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 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 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.

Objective:To investigate the clinicpathological, immunohistochemical and molecular genetic features of malignant mixed mesodermal tumor (MMMT) in the female reproductive system.Methods:To analyze its histopathological characteristics, we performed a retrospective review of the MMMT cases diagnosed at PLA General Hospital, Beijing, China during 2005-2019 using its surgical and pathological databases. EnVision immunohistochemical staining was used to detect the expression of ER, PR, p16, p53 and MMR proteins.Results:Fifty cases were conformed to the diagnosis, including 29 cases originated in the uterus, 16 cases in ovary, 4 cases of synchronous occurrence in uterus and ovary, 1 case in cervix. The tumor was histologically composed of two components, namely carcinoma and sarcoma ones, with clear borderline or blend mutually. The proportion of cancer component in the whole tumor ranged from 5%-90%. The proportion of carcinoma was more than 50% in 76% of the cases, and less than 50% in 24% of cases, including 2 cases with<10% of carcinoma. In the cases of primary uterine MMMT, the main carcinoma type was high grade endometrioid carcinoma (55%, 16/29). In ovarian MMMT, the main carcinoma type was serous carcinoma (12/16), while that of cervical MMMT was squamous cell carcinoma. The others were clear cell carcinoma or the undifferentiated carcinoma. There was one carcinoma type in most cases, only 7 cases had two carcinoma types. Homologous sarcomas, including stromal sarcoma, leiomyosarcoma and high-grade spindle cell sarcomas, were more commonly found in uterine MMMT (72.4%, 21/29). While heterogenic sarcomas, including chondrosarcoma, osteosarcoma and rhabdomyosarcoma, were more commonly noted in ovarian MMMT (12/16) than MMMT of other sites. There were 10 cases that consisted of two types of sarcomas. The synchronous MMMT of uterus and ovary had similar morphology and the types of carcinoma and sarcoma. The tumor cells that spread or metastasized to lymph node, omentum, intestinal wall or skin were all carcinoma cells, and were morphologically consistent with the original tumors. Immunohistochemically, ER and PR were both negative (23/25 in uterine, 8/10 in ovarian tumors). p16 was strongly positive (11/11 in uterine tumors, and 6/6 in ovarian tumors), with similar expression patterns in the carcinoma and sarcoma components. p53 showed mutant-type staining (64%, 21/33) and expressed synchronously in carcinoma and sarcoma components. p53 mutation was found in 35% cases of endometrial carcinoma and 46.7% cases of non-endometrial carcinoma. p53 mutation was also found in only 31.8% cases of heterogenic sarcomas, but in 50% of non-heterogenic sarcomas. Twenty-eight cases (28/33, 85%) presented intact mismatch repair proteins, while 5 cases (5/33, 15%) presented deficient mismatch repair proteins.Conclusions:MMMT in female reproductive system is a rare high-grade biphasic tumor with complex and diverse morphology. The immunohistochemical features are characterized by negative ER/PR and strongly positive p16, mostly mutant p53 and proficient mismatch repair proteins. The patients with a high FIGO stage have worse prognosis.

Objective:To investigate the expression of PD-L1, CD4, CD8 and CXCL-13 in cervical carcinoma, and their clinicopathological significance was analyzed.Methods:A total of 77 patients with cervical carcinoma in the Seventh Medical Center, PLA General Hospital from January 2019 to December 2021 were included. All patients received radical surgical resection in the Seventh Medical Center of Chinese PLA General Hospital. The expression of PD-L1, CD4, CD8 and CXCL-13 was detected by immunohistochemical (IHC) method. The correlation between IHC markers and patients′ clinicopathological parameters was analyzed.Results:There were 59 cases of squamous cell carcinoma and 18 cases of adenocarcinoma (ranging from 29 to 69 years) with an average of (49.4±9.8) years. PD-L1 was expressed in different degrees in cervical squamous cell carcinoma and adenocarcinoma (χ2=4.975, P=0.026); CD4 +, CD8 +and CXCL-13 +tumor infiltrating lymphocytes (TIL) were observed in the carcinoma cell nests and peritumoral stroma. PD-L1 expression in cervical carcinoma was moderately correlated with the number of CD4 +TIL in the carcinoma nests, and the number of CD8 +, CXCL-13 +TIL infiltration in the carcinoma nests and stroma, but not to the patient′s age, histologic differentiation, presence or absence of vascular invasion, presence or absence of lymph node metastasis and FIGO stage ( P>0.05). Conclusions:The high expression of PD-L1 in cervical carcinoma tissues is closely related to the number of TIL in the carcinoma nests and peritumor stroma, suggesting that they may have important reference value for predicting the response to immunotherapy in patients with cervical carcinoma.