Objective To discuss the improvement for the verification of inter-day precision of PS activity assay and the verification protocol for limits of quantitative of FⅧ:C and FⅨ:C assay,aiming at the problems in the performance verification of anticoagulant protein S(PS),coagulation factor Ⅷ activity(F Ⅷ:C)and coagulation factor Ⅸ activity(F Ⅸ:C)assay.Methods SYSMEX CN-6000 and supporting reagents were used,and low-and high-value quality control(QCs)were used as the study samples.Following the American Clinical and Laboratory Standards Institute(CLSI)document EP15-A3,an inter-day precision verification study of the PS activity assay was performed with three reagent preparation methods designed(specification-required method,instrument's manual method and improved method).The specification-required method was carried out completely according to the specification,and the instrument manual method involved allowing the required reagents to stand in the device for 30 minutes based on the specification-required method,and the improved method mixed and dispensed the reagents needed to be based on the specification-required method.To study the bottle variation of the same batch of reagents for the same sample,the acceptable range of external quality assessment(EQA)of the National Center for Clinical Laboratories(NCCL)was used as the evaluation standard.Following the CLSI EP25 document,the PS activity assay reagent onboard stability verification was performed with the specification-required method and the improved method.Following the CLSI EP17-A2 document,WS/T 514-2017"Establishment and Verification of Detection Capability for Clinical Laboratory Measurement Procedures"and the International Committee for Standardization in Hematology(ICSH)guidelines,the LoQ for FVIII:C and FIX:C assays was verified.The verification results were passed if the requirements of the specification were met.Results The verification result of inter-day precision(CVWL:12.9%～21.6%)of PS activity assay according to the specification method and the instrumental manual method exceeded the requirements of the specification(<10%CVWL at high levels,<20%CVWL at low levels).The results of inter-day precision verification with the improved method(CVWL:2.9%and 4.5%)were consistent with the requirements of the specification.The bottle variation exceeded the acceptable range of EQA.The improved method corrected the reagent on-board stability verification results of reagents(relative deviations of-4.24%～9.97%)by the specifications(high levels<10%,low levels<20%).The LoQ verification results for FⅧ:C and FⅨ:C assay were by the product specifications(FⅧ:C:0.75%～1.46%and 0.74%～1.40%;FⅨ:C:0.71%～1.27%and 0.70%～1.32%).Conclusion An improved method to improve the inter-day precision of PS activity detection is provided,and LoQ verification protocol for F Ⅷ:C and FⅨ:C assay is provided for clinical laboratory reference.

Objective To discuss the improvement for the verification of inter-day precision of PS activity assay and the verification protocol for limits of quantitative of FⅧ:C and FⅨ:C assay,aiming at the problems in the performance verification of anticoagulant protein S(PS),coagulation factor Ⅷ activity(F Ⅷ:C)and coagulation factor Ⅸ activity(F Ⅸ:C)assay.Methods SYSMEX CN-6000 and supporting reagents were used,and low-and high-value quality control(QCs)were used as the study samples.Following the American Clinical and Laboratory Standards Institute(CLSI)document EP15-A3,an inter-day precision verification study of the PS activity assay was performed with three reagent preparation methods designed(specification-required method,instrument's manual method and improved method).The specification-required method was carried out completely according to the specification,and the instrument manual method involved allowing the required reagents to stand in the device for 30 minutes based on the specification-required method,and the improved method mixed and dispensed the reagents needed to be based on the specification-required method.To study the bottle variation of the same batch of reagents for the same sample,the acceptable range of external quality assessment(EQA)of the National Center for Clinical Laboratories(NCCL)was used as the evaluation standard.Following the CLSI EP25 document,the PS activity assay reagent onboard stability verification was performed with the specification-required method and the improved method.Following the CLSI EP17-A2 document,WS/T 514-2017"Establishment and Verification of Detection Capability for Clinical Laboratory Measurement Procedures"and the International Committee for Standardization in Hematology(ICSH)guidelines,the LoQ for FVIII:C and FIX:C assays was verified.The verification results were passed if the requirements of the specification were met.Results The verification result of inter-day precision(CVWL:12.9%～21.6%)of PS activity assay according to the specification method and the instrumental manual method exceeded the requirements of the specification(<10%CVWL at high levels,<20%CVWL at low levels).The results of inter-day precision verification with the improved method(CVWL:2.9%and 4.5%)were consistent with the requirements of the specification.The bottle variation exceeded the acceptable range of EQA.The improved method corrected the reagent on-board stability verification results of reagents(relative deviations of-4.24%～9.97%)by the specifications(high levels<10%,low levels<20%).The LoQ verification results for FⅧ:C and FⅨ:C assay were by the product specifications(FⅧ:C:0.75%～1.46%and 0.74%～1.40%;FⅨ:C:0.71%～1.27%and 0.70%～1.32%).Conclusion An improved method to improve the inter-day precision of PS activity detection is provided,and LoQ verification protocol for F Ⅷ:C and FⅨ:C assay is provided for clinical laboratory reference.

Cardial troponin I(cTnI)is the preferred serological marker for the diagnosis of myocardial injury.cTnI detection is based on antibody sandwich immunoassay.The epitopes of cTnI antigen targeted by detecting and capturing antibodies in different detection reagents are inconsistent,which easily leads to the heterogeneity of cTnI detection results.Endogenous interfering factors such as cTnI autoantibody,heterophile antibody,rheumatoid factor,ect,which can seriously interfere with the results of cTnI detection,and affecting the clinical diagnosis,treatment and prognosis of myocardial injury diseases.In this paper,the research progress of antibody sandwich immunoassay for cTnI and interference of endogenous factors on cTnI detection and solutions are reviewed to provide theoretical basis for differential diagnosis of abnormal cTnI detection results in clinical practice.

Objective:To construct four retroviral plasmids for differential expression of green fluorescent protein (GFP) based on different terminal sequences of internal ribosome entry site (IRES) and provide reference for subsequent flow analysis or imaging.Methods:Based on the fact that the transcription efficiency of encephalomyocarditis virus IRES depends on its terminal sequence, IRES and enhanced GFP (EGFP) were fused into four fragments with different connection modes by overlapping PCR, and then cloned into retroviral plasmid pMSCV-NGFR. NGFR fragment was amplified by PCR and inserted in front of the retroviral plasmids pMSCV-IRES(1-4)-EGFP. These retrovirus plasmids pMSCV-NGFR-IRES(1-4)-EGFP were transfected into 293T cells. The expression ratio and mean fluorescence intensity (MFI) of EGFP were analyzed, and the expression of NGFR was also detected.Results:Four retroviral plasmids pMSCV-NGFR-IRES(1-4)-EGFP were successfully constructed. No significant difference in the expression efficiency of EGFP at 24 or 48 h was observed in 293T cells transfected with the four different retroviral plasmids, but there was significant difference in fluorescence intensity. Moreover, the expression of NGFR was not significantly different, indicating that the addition of different nucleotide sequences between IRES and EGFP would make a significant difference in the fluorescence intensity of EGFP.Conclusions:The expression intensity of EGFP was affected by the sequence between IRES and EGFP. Retroviral plasmids expressing EGFP of different intensity could meet different experimental requirements.

Objective: To assess the association of single nucleotide polymorphisms (SNPs) in SLCO1B3 gene with prognosis of breast cancer (BC) patients treated with neoadjuvant chemotherapy of TA regimen (taxane and antharcycline drugs). Methods: 439 female BC patients were recruited and treated with neoadjuvant chemotherapy of TA regimen. A blood sample (2 ml) of peripheral blood was collected from each patient before chemotherapy. Tagging SNPs (tag-SNPs) were selected. We investigated the association of tag-SNPs with prognosis, by Sequenom Mass ARRAY system platform, characterizing tag-SNPs. The hazard ratio (HR) and 95% confidence interval (CI) for progression or death were calculated by multivariable-adjusted Cox regression model. Results: Seven tag-SNPs (rs11045689, rs200104106, rs3764006, rs3834935, rs4149117, rs7305323 and rs73241801) were selected for study. Compared with individuals carrying the rs11045689 GG genotype, individuals carrying rs11045689 AA genotype performed worse PFS and OS, with the HR (95% CI) for progression being 1.39 (1.11~1.75) and the HR (95% CI) for death being 1.38 (1.04~1.83). Compared with individuals carrying the rs73241801 CC genotype, individuals carrying rs73241801 TT genotype performed better OS (P=0.041), with the HR (95% CI) for death being 0.65 (0.44~0.94). The number of risk allele was significantly associated with PFS (P=0.012) and OS (P=0.017) of BC patients by accumulation analysis. Compared with individuals carrying one or less than one risk allele, individuals carrying four risk alleles performed worse PFS and OS, with the HR (95% CI) for progression being 1.37 (1.09~1.72) and the HR (95% CI) for death being 1.36 (1.02~1.81). Conclusion The variations of rs11045689 and rs73241801 in SLCO1B3 gene were significantly associated with prognosis of BC patients treated with neoadjuvant chemotherapy of TA regimen, which might serve as biomarkers for predicting prognosis of BC patients treated with neoadjuvant chemotherapy.

Objective To assess the association of single nucleotide polymorphisms ( SNPs) in SLCO1B3 gene with prognosis of breast cancer (BC) patients treated with neoadjuvant chemotherapy of TA regimen (taxane and antharcycline drugs). Methods 439 female BC patients were recruited and treated with neoadjuvant chemotherapy of TA regimen.A blood sample (2 ml) of peripheral blood was collected from each patient before chemotherapy. Tagging SNPs (tag?SNPs) were selected. We investigated the association of tag?SNPs with prognosis, by Sequenom Mass ARRAY system platform, characterizing tag?SNPs. The hazard ratio ( HR ) and 95% confidence interval ( CI ) for progression or death were calculated by multivariable?adjusted Cox regression model. Results Seven tag?SNPs ( rs11045689, rs200104106, rs3764006, rs3834935, rs4149117, rs7305323 and rs73241801) were selected for study. Compared with individuals carrying the rs11045689 GG genotype, individuals carrying rs11045689 AA genotype performed worse PFS and OS, with the HR (95% CI) for progression being 1.39 (1.11～1.75) and the HR (95% CI) for death being 1.38 ( 1.04～1.83). Compared with individuals carrying the rs73241801 CC genotype, individuals carrying rs73241801 TT genotype performed better OS (P＝0.041), with the HR (95% CI) for death being 0.65 (0.44～0.94). The number of risk allele was significantly associated with PFS (P＝0.012) and OS (P＝0.017) of BC patients by accumulation analysis. Compared with individuals carrying one or less than one risk allele, individuals carrying four risk alleles performed worse PFS and OS, with the HR (95%CI) for progression being 1.37 (1.09～1.72) and the HR ( 95% CI) for death being 1.36 (1.02～1.81). Conclusion The variations of rs11045689 and rs73241801 in SLCO1B3 gene were significantly associated with prognosis of BC patients treated with neoadjuvant chemotherapy of TA regimen, which might serve as biomarkers for predicting prognosis of BC patients treated with neoadjuvant chemotherapy.

Objective To assess the association of single nucleotide polymorphisms ( SNPs) in SLCO1B3 gene with prognosis of breast cancer (BC) patients treated with neoadjuvant chemotherapy of TA regimen (taxane and antharcycline drugs). Methods 439 female BC patients were recruited and treated with neoadjuvant chemotherapy of TA regimen.A blood sample (2 ml) of peripheral blood was collected from each patient before chemotherapy. Tagging SNPs (tag?SNPs) were selected. We investigated the association of tag?SNPs with prognosis, by Sequenom Mass ARRAY system platform, characterizing tag?SNPs. The hazard ratio ( HR ) and 95% confidence interval ( CI ) for progression or death were calculated by multivariable?adjusted Cox regression model. Results Seven tag?SNPs ( rs11045689, rs200104106, rs3764006, rs3834935, rs4149117, rs7305323 and rs73241801) were selected for study. Compared with individuals carrying the rs11045689 GG genotype, individuals carrying rs11045689 AA genotype performed worse PFS and OS, with the HR (95% CI) for progression being 1.39 (1.11～1.75) and the HR (95% CI) for death being 1.38 ( 1.04～1.83). Compared with individuals carrying the rs73241801 CC genotype, individuals carrying rs73241801 TT genotype performed better OS (P＝0.041), with the HR (95% CI) for death being 0.65 (0.44～0.94). The number of risk allele was significantly associated with PFS (P＝0.012) and OS (P＝0.017) of BC patients by accumulation analysis. Compared with individuals carrying one or less than one risk allele, individuals carrying four risk alleles performed worse PFS and OS, with the HR (95%CI) for progression being 1.37 (1.09～1.72) and the HR ( 95% CI) for death being 1.36 (1.02～1.81). Conclusion The variations of rs11045689 and rs73241801 in SLCO1B3 gene were significantly associated with prognosis of BC patients treated with neoadjuvant chemotherapy of TA regimen, which might serve as biomarkers for predicting prognosis of BC patients treated with neoadjuvant chemotherapy.

Objective To study the function of gene 0955 in Streptococcus suis type 2 98HAH33. Methods Growth condition of wild-type, mutant and complemented strains of Streptococcus suis type 2 98HAH33 was compared at different stages. Differences in adhesion ability to host cells and anti-phagocyto-sis among these strains were compared by using bacterial adhesion test and analyzing their survival rates in blood. Mouse and piglet models were used to evaluate their virulence. Results The growth of the mutant and the complemented strains was slightly slower than that of the wild type strains in logarithmic growth phase, but no significant difference was found in plateau phase. Bacterial adhesion test showed that gene 0955 might encode a new adhesion factor of Streptococcus suis type 2 98HAH33. Blood bactericidal test sug-gested that gene 0955 was not associated with anti-phygocytosis. Animal experiments showed that gene 0955 might be a novel virulence gene of Streptococcus suis type 2 98HAH33. Conclusion Gene 0955 might en-code a novel adhesion factor and virulence factor of Streptococcus suis type 2 98HAH33.

Objective To study the mechanism of carboxypeptidase E ( CPE ) in promoting the migration of lymphocytes and their subsets through vascular endothelial cells. Methods CRISPR/Cas9 technology was used to prepare cpe gene-knockout MS1 (Cpe-/-MS1) cells. Adhesion ability of lymphocytes to MS1 and Cpe-/-MS1 cells was analyzed with adhesion assay. Expression of adhesion molecules on these cells were detected by RT-PCR and flow cytometry. Transwell model was used to compare the difference in the transmigration of lymphocytes and their subsets through MS1 and Cpe-/-MS1 cells. Results Cpe-/-MS1 cells were successfully obtained. Under the stimulation of TNF-α, the adhesion ability of lymphocytes to MS1 cells was much better than that of Cpe-/-MS1 cells. Moreover, adhesion molecules expressed on MS1 cells were significantly more than those on Cpe-/-MS1 cells. The percentages of lymphocytes and their sub-sets that transmigrated through MS1 cells were significantly higher than those through Cpe-/-MS1 cells. Con-clusion CPE involved in the adhesion of lymphocytes to vascular endothelial cells and the transmigration of them through vascular endothelial cells, which was of great significance for understanding the migration of lymphocytes across vascular endothelial cells to peripheral lymph nodes.

Objective To explore the expression of S100A14 in breast cancer tissue, and the EGF and S100A14 feedback regulatory mechanism. Methods S100A14 mRNA level in 52 cases of of breast cancer and adjacent normal tissue was detected by quantitative real?time PCR. S100A14 protein in 21 cases of breast cancer and adjacent normal tissue was detected by Western blot. S100A14 mRNA after EGF treatment was detected by RT?PCR and real?time PCR. The levels of S100A14, p?ERK and t?ERK were detected by Western blot. Knocking down S100A14 expression was performed by siRNA technology. Results The levels of S100A14 mRNA and protein were significantly increased in breast cancer tissues ( P<0.05 for both) . The high expression of S100A14 was related with the recurrence of breast cancer patients ( P=0.038). S100A14 mRNA level was significantly up?regulated in the MDA?MB?453 cells (1.50±0.11) and MCF?7 cells (1.40±0.03) after 1 ng/mL EGF treatment, and 1.66±0.08 and 1.71±0.17 in the MDA?MB?453 cells after 10 ng/mL EGF treatment, significantly higher than that of the control group (1.00±0.09 and 1.00±0.03) (P<0.05 for both). In the TD47 cells, the S100A14 mRNA levels in the control, 1 ng/ml EGF and 10 ng/ml EGF + U0126 treatment groups were 1. 00 ± 0. 04, 1. 56 ± 0. 04 and 1. 00 ± 0. 10, respectively ( P<0.05) . Conclusions The expression of S100A14 mRNA and protein is promoted by EGF through p? ERK signaling pathway in breast cancer cells. There may be a feedback loop between EGF and S100A14.