College of American Pathologists (CAP) and International Standard Organization (ISO)15189 accreditation provides requirements to the verification and validation tests for clinical molecular diagnostic assays.The article will introduce the requirements in these aspects including the differences between the two accreditation systems,the differences between the verification and validation tests,the essential factors and standardized framework of the verification and validation tests for clinical molecular diagnostic assays.

The enzymatic activity of monocytes was studied with light microscopic semi-quantitive analysis and microspectrophotometer, including B-glucuronidase, acid phosphatase, N-acetyl-?-glucosaminidase, peroxidase and ?-naphthyl acetate esterase in peripheral blood smears prepared by hcparinazed micropipet centrifugal method in some normal donors and patients. The data of normal groups have been obtained. The results tested by microspectrophotometry have indicated that the larger the area of monocyte was, the stronger the five kinds of enzymatic activity in monocyte was. In comparison with the normal groups, there were significant alterations of the different kinds of enzymatic activity of monocytes in the patients with cir- rhosis, systemic lupus erythematosus, pemphigus, untreated malignant tumor and radiotheraped tumor respectively. The significance of the results described above have been discussed in the paper.

Myeloproliferative neoplasms ( MPN ) are clonal hematopoietic stem cell diseases characterized by proliferation of one or more myeloid cell lineages in the bone marrow and increased mature and immature cells inperipheral blood.In recent years , several molecular markers have been discovered , such as JAK2 (JAK2 V617F and JAK2 exon12),MPL515 and TET2 mutations and so on.The discoveries provide important significance of better understanding of the pathogenesis in MPNs and are helpful for the purposes of both diagnosis and therapy.However , diagnosis remains difficult in the remaining 30%-45% of patients who lack JAK2 or MPL mutations.Recently as was reported , the identification of CALR mutations will partly address a gap above and may be a new biomarker in the molecular diagnosis of MPNs .In this review,the discovery of CALR mutations in MPN will be introduced.

Over the past several years, mass spectrometry technology has become the important method of choice for the discovery of new biomarkers.Because the features of mass spectrometry-based proteomics including sensitivity, high throughput, speed, combined with advanced bioinformatics allow for the rapid analysis of a bunch of proteins simultaneously.It has become a powerful laboratory tool in clinical study.However recent studies showed that critical comments were made on the poor reproducibility,statistical analysis of the data et al.This article focused on challenges of study design, mass spectrometry technology and biological relevance associated its application of mass spectrometry based proteomics in serum or plasma.

microRNAs as novel tumor biomarkers exert important clinical value in early screening and antidiastole of human cancers .They show a broad prospect in clinical application , particularly due to their low invasive and high sensitivity .This review focused on the application of circulating microRNA for diagnosis and monitoring treatment in colorectal cancer based on the recent findings .Meanwhile, the defects in clinical practice by using microRNAs as tumor biomarkers and the major causes which could constraint their application, especially the unreliable detection result caused by many factors , have been discussed.

p27kip1 is an important negatively regulator of cell cycle progression and plays a central role in the pathogenesis of a member of tumors including breast cancer. In breast cancer cells, the level of p27kip1 expression usually decreases during tumor development and progression, in addition, cytoplasm mislocalization of p27kip1 has been reported, but less is known about the exact molecular mechanisms. Studies have indicated that phosphorylation is the key regulation way, several signal transduction pathways are involved in the regulation of the expression and distribution of p27kip1. To further understand the mechanism, the disparity of the interacting protein profiling between tumor cells and normal cells must be identified first. Including cyclins, cyclin-depend kinases, CRM1, jab1, SKP2, p27kip1 has various interacting molecules. There are also several interacting molecules especially for breast cancer cells. It seems that different protein profiling cause the different expression and intracellular distribution in different cell cycle phase. So, disparity of the p27kip1 protein profiling may be the main mechanism of its down-expression and mislocalization in breast cancer cells.

Objective:Tumor size,lymphnode metastasis,hormone level and so on have been the prognosis predictors of breast cancer,but they are not ideal. This study is to investigate the influence of the expression of p27Kip1 in early breast cancer prognosis and its relationship with clinical feature. Methods:Archival early breast carcinomas (n=106),were analyzed for p27Kip1 expression by immunohistochemistry EnVision system using specific mono-clonal antibody. Results:The p27Kip1 protein nucleus expression was low in 61 (57.55%) of the 106 cases in early breast carcinomas,which has statistic difference from the high expression in survival rate (P0.05),but correlated with lymph-node metastasis and histological grade (P

Objective Under the ISO15189 and America Association of Pathologists ( CAP ) laboratory accreditation system, to establish the performance verification standards for detecting hepatitis B virus resistance gene by sanger sequencing.Methods 25 cases of HBV drug resistance outpatients and inpatients were collected from August 2012 to December in Hepatitis Clinic of Huashan Hospital Affiliated to Fudan University.Analytical performance parameters including analytical sensitivity, precision, accuracy, analytical specificity, reference range/reportable range, etc were evaluated.Sequencing quality evaluation parameters included fluorescence signal intensity overall sequencing chromatogram, signal to noise ratio, trace scores and QV value.Results 10%-20% mutation could be detected under wild-type background. The methool had good precision and accuracy.No obvious interference and cross contamination were observed.Conclusions Performance validation of the sequencing should combine with the practical application.Especially in view of the different detection subjects, and appropriately adjusted to meet the clinical needs.Detection of hepatitis B virus resistance gene by the in the test method in this study can be used in clinical detection.

Objective To evaluate the DLC-l gene promoter methylation in acute lymphoblastic leukemia(ALL)in children by methylation specific-PCR(MSP),explore the its prognostic value.Methods Both pyrosequecing and MSP was used to detect DLC-1 gene methylation in bone marrow samples from 34 children with acute lymphoblastic leukemia,5 normal bone marrow samples and acute leukemia T cell line 6T-CET.DLC-1 mRNA expression in cells with、or without treatment with 5-aza-2'-deoxycytidine wag investigated by real time RT-PCR Results MSP analysis showed that DLC-1 promoter methylation Was identified in 21 of bone marrow samples from ALL patients,but was absent in 5 normal bone marrow specimens.These results were completely agreement with pyrosequencing analysis.In additional,the latter can give the quantitative analysis of methylation status in specific CpG sites.No association Was observed between DLC-1 methylation and patient’S clinical-biologic characteristics including age,gender,WBC count,FAB classification,immunology typing and clinical typing(P>0.05)In 18 ALL patients achieving complete remission(CR)witIl DLC-1 methylation,14 relapsed in 12 monthhs,while only 4 relapsed in 1 patients without DLC-1 gene metllylation.Treatment of the cell line 6T-CEM harboring methylation with 5-aza-2'-deoxycytidine increased DLC-1 expression in dose dependent manner. Conclusions This is the first report of high frequency of promoter methylation of DLC-1 in ALL It shows that DLC-1 methylation iS useful for predicting relapse of ALL.Pyrosequencing assay Call provide a sensitive,easy-to-use method for quantitative assessment of DLC-1 methylation.

BACKGROUND:Studies on high glucose exposure in human periodontal ligament cel s usual y focus on the biological behaviors, pathways and secretory factors, but whether the metabolic memory is involved is little known. OBJECTIVE:To investigate the metabolic memory of high glucose exposure in human periodontal ligament cel s. METHODS:Human periodontal ligament cel s were primarily cultured and identified. Cel s at 5-8 passages were selected and randomized into four groups. Group A (controls):DMEM containing 5.5 mmol/L glucose for 8 days;group B (5-day memory group):DMEM containing 35 mmol/L glucose for 3 days and DMEM containing 5.5 mmol/L glucose for 5 days;group C (3-day memory group):DMEM containing 35 mmol/L glucose for 5 days and DMEM containing 5.5 mmol/L glucose for 3 days;group D (8-day high glucose group):DMEM containing 35 mmol/L glucose for 8 days. The cel proliferation was detected by cel counting kit-8, the cel apoptosis was determined by flow cytometry, and the levels of total proteins and alkaline phosphatase were investigated using ELISA. RESULTS AND CONCLUSION:Compared with the control group, the cel proliferation in the other three groups was significantly reduced (P<0.05), the number of apoptotic cel s was significantly increased, while the levels of total proteins and alkaline phosphatase were significantly decreased (P<0.05). These results suggest that high glucose causes persistent changes in human periodontal ligament cel s by inhibiting cel viability, increasing the apoptosis and downregulating the levels of the total proteins and alkaline phosphatase