesBlood samples from 804 apparently D-negative samples were observed,including 515 Rh D-negative unrelated Chinese pregnant women and 21 true Rh-negative transfusion recipients referred to the Anhui Provincial Hospital and the First Affiliated Hospital of Anhui Medical University during the period of January 1,2008 to January 1,2017.The alloimmunisation status of 21 true Rh-negative recipients who transfused with DEL red blood cells was investigated for the immunogenicity of DEL red blood in Rh D-negative recipients.ImmunohematologyABO and Rh D typing of maternal peripheral blood and neonatal umbilical cord blood were performed with commercial reagents.Rh C/c and Rh E/e antigens was determined by immediate-spin tube test with the monoclonal anti-C,c,E,e in saline.Antenatal antibody screening and identification were tested by an IAT with gel test system and a tube method with the PEG-IAT method,respectively.The titration of anti-D was performed against a pool of papain-treated D+C-E+c+e-RBCs by using IAT.Blood hemoglobin(Hb)and the bilirubin level were measured for the newborn in this study.Adsorption and elution testsAdsorption-elution was performed on the investigated blood samples to test for DEL phenotype.The adsorption-elution procedure was also performed on 142 pregnant women with DEL phenotype for D epitope mapping with specific human anti-D Mo Abs aimed at D antigen epitopes 1-9(ep D1-6 and ep D8-9).RHD genotypingAmplification of RHD exonsWe devised RHD-specific primers based on intron sequences to amplify genomic DNA for RHD exons 1 through 10.PCR was performed using a commercially available system.Amplified DNA products were visualized by electrophoresis in a 1.5% agarose gel with ethidium bromide staining and photographed under UV light.DNA sequencingThe complete RHD exons 1 to 10 were amplified according to Gassner and his colleagues,with cycle-sequencing kits.Sequence determination of amplicons was performed on both strands with an ABI PRISM 3730 automated sequencer.Determine of RHD-CE-D hybrid allelesPCR-SSP was performed with commercially available typing kits(CDE,weak D,RHd,Innotrain).Kit �CDE� is capable of properly identifying RHD-CE-D hybrid alleles by detecting RHD-specific DNA sequences in the 5'-untranslated region and exons 2,3,4,5,6,7,9,and 10 of RHD and RHC,RHc(intron1 and exon 2)and RHE,RHe(exon 5)of RHCE as described previously.Results:Serologic phenotypesIn this study,a total of 221 DELs(including 142 pregnant women)were found among the samples from 804 D-negative HAN individuals.DEL was found to occupy 27.5% of the total D-negative samples.96.4%(213/221)were associated with antigens C while the majority exhibited a Ccee phenotype.The frequencies of phenotypes Rh C/c and E/e in the population sample were Ccee(81.00%)?CCee(10.86%)? CCEe(0.90%)?Cc Ee(3.62%)? cc Ee(3.17%)?cc EE(0%)and ccee(0.45%),respectively.Molecular characterization of pregnant women with DEL phenotypesA total of 130(91.5%)samples were determined to have the RHD1227 A allele.Seven sample lacked RHD exon 4�9,four sample lacked exons 2�5,one lacked exons 4�7.According to RHD/CE PCR-SSP,seven RHD-CE(4�9)-D,four RHD-CE(2�5)-D and one RHD-CE(4�7)-D for RHD-CE-D hybrid alleles as judged by testing for the RHD 5'-untranslated region and exons 2,3,4,5,6,7,9,and 10.Ep D mapping of pregnant women with DEL phenotypeRBCs of the 130 samples carrying RHD(K409K)allele were found to express a normal D-positive epitopes pattern.The twelve samples with RHD-CE-D hybrid alleles were associated with a partial D antigen and lacking of certain proportion of normal D epitopes.Samples with the three different hybrid alleles demonstrated a prominent qualitative D antigen alteration.Alloanti-D immunisation analysisSix cases of alloanti-D immunized women were discovered at our transfusion department during the entire study period.The six pregnant women who were initially typed as D-negative had anti-D titers ranged from 4 to 128.None of them had a history of blood transfusion.All the 6 pregnant women had a history of gestations or parturitions and carry with D-positive fetus.DNA sequencing and ep D mapping showed that all the 6 cases carried with RHD-CE-D hybrid alleles and lost of some Rh D epitopes.Two HDFN occurred in the 6 cases and one case had severe HDFN whose peak bilirubin concentration was 416 umol/L and the alloanti-D titer up to 128.During the study period 21 true Rh-negative recipients were detected for the immunogenicity of DEL red blood in Rh D-negative recipients.During the study period 54 DEL RBC units were issued to 36 true Rh-negative recipients.Anti-D was not detected in their serum before or after DEL transfusion.Exposure to DEL RBCs did not increase anti-D alloimmunization.Conclusion:1.The allele is polymorphism of DEL phenotype in Anhui province of Chinese Han population.2.The data indicated that different DEL genotypes code either for complete or partial D antigen expression.3.Alloanti-D immune response by DEL is a rare event in true D-negative recipients.Therefore,we should continue to deem DEL as D-negative for donors in Chinese so as to save the rare D-negative blood donor pool.4.To avoid anti-D alloimmunization,individuals with partial DEL phenotype should be treated as if they were Rh D-negative: such recipients should receive only Rh D-negative RBCs.5.Antenatal Rh D immune globulin prophylaxis is necessary for partial DEL women.Meanwhile,if pregnant women with complete DEL phenotype do not need Antenatal Rh D immune globulin prophylaxis.