Determination for KIR genotype and allele copy number via real-time quantitative PCR method.

Killer cell immunoglobulin-like receptor (KIR) and human leukocyte antigen (HLA) play crucial roles in regulating NK cell activity. Here, we report a real-time quantitative PCR (qPCR) to genotype all KIR genes and their copy numbers simultaneously. With 18 pairs of locus-specific primers, we identified KIR genes by Ct values and determined KIR copy number using the 2-∆Ct method. Haplotypes were assigned based on KIR gene copy numbers. The real-time qPCR results were consistent with the NGS method, except for one sample with KIR2DL5 discrepancy. qPCR is a multiplex method that can identify KIR copy number, which helps obtain a relatively accurate haplotype structure, facilitating increased KIR research in laboratories where NGS or other high-resolution methods are not available.

The characteristic of HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3/4/5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1 alleles in Zhejiang Han population.

The Zhejiang Han population, a subgroup of the Southern Han ethnic group, resides in Zhejiang Province, situated on the southeast coast of China. In this study, we conducted HLA genotyping for 813 voluntary umbilical cord blood donors from the Zhejiang Han population, targeting 11 HLA loci, namely HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3/4/5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1, using the next-generation sequencing method. Our analysis of the alleles and haplotypes revealed a high degree of polymorphism within these loci. A total of 289 unique HLA alleles were identified, with the HLA-B locus exhibiting the most significant diversity, while HLA-DRB4 displayed the lowest variation. Due to the inherent limitations of the sequencing method, some unresolvable alleles in the specific loci, such as HLA-DRB1, HLA-DPA1, and HLA-DPB1, were assigned as G group designation. In our comprehensive analysis across all 11 HLA loci, a total of 1204 haplotypes were estimated. The distribution of these alleles was similar to those of the Chinese Southern Han population while highly different from the Caucasian population. These findings contribute to a deeper understanding of the genetic characteristics of HLA loci within the Chinese Southern Han population.

[Sequence analysis and identification of a novel HLA-DPB1*02:01:69 allele by third-generation sequencing].

OBJETIVE: To analyze the sequence of a novel HLA-DPB1 allele in an individual. METHODS: A individual identified from the database of blood donors for matched platelet transfusion at the Blood Center of Zhejiang Province in May 2022 was selected as the study subject. HLA genotype of the individual was determined by next-generation sequencing (NGS) on an Ion Torrent S5 platform. The sequence of the HLA-DPB1 locus was also determined by NGS on an Illumina Miseq platform and third-generation sequencing using Oxford Nanopore MinION. This study was approved by the Blood Center of Zhejiang Province (Ethics No. 2021-001). RESULTS: A novel HLA-DPB1*02 allele was identified in the specimen, for which the closest genotype was HLA-DPB1*02:new,17:01:01G, with the variant located in exon 3. Meanwhile, the NGS also revealed a novel HLA-DPB1*17 allele, with the closest genotype being HLA-DPB1*02:01,17:new. Both the HLA-DPB1*17:01:01:01 and HLA-DPB1*02 alleles were identified by third-generation sequencing. Compared with the HLA-DPB1*02:01:02:01 allele, the novel allele had a G>A variation at position 369 in the exon 3, which however did not result in amino acid change. CONCLUSION: A novel HLA-DPB1 allele has been identified and validated by both NGS and TGS, which has been named as HLA-DPB1*02:01:69 by the World Health Organization Committee on Nomenclature of Factors of the HLA System.

Prevalence and Residual Risk of HIV in Volunteer Blood Donors of Zhejiang Province, China, from 2018 to 2022.

Background: Blood safety levels have been significantly improved since the implementation of nucleic acid amplification technology (NAT) testing for blood donors. However, there remains a residual risk of transfusion transmission infections. This study aimed to evaluate the prevalence of HIV and its residual risk transmission among volunteer blood donors of Zhejiang Province, China, for five years after NAT implementation. Materials and Methods: All specimens and information were collected from voluntary unpaid donors at all blood services in Zhejiang Province, China, from January 2018 to December 2022. The HIV antibody or antigen and HIV RNA were detected using enzyme-linked immunosorbent assay and NAT, respectively. The HIV residual risk transmission was calculated using the incidence or window period model. Results: A total of 3,375,678 voluntary blood donors were detected, revealing an HIV prevalence of 9.92/100000. The HIV prevalence of blood donors in 12 blood services in Zhejiang Province was 6.11, 6.98, 7.45, 8.21, 8.36, 8.94, 9.04, 9.66, 9.73, 10.22, 11.80, and 12.47 per 100000 donors, without statistically significant difference observed among the services (p > 0.05). The HIV prevalence of males (15.49/100000) was significantly higher compared to females (1.95/100000; p < 0.05). There was an insignificant difference in HIV prevalence among blood donors of all different age groups (p > 0.05), but the HIV prevalence in the 26-35 age group and 18-25 age group was significantly higher compared to the 36-45 age group (p < 0.05). The difference in HIV prevalence between first-time blood donors (13.65/100,000) and repeat blood donors (6.78/100,000) was statistically significant (p < 0.05). From 2018 to 2022, the HIV residual risk in blood transfusion transmission was 0.266/100000. Conclusion: The prevalence of HIV among blood donors in Zhejiang Province, China, is associated with age, gender, and times of blood donation. The HIV residual risk in blood transfusion transmission remains low in the province, and increasing the rate of repeat blood donors is beneficial to improve blood safety.

HLA-A*02:06 allele may be susceptible to myelodysplastic syndrome in Zhejiang Han population, China.

The association between HLA loci and haematological malignancy has been reported in certain populations. However, there are limited data for HLA loci at a high-resolution level with haematological malignancy in China. In this study, a total of 1115 patients with haematological malignancies (including 490 AML, 410 acute lymphoblastic leukaemia (ALL), 122 myelodysplastic syndrome [MDS] and 93 non-Hodgkin's lymphoma [NHL]) and 1836 healthy individuals as a control group in the Han population of Zhejiang Province, China, were genotyped for HLA-A, HLA-C, HLA-B, HLA-DRB1 and HLA-DQB1 loci at high resolution. The possible association between HLA alleles and haplotypes and haematologic malignancy was analysed. The allele frequencies (AFs) of HLA-A*02:05, HLA-A*02:06, HLA-A*32:01, HLA-B*35:03, HLA-B*54:01, HLA-B*55:07, HLA-DRB1*04:05, HLA-DRB1*15:01, HLA-DQB1*04:01 and HLA-DQB1*06:02 in the MDS patients were much higher than those in the control group (P < 0.05), while the AFs of HLA-C*07:02, HLA-DRB1*03:01, HLA-DRB1*14:54, HLA-DQB1*02:01 and HLA-DQB1*05:03 were obviously lower than those in the control group (p < .05). Interestingly, the differences in these HLA alleles in patients with MDS were not significant after applying Bonferroni correction (Pc > .05), except for HLA-A*02:06 (Pc < .01). There were 13, 6 and 10 HLA alleles with uncorrected significant differences (p < .05) among patients with AML, ALL and NHL, respectively, compared with those in the control group, but the differences in these HLA alleles were not significant after correction (Pc > .05). Compared to those of the control group, there were some haplotypes over 1.00% frequency in patients with AML, MDS and NHL patients with uncorrected significant differences (p < .05). However, none of them showed a significant difference after correction as well (Pc > .05). The study reveals that HLA-A*02:06 may lead to susceptibility to MDS, but none of the HLA alleles were associated with AML, ALL or NHL after correction. These data will help to further understand the role of HLA loci in the pathogenesis of haematological malignancy in China.

[Study of the molecular characteristics of a Bweak phenotype due to a novel c.398T>C variant of the ABO gene].

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.

The impact of nucleic acid testing to detect human immunodeficiency virus, hepatitis C virus, and hepatitis B virus yields from a single blood center in China with 10-years review.

BACKGROUND: Since 2010, the Blood Center of Zhejiang province, China, has conducted a pilot nucleic acid amplification testing (NAT) screening of blood donors for Hepatitis B virus (HBV), Hepatitis C virus (HCV), and Human immunodeficiency virus (HIV). This study aims to assess the results of NAT testing over 10 years to establish the effects and factors influencing NAT yields of HBV, HCV, and HIV. METHODS: Blood donations from seven different blood services were screened for HBV DNA, HCV RNA, and HIV RNA using 6 mini pools (6MP) or individual donation (ID)-NAT method between August 1, 2010, and December 31, 2019, at the NAT centralized screening center. We compared 3 transcription-mediated amplification (TMA) assays and 2 polymerase chain reaction (PCR) assays. Further, HBV, HCV, and HIV NAT yields were calculated and donor characteristics and prevalence of HBV NAT yields analyzed. Donors with HCV and HIV NAT yield were also followed up. RESULTS: 1916.31 per million donations were NAT screening positive overall. The NAT yields for HBV, HCV, HIV and non-discriminating reactive were 1062.90 per million, 0.97 per million, 1.45 per million, and 850.99 per million, respectively, which varied in the seven blood services and different years. HBV NAT yields were higher than those of HCV and HIV and varied across demographic groups. Risk factors included being male, old age, low education level, and first-time donors. We found no differences in NAT yields of HBV, HCV, and HIV between the 3 TMA and 2 PCR assays; nonetheless, statistically, significant differences were noted between the five assays. CONCLUSION: In summary, NAT screening in blood donations reduces the risk of transfusion-transmitted infections and shortens the window period for serological marker screening. Therefore, a sensitive NAT screening method, ID-NAT workflow, and recruitment of regular low-risk donors are critical for blood safety.

[Analysis of loss of heterozygosity at HLA loci in a patient with leukemia].

OBJECTIVE: To detect loss of heterozygosity (LOH) at human leukocyte antigen (HLA) loci in a Chinese patient with leukemia after haploidentical hematopoietic stem cell transplantation. METHODS: HLA genotyping was carried out on peripheral blood, hair follicle and buccal swab samples derived from the patient after the transplantation as well as peripheral blood samples from his parents by using PCR-sequence specific oligonucleotide probe method and PCR-sequence based typing method. Short tandem repeat (STR) loci were detected by using a 23 site STR assay kit and a self-developed 6 STR loci assay for the HLA regions. RESULTS: After the transplantation, the HLA genotype of the peripheral blood sample of the patient was identical to his father. The patient was HLA-A*02:01,24:02, C*03:03,03:04, B*13:01,15:01, DRB1*08:03,12:02, DQB1*03:01,06:01 for his hair follicle specimen. However, homozygosity of the HLA loci was found in his buccal swab sample. Only the HLA-A*24:02-C*03:03-B*15:01-DRB1*08:03-DQB1*06:01 haplotype from his father's was present, while the HLA-A*02:01-C*03:04-B*13:01-DRB1*12:02-DQB1*03:01 haplotype from his mother was lost. After the transplantation, the alleles of the 23 STR sites in the patient's peripheral blood sample were consistent to his father, with no allelic loss detected in his buccal swab sample. However, at least 4 STR loci in the HLA region were lost in his buccal swab sample. CONCLUSION: LOH at the HLA loci has been detected in the buccal swab sample of a patient with leukemia who received haploidentical hematopoietic stem cell transplantation.

Prevalence of Treponema Pallidum Antibody among Volunteer Blood Donors in China.

Background: Infection with syphilis is still a major public health problem. The precise data for syphilis seroprevalence in the populations will help to develop a strategy for prevention and treatment of it. However, the data for syphilis prevalence in continuous years among volunteer blood donors in China is rare. Methods: A retrospective study for Treponema pallidum (TP) antibody in blood donors was conducted from January 2010 to December 2019 at the Blood Center of Zhejiang Province, China. TP antibody was detected with two different reagents using enzyme-linked immunosorbent assay and the only sample which was reactive in the two reagents was defined as seropositive. Results: A total of 992,646 volunteer blood donors were analyzed and the positive rate of TP antibody in the blood donors was 0.43%. From 2010 to 2019, the positive rates of TP antibody were 0.53%, 0.51%, 0.51%, 0.43%, 0.36%, 0.18%, 0.11%, 0.12%, 0.11%, and 0.10%, respectively. The positive rates of TP antibody were significantly different among blood donor age group (p < 0.001), with the highest positive rate in 45-54-years-old group (0.93%). The positive rates of TP antibody in male and female blood donors were 0.44% and 0.41%, respectively. The positive rate was 0.57% among the first-time blood donors, which was significantly higher than that of the repeat blood donors (0.17%). The positive rate of TP antibody in blood donors decreased gradually with the increase of educational level. Conclusion: The syphilis seroprevalence is low in the blood donors of the Hangzhou area, and the positive rate of blood donors is associated with age, educational level, and times of blood donation. Increasing the number of repeat blood donations is helpful to improve blood safety.

The combinatorial diversity of KIR and HLA class I allotypes in Peninsular Malaysia.

Killer cell immunoglobulin-like receptors (KIRs) interact with polymorphic human leucocyte antigen (HLA) class I molecules, modulating natural killer (NK) cell functions and affecting both the susceptibility and outcome of immune-mediated diseases. The KIR locus is highly diverse in gene content, copy number and allelic polymorphism within individuals and across geographical populations. To analyse currently under-represented Asian and Pacific populations, we investigated the combinatorial diversity of KIR and HLA class I in 92 unrelated Malay and 75 Malaysian Chinese individuals from the Malay Peninsula. We identified substantial allelic and structural diversity of the KIR locus in both populations and characterized novel variations at each analysis level. The Malay population is more diverse than Malay Chinese, likely representing a unique history including admixture with immigrating populations spanning several thousand years. Characterizing the Malay population are KIR haplotypes with large structural variants present in 10% individuals, and KIR and HLA alleles previously identified in Austronesian populations. Despite the differences in ancestries, the proportion of HLA allotypes that serve as KIR ligands is similar in each population. The exception is a significantly reduced frequency of interactions of KIR2DL1 with C2+ HLA-C in the Malaysian Chinese group, caused by the low frequency of C2+ HLA. One likely implication is a greater protection from preeclampsia, a pregnancy disorder associated with KIR2DL1, which shows higher incidence in the Malay than in the Malaysian Chinese. This first complete, high-resolution, characterization of combinatorial diversity of KIR and HLA in Malaysians will form a valuable reference for future clinical and population studies.