Objective To develop an improved enzymatic method for assaying creatinine in serum and urine. Methods Cratinine iminhydrolase catalyzes the conversion of creatinine to N-methylhydantoin and ammonia,the later combines with 2-oxoglutarate and NADPH in the presence of glutamate dehydrogenase.Before beginning the reaction sequence,endogenous ammonia is removed by an “ammonia scavenger system”that involves the same auxiliary reaction.The NADPH and 2-Oxoglutarate consumed are restored through a reaction catalyzed by isocitrate dehydrogenase.The activity of this strictly magnesium-dependent enzyme is completely blocked by magnesium-complexing reagent that acts promptly whenever the reaction has started with creatinine iminohydrolase as startor.The reaction is monitored kinetically by measuring the NADPH decrease via its absorbance at 340nm.Results The method affords a simple,rapid,and sensitive assay with good precision and extended linearity.Test results compare closely with the HPLC precedure.The proposed method is not subject to interference from several metablites or from the drugs used commonly in clinics.Conclusion The method is easily automated and is suitable for routine work in clinical laboratories.

Objective To investigate the clinical significance of the electrocardiogram (ECG) to cardiac involvements in Systemic lupus erythematosus (SLE). Methods 80 cases without sign and syndrome of cardiac involvements were analyzed retrospectively in our hospital between January 2004 and April 2009.Results In 80 cases without sign and syndrome of cardiac involvements, 46 cases had ECG changes,with the positive rate being 57.5% . In the next echocardiography examination, 39 cases had heart struction or function change,with the positive rate being 48.8% . Conclusion For SLE patients, ECG examinations can find the change,and in the next echocardiography examination, cardiac involvements degree can be sure. It can provid an early important information for diagnose and treatment.

Objective:To investigate the clinical value of indocyanine green(ICG) fluorescence imaging in complex laparoscopic cholecystectomy.Methods:The data of 96 patients with complicated gallbladder stones with cholecystitis and cholecystitis who underwent laparoscopic cholecystectomy(LC) from July 2018 to August 2020 in Hepatobiliary and Pancreatic Surgery of Huangshi Central Hospital of Edong Healthcare Group were retrospectively analyzed. Before operation, the patients were divided into experimental group( n=44) and control group( n=52) according to whether indocyanine green was injected intravenously. Seven hours before operation, the experimental group was injected with 2.5 mg indocyanine green, the experimental group underwent LC under guidance of ICG fluorescence imaging technology. The control group underwent conventional LC. The recognition rate of common bile duct and cystic duct, complete anatomy time of gallbladder triangle, operation time, intraoperative blood loss, bile duct injury and residual stone rat were compared. The measurement data obeying normal distribution was expressed by ( Mean± SD), and the t test was used comparison between groups, and the chi-square test or Fisher exact probability was used comparison between enumeration data. Results:The operation was successfully performed in both groups, In the control group, 1 case was converted to laparotomy, There was no perioperative death. Before the incision of the serosa of the triangle of the gallbladder, In the experimental group, the common bile duct recognition rate was 84.1%(37/44), the recognition rate of cystic duct was 72.7%(32/44). In the control group, the common bile duct recognition rate was 26.9%(14/52), the recognition rate of cystic duct was 28.8% (15/52). There were statistically significant differences in the recognition rate of common bile duct and cystic duct between the two groups ( P< 0.05). In experimental group, the time of complete dissection of gallbladder triangle, the operation time, the intraoperative blood loss were (30.2±8.6) min, (48.2±9.8) min, (16.3±5.2) mL, and (46.7±13.9) min, (65.2±15.4) min, (26.1±11.3) mL in the control group, there were statistically significant difference in the above indicators between experimental group and control group( P<0.05). There was no extrahepatic bile duct injury and residual stones in the experimental group. In the control group, there was 1 case of right posterior hepatic duct injury, 2 cases of common bile duct injury and 1 case of residual gallstone. There was no significant difference in extrahepatic bile duct injury and postoperative stone residual rate between the two groups ( χ2=3.532, P=0.081). Conclusion:ICG fluorescence navigation is helpful for early identification of common bile duct and cystic duct in laparoscopic complex cholecystectomy, which can avoid iatrogenic bile duct injury and has good clinical value.

BACKGROUND:Whether determination of tacrolimus blood concentration by different immunoassay methods can influence predictive ability to immunosuppressive effects and toxicity, and whether it can be more sensitive to reflect blood concentration in patients with renal dysfunction are worthy of studying.
OBJECTIVE:To analyze the correlation of tacrolimus (FK506) concentrations determined by enzyme-multiplied immunoassay technique (EMIT) and enzyme linked immunosorbent assay (ELISA) in combination with renal function parameters.
METHODS:133 clinical blood samples were col ected. EMIT and ELISA techniques were used to determine the FK506 concentration. The correlation of two determination methods were analyzed, combined with renal function. RESULTS AND CONCLUSION:In patients with renal dysfunction, the mean results and standard deviation mensurated by ELISA were higher than those by EMIT. For blood concentration in 5-20μg/L by ELISA, the incidence of renal dysfunction occurred less than by EMIT. The overal mean results of blood concentration for two methods appeared no significant difference (r=0.904 5, P>0.05). When the concentration was less than 2.0μg/L, the concentration results by EMIT were higher than those by ELISA (P<0.01). When the concentration was more than 2.0μg/L, there was no significant difference between two determination methods (P>0.05). These findings indicate that EMIT and ELISA has good correlation, which are both suitable for clinical routine determination of plasma concentration. It is not recommended for applying EMIT method to determine low blood concentrations (<2.0μg/L). The reference range of concentration should be compartmentalized depending on combination of determination methods and renal function.

Objective To compare complications incidence rates of 8-core and 12-core prostate biopsies guided with transrectal ultrasound retrospectively,and to study intervention ways to decrease complications.Methods The data of 260 consecutive patients undergoing first-time transrectal ultrasound-guided biopsy were analyzed.132 patients underwent 8-core biopsy and 128 patients underwent 12-core biopsy.Results In 8-core group,there were 7 cases of infections,15 cases of gross hematuria,7 cases of rectal bleeding and 6 cases of acute urinary retention,and in 12-core group,5 cases of infections,19 cases of gross hematuria,9 cases of rectal bleeding and 7 cases of acute urinary retention in 12-core group.There are no statistical differences of complications incidence rate between two groups (P ＞ 0.05).when prostate volume ＞ 45 ml,detection rate of prostate cancer in 12-core group was significantly higher than that in 8-core group.Conclusions There are no statistical differences of complications incidence rate between 8-and 12-core prostate biopsy.Homologous measure must be taken to reduce complications.12-core prostate biopsy is recommended as prostate volume ＞ 45 ml.

Objective To investigate the changes of CD4~+ T cell subset in the role of immuno-pathogenesis of type 1 diabetes mellitus(T1DM). Methods Real-time PCR was used to evaluate the ex-pression levels of transcriptional factors (T-bet, GATA-3, Foxp3, ROR-γt), cytokines (IFN-γ, IL-4, IL-10, IL-17A) and negative regulatory factors (CTLA-4, GITR) mRNA from CD4~+ T cells. The proportions of Th1, Th2, Tr and Th17 cells in peripheral blood were detected by flow cytometric analysis. Plasma cyto-kine (IFN-γ, IL-4, TGF-β and IL-6) concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Results (1) The proportions of Th1 cells in peripheral blood from children with T1DM were siguificanfly increased than that of healthy controls, and proportions of Th2 were decreased (P < 0.01). There were no significant differences between diabetic patients and healthy controls regarding the proportions of Tr cells and Th17 cells(P >0.05). (2) Transcription levels of T-bet and IFN-γ mRNA were significantly up-regulated, while GATA3 and IL-4 were significantly down-regulated in children with T1DM. The mRNA expression levels of Tr negativity regulatory factors such as IL-10, CTLA-4 and GITR were lower in CD4~+ T cells from children with TIDM compared with the controls(P <0.01). There were no statistically differences to be observed in mRNA expression levels of ROR-γt and IL-17A genes between two groups(P > 0.05).(3) In comparison with controls, serum concentrations of IFN-γ or IL-4 were remarkable increased or de-creased respectively (P < 0. 01), while TGF-β and IL-6 did not change in children with T1DM (P > 0.05). Conclusion The Th1/Th2 imbalance might be play an important role in immunopathogenesis of T1DM. Functional deficiency of Tr cell might further exacerbate Th1/Th2 imbalance and lead to disturbance of cellu-lar immune response.

[Summary] The proportions of CD19+CD24 hi CD38 hi regulatory B cells ( Breg ) and CD4+CD25+Treg in peripheral blood of children with type 1 diabetes mellitus ( T1DM) were detected by flow cytometric analysis. The concentration of interleukin 10 (IL-10) protein was measured by ELISA. The mRNA expression levels of Foxp3, cytotoxic T lymphocyte-associated antigen-4 ( CTLA-4 ) , and glucocorticoid-induced tumor necrosis factor receptor ( GITR) in CD4+T cells and IL-10 in CD19+ B cells were evaluated using real-time PCR. The results showed that the proportions of CD19+CD24hiCD38hiBreg in peripheral blood, the mRNA and protein levels of IL-10 in B cells from patients with T1DM were lower than those from healthy controls (P<0. 05). The mRNA expression levels of Treg associated factors such as Foxp3, CTLA-4, and GITR were lower in CD4+ T cells from children with T1DM compared with the controls(P<0. 05). These results suggest that Breg cell deficiency and dysfunction might be one of the important factors causing cellular immune dysfunction in patients with T1DM.

Objective To investigate the effects of polycyclic aromatic hydrocarbons ( PAHs) on regulatory T ( Treg) cells in newborns.Methods Blood samples were taken from the umbilical cord of sev-en newborn babies.CD4+CD25+T cells were isolated and treated with or without 300 nmol/L of phenan-threne for 72 hours in the presence of 100 IU/ml of IL-2.The expression of forkhead box P3 (Foxp3), sig-nal transducer and activator of transcription 3 (STAT3) and STAT5 were analyzed by 8-color flow cytometry. RT-PCR was performed to detect the expression of DNA ( cytosine-5 )-methyltransferase 1 ( DNMT1 ) , DNMT3a, DNMT3b and IL-4 at mRNA level.Pyrosequencing in combination with bisulphite sequencing was used to evaluate the methylation within the promoter and the Treg-specific demethylated region ( TSDR) of Foxp3 locus.Treg cells were cultured for 7 hours with autologous Tresp at Tresp/Treg ratios of 1 ∶1, 2 ∶1, 4 ∶1 and 8 ∶1 and stimulated with anti-CD3/CD28 beads and IL-2 for the evaluation of the immunosuppres-sive activities of Treg cells.Results (1) PAHs inhibited the expression of Foxp3 and the function of Treg cells collected from newborns.(2) PAHs significantly decreased the expression of STAT5 and Foxp3, but increased the expression of STAT3 (P<0.05).(3)PAHs enhanced the methylation of the promoter and the TSDR within Foxp3 gene.(4) The transcription levels of DNMT1, DNMT3a and DNMT3b in PAHs treated group were significantly higher than those of the control group (P<0.05).(5) More IL-4 was secreted by PAHs treated CD4+CD25+T cells, indicating that IL-4 was negatively correlated with STAT5, but positively correlated with STAT3.Conclusion PAHs decreased the number and inhibited the function of Treg cells in newborns.The possible mechanism might be related to the abnormal expression of STAT3 and STAT5 in-duced by IL-4 as well as the methylation within Foxp3 gene.

Objective To investigate the possible factors for differentiation affecting of neonatal regulatory T cells(Treg). Methods Umbilical cord blood was collected from 200 newborns. Treg number was detected by DNA demethylation in the Foxp3 of Treg - cell - specific demethylatedregion(TSDR)based on high resolution melting anal-ysis(HRMA),concentrations of 7,8 - dihydroxy - 9,10 - epoxy - benzo(a)pyrene(BPDE - DNA)adducts and interleukin - 4( IL - 4)in the supernatants of cord blood by enzyme - linked immunosorbent assay( ELISA),and follow - up questionnaires were carried out till 1. 0 - 1. 5 years,for recurrent wheezing or stubborn eczema in infants and related information on parental history of atopic diseases. Results (1)In wheezing group[(0. 48 ± 0. 05)% ]and ec-zema group[(0. 76 ± 0. 05)% ],the number of Tregs was significantly lower compared with that of the asymptomatic group[(1. 14 ± 0. 08)% ](t = 2. 62,2. 83,all P ﹤ 0. 05);the number of Treg in parental history of atopic group was significantly lower than that of the non - atopic group(P ﹤ 0. 05);but the Treg numbers in the non - atopic group was still lower than that of the asymptomatic group(P ﹤ 0. 05).(2)The concentrations of BPDE - DNA adducts in the wheezing group[(236. 30 ± 6. 59)ng/ L]and the eczema group[(173. 40 ± 7. 38)ng/ L]were higher than those of the asymptomatic group[(111. 01 ± 3. 36)ng/ L](t = 10. 35,6. 53,all P ﹤ 0. 05),while BPDE - DNA adduct concen-trations in the atopic group with parental history of wheezing or eczema in infants were lower than those of the non -atopic group(P ﹤ 0. 05).(3)The concentrations of IL - 4 in the wheezing or eczema group in the supernatants of cord blood was higher than the asymptomatic group(P ﹤ 0. 05). Conclusions Neonatal genetic factors and BPDE - DNA adducts could affect Treg differentiation,which are probably the reasons for the formation of allergic diseases.

BACKGROUND: Commutable reference materials (RMs) are suitable for end-users for evaluating the metrological traceability of values obtained using routine measurement systems. We assessed the performance of 6 routine measurement systems with validated secondary RMs. METHODS: We tested the homogeneity, stability, and commutability of 5 minimally processed human serum pools according to the standard guidelines. The serum pools were assigned values as per the reference procedure of the United States Centers for Disease Control and were used to evaluate the trueness of results from 6 commercial measurement systems based on enzymatic methods: 3 glucose oxidase (GOD) and 3 hexokinase (HK) methods. RESULTS: The prepared RMs were validated to be sufficiently homogenous, stable, and commutable with the patient samples. Method bias varied for different systems: GOD01, -0.17 to 2.88%; GOD02, 1.66 to 4.58%; GOD03, -0.17 to 3.14%; HK01, -3.48 to -0.85%; HK02, -3.83 to -0.11%, and HK03, -1.82 to -0.27%. CONCLUSIONS: We observed that the prepared serum glucose RMs were qualified for trueness assessment. Most of the measurement systems met the minimal quality specifications.
Blood Chemical Analysis/instrumentation/*standards ; Blood Glucose/*analysis ; Glucose Oxidase/metabolism ; Hexokinase/metabolism ; Humans ; Reagent Kits, Diagnostic ; Reference Standards ; Regression Analysis