Objective To develop a candidate reference method for the measurement of creatinine in human serum based on isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS). Methods An isotopically labeled internal standard [<'2>H<,3>] creatinine was added to the serum sample and equilibrated with the endogenous creatinine. The samples were treated with anhydrous ethanol to remove proteins by precipitation. After being washed with chloroform for further clean-up, the samples were analyzed by LC/MS/MS. Serum creatinine was quantified by a bracketing calibration. Results The within-run, between-run and total coefficients of variation ranged from 0.52% to 0.61%, 0.11% to 0.59% and 0.61% to 0.83%, and the averages were 0.57%, 0.43% and 0.73%, respectively. The analytical recoveries ranged from 99.09% to 101.13% with an average of 100.3%.The results of analyzing the certified reference material SRM 909b (Level Ⅰ and Ⅱ) and SRM 967b showed biases of less than 0.4%. Conclusions An ID-LC/MS/MS method for measuring serum creatinine has been developed. The method is highly precise and accurate and may be used as a candidate reference method for serum creatinine measurements.

Objective To develop a candidate reference method for the measurement of serum glucose based on isotope dilution liquid chromatography tandem mass spectrometry(ID-LC/MS/MS)Methods An internal standard [~(13)C_6]glucose was added to serum samples and equilibrated with endogenous glucose.Serum proteins were removed by a precipitation with anhydrous ethanol.Serum glucose and the internal standard were then reacted with 1-phenyl-3-methyl-5-pyrazolone and the formed derivatives were analyzed by liquid chromatography tandem mass spectrometry with multiple reaction monitoring(MRM).The method was calibrated with bracketing calibrators and serum glucose concentrations were calculated by comparing the peak area ratios of samples with that of the calibrators.Results The within-run,between-run and total coefficients of variation averaged 0.36%,0.47%and 0.61%,respectively.The analytical recoveries ranged from 99.0% to 100.9%.Results of analyzing the certified reference material SRM 965a showed an average biases of-0.20%.Conclusions An ID-LC/MS/MS method for measuring serum glucose has been developed.The method is highly precise and accurate and may be used as a candidate reference method.

Objective To develop a candidate reference method for the measurement of urea in human serum based on isotope dilution/gas chromatography/mass spectrometry.Methods [13C,15N2]Urea used as internal standard Was added to the serum sample and equilibrated with endogenous nonlabeled urea.The serum samples were treated with anhydrous ethanol to emove proteins by precipitation.The serum urea and labeled urea were converted into a trimethylsilyl derivative of 2-hydroxypyrimidine and analyzed by gas chromatography/mass spectrometry system with selected ion monitoring.The concentration of serum ureaWas calculated by the theory of bracketing method.Results The average value of within-run oefficient of vailation(CV),between-run CV and total CV of the procedure were 0.38%(ranged from 0.12%to 0.47%),O.62%(ranged form 0.49% to 0.87%)and 0.73%(ranged from 0.51% to 0.93%).Respectively.The analytical recoveries ranged from 99.37% to 100.95%.The resuhs of analyzing the certified refefence material SRM909b(Level Ⅰand Ⅱ)showed a bias less than 0.2%.Conclusion The procedure for measuring urea in serum is a highly accurate and precise method and can be used as a candidate reference method for serum urea assays.

Objective To evaluate the matrix effects in serum urea measurements of external quality assessment(EQA)materials and commercial reference materials(calibrators or controls)on enzymatic methods and to verify the trueness of the enzymatic methods.Methods The Clinical and Laboratory Stadards Institute(CLSI)EP 14-A2 protocol was used for the evaluation of matrix effect.An isotope dilution gas chromatography mass spectrometry method was used as the comparative method.Twenty five fresh patient serum samples,15 EQA materials and 13 calibrators or controls were analyzed with 7 enzymatic methods (evaluated methods)and the comparative method and results were processed according to the protocol. The trueness of the evaluated methods were also assessed by comparing the fresh sample results obtained with the evaluated and comparative methods.Results Eight of 15 EQA materials and 3 of 13 calibrators or controls showed no matrix effects on all the 7 routine methods.One processed sample showed matrix effect on all the routine methods.Method dependent matrix effects of other materials were observed on other materials.Calibration biases were also observed on some enzymatic methods.Conclusions Matrix effects and calibration bias have been observed in serum urea measurements.Continued efforts are needed for improving the accuracy and the comparability of serum urea measurements.

Objective To develop a new isotope dilution gas chromatography mass spectrometry method (ID/GC/MS) for the measurement of serum cholesterol.Methods Serum was mixed with an isotope labeled internal standard ([3,4-~(13)C]-cholesterol) and treated with alcoholic sodium hydroxide to hydrolyze cholesterol ester to cholesterol.Cholesterol and internal standard was extracted and derived by N, O-Bis(trimethylsilyl) trifluoroacetamide to trimethylsilyl ethers.The derivation products were analyzed by capillary column GC combined with electron impact MS using scan and selected ion monitor (SIM) modes. Signals of cholesterol internal standard were corrected for the contributions from cholesterol and the signal ratio of cholesterol to internal standard for the calibrators were linearly regressed against cholesterol concentrations.The resulted regression equation was used for the calculation of serum cholesterol concentrations.Results The new ID/GC/MS method showed a mean within-run coefficient variance (CV) of 0.04%-0.81%.Comparison with two levels of standard reference material (SRM1951a) of National Institute of Standards and Technology (NIST) displayed a bias of 0.19% and 0.90% respectively.Conclusion A time-gaining ID/MS method has been established that is highly precise and accurate and can be used for the measurement of serum cholesterol.

Objective:To analyze the type and frequency of thalassemia gene mutation in children aged 0 to 18 years in Chengdu.Methods:A total of 568 children from Chengdu, who were initially positive for thalassemia during screening from September 2018 to July 2021, were recruited. Among them, there were 308 males and 260 females. The type of mutation and distribution of α and β types of thalassemia in this cohort was analyzed utilizing PCR reverse dot blot.Results:Among the 568 children, 356 were genetically diagnosed as thalassemia, with a total positive rate of 62.68%. Among them, there were 140 cases of α-thalassemia with a positive rate of 24.65%, and 202 cases of β-thalassemia with a positive rate of 35.56%. There were 14 carriers of α-complex β-thalassemia gene, and the positive rate was 2.46%. Among these cases, the types of α-thalassemia gene mutation were mainly αα/--sea (79.29%), αα/-α3.7 (7.86%), and-α3.7/--sea (7.14%) genotypes, accounting for 94.29% of all types. In the 202 β-thalassemia patients, 199 heterozygous mutations were identified, mainly including cd17(A?T) (36.13%), cd41-42(-TCTT) (32.68%), IVS-2-654(C?T) (20.79%), and accounting for 88.61% of all types of gene mutation, and 3 compound heterozygous mutations were detected. α-complex β-thalassemia was detected in 14 patients, including cd41-42(-TCTT)/-α3.7, VS-2-654(C?T)/--sea, cd17(A?T)/-α3.7 and cd41-42(-TCTT)/--sea, which accounting for 57.14% of all types of gene mutation. Our results showed that there is no sex difference between α and β thalassemia in Chengdu area, whereas the prevalence of α combined with β thalassemia is higher in males ( P=0.003). Conclusions:The type of α-thalassemia mutation in Chengdu is mainly αα/--sea, whereas β-thalassemia with cd17 (A?T) mutations and α-complex β-thalassemia are more frequent in males. This study provides a reference for the formulation of prevention and control strategies for thalassemia in Chengdu.

OBJECTIVE:To evaluate the efficacy and safety of Yupingfeng powder combined with second- generation antihistamines versus second-generation antihistamines for chronic urticaria(CU)systematically,and to provide evidence-based reference for clinical treatment for CU. METHODS:Retrieved from PubMed,Embase,The Cochrane Library,CJFD,VIP and CBM,RCT about therapeutic efficacy(total response rate,cure rate,recurrence rate)and safety(the incidence of ADR)of Yupingfeng combined with second-generation antihistamines(trial group)versus second-generation antihistamines(control group) in the treatment of CU were collected. The data extraction was performed for included clinical studies,and Meta-analysis was performed by using Rev Man 5.3 statistical software after quality evaluation with Cochrane Handbook 5.1.0 evaluation criteria. RESULTS:A total of 34 RCTs were enrolled,involved 3 405 patients in total. Results of Meta-analysis showed that the total response rate [OR=4.02,95%CI(3.03,5.34),P<0.001],cure rate [OR=2.25,95%CI(1.95,2.60),P<0.001] and recurrence rate [OR=0.33,95%CI(0.26,0.42),P<0.001] of trial group were significantly better than those of control group,with statistical significance. There was no statistical significance in the incidence of ADR between 2 groups [OR=0.98,95%CI(0.71,1.37),P=0.92]. CONCLUSIONS:For CU therapy,Yupingfeng powder combined with second-generation antihistamines is better than second-generation antihistamines alone in improving total response rate and cure rate,reducing recurrence rate,both have similar safety.

Objective To evaluate individualized treatment of aseptic femoral nonunion after interlocking intramedullary nailing based on the morphology of each nonunion and nailing stability in each specific patient.Methods This study reviewed 108 patients who had been treated and followed up for more than one year for aseptic femoral nonunion following interlocking intramedullary nailing between February 2012 and February 2016.They were 89 men and 19 women,aged from 23 to 65 years (average,45.5 years).The classification and corresponding treatments were as follows:Type Ⅰ (15 cases),characterized by callus (+)/bone defect (-) and nailing stability (+),were treated by augmentation plating;Type Ⅱ (43 cases),characterized by callus (+)/bone defect (-) and nailing stability (-),were treated by exchange for larger nailing/larger nailing with poller screws;Type Ⅲ (23 cases),characterized by callus (-)/bone defect (+) and stability of nailing (+),were treated by augmentation plating with bone grafting;Type ⅣV (27 cases),characterized by callus (-)/bone defect (+) and stability of nailing(-),were treated by double plating and bone grafting.The healing of bone nonunion,complications and assessments for bone and function were followed up.Results All these patients received follow-up for 12 to 14 months (average,12.5 months).All the nonunions were healed with no postoperative complications.Bone healing was achieved after 4 to 8 months (average,5.5 months).The good to excellent rates for bone and function were 100％.Conclusion To achieve better surgical outcomes,the treatment of aseptic femoral nonunion after interlocking intramedullary nailing should be individualized according to the morphology of each nonunion and nailing stability in each specific patient.

Medical ethics has a long history and rich connotations.It has developed from the simple "medical morality" of ancient times to the modem medical ethics.The basic principles of medical ethics include autonomy,non-maleficence,beneficence,justice,and so on.Researchers often conduct clinical researches in the balance between achievements and ethical norms.Clinical researchers of surgery should have a deep understanding of medical ethical principles and strictly abide by medical ethics.Ethics committee should strictly perform their duties and play the role of inspection and supervision.Modem medical knowledges should be popularized throughout the society to make clinical research correctly understood.Adhering principles of ethics first,people orientation and cooperation practice,with patients' benefit as evaluation criteria,balance of surgical "Dao" and "Shu" can be achieved.

Objective:To investigate the effects of caspase-11 non-canonical inflammasome on the Leptospira interrogans ( L. interrogans)-induced secretion of inflammatory cytokines in J774A.1 cells. Methods:Murine mononuclear macrophage cells (J774A.1) were infected with L. interrogans strain 56601. Expression of caspase-11, IL-1β, IL-1α and IL-18 at mRNA level in J774A.1 cells were detected by real-time RT-PCR. The levels of caspase-11, IL-1β, IL-1α and IL-18 in the culture supernatants of J774A.1 cells were detected by ELISA. Results:Real-time RT-PCR showed that caspase-11 expression at mRNA level was 5.12, 14.21, 8.94, 14.06, 18.58 and 0.93 times of that in uninfected cells after 1, 2, 4, 8, 12 and 24 h of L. interrogans infection, and respectively decreased to 0.10, 0.07, 0.10, 0.09, 0.07 and 0.45 times after caspase-11 inhibitor intervention ( P<0.05). Expression of IL-1β, IL-1α and IL-18 at mRNA level was significantly increased after infection ( P<0.05). After the intervention with caspase-11 inhibitor, IL-1β mRNA decreased to 0.05, 0.03, 0.02, 0.05, 0.06 and 0.02 times ( P<0.05); IL-1α mRNA decreased to 0.14, 0.07, 0.15, 0.10, 0.03 and 0.06 times ( P<0.05); IL-18 mRNA decreased to 0.08, 0.10, 0.16, 0.18, 0.10 and 0.07 times ( P<0.05). ELISA results showed that the expression of caspase-11, IL-1β, IL-1α and IL-18 at protein level was significantly increased. After the intervention with caspase-11 inhibitor, caspase-11 level decreased to 43.07, 41.64, 51.96, 86.56, 105.36, and 129.95 pg/ml ( P<0.05); IL-1β level decreased to 15.01, 14.19, 68.02, 31.20, 173.13 and 104.98 pg/ml ( P<0.05); IL-1α level decreased to 12.14, 15.40, 38.01, 21.97, 24.48 and 27.09 pg/ml ( P<0.05); IL-18 level decreased to 96.27, 102.21, 85.34, 116.28, 155.36 and 114.03 pg/ml ( P<0.05). Conclusions:Caspase-11 non-canonical inflammasome was involved in the mediation of IL-1β, IL-1α and IL-18 secretion in mouse mononuclear macrophages after L. interrogans infection.