Two decades of voluntary nonremunerated blood donation in Shenzhen, China.

BACKGROUND: As an emerging metropolis with population expansion from 2 million to 10 million from 1993 to 2012, the clinical demand for blood in Shenzhen has increased 20 times. To deal with this big challenge, Shenzhen utilized voluntary nonremunerated blood donation (VNRBD) in 1993 for the first time in China. After two decades of efforts, Shenzhen has achieved self-sufficiency in its blood supply and guaranteed its blood security by nonpaid blood donation. STUDY DESIGN AND METHODS: We summarized the strategies to achieve self-sufficiency and security in the blood supply in Shenzhen during two decades, including the legal construction of VNRBDs and the continuously improving strategies to recruit and retain nonpaid donors. The collection data of whole blood (WB) and apheresis platelet (PLT) donations were retrieved, and donor demographic and donation characteristics were analyzed. RESULTS: From 1993 to 1998, paid and nonpaid blood donations coexisted in Shenzhen. From the year 1999, all WB for clinical use came from VNRBDs. From 1999 to 2012, the donors who chose to donate 400 mL each time and repeat and regular donors increased sharply to meet the fast growth of clinical demand. From the year 2005, the clinical demand for PLTs was entirely satisfied by nonpaid donations. CONCLUSIONS: After two decades of practice, we believe that the legal regime of VNRBD is fundamental guarantee for long-term self-sufficiency and security in the blood supply. In addition, strengthening the publicity to improve the public's awareness and improving donation services and measures to recruit more nonpaid donors and retain repeat and regular donors are very important.

Discovery of the novel HLA-A*11:452N allele by next-generation sequencing in a Chinese individual.

HLA-A*11:452N differs from A*11:01:01:01 by a single nucleotide exchange in exon 1.

Genomic full-length sequence of the HLA-B*40:86 allele identified in a Chinese individual.

HLA-B*40:86 differs from B*40:06:01:03 by a single nucleotide exchange in exon 3.

[Establish a Graded Method to Avoid HLA Class I Antibodies Corresponding Antigen and Combining HLAMatchmaker Application in Improving the CCI Value after Platelet Transfusion for Patients with IPTR].

OBJECTIVE: To establish a graded method to avoid mean fluorescence intensity (MFI) threshold of HLA Class I antibodies corresponding antigen, and the HLAMatchmaker program has been used to select the minimum mismatch value of donor-patient epitopes. Evaluate the application value of combining both methods in selecting HLA compatible platelets (PTL) for patients with immune platelet transfusion failure (IPTR) in improving platelet the corrected count increment (CCI). METHODS: A total 7 807 PLT cross-matching compatible were performed by the solid-phase red cell adherence (SPRCA) method for 51 IPTR patients. The Luminex single antigen flow cytometry was used to detect HLA Class I antibodies in patients, and detected the MFI value for different specificity antigens of HLA Class I antibodies, was graded into strong positive group (MFI>4 000, level 1), medium positive group (1 000< MFI≤4 000, 2), weak positive group (500< MFI≤1 000, 3), and one negative control group (MFI≤500). The results of 7 807 SPRCA their negative/positive reaction wells were enrolled and statistically analyzed in different grades and the four groups, the statistical differences between the four groups were compared. Multiple applications for the select HLA Class I compatible donor events were made for patients in two cases, and HLAMatchmaker program was used to calculate the number of HLA Class I epitopes mismatches between the donors and patients. The donor with the minimum number of epitopes mismatches was selected, while avoiding the corresponding antigens of HLA Class I antibodies in levels 1 and 2, the provision of HLA compatible platelets for IPTR. After the transfusions, the CCI value of the platelet transfusion efficacy evaluation index was calculated, and the clinical evaluation of the transfusion effect was obtained through statistical analysis. RESULTS: There were statistically significant differences in the positive results of SPRCA immunoassay among the strong positive group, medium positive group, and weak positive group of 51 IPTR patients with different specific of HLA -I class antibodies and corresponding antigens(all P <0.001). The positive results showed a range from high to low, with strong positive group>medium positive group>weak positive group. There were a statistical difference among between the strongly positive or moderately positive groups and the negative control group(P <0.001). There was no statistical difference between the weakly positive group and the negative control group(P >0.05). The strong positive group was set as the corresponding specific HLA Class I site corresponding antigen grade 1 avoidance threshold, the medium positive group as the grade 2 avoidance thresholds, and the weak positive group as the grade 3 avoidance threshold. In the case of donor platelet shortage, it is not necessary to avoid the weak positive group. Avoiding the strategy of donor antigens and HLAMatchmaker program scores ≤7 corresponding to HLA Class I antibodies of levels 1 and 2, with CCI values>4.5×109/L within 24 hours, can obtain effective clinical platelet transfusion conclusions. CONCLUSION: When selecting HLA Class I compatible donors for IPTR patients, the grading avoids HLA Class I antibodies corresponding to donor antigens, and the donor selection strategy with the minimum scores of HLAMatchmaker program is comprehensively selected. The negative result confirmed by platelet cross-matching experiments has certain practical application value for improving platelet count in IPTR patients.

Genomic sequence of the novel HLA-A*26:206:02N allele, identified in a patient with hepatitis B infection.

HLA-A*26:206:02N differs from A*26:01:01:01 by a single nucleotide exchange in exon 3.

Demographic Factors Among HIV Confirmed Blood Donors from 2013 to 2021 in Shenzhen.

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.

[Establishment of a large-scale bi-directional sequencing and genotyping platform for MICA gene exons 2 to 4].

OBJECTIVE: To establish a stable and large-scale bi-directional sequencing platform for genotyping MICA gene exons 2 to 4, and to analyze single nucleotide polymorphisms(SNP) of the region. METHODS: Primers for particular alleles of MICA gene exons 2 to 5 were designed. Optimal conditions for PCR amplification and sequencing reaction were explored. A commercialized one-way sequencing kit for MICA allele was used as a parallel control. Four samples carrying a MICA *010 allele were subjected to cloning and haplotype sequencing. RESULTS: Results of MICA allele typing of 100 samples for a parallel control group were confirmed by the establish method. Twenty-two SNP in MICA gene exons 2 to 4 were detected in Chinese population. Two novel allelic sequences were accepted by GenBank and IMGT/HLA database and officially named as MICA*065 and MICA*066 by the WHO Nomenclature Committee. A novel SNP in MICA gene intron 3 was discovered, with allelic sequence submitted to GenBank and IMGT/HLA database. CONCLUSION: The bi-directional sequencing genotyping platform may be applied for large-scale study of MICA allelic polymorphisms, tissue typing, organ transplantation and disease research.

[Study on the Relationship between the Level of Soluble HLA-E Molecules in Plasma and Gene Polymorphism and Leukemia].

OBJECTIVE: 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. METHODS: 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. RESULTS: The level of plasma sHLA-E of 103 leukemia patients was significantly higher than that of 113 healthy blood donors (P<0.001); And the level of plasma sHLA-E in 77 myeloid leukemia patients was also significantly higher (P<0.001). The percentage of patients with plasma sHLA-E concentration of 0-199 ng/ml in leukemia and myeloid leukemia patients was 37.86% and 32.47%, respectively, which was significantly lower than 53.98% of healthy donors, the difference was statistically significant (P<0.05, P<0.01); While, when the plasma sHLA-E concentration was more than 400 ng/ml, the percentage was 33.01% and 36.36%, respectively, which was significantly higher than 13.28% of healthy donors, the difference was also statistically significant (P=0.001, P<0.001). There was no significant difference in the level of plasma sHLA-E among different HLA-E genotypes (P>0.05), whether healthy blood donors or leukemia patients. CONCLUSION: The level of plasma sHLA-E in patients with leukemia (especially myeloid leukemia) is significantly higher than that of healthy blood donors, but different HLA-E genotypes do not affect the level of plasma sHLA-E. A cut-off value for the concentration of plasma sHLA-E (recommended risk value >400 ng/ml) can be set to assess the risk of certain pre-leukemia patients.

Correlation of Human Leukocyte Antigen-E Genomic Polymorphism with Leukemia and Functional Study of Human Leukocyte Antigen-E Different Type Promoters.

Human leukocyte antigen (HLA)-E is one of the least polymorphic nonclassical major histocompatibility complex (MHC) I genes; its nucleotide variability can affect immune response. In this study, we assess the correlation between HLA-E polymorphism and leukemia and further study the transcriptional activity of promoter variation at nucleotide position-26. A total of 142 healthy blood donors and 111 leukemia patients were collected. The genomic sequence of HLA-E was amplified by high-fidelity reaction system and identified by Sanger and cloning sequencing. The dual luciferase reporter gene assay was used to detect the transcription activity of promoter variation at nucleotide position-26. In the HLA-E genomic sequence results, a total of 16 alleles and 32 genotypes were detected; the HLA-E*01:01:01:06 allele had a significantly lower frequency in leukemia patients than in healthy participants (p = 0.026 < 0.05). And the HLA-E*01:03:02:01, *01:03:02:01 genotype showed the greatest difference in frequency between the two groups of participants (p = 0.028 < 0.05). Eight HLA-E alleles were first reported worldwide in Chinese individuals. The results of the dual luciferase reporter gene experiment showed that the transcription activity of the mutant-type promoter (HLA-E*01:01:01:06 with "T" allele at nucleotide position-26) was significantly lower compared with the wild-type promoter (HLA-E*01:01:01:01 with "G" allele at nucleotide position-26) (p = 0.0242 < 0.05). HLA-E*01:01:01:06 allele has a protective effect against leukemia through decreasing transcription activity by "T" variation at nucleotide position-26.

[Molecular polymorphism Analysis on CD36 Deficiency among Platelet Blood Donors in Shenzhen].

OBJECTIVE: To analyze the molecular polymorphisms of CD36 among 58 blood donors with CD36 deficiency and compare with CD36 positive controls. METHODS: A total of 58 donors with CD36 deficiency during a screening conducted in the laboratory from September 2019 to December 2020 were enrolled as the test group, including 39 males and 19 females, while 120 platelet donors with CD36 positive were randomly selected as the controls, including 76 males and 44 females. All of the subjects were Han nationality. The PCR-SBT method was used to detect coding region of CD36 gene, and molecular mutations were compared with those CD36 positive controls. RESULTS: Among the 58 donors with CD36 deficiency, mutations appears in 32 individuals. The detection rate for type I was 71.43% (5/7), and type II was 51.92% (27/52), while among the 120 controls, mutations appears in 12 donors (10%). In the CD36 antigen-deficient donors, 16 variations were found, in which 329-330 del AC with the highest frequency accounted for 20.69%, followed by 1228-1239 del ATTGTGCCTATT(15.52%) and 1156 C>T(10.34%). Two variations, 198-205 del GATCTTTG and 220 C>T, led to premature termination of translation; four mutations, 329-330 del AC, 560 ins T, 1011-1049 39bp dupl and 1343-1344 ins TCTT, caused translation frame shift; 1228-1239 del ATTGTGCCTATT led to deletion of four amino acids (Ile-Val-Pro-Ile) at sites 410-413 of the peptide chain. The 1140 T>A and 1275 G>A were synonymous mutations, and the other 7 mutations resulted in the substitution of single nucleotide. The platelet expression in the donors of CD36 positive with 329-330 del AC or 1228-1239 del ATTGTGCCTATT mutation (heterozygote) was lower than those CD36 positive individuals without mutations (homozygote). CONCLUSION: Multiple gene mutations in the CD36 coding region may cause CD36 deficiency, and the heterozygous individuals with mutations may lead to CD36 antigen reduction or deletion. Mutation is not detected in 44.83% of CD36 deficient individuals, there may be some other reasons for the CD36 antigen deficiency.

Establishment of Rapid Detection Methods for rs76971248 Related to Leukemia.

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 (p < 0.05). And the frequency of the G/G genotype in leukemia patients was significantly higher than that in healthy blood donors (p < 0.05). 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.

[Recombination of HLA haplotypes in Guangdong Han nationality].

OBJECTIVE: To analyze the human leukocyte antigen complex class I (-A, -B & -C) and class II (-DRB1 & -DQB1) linked haplotypes of Guangdong Han nationality and to study the recombination events of five classical loci in the inheritance of HLA haplotypes. METHODS: A total of 939 peripheral blood samples were collected from 198 families in Guangdong Han nationality who came to our center for HLA typing from 2000 August to 2009 December. HLA-(A, B & DRB1) and HLA-(C & DQB1) alleles were typed by low-resolution polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSO) and PCR-sequence specific primers (PCR-SSP) methods respectively. Then the recombination sites were analyzed by familial study. The samples of 52 individuals from the families with exchange recombination were analyzed by the sequence-based typing (SBT) to judge whether the recombination was interallelic or interlocus exchange. RESULTS: Among 543 offspring individuals of 198 families in Guangdong Han nationality, 9 individuals with HLA-A-C-B-DRB1-DQB1 linked haplotypes had a recombination rate of 1.657%. Among 9 HLA haplotypes recombined families, 3 of them were found to have a crossover between HLA-A and -Cw loci and 6 of them a crossover between HLA-B and -DRB1 loci. Four of these recombination events occurred in the most common haplotypes A*3303-Cw*0302-B*5801-DRB1*0301-DQB1*0201 of Guangdong Han nationality. Among 9 cases of recombination, 5 of them were formed by a crossover between maternal chromosomes and 4 cases a crossover between paternal chromosomes. Three individuals with an exchange between A/Cw loci were all females. Among 6 cases with an exchange between B/DRB1 loci, 5 of them were males and 1 case was female. CONCLUSION: During the inheritance, recombination of HLA linked haplotype mainly occurred between A/Cw loci and B/DRB1 loci, the recombination is related to the haplotype-specificity and sex-specificity.

[Cloning and sequencing HLA-A and -B genomic DNA and analyzing polymorphism in regulatory regions in Chinese Han individuals].

In the present study, a high-resolute method for cloning and sequencing genomic full-length HLA-A and -B using 20 Chinese Han individuals was established. We detected 10 HLA-A allele sequences 4.2 kb in length and 6 HLA-B allele sequences 3.7 kb in length, and the sequences included all exons, all introns, 5'promoter, and 3'UTR of the two genes. All sixteen sequences have been submitted to GenBank and IMGT/HLA database. A*1153 is a novel allele, and the introns of B*151101 are firstly reported here. The 5'promoter and 3'UTR sequences of 5 HLA-A alleles and 2 HLA-B alleles are also firstly disclosed, and all other alleles have extended the genomic full length sequences released in IMGT/HLA database. The polymorphic structures of upper 5'promoter and downstream 3'UTR, which were uncovered in IMGT/HLA database, are firstly depicted in Chinese Han individuals. Twenty-six single nucleotide polymorphisms (SNPs) and one 3 bp-insertion/deletion (Indel) were located in the upper 5'promoter and 14 SNPs were located in the 3'UTR of HLA-A. In addition, five SNPs and one 1 bp-indel were located in the upper 5'promoter and 5 SNPs were located in the 3'UTR of HLA-B. Through analyzing the phylogenetic trees of 5'promoter, exons and 3'UTR of the two genes, we found that the evolution history of regulatory regions and exons is different between the two genes. The regulatory regions are tightly linked with exons in most of HLA-A alleles excluding A*24020101. On the contrary, recombinant events may occur frequently between regulatory regions and exons in most HLA-B alleles.

[Single nucleotide polymorphisms in 22 HLA-Cw alleles in Chinese Han population].

To analyze the molecular genetic polymorphism of full-length HLA-Cw gene, a total of 28 samples with known genotypes from Chinese Han population were amplified by long-range PCR using high-fidelity Pfu polymerase. A fragment 4.5 kb in length of HLA-Cw gene was subjected to cloning and haplotype sequencing. The single nucleotide polymorphisms (SNPs) in all segments of the whole region of HLA-Cw gene were analyzed. As a result, we detected 22 different HLA-Cw alleles in 28 samples, all of which were submitted to GenBank and the IMGT/HLA Database. Among the 22 HLA-Cw alleles, the intronic sequences of Cw*030301, Cw*0706 and Cw*140201 were firstly elucidated. The novel intronic sequence and the SNPs information may help to design allele-specific primers for accurate sequence-based typing (SBT) and to avoid allele dropout events in SBT test. We aligned all the diploid sequences using ClustalX program and imported them into Dnasp4.0 to calculate polymorphism in all coding- and non-coding regions. We found 244 SNPs and 10 insertion/deletions (Indels). According to the analysis of polymorphism level, phylogenetic trees and frequency spectrum, we proposed that the evolution of intron 4 and exon 5 was under balancing selection. Selection on these segments indicated that they may be functionally important in evolution of HLA-Cw gene. The full-length sequences obtained and related SNPs information can be used as resources of markers for high-resolution typing, complex diseases association studies and human evolution.

A novel HLA-E allele, HLA-E*01:03:01:05, identified in a healthy Chinese blood donor.

HLA-E*01:03:01:05 differs from E*01:01:01:01 by a single nucleotide exchange in nucleotide -33(T->C).

A novel HLA-E allele, HLA-E*01:12:01:01, identified in a healthy Chinese blood donor.

HLA-E*01:12:01:01 differs from HLA-E*01:01:01:01 by a single nucleotide in exon 5 changing 276 proline to glutamine.