Objective To explore the relationship between K-ras gene and cholangiocarcinoma by detecting the K-ras gene mutation of bile duct tissues.Method We studied all the patients who presented to our hospital from June 2010 to June 2012 with stenosis of the bile duct.There were 17 cases of cholangiocarcinoma and 17 cases of benign stenosis.From the DNA extracted from the paraffin tissues,we used the HRM assay for K-ras gene mutation.Result We found that the HRM method and the DNA typing had exactly the same result for the DNA content which confirmed the effectiveness of the HRM assay.Of note,the K-ras mutation rate was found to be significantly higher in the cholangiocarcinoma cases (11/17) when compared with the benign cases (3/17).Conclusion The mutation of the K-ras gene was closely related to cholangiocarcinoma.Our results suggest a new way to diagnosis cholangiocarcinoma.

Objective To analyze the perioperative complications and prognosis of surgical treatment of pancreatic ductal stones.Methods 60 patients diagnosed to have pancreatic ductal stones and were admitted into our hospital from January 2009 and December 2014 were enrolled into this retrospective study.There were 47 males and 13 females.The average age was (44.1 ± 12.0).Results All patients underwent surgical operation and there was no perioperative death.Perioperative complications occurred in 7 patients (11.7％), including pancreatic fistula in 4 patients, delayed gastric emptying in 1 patient, abdominal infection in 1 patient, anastomotic bleeding in 1 patient.Eight patients developed elevated amylase levels.The fasting time was 3.4 ± 1.2 days and the hospital stay was 10.0 ±4.0 days.The follow-up rate was 88.3％ (53/60), at a mean follow-up of 27 months.In 51 patients who presented with abdominal pain, 43 patients (84.3％) had complete pain relief and 7 patients (13.7％) had partial pain relief.In 14 patients with preoperative diabetes mellitus, 1 patient had improvement and 2 patients recovered from diabetes.Two additional patients were diagnosed to have diabetes two years after the operation.Of the 2 patients who were shown to have pancreatic cancer on histopathological study, 1 patient was lost to follow-up while another patient died at 27 months after the operation.The patients diagnosed to have epithelial dysplasia were followed-up and none developed pancreatic cancer.Conclusions The relationship between chronic pancreatitis and pancreatic ductal stones was very close.Chronic pancreatitis also caused exocrine and endocrine dysfunction.Surgical operation plays an important role in symptomatic relief and in delaying pancreatic dysfunction.

Objective To investigate the value of initial absolute lymphocyte counts (ALC) in predicting the prognosis of children with primary immune thrombocytopenia(ITP).Methods The initial clinical data of 214 children with incipient ITP were retrospectively analyzed and followed up for more than one year.Statistical analysis was made to find out the influence factors for the prognosis of patients with ITP in children to evaluate the predictive value of ALC on assessing the prognosis of ITP in children,and to analyze the association between ALC and outcomes and the clinical value of prognostic stratification.Results The remission rate of children with ITP was 71.5％ in 12 months.Multivariate unconditioned Logistic analysis showed that disease duration,the infection history,response to treatment and ALC were independent risk factors for chronic ITP(all P ＜ 0.05).Cutoff value of initial ALC was 3.005 × 109/L,sensitivity and specificity of value were 71.9％,83.6％.Three months and 12 months no remission rate of ITP in children between ALC ＞ 3.005 × 109/L and ≤ 3.005 × 109/L had statistical significant difference (24.2％ vs 64.9％,8.3％ vs 54.2％,x2 =42.13,P ＜0.001).There was no statistical difference in therapy efficacy between 2 groups(x2 =5.098,P ＞ 0.05).Remission rate between the ≤5 years old group and ＞ 5 years old group was statistical difference based on age stratification(x2 =22.371,22.177,all P ＜ 0.01).ITP in children were stratified into low risk group,intermediate risk group,high risk group based on the initial ALC and infection history,with remission rates in 3 month respectively 75.8％,54.1％,22.8％,respectively,and 91.6％,68.4％,31.6％ in 12 months,respectively;there were statistically significant differences (x2 =44.867,68.802,all P ＜ 0.001).Conclusions Initial ALC is an independent effecting factor in the prognosis of children with ITP.Initial ALC could be used for stratifying patients with the infection history before 6 weeks and supposed to be a predictive index for the prognosis of ITP in children.

Objective To establish a CVS-11 pseudovirus particles ( pp)-based assay for detec-tion of neutralizing antibody against rabies virus. Methods An improved rapid fluorescence focus inhibition test ( RFFIT) for detection of neutralizing antibody against rabies virus ( RVNA) was established based on the CVS-11 pseudovirus expressing a luciferase reporter gene. Forty-six human serum samples were analyzed with the improved RFFIT and the results were compared with those by using standard RFFIT. Moreover, the improved RFFIT was used to detect the titers of RVNA in 91 serum samples collected from pet dogs and pet-breeders in Beijing. Results The coincidence rate of the improved RFFIT and the standard RFFIT was 100% regarding to the analysis of 46 human serum samples and 5 negative reference serum samples. Moreo-ver, the RVNA titers of all serum samples obtained with CVS-11 pseudovirus-based assay showed a signifi-cant high correlation with those obtained with standard RFFIT (n=46, r=0. 94, P<0. 000 1). All of the 91 serum samples collected from pet dogs and pet-breeders in Beijing were positive for RVNA as indicated by the improved RFFIT with a mean titer of 33. 01 IU/ml. Conclusion We established an improved RFFIT based on the CVS-11 pp expressing luciferase reporter gene, which might be used as a reliable alternative RFFIT for measuring RVNA titer. Analysis of the 91 serum samples collected in Beijing with the improved RFFIT showed that all samples were positive for RVNA.

Objective To determine anatomic parameters related to the surgery of lumbar cortical bone trajectory of normal adults and the feasibility of screw application in cortical bone and its specification and dimension.Methods Lumbar 3D image data of 80 adults between 18 and 40 years old (40 for each gender) were randomly selected as subjects of our research.With the help of the imaging software for 3D interactive viewing,the ideal starting point was supposed to be the junction of the center of the superior articular process and 1 mm below the inferior border of the transverse process.The diameter,length,lateral angle to the vertebral horizontal plane,cephalad angle to the vertebral sagittal plane of the trajectory and the horizontal distance from the insertion point to the lateral edge of the vertebral plate were measured.Differences of anatomic parameters for each gender,side and segment were analyzed.Results Differences of anatomic parameters on both sides of each segment had no statistical significance.At the ideal trajectory,the mean screw length was 37.56±2.41 mm at L1,38.72±2.36 mm at L2,39.51 ±2.51 mm at L3,39.78± 2.87 mm at L4 and 38.83±2.74 mm at L5.The mean screw diameters from L1 to L5 were 6.04±1.23 mm,6.17±1.24 mm,7.15±1.22 mm,8.02± 1.41 mm and 8.68± 1.42 mm respectively.However,differences of ideal entry angle of L1 to L5 had no statistical significance.The mean lateral angle from L1 to L5 were 8.46°±2.11°,9.37°±2.84°,9.62°±2.16°,9.53°± 1.98°,9.04°± 1.97°,while the mean cephalad angle to the vertebral sagittal plane from L1 to L5 were 26.49°±4.97°,25.94°±4.56°,26.42°±4.42°,26.29°±3.48°,26.89°±3.69°.The mean distance from the insertion point to the lateral edge of the vertebral plate gradually increased from L1 to L5,which were 1.19±0.75 mm,1.54±1.08 mm,2.01±1.45 mm,3.49±1.52 mm,4.47±1.32 mm respectively.The screw diameters of each segment for men were greater than those for women.Conclusion The common length of screw for lumbar cortical bone trajectory of normal adults might be from 35 mm to 40 mm.The safe upper limits of the screw diameter were 5.5 mm at L1,5.5-6.0 mm at L2,6.5-7.0 mm at L3,7.5 mm at L4 and 8 mm at L5.The average lateral angle of all 5 lumbar segments was 9.20°± 2.11° and the average cephalad angle was about 26.41°±4.22°.

Objective To explore the value of urine formaldehyde test in the diagnosis of Alzheimer's disease (AD),and the influential factors of urine formaldehyde level in AD patients.Methods A total of 52 AD patients and 53 cognitively normal controls were recruited in a cohort study.All subjects were no less than 65 years old,and those with acute infection,or dysfunction in heart,liver or kidneys were excluded.The impact of age,gender,onset age,MMSE score,NPI score,MTA score,and ApoE ε4 gene on urine formaldehyde of AD patients were analyzed by multiple regression analysis.Results Urine formaldehyde level of AD group was statistically higher than that of cognitively normal control group ((13.27±4.16)μmol/L vs (10.76±4.47)μmol/L,t=2.99,P=0.15).Urine formaldehyde of AD patients was statistically negatively correlated with MMSE score (β=-0.35,P=0.03) and MTA score (β=-0.38,P=0.02).The impact of onset age,neuropsychiatric disorders and ApoE ε4 gene on urine formaldehyde of AD patients was not statistically significant(all P>0.05).Conclusion Urine formaldehyde level is worthwhile to be explored as a marker in AD diagnosis and severity assessment.

Objective:To explore and evaluate the accuracy, conformity, and clinical application value of digital surgical techniques in guiding 125I seed implantation in the brachytherapy of deep head and neck tumors. Methods:Twelve patients with deep head and neck tumors who received brachytherapy of radioactive 125I seed implantation were selected for the study.The locations of the implantation needles and seeds were designed based on CT images before the operation.A digital positioning model was built according to the treatment plan to accurately import the planned locations of implantation needles into the surgical navigation system.Meanwhile, an individualized puncture guidance template was designed and printed according to the patients′ facial profiles as well as the locations and directions of the implantation needles.During the operation, the template was put in place under the guidance of the navigation system and meanwhile, the implantation needle puncture and radioactive seed implantation were conducted under the joint guidance of the visual needle path and real-time implantation needle locations in the navigation system and template guidance holes.The locations of the implantation needle and the seeds were validated by CT scan and the dose distribution in target areas was calculated.Adverse reactions such as hematoma, pain, infection, nonunion of puncture sites, and tumor cell implantation were observed during and after the operation. Results:All 12 patients successfully received implantation needle puncture and radioactive seed implantation under the guidance of the digital surgical techniques, achieving excellent effects of real-time visualization guidance.Meanwhile, the locations and number of the implantation needles and seeds were consistent with the treatment plan and were distributed evenly.Furthermore, according to postoperative verification, D90 ranged from 83.7 Gy to 131.0 Gy, with an average of 107.5 Gy; V100 was 89.6%-99.3%, with an average of 94.6%, and V150 ranged from 40.2% to 58.9%, averaging 47.8%.No serious adverse reactions were observed during and after the operation. Conclusions:With digital surgical techniques, the surgical navigation system and 3D-printed individualized puncture guidance template jointly guided the implantation needle puncture and 125I seed implantation, improving the accuracy and conformity of the brachytherapy.Therefore, they have clinical application value in head and neck brachytherapy, especially in deep areas with complex anatomical structures.

Objective To investigate the clinical and laboratory characteristics of patients with chronic lymphocytic leukemia (CLL). Methods 503 patients with CLL admitted from October 1998 to February 2015 were retrospectively analyzed. Baseline characteristics were compared using Chi-square test and Kaplan-Meier methodology was undertaken for survival analyses. Results The median age was 58 years (26-86 years):335 cases were male and 168 cases were female. 204 cases (40.5%) were at the clinical stage of Binet A, followed by Binet B (148 cases, 30.1%) and Binet C (151 cases, 29.3%). 108 cases (21.1%) had anemia at diagnosis, while 113 cases (26.5 %) had an elevated level of lactate dehydrogenase and the expression of CD38 was detected among 100 cases (29.1 %). Clonal abnormalities were observed using fluorescence in situ hybridization (FISH) analysis. Those involving 13q deletion were the most frequent (156 cases, 47.3 %), followed by IgH translocation (22.4 %), trisomy 12 (21.2 %) and 17p deletion (14.5 %). The mutational status of immunoglobulin heavy chain variable region was determined among 230 cases, 165 cases (71.7%) of which were found to be with mutated status. The most frequently encountered gene was V4-34 (28 cases, 12.4 %). The median progression-free survival (PFS) was 89.0 months (95 %CI 75.0-103.0 months), while the median overall survival was 129.0 months (95 %CI 106.9-151.1 months). Conclusion Compared with patients in the western world, CLL patients in this study are younger at diagnosis and have longer overall survival, which, to some extent, could reflects the characteristics of CLL patients in China.

Objective: To study the relationship between platelet activation and the degree of bleeding in patients with primary immune thrombocytopenia (ITP) . Methods: 43 patients with ITP were assessed based on ITP-BAT bleeding grading system. Platelet membrane glycoproteins (GP) Ⅰb, GPⅡb/Ⅲa and P-selectin expression were detected by flow cytometry analysis with and without adenosine diphosphate (ADP) stimulation. Association of platelet activation with platelet count, immature platelet fraction (IPF) , bleeding severity were evaluated. Results: GPⅡb/Ⅲa and P-selection expressions on unstimulated platelet in ITP patients were higher than those in healthy controls (65.69±10.73 vs 7.16±0.99, t=4.130, P<0.001; 15.43±1.41 vs 12.55±1.03, t=2.070, P=0.043, respectively) , and GPⅠb expression was lower than that in healthy controls (240.11±24.93 vs 295.11±22.15, t=2.417, P=0.020) . Comparatively to healthy individuals, following ADP stimulation, GPⅡb/Ⅲa expression in ITP patients increased (133.96±12.17 vs 39.67±4.99, t=5.256, P<0.001) , whereas GPⅠb and P-selection expressions decreased (37.09±3.94 vs 109.77±23.66, t=3.901, P<0.001; 149.06±19.14 vs 205.73±21.00, t=2.070, P=0.043, respectively) . ADP-stimulated GPⅠb, ADP-stimulated and unstimulated P-selection, proportion of GPⅠb, P-selection levels with/without ADP stimulation were significantly associated with platelet counts (P<0.05) . ADP-stimulated P-selection and proportion of P-selection levels with/without ADP stimulation were significantly associated with IPF (P<0.05) . There were significant differences in the expressions of unstimulated P-selection, ADP-stimulated P-selection, ADP-stimulated GPⅠb stratified by bleeding grades (P<0.05) . The ratios of P-selection, GPⅡb/Ⅲa and GPⅠb with/without ADP stimulation in ITP patients were significantly different among various bleeding grades (P<0.05) . Higher proportion of GPⅠb with/without ADP stimulation was associated with higher risk of bleeding (OR=3.05, P=0.011) . Lower proportion of GPⅡb/Ⅲa and P-selection with/without ADP was associated with higher risk of bleeding (OR=0.32, P=0.023; OR=0.04, P=0.006, respectively) . Conclusion Platelet activation index could accurately assess the degree of bleeding in patients with ITP, and also be used as the observation index and reference index for treatment.

OBJECTIVETo explore the genetic etiology of a patient with classic maple syrup urine disease (MSUD).

METHODSNext-generation sequencing (NGS) was used to screen the exons of BCKDHA, BCKDHB, DBT and DLD genes. Suspected mutations were verified by Sanger sequencing. Bioinformatic analysis was carried out to predict the influence of mutations on the protein structure and function.

RESULTSNGS and Sanger sequencing have detected a c.550delT mutation in exon 5 of the BCKDHB gene in the mother and a c.1046G>A mutation in exon 10 of the BCKDHB gene in the father, while no mutation was found with BCKDHA, DBT and DLD genes. Among these, the c.550delT is a novel mutation. Bioinformatic analysis suggested that the two mutations both located in a highly conserved region and may decrease the activity of branched-chain α-ketoacid dehydrogenase complex through alternation of its structure.

CONCLUSIONThe compound heterozygous mutations c.550delT and c.1046G>A of the BCKDHB gene probably underlie the clinical manifestations of the patient with classic MSUD.