Objective To construct mice hepatocellular carcinoma models with different tumor immune microenvironments(TIME)and explore the differences.Methods H22 and hepa1-6 were used to construct subcutaneous transplantation tumor model of C57 mice as homologous hepatocellular carcinoma cell lines(denoted as H22 group and hepal-6 group,each n=8),and the differences of TIME were evaluated.Immunohistochemistry was used to detect and quantify the infiltration of T cells,CD4+T cells,CD8+T cells,regulatory T cells and B cells in TIME.Flow cytometry was performed to detect the differences of composition of immune cell subpopulations in peripheral blood and tumor parenchyma.Gene expression profile characteristics of tumor tissue were analyzed based on high-throughput transcriptome sequencing technology,and enrichment analyses of immune-related signaling pathways were evaluated combined with gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG).Results H22 group showed cold and hepa1-6 group showed hot TIME characteristics.The number of T cells,CD4+T cells and CD8+T cells in tumor tissue of H22 group were all lower,while the proportion of T cells,CD4+T cells and CD8+T cells in peripheral blood were all higher than those of hepa1-6 group(all P＜0.05).Compared with H22 group,up-regulated genes of tumor tissue in hepa1-6 group expressed significantly enriched in tumor immune activation-related signaling pathways.Conclusion H22 and hepa1-6 hepatocellular carcinoma models showed distinct TIME characteristics of cold and hot tumors,respectively,and the amount of immune cells in tumor tissue of the former were significantly lower than those in the latter.

Objective To construct mice hepatocellular carcinoma models with different tumor immune microenvironments(TIME)and explore the differences.Methods H22 and hepa1-6 were used to construct subcutaneous transplantation tumor model of C57 mice as homologous hepatocellular carcinoma cell lines(denoted as H22 group and hepal-6 group,each n=8),and the differences of TIME were evaluated.Immunohistochemistry was used to detect and quantify the infiltration of T cells,CD4+T cells,CD8+T cells,regulatory T cells and B cells in TIME.Flow cytometry was performed to detect the differences of composition of immune cell subpopulations in peripheral blood and tumor parenchyma.Gene expression profile characteristics of tumor tissue were analyzed based on high-throughput transcriptome sequencing technology,and enrichment analyses of immune-related signaling pathways were evaluated combined with gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG).Results H22 group showed cold and hepa1-6 group showed hot TIME characteristics.The number of T cells,CD4+T cells and CD8+T cells in tumor tissue of H22 group were all lower,while the proportion of T cells,CD4+T cells and CD8+T cells in peripheral blood were all higher than those of hepa1-6 group(all P＜0.05).Compared with H22 group,up-regulated genes of tumor tissue in hepa1-6 group expressed significantly enriched in tumor immune activation-related signaling pathways.Conclusion H22 and hepa1-6 hepatocellular carcinoma models showed distinct TIME characteristics of cold and hot tumors,respectively,and the amount of immune cells in tumor tissue of the former were significantly lower than those in the latter.

【Objective】 To investigate the immune status of blood donors in Yangzhou area after SARS-COV-2 vaccinating. 【Methods】 Among 112 voluntary blood donors from August 29 to September 22, 2021, 111 were vaccinated with SARS-COV-2 vaccine.IgM antibody(by enzyme-linked immunocapture method), IgG antibody(by indirect method of combined immunoassay)and IgG antibody titer were detected. 【Results】 A total of 99.11% (111/112 ) voluntary blood donors were vaccinated, two-shot(n=103), one-shot(n=1) and three-shot (n=7) accounting for 91.96%, 0.89% and 6.25%, respectively.Eighty-eight (78.57%) were positive for IgG antibodies, and 14 (12.5%) were positive for IgM antibodies.No statistically significant difference was found in IgG and IgM positive yielding between males and females (P>0.05). The proportion (0.89%, 1/112) of positive IgM in blood donors with blood type A was significantly lower than that of other blood types (P<0.01). The IgG antibody titer of blood donors maintained rather high level within 6 months after vaccinating.47.66% of the donors presented antibody titer more than 160, and 5.60% had IgM antibody been detected within 1 month after vaccinating. 【Conclusion】 At present, the SARS-COV-2 vaccination effect in China is generally good.Since IgG antibodies cannot be detected after 6 months, it is suggested to perform IgG antibody testing for donors who have completed the second dose for more than 6 months.For those IgG antibody negative, booster shots should be conducted.For donors with high IgG antibody titer, their plasma may be considered to replace with COVID-19 convalescent plasma for the treatment of patients with rapid disease progression, or severe/critically ill patients diagnosed with COVID-19, so as to avoid the risk of COVID-19 re-spreading during convalescent plasma collection in blood centers. For blood donors with positive IgM antibodies, it is recommended to follow up the NAT results to minimize the risk of transmission.

Objective To compare two different methods to detect the differences of gene mutation rate, sensitivity, specificity and coincidence rate of epidermal growth factor receptor (EGFR) in non-small-cell lung cancer (NSCLC) so as to assess the clinical value of qRT-PCR method and its environmental-friendly technologyplatforms.One uses environmental fixative poly hydroxyl acrylic acid and green transparent liquid dewaxing Van-clear alone or in combination to replace the traditional fixative 4％ (volume fraction) neutral buffered formalin and the traditional transparent dewaxing liquid xylene in application of quantitative real-time polymerase chain reaction (qRT-PCR).The other uses traditional reagents in direct sequencing.Methods We selected 91 cases of primary NSCLC specimens resected between May 2013 and March 2016 in Zhongshan Bo`ai Hospital and Zhongshan Hospital of Traditional Chinese Medicine.Five samples were taken from the same tumor lesion.We used a random number table to randomly divide these samples into Groups A, B , C, D, and E.Group A received direct sequencing method in detection of EGFR gene mutations.Besides, during the experiment, 4％ neutral buffered formalin was used for fixing, and xylene transparent dewaxing was used to make slices for DNA extraction dewaxing.Group B received qRT-PCR method to detect EGFR gene mutations.Meanwhile, during the experiment, 4％ neutral buffered formalin was used for fixing, and xylene transparent dewaxing was used to make slices for DNA extraction dewaxing.Group C received qRT-PCR method in detection of EGFR gene mutations.At the same time, during the experiment, polyhydroxy acrylic acid was used for fixing, and xylene transparent dewaxing was used to make slices for DNA extraction dewaxing.Group D received qRT-PCR method to detect EGFR gene mutations.In the meantime, 4％ neutral buffered formalin was used for fixing, Van-clear transparent dewaxing was used to make slices for DNA extraction dewaxing.Group E received qRT-PCR method in detection of EGFR gene mutations.In addition, during the experiment, polyhydroxy acrylic acid was used for fixing, and Van-clear transparent dewaxing was used to make slices for DNA extraction dewaxing.In addition, during the experiment, polyhydroxy acrylic acid was used for fixing, and Van-clear transparent dewaxing was used to make slices for DNA extraction dewaxing.The mutations of Exons 18, 19, 20, and 21 in EGFR genes were respectively determined in the five groups of NSCLC.Results ① Groups B, C, D, E and A did not significantly differ in the percentage of people with mutations or target site mutation rates of EGFR genes in NSCLC (P> 0.05).② The detection results of EGFR target site mutation in Groups B, C, D, E and A had good sensitivity, strong specificity, and high compliance rate.Conclusion The green transparent liquid dewaxing Van-clear alone or in combination to replace the traditional fixative 4％ neutral buffered formalin and the traditional transparent dewaxing liquid xylene in the application of qRT-PCR so as to detect EGFR gene mutations in NSCLC has good consistent results compared with the method that uses traditional reagents in direct sequencing.It has the significance and value in clinical application.

Background and purpose: Adequate tissue ifxation, transparent dewaxing is an important step of hematoxylin eosin (HE) staining and lfuorescence in situ hybridization (FISH) in detection of breast cancerHER-2 gene. The purpose of this study was to make a comparison between poly hydroxyl acrylic acid which is an environmen-tally friendly ifxation liquid and 4% neutral buffered formaldehyde in tissue ifxation for HE staining and FISH to detect theHER-2 gene in the breast cancer tissue sections. The study aimed to evaluate the feasibility of replacing 4% buffered formaldehyde, a traditional ifxation liquid, with the poly hydroxyl acrylic acid, an environmentally friendly ifxation lfuid.Methods:This project was performed on tissue samples collected from 69 cases of breast cancer, 41 cases of breast ifbroadenoma, 40 cases of uterine leiomyoma, 25 cases of cervical tissue, 25 cases of placenta obtained from the outpatient and inpatient departments of Zhongshan Boai Hospital from Mar. 2011 to Jan. 2015, from each of which two samples were drawn and two blocks of each specimen were divided into two groups randomly. Then one group was ifxed with 4% neutral buffered formaldehyde and made into 200 sections by HE while the other group was ifxed with poly hydroxyl acrylic acid and made into another 200 sections. The slice level of the two groups was determined by the staining condition of the sections, and SPSS 19.0 was employed to compare the excellent and good rate of HE staining. Additional 69 sections were produced with two groups of breast invasive ductal carcinoma tissues, and SPSS 19.0 was used to detect the ampliifcation ofHER-2 gene by FISH.Results:First, the number of best-quality slices stained with HE ifxed separately by poly hydroxyl acrylic acid and 4% neutral buffered formaldehyde was 155 and 166, respectively. The number of excellent pieces was 41 and 33, respectively, while the number of mediocre pieces was 3 and 1 with bad pieces being 1 and 0, respectively. The excellent and good rates of HE staining were 98% and 99.5%, respectively. There was no significant difference between the two groups (χ2=1.33,P>0.05).Second, the positive rates of the tis-sue slices by FISH ifxed separately by poly hydroxyl acrylic acid and 4% neutral buffered were 26.09% and 23.19%, respectively. There was no signiifcant difference between the two groups (χ2=0.50,P>0.05).Conclusion:The results obtained with HE staining and FISH using poly hydroxyl acrylic acid as a ifxation liquid are not signiifcantly different from those using 10% neutral buffered formaldehyde. Therefore, poly hydroxyl acrylic acid meets the requirements of environmental protection, and thus has the potential to be promoted and widely used.

Objective:To observe the difference of the human telomeres RNA component (hTERC) genes’amplification in the cervical tissue by applying the environment-friendly fixative poly hydroxy acrylic acid and the transparent dewaxing solution Van-clear separately or jointly to replace the traditional fixative 4% (volume fraction)neutral buffered formalin and the conventional transparent dewaxing solu-tion xylene in the use of fluorescence in situ hybridization (FISH)for detection.Methods:In the study, 255 cases of cervical tissue specimens submitted by the Department of Gynecology in Zhongshan Boai Ho-sipital were collected from Mar.2013 to Apr.2015.Four samples were taken from the same lesion site. All the cases were divided into 4 groups and named group A,B,C,and D.Group A used 4% neutral buffered formalin fixed and xylene dewaxing to make slices.Group B used poly hydroxy acrylic fixed and xylene dewaxing to make slices.Group C used 4% neutral buffered formalin fixed and Van-clear trans-parent to make slices.Group D used poly hydroxy acrylic fixed and Van-clear transparent dewaxing to make slices.The amplification of hTERC genes in the four groups of cervical specimens was also detected by FISH technique.Results:When the hTERC genes were detected by FISH method under the fluore-scence microscope,it was obvious that the tissue profile and the background of group A,B,C and D were all clear.The probe was fixed in the accurate position so that the bright red or green fluorescence signals were easily found in these four groups.Compared with the positive rate of group A,there was no statistical significance in that of group B,C and D (P>0.05).At the same time,the coincidence rate of the FISH results was high,which showed that the new environment-friendly reagent had no significant difference in the detection of cervical hTERC genes by FISH technique.Conclusion:It is possible for the environment-friendly reagent poly hydroxy acrylic acid and Van-clear to replace 4%neutral buffered for-malin and xylene separately or jointly to detect the cervical hTERC genes by adopting FISH technique.