Flow cytometry (FCM) is one of the most advanced cell quantitative analysis technology.Today,flow cytometry has been extensively and intensively used in medicine and other science,ranging from basic research to clinical diagnosis With the rapid development of science and technology of China,the application of flow cytometry in clinical research and diagnosis has also made significant progress.

Objective To discuss Foxp3 expression in peripheral blood from the patients with various stages of gastric cancer by SYBR Green I real-time PCR assay.Methods Foxp3 standard was prepared by conventional RT-PCR and employed to generate quantitative PCR standard curve.The precision,sensitivity and specificity of the assay were measured.SYBR Green I real-time PCR assay was adopted to detect and quantify Foxp3 expression in peripheral blood from 40 gastric cancer patients and 8 normal controls.Results The coding sequence (CDS) of full length Foxp3 was obtained and used as standards.The dynamic range of standard curve of real-time PCR assay was 102-108 copies.The mean coefficient of variation (CV) of inter-and intra-assay was less than 5%.The minimum detectable limit was 280 copies of Foxp3 mRNA.Foxp3 expression level in gastric cancer was significantly higher than that in normal control (P

Objective To explore the levels of serum chemokine in the patients with gastric cancer using cytometric beads array (CBA) and to find out the laboratory evidence for gastric cancer immunotberapy.Methods Forty-five patients with gastric cancer,thirty patients with benign gastric diseases and forty healthy controls were included.The chemokine levels of interleukin-8 (IL-8),regulated upon activation normal T-cell expressed and secreted (RANTES),monokine induced by IFN-γ (MIG),monocyte ehemoattractant protein-1(MCP-1) and interferon-γ induced protein-10 (IP-10) in serum of these three groups were measured using CBA ,then these data were analyzed using BD CBA analysis software.Results The levels of serum chemokines in healthy controls were (176.4±20.7) μg/L for IP-10,(111.3±17.2) μg/L for MCP-1,(503.9±47.2) μg/L for MIG,(472.4±116.7) μg/L for IL-8,respectively.In gastric cancer patients,the levels were (266.6±24.7) μg/L for IP-10,(100.4,70.8-193.5) μg/L for MCP-1,(1614±275.4) μg/L for MIG,(500.0±164.8) μg/L for IL-8.The levels of serum chemokines in patients with benign gastric disease were (207.9±31.7) μg/L for IP-10,(121.2±23.6) μg/L for MCP-1,(514.5±63.0) μg/L for MIG,(480.2±134.8) μg/L for IL-8,respectively.The levels of IP-10 and MIG among these three groups were significantly different (IP-10:F = 3.52,P < 0.05,MIG:F = 9.27,P < 0.01).The levels of IP-10 were elevated in the cancer patients compared with healthy controls (P < 0.05),while the levels of MIG were elevated significantly in the cancer patients compared with healthy controls and benign disease controls(t=3.29,P < 0.01,t=2.84,P<0.01).Conclusions CBA is a novel method with convenience,sensitivity and accuracy for the detection of serum ehemokines.The concentration of IP-10 and MIG in gastric cancer patients are elevated,indicating its implication in cancer pathogenesis and metastasis.Measurement of serum chemokines will lay the groundwork for therapeutic strategy in gastric cancer by inhibiting and anti-chemokine.

Objective To evaluate the consistency and accuracy among 3 brands of flow cytometers (BriCyte E6,BD FACSCanto Ⅱ and Beckman Coulter FC 500) in the detection of lymphocyte subsets.Methods According to the methodology,the BriCyte E6 was compared with 2 flow cytometers commonly used in clinical detection.Seventy-three cases (40 male and 33 female) of anticoagulation peripheral blood specimens were collected in the clinical laborartory department of Xinhua Hospital in July 2015 and the percentage (％) and absolute number (#) of the lymphocyte subsets were detected by 3 different flow cytometers within samples collected 4 h.Results There were good consistency among the 3 flow cytometers (R2 ＞0.95,R2 from 0.969 5 to 0.992 4) in the detection of lymphocyte subsets percentage,so did in the detection of absolute number (R2 ＞ 0.95,R2 from 0.969 1 to 0.993 3).As to the precision evaluation,in the detectionof CD8％,T#,CD4+ T# and CD8+ T#,BriCyte E6 achieved a low CV％ compared with FACSCanto Ⅱ and FC 500 (Friedman statistics are 16.720,11.840,15.760 and 15.430,P =0.000 2,0.027,0.000 4,0.000 4,respectively).In the detection of T％,CD4％,NK％,B％,NK#,B#,there was no significant difference among the 3 flow cytometers (Friedman statistics are 4.242,3.916,0.852,2.595,1.835 and 0.578,P =0.119 9,0.141 2,0.653 2,0.273 3,0.399 6,0.749 0,respectively).Conclusions The 3 flow cytometers have a good consistency in the detection of lymphocyte subsets.BriCyte E6 may be an alternative or complement of existing flow cytometers.

Objective To investigate the expression of galectin-10 in CD4+CD25+CD127low/-regulatory T cells(Treg) and the correlation between expressions of galectin-10 and Foxp3.Methods CD4+CD25+CD127low/-Treg cells and CD4+CD25-CD127+ T cells were isolated from PBMC of healthy volunteers and lymph nodes of gastric cancer patients by flow cytometry.Following RNA micro-extraction RT-PCR analysis was used to detect mRNA expression of galectin-10 and Foxp3.Results The mRNA expression of galectin-10 and Foxp3 were dominant in Treg cells,whereas nearly no expression in CD4+CD25-CD127+ T cells.The results from PBMC of healthy volunteers and lymph nodes of gastric cancer patients were consistent between.Conclusion The expression of galectin-10 was obviously high in Treg cells,so it may become a novel marker for phenotypic characterization of Treg cells.

Objective The aim of the study WSfl to establish the method using CDl27 as the new biomarker to identify regulatory T cells(Treg cells)and apply the CD 127 to detect the Treg cells in patients with gastric cancer.Methods The phenotypes of Treg cells were analyzed using five-color flow cytometry method Foxp3.FITC/CD127-PE/CD4-PerCP/CD25-APC/CD3-PC7.The mRNA and protein expression of Foxp3 in isolated CD+4 CD25high CD127-/low Treg cells were detected.The relationships between Foxp3 and CD127 protein expression in CD4+ T cells from aduh human peripheral blood were investigated.PBMCs,Ascltes,turnor-infihration lymphocyte and tumor-draining lymph nodes in 35 patients with gastric cancer and PBMCs in 20 normal healthy donors were evaluated for the proportion of Treg ceils,as well as the percentage ot the total CD +4 cells.Results CD4+ CD25 high CD-127 low ceils expressed the high Foxp3 in protein level(87.1%)and mRNA level.Within the CD4+CD+25 population,there was a significant correlation between Foxp3 and the CD127low phenotype(r=0.985,P<0.01).Compared with healthy olunteers,patients with gastric malignancies had a higher proportion of CD4+4 Cdhigh 25 CD-low127 cells in peripheral blood(t=2.542,P<0.05).The Dereentages of Treg cells were more abundant in ascites(t=2.357,P<0.05),TIL(t=6.174,P<0.01) and tumor-draining lymph nodes(t=5.481,P<0.01)of individuals with gastric cancer than that in their blood.There were significant differences in the prevalence of Treg ceils between the early and advanced disease stages in gastric cancer[(6.04±2.31)%in stageⅠ+Ⅱ VB(10.16±2.29)% Ⅲ+Ⅳ,t=2.473,P<0.05].Conclusions The CD127 biomarker can be used to selectively enrich human Treg cells for in vitro functional studies.The populations of CD4+ CD high25 CD127-/low Treg cells increased ith tumor stage in individuals with gastric cancer.

Objective To investigate the populations of CD+4CD25high Foxp3+ regulatory T cells(Treg cells)and mRNA expression of Foxp3 in peripheral and the marginal region of tumor from patients with gastric cancer,and to evaluate the prevalence of Treg cells from patients with gastric cancer in relation to the TNM stages.Methods PBMC,Ascites,tumor-infiltration lymphocyte and tumor-draining lymph nodes in 47 patients with gastric cancer(TNM I11,Ⅱ18,Ⅲ10,Ⅳ8)and PBMC in 20 normal healthy donors were evaluated for the proportion of Treg cells,as a percentage of the total CD4+ cells,by flow cytometric analysis.Levels of mRNA for Foxp3 were measured witll a real-time quantitative PCR Results The percentage of CD4+CD25high Foxp3+ Treg cells in PBMC for cases of gastric cancer were significantly higher than those for healthv donors(P<0.05).The percentage of Treg cells were more abundant in ascites(P< O.05),1rIL(P<0.01)and tumor-draining lymph nodes(P<0.01)of individuals with gastric cancer than in their blood.And the Foxp3 mean fluorescent intensity (MFI) of Treg cells in TIL and tumor draining lymph nodes with gastric caners were significantly higher than PBMC and ascites(P<0.05).The proportion 0f Treg cell in patients of gastric cancers with stage I,II,Ⅲ,Ⅳ were(5.72±1.95)%,(6.45± 2.23)%,(9.58±3.13)%,(11.70±2.30)%,respectively.There were significant correlation between Drevalenee of Treg cells and disease stages in gastric cancer(r=0.784,P<0.01).Foxp3 mRNA levels were higher in PBMC from the group with gastric cancers than in the group with healthy donors.Foxp3 mRNA levels were correlated with TNM stages(r=0.623,P<0.05).Conclusions Our results suggest that the populations of CD4+CDhigh25 Foxp3+Treg cells and Foxp3 mRNA levels in patients with gastric cancer are signifieantlv higher in comparison to those in control donors.In addition,the expression of Fox：p3 mRNA increased with tumor stage.The increased prevalence of Treg cells may be one of the explanations for impaired cell-mediated immunity in cancer bearing hosts.

Objective To establish a five-color flow cytometric immunophenotyping protocol and valuate its clinical application in leukemia and lymphoma. Methods Samples from 73 cases of acute leukemia and 30 cases of lymphoma were analyzed using a comprehensive antibody panel with five-color combination. The antibody combination CD7/CD33/CD19/CD13/CD45 and MPO/cCD79a/cCD3/CD117/CD45,composed of different lineage distinctive and lineage sensitive markers of B, T and myeloid cells, were applied to determine the lineage originality of leukemia and lymphoma celia. The markers used in the second step analysis were based on the findings of the first step. Results Five-color compensation can be performed automatically and easily with the advanced digital compensation program. The results showed that the expression ratio of CD19 in B-ALL was 100% (27/27), cCD79, pesitivity was 92.6% (25/27) and CD7,cCD3 was expressed in all T-ALL cases (8/8). The B-ALL could be staged depending on the expression level of CD34 ,CD10 ,cμ,sIgM and the T-ALL could be dearly staged dependent on the expression level of CD2,CD1a,,CD4,CD4,CD8. The expression ratio of cMPO, CD117 was 89. 5% (34/38) and 81.6% (31/38) in AML respectively, however CD7 was also expressed in 26.3% (10/38) of AML cases. Combined with morphology, immanophenotypes could be used for diagnosing AML with subtyping. The CD19/SSC gating can be used for immunophenotyping of B cell lymphoma with differential diagnosis. The number of robes required was significantly reduced with our panel from 12 tubes of 3-color to 6 tubes of 5-color. Implementation of the five-color protocol had resulted in 20% reduction in reagent costs. Conclusions The application of fivecolor staining protocol significantly improve the sensitivity and accuracy of measurement of immunophenotypes of acute leukemia and lymphoma. It reduces the cost and can be widely applied in the clinical laboratory diagnosis.