Yun‐Ping Xu

Background: Immune‐mediated platelet transfusion refractoriness (IPTR) remains a major clinical challenge, with immune alloimmunization against HLA and HPA as the predominant causes. Although HLA cross‐reactive groups (CREGs) provide a practical alternative to allele‐level matching, methods for simultaneous HLA‐CREG and HPA genotyping remain limited despite their potential to improve platelet matching. Methods: Specific U‐anchoring primers (UAPs) and amplification refractory mutation system (ARMS) primers were designed and optimized based on one or more SNPs within HLA‐CREG subgroups and HPA alleles. Reaction conditions were optimized, and analytical performance was evaluated. A total of 200 donor DNA samples were tested and compared with NGS and a commercial HPA sequencing kit, with discrepant results verified by Sanger sequencing. Results: We developed and optimized a novel multiplex real‐time quantitative PCR (qPCR) assay integrating UAPs and ARMS primers for simultaneous detection of HLA‐A/B CREGs and major HPA alleles in 12 reaction tubes. The assay demonstrated an overall limit of detection (LOD) of 0.838 ng/μL and the intra‐/inter‐assay coefficients of variation were both below 5%, indicating high reproducibility. HLA‐CREG typing results were 100% concordant with next‐generation sequencing. Sixteen discrepant HPA genotyping results were identified compared with the commercial kit, all of which were confirmed by Sanger sequencing and were consistent with the multiplex qPCR assay. Conclusions: This multiplex qPCR assay enables rapid, accurate, and cost‐effective simultaneous detection of HLA‐CREGs and HPA alleles, providing a practical tool for comprehensive platelet compatibility assessment and improved transfusion matching.

The novel HLA‐A*33:03:68 allele differs from HLA‐A*33:03:01:01 by 1 variation in exon 3.

The novel HLA‐A*02:1144 allele differs from HLA‐A*02:03:01:01 by 3 nucleotides in exon 7.

Introduction: The human leukocyte antigen (HLA) system plays a critical role in the human immune system and is strongly associated with immune recognition and rejection in organ transplantation. HLA typing method has been extensively studied to increase the success rates of clinical organ transplantation. However, while polymerase chain reaction sequence-based typing (PCR-SBT) remains the gold standard, cis/trans ambiguity and nucleotide sequencing signal overlay during heterozygous typing present a problem. The high cost and low processing speed of Next Generation Sequencing (NGS) also render this approach inadequate for HLA typing.Methods and materials: To address these limitations of the current HLA typing methods, we developed a novel typing technology based on nucleic acid mass spectrometry (MS) of HLA. Our method takes advantage of the high-resolution mass analysis function of MS and HLAMSTTs (HLA MS Typing Tags, some short fragment PCR amplification target products) with precise primer combinations.Results: We correctly typed HLA by measuring the molecular weights of HLAMSTTs with single nucleotide polymorphisms (SNPs). In addition, we developed a supporting HLA MS typing software to design PCR primers, construct the MS database, and select the best-matching HLA typing results. With this new method, we typed 16 HLA-DQA1 samples, including 6 homozygotes and 10 heterozygotes. The MS typing results were validated by PCR-SBT.Discussion: The MS HLA typing method is rapid, efficient, accurate, and readily applicable to typing of homozygous and heterozygous samples.

Background: New HIV (Human immune deficiency virus) infections are continuously increasing in China and it remains a huge challenge to blood donation. As access to health services has affected by COVID-19 (Corona virus disease 2019) pandemic, a drop in new diagnoses (especially HIV) was observed worldwide. Methods: During 2013– 2021, 735,247 specimens from unpaid blood donors collected by Shenzhen Blood Center underwent ELISA (Enzyme -linked immunosorbent assay) and NAT (Nucleic acid test). Samples with reactivity results were sent to the Shenzhen Center for Disease Control and Prevention for WB (Western blot). All data were statistically analyzed by the Chi-Square test. Results: From 2013 to 2021, the prevalence of HIV among male blood donors was higher than in females (P < 0.01). During the COVID-19 pandemic, the prevalence of HIV among repeat blood donors decreased significantly compared to 2019 (P < 0.05), and the characteristics of blood donors changed in 2020 compared to 2019 and 2021. Conclusion: The high proportion of female blood donors would help prevent HIV from getting into the blood supply. The COVID-19 pandemic affected the demographics of blood donors as well as the prevalence of HIV among repeat blood donors. An increased number of repeat blood donors can help decrease the risk of HIV transfusion transmission during the epidemic.

To explore the relationship between the level of soluble HLA-E (sHLA-E) molecules in plasma and gene polymorphism and leukemia in Shenzhen of China. Enzyme-linked immunosorbent assay was used to detect sHLA-E level in plasma of 103 leukemia patients and 113 healthy blood donors. PCR-SBT was used to identify the HLA-E genotype of 73 leukemia patients and 76 healthy blood donors. The level of plasma sHLA-E of 103 leukemia patients was significantly higher than that of 113 healthy blood donors (P400 ng/ml) can be set to assess the risk of certain pre-leukemia patients.

Background. The HLA-E gene is a member of the HLA-I gene family. Its genetic polymorphism is regarded as associated with numerous diseases. Establishing a rapid and accurate detection method of disease-related SNP sites in HLA-E is particularly important. Methods. Blood samples from 226 healthy blood donors and 228 leukemia patients were collected, and DNA was extracted. Three typing methods based on PCR-sequence-based typing, TaqMan genotyping, and high-resolution melting curve were established to identify rs76971248 (G>T). The Chi-square test was used for statistical analysis by SPSS. Results. Three methods based on PCR-SBT, TaqMan genotyping, and HRM were all able to identify rs76971248. The software for analyzing the results of HLA-E sequencing was easy to use, and the results were accurate. The frequency of rs76971248 in different types of leukemia patients was significantly lower than that in healthy blood donors ( ). And the frequency of the G/G genotype in leukemia patients was significantly higher than that in healthy blood donors ( ). Conclusions. For the screening of known SNP sites in large-scale populations, among the three methods, the TaqMan genotyping method had the advantage of shortest time consumption, simplest operation, and greatest specificity, which was the most appropriate method for this experiment. The analysis software for HLA-E gene sequencing needed to be further optimized. rs76971248 had a protective effect against leukemia. And the G/G genotype was a risk factor for leukemia.