Objective: To investigate the impact of RHAG 808A variant, commonly identified in the Chinese population, on RhAG protein, RhD and RhCE antigens expression through in vivo and in vitro expression analysis. Methods: A missense mutation of RHAG gene (c. 808G>A, p. Val270Ile) with high frequency was found in KMxD database. Bioinformatics analysis was performed using Polyphen-2 and Provean software. High resolution melting (HRM) method was utilized to screen for the variant carriers in the blood donors. The expression of RhAG protein, RhD and RhCE antigens on the surface of red cells of variant carriers were detected via flow cytometry. Wild-type and mutant vectors of RHAG were constructed and transfected into HEK 293T cells for in vitro expression analysis. Then, the expression of RhAG protein, RhD and RhCE antigens were analyzed by flow cytometry. Results: Polyphen-2 and Provean software suggested that the amino acid change (p. Val270Ile) of RhAG protein may be harmful or neutral respectively. Among the 999 blood donors from Guangzhou Blood Center, 4 homozygous carriers and 99 heterozygous carriers of RHAG 808A mutant allele were identified. The frequency of this allele was 5.4% (107/1 998). No significant differences in RhAG protein, RhD and RhCE antigens expression level was identified between the homozygous carriers, heterozygous carriers of RHAG 808A variant allele and the wild-type individuals. In vitro analysis for antigen expression study obtained the similar results. Conclusion: The RHAG 808A variant allele commonly identified in the Chinese population has no effect on the expression of RhAG protein, RhD and RhCE antigens, so the variant should be a population polymorphism site.

【Objective】 To identify the specificity of alloantibody against high-frequency antigens in one case suffering with severe hemolytic diseases of the fetus and newborn (HDFN) and to screen for matching blood for transfusion. 【Methods】 The HDFN test and the antibody serological identification tests in the mother were performed. Several common high frequency antigens of maternal red blood cells (RBCs) were determined. IgG subtype coated on the RBCs of the newborn was determined. The phagocytic efficiency of the antibody was tested using the monocyte phagocytosis of sensitized erythrocyte by flow cytometry in vitro. Sanger sequencing of DI gene was performed in the mother, father and mother’s brother. The diluted maternal plasma was used for large scale screening of matching blood using IAT in Coomb’s gel card. 【Results】 Di(b-) phenotype was identified in the mother of the newborn and anti-Di^b (titer: 512) related HDN was detected in the newborn. IgG1 and IgG2 subtypes of anti-Di^b were detected and the rate of monocyte phagocytosis was 88.83%(74.7/84.09). The compatible blood was not detected in the maternal relatives. Subsequently, the newborn received the matching RBCs of two Di(b-) donors identified from 5 520 blood donors and discharged from the hospital. We screened out 17 Di(b-) donors out of 51 334 blood donors, indicating that the distribution frequency of Di(b-) among blood donors in Guangzhou was about 0.033% (17/51 334). 【Conclusion】 By serology and molecular biology methods, the newborn was identified with HDFN caused by anti-Di^b, and an effective large-scale screening method for Di (b -) rare blood types was established to find matching blood, which supported the establishment of rare Di(b-) blood database.

Objective:To explore the prevalence and independent associated factors of vascular calcification (VC) in non-dialysis chronic kidney disease (CKD) patients of stage 3-5.Methods:It was a single-center cross-sectional observational study. Non-dialysis stage 3-5 CKD patients ≥18 years old who were admitted to the Department of Nephrology, the First Affiliated Hospital of Sun Yat-sen University from May 1, 2022 to December 31, 2022 with VC evaluation were enrolled. The patients' general information, laboratory examination and imaging data were collected. Coronary artery calcification (CAC), thoracic aorta calcification (TAC), abdominal aorta calcification (AAC), carotid artery calcification and aortic valve calcification (AVC) were evaluated by cardiac-gated electron-beam CT (EBCT) scans, lateral lumbar x-ray, cervical macrovascular ultrasound and echocardiography, respectively. The differences in clinical data and the prevalence of VC at different sites of patients with different CKD stages were compared, and the prevalence of VC at different sites of patients in different age groups [youth group (18-44 years old), middle-aged group (45-64 years old) and elderly group (≥65 years old)] and patients with or without diabetes were compared. Multivariate logistic regression analysis was used to analyse the independent associated factors of VC for different areas.Results:A total of 206 patients aged (51±14) years were included, including 129 (62.6%) males. There were 44 patients with CKD stage 3 (21.4%), 51 patients with CKD stage 4 (24.8%), and 111 patients with CKD stage 5 (53.9%). CKD was caused by chronic glomerulonephritis [104 cases (50.5%)], diabetic kidney damage [35 cases (17.0%)], hypertensive kidney damage [29 cases (14.1%)] and others [38 cases (18.4%)]. Among 206 patients, 131 (63.6%) exhibited cardiovascular calcification, and the prevalence of CAC, TAC, AAC, carotid artery calcification, and AVC was 37.9%, 43.7%, 37.9%, 35.9% and 9.7%, respectively. The overall prevalence of VC in young, middle-aged and elderly patients was 24.6%, 73.6% and 97.4%, respectively. With the increase of age, the prevalence of VC in each site gradually increased, and the increasing trend was statistically significant (all P<0.001). The overall prevalence of VC in CKD patients with diabetes was 92.5% (62/67), and the prevalence of VC at each site in the patients with diabetes was significantly higher than that in the patients without diabetes (all P<0.001). Multivariate logistic regression analysis revealed that age (every 10 years increase, OR=2.51, 95% CI 1.77-3.56, P<0.001), hypertension ( OR=5.88, 95% CI 1.57-22.10, P=0.009), and diabetes ( OR=4.66, 95% CI 2.10-10.35, P<0.001) were independently correlated with CAC; Age (every 10 years increase, OR=6.43, 95% CI 3.64-11.36, P<0.001) and hypertension ( OR=6.09, 95% CI 1.33-27.84, P=0.020) were independently correlated with TAC; Female ( OR=0.23, 95% CI 0.07-0.72, P=0.011), age (every 10 years increase, OR=3.90, 95% CI 2.42-6.29, P<0.001), diabetes ( OR=5.37, 95% CI 2.19-13.19, P<0.001) and serum magnesium ( OR=0.01,95% CI 0-0.35, P=0.014) were independently correlated with AAC. Moreover, age and diabetes were independently correlated with carotid artery calcification, AVC and overall VC Conclusions:The prevalence of VC in non-dialysis CKD patients of stage 3-5 is 63.59%, of which CAC reaches 37.9%, TAC is the most common one (43.7%), while AVC is the least one (9.7%). Age and diabetes are the independent associated factors for VC of all sites except TAC, while hypertension is an independent associated factor for both CAC and TAC.

【Objective】 To solve the difficulty of RhD blood group typing in a patient with double population(DP) of red blood cells for RhD antigen by serological and genotyping analysis. 【Methods】 Separation of the two populations of red blood cells of the patient was performed using capillary centrifugation method. ABO, RhD and RhCE typing, direct anti-human globulin test (DAT), irregular antibody screening, antibody identification and blood crossmatching of the patient were conducted using the standard serological methods. The hybrid Rhesus zygosity analysis of the RHD gene was performed by PCR-RFLP method. RHD and RHCE genotype of the patients were identified by PCR-SSP method. 【Results】 The patient was B type but with DP of red blood cells for RhD, Rhc and RhE antigens. DAT of the patient was positive and the alloanti-D was detected in serum. The RHD zygosity was D-/D- homozygote. PCR-SSP testing showed the RHD gene deletion (RHD * 01N. 01/01N.01 genotype) and Ccee of RHCE genotype in the patient, which was consistent with RHD zygosity analysis. 【Conclusion】 This is a special case with D-negative phenotype which was wrongly detected as D-positive type after D-positive red blood cells transfusion in emergency. When the DP of red cells for D antigen encountered like this case, the RhD typing can be accurately determined by using RHD genotyping analysis to provide strong evidence to the clinical blood transfusion.

Objective:To explore the clinical and histopathologic features of lupus nephritis (LN) patients with positive antineutrophil cytoplasmic antibody (ANCA), so as to provide more theoretical basis to recognize and treat this disease.Methods:Clinical data of biopsy-proven LN patients with ANCA test in the First Affiliated Hospital of Sun Yat-sen University from November 1, 2002 to September 11, 2020 were collected and analyzed retrospectively. The difference of clinical data, laboratory examination, and pathological examination of renal biopsy between ANCA-positive group and ANCA-negative group. The clinicopathological characteristics between different ANCA serotypes were compared.Results:A total of 1 304 patients with LN confirmed by renal biopsy and ANCA test results were enrolled. Eighty ANCA-positive patients from 1 304 LN patients were screened. There are 55(68.8%) ANCA-positive LN patients with positive anti-myeloperoxidase antibodies (MPO). There were 14(17.5%) ANCA-positive LN patients with positive anti-proteinase 3 antibodies (PR3), and 11(13.8%) ANCA-positive patients with double positive antibodies of MPO and PR3. ANCA-positive LN patients had significantly higher serum creatinine [135.5(68.0, 361.8) μmol/L vs 88.0(64.0, 165.0) μmol/L, P=0.004] and blood urea nitrogen [12.35(6.35, 21.18) mmol/L vs 8.60 (5.50, 15.70) mmol/L, P=0.026] as well as lower estimated glomerular filtration rate [45.70(13.83, 84.10) ml·min -1·(1.73 m 2) -1 vs 66.75(38.43, 96.22) ml·min -1·(1.73 m 2) -1, P=0.001] than ANCA-negative patients (stratified sampling of 160 patients). ANCA-positive LN patients had higher chronicity index than ANCA-negative LN patients [3(2, 7) vs 2(0, 5), P=0.006]. There were statistically significant difference in hemoglobin, serum creatinine and estimated glomerular filtration rate among ANCA-positive group, ANCA-negative group, and MPO-ANCA and PR3-ANCA double positive group. MPO-ANCA and PR3-ANCA double positive LN patients had the lowest hemoglobin and estimated glomerular filtration rate, and highest serum creatinine among the three groups (all P<0.05). Conclusions:ANCA-positive LN patients have worse renal function and higher renal histological chronicity index than ANCA-negative LN patients, especially for patients with double positive MPO-ANCA and PR3-ANCA. More stringent monitoring and therapy may be needed in this subgroup of LN patients.

【Objective】 To identify the antibody specificity in a pregnant women who had no history of blood transfusion but presented the antibodies against high-frequency antigens. 【Methods】 ABO, RhD blood group antigens were identified by saline. Antibody screening and identification were performed by saline and indirect Coomb’s technique. Further antibody identification tests were conducted using papain, trypsin and chymotrypsin-treated cells. Antibody titer in serum was tested. PCR amplification and sequencing analysis of 16 exons of ABCG2 gene were conducted. 【Results】 The blood type of the patient were B, RhD positive. The serum reacted with antibody screening/identified cells by indirect antiglobin test(both 2+ ) but not by saline. The agglutination was enhanced after papain treatment (4+ ), but remained unchanged after trypsin and chymotrypsin treatment (2+ ). The IgG titer was 1∶2. The sequencing analysis of ABCG2 gene revealed a homozygous nonsense mutation(c.376C>T, p. Gln126X) in exon 4 of the women. 【Conclusion】 In this case, the development of anti-Jr^a in Jr(a-) mother was stimulated by mother-child serology incompatibility during pregnancy.

【Objective】 To establish an experimental method for detecting phagocytosis of sensitized red blood cells in vitro by flow cytometry. 【Methods】 Mononuclear cells were isolated from the peripheral blood of blood donors and cultured in a cell incubator for 1 hour, and then adherent monocytes were isolated and obtained. Di^b-positive red blood cells (RBCs) were labeled with PKH26 and then sensitized with IgG anti-Di^b. The sensitized RBCs were added to monocytes for in vitro phagocytosis assay. Monocytes were labeled with FITC anti-human CD14, then phagocytosis was measured by flow cytometry, and the phagocytic efficiency was calculated. The method was used to detect the phagocytic efficiency of monocytes on human IgG anti-D sensitized RBCs with different titers. 【Results】 The phagocytic efficiency of monocytes was averaged at 5% (1.2%~7.6%, SD 3.30) versus 81% (71.4%~92.7%, SD 8.65) in the negative versus positive control group, respectively. Phagocytic activity of monocytes mediated by anti-D was correlated with the antibody titer. The phagocytosis efficiency was within 10% when the antibody titer was lower than 32 and increased sharply when the titer was between 32 to 128, it entered a plateau and stabilized at 80% at the titer above 256. 【Conclusion】 A detection platform for detecting phagocytosis-sensitized RBCs in vitro by flow cytometry has been successfully established. It can be used to assess the clinical significance of red blood cell allotype or autologous IgG antibodies.

Background: Resistance to antibiotics is the major cause for failure of Helicobacter pylori (Hp) eradication therapy. Therefore, exploring new eradication regimen has become a hotspot of research. Aims: To investigate the efficacy, safety and optimal dose of antofloxacin-based bismuth quadruple therapy for first-line Hp eradication. Methods: Four hundred patients with Hp infection and naive to eradication therapy were prospectively recruited from January 2019 to December 2019 at the 900th Hospital of Joint Logistics Support Force, PLA and were randomly divided into four groups: low-, normal-, and high-dose antofloxacin groups and control group, 100 cases in each group. Patients in low-, normal-, and high-dose antofloxacin groups received antofloxacin 100 mg, 200 mg, and 300 mg qd, respectively, pantoprazole 40 mg bid, bismuth potassium citrate 220 mg bid, and amoxicillin 1 000 mg bid for 14 days; patients in control group received levofloxacin 500 mg qd and the other three drugs with same dose and frequency for 14 days. Adverse events during treatment were recorded. Hp eradication was confirmed by

Objective:To explore the relationship between body mass index (BMI) and clinicopathological features and prognosis of gallbladder cancer.Methods:The clinical and follow-up data of three hundred and eighty-six patients of gallbladder carcinoma were retrospectively, who were treated from January 2008 to December 2013 in the Department of Hepatobiliary Surgery, Eastern Hepatobiliary Hospital, Second Military Medical University. According to the guidelines for prevention and control of overweight and obesity in Chinese adults, the patients were divided into three groups: normal weight group(BMI<23.5 kg/m 2, 239 cases, accounting for 61.9%), overweight group (23.5 kg/m 2≤BMI<27.5 kg/m 2, 127 cases, accounting for 32.9%) and obesity group(BMI≥27.5 kg/m 2, 20 cases, accounting for 5.18%). The clinicopathological factors(gender, age, diabetes mellitus, hypertension, gallbladder related diseases, jaundice, tumor location, TMN, postoperative days, tissue differentiation, liver invasion, intraoperative blood transfusion, complications) of the three groups were compared, and the relationship between BMI and 5-year survival rate was analyzed. Measurement data with normal distribution were indicated as mean±standard deviation( Mean± SD), measurement data with skewed distribution were represented as M( P25, P75). Nonparametric rank sum test was used for measurement data. Categorical variables were compared by the chi-square test or Fisher probability method. The survival curve was drawn by the Kaplan-Meier method. The univariate analysis and multivariate analysis of prognosis were respectively done using the Log-rank test and COX regression model. Results:The median survival time of 386 patients with gallbladder cancer was 12.1 months. The overall survival rates of 1, 3 and 5 years were 51.8%, 25.2% and 16.8%, respectively. Univariate survival analysis showed that age, jaundice, accidental gallbladder cancer, tumor location, TMN, surgical method, tissue differentiation, liver invasion, intraoperative blood transfusion, and complications affected the 5-year survival rate ( χ2=12.24, 30.87, 37.01, 7.92, 104.23, 118.76, 12.05, 49.12, 6.85, 12.24, P<0.05). BMI was related to hypertension, but it had no significant effect on the 5-year survival rate. However, with the increase of BMI, the 5-year survival rate increased (16.3% vs 16.7% vs 23.3%, P=0.774). Multivariate survival analysis showed that surgical method( OR=1.441, 95% CI: 1.219-1.705), liver invasion( OR=1.625, 95% CI: 1.264-2.091), M stage( OR=1.664, 95% CI: 1.070-2.587), and N stage( OR=1.511, 95% CI: 1.218-1.875) were independent risk factors for prognosis in this group of patients ( P<0.05), and BMI was not an independent risk factor ( P=0.901). Conclusions:BMI has no significant effect on the prognosis of patients with gallbladder cancer. Obese patients with gallbladder cancer do not need to wait for weight loss before surgery.

Objective To compare the tumor characteristics and survival between postoperative incidentally discovered gallbladder cancer (ID-GBC) and preoperatively suspected gallbladder cancer (PS-GBC).Methods The data of 276 GBC patients who underwent surgical resection with curative intent between January 2004 and December 2014 at the Eastern Hepatobiliary Surgery Hospital were retrospectively analyzed.Results The 1-,3-,and 5-year cumulative survival rates of the ID-GBC group (88.8％,52.2％,and 33.0％,respectively) were significantly better than those in the PS-GBC group (57.5％,25.7％,and 16.6％,P ＜ 0.05).In the ID-GBC group,multivariate analysis revealed that T staging,hepatic invasion and time interval from cholecystectomy to re-operation were independent prognostic factors.The overall survival (OS) in the group with the time interval within 2 weeks was significantly better than those in the other two groups (both P ＜ 0.05).However,there were no significant differences in OS between the groups with the time interval of 2 weeks to 1 month and more than 1 month (P ＞ 0.05).Conclusions Postoperative ID-GBC had significantly better survival outcomes than PS-GBC.Reoperation within two weeks in patients with ID-GBC is a good strategy.