Expert consensus on the resolution level of the methods for HLA genotyping / 中国输血杂志

Human leukocyte antigen (HLA) genotyping has been widely used in establishing the database of the donors for hematopoietic stem cell, HLA matching selection between the donor and recipient, establishing the database of platelet donors with known HLA genotype, diagnosis of HLA association with diseases, genetics study and other scientific research. With the increasing number of HLA alleles and the development of HLA genotyping technology, there are some controversy in the definition of resolution for HLA genotyping, the description of the results for HLA genotyping between donor and recipient and the determination of HLA matching level. In order to improve the normalization of the definition and interpretation of HLA genotyping resolution, this expert consensus is formulated by many experts from the fields of HLA genotyping in the laboratory and clinical transplantation according to the relevant domestic and foreign literature and clinical practice. The definition of the resolution of HLA genotyping, the methods of HLA genotyping, the description of the results between donor and recipient and HLA matching determination are summarized, which will further standardize HLA genotyping technology and ensure the accuracy of the results for HLA genotyping.

Allele frequency polymorphism of Duffy, Kidd, Diego blood group system: a study in Dongxiang ethnic group in Gansu Province / 中国输血杂志

【Objective】 To investigate the allele frequencies polymorphic distribution of Duffy, Kidd and Diego blood group systems in Dongxiang ethnic group in Gansu Province. 【Methods】 Blood samples of 100 unrelated blood donors were randomly selected from Dongxiang ethnic group in Gansu from January to December 2017. Allelic typing of Duffy, Kidd and Diego blood groups was performed by fluorescence PCR. 【Results】 The allele frequencies of Duffy, Kidd, and Diego blood group systems of Dongxiang ethnic group were 0.835 for Fy*01, 0.165 for Fy*02, 0.570 for Jk*01, 0.430 for Jk*02, 0.020 for DI*01, 0.980 for DI*02, respectively. No Fy(a-b-), Jk(a-b-), Di(a+b-) rare phenotypes were found. The antigen incompatibility rates of Fya/Fyb, Jka/Jkb, Dia/Dib of Duffy, Kidd, and Diego blood group systems were 23.76%, 37.01% and 3.84%, respectively. 【Conclusion】 The allele frequencies distribution of Duffy, Kidd and Diego blood group systems in Dongxiang ethnic group in Gansu were polymorphic and has unique ethnic distribution characteristics.

Molecular analysis of the new allele 803delC of subtype B / 中国输血杂志

【Objective】 To analyze the serological characteristics and molecular mechanism of a novel B subtype allele 803delC. 【Methods】 ABO blood group was detected by serological method. Sequence-specific primer polymerase chain reaction (PCR-SSP) was used to detect ABO blood group genes. The coding region of exon 1-7 of ABO gene was detected by Sanger sequencing to determine the mutation site. 【Results】 Serological identification of patients was with forward O-type and reverse B-type. The result of PCR-SSP genotyping was A/O. There was A gene, which was not consistent with serological results. Further Sanger double-strand sequencing revealed that the C-base was deleted at position 803 of exon 7 on the basis of ABO*B. 01/ABO*O. 01.01. The mutation eventually leads to the amino acid substitution of p. Ala268Gly and p. Phe269Ser and the production of new open reading frame starting at position 269, with the new open reading frame No.20 amino acid being stop codon, resulted in the termination of B gene expression. Further single-strand sequencing of the ABO gene revealed that the mutation was located in the ABO*B. 01 gene. The mutation was submitted to the NCBI database with the number OR343908. 【Conclusion】 A new ABO allele leading to B variant has been found in Chinese population. Genetic detection can be used to identify the ambiguous blood group with discrepancy between forward and reverse blood grouping.

Causes of HLA-A2 reactive antibodies and analysis of their eplets / 中国输血杂志

【Objective】 To analyze differences of eplets between the patient who generated HLA allele-specific antibodies after platelet transfusion with donors. 【Methods】 The HLA genotypes of the patient and donors were detected by PCR-SBT, and the Luminex single antigen beads coating was used to screen HLA-Ⅰ antibodies in the patient’s serum. HLA Matchmaker was utilized to analyze different amino acids and eplets. 【Results】 The patient carried HLA-A*02∶03 allele, and HLA-A2 antibodies were found in his serum after platelet transfusion (A*02∶01, A*02∶06, and A*02∶07). Sequence alignment showed that the patient′s A*02∶03 has a difference in position 149, which resulted in a different eplet between A*02∶03 and A*02∶01, A*02∶06, A*02∶07 and then induced the production of antibodies. 【Conclusion】 HLA antibodies are specific for HLA epitopes that have structural differences due to amino acid differences between HLA alleles, suggesting that high-resolution typing of HLA-A, -B need to be conducted in patients and donors, and the acceptable mismatch of HLA should be determined based on epitopes rather than antigens, so as to reduce alloimmune response and improve platelet count after transfusion.

The fliL gene significantly affects the motility and sporulation abilities of Clostridioides difficile / 生物工程学报

Flagella are the main motility structure of Clostridioides difficile that affects the adhesion, colonization, and virulence of C. difficile in the human gastrointestinal tract. The FliL protein is a single transmembrane protein bound to the flagellar matrix. This study aimed to investigate the effect of the FliL encoding gene flagellar basal body-associated FliL family protein (fliL) on the phenotype of C. difficile. The fliL gene deletion mutant (ΔfliL) and its corresponding complementary strains (: : fliL) were constructed using allele-coupled exchange (ACE) and the standard molecular clone method. The differences in physiological properties such as growth profile, antibiotic sensitivity, pH resistance, motility, and spore production ability between the mutant and wild-type strains (CD630) were investigated. The ΔfliL mutant and the : : fliL complementary strain were successfully constructed. After comparing the phenotypes of strains CD630, ΔfliL, and : : fliL, the results showed that the growth rate and maximum biomass of ΔfliL mutant decreased than that of CD630. The ΔfliL mutant showed increased sensitivity to amoxicillin, ampicillin, and norfloxacin. Its sensitivity to kanamycin and tetracycline antibiotics decreased, and the antibiotic sensitivity partially returned to the level of CD630 strain in the : : fliL strain. Moreover, the motility was significantly reduced in the ΔfliL mutant. Interestingly, the motility of the : : fliL strain significantly increased even when compared to that of the CD630 strain. Furthermore, the pH tolerance of the ΔfliL mutant significantly increased or decreased at pH 5 or 9, respectively. Finally, the sporulation ability of ΔfliL mutant reduced considerably compared to the CD630 strain and recovered in the : : fliL strain. We conclude that the deletion of the fliL gene significantly reduced the swimming motility of C. difficile, suggesting that the fliL gene is essential for the motility of C. difficile. The fliL gene deletion significantly reduced spore production, cell growth rate, tolerance to different antibiotics, acidity, and alkalinity environments of C. difficile. These physiological characteristics are closely related to the survival advantage in the host intestine, which is correlated with its pathogenicity. Thus, we suggested that the function of the fliL gene is closely related to its motility, colonization, environmental tolerance, and spore production ability, which consequently affects the pathogenicity of C. difficile.

Using Next-Generation Sequencing Technology to Confirm the HLA Rare Alleles Detected by PCR-SSOP / 中国实验血液学杂志

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.

Potential unreliability of ALK variant allele frequency in the efficacy prediction of targeted therapy in NSCLC / 医学前沿

Anaplastic lymphoma kinase (ALK) is the most common fusion gene involved in non-small cell lung cancer (NSCLC), and remarkable response has been achieved with the use of ALK tyrosine kinase inhibitors (ALK-TKIs). However, the clinical efficacy is highly variable. Pre-existing intratumoral heterogeneity (ITH) has been proven to contribute to the poor treatment response and the resistance to targeted therapies. In this work, we investigated whether the variant allele frequencies (VAFs) of ALK fusions can help assess ITH and predict targeted therapy efficacy. Through the application of next-generation sequencing (NGS), 7.2% (326/4548) of patients were detected to be ALK positive. On the basis of the adjusted VAF (adjVAF, VAF normalization for tumor purity) of four different threshold values (adjVAF < 50%, 40%, 30%, or 20%), the association of ALK subclonality with crizotinib efficacy was assessed. Nonetheless, no statistical association was observed between median progression-free survival (PFS) and ALK subclonality assessed by adjVAF, and a poor correlation of adjVAF with PFS was found among the 85 patients who received first-line crizotinib. Results suggest that the ALK VAF determined by hybrid capture-based NGS is probably unreliable for ITH assessment and targeted therapy efficacy prediction in NSCLC.

Prognostic Predictive Value of ITPKB Mutation's Variant Allele Frequency in Diffuse Large B-cell Lymphoma / 肿瘤防治研究

Objective To investigate the correlation between ITPKB mutation's variant allele frequency (VAF) and prognosis of diffuse large B-cell lymphoma (DLBCL). Methods This study included 155 patients with DLBCL initially diagnosed in the People's Hospital of Xinjiang Uygur Autonomous Region from June 2014 to December 2020. Paraffin-embedded tumor tissue specimens were obtained, and tumor tissue DNA was extracted. A total of 475 hotspot genes including ITPKB were detected by the next generation sequencing to analyze the relationship of the VAF of high-frequency mutant gene with progression-free survival (PFS) and overall survival (OS). Results The mutation frequency of ITPKB was 18.71%. The PFS was significantly shorter in the patients with ITPKB mutations than in those without mutations (37 months vs. 108 months; HR=1.643, 95%CI: 0.920-2.934, P=0.093). The R-language based web tool was used to find the best VAF cutoff to differentiate prognosis. The patients were divided into two groups (VAF High vs. VAF Low+Wt) according to their VAF values. The optimal VAF threshold for ITPKB was 27.48% (HR=3.480, 95%CI: 1.70-7.13, P=0.00027). Multivariate Cox analysis was conducted using clinical indicators such as age, gender, COO classification, IPI, and LDH, and the results showed that PFS was associated with high ITPKB VAF (≥28%) (HR=3.592, 95%CI: 1.738-7.425, P < 0.001) which was an independent adverse predictor of PFS. Conclusion The high load of ITPKB mutation is an independent risk factor for the PFS of patients with DLBCL, and the VAF of ITPKB mutation has a prognostic predictive value for patients with DLBCL.

Matrix metalloproteinases and their gene polymorphism in young ST-segment elevation myocardial infarction

Background: Genetic polymorphism in MMPs are associated with multiple adverse CV events. There is little evidence regarding role of MMPs and their genetic polymorphisms in young (<50 years) STsegment elevation myocardial infarction (STEMI) patients. Methods: This study included 100 young (18e50 years) STEMI patients and 100 healthy controls. Serum levels of MMP-3, MMP-9 and TIMP were estimated for both patients as well as controls. Additionally, genetic polymorphisms in the MMP-9 gene (_x0001_1562 C/T and R279Q) & MMP-3 gene (5A/6A-1612) was evaluated. All these patients were followed up for one year and major adverse cardiac events (MACE) were determined. Results: Serum levels of MMP-3 (128.16 ± 115.81 vs 102.3 ± 57.28 ng/mL; P ¼ 0.04), MMP-9 (469.63 ± 238.4 vs 188.88 ± 94.08 pg/mL; P < 0.0001) and TIMP (5.84 ± 1.93 vs 2.28 ± 1.42 ng/mL; P < 0.0001) were significantly higher in patients as compared to controls. Additionally, patients with genetic polymorphisms in the MMP genes (5A/5A, 6A/6A and the AG genotypes) had an increased risk of STEMI. Patients with MACE had significantly higher levels of MMP-9 (581.73 ± 260.93 vs 438.01 ± 223.38 pg/mL; P ¼ 0.012). A cutoff value of 375.5 pg/mL of MMP-9 was best able to discriminate patients with STEMI and MACE with sensitivity of 77.3% and specificity of 57%. Conclusion: Novel biomarkers such as MMP-3, MMP-9 and TIMP and their genetic polymorphism are associated with the susceptibility for STEMI in young individuals. Higher MMP-9 levels in STEMI patients with MACE suggests its potential role in predicting cardiac remodeling and left ventricular dysfunction

Frequency of Rh and K antigens in blood donors in Riyadh

ABSTRACT Objectives: Investigate the prevalence of Rh and the K antigens and their phenotypes in the red blood cells of blood donors in Riyadh, Saudi Arabia. Methods: This is a retrospective study. The five principal Rh antigens (D, C, c, E, e) and the Kell antigen from the Kell blood group were tested in 4,675 random samples collected from four blood bank centers in Riyadh. Data were collected for seven weeks (from January 4, 2019 to February 28, 2019). Antigens were tested using the TANGO Optimo system. Results: We found that approximately 86% of the donors had the D antigen, 66% had C, 78% had c, 26% had E, 97% had e and 14% had K. The most common Rh phenotypes were R1r (31%) and R1R1 (22%). Conclusion: The differences in the results between the study population and other populations, such as Caucasian, Indian and African populations indicate the importance of establishing a population-specific database.

Polymorphism of KIR2DL4 gene among northern Chinese Han population / 中国输血杂志

【Objective】 To investigate the polymorphism of KIR2DL4 gene in northern Chinese Han population. 【Methods】 A total of 327 DNA samples were isolated by magnetic beads from unrelated individuals of northern Chinese Han population. The coding sequence (CDS) of KIR2DL4 were amplified using four pairs of KIR2DL4-specific PCR primers developed by our own KIR sequencing-based typing patent, and each exon of KIR2DL4 carried by the four PCR amplicons was then subjected to DNA Sanger sequencing in both directions. The genotype of each sample was assigned using the Assign 4.7 software. 【Results】 Among 327 individuals, 8 different kinds of KIR2DL4 alleles were detected, with observed frequencies ranked as KIR2DL4*00102 [77.1%(252/327)], *00501 [35.8%(117/327)], *011 [20.2%(66/327)], *00602 [12.5%(41/327)], *00801 [8.6%(28/327)], *00103 [4.9%(16/327)], *00503 [1.5%(5/327)] and *00504 [0.9%(3/327)]. In this study, the 10A type alleles which can encode a full membrane-bound receptor include 2DL4*00102, *00103, *00501, *00503, *00504 and *00602, whereas the 9A type alleles which produce truncated forms of receptor include 2DL4*00801 and *011. The observed frequencies for 10A and 9A type KIR2DL4 alleles were 97.6% (319/327) and 27.8% (91/327), respectively. The ratio of 10A to 9A type was 3.5: 1. The observed frequencies of KIR2DL4 alleles in northern Chinese Han population showed no significant difference compared with southern Chinese Han population (P>0.05). 【Conclusion】 The allelic diversity of KIR2DL4 elucidated in the present study can provide valuable data for KIR-associated disease studies and human evolution.

Serological and molecular study of 20 ABO subgroup samples in Shenyang, including identification of 2 ABO new alleles / 中国输血杂志

【Objective】 To study the serological characteristics and genetic background of 20 samples with ABO blood group discrepancies in Shenyang. 【Methods】 Serological test, polymerase chain reaction-sequence specific primer (PCR-SSP) and sequencing of the full coding of ABO gene and the Intron 1 were conducted in 20 samples with ABO blood group discrepancies. 【Results】 Ten subtypes (Am, Bw, Bx, B3,, A2B, AxB, A2Bw, A2Bx, AwB and ABw) were detected in 20 samples, with AB subtype as the dominant. Sixteen ABO alleles were found, including 5 common alleles (A1.01, A1.02, B. 01, O. 01.01 and O. 01.02), nine rare alleles (AW.37, BW.03, BW.08, B3.07, cisAB.02, cisAB.03, cisAB.06, BA.04 and O. 01.04) and two novel alleles (AM.03 and cisAB.07). The AM.03 allele had a nucleotide change at position 912 (C to A) compared with A1.02 allele, which resulted in an amino acid substitution (S304R). The cisAB.07 allele was observed a missense mutation at position 797 (T to C) which resulted in an amino acid substitution (M266R) compared with B. 01 allele. The serologic had been changed, and both A antigen and B antigen were expressed. 【Conclusion】 The study revealed the genetic background of 20 samples with ABO blood group discrepancies, and two new alleles as ABO*AM.03 (912 C to A) and ABO*cisAB.07 (797 T to C) were first reported.

Analysis and prospect of platelet compatible transfusion / 中国输血杂志

Platelet compatible transfusion can effectively solve the immune mediated platelet transfusion refractoriness (PTR), save platelet resources and improve blood safety. This paper comments and prospects the compatibility modes of HLA, HPA and CD36, HLA antibody titer, antigen immunogenicity and the development of platelet compatible transfusion. The pattern of HLA compatible platelets involves the matching in the alleles, antigens and epitopes levels, respectively, as well as avoidance donor specificity antibody (DSA) method. While setting the mean fluorescence intensity (MFI) threshold of avoidance DSA needs to be explored when using the DSA prediction method. Allele specific HLA antibodies can be found in the patients with PTR. Therefore, the patients and donors should be genotyped for HLA-A, -B loci at high-resolution level in order to avoid allele specific HLA antibodies. The immunogenicity of various antigens or epitopes at HLA-A and -B loci are different. Selecting donor platelets with low antigen expression or low immunogenicity may be a way of HLA compatible platelets. As the probability and type of HPA antibody production are different in the various populations, the approaching of compatibility HPA involves allele matching and avoidance DSA. As to CD36, the compatibility mode mainly refers to avoidance DSA, which means blood donors with CD36 antigen type Ⅰdeficiency are preferentially selected, and then those with CD36 antigen type Ⅱ deficiency. In the future, more attention should be paid to the scale up of database capacity and update of the information construction. The time waiting for compatible platelets transfusion in clinical could be significantly shortened if the requiring and matching are only conducted within the inventory and candidate platelets.

Analysis of HLA allele-specific antibodies and Eplets in patients with platelet transfusion refractoriness / 中国输血杂志

【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.

Polymorphism study of HLA-A, B, DRB1 alleles in patients with chronic renal failure, in Shandong Peninsula / 中国输血杂志

【Objective】 To investigate the association between HLA-A, B, DRB1 alleles and chronic renal failure (CRF) in Han population of Shandong Peninsula. 【Methods】 Sequence specific oligonucleotide probe-polymerase chain reaction (PCR-SSO) was used to genotype 880 patients with CRF in the Han population of Shandong Peninsula. The allele frequencies of HLA-A, B and DRB1 were compared with 865 hematopoietic stem cell voluntary donors, and the association between HLA gene polymorphism and CRF was analyzed. 【Results】 A total of 33 HLA-A alleles, 76 HLA-B alleles and 39 HLA-DRB1 alleles were detected in the study group. DRB1*11∶01 (6.70% vs 4.45%) and DRB1*12∶02 (8.69% vs 5.90%) in CRF group were significantly higher than those in the control(Pc<0.05), and B*15∶11 (1.82% vs 3.64%) among CRF group was significantly lower compared with the control(Pc<0.05). The frequency of three loci haplotypes A*30∶01 -B*13∶02 -DRB1*07∶01 (16.61% vs 7.61%), A*33∶03 -B*58∶01 -DRB1*03∶01 (4.57% vs 1.62%) and A*02∶07 -B*46∶01 -DRB1*09∶01 (4.06% vs 1.09%) in CRF patients were significantly higher than that of the control(Pc<0.05), which were strongly correlated with CRF. 【Conclusion】 The data on the association of HLA-A, B, DRB1 alleles and haplotype polymorphisms with CRF in Shandong Peninsula has been obtained in this study. DRB1*11∶01 and DRB1*12∶02 may be the susceptibility risk factors for development of CRF, and B*15∶11 may be protective genes against development of CRF, and A*30∶01 -B*13∶02 -DRB1*07∶01, A*33∶03 -B*58∶01 -DRB1*03∶01 and A*02∶07 -B*46∶01 -DRB1*09∶01 may be the susceptible haplotypes in Han population of Shandong Peninsula.

Distribution and molecular basis of A2 subtype in Guangzhou blood donors / 中国输血杂志

【Objective】 To investigate the distribution and molecular basis of A2 subtype among blood donors in Guangzhou. 【Methods】 Whole blood samples of 793 group A blood donors in Guangzhou Blood Center were randomly collected. A2 subtype was screened by anti-A1 lectin in 96-well plate, and ABO typing was confirmed by the tube test. Then, the genomic DNA of A2 subtype blood donors was extracted, and the exons 6-7 of ABO gene were sequenced to determine the genotype of A2 subtype. 【Results】 Among 793 group A blood donors, there were 13 cases of A2 subtype, and the frequency in group A was 1.64%(13/793). The sequencing results of exons 6-7 of ABO gene showed that 10 of the 13 A2 subtypes carried A2 alleles, which were ABO*A2.05/ ABO*O.01.01 (n=5), ABO*A2.05/ ABO*O.01.02(n=2), ABO*A2.01/ABO*O.01.01(n=1) and ABO*A2.01/ ABO*O.01.02(n=2), respectively. The other 3 cases only carried ABO*A1.02 alleles. 【Conclusion】 The main genotype of A2 subtype in Guangzhou is ABO*A2.05, followed by ABO*A2.01.

The establishment of a high-resolution melting curve genotyping method for ABCG2*376T allele of Jr(a-) and its distribution frequency study / 中国输血杂志

【Objective】 To establish a high-throughput detection method for ABCG2*376T allele of Jr(a-), and apply it to the study of the frequency of this allele in the Chinese population. 【Methods】 The specific primers were designed and synthesized, the sample carrying homozygous ABCG2*376T alleles, obtained in the previous study, was used as the homozygous positive control, and the sample carrying heterozygous allele as the heterozygous positive control. The wild-type sample was used as a negative control, and a high-resolution melting curve(HRM) method for detecting this allele was established. The established method was used to screen DNA samples from blood donors in Guangzhou, and the samples carrying ABCG2*376T alleles were sequenced to confirm the accuracy of the HRM method. 【Results】 A HRM method, which can detect ABCG2*376T allele and accurately type homozygotes and heterozygotes at the same time, had been established successfully. Fifteen individuals with heterozygous alleles were screened out of 1 560 blood donors in Guangzhou, while none homozygous allele was detected. 【Conclusion】 The HRM method can be used to accurately screen and type ABCG2*376T allele. The frequency of this allele in Chinese population is about 0.48%(15/3120).

Identification of pregnant women with DFR genotype and discussion of prenatal examination strategy / 中国输血杂志

【Objective】 To identify three cases of pregnant women with the D variant phenotype using serological and molecular tests, and discuss the strategy of prenatal examination. 【Methods】 The peripheral blood samples from three pregnant women with the D variant phenotype were collected. RhD variant phenotype was determined using routine serological methods with two different kinds of monoclonal anti-D. The serological characteristic for the epitope of D antigen was further analyzed using the commercial panel anti-D reagents (D-Screen, Diagast). The hybrid RHD-CE-D allele was analyzed by the Multiplex Ligation-dependent Probe Amplification (MLPA) assay and polymerase chain reaction with sequence specific primers (PCR-SSP) method. Further Sanger sequencing of RHD gene exons was also performed. 【Results】 DFR phenotype was primarily determined by serological characteristic for the epitope of D antigen. RHD*DFR2/01N.01(n=2) and RHD*DFR1/1227A(n=1) genotypes were identified by the MLPA assay, PCR-SSP and Sanger sequencing. 【Conclusion】 Two pregnant women with RHD*DFR2/01N.01 genotype should be treated as D negative patients clinically, while the pregnant woman with RHD*DFR1/1227A genotype can be treated as Asia type DEL to avoid unnecessary antibody screening and anti-D prophylaxis.

Interpretation of results obtained using real-time fluorescence PCR based on multiplex PCR fragment analysis for ABCB1 tri-allele / 四川精神卫生

ObjectiveTo resolve the problems related to the abnormal interpretations of real-time fluorescence polymerase chain reaction （PCR） results for tri-allele， to formulate the interpretation methods of real-time fluorescence PCR by referring to multiplex PCR fragment analysis， so as to obtain an accurate， simple and cheap detection method for ABCB1 tri-allele. MethodsA total of 2 794 DNA samples were collected from Xi'an Mental Health Center from March 2020 to March 2021， and 5% of which were selected as experiments. Real-time fluorescence PCR method and multiplex PCR fragment analysis method were performed respectively. According to the comparison of Ct values of PCR curves and the comparison of base peak intensity in multiplex PCR fragment analysis， comparison and analysis were conducted on the interpretation results of the two methods， and samples with different interpretation results were verified， thereafter， PCR interpretation method was formulated. ResultsA total of 139 samples were collected， of which 120 alleles and 19 tri-allele were detected. The results of allele detection by two methods were absolutely consistent. In combination with the results of multiplex PCR fragment analysis， a method for the interpretation of real-time fluorescence PCR for 19 cases of tri-allele was developed as follows： when ∣∣Ct.G－Ct.T∣－∣Ct.G－Ct.A∣∣ in amplification curve was less than 3， the interpretation results were the combination of the base pairs with small Ct values； when ∣∣Ct.G－Ct.T∣－∣Ct.G－Ct.A∣∣ was greater than or equal to 3， the interpretation results were homozygotes from the base pairs with minimum Ct values. According to the interpretation method， the results of real-time fluorescence PCR were revised， and it showed 1 case of G/G， 1 case of A/A， 4 cases of T/G， 5 cases of T/A and 8 cases of T/T， which were consistent with the results of multiplex PCR fragment analysis. ConclusionReferring to the multiplex PCR fragment analysis method， the interpretation of ABCB1 tri-allele by real-time fluorescence PCR is developed， and the two interpretation methods are in good agreement.

Identification of Murrayae Folium et Cacumen by Multiplex Allele-Specific PCR / 中国实验方剂学杂志

ObjectiveTo establish a polymerase chain reaction（PCR） method to accurately discriminate the crude materials of Murrayae Folium et Cacumen， Murraya exotica and M. paniculata. MethodBased on the difference in chloroplast genome sequences of M. exotica and M. paniculata， species-specific identification primers P03 and P04 of M. exotica and M. paniculata were designed according to single nucleotide polymorphism （SNP） on the chloroplast genome. A multiplex allele-specific PCR identification method was established for the identification of M. exotica and M. paniculata following the optimization of annealing temperature， number of cycles， and primer concentration ratio. The established PCR method for identification was explored and verified in terms of tolerance and feasibility by investigating the type of Taq polymerases and PCR system model. ResultIn this multiplex allele-specific PCR identification method， about 330 and 230 bp of specific fragments were amplified from DNA templates of M. exotica and M. paniculata， respectively， under the following conditions：cycle number of 31， annealing temperature of 60 ℃， and primer concentration ratio of P03 and P04 of 1∶2. Consistent results were obtained for samples from different sources. ConclusionThe multiplex allele-specific PCR identification method established in this study can accurately identify the origin of Murrayae Folium et Cacumen， which can be used for the simultaneous identification of M. exotica and M. paniculata by the length of fragments in a single identification assay.