Objective To investigate the serological characteristics and gene mutation mechanism of RhD variant blood donors in Zhengzhou.Methods From January 2023 to December 2023,1 619 RhD-negative blood donors sent to our labora-tory were selected for the study,and RhD negative confirmation test and RhCE phenotype detection were applied by tube method and microcolumn gel indirect antiglobulin test method.RHD gene amplification and Sanger sequencing were used to detect RhD variant sample genotypes.Results A total of 69 cases of RhD variants were detected in the RhD negative con-firmation test,with a proportion of 4.26%(69/1 619).The RhCE phenotypes were ccEe,Ccee,CcEe and CCee.There were 17 genotypes and 15 phenotypes of the D variant.The RHD?weak partial 15 allele was the most frequent(33 cases),with a frequency of 47.83%(33/69),and the main phenotype was the ccEe.This was followed by the RHD?DVI.3 allele in 20 ca-ses with a frequency of 28.99%(20/69)and the predominant phenotype was Ccee.The RHD?weak partial 15/RHD?01EL.01 heterozygote was found in 3 cases with a frequency of 4.35%(3/69),all with the CcEe phenotype.Other rare genotypes were present in 13 cases with a frequency of 18.84%(13/69).Antibody screening was positive in 3 cases with a frequency of 4.35%(3/69).Two cases of female blood donors,both with history of pregnancy and childbirth,were identified as anti-D;one case of male donor was anti-M.Conclusion The RHD?weak partial 15 genotype was the most common among the RhD variants in blood donors in Zhengzhou,followed by the RHD?DVI.3 genotype.It plays an important role in guarantee-ing the safety of blood supply and guiding precision transfusion.

Objective To analyze the application of serological test results in the diagnosis and treatment of anti-M-in-duced hemolytic disease of the fetus and newborn(HDFN),and to explore HDFN prevention strategies.Methods The se-rological test results of 12 cases of HDFN caused by anti-M diagnosed in our laboratory from January 2017 to December 2023 were retrospectively analyzed,including blood group identification of mothers and children,serum total bilirubin/hemoglo-bin/antibody titer test,and three hemolysis tests in newborns.Clinical data of the children and mothers were collected,in-cluding pregnancy history,blood transfusion history,prenatal antibody testing,history of intrauterine blood transfusion and gestational week of delivery,and the prognosis of the children was followed up.Results All 12 cases of fetal neonatal he-molytic disease due to anti-M were RhD+MN phenotype newborn born to RhD+NN mother,with maternal-fetal incompati-blility in MN blood groups.In the ABO blood group system,ABO incompatibility between mother and child accounted for 41.7%(5/12).None of the mothers had a history of blood transfusion,and the median titer of the test at 4℃was 32,and the median titer at 37℃was 4.The mothers of 3 cases had a history of multiple intrauterine blood transfusions,with an inci-dence of 25%(3/12).One case had an abnormal first pregnancy,with an incidence of 8.3%(1/12),and seven cases had an abnormal pregnancy with a miscarriage,with an incidence of abnormal pregnancy and birth history of 58.3%(7/12).There were 6 cases of premature labor,with an incidence of 50%(6/12).The mothers in three cases underwent regular ob-stetric examination and the specificity of the antibodies was determined,accounting for 25%(3/12).Twelve children had free antibodies with a median titer of 6 at 4℃and 2 at 37℃.Two children had anti-M antibodies that were not reactive at 37℃,with a negative rate of 16.7%(2/12).The positive rate of DAT and elution test was respectively 8.3%(1/12)and 16.7%(2/12)in the children.The median minimum hemoglobin value was 75 g/L,and all 12 children received blood transfusions.The median peak total bilirubin value was 157.5 μmol/L,and none of them reached the threshold for blood ex-change.The rate of delayed anemia was16.7%(2/12),the postnatal mortality rate was8.3%(1/12),and 11 children was free of growth and neurodevelopmental delay in prognosis.Conclusion Anti-M can cause severe HDFN,which can also oc-cur in primigravida.The intensity of antibody titer does not correlate with the severity of the disease,and it is prone to cause delayed anemia,which should be monitored regularly according to the serological characteristics of anti-M and clinical symp-toms,and should be treated timely.

Objective:To serologically and genotypically analyze the pedigree of a case with a new allele c.175_176insGA of B el subtype and preliminarily explore the molecular mechanism of weak expression of glycosyltransferase B. Method:In the descriptive study,a 23-year-old male voluntary blood donor and his family members were selected for the study. The ABO and Le blood types of the proband and his family members was identified by the test tube method. The agglutination inhibition test was applied to detect the B and H antigens in saliva, and the Sanger sequencing and PacBio (Pacific Bioscience) third-generation haplotype sequencing were performed on the study subjects to identify genotypes. Finally, Expasy software were applied to amino acid translation of DNA sequences and prediction of protein length after gene alteration. ORF finder was applied to predict alternative start codons as well as open reading frames of mRNA, and protein expression mechanisms were analyzed.Results:The proband and her sister were B el subtype, her mother was AB el subtype, her father was normal O type, and all members of the family were Le(a+b+) phenotype. Sanger sequencing results showed that a new allele of c.175_176insGA was found in exon 4 of the proband, her mother, and her sister. Third-generation haplotype sequencing detected the haplotypes of the family members, which revealed that the proband was ABO*O.01.02/ABO*BEL.NEW (c.175_176insGA), the father was ABO*O.01.02/ABO*O.01.02, the mother was ABO*A1.02/ABO*BEL.NEW (c.175_176insGA), and the sister was ABO*O.01.02/ABO*BEL.NEW (c.175_176insGA). Analysis of the protein expression mechanism indicated that although the new allele of ABO*BEL.NEW was presumed to cause a frameshift mutation and result in a premature stop codon p.Asp59Glu*fs20 in exon 5, encoding an inactive glycosyltransferase, an alternative start codon could be utilized to initiate translation of B el subtype functional glycosyltransferase. Conclusion:Expression of the new allele of B el subtype is associated with the translation of B el subtype glycosyltransferase initiated by alternative start codons.

【Objective】 To verify the results of HBV DNA and HCV RNA screening under different brands of vacuum collection tubes for blood samples, storage temperature and storage time. 【Methods】 Experiment 1 was conducted as follows: blood samples were collected simultaneously from 52 voluntary blood donors using two brands(divided into group A and group B) of vacuum collection tubes for blood samples. The plasma separation of group A and group B were compared, and the effects of storage time on the NAT yield of HBV DNA and HCV RNA were statistically analyzed. Experiment 2 was conducted as follows: the effects of different storage temperature, time and tubes on the NAT yield of HBV DNA and HCV RNA samples with low viral load in group A and B were verified and compared in the simulated phlebotomy condition. 【Results】 In Experiment 1: After centrifugation, blood plasma layer and cells layer were separated completely in group A(100%, 52/52), but one sample was not well separated in group B(1/52, 1.92%). After 4 to 10 h after collection, blood samples of two groups were centrifuged and screened for HBV DNA, HCV RNA within 24 h. No positive samples were yielded and the Ct values of internal control(IC-DNA and IC-RNA) were uniform. In Experiment 2: Whole blood samples, stored for either 4 h or 6~10 h at 4 ℃ or 25℃ before centrifugation, showed no difference on the NAT-yield of HBV DNA nor HCV RNA samples with low viral load(P>0.05). Ct values of HBV DNA and HCV RNA of group A was similar to those of group B as centrifuged samples were stored for 24 h or 72~104 h at 4℃(P>0.05), but all increased as the storage time prolonged. Ct values of HBV DNA in group A increased from 33.45±0.29(24 h) to 33.82±0.08(72~104 h) and HCV RNA from 35.21±0.20 to 36.12±0.43; HBV DNA from 33.46±0.25 to 34.30±0.60 and HCV RNA from 35.47±0.24 to 36.49±0.51 in group B. 【Conclusion】 Under certain laboratory condition, different storage time, storage temperature and tubes shed few effect on the NAT-yield of HBV DNA and HCV RNA samples with low virus loads. However, it is suggested that the blood sample be detected within 72 h after centrifugation at 4 ℃ storage.

【Objective】 To evaluate the residual risk of hepatitis C virus (HCV) in blood screening among voluntary blood donors in Zhengzhou. 【Methods】 The ELISA and NAT screening results of 497 171 voluntary blood donors in Zhengzhou from January 2019 to December 2020 were collected through the information management system of our blood center.The residual risk of HCV was assessed using the Prevalence-Window Period Residual Risk Model. 【Results】 The residual risk among repeated and first-time blood donors was 1∶132 280 (95% CI: 1∶95 520~1∶188 820) and 1∶44 090 (95% CI: 1∶31 840~1∶62 940), respectively. The overall residual risk of blood donors screening was 1∶68 540 (95% CI: 1∶65 910~1∶130 290). The reactive rate of HCV screening in first-time blood donors (0.144%, 334/231 168) was significantly higher than that in repeated blood donors (0.014%, 36/266 003) (P<0.05), and the reactive rate of repeated blood donors in 2019 (0.019%, 26/135 267) was significantly higher than that in repeat blood donors in 2020 (0.008%, 10/130 736) (P<0.05). 【Conclusion】 The residual risk of HCV among voluntary blood donors in Zhengzhou is low.The publicity and recruitment should be further strengthened to establish a stable team of voluntary blood donation, and health consultation and physical examination should also be strengthened to further reduce the residual risk of blood transfusion.

【Objective】 To explore the quantitative value of key points for ABO blood group initial screening in fixed blood donation sites, so as to provide reference for standardized testing process of sliding method. 【Methods】 Several groups of experiments were carried out to illustrate the optimal conditions, including the serum dosage of monoclonal reagent, red blood cell dosage of blood sample, and reaction time in ABO blood group initial screening, by sliding method, and the quantitative value of key points of sliding method was preliminarily determined. Blood typing tests of 310 blood donor samples including type A, B, O, AB, subtype A and subtype AB were conducted to evaluate the effects of quantitative value of key points in the initial screening procedure. The test tube method would be conducted if the results are inconsistent with the fully automated blood grouping analyzer. The ABO subtypes suspected are identified by serological and molecular biological methods. 【Results】 The quantitative value of key points in initial screening procedure of sliding methods was as follows: 2 drops of reagent serum, 5-10 μL of whole blood and 3 minutes of reaction time. The concordance rate of ABO blood type screening comparison experiment in 310 blood donors was 100%. 【Conclusion】 ABO blood group initial screening by sliding method with quantitative value can effectively standardize the pre-donation blood type screening in fixed blood donation sites, and can meet the requirements of ABO blood group initial screening.

【Objective】 To explore the factors affecting NAT reactive blood donors re-entry, so as to provide data support for formulation of scientific and reasonable strategy. 【Methods】 The basic data and laboratory test results of 174 NAT reactive returning blood donors from January 2019 to August 2021 were collected and statistically analyzed by logistic regression. 【Results】 Among 174 HBV DNA reactive blood donors applying for re-entry, 81 (46.6%) were eligible for re-entry. Blood donation type and deconstructed Ct value were independent influencing factors of blood donors’ re-entry (P<0.05). The Ct value of minipool and deconstruction test showed significant affection on the re-entry (P<0.05). Donors with minipool-Ct-value exceeding deconstructed-Ct-value had a low likelihood of success with re-entry(P>0.05). No significant difference was observed in Ct values of deconstruction test, first re-entry test and second re-entry test (P<0.05). 【Conclusion】 In view of the low re-entry rate of NAT reactive blood donors, it is necessary to establish a set of safety criteria to lessen workloads. Donors with exceeding minipool-Ct-values, repeat reactive by two NAT reagents, failure in the first re-entry test are suggested to be deferred permanently.