【Objective】 To explore the effectiveness of using donor plasma reinfusion to flush the leukoreduction system (LRS) chamber during the final reinfusion phase with the Trima Accel automated blood collection system in preventing the reduction of CD4+ and CD8+ T lymphocytes. 【Methods】 A longitudinal and cross-sectional study was designed. CD4+ count<200 cells/μL and CD8+ count<125 cells/μL were considered as the criteria for deficiency. Eighteen first-time platelet donors were followed up. The lymphocyte count was measured at 0, 3-6 and 7-14 times of blood donation in the last 300 days. 170 healthy blood donors who have not donated blood were selected as the control group. According to the cut-off point(October 2021), 88 blood donors who mainly used automatic blood collection system to donate platelet apheresis in the last 365 days(median blood donation times ≥17.5)were divided into three groups(A, B and C)and blood samples were obtained. The time for Groups A, B and C started donating platelet apheresis were as follows: Group A: before October 2019, Group B: from October 2019 to September 2021, Group C: after October 2021. Blood samples were analyzed to obtain blood counts including CD4 + and CD8 + T lymphocytes. Blood samples were analyzed to obtain blood cell counts including CD4+ and CD8+ T lymphocytes. Through a comparative analysis, this study aimed to determine if there are any statistical differences in the detection indices between the follow-up groups with varying frequencies of blood donation, the control group, and groups A, B, and C. This approach was employed to infer the efficacy of donor plasma reinfusion in flushing the leukoreduction system (LRS) chamber for preventing the decline of CD4+ and CD8+ T lymphocytes. 【Results】 Eighteen first-time blood donors who were converted to regular platelet apheresis donors did not show a decrease of CD4 + and CD8 + T lymphocytes in the 5 th and 11 th blood donation (median number of blood donation), and there was no significant difference between the above indexes and those in the 0 th blood donation. Among the previous frequent blood donors, the CD4+ and CD8+ T lymphocyte counts in Group B and Group C are both higher than the standard value, showing no statistical difference from the control group. Among regular blood donors, the CD4+ and CD8+ T lymphocyte counts in groups B and C were higher than the criteria values, and had no statistical difference compared to the control group.The CD4+ T lymphocyte count in Group A was normal, with only one donor in Group A having a CD8+ T lymphocyte count below 125 cells/μL. This donor has donated 281 times of platelet apheresis, and the group he belongs to has started blood donation 2-21 years(median of 5 years) before the adjustment of reinfusion mode. The CD4+ and CD8+ T lymphocyte counts in Group A showed significant differences compared to the control group, with median counts (Group A/Control Group) of 359/521 and 257/372, respectively, P<0.001. In Group A, 0%(0/35) had a CD4+ count below 200 cells/μL, and 2.85%(1/35) of donors had a CD8+ count below 125 cells/μL, which was far lower than the proportion of CD4+ and CD8+ T cell deficiency found in regular apheresis donors by John M. Gansner and Mahboubeh Rahmani. The study showed that the adjustment of the plasma reinfusion mode did not further reduce the T lymphocyte counts in blood donors, but instead further restored the T lymphocyte counts in regular blood donors. This indicated that after the adjustment of plasma reinfusion mode, blood donors might not have lost CD4+ and CD8+ T lymphocytes during blood donation, or only lost a small amount, and can recover even if they donate platelet apheresis frequently. 【Conclusion】 Trima Accel automated blood collection system has a good effect on preventing CD4 + and CD8 + T lymphocytes from being reduced by flushing the LRS chamber with donor plasma.

【Objective】 To detect the anti-SARS-CoV-2 antibody levels in blood donors in Guangzhou, so as to provide laboratory data support for the collection and clinical use of convalescent plasma. 【Methods】 Anti-SARS-CoV-2 antibodies were measured by ELISA in qualified donors. Among them, 326 donors who gave blood in February 2023 were tested for IgG antibodies, 444 donors were tested for neutralizing antibodies. In July 2023, 398 donors were tested for IgG and IgM. 【Results】 399 of 724 blood samples diluted with normal saline (1∶160) were IgG reactive, with a reactive rate of 55.11%. Chi-square test showed that there was a significant difference in the reactive rate of IgG among samples collected at different times (25.46% in February vs 79.40% in July, χ²=210.74, P<0.01, 95%CI: 7.97, 15.98), but there was no significant difference in the reactive rate between different genders and different age groups. IgM was detected in 5 of 398 blood samples, with a reactive rate of 1.26%. The IgG test results of these five blood donors were all reactive, whereas the nucleic acid test results were negative. Neutralizing antibody was detected in 440 of 444 blood samples, with a reactive rate of 99.10%, and 71.59% of the reactive donors had a neutralizing antibody level of 10 μg/mL or more. 【Conclusion】 Blood donors in Guangzhou have a high level of SARS-CoV-2 antibody, which is sufficient to provide convalescent plasma for clinical treatment.

【Objective】 HLA-DRB1 * 11:01, as a class HLA-Ⅱ gene, was reported to be associated with spontaneous clearance of HCV in Han and Li population. Our study was to investigate the effects of viral selection pressure and CD4+T cell epitope on the natural outcome of HCV infection in HLA-DRB1 * 11:01 positive infected patients. 【Methods】 The positive selection sites and population growth of E1E2 and NS3 genes of common HCV 6a in HLA-DRB1 * 11:01 positive and negative groups in Guangdong were respectively analyzed. The peptide library covering the conserved regions of common HCV genotypes was used to stimulate HCV spontaneous clearance group and chronic infection group using ELISPOT method. Reactive peptides were obtained according to the number of spot-forming cells per well and the frequency of occurrence in different groups. 【Results】 The positive selection sites (PSSs) of E1E2 and NS3 of common HCV 6a in HLA-DRB1 * 11:01 negative group were greater than those in HLA-DRB1 * 11:01 positive group. Furthermore, the number of PPSs in CD4+T cell peptide in HLA-DRB1 * 11:01 negative group were also greater than those in HLA-DRB1 * 11:01 positive group; Both groups of HCV 6a had a population growth in Guangdong, and the expansion trend of HLA-DRB1 * 11:01 negative group was significantly higher than that of HLA-DRB1 * 11 :01 positive group. Compared to HCV chronic infection group, the response rate of HCV spontaneous clearance group to five peptides (C-52 E2691-707, C-119 NS31545-1560, C-134 NS4A1669-1684, C-154 NS4B1912-1927, C-159 NS4B1929-1944) was higher. However, the HCV chronic infection group showed a higher response rate to two of the peptides(C-111 NS31497-1512, C-130 NS31650-1665). When HLA-DRB1 * 11:01 typing was considered, there was no significant difference in HCV-specific immune response generated by PBMCs between HLA-DRB1 * 11:01 positive and HLA-DRB1 * 11:01 negative groups. 【Conclusion】 This study revealed the relationship between viral selection pressure of HLA-DRB1 * 11:01 HCV infected persons and CD4+T cell antigen epitopes. At the same time, CD4+ T cell antigen epitopes of HCV pan-genotype were obtained, providing basic data for the development of T cell vaccine suitable for HCV pan-genotype.

Blood screening is essential for blood safety. Traditional methods are not very effective in unknown pathogen testing. The blood metagenomic analysis with high-throughput sequencing as the main method is not limited to a particular microbe but detects all microbes in the sample (total genome), which has advantages that traditional methods cannot compare with. With the increasingly maturation of technology and the gradual reduction of cost, metagenomic sequencing has been applied more and more widely. In this paper, the concept and research procedures of metagenomics, the current status and future prospect of metagenomics sequencing technology in the field of blood screening are reviewed.

【Objective】 To study the CD4 T cell epitopes in Core and NS3 protein of genotype 1(GT1) and 6(GT6) of hepatitis C virus(HCV). 【Methods】 A total of 298 overlapping peptides(16-mer) spanning Core and NS3 protein of GT1 and GT6 HCV were synthesized. Peripheral blood mononuclear cells(PBMCs) from 17 HCV+ and 7 healthy blood donors were stimulated by peptide pools, followed by evaluating T cell response by IFN-γ ELISPOT, by which 21 peptides with positive results were found. These peptides were further applied to individually stimulate 20 HCV+ and 18 healthy PBMCs. The differences of responsive frequencies to the 21 positive peptides between the two study groups were compared. 【Results】 Pooled and individual peptide stimulation tests showed that HCV+ PBMCs were responsive to the stimulation of 5 peptides(GT1 NS31273-1288 and NS31315-1330; GT6 NS31033-1048, NS31087-1102 and NS31351-1366), with a responsive frequency ranging 18.9%-27.0%. In contrast, healthy PBMCs were not or low responsive(0%-4.0%) to these five peptides. The responsive frequencies were statistically different between the two groups(P<0.05). No reported epitopes in IEDB were found identical with these 5 peptides via sequence alignment. 【Conclusion】 Our study identified novel CD4 T cell epitopes in NS3 protein of GT1 and GT6 HCV, which has potential application value for the research and development of HCV vaccine.

【Objective】 To learn the situation of the evolution process of HCV virus population and the selection pressure of HCV NS5B in intravenous drug users (IDUs) in Guangdong. 【Methods】 141 blood samples from hepatitis C virus (HCV) RNA-positive blood donors and 58 from HCV patients in Guangdong were randomly collected for HCV NS5B sequence amplification, combined with HCV NS5B sequences from blood donors and IDUs obtained by sequencing previously(between 2009 and 2011). Homology analysis was performed by Molecular Evolutionary Genetics Analysis (MEGA) software, evolutionary analysis were performed by Bayesian Evolutionary Analysis Sampling Trees (BEAST) software package. Selection pressure analysis was performed on sequences isolated from IDUs by Datamonkey online software package with Mixed Effects Model Evolution (MEME) method, and the population expansion of species were analyzed using Tajima and Fu neutrality test by Arlequin software. 【Results】 The comparison results of internal homology among different subtypes of IDUs in this group were as follows : HCV-3b had the highest homology (97%), followed by HCV-3a (96%), HCV-6a (95%) and HCV-1b (94%); HCV evolution rate analysis showed that HCV-1b had the fastest evolution rate [2.17E-03 substitutions/site/year (y/y/y)], followed by HCV-3b (2.12E-0 y/y/y), HCV-3a (1.58E-03 y/y/y) and HCV-6a (1.28E-03 y/y/y). The analysis on effective population of HCV: 1980~1990 was rapid growth period for HCV-6a, 1990~1995 period for HCV-1b, and 2000~2007 period for HCV-3a. HCV population genetic characteristics was as follows: HCV-1b, 3a, 3b and 6a experienced population expansion, among which 3a and 3b were the most obvious. As to the analysis of HCV selection pressure, two positive selection sites (235 and 243)were found in the 339 nucleotide fragment of the NS5B sequence in injecting drug users, but mutation only occurred at position 316 [mutation rate 1.24% (14/1 130)] among 5 direct antiviral drug (DAA) sites in this gene. 【Conclusion】 The evolution of HCV-3b in Guangdong has showed an obvious trend of population expansion, with a high proportion and homology especially in the local IDUs. HCV-3b should be the focus of HCV prevention and control in this region. Given that the positively selected sites of the HCV NS5B gene region of IDUs in Guangdong are non-DAA binding sites, DAA is expected to demonstrate a good effect on these patients.

【Objective】 To investigate the CMV-IgG positive yeild among blood donors in Guangzhou and explore the differences in the efficacy of three test reagents, aimed at improving blood safety and service capacity of blood centers. 【Methods】 A total of 630 blood samples from eligible blood donors from July to October 2020 in our center were randomly selected and screened for CMV-IgG by one ELISA reagent.Among them, 180 samples were tested in parallel using three reagents (two ELISA reagents and one ECLIA reagent), and those tested negative were conducted quantitative CMV-DNA detection.The test results of different reagents were compared and analyzed. 【Results】 Out of the 630 samples, a total of 598 positive samples were screened out, including 180 samples yielded by three reagents, 171 and 175 by the two ELISA reagents, respectively, and 175 by ECLIA.The results given by three reagents were consistent (Kappa>0.4), and no significant difference in the positive yeild by three reagents was found.In the 180 samples, 11 were negative, among which 3, 2 and 6 samples were negative by all three reagents, two reagents and one reagent (ELISA), respectively.All the 11 samples were tested negative for CMV-DNA. 【Conclusion】 The yeild of positive CMV-IgG in blood donors was 94.9% (598/630), suggesting a high prevalence of CMV in Guangzhou. CMV serologically negative blood should be considered when providing blood products to immunocompromised patients to improve the safety of recipients.The detection results of ELISA reagents and ECLIA reagent for CMV- IgG are consistent, but ECLIA reagent has better detection efficacy.

Guided bone regeneration technology applied in alveolar bone defect regeneration is based on the barrier function and space maintenance of the barrier membrane. Therefore, traditional development strategies for barrier membranes focus on their physical barrier function, degradation characteristics and biocompatibility to avoid immunogenicity. However, not only does the barrier membrane passively block connective tissue, it is recognized as a “foreign body”that triggers a persistent host immune response, known as a foreign body reaction. The theories of osteoimmunology reveal a close relationship between the immune system and bone system and emphasize the role of immune cells in bone tissue-related pathophysiological processes. Based on these findings, we propose a novel development strategy for barrier membranes based on immune microenvironment regulation: by manipulating mechanical properties, surface properties and physiochemical properties, barrier membranes are endowed with an improved immunomodulation ability, which helps to regulate immune cell reactions to induce a favorable local immune microenvironment, thus coordinating osteogenesis and osteoclastogenesis as well as barrier membrane degradation to increase the efficiency of barrier membranes in GBR applications. In this paper, we review the development of barrier membranes and their close relationship to the immune microenvironment concerning bone regeneration and membrane degradation. Additionally, the outcomes of research on barrier membranes based on the regulation of the immune microenvironment have been summarized to improve the osteogenesis efficiency of barrier membranes and solve the problem of the regeneration and repair of bone defects, especially alveolar bone defects.

【Objective】 To investigate the correlation of peripheral myeloid-derived suppressor cells (MDSC) with hepatitis c virus (HCV) infection. 【Methods】 109 voluntary blood donors who donated blood during February 2018 to September 2020 at Guangzhou Blood Center were recruited in this study. They were assigned to chronic hepatitis c (CHC) group (n=48), spontaneous clearance (SC) group (n=29) and healthy donors (control) group (n=32) according to the results of anti-HCV and HCV RNA tests. Blood samples were drawn from the participants and peripheral blood mononuclear cells (PBMC) were freshly isolated, followed by staining with fluorescently-labeled antibody against cell surface markers of MDSC, which were then applied to the detection of monocytic- (M) and polymorphonuclear (PMN)-MDSC by flow cytometry. Parameters for liver function including alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT), total bilirubin (TBIL) and direct bilirubin (DBIL) were also measured. One-way ANOVA tests were applied to compare the differences of M- and PMN-MDSC and liver function between three study groups. For pairwise comparisons, P values were adjusted for multiple comparisons by Bonferroni correction (Pc). 【Results】 The frequencies of M-MDSC (%) in CHC, SC and HC were 1.39±0.86, 0.85±0.63 and 0.57±0.23, respectively (P<0.01). Specifically, CHC presented significantly higher level of M-MDSC than SC (Pc<0.01) and control (Pc<0.05). There was no significant difference in the frequencies of PMN-MDSC among the three study groups (0.81±0.54 vs 0.65±0.40 vs 0.62±0.29, P>0.05). In addition, AST (34.4±19.2 vs 23.0±7.78 U/L) and GGT (40.8±31.4 vs 22.3±7.40 U/L) level were higher in CHC compared with control (Pc<0.05 and Pc<0.01, respectively). 【Conclusion】 The level of peripheral M-MDSC was significantly elevated in chronic HCV infected donors, which would related to the progression of chronicity after HCV infection.

ObjectiveTo investigate the application of next-generation sequencing (NGS) in determining the full-length sequence and baseline resistance-associated substitution (RAS) of hepatitis C virus (HCV) subtype 3b. MethodsNucleic acid was extracted from plasma of a HCV RNA-positive blood donor, and after sequence-independent amplification, a sequencing library was constructed and NGS was performed using Illumina Hiseq. Bioinformatics methods were used to analyze full-length HCV sequence, viral genotype, and baseline RAS to direct-acting antivirals (DAAs). ResultsA total of 8.4 Gb data with more than 56 million reads were obtained. The full-length HCV sequence was obtained by bioinformatics analysis, with an average sequencing depth of 488 007 and a genotype of 3b subtype. A total of 12 RASs were identified in HCV amino acid sequence, i.e., Y56H, Q80K, Q80R, and A156G located in NS3, M28G, Q/A30G, Q/A30K, L31F, L31M, and Y93H located in NS5A, and S282T and V321A located in NS5B, among which Q/A30K and L31M located in NS5A had high frequencies of 99.16% and 98.37%, respectively, while the other 10 RASs had low frequencies of ＜0.5%. ConclusionNGS can be used to determine the full-length sequence and genotype of HCV subtype 3b and identify baseline RASs, which has great significance in the epidemiological study of HCV subtype 3b and the development of DAA treatment regimens.