A puerpera with a obstetric history of gravida 2, para 2, underwent blood typing due to the presence of agglutination reactions in her serum against all tested red blood cells. She was found to be blood type O and her RhD phenotype was identified as CcDEe through serological testing. The reaction agglutination intensity between her serum and 26 O-type blood cells from the panel was 2+. Whole genome sequencing was performed, yielding data on 4014 single nucleotide polymorphisms (SNPs) and 958 insertion/deletion (INDEL) loci across 50 genes responsible for encoding blood group systems. Among these, only a single SNP , rs72552713 was predicted to be a highly harmful variant, which is the c.376C>T variation in the ABCG2 gene encoding JR blood group antigen, leading to the premature stop codon (p.Gln126Ter). The c.376C>T variation has been named the ABCG2*01N.01 by the working party on Red Cell Immunogenetics and Blood Group Terminology of International Society of Blood Transfusion. The postpartum woman was found to have the Jr(a-) phenotype. Whole genome sequencing can accurately determine the antigens of blood group systems in some difficult specimens.

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.
Female ; Animals ; ABO Blood-Group System/genetics* ; Phenotype ; Genotype ; Exons ; Alleles

【Objective】 To explore the influence of anti-HLA-Ⅰ with different mean fluorescence intensity (MFI) on the efficacy of HLA-A and -B gene matching platelet transfusion, so as to provide scientific data for clinical platelet gene matching transfusion strategy. 【Methods】 A total of 81 PTR patients had applied for HLA-Ⅰgene matched platelets from the platelet gene database established by our laboratory, and 28 (MFI <5 000) of them needed further avoiding of partial donor-specific antibodies and they were enrolled as the research subjects. According to the platelet MFI value of HLA-Ⅰ antibody-targeting antigen, they were divided into negative transfusion group (MFI <500) (group A) and positive transfusion groups (MFI≥500) ; the latter were further divided into group B (500≤MFI <1 000), group C (1 000≤MFI <3 000) and group D (MFI≥3 000) according to MFI value. Corrected count increment (CCI) in platelet count was used to compare the platelet transfusion effect in 4 groups. 【Results】 Among 28 platelet recipients with MFI <5 000, 19(67.86%) patients successfully received 72 effective transfusions. The first CCI (×10⁹/L) in groups A, B, C and D were 10.27±7.46, 7.58±4.75 (P>0.05), 17.36±7.63 (P>0.05) and -0.77±2.30 (P<0.05), respectively. There was no statistical difference among group A, B and C. 【Conclusion】 The application of HLA-Ⅰ gene matching platelets in PTR patients can adjust the MFI threshold(<2 000) appropriately according to the patient′s condition without compromising the platelet transfusion effect.

【Objective】 To analyze the specificity of HLA class-Ⅰ antibody in the patients received HLA-matched platelet transfusions and estimate the relative immunogenicity of HLA-Ⅰ antigens. 【Methods】 The samples from 96 patients who suffered from platelet transfusion refractorines(PTR) and applied for transfusion with genotype-matched platelet were collected. The specificity of HLA I antibody was detected by Luminex technique, and the antibody expression level was analyzed according to MFI. The mismatch rate of HLA antigen and relative immunogenicity of the population were estimated according to the allele frequency distribution of HLA-A and B loci as well as the yielding frequency of antibody. 【Results】 HLA-Ⅰ antibodies were detected in all 96 patients, with varied species of antibodies. The average positive yielding rates of antibodies corresponding to HLA-A, -B and -C magnetic bead coated antigens (97 in total) were 0.38, 0.47 and 0.28, respectively. Among the HLA-A and -B loci in the Zhejiang population, HLA-A2, A11, A24 and HLA-B60, B46, B58 were the antigens with higher frequency, and their relative immunogenicity was 0.403, 0.283, 0.342, and 0.100, 0.067, 0.178, respectively. 【Conclusion】 The specificity of HLA-Ⅰ antibodies in PTR patients is different, which confirms that the relative immunogenicity differs by HLA-A and -B antigens.

【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.

OBJECTIVE: To explore the molecular basis for an individual with Bw subtype. METHODS: Routine serological reactions were used to determine the surface antigens of erythrocytes and antibodies in serum. PCR-sequence-based typing (PCR-SBT) was used to analyze the coding regions of the ABO gene and erythroid-specific regulatory element in its intron 1. Amplicons for exons 5 to 7 containing the variant site were subjected to TA cloning for the isolation of the haploid and verification of the sequence. The 3D structure of mutant protein was predicted with Pymol software. Changes of amino acid residues and structural stability were also analyzed. RESULTS: Serological assay showed that the individual had weakened B antigen and anti-B antibody in his serum. His genotype was determined as ABO*B.01/ABO*O.01.01. Sequencing of the entire coding region of the ABO gene identified an additional heterozygous c.734C/T variant. No variant was found in the erythroid-specific regulatory element of intron 1. Haploid cloning and isolation has obtained an ABO*O.01.01 allele and a ABO*B.01 allele containing a c.734T variant, which has led to substitution of Thr by Ile at position 245 in the functional center of glycosyltransferase. Based on the 3D structure of the protein, the residues binding with the mutation were unchanged, but the bonding distance between the hydrogens was changed with the amino acid substitution. Meanwhile, the connections with water molecules were increased. CONCLUSION The c.734C>T variant of the GTB gene can lead to an amino acid substitution in the functional center of the enzyme, which in turn may affect the stability of glycosyltransferase B protein and reduceits enzymatic activity.
ABO Blood-Group System/genetics* ; Alleles ; Exons/genetics* ; Genotype ; Glycosyltransferases/genetics* ; Humans ; Male ; Phenotype

【Objective】 To analyze the positive rate of antibodies against human leukocyte antigen(HLA)and MHC class I chain-related gene A(MICA) in the convalescent plasma from individuals recovered from COVID-19. 【Methods】 HLA-Ⅰ, -Ⅱ and MICA antibodies were screened simultaneously by Luminex platform. The specificity of HLA-Ⅰ and -Ⅱ antibodies was identified by single antigen reagents.The positive rate of antibody in different groups were compared by Chi-square test. 【Results】 A total of 88 cases of convalescent plasma were collected, among which the positive rates of HLA-Ⅰ, -Ⅱ and MICA antibodies were 18.19%, 19.32% and 10.23%, respectively, and 64 individuals (72.73%) were negative for HLA-Ⅰ and -Ⅱ antibodies. 95 blood donors were randomly selected as the control group, and the positive rate of HLA-Ⅰ, -Ⅱ and MICA antibodies were 8.42%, 13.68% and 10.53%, respectively, and 76 individuals(80.00%) were negative for HLA-Ⅰ and -Ⅱ antibodies. There were no significant difference in the positive rates of HLA-Ⅰ, -Ⅱ and MICA antibodies between convalescent individuals and control group. The specificity of HLA antibody to epitopes was different in each convalescent individual with positive HLA antibodies, and most antibodies were targeted to the epitopes of multiple HLA alleles. 【Conclusion】 A certain proportion of HLA antibody was found in the convalescent plasma of individuals recovered from COVID-19. Therefore, HLA antibody screening is helpful to improve the safety of transfusion.

Objective:To investigate the effect of virus inactivation on weak positive result of 2019 novel coronavirus(2019-nCoV) nucleic acid test.Methods:A retrospective study was conducted on the nasopharyngeal swabs of three patients with positive PCR nucleic acid test for 2019-nCoV at different concentrations in the Second affiliated Hospital of Zhejiang University Medical College from January to February 2020.The virus in nasopharyngeal swab specimens were inactivated by water bath at 56 ℃ for 30 min, dry bath at 56 ℃ for 60 min and dry bath at 60 ℃ for 30 min respectively. After treatment, these samples RNA were extracted and then detected by three new commercial quantitative real-time polymerase chain reaction reagent kits for 2019-nCoV.Cycle threshold (Ct) value was used to evaluate the effect of virus inactivation on nucleic acid detection of 2019-nCoV.Results:There was no significant difference between the groups before and after inactivation. Ct values of ORF1ab gene before inactivation were 23.28±0.28, 25.25±0.25, 28.93±0.44, 32.06±0.47, 35.20±0.38, 32.89±0.38, 36.24±0.23, 33.30±0.46, and those after inactivation were, group 1:23.60±0.20, 27.29±0.30, 31.83±0.51, 37.41±0.46, group 2: 24.25±0.34, 27.18±0.42, 31.84±0.61, 34.99±1.01, 34.89±0.45,group 3: 23.37±0.17, 26.89±0.52, 32.05±0.50.Ct value of N gene before inactivation were 24.38±0.09, 26.64±0.11, 30.35±0.12, 33.29±0.33, 36.93±0.11, 34.50±0.12, 35.63±0.12, those after inactivation were, group 1: 24.66±0.11, 28.52±0.14, 32.71±0.14, 37.00±0.13;group 2: 25.41±0.10, 28.79±0.15, 33.29±0.28; group 3: 23.37±0.11, 28.68±0.11, 33.54±0.13, 37.18±0.23(ORF1ab gene: t=-1.416; N gene: t=-1.379, P>0.05). There was no significant difference among the three inactivation groups, the specific Ct values are shown above(ORF1ab gene: t=-0.460; N gene: t=-0.132, P>0.05). However, the Ct values of the inactivated groups (1,2,3) and the non-inactivated group at different dilution times were different (10 ×:Ct value of ORF1ab was 25.25±0.25 in the non-inactivated group, and 27.29±0.30, 27.18±0.42 and 26.89±0.52 in the inactivated group1,2 and 3, t(ORF1ab)=-7.327, P<0.01.Ct value of N gene in the non-inactivated group was26.64±0.11, those in inactivated group 1, 2 and 3 were 28.52±0.14, 28.79±0.15 and 28.68±0.11, respectively, t (N)=-19.340, P<0.01. 100 ×:Ct value of ORF1ab was 28.93±0.44 in the non-inactivated group, and 31.83±0.51,31.84±0.61 and 32.05±0.50 in the inactivated group1,2 and 3, t (ORF1ab)=-9.462, P<0.01. Ct value of N gene in the non-inactivated group was 30.35±0.12, those in the inactivated group 1, 2 and 3 were 32.71±0.14, 33.29±0.28 and 33.54±0.13, respectively, t (N)=-18.583, P<0.01. The positive detection rate of the non-inactivated group (7/11, 8/11, 5/11) was significantly different from that of the inactivated group (inactivated group 1:4/11, 4/11, 3/11, inactivated group 2:3/11, 3/11, 3/11, and inactivated group 3:3/11, 3/11, 2/11) ( Z=-2.670, P<0.01). There were no significant difference among the inactivated groups(inactivated group 1:4/11, 4/11, 3/11, inactivated group 2:3/11, 3/11, 3/11, inactivated group 3:3/11, 3/11, 2/11) ( Z=4.413, P>0.05) and among the three reagents(reagent 1:7/11, 4/11, 3/11, 3/11, reagent 2:8/11, 4/11, 3/11, 3/11, reagent 3:5/11, 3/11, 3/11, 2/11)(χ 2=1.199, P>0.05). Conclusion:The virus inactivation can degrade the nucleic acid of the 2019-nCoV, resulting in the decrease of the Ct value and the false negative results of the low-concentration specimens.

Objective To investigate the renoprotective effect of aspirin-triggered lipoxins(ATL)on kidney of mice with acute kidney injury(AKI).Methods Eighty-eight male specific pathogen-free(SPF)C57BL/6J mice were randomly divided into lipopolysaccharide(LPS)groups(including 2 h group,4 h group,8 h group,12 h group, 24 h group),ATL+LPS(including 2 h group,4 h group,8 h group,12 h group,24 h group)and normal control group according to random numble table,and each group had 8 mice.The mice in LPS groups were given LPS intraperitoneal injection to establish AKI animal models,while the mice in ATL+LPS groups were given ATL intraperitoneal injection 30 minutes before LPS intraperitoneal injection.The enzyme linked immunosorbent assay was used to test the serum creatinine(Scr),serum urea nitrogen(BUN),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and urine neutrophil gelatinase-associated lipocalin(NGAL),kidney injury molecule-1(KIM-1),cysteine-rich protein-61 (Cyr61)and netrin-1 levels of mice.Results The kidney tissue injury scores of mice of ATL+LPS group[4 h:(22.32 ± 1.04)scores,8 h:(31.11 ± 1.86)scores,12 h:(18.22 ± 0.92)scores,24 h:(20.87 ± 3.18)scores] were lower than those of LPS group at the corresponding time points[4 h:(35.47 ± 2.27)scores,8 h:(52.28 ± 2.82) scores,12 h:(54.99 ± 4.56)scores,24 h:(53.41 ± 4.76)scores],and the differences were statistically significant(all P<0.01).The values of Scr,BUN,TNF-α and IL-1β in ATL+LPS group[Scr 8 h:(143.07 ± 5.02)μmol/L, BUN 12 h:(33.07 ± 3.52)mmol/L,TNF-α 4 h:(196.33 ± 14.181)ng/L and 8 h:(221.77 ± 10.11)ng/L,IL-1β 4 h:(50.25 ± 2.67 ng/L)]were lower than those in LPS group at the corresponding time points[Scr 8 h:(227.43 ± 11.17)μmol/L,BUN 12 h:(59.68 ± 3.84)mmol/L,TNF-α 4 h:(267.87 ± 26.48)ng/L and 8 h:(334.78 ± 21.08)ng/L,IL-1β 4 h:(89.45 ± 5.87)ng/L],and the differences were statistically significant(all P<0.01). The urine NGAL[4 h:(56.76 ± 4.01)μg/L,8 h:(65.44 ± 7.81)μg/L],KIM-1[8 h:(78.19 ± 9.48)μg/L] and netrin-1[8 h:(40.12 ± 2.01)ng/L,12 h:(36.87 ± 2.87)ng/L]of mice in ATL+LPS group were lower than those in LPS group at the corresponding time points[NGAL 4 h:(168.77 ± 10.77)μg/L,8 h:(155.33 ± 8.26) μg/L;KIM-1 8 h:(124.73 ± 13.47)μg/L;netrin-1 8 h:(89.17 ± 2.74)ng/L,12 h:(81.11 ± 3.88)ng/L],and the differences were statistically significant(all P<0.01).Conclusions ATL can treat LPS-induced AKI and play a renoprotective role in the kidney.

The objective of this study was to investigate the regulatory effects of TGF-β1 on CCL3/4 expression and inflammation-related pain during intervertebral disc degeneration (IVDD). TGF-β1 and CCL3/4 expression patterns in different degenerative human nucleus pulposus (NP) tissues were measured by qPCR and immunohistochemistry (IHC), and the effects of TGF-β1 on CCL3/4 expression were measured by qPCR, ELISA and immunofluorescence. The roles of NF-κB and MAPK in TGF-β1-mediated CCL3/4 promoter activity were studied using siRNAs, western blotting and qPCR. After establishing an IVDD rat model in vivo, we administered intradiscal injections of TGF-β1. The effects of TGF-β1 on IVDD were determined by MRI and histological analyses, and the effects of TGF-β1 on dorsal root ganglion (DRG) inflammation and pain development were determined by IHC staining and pain-behavior testing, respectively. TGF-β1 and CCL3/4 expression was elevated in degenerative NP tissue. CCL4 expression was significantly inhibited by TGF-β1 treatment. Pharmacological inhibition or siRNA knockdown of the ERK1/2 signaling attenuated TGF-β1-mediated suppression of CCL4 expression. In vivo, TGF-β1 injection inhibited the development of degenerative features in the IVDD model. Moreover, TGF-β1 prevented the inflammatory response and pain development. The results of this study show that TGF-β1 downregulates CCL4 expression through ERK1/2 signaling activation in NP cells. Furthermore, TGF-β1 can prevent degenerative processes, inhibit inflammatory responses in the DRG and prevent pain development in the IVDD rat model. The results of this study indicate that TGF-β1 may represent a therapeutic target for the control of inflammation-related pain associated with IVDD.
Animals ; Blotting, Western ; Diagnosis-Related Groups ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique ; Ganglia, Spinal ; Humans ; Immunohistochemistry ; Inflammation ; Intervertebral Disc Degeneration* ; Intervertebral Disc* ; Magnetic Resonance Imaging ; Models, Animal* ; Rats* ; RNA, Small Interfering