OBJECTIVE:To investigate the polymorphism and genotype frequency of HPA gene in platelet donors in Shenzhen area, and to explore its application in clinical platelet transfusion. METHODS:The blood samples of 461 platelet donors aged 18-55 years old were genotyped for HPA-1~6,-10,-15 and -21 using multiple fluorescent PCR. Platelet-specific antibodies were detected according to the instructions of PAKPLUS kit. RESULTS:The a and b alleles of HPA-3 and HPA-15 genes showed high frequency distribution, and their gene frequencies were HPA-3a (54.23%), HPA-3b (45.77%), HPA-15a(55.42%),HPA-15b(44.58%), respectively, and their dual antigen mismatch rate was the highest (37.32%, 37.20%). In HPA-21, the genotype distribution between male and female was statistically significantly different( P < 0.05). Among the patients with positive PAKPLUS test, 35.59% of patients were positive for platelet membrane glycoprotein antibody. CONCLUSION:Some HPA antigens in Shenzhen area have high mismatch rates and significant regional differences. The investigation of HPA genes and antibodies diversity laid the foundation for establishing a local platelet donor and recipient HPA information database and addressing platelet alloimmunity issues.

OBJECTIVE:To study the genetic polymorphisms of short-tandem repeats (STR) for the D13S317 locus among an ethnic Han Chinese population and verify a novel tri-allelic pattern identified for the locus.METHODS:A total of 378 paternity test cases from Guangdong Forensic Authentication Institute from October 17, 2017 to December 28, 2017 were selected as the study subjects. A GlobalFilerTM Express kit was used for the STR genotyping. Samples suspected for having a novel tri-allelic pattern were verified with a PowerPlex 21 kit. Potential variant of the primer-binding region and flanking sequences underlying the tri-allelic pattern was excluded by molecular cloning and sequencing.RESULTS:Six alleles were detected for the D13S317 locus, with the characteristic distribution frequencies being 8 (29.1%), 9 (13.1%), 10 (15.21%), 11 (24.21%), 12 (13.89%) and 13 (3.44%), respectively. In one of the families, the D13S317 locus of the proband was suspected to harbor a triband allele (8, 9, 10). A re-test has confirmed the result of initial test. Molecular cloning and sequencing analysis of the D13S317 locus in the proband and his daughter has failed to find allelic variants in the primer-binding region and flanking sequence, which has confirmed the novel tri-allelic pattern for the locus.CONCLUSION:A novel type 2 tri-allelic pattern (8, 9, 10) at the D13S317 locus has been identified among the ethnic Han Chinese population. The pattern has not been transmitted to the female offspring, and has been included in the international STRBase database for the first time.

OBJECTIVE:To investigate the accuracy of next-generation sequencing technology (NGS) in detecting the polymorphisms of HLA-DRB1, DQB1, DQA1, DRB3, DRB4, DRB5, DPA1 and DPB1 alleles in randomly-selected unrelated healthy individuals from Shenzhen Han population, investigate the potential reason for HLA-DRB1 allele dropout in routine NGS, and establish an internal quality control system.METHODS:NGS-based HLA class II genotyping was performed on 1 012 samples using the MiSeqDxTM platform. The suspected missed alleles indicated by the quality control software and HLA-DRB1 homozygotes were confirmed by PCR-SSOP or PCR-SBT methods.RESULTS:A total of 139 alleles were detected, including HLA-DRB1(45), DRB3(7), DRB4(5), DRB5(7), DQA1(17), DQB1(21), DPA1(10) and DPB1(27). HLA-DRB1*09:01(17.09%),15:01(10.72%); DRB3*02:02(25.99%),03:01(10.18%); DRB4*01:03(36.46%); DRB5*01:01(15.42%); DQA1*01:02(20.01%),03:02(17.19%); DQB1*03:01(19.47%),03:03(17.98%), 05:02(11.66%), 06:01(10.67%); DPA1*02:02(54.45%), 01:03(31.18%) and DPB1*05:01(39.13%), 02:01(16.90%) alleles were the most common alleles in Shenzhen Han population (frequencies >10%). There was no statistical difference between the gene frequencies of HLA-DRB1 and DQB1 loci in our study. The HLA Common and Well-Documented Alleles in China (CWD2.4) (χ2=12.68, P >0.05). 94 cases of HLA-DRB1 homozygous samples detected by NGS were retested by PCR-SSOP or SBT method, and one case of allele dropout at HLA-DRB1 locus was found. SBT method confirmed that the allele of DRB1*04:03 was missed. The laboratory internal quality control system was established. Two cases of new alleles were detected and named by WHO Nomenclature Committee for Factors of the HLA System.CONCLUSION:The HLA genotyping results based on NGS showed a significantly lower ambiguity rate. The HLA class II alleles exhibit genetic polymorphism in the Han population of unrelated healthy individuals in Shenzhen. The independent method based on NGS in clinical histocompatibility testing has limitations and requires internal quality control strategies to avoid allele-dropout events.

ABSTRACTThe 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*33:03:68 allele differs from HLA-A*33:03:01:01 by 1 variation in exon 3.

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

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

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

HLA-B*58:01:40 differs from HLA-B*58:01:01 by a single nucleotide change in exon 3, 507 C- > T (codon 145.3 CGC- > CGT).

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.

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

OBJECTIVE:To confirm the HLA genotypes of the samples including 4 cases of magnetic bead probe HLA genotyping result pattern abnormality and 3 cases of ambiguous result detected by PCR sequence-specific oligonudeotide probe (SSOP) method.METHODS:All samples derived from HLA high-resolution typing laboratory were detected by PCR-SSOP. A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality and 3 samples of ambiguous result were further confirmed by PCR sequence-based typing (SBT) technology and next-generation sequencing (NGS) technology.RESULTS:A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality were detected by PCR-SSOP method. The results of SBT and NGS showed that the HLA-A genotype of sample 1 did not match any known genotypes. NGS analysis revealed that the novel allele was different from the closest matching allele A*31:01:02:01at position 154 with G>A in exon 2, which resulting in one amino acid substitution at codon 28 from Valine to Methionine (p.Val28Met). The HLA-C genotype of sample 2 was C*03:119, 06:02, sample 3 was C*03:03, 07:137, and sample 4 was B*55:02, 55:12. A total of 3 samples with ambiguous result were initially detected by PCR-SSOP method. The re-examination results of SBT and NGS showed that the HLA-B genotype of sample 5 was B*15:58, 38:02, sample 6 was DRB1*04:05, 14:101, and sample 7 was DQB1*03:34, 05:02. Among them, alleles C*03:119, C*07:137 and DRB1*14:101 were not included in the Common and Well-documented Alleles (CWD) v2.4 of the Chinese Hematopoietic Stem Cell Donor Database.CONCLUSION:The abnormal pattern of HLA genotyping results of magnetic probe by PCR-SSOP method suggests that it may be a rare allele or a novel allele, which needs to be verified by sequencing.

HLA‐C*08:99 differs by one non‐synonymous nucleotide from C*08:01:01 in exon 5, codon 288 GTT>ATT.

HLA‐C*08:236N differs from C*08:01:01 by a single nucleotide exchange in exon 5 at position 1991.

OBJECTIVE To establish a reliable sequence-based typing method for KIR2DS4 and study its allele polymorphism in Chinese Han population. METHODS Using PCR-SSP method to detect the positive or negative of KIR2DS4 gene in 222 random Chinese Han individuals, and then using the method of high fidelity and long-fragment PCR-SBT to amplify, sequence and genotype the exons 4 and 5 of KIR2DS4 positive individuals. RESULTS We successfully amplified the fragment with 3.2 kb length contains exons 4 and 5 of KIR2DS4 and detected the KIR2DS4 allele frequency in Chinese Han population. 209 KIR2DS4 positive individuals were detected, and the positive rate is 94.1%. By sequence-based typing, we identified 12 genotypes and 7 alleles of KIR2DS4. The 6 known alleles and their detection frequency is as follows: KIR2DS4* 00101/011 (180, 81.1%), KIR2DS4* 010 (53, 23.9%), KIR2DS4* 004 (34, 15.3%), KIR2DS4* 003 (15 and 6.8%), KIR2DS4* 006 (2, 0.9%) and KIR2DS4* 015 (1, 0.5%). In this study, we found a new allele, KIR2DS4* 016, with the difference in exon 5 comparing its most similar allele KIR2DS4* 010. In the exon 5 of KIR2DS4* 010, there is a 22bp-deletion, while the exon 5 of KIR2DS4* 016 is normal. This is not a rare allele because it was detected 3 times in studied population and with the frequency of 1.4%. The sequence of the new allele sequence has been submitted to GenBank (accession no.: KC414890) and the IPD -KIR database (submission no.: IWS40001804), and was nominated by WHO nomenclature committee for HLA system. CONCLUSION In this study, a sequence-based typing method for KIR2DS4 was established, and the polymorphism data of KIR2DS4 in Chinese Han population was enriched by studying the allele polymorphism and new allele.

The HLA‐A*31:188N allele differs from A*31:01:02:01 by a single nucleotide deletion in exon 3.

The HLA-DRB3*02:02:19 allele differs from DRB3*02:02:01:02 by a single nucleotide change in exon 2.

HLA‐DPB1*1104:01 differs from HLA‐DPB1*540:01 by a single nucleotide change in exon 2.

HLA-DPB1*1104:01 differs from HLA-DPB1*540:01 by a single nucleotide change in exon 2.

The HLA-DPA1*02:33 allele differs from DPA1*02:02:02:04 by two nucleotide change in exon 4.