Objective To compare the success rates of different culture methods for hemorrhagic amniotic fluid.Methods Thirty-one hemorrhagic amniotic fluid samples from pregnant women who were subjected to chromosomal examination at Anhui Maternal and Child Health Hospital were collected from January 2021 to December 2022.Two culture methods,the slide in situ culture box method(re-ferred to as the slide method)and the plastic bottle culture combined with slide in situ culture box method(referred to as the combined culture method),were used for cell cultivation.All the cells were harvested and stained with Giemsa staining,then the number of eligi-ble karyotype was counted and the success rates were compared between the two methods.Results Among the 31 cases of the slide method,21 were successfully cultured with a success rate of 67.7％.For the combined culture method,all the 31 cases were success-fully cultured with a success rate of 100％.The success rate of the combined culture method was significantly higher than that of the slide method(P＜0.05).Of 31 bloody amniotic fluid samples,three cases were fresh bleeding,and an average number of eligible kary-otype was 8 in the slide method and 32 in the combined culture method.Twenty-eight cases were old bleeding,and an average number of eligible karyotype was 13 in the slide method and 53 in the combined culture method.The number of eligible karyotype in the com-bined culture method was significantly higher than that of the slide method(P＜0.05).Conclusion The combined culture method is suitable for the cultivation of hemorrhagic amniotic fluid samples,and should be worthy of promotion in the clinics.

Objective:To establish a rapid method to detect the minimum inhibitory concentration (MIC) of imipenem in Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) based on ompK36 gene′s GD mutation. Methods:This was a methodological evaluation study. A total of 258 isolates of Klebsiella pneumoniae were collected from Lishui Municipal Central Hospital from March 2011 to December 2019. Porin gene ompK36 and carbapenemase genes blaKPC, blaNDM, blaIMP and blaOXA-48 were amplified by PCR and confirmed by sequencing. The MIC was detected and confirmed by microbroth dilution susceptibility test, and the corresponding patterns of genotype and MIC were constructed. Based on the patterns, a method for rapid detection of imipenem MIC by real-time fluorescence PCR (RT-PCR) was designed and established. The 159 isolates of non-repetitive Klebsiella pneumoniae collected by Lishui Disease Prevention and Control Center (CDC) from 2017 to 2019 were used for further verification. The sensitivity and specificity were calculated by fourfold table. Kappa test was used to compare the consistency between RT-PCR and microbroth dilution susceptibility test. Results:Among 258 isolates, 109 isolates did not carry carbapenemase gene, 65 isolates carried ompK36 gene GD mutation, 127 isolates carried blaKPC, 15 isolates carried blaNDM, 9 isolates carried blaIMP, and blaOXA-48 was not detected. With mircobroth dilution susceptibility test as the standard, there were 3 corresponding patterns between the drug resistance gene and the imipenem MIC of Kp: when all the 4 carbapenemase genes were negative, MIC≤1 mg/L, the sensitivity was 100% (107/107) and the specificity was 98.4% (125/127); when blaKPC was positive and ompK36 gene GD mutation was negative, 4 mg/L≤MIC≤16 mg/L, the sensitivity was 88.2% (60/68) and the specificity was 98.8% (164/166); when blaKPC and ompK36 gene GD mutation were both positive, MIC≥32 mg/L, the sensitivity was 96.6% (57/59) and the specificity was 96.6% (169/175). RT-PCR detected blaKPC, blaNDM, blaIMP, blaOXA-48 genes accurately.The RT-PCR results of ompK36 gene GD mutation in the KPC-producing isolates were 100% consistent with the sequencing results. In the 159 isolates from Lishui CDC, the sensitivity and specificity of imipenem MIC detected by RT-PCR were higher than 95% in all 3 patterns with mircobroth dilution susceptibility test as the standard, and Kappa value was 0.971. Conclusion:The RT-PCR based on ompK36 gene GD mutation was helpful to quickly determine the MIC range of imipenem in KPC-Kp.

BACKGROUND:There is an internal relationship between hyperhomocysteinemia and vascular calcification.However,the pathogenesis of hyperhomocysteinemia promoting vascular calcification is still unclear. OBJECTIVE:To investigate the role of bone morphogenetic protein-2 in hyperhomocysteinemia-induced vascular calcification. METHODS:Human carotid wax samples were divided into a calcified group(n=29)and a non-calcified group(n=13)according to the presence or absence of calcified plaque.Sixteen ApoE-/-mice were randomly divided into a control group and a hyperhomocysteinemia group,with 8 mice in each group.Bone morphogenetic protein-2 vector was used to transfect rat thoracic artery smooth muscle A7r5 cells,and gradient concentration of homocysteine(50,100,200,and 400 μmol/L)was utilized to treat A7r5 cells.Calcification was detected by alizarin red staining and hematoxylin-eosin staining.The interaction of bone morphogenetic protein 2 with Runt-related transcription factor 2 was detected by immunofluorescence,and the expressions of bone morphogenetic protein 2,Runt-related transcription factor 2,and α-smooth muscle actin were detected by immunohistochemistry and western blot assay. RESULTS AND CONCLUSION:(1)Human carotid artery tissue staining revealed that compared with the non-calcification group,inflammatory cells increased and calcification positive rate increased in the calcification group(P<0.05).Compared with the non-calcification group,the expressions of bone morphogenetic protein-2 and Runt-related transcription factor 2 were up-regulated,and the expression of α-smooth muscle actin was decreased in the calcification group(all P<0.05).(2)The staining of mouse arterial specimens exhibited that,the positive rate of calcified area in the hyperhomocysteinemia group was significantly higher than that in the control group(P<0.05);serum homocysteine level in the hyperhomocysteinemia group was significantly higher than that in the control group(P<0.05).Compared with the control group,the expressions of bone morphogenetic protein-2 and Runt-related transcription factor 2 were up-regulated,and the expression of α-smooth muscle actin was decreased in the hyperhomocysteinemia group(all P<0.05).(3)A7r5 cell culture analysis demonstrated that with the increase of homocysteine concentration gradient,the degree of calcification,the content of bone morphogenetic protein-2 and Runt-related transcription factor 2 protein in A7r5 cells increased(P<0.05),and the content of α-smooth muscle actin protein decreased(P<0.05).(4)The A7r5 cell culture analysis of overexpressed bone morphogenetic protein 2 showed that the calcification degree of the overexpressed bone morphogenetic protein 2 group was increased compared with the corresponding control group,the β-sodium glycerophosphate group,and the homocysteine group.RUNt-related transcription factor 2 expression up-regulated(P<0.05)and α-smooth muscle actin expression down-regulated(P<0.05).(5)The expression of bone morphogenetic protein 2 increased in A7r5 cells cultured with homocysteine in calcified medium,and the expression of Runt-related transcription factor 2 increased with the increase of bone morphogenetic protein 2 expression.(6)The results confirm that bone morphogenetic protein-2 is a key target gene in the regulation of smooth muscle cell phenotypic transformation resulting in vascular calcification by hyperhomocysteinemia.Targeted regulation of bone morphogenetic protein-2 reduces hyperhomocysteinemia-induced vascular calcification.

Objective:To establish a method for determintation of chlorogenic acid and linarin in Yejuhua granules by HPLC.Methods:We applied HPLC methods. The Kromasil 100-5 C18 column (250 mm×4.6 mm,5 μm) was used, the mobile phase was acetonitrile-0.4%H 3PO 4 solution (gradient elution), the flow rate was 1.0 ml/min, the dection wavelenghth was 334 nm and the column temperture was 32 ℃. Results:Chlorogenic acid and buddleoside had good linearity in the ranges of 0.30-1.50 μg ( r2=0.999 1) and 0.12-0.62 μg ( r2=0.999 8), respectively. The average recoveries were 99.70% and 96.67%, with RSD<2%, respectively. Conclusion:The method is simple, rapid, reliable, efficient, and can be used for determination of chlorogenic acid and buddleoside in Yejuhua Granules.

Objective:To evaluate the clinical value of high-definition intelligent endoscopy (iSCAN) combined with stroboscopy in identifying vocal cord leukoplakia.Methods:Seventy-nine patients with vocal cord leukoplakia who underwent CO 2 laser laryngeal microsurgery and diagnosed by histopathology were recruited between October 2020 to August 2021. The morphological features, microvascular morphology and mucosal waves were observed by stroboscope; SPSS 20.0 software was used for statistical analysis. Results:There were 79 patients with a total of 119 lesions (56 on left and 63 on right).Pathological examination showed that 51 sides of the vocal cords were malignant lesions (severe dysplasia, carcinoma in situ and invasive carcinoma), and 68 sides were benign lesions.Under stroboscopy, 69 sides of mucosal wave were normal or slightly decreased, and 50 sides were severely decreased or disappeared.The decrease degree of mucosal wave was positively correlated with malignant lesions ( ρ=0.687, P<0.001).Under iSCAN endoscopy, there was a positive correlation between the morphological changes of microvessels at the lesion site (vertical) and the malignant lesion ( ρ=0.687, P<0.001).Univariate analysis showed that lesion size, thickness, uneven color, granular elevation, peripheral erythema and asymmetry were positively correlated with malignant lesions ( ρ=0.530, 0.401, 0.538, 0.315, 0.497, 0.281, P<0.05).Logistic regression analysis showed that the risk of pathological malignancy with large lesions was 5.437 times higher than those of small lesions, the vertical vascular changes under iSCAN were 8.711 times higher than that of normal vascular morphology, and the severe reduction or disappearance of mucosal waves was 9.12 times higher than that of normal or mild reduction of mucosal waves. Conclusion:ISCAN can be combined with staphyloscopy to comprehensively observe and evaluate the changes of vocal cord morphology, submucosal microvessels and mucosal wave of vocal cord in patients with vocal cord leukoplosis, thus improving the ability to distinguish benign and malignant lesions.

Objective:To evaluate the best parameter of predicting the operation time and clearance of flexible ureteroscopic lithotripsy through comparing correlations between three stone burden parameters (diameter, area, volume) and the operation time or clearance retrospectively.Methods:Clinical data and CT images of 70 patients who performed flexible ureteroscopic lithotripsy because of single kidney stone in our center from January 2018 to December 2019 were retrospectively reviewed. There were 46 males and 24 females; their age was (47±12) years old. Stones were located on the left side in 28 cases and right side in 42 cases; 32 cases in the renal pelvis , 29 cases in the lower calyx, 6 cases in the middle calyx and 3 cases in the upper calyx. The free software ITK-SNAP 3.6.0 to segment kidney stones in 3D models with the CT image was used. The stone volume was calculated automatically after the segment. The largest section of the stone on the CT coronal plane was selected to measure the maximum length (D) and width (d) of the stone, the maximum diameter of the stone was D, and the stone area was calculated using the formula 0.25πDd. The patients were divided by the operation clearance into total clearance group and partial clearance group. The correlations between three stone burden parameters (volume, diameter, area) and operation time or clearance of the flexible ureteroscopic lithotripsy were compared. Simple linear regression model was also applied to compare three measurement methods. Then other factors which may affect the operation time was evaluated with the stepwise linear regression model, such as stone component and location.Results:The median operation time was 63(50, 84)min. Of 70 cases, 47 cases were in the stone-free group, with stone volume 633(248, 1 087)mm 3, maximum diameter 15(10, 19)mm, and area 82(49, 186)mm 2. 23 cases were in the non stone-free group, with volume 696(408, 1 418)mm 3, maximum diameter 15(12, 20)mm, area 105(73, 201)mm 2. There was no difference between the two groups in volume, maximum diameter and area of stones (all P>0.05). The stone-free rate of the diameter >2 cm group was 55% (6/11), ≤2 cm group was 70% (41/59). There was no significant difference between the two groups. Correlation between stone volume and operation time is the best. The correlation coefficient of stone volume is 0.58, of stone diameter is 0.33, of stone area is 0.34. Coefficients of determination of the stone volume linear regression is the best, too. R square of stone volume is 0.36, of stone diameter is 0.17, of stone area is 0.22. Forward stepwise regression model shows stone volume is the most important parameter which correlate with operation time. None of stone volume, diameter or area has significant correlation with the clearance of stone. Conclusion:Stone volume is the best predictive parameter of the stone burden because it has the best correlation with the operation time of the flexible ureteroscopic lithotripsy of the single kidney stone.

OBJECTIVE: To explore the value of in situ amniocyte culture for prenatal diagnosis. METHODS: 2716 amniotic fluid samples were cultured in situ on slides. After the culture, the slides were stained, photographed and analyzed. RESULTS: All samples were successfully analyzed, with the success rates for primary culture and subculture being 98.42% and 1.58%, respectively. 224 samples (8.25%) were detected with chromosomal aberrations, which included 125 cases with trisomy 21, 31 with trisomy 18, 3 with trisomy 13, 4 with 45,X, 17 with 47,XXY, 5 with 47,XYY, 1 with 48,XXY,+18, 1 with 48,XXYY, 26 with structural chromosomal aberrations, and 11 with mosaicisms for aneuploidies. CONCLUSION In situ amniocyte culture is stable and has a high success rate, and is capable of identifying true and false mosaicisms, which can improve the accuracy of prenatal diagnosis.

Objective To investigate the outcomes of fetuses with hemolytic anemia caused by red cell alloimmunization following intrauterine transfusion (IUT),and to analyze the influence of hydrops fetalis on IUT treatment.Methods A retrospective analysis was conducted on 70 fetuses,who were admitted to the Fetal Medicine Center,the First Affiliated Hospital of Sun Yat-sen University from January 2005 to May 2018,with hemolytic disease requiring IUT.Clinical data of the fetuses and the gravidas were collected and divided into hydrops group (17 cases) and non-hydrops group (53 cases) based on their conditions before IUT.Results of routine blood tests before and after the first IUT,gestational age at the first IUT,prognosis and outcomes of the fetuses were compared between two groups.t-test,rank-sum test,Chi-square test (or Fisher's exact test) and multivariant logistic regression analysis were used for data analysis.Results Totally,the 70 fetuses underwent 231 times of IUT.Compared with the non-hydrops group,the hydrops group had a significantly increased incidence of severe anemia [14/17 vs 47.2％ (25/53),x2=6.458,P=0.011],but decreased hemoglobin [(38.5 ± 21.4) vs (68.7± 19.3) g/L,t=5.471,P＜0.001] and hematocrit level [0.110 (0.044-0.246) vs 0.222 (0.077-0.299),Z=-4.390,P＜0.001] before the first IUT.After the IUT,the survival rate of the fetuses in hydrops group was significantly lower than that of the non-hydrops group [11/15 vs 94.3％ (50/53),P=0.038].There was no significant difference in gestational age at birth,birth weight,neonatal hemoglobin level at birth,the incidence of exchange transfusion,the number of blood transfusions required or the incidence of severe neonatal complication between the two groups (all P＞0.05).Logistic regression analysis indicated that the fetal hydrops was an independent risk factor for fetal survival (OR=12.8,95％CI:1.2-136.4,P=0.035).Conclusions Hydrops fetalis might reduce the survival rate of fetal hemolytic disease after 1UT.

Objective: To analyze the molecular epidemiological characteristics of Escherichia coli producing NDM-5 carbapenemase in the neonatal department of our hospital. Methods: Three carbapenem-resistant Escherichia coli strains（E1, E2 and E3） isolated from neonatal ward of our hospital from August to September of 2017 were collected. Vitek 2 Compact system combined with K-B disk method was used for drug sensitivity test. The resistance genes were detected by PCR amplification. Plasmid replicon typing was detected by PCR. Plasmid conjugation tests were performed to explore the conjugating transfer of plasmids in the three strains. The homology of the three strains was analyzed by multiple locus sequence typing (MLST) and pulse field gel electrophoresis (PFGE). Results: Drug susceptibility test showed that the three bacteria were resistant to most β-lactam antibiotics except Aztreonam, and resistant to quinolones and SMZ-TMP, but sensitive to aminoglycosides drugs. PCR and sequencing results indicated that the three strains carried bla SHV gene and extended-spectrum beta-lactamase gene (bla SHV , bla TEM and bla CTX-M ). The plasmid replicon type was IncX3. Transfer test of E1 strain was successful. MLST results indicated that all the three strains were ST1642 type. MLST and PFGE results indicated that the bands of the three bacteria were identical. Conclusion Both NDM-5 carbapenemase and extended-spectrum beta-lactamase were detectable in the three strains of carbapenem-resistant bacteria from neonatal department. MLST and PFGE results suggested that the three strains were from the same clonal source.

Objective To determine the correlation between phosphorylation extracellular signal-regulated kinase (p-Erk) and monopolar spindle 1 (Mps1) in colorectal cancer patients with the BRAF V600E mutation or not, and to explore the relationship between the expression of p-Erk and Mps1 with the clinicopathological features and prognosis of colorectal cancer patients. Methods Two hundred and eighty-eight paraffin-embedded tissue sections containing both the carcinoma and its adjacent non-neoplastic colorectal tissue were collected from January 2009 to June 2015 in the First Hospital of Shanxi Medical University. BRAF mutation was detected by Sanger sequencing. Kaplan-Meier survival analysis was used to analyze the correlation between BRAF V600E and prognosis. Immunohistochemistry was used to detect the expression level of p-Erk and Mps1 in colorectal cancer with wild type or BRAF V600E mutation. The correlation between p-ERK and Mps1 expression was analyzed by using linear regression analysis. Results The BRAF V600E mutation rate was 5.2 % in colorectal carcinomas. In addition, the poorly differentiated tumours and mucinous tumours had higher incidence of BRAF mutations than well differentiated tumours and non-mucinous tumours respectively [14.3 % (9/63) vs. 2.7 % (6/225),χ 2= 11.208,P = 0.001; 25.0 % (6/24) vs. 3.4 %(9/264),χ 2=16.630,P <0.001). The positive rate of p-Erk and Mps1 in colorectal carcinomas with BRAF V600E was significantly higher than that in colorectal carcinomas with BRAF WT (14/15 vs. 3/15, P <0.05; 15/15 vs. 3/15, P < 0.05]. It was found that the p-Erk expression correlated positively to the Mps1 expression (R2= 0.419,P < 0.001). The expressions of p-Erk protein in poorly differentiated adenocarcinoma was significantly higher and mucinous adenocarcinoma than those in high differentiated adenocarcinoma and non-mucinous adenocarcinoma (χ 2= 6.679, P = 0.01; χ 2= 5.735, P = 0.017), as well as in the group with lymph node metastasis than without lymph node metastasis (χ 2=5.436, P =0.02). Positive rate of Mps1 in poorly differentiated carcinoma was higher than that in well differentiated adenocarcinoma of colorectal carcinomas (χ 2=7.950, P =0.009). The Kaplan-Meier survival analysis indicated that patients with BRAF V600E had a worse survival rate than BRAF WT patients. Conclusions BRAF V600E may play an important role in specific pathological kinds of colorectal carcinomas, which is expected to be an independent prognostic factor. The expression of p-Erk is significantly correlated with Mps1 in colorectal carcinomas, suggesting that Mps1 may become a new potential target for targeted therapy.