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Objective To elucidate the prediction ability of monocyte monolayer assay(MMA) used in hemolytic disease of fetus and newborn(HDFN) caused by IgG anti-M. Methods Plasma from eight pregnant women containing IgG anti-M were collected, and were divided into two groups(4 cases with HDFN, with severe clinical symptoms such as fetal hydrops, and 4 cases without HDFN) according to the clinical outcomes. M antigen positive cells were sensitized with dithiothreitol(DTT) treated plasma from eight pregnant women respectively. MMA was performed by coincubation with monocytes and sensitized M cells, along with negative and positive control set up. T-test was conducted to compare the difference in phagocytic efficiency between two groups. Results The phagocytic efficiency in group with HDFN were 15.37%, 13.05%, 9.17% and 24.50% respectively, with the mean value of 15.52%, while the group without HDFN were 8.74%, 11.07%, 5.12% and 6.23% respectively, with the mean value of 7.79%.There was no significant difference in phagocytic efficiency between two groups(P>0.05). The mean values of both groups were not significantly different from the negative control(P>0.05), but both were significantly lower than positive control(P

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-Dib (titer: 512) related HDN was detected in the newborn. IgG1 and IgG2 subtypes of anti-Dib 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-Dib, 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.

ObjectiveTo establish a digital PCR system for accurate and sensitive quantitative detection of Helicobacter pylori (Hp). MethodsSpecific primer and probe were designed based on the Hp 16S rRNA sequence, and reference bacteria was used to conduct specificity testing. The sensitivity was detected using gradient diluted DNA as templates. The accuracy was detected using different concentration templates with multiple repeated tests. The gradient method was used to set the primer concentration and annealing temperature for optimizing reaction conditions.ResultsThe designed primers and probes could detect Hp 16S rRNA genes specifically without the interference from Escherichia coli and other bacteria. The optimal reaction temperature for the detection was 57.1 ℃, and the optimal primer concentration was 550 nmol/L. The detection limit was 0.35 copies/μL, the coefficient of variation (CV) of the detection was < 10%, and the coefficient of determination for the linear regression between dilution ratio and DNA concentration was 0.9968. ConclusionThe established digital PCR system is specific, sensitive, and highly accurate for Hp detection.

【Objective】This study aimed to investigate and analyze the correlation between the quality of fruit-bearing shoots in 'Xianjinfeng' litchi and fruit setting and fruit quality. Tthe effects of different quality fruit-bearing shoots on fruit setting and quality were compared. The quantitative standard of high quality fruit branches was put forward to provide reference for the development of strong fruit branches in production.【Method】During the key phenological period, the quality indicators of fruit-bearing shoots in 'Xianjinfeng' litchi, including length, diameter, and number of leaves, were measured and statistically analyzed. Number of initial fruit setting and final fruit setting with fruit quality were also assessed. Violin plots, Pearson correlation coefficients, and one-way ANOVA were utilized to analyze the distribution, correlation, and significance of the data, as well as to compare the differences in fruit setting and quality among fruit-bearing shoots of different qualities.【Result】The length, diameter, and number of leaves of fruit-bearing shoots in 'Xianjinfeng' litchi were concentrated within the ranges of 10.2-14.5 cm, 0.4-0.5 cm, and 28-38 leaves, respectively. The number of initial fruit setting exhibited a significant positive correlation with the diameter of the mother branches, while the final fruit setting rate showed a significant positive correlation with both length and diameter. The correlation between the length, diameter, and number of leaves of fruit-bearing shoots and the same fruit quality traits was generally consistent, either positively or negatively correlated. Notably, single fruit weight demonstrated a significant positive correlation with diameter, while TSS (Total Soluble Solids) and TSS/TA (Total Soluble Solids/Titratable Acidity) showed a significant positive correlation with the number of leaves. Fruit-bearing shoots with a length greater than 14.5 cm exhibited the highest initial fruit setting number (16.95 fruits) and final fruit setting rate (20.73%). Fruit-bearing shoots with a diameter greater than 0.5 cm exhibited the highest initial fruit setting number (15.48 fruits), final fruit setting rate (28.03%), single fruit weight (26.28 g), and edible rate (70.95%). Fruit-bearing shoots with more than 38 leaves exhibited the highest TSS (18.36%) and TSS/TA (250.81).【Conclusion】Based on the findings, a robust standard for fruit-bearing shoots in 'Xianjinfeng' litchi was determined as having a length greater than 14.5 cm, diameter greater than 0.5 cm, and number of leaves greater than 38.

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 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).

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. Dib-positive red blood cells (RBCs) were labeled with PKH26 and then sensitized with IgG anti-Dib. 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.

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-Jra in Jr(a-) mother was stimulated by mother-child serology incompatibility during pregnancy.