OBJECTIVE: To analyze the distribution frequency and characteristics of HLA-A and HLA-B loci in patients with immune-mediated Platelet transfusion refractoriness (iPTR) in order to provide data support for investigating HLA gene matching strategies for platelet transfusion and improving transfusion efficacy. METHODS: A total of 532 iPTR patients who applied for gene matched platelet at the Blood Center of Zhejiang Province between January 2020 and June 2024 were selected as the study subjects. Genomic DNA was extracted from peripheral blood samples from the patients, and the HLA-A and HLA-B loci were detected simultaneously using a PCR-sequence specific oligonucleotide probe method (PCR-SSO) and PCR-sequence based typing (PCR-SBT). Statistical methods were used to analyze the distributions of HLA-A and HLA-B antigens and genotypes on the platelet surface of the patients. An analysis of the differences was conducted to compare the results with the Common and Well-Documented (CWD) allele in the Chinese population from the China Marrow Donor Program (CMDP) database. This study was approved by the Medical Ethics Committee of the Blood Center of Zhejiang Province (Ethics No.: Provincial Blood Center Ethics Review 2023Yan No.004). RESULTS: Among the 532 iPTR patients, 19 HLA-A antigens (including 32 HLA-A alleles) and 37 HLA-B antigens (including 64 HLA-B alleles) were detected. The antigens with the highest frequencies were A2, A11, A24, and B46, B60, B58, with the combined distribution frequency of the top three antigens reaching 71.43% and 36.94%, respectively. The most prevalent alleles of the HLA-A and HLA-B loci were A*11:01, A*24:02, A*02:07 and B*46:01, B*40:01, B*58:01. The frequencies of common alleles A*01:01, A*02:07, A*11:02, A*30:01 and B*13:02, B*27:04, B*40:01, B*44:03, B*46:01 showed significant differences (P < 0.05) compared to the normal population in the CWD table (version 2.4) of CMDP. CONCLUSION The HLA-A and HLA-B genes of the iPTR patients showed great divergence, and the distribution frequencies of certain alleles have differed significantly from those of the normal population. This study has provided genetic data for exploring the molecular mechanism underlying iPTR, which is of significant clinical importance for searching HLA gene matched donors.
Humans ; HLA-B Antigens/genetics* ; Alleles ; Female ; HLA-A Antigens/genetics* ; Male ; Platelet Transfusion/adverse effects* ; Gene Frequency ; Middle Aged ; Adult ; Genotype ; Aged ; Adolescent ; China

A 90-year-old female patient with novel coronavirus infection, severe pneumonia, and no history of blood transfusion andtransplantation.The mixed appearance phenomenon appeared in the admission blood group identification, and was sent the sample to our laboratory for difficult blood group identification. In the tube saline method, the patient′s red blood cells were positively reacted with 2 monoclonal anti-A and 5 human anti-A reagents.In the microcolumn gel method, the patient′s red blood cells showed 2 positive and 2 negative reactions with monoclonal anti-A and 5 positive and 1 negative reactions with human anti-A. The patient ′s red blood cells showed negative reaction with peanuts in phytohemagglutinin, and positive reaction with double flower lentils, wild soybeans and a string of purples. The patient ′s red blood cells treated with papain showed negative reaction with all monoclonal anti-A reagents, human anti-A and phytohemagglutinin. The patient ′s ABO gene was sequenced as ABO * B.01/O.01.02, but C1GALT1C1 gene mutation was not founded in the gDNA of the whole blood sample.It is speculated that the exposure of Tn antigen on the patient ′s red blood cells leads to red blood cells polyagglutination, resulting in ABO blood group inconsistency.

A 90-year-old female patient with novel coronavirus infection, severe pneumonia, and no history of blood transfusion andtransplantation.The mixed appearance phenomenon appeared in the admission blood group identification, and was sent the sample to our laboratory for difficult blood group identification. In the tube saline method, the patient′s red blood cells were positively reacted with 2 monoclonal anti-A and 5 human anti-A reagents.In the microcolumn gel method, the patient′s red blood cells showed 2 positive and 2 negative reactions with monoclonal anti-A and 5 positive and 1 negative reactions with human anti-A. The patient ′s red blood cells showed negative reaction with peanuts in phytohemagglutinin, and positive reaction with double flower lentils, wild soybeans and a string of purples. The patient ′s red blood cells treated with papain showed negative reaction with all monoclonal anti-A reagents, human anti-A and phytohemagglutinin. The patient ′s ABO gene was sequenced as ABO * B.01/O.01.02, but C1GALT1C1 gene mutation was not founded in the gDNA of the whole blood sample.It is speculated that the exposure of Tn antigen on the patient ′s red blood cells leads to red blood cells polyagglutination, resulting in ABO blood group inconsistency.

A puerpera with a obstetric history of gravida 2, para 2, underwent blood typing due to the presence of agglutination reactions in her serum against all tested red blood cells. She was found to be blood type O and her RhD phenotype was identified as CcDEe through serological testing. The reaction agglutination intensity between her serum and 26 O-type blood cells from the panel was 2+. Whole genome sequencing was performed, yielding data on 4014 single nucleotide polymorphisms (SNPs) and 958 insertion/deletion (INDEL) loci across 50 genes responsible for encoding blood group systems. Among these, only a single SNP , rs72552713 was predicted to be a highly harmful variant, which is the c.376C>T variation in the ABCG2 gene encoding JR blood group antigen, leading to the premature stop codon (p.Gln126Ter). The c.376C>T variation has been named the ABCG2*01N.01 by the working party on Red Cell Immunogenetics and Blood Group Terminology of International Society of Blood Transfusion. The postpartum woman was found to have the Jr(a-) phenotype. Whole genome sequencing can accurately determine the antigens of blood group systems in some difficult specimens.

OBJECTIVE: To explore the molecular mechanism for an individual with Bweak subtype. METHODS: Serological methods were used to identify the proband's phenotype. In vitro enzyme activity test was used to determine the activity of B-glycosyltransferase (GTB) in her serum. The genotype was determined by PCR amplification and direct sequencing of exons 5 to 7 and flanking sequences of the ABO gene. T-A cloning technology was used to isolate the haploids. The primary physical and chemical properties and secondary structure of the protein were analyzed with the ProtParam and PSIPRED software. Three software, including PolyPhen-2, SIFT, and PROVEAN, was used to analyze the effect of missense variant on the protein. RESULTS: Serological results showed that the proband's phenotype was Bweak subtype with anti-B antibodies presented in her serum. In vitro enzyme activity assay showed that the GTB activity of the subject was significantly reduced. Analysis of the haploid sequence revealed a c.398T>C missense variant on the B allele, which resulted in a novel B allele. The 398T>C variant has caused a p.Phe133S substitution at position 133 of the GTB protein. Based on bioinformatic analysis, the amino acid substitution had no obvious effect on the primary and secondary structure of the protein, but the thermodynamic energy of the variant protein has increased to 6.07 kcal/mol, which can severely reduce the protein stability. Meanwhile, bioinformatic analysis also predicted that the missense variant was harmful to the protein function. CONCLUSION The weak expression of the Bweak subtype may be attributed to the novel allele of ABO*B.01-398C. Bioinformatic analysis is helpful for predicting the changes in protein structure and function.
Female ; Animals ; ABO Blood-Group System/genetics* ; Phenotype ; Genotype ; Exons ; Alleles

【Objective】 To analyze the specificity and Eplets of HLA allele-specific antibody in patients with platelet transfusion refractoriness (PTR). 【Methods】 HLA-A and B genotypes were detected by PCR-SBT, and HLA-Ⅰ antibodies in patients′ serum were detected by Luminex single antigen beads coating method. IPD-IMGT/HLA Database was used to find the differential amino acids of allele-specific antibodies, and HLA Eplet database was used to analyze the registry Eplets. 【Results】 HLA allele-specific antibodies were found in 12 out of 82 patients with PTR.After sequence alignment, a total of 18 differential amino acids were found, such as 19E>19K, 166D>116E, 167G>167W and so on. Among these differential amino acids, 16 registry Eplets were retrieved such as 19E>19K, 95I>95L, 113YD>113HD and so on.The amino acid substitution of 166DG>166EW, 70Q>70H, 67S>67Y, 94I>94T, 82LR>82RG, and 211G>211A may form new Eplets that have not been registered.The antigens of A11, A24, B15, B27 and B38 can be further subdivided into HLA narrow specific antigens. 【Conclusion】 It was found that there were HLA allele-specific antibodies in patients with PTR, suggesting that high-resolution typing of HLA-A, B should be carried out for these patients and platelet donors in HLA compatible transfusion of PTR.

【Objective】 To explore the influence of anti-HLA-Ⅰ with different mean fluorescence intensity (MFI) on the efficacy of HLA-A and -B gene matching platelet transfusion, so as to provide scientific data for clinical platelet gene matching transfusion strategy. 【Methods】 A total of 81 PTR patients had applied for HLA-Ⅰgene matched platelets from the platelet gene database established by our laboratory, and 28 (MFI <5 000) of them needed further avoiding of partial donor-specific antibodies and they were enrolled as the research subjects. According to the platelet MFI value of HLA-Ⅰ antibody-targeting antigen, they were divided into negative transfusion group (MFI <500) (group A) and positive transfusion groups (MFI≥500) ; the latter were further divided into group B (500≤MFI <1 000), group C (1 000≤MFI <3 000) and group D (MFI≥3 000) according to MFI value. Corrected count increment (CCI) in platelet count was used to compare the platelet transfusion effect in 4 groups. 【Results】 Among 28 platelet recipients with MFI <5 000, 19(67.86%) patients successfully received 72 effective transfusions. The first CCI (×10⁹/L) in groups A, B, C and D were 10.27±7.46, 7.58±4.75 (P>0.05), 17.36±7.63 (P>0.05) and -0.77±2.30 (P<0.05), respectively. There was no statistical difference among group A, B and C. 【Conclusion】 The application of HLA-Ⅰ gene matching platelets in PTR patients can adjust the MFI threshold(<2 000) appropriately according to the patient′s condition without compromising the platelet transfusion effect.

【Objective】 To analyze the specificity of HLA class-Ⅰ antibody in the patients received HLA-matched platelet transfusions and estimate the relative immunogenicity of HLA-Ⅰ antigens. 【Methods】 The samples from 96 patients who suffered from platelet transfusion refractorines(PTR) and applied for transfusion with genotype-matched platelet were collected. The specificity of HLA I antibody was detected by Luminex technique, and the antibody expression level was analyzed according to MFI. The mismatch rate of HLA antigen and relative immunogenicity of the population were estimated according to the allele frequency distribution of HLA-A and B loci as well as the yielding frequency of antibody. 【Results】 HLA-Ⅰ antibodies were detected in all 96 patients, with varied species of antibodies. The average positive yielding rates of antibodies corresponding to HLA-A, -B and -C magnetic bead coated antigens (97 in total) were 0.38, 0.47 and 0.28, respectively. Among the HLA-A and -B loci in the Zhejiang population, HLA-A2, A11, A24 and HLA-B60, B46, B58 were the antigens with higher frequency, and their relative immunogenicity was 0.403, 0.283, 0.342, and 0.100, 0.067, 0.178, respectively. 【Conclusion】 The specificity of HLA-Ⅰ antibodies in PTR patients is different, which confirms that the relative immunogenicity differs by HLA-A and -B antigens.

OBJECTIVE: To explore the molecular basis for an individual with Bw subtype. METHODS: Routine serological reactions were used to determine the surface antigens of erythrocytes and antibodies in serum. PCR-sequence-based typing (PCR-SBT) was used to analyze the coding regions of the ABO gene and erythroid-specific regulatory element in its intron 1. Amplicons for exons 5 to 7 containing the variant site were subjected to TA cloning for the isolation of the haploid and verification of the sequence. The 3D structure of mutant protein was predicted with Pymol software. Changes of amino acid residues and structural stability were also analyzed. RESULTS: Serological assay showed that the individual had weakened B antigen and anti-B antibody in his serum. His genotype was determined as ABO*B.01/ABO*O.01.01. Sequencing of the entire coding region of the ABO gene identified an additional heterozygous c.734C/T variant. No variant was found in the erythroid-specific regulatory element of intron 1. Haploid cloning and isolation has obtained an ABO*O.01.01 allele and a ABO*B.01 allele containing a c.734T variant, which has led to substitution of Thr by Ile at position 245 in the functional center of glycosyltransferase. Based on the 3D structure of the protein, the residues binding with the mutation were unchanged, but the bonding distance between the hydrogens was changed with the amino acid substitution. Meanwhile, the connections with water molecules were increased. CONCLUSION The c.734C>T variant of the GTB gene can lead to an amino acid substitution in the functional center of the enzyme, which in turn may affect the stability of glycosyltransferase B protein and reduceits enzymatic activity.
ABO Blood-Group System/genetics* ; Alleles ; Exons/genetics* ; Genotype ; Glycosyltransferases/genetics* ; Humans ; Male ; Phenotype

【Objective】 To analyze the positive rate of antibodies against human leukocyte antigen(HLA)and MHC class I chain-related gene A(MICA) in the convalescent plasma from individuals recovered from COVID-19. 【Methods】 HLA-Ⅰ, -Ⅱ and MICA antibodies were screened simultaneously by Luminex platform. The specificity of HLA-Ⅰ and -Ⅱ antibodies was identified by single antigen reagents.The positive rate of antibody in different groups were compared by Chi-square test. 【Results】 A total of 88 cases of convalescent plasma were collected, among which the positive rates of HLA-Ⅰ, -Ⅱ and MICA antibodies were 18.19%, 19.32% and 10.23%, respectively, and 64 individuals (72.73%) were negative for HLA-Ⅰ and -Ⅱ antibodies. 95 blood donors were randomly selected as the control group, and the positive rate of HLA-Ⅰ, -Ⅱ and MICA antibodies were 8.42%, 13.68% and 10.53%, respectively, and 76 individuals(80.00%) were negative for HLA-Ⅰ and -Ⅱ antibodies. There were no significant difference in the positive rates of HLA-Ⅰ, -Ⅱ and MICA antibodies between convalescent individuals and control group. The specificity of HLA antibody to epitopes was different in each convalescent individual with positive HLA antibodies, and most antibodies were targeted to the epitopes of multiple HLA alleles. 【Conclusion】 A certain proportion of HLA antibody was found in the convalescent plasma of individuals recovered from COVID-19. Therefore, HLA antibody screening is helpful to improve the safety of transfusion.