Ambiguous results of the HLA-DPB1 genotyping results: Based on AllType NGS 11-loci sequencing reagent / 中国输血杂志

【Objective】 To statistically analyze the characteristics of ambiguous results of HLA-DPB1 genotyping given by AllType NGS 11-loci sequencing reagent via two next generation sequencing platforms, i. e. Ion Torrent S5 and Illumina Miseq. 【Methods】 A total of 434 samples from patients or donors were genotyped for HLA-DPB1 locus using AllType NGS 11-loci sequencing reagent from One Lambda company; 336 samples of them were sequenced via the Ion Torrent S5 platform and other 98 samples were sequenced via the Illumina Miseq platform. All 434 samples were genotyped for HLA-DPB1 gene simultaneously using PCR-SSO flow fluorescent bead method. The ambiguous genotypes of HLA-DPB1*13∶01∶01/107∶01 were distinguished by Sanger sequencing. The HLA-DPB1 genotype results by NGS method were assigned by TypeStream Visual professional software, and the ratio of ambiguous combination was calculated by direct count method. 【Results】 Ambiguous results were found in 357 out of 434 samples, accounting for 82.3% (357/434) when HLA-DPB1 allele was assigned to the third field using NGS method. Ambiguous results with 45 types were given in 275 out of 336 samples by the Ion Torrent S5 platform, accounting for 81.8% (275/336) and 82(with 27 types) out of 98 samples by the Illumina Miseq platform, accounting for 83.7% (82/98). All samples were re-genotyped for HLA-DPB1 gene by PCR-SSO, and none HLA-DPB1 allele had been missed by NGS. A total of 43 ambiguous alleles in HLA-DPB1*13∶01∶01/107∶01 involving 41 samples were distinguished by Sanger sequencing; HLA-DPB1*13∶01∶01 were detected in 25 (58.1%, 25/43) and HLA-DPB1*107∶01 in 18 (41.9%, 18/43). 【Conclusion】 There were still a high proportion of HLA-DPB1 ambiguous combinations using the AllType NGS 11-loci sequencing reagent. Sequencing exon 1 of HLA-DPB1 gene by Sanger sequencing can resolve part of the ambiguous results in HLA-DPB1*13∶01∶01/107∶01 alleles.

Study of the molecular characteristics of a Bweak phenotype due to a novel c.398T>C variant of the ABO gene / 中华医学遗传学杂志

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.

Predictive value of D-dimer for deep venous thrombosis of lower extremity in adult burn patients / 中华烧伤杂志

Objective: To investigate the predictive value of D-dimer for deep venous thrombosis (DVT) of lower extremity in adult burn patients. Methods: A retrospective case series study was conducted. The clinical data of 3 861 adult burn patients who met the inclusion criteria and were admitted to the Department of Burns of Zhengzhou First People's Hospital from January 1, 2015 to December 31, 2019 were collected. The patients were divided into DVT group (n=77) and non-DVT group (n=3 784) according to whether DVT of lower extremity occurred during hospitalization or not. Data of patients in the two groups were collected and compared, including the gender, age, total burn area, D-dimer level, with lower limb burn and inhalation injury or not on admission, with sepsis/septic shock, femoral vein indwelling central venous catheter (CVC), history of surgery, and infusion of concentrated red blood cells or not during hospitalization. Data were statistically analyzed with independent sample t test, Mann-Whitney U test, and chi-square test. The indicators with statistically significant differences between the two groups were analyzed with multivariate logistic regression analysis to screen the independent risk factors for DVT of lower extremity in 3 861 adult burn patients. The receiver operating characteristic (ROC) curve of the independent risk factors predicting DVT of lower extremity in 3 861 adult burn patients were drawn, and the area under the curve (AUC), the optimal threshold value, and the sensitivity and specificity under the optimal threshold value were calculated. The quality of the AUC was compared by Delong test, and the sensitivity and specificity under the optimal threshold value were compared using chi-square test. Results: There were no statistically significant differences in gender, occurrence of sepsis/septic shock or history of surgery during hospitalization between patients in the two groups (P>0.05), while there were statistically significant differences in age, total burn area, D-dimer level, lower limb burn and inhalation injury on admission, and femoral vein indwelling CVC and infusion of concentrated red blood cells during hospitalization between patients in the two groups (t=-8.17, with Z values of -5.04 and -10.83, respectively, χ2 values of 21.83, 5.37, 7.75, and 4.52, respectively, P<0.05 or P<0.01). Multivariate logistic regression analysis showed that age, total burn area, and D-dimer level were the independent risk factors for DVT of lower extremity in 3 861 adult burn patients (with odds ratios of 1.05, 1.02, and 1.14, respectively, 95% confidence intervals of 1.04-1.06, 1.00-1.03, and 1.10-1.20, respectively, P<0.05 or P<0.01). The AUCs of ROC of age, total burn area, and D-dimer level for predicting DVT of lower extremity in 3 861 adult burn patients were 0.74, 0.67, and 0.86, respectively (with 95% confidence intervals of 0.68-0.80, 0.60-0.74, and 0.83-0.89, respectively, P values<0.01), the optimal threshold values were 50.5 years old, 10.5% total body surface area, and 1.845 mg/L, respectively, the sensitivity under the optimal threshold values were 71.4%, 70.1%, and 87.0%, respectively, and the specificity under the optimal threshold values were 66.8%, 67.2%, and 72.9%, respectively. The AUC quality and sensitivity and specificity under the optimal threshold value of D-dimer level were significantly better than those of age (z=3.29, with χ2 values of 284.91 and 34.25, respectively, P<0.01) and total burn area (z=4.98, with χ2 values of 326.79 and 29.88, respectively, P<0.01), while the AUC quality and sensitivity and specificity under the optimal threshold values were similar between age and total burn area (P>0.05). Conclusions: D-dimer level is an independent risk factor for DVT of lower extremity in adult burn patients, its AUC quality and sensitivity and specificity under the optimal threshold value are better than those of age and total burn area, and it has good predictive value for DVT of lower extremity in adult burn patients.

Value of cerebral hypoxic-ischemic injury markers in the early diagnosis of sepsis associated encephalopathy in burn patients with sepsis / 中华烧伤杂志

Objective: To explore the value of cerebral hypoxic-ischemic injury markers in the early diagnosis of sepsis associated encephalopathy (SAE) in burn patients with sepsis. Methods: A retrospective case series study was conducted. From October 2018 to May 2021, 41 burn patients with sepsis who were admitted to Zhengzhou First People's Hospital met the inclusion criteria, including 23 males and 18 females, aged 18-65 (35±3) years. According to whether SAE occurred during hospitalization, the patients were divided into SAE group (21 cases) and non-SAE group (20 cases). The gender, age, deep partial-thickness burn area, full-thickness burn area, and acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) scores of patients were compared between the two groups. The serum levels of central nervous system specific protein S100β and neuron specific enolase (NSE) at 12, 24, and 48 h after sepsis diagnosis (hereinafter referred to as after diagnosis), the serum levels of interleukin-6 (IL-6), IL-10, tumor necrosis factor α (TNF-α), Tau protein, adrenocorticotropic hormone (ACTH), and cortisol at 12, 24, 48, 72, 120, and 168 h after diagnosis, and the mean blood flow velocity of middle cerebral artery (VmMCA), pulsatility index, and cerebral blood flow index (CBFi) on 1, 3, and 7 d after diagnosis of patients in the two groups were counted. Data were statistically analyzed with chi-square test, analysis of variance for repeated measurement, independent sample t test, and Bonferroni correction. The independent variables to predict the occurrence of SAE was screened by multi-factor logistic regression analysis. The receiver operating characteristic (ROC) curve was drawn for predicting the occurrence of SAE in burn patients with sepsis, and the area under the curve (AUC), the best threshold, and the sensitivity and specificity under the best threshold were calculated. Results: The gender, age, deep partial-thickness burn area, full-thickness burn area, and APACHE Ⅱ score of patients in the two groups were all similar (χ2=0.02, with t values of 0.71, 1.59, 0.91, and 1.07, respectively, P>0.05). At 12, 24, and 48 h after diagnosis, the serum levels of S100β and NSE of patients in SAE group were all significantly higher than those in non-SAE group (with t values of 37.74, 77.84, 44.16, 22.51, 38.76, and 29.31, respectively, P<0.01). At 12, 24, 48, 72, 120, and 168 h after diagnosis, the serum levels of IL-10, Tau protein, and ACTH of patients in SAE group were all significantly higher than those in non-SAE group (with t values of 10.68, 13.50, 10.59, 8.09, 7.17, 4.71, 5.51, 3.20, 3.61, 3.58, 3.28, 4.21, 5.91, 5.66, 4.98, 4.69, 4.78, and 2.97, respectively, P<0.01). At 12, 24, 48, 72, and 120 h after diagnosis, the serum levels of IL-6 and TNF-α of patients in SAE group were all significantly higher than those in non-SAE group (with t values of 8.56, 7.32, 2.08, 2.53, 3.37, 4.44, 5.36, 5.35, 6.85, and 5.15, respectively, P<0.05 or P<0.01). At 12, 24, and 48 h after diagnosis, the serum level of cortisol of patients in SAE group was significantly higher than that in non-SAE group (with t values of 5.44, 5.46, and 3.55, respectively, P<0.01). On 1 d after diagnosis, the VmMCA and CBFi of patients in SAE group were significantly lower than those in non-SAE group (with t values of 2.94 and 2.67, respectively, P<0.05). On 1, 3, and 7 d after diagnosis, the pulsatile index of patients in SAE group was significantly higher than that in non-SAE group (with t values of 2.56, 3.20, and 3.12, respectively, P<0.05 or P<0.01). Serum IL-6 at 12 h after diagnosis, serum Tau protein at 24 h after diagnosis, serum ACTH at 24 h after diagnosis, and serum cortisol at 24 h after diagnosis were the independent risk factors for SAE complicated in burn patients with sepsis (with odds ratios of 2.42, 1.38, 4.29, and 4.19, 95% confidence interval of 1.76-3.82, 1.06-2.45, 1.37-6.68, and 3.32-8.79, respectively, P<0.01). For 41 burn patients with sepsis, the AUC of ROC of serum IL-6 at 12 h after diagnosis for predicting SAE was 0.92 (95% confidence interval was 0.84-1.00), the best threshold was 157 pg/mL, the sensitivity was 81%, and the specificity was 89%. The AUC of ROC of serum Tau protein at 24 h after diagnosis for predicting SAE was 0.92 (95% confidence interval was 0.82-1.00), the best threshold was 6.4 pg/mL, the sensitivity was 97%, and the specificity was 99%. The AUC of ROC of serum ACTH at 24 h after diagnosis for predicting SAE was 0.96 (95% confidence interval was 0.89-1.00), the best threshold was 14.7 pg/mL, the sensitivity was 90%, and the specificity was 94%. The AUC of ROC of serum cortisol at 24 h after diagnosis for predicting SAE was 0.93 (95% confidence interval was 0.86-1.00), the best threshold was 89 nmol/L, the sensitivity was 94%, and the specificity was 97%. Conclusions: Serum Tau protein, ACTH, and cortisol have high clinical diagnostic value for SAE complicated in burn patients with sepsis.

Construction and application of platelet virtual matching information system / 中国输血杂志

【Objective】 To construct a platelet digital matching information system (PMIS). 【Methods】 The framework of PMIS was designed and the main functions were developed. The information system was connected with the information modules of clinical application, laboratory testing, blood donation service, blood inventory and distribution. Further, the preliminary application of this system will be carried on in clinical. 【Results】 The PMIS had been successfully developed and 5048 blood donors with HLA and HPA genotypes were registered in the system. A total of 306 patients applied for matching and 16.5% of them received compatible platelet reports immediately from the inventory bloods, with the median waiting time of all matching as 3 days, which was significantly shorter than that of the manual method (3.8±3.1 days vs 5.4±5.4 days). 【Conclusion】 The developed PMIS has realized the whole process management of blood donors and patients, which is helpful to improve the platelet matching level.

Analysis of HLA-Ⅰ antibody specificity and estimation of antigen immunogenicity: 96 patients recieved genotype-matched platelet transfusions / 中国输血杂志

【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.

Correlation between the quantitative intensity of HLA-Ⅰ gene and its antibody and the clinical transfusion effect of matching platelets / 中国输血杂志

【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.

Analysis of loss of heterozygosity at HLA loci in a patient with leukemia / 中华医学遗传学杂志

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.

Cerebellar ataxia, neuropathy, vestibular areflexia syndrome with replication factor C subunit 1 gene mutation: a case report and literature review / 中华神经科杂志

Objective:To investigate the clinical and genetic characteristics of cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) with replication factor C subunit 1 (RFC1) gene mutation to improve the understanding of this disease.Methods:A case of CANVAS diagnosed in the Peking University Third Hospital in January 2021 was reported. Detailed genetic analyses of ataxia were performed with DNA extracted from the peripheral blood of the patient. Studies including pathogenic variants of RFC1 gene causing CANVAS were reviewed and the clinical and genetic characteristics of the disease were summarized.Results:The patient was a 51-year-old female with the prominent manifestation of progressive walking instability. And the clinical data met the diagnostic criteria of CANVAS. The genetic tests excluded other hereditary ataxia mutations and identified the biallelic expansion of the pathogenic variant structure (AAGGG)exp repeat amplification in RFC1 gene. A total of 14 studies on CANVAS with RFC1 gene mutation were reviewed. The overall mutation rate of RFC1 gene in CANVAS was 68%-100%, and it varied in sporadic and familial CANVAS. And the mutation had ethnic differences.Conclusions:Among adult patients with late-onset ataxia, the combination of brain magnetic resonance imaging, electrophysiology tests and vestibular function examination is beneficial to the identification of CANVAS. And the genetic test of RFC1 gene has significant value in the diagnosis of this disease. This patient with CANVAS expands the disease spectrum of ataxia in China, and confirms that RFC1 gene mutation is of great significance in the screening of ataxia disorders in the Chinese population.

Study of the distribution of KIR3DL2 alleles among ethnic Han Chinese from Zhejiang / 中华医学遗传学杂志

OBJECTIVE@#To study the distribution of KIR3DL2 alleles among ethnic Han Chinese from Zhejiang.@*METHODS@#Genomic DNA was extracted by using a magnetic bead method. The full sequence of the KIR3DL2 gene was amplified with four pairs by PCR primers. The coding regions of 208 unrelated ethnic Han Chinese blood donors were analyzed using a BigDye Terminator v3.1 Sequencing Kit. The genotypes were assigned based on the nucleotide polymorphism of the KIR3DL2 gene.@*RESULTS@#Among the 208 samples, 133 were KIR3DL2 heterozygotes and 75 were homozygotes. Forty six KIR3DL2 genotypes were detected. Respectively, 70, 33 and 23 individuals were found to have a KIR3DL2*00201/KIR3DL2*00201, KIR3DL2*00201/KIR3DL2*00701, and KIR3DL2*00201/KIR3DL2*01001 genotype. Twenty-two KIR3DL2 alleles were discovered, and the frequencies of KIR3DL2*00201, KIR3DL2*00701 and KIR3DL2*01001 were 57.45%, 13.46% and 9.13%, respectively.@*CONCLUSION@#The distribution of KIR3DL2 alleles among ethnic Han Chinese in Zhejiang has been determined and fits the criteria for genetic polymorphism.

Identification of a glycosyltransferase allele associated with Bw subtype and analysis of the protein structure / 中华医学遗传学杂志

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.

Molecular characterization of a recombination allele of ABO blood group / 中华医学遗传学杂志

OBJECTIVE@#To analyze the molecular characteristics of a recombinant allele of the ABO blood group.@*METHODS@#The ABO phenotype was determined with the tube method. The coding regions of the ABO and FUT1 genes were analyzed by PCR-sequence based typing. The ABO alleles of the proband were determined by allele-specific primer sequencing. The full sequences of the ABO gene of the proband and her mother were determined through next generation sequencing.@*RESULTS@#The red blood cells of the proband did not agglutinate with anti-H, and the sequence of the FUT1 gene was homozygous for c.551_552delAG.The proband was thereby assigned as para-Bombay. Bi-directional sequencing also found that she was heterozygous for c.261G/del,467C>T,c.526C>G,c.657C>T,c.703G>A,c.796C>A,c.803G>C and c.930G>A of the coding regions of the ABO gene. Allele-specific primer sequencing also found her to carry a ABO*A1.02 allele and a recombinant allele from ABO*O.01.01 and ABO*B.01. The recombination site was located between nucleotide c.375-269 and c.526, and the allele was maternally derived.@*CONCLUSION@#An recombinant allele of the ABO gene has been identified, which has originated from recombination of ABO*O.01.01 with the ABO*B.01 allele.

HLAⅠ-matched transfusion for immune-mediated platelet transfusion refractoriness / 中国输血杂志

【Objective】 To establish the HLA-A, -B genotype-matched transfusion strategy for immune-mediated PTR patients based on donor HLA genotyping database, so as to improve the transfusion efficacy. 【Methods】 The serologic cross-match was used to screen immune PTR primarily. 35 PTR patients screened out were subjected to HLA-match. 24 patients were tested for HLA-A, -B genotyping and antibodies against platelet HLA classⅠ, and then received a total of 83 HLA-typed platelet transfusions, based on patient platelet genotype, donor specific antibody (DSA)(priority), and HLA-A, -B cross-reactive groups (CREGs) principle(lower priority). The other 11 patients received a total of 55 HLA-A/B-matched transfusions according to CREGs principle. The clinical information and transfusion outcome were followed up, and the corrected count increment (CCI) was calculated and statistically analyzed. 【Results】 A total of 453 ABO serological cross-matching tests were performed for 35 PTR patients, with 12.94 tests (453/35) per patient, an average of 4.21 (1908/453) donors per test and positive rate of 69.86% (1333/1908). 23 out 24(95.83%) patients, subjected to HLA class I antibody, were positive and each carried (44.37 ± 22.31) kinds of specific antibodies. According to the fluorescence intensity of the antibody in the patient′s serum, the antibody was strongly positive in 17(73.91%) cases, positive 20(86.96%) and weakly positive 23(100%). After 138 HLA-matched transfusions, the first mean CCI value was 14.08 ± 11.12 (23.95 ± 21.28 h), which was significant higher than 1 hour CCI (>7.5 effective) or 24 hours CCI (> 4.5 effective). The responses of DSA avoidance group (CCI of 1st =15.56±11.00)was significant higher than that of non-DSA avoidance group(CCI of 1st =11.86±12.00)(t=2.045, P<0.05). 49.28% of the patients had one or more non-immune factors during platelet transfusion. 【Conclusion】 The HLA-matched platelet transfusion is feasible to prevent and improve immune-mediated PTR. For patients with multiple blood transfusions and positive platelet antibodies, DSA avoidance and CREGs principle combined transfusion strategy can significantly improve the efficacy of blood transfusion and provide accurate platelet transfusion for the clinical.

Detection of anti-HLA and anti-MICA in convalescent plasma from individuals recovered from COVID-19 / 中国输血杂志

【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.

Effect of two kinds of surface pretreatments on bending strength to the modified polyetheretherketone / 华西口腔医学杂志

OBJECTIVE@#To evaluate the effect of sandblasting or acid etching on the three-point bending strength to the modified polyetheretherketone (PEEK).@*METHODS@#Forty-eight bars (15 mm×2 mm×1 mm) of specimens were fabricated from the modified PEEK (BioHPP). They were randomly divided into the following groups: A, B, C and D groups, which were blasted with alumina particles; E, F, and G groups, which were etched with 98% concentrated sulfuric acid; and control group H. The sand blast pressure of groups A, B and C was 0.2 MPa, and the grain sizes of the sand blasted were 120, 50, and 250 µm, respectively. Group D was blasted with 120 µm particle size at 0.7 MPa pressure. Groups E, F and G were acid etched for 60, 120, and 300 s, respectively. No surface treatment was conducted in group H. After all the specimens were processed, one sample was randomly selected from each group to observe its surface morphology under a scanning electron microscope (SEM), and the other specimens were tested for their three-point bending strength. SPSS 22.0 software was used to analyze the experimental data and to test whether the difference was statistically significant.@*RESULTS@#SEM observation showed that the surface morphology of the specimen changed after the treatment and revealed different degrees of cracks, pits, or voids. The three-point bending test indicated that the strength of the specimens treated with sandblasting or concentrated sulfuric acid decreased compared with that of the control group (P0.05). The strength of group D was lower than that of group A at the same particle size (P0.05).@*CONCLUSIONS@#The bending strength of BioHPP could be significantly decreased by surface sand blasting or concentrated sulfate etching as the sandblasting pressure increased, but the bending strength did not decrease as sand particle size and acid etching time changed.

Key technologies in digital breast tomosynthesis system:theory, design, and optimization / 南方医科大学学报

OBJECTIVE@#To develop a digital breast tomosynthesis (DBT) imaging system with optimizes imaging chain.@*METHODS@#Based on 3D tomography and DBT imaging scanning, we analyzed the methods for projection data correction, geometric correction, projection enhancement, filter modulation, and image reconstruction, and established a hardware testing platform. In the experiment, the standard ACR phantom and high-resolution phantom were used to evaluate the system stability and noise level. The patient projection data of commercial equipment was used to test the effect of the imaging algorithm.@*RESULTS@#In the high-resolution phantom study, the line pairs were clear without confusing artifacts in the images reconstructed with the geometric correction parameters. In ACR phantom study, the calcified foci, cysts, and fibrous structures were more clearly defined in the reconstructed images after filtering and modulation. The patient data study showed a high contrast between tissues, and the lesions were more clearly displayed in the reconstructed image.@*CONCLUSIONS@#This DBT imaging system can be used for mammary tomography with an image quality comparable to that of commercial DBT systems to facilitate imaging diagnosis of breast diseases.

Improved survival of heterotopic heart transplant overexpressing IDO rat bone mesenchymal stem cells / 中国免疫学杂志

Objective:To explores the improvement in survival mechanism of a rat model of enterocoelia heterotopic heart transplant with rat bone marrow mesenchymal stem cells( BMSCs) IDO-overexpressing. Methods:IDO-overexpressing rat BMSCs were produced through transfection of rat BMSCs with IDO gene carried by the lentiviral vector GV308. A rat model of enterocoelia heterotopic heart transplantation was established. This rat model received a cell treatment via its tail veins, as follows: ①Echocardiography was employed to detect the functional changes in the transplanted heart.②The fluorescence intensity of the different parts of the transplanted heart was evaluated using a body imaging system for small living animals.③Receptors rat spleens cells were obtained and used for a flow cytometric detection of the expression levels of CD40,CD86,CD80,MHCⅡ,CD274,CD45RA,CD45RA+CD45RB,and Treg cells. ④A transplanted heart was obtained after injection to evaluate inflammatory cell infiltration through HE staining.⑤Liquid phase chips were used to detect changes in the serum factors IL-1ɑ,IL-4,IL-1β,IL-2,IL-10,IFN-γ,IL-18,TGFβ1, TGFβ2 and TGFβ3 in after injection cells. Results:①After the rat heterotopic heart transplantation model and the corresponding cell treatment were established,after over-expressed IDO-BMSCs treatment 2 days the EF and FS were higher in the transplanted heart than other groups.②The fluorescence intensity of the parts of the transplanted heart was highest in the IDO-BMSC overexpression group as revealed by small animal living body evaluation. ③Two days after the interventions, spleen cells in the over-expressed IDO-BMSCs group showed reduced expression levels of CD40,CD86,CD80,MHCⅡ,CD45RA,CD45RA+CD45RB and increased expression levels of CD274 and Treg cells as revealed by flow cytometry.④Liquid phase chips were used to examine the serum obtained from each group 2 days after the intervention,and the results showed that the expression levels of IL-1α,IL-4,IL-1β,IL-2,IFN-γand IL-18 in the IDO-BMSC overexpression group decrease. By contrast,the expression levels of IL-10,TGFβ1,TGFβ2 and TGFβ3 increase. HE staining results demonstrate that inflammatory cell infiltration was lower in IDO-BMSC overexpression group than in other groups. Conclusion:IDO-overexpressing BMSCs improve the survival of a transplanted heart through effective adjustment of immune DC and T cells,as well as cell factors.

Application of recombinant GPⅢa combined Luminex beads for the detection of HPA-1a antibody / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To generate recombinant GPⅢa as an alternative source for HPA-1a antigen and combine it with Luminex xMAP beads for the detection of HPA-1a-specific alloantibody.</p><p><b>METHODS</b>The full coding region of ITGB3 gene was amplified and ligated with pcDNA3.1. The recombinant plasmid was transfected into CHO cells, and those with stable expression were screened with G418. Expressed protein was identified and coupled with Luminex xMAP beads, which were then reacted with sera samples. Subsequently, phycoerythrin-labeled anti-species IgG antibody was added to the reaction wells and the median fluorescence was determined on a Luminex-100 analyzer.</p><p><b>RESULTS</b>DNA sequencing confirmed that the cloned ITGB3 gene was HPA-1aa. The recombinant GPⅢa was coupled with Luminex xMAP beads. The sensitivity of Luminex beads assay to detect HPA-1a antibody was dilution 1/32 (3.125 U/mL). The Luminex beads assay could specifically identify the HPA-1a antibody from the test sera, and the results were consistent with that of monoclonal antibody-specific immobilization of platelet antigens (MAIPA) technology. Cross-reactivity was not observed with the samples containing HLA, ABO and other HPA antibodies (HPA-3a and HPA-5b). The results illustrated that to detect HPA antibody with Luminex xMAP beads technology is feasible.</p><p><b>CONCLUSION</b>Recombinant GPⅢa was successfully obtained and used to establish a Luminex technology-based method for the detection of HPA antibodies.</p>

Study of the molecular basis for an individual with Bel variant due to deletion of B glycosyltransferase gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the molecular basis of an individual with Bel variant of the ABO blood group.</p><p><b>METHODS</b>The ABO antigen and serum antibody of the individual were detected by serological method. All coding regions and flanking introns of the ABO gene were amplified with PCR and sequenced bidirectionally. The haplotypes of the individual were analyzed by cloning and sequencing. A three dimensional model of the mutant protein was constructed and analyzed.</p><p><b>RESULTS</b>The individual has expressed a very weak B antigen on its red blood cells by absorption and elution testing, which was identified as a Bel variant phenotype. The heterozygous sites in exon 6 (261del/G) and exon 7 (297A/G, 484del/G, 526C/G, 657C/T, 703G/A, 796C/A, 803G/C, 930G/A) of the coding region of the ABO gene were identified by direct sequencing. Haplotype analysis showed that the individual has carried an O01 allele and a novel B allele. The sequence of the novel B allele was identical to B101 except for a del G at nucleotide position 484 (484delG), which was nominated as B120 by the Blood Group Antigen Gene Mutation Database (dbRBC NCBI). The 484delG mutation of the B allele has led to a reading frame shift and created a premature terminal codon for the glycosyltransferase (GT) enzyme. Prediction of the 3D structure suggested that the GT enzyme has become an incomplete protein only with its N-terminal region.</p><p><b>CONCLUSION</b>The 484delG mutation of the glycosyltransferase B gene has probably abolished or reduced the enzymatic activity and resulted in the Bel variant phenotype.</p>

Influence of projection data correction on digital breast tomosynthesis imaging / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of detector performance during digital breast tomography (DBT) projection data acquisition on reconstructed image quality.</p><p><b>METHODS</b>With reference to the traditional detector data correction method and the specific data acquisition pattern in DBT imaging, we utilized dark field correction, light field and its gain correction for processing the projection data collected by the detector. The reconstructed images were evaluated using iterative reconstruction method based on total generalized variation (TGV).</p><p><b>RESULTS</b>In physical breast phantom experiment, the proposed method resulted in a reduced Heel effect caused by nonuniform photon number. The reconstructed DBT images after correction showed obviously improved image quality especially in the details with a low contrast.</p><p><b>CONCLUSION</b>The dark field correction, light field and its gain correction process for DBT image reconstruction can improve the image quality.</p>