ObjectiveTo facilitate the establishment of fully automatic clinical diagnostic nucleic acid assay,a study was carried out for developing a fast and convenient method of extracting highly purified viral nucleic acid from human blood with magnetic beads.Method Five KHB (Shanghai Kehua Biological Engineering Company Limited) internal strong positive HBV serum samples were used in the process,for the method development,screening and selecting the magnetic beads based on magnetism,suspension power and extraction effects of HBV DNA,the optimization of the selected key components of extraction reagents were performed,which including the quantity of magnetic beads( 1 - 1.75 μl/ml),the buffer pH of lysis (4 -9),washing conditions with or without alcohol,elution for proliferation with or without magnetic beads,and etc.The evaluation for the extraction method was carried out including linearity,precision,sensitivity,specificity,anti-interference and resistance to contamination.The magnetic beads method was compared with domestic mature boiling method in a quantitative test with 100 HBV serum samples,the magnetic beads method was also compared with the interntional Gold standard Roche COBAS Tagman test method ( Roche method) in a quantitative test with 50 HBV serum samples and the results were statistically compared for their linear regression.ResultsOptimization experiment result showed superparamagnetic beads A was the best among the candidates ( C,B,D) in terms of their physical properties and extraction effects of DNA.The optimum condition of the assay were as follow:magnetic beads best used at 1.25 μl/ml,buffer pH 7 - 8 of lysis,ethanol was eliminated from washing buffer,proliferation can be carried out with magnetic beads.The parallel comparison experiments with five KHB internal strong positive HBV serum sample and gradient diluted serum sample between beads method and boiling method were carried out.The results showed that the linear range of beads method was 4 × 101 -4 × 108 IU/ml and that of boiling method was 4 × 102 -4 × 108 IU/ml.The sensitivity for beads method was 30 IU/ml and that of boiling method was 100 IU/ml.The precision CV of beads method was 6.0％ and boiling method was 11.9％.The false positive rate was 0 for beads method vs 3.1％ for boiling method.The anti-interference ability experiment showed that when sample contained sodium heparin concentration exceed 62.5 U/ml, there was significant drop of HBV DNA detection with boiling method and no HBV DNA was detected when sodium heparin concentration in the sample reached 1000 U/ml.However,sample sodium heparin concentration had no effect on HBV DNA detection with beads method.The comparison study between beads method and boiling method was carried out by use of 100 clinical HBV positive serum samples.The result showed that the sensitivity for beads method was 86％ vs boiling method 74％.The correlation between the two method was R2 =0.948,P ＜ 0.05.The comparison study between beads method and Roche method was carried use of 50 clinical HBV DNA positive serum samples and the result showed that sensitivity for beads method was 95％ (49/50) vs Roche method was 100％ (50/50).The correlation was R2 =0.963,P ＜ 0.05.ConclusionsThe established method for automatic nucleic acid extraction with magnetic bead displayed characteristic of high yield nucleic acid extraction,broader testing range,accurate quantification and convenient in operation.This can be one choice for nucleic acid extraction and applicable for clinical automatic diagnostic system.( Chin J Lab Med,2012,35:843 -850 )

Objective To improve the diagnostic accuracy by analyzing the computed tomography ( CT) characteristic performance of mucoepidermoid carcinoma of the lung. Methods CT findings 15 cases proved by pathology of mucoepidermoid carcinoma of the lung were retrospectively analyzed,and CT characteristic performance were studied by reviewing the literatures. Results The mucoepidermoid carcino-ma were located in right bronchial of 8 cases,left bronchial of 6 cases,peripheral part of the lung of 1 case. Calcification was seen in 7 mas-ses. Most masses density was not homogeneous at plain scanning. Venous phase have significantly enhanced compared with arterial phase. Ten cases showed obstructive pneumonia and atelectasis,3 cases with mediastinal lymph node metastases. Conclusion Multiple mucoepidermoid carcinoma of the lung grows extension of the long axis of bronchial,calcification be seen in most of patients. The enhanced mode of the lesions appeared as continuous progressive enhancement,some were illustrated with post obstructive pneumonia or atelectasis,CT have a certain value of diagnosis and diffenertial diagnosis.

Objective To evaluate the CT and MRI imaging features of primary ventricular lymphoma ,so as to provide a reference for the diagnosis and differential diagnosis of the disease .Methods The clinical data and imaging findings of primary ventricular lymphoma in one case was retrospectively analyzed with literature review and compared with primary lymphoma in parenchymal brain .Results Slightly high density tumor presented with regular shape in the trigone of lateral ventricles on CT findings , and slightly low signal intensity on T 1 and slightly high signal intensity on T 2 appeared on MRI findings with small cystic necrosis in tumour .An enhancement scan showed obviously enriched, and there was perilesional edema.After 14 day’s dehydration treatment, rechecked indicated that the lesions were significantly en-larged, the shape were irregula , the area of cystic necrosis was enlarged , heterogeneous enhanced scanning were enhanced , perilesional ede-ma became more heavier , and the imaging feature of “closed fist” appeared .It was proved to be diffuse large B cell lymphoma with surgical pathologic examination , and there was no lymphoma in other regions of the body with whole body PET-CT examination , which was clinically diagnosed as primary ventricular lymphoma .Conclusion Primary ventricular lymphoma is rarely reported ,and it has similar imaging mani-festations with primary lymphoma in parenchymal brain .The combination of multiple imaging modalities will be helpful to diagnosis and dif -ferential diagnosis for the suspected patients .

Objective To assess the sequence variations in preS/S regions of occult hepatitis B virus (OHB) and their relationship to severe chronic hepatic injury. MethodsWe collected samples from HBsAg negative patients, and evaluated their HBV-DNA by nest-PCR. HBV-DNA positive samples were used for analysis of preS/S region by PCR sequencing. Results Sixty-nine cases with HBV-DNA were identified in 468 cases without HBsAg. The positive percents were 16％, 8.7％, 36.4％, 18.3％ and 0％in group of only HBcAb positive, only HBeAb positive, only HBeAg positive, both HBcAb and HBsAb positive and all indexes negative, respectively. The level of HBV-DNA of OHB was significant lower than that in HBsAg positive patients. Compared with HBsAg positive controls, preS/S deletion, M1I and Q2K in preS2 region, Q129N/R/P,G185R and S210R in S region were more common in OHB. Moreover, M1I and Q2K in preS2 region, G185R and S210R in S region in OHB with severe chronic hepatic injury were more common that those in OHB without severe chronic hepatic injury. Compared with HBsAg positive patients with severe chronic hepatic injury, the level of HBV-DNA was lower, while the frequency of M1I and Q2K mutation in preS2 region, G185R and S210R in S region were more common in OHB patients with severe chronic hepatic injury. ConclusionThe virological factors were different between OHB and HBsAg positive patients. The M1I and Q2K in preS2 region, G185R and S210R in S region might be useful for prognosis evaluation of OHB patients.

Objective To investigate clinical efficacy of Easy First strategy in laparoscopic pancreaticoduodenectomy (PD) for borderline resectable pancreatic cancer.Methods The clinical data of 9 patients with borderline resectable pancreatic cancer who were admitted to the Sir Run Run Shaw Hospital of Zhejiang University (8 patients) and Zhejiang Provincial People's Hospital (1 patient) from June 2013 to March 2015 were retrospectively analyzed.Nine patients underwent laparoscopic pancreaticoduodenectomy based on the Easy First strategy (to sequentially dissect and amputate jejunum,stomach,hepatoduodenal ligament,common bile duct,main portal vein,head of pancreas,second segment and third segment of the duodenum,uncinate process and neck of pancreas).The operation time,volume of intraoperative blood loss,postoperative pathological examination,complications and duration of hospital stay were recorded.Patients were followed up once every 3 months by outpatient examination and telephone interview up to June 2015.Results Of 9 patients,4 received totally laparoscopic PD (2 received partial resection and repair of portal venous wall),1 received laparoscopic assisted resection and digestive tract reconstruction,and 4 received laparoscopic transection of jejunum,bile tract and stomach and conversion to open surgery for resection and digestive tract reconstruction (1 with severe adhesion between tumor and portal vein,3 with bleeding due to dissection of neck and unciform process of pancreas).The operation time and volume of intraoperative blood loss in all patients and in 4 patients with totally laparoscopic PD were (404 ± 49) minutes and (456 ± 348) mL,(395 ± 61) minutes and (188 ± 25) mL,respectively.Of 9 patients,5 with postoperative complications were cured without perioperative death,including 2 with Grade B pancreatic fistula,1 with biliary leakage,1 receiving reoperation due to gastric stump bleeding at postoperative day 7 and 1 with abdominal infection.The extubation time of right drainage tube and left drainage tube was (9 ± 5) days and (11 ± 4) days,respectively.The duration of hospital stay was (24 ± 10)days.All patients were diagnosed as with pancreatic cancer by pathological examinations with the tumor diameter of (3.2 ± 0.8) cm.The number of harvested lymph nodes in all patients and in 4 patients with totally laparoscopic PD were 16.8 (range,6.0-25.0) and 19.8 (range,15.0-25.0).All the patients were followed up for mean time of 12 months (range,4-24 months),including 1 death at postoperative month 3,1 with tumor survival of 20 months and others with tumor-free survival.The postoperative survival time of 4 patients was more than 18 months at the end of follow-up.Conclusion Easy First strategy in laparoscopic PD is safe,feasible and practical for borderline resectable pancreatic cancer.

Objective: Our aim was to study the role of telomerase activation in the course of cervical carcinogenesis and progression.Methods:Telomeric repeat amplification protocol(TRAP) assay was used to measure telomerase activity in tissue samples with various cervical conditions:40 with cervical cancer, 50 with cervical intraepithelial neoplasia(CIN), 20 with normal cervice. Results:The positive rate of telomerase activity was 95.0%,44.0%, and 10.0% in cervical cancer, CIN, and normal cervices, respectively, which was significantly higher in cervical cancer than that in CIN and normal cervices, so was that in CIN than that in normal cervices (P<0.01) . The positive rate was 22.2%, 37.5%, and 75.0% in CINⅠ,Ⅱ, and Ⅲ, respectively, which was significantly higher in CINⅢ than that in CIN Ⅱand CINⅠ (P<0.01).Conclusion:Telomerase activation may relate to cervical carcinogenesis, which correlates well with the grade of cervical lesions.

Objective: To summary the experience of 233 cases of laparoscopic pancreaticoduodenectomy (LPD) performed by a single surgical team. Methods: Data of patients undergoing LPD from September 2012 to October 2016 were reviewed. There were 145 males and 88 females with the mean age of(60.3±13.0)years old, ranging from 19 to 92 years old, and the mean body mass index of (22.8±3.5)kg/m^2, ranging from 16.3 to 36.8 kg/m². There were 195 patients with clinical manifestation and 54 patients who had the history of abdominal surgery. Results: LPD were performed on 233 patients by same surgical team consecutively. The mean operative time was(368.0±57.4)minutes. Mean blood loss was(203.8±138.6)ml. The postoperative morbidity rate was 33.5%, with 6.9% of grade B or C pancreatic fistula and 9.9% of bleeding. The reoperation rate was 5.6%. The mortality during 30 days after operation was 0.9%. Mean postoperative hospital stay was (18.1±11.2)days. Mean tumor size was (3.9±2.4)cm, and the mean number of lymph nodes harvested was 21.3±11.9.One hundred and sixty-three patients were diagnosed as malignant tumor, including pancreatic adenocarcinoma(n=84), cholangiocarcinoma(n=17), ampullary adenocarcinoma(n=55), duodenal adenocarcinoma(n=5), gastric cancer(n=1)and duel cancer (n=1) located in distal stomach and duodenum. Conclusion The key point to make laparoscopic pancreaticduodenectomy a routine safe procedure is to operate the procedure under skilled hands in selected patients via suitable surgical approaches.

【Objective】 To develop and verify a kinetic method for the determination of prekallikrein activator (PKA) content. 【Methods】 The optimal reaction conditions were determined by comparing the factors of pH and ionic strength of different sample dilution buffers, incubation time of each procedure, and incubation temperature. The accuracy, specificity, precision, linearity, stability and durability of the method were validated. 【Results】 The sample was diluted with 0.05 mol/L Tris-HCl buffer (pH8.5, containing 0.15 mol/L NaCl) and incubated by prekallikrein (PK) at 37℃ for 20 min. After that, the substrate S-2302 was added. Within 10 min before the measurement, the absorbance change rate reached △A405/min. The validation results indicated that the linear range of the method was (0.5~4.0)IU/mL, while the recovery of calibration standard was 96.9%~103.7% with the R² value more than 0.99. The specificity test showed that human serum albumin, excipients of intravenous human immunoglobulin (pH4), low pH and protein content had no significant effect on the detection of PKA, The recovery rates of standard sample solution in the specificity experiment were 98.0% (0.9% sodium chloride solution), 95.3% (0.46% sodium caprylate solution), 96.7% (10% maltose solution, pH4.0), 94.0%(20%BSA), and 94.0%(5%BSA, pH4.0), respectively. The accuracy and precision of the method can meet requirements in the range between 0.5 and 4.0 IU/mL. The inter-batch recovery rate of quality control samples were between 96.4%~109.5% with the coefficients of variation(CV) between 0.2%~6.9%, while the intra-batch recovery rate were between 101.5%~102.9% with the CV between 2.6%~5.9%. The linearity, accuracy and precision of the assay can meet the requirements when PK and S-2302 were placed at room temperature for less than 6 hours, with the recovery rate of quality control samples between 94.9%~109.9%. The end-point method and kinetic method were used to determine the PKA in 20 batches of human serum albumin, and the consistency showed that there was no significant difference between the two methods(P>0.05). 【Conclusion】 A kinetic method for determination of PKA content with good linearity, specificity, accuracy, precision, stability and durability has been established. Compared with the method in ChP, the new method is more convenient, accurate and rapid to determine the content of PKA in human albumin and human immunoglobulin (pH4) for intravenous injection.