Effect of the DNA isolation method on the results of multiple fluorescent PCR and DNA fragment analysis

INTRODUCTION: The quality of the nucleic acids has tremendous effects on the results of molecular diagnostic tests. METHODS: The authors have isolated DNA from amniotic fluid samples by using rezin-binding and silica adsorption methods. In the case of the rezin-binding technique they used both frozen and fresh samples. The quality of the isolated DNA was compared by fluorescent PCR and DNA fragment analysis. RESULTS: With the use of the rezin-binding method on frozen samples the authors obtained more PCR fragments following the fluorescent PCR amplification. The gender and the aneuploidy screening is easier from the electrophoretogramms using better quality DNA. Out of the three studied DNA isolation method the silica adsorption seemed to be the best for the fluorescent-PCR and DNA fragment analysis. DISCUSSION: The authors found the silica adsorption method is more suitable for DNA isolation from amniotic fluids for fluorescent PCR and DNA fragment analysis amplification. They suppose silica adsorption DNA isolation results in a total removal of the PCR inhibitors.     

Continuing care of patients with cardiovascular risk in general practice: patients with dyslipidemia and their care

INTRODUCTION: The role of dyslipidemia is essential in the development of atherosclerosis, therefore continuing care of dyslipidemic patients is an extremely important task in cardiovascular prevention. AIMS: This study was aimed to investigate continuing care of dyslipidemic patients in general practices. Method of the study was a questionnaire survey, 397 patients of 39 general practices were involved into the study. RESULTS: The cause of their continuing care was hypercholesterolemia in 91.7%, hypertriglyceridemia in 69%, decreased HDL-C in 18.4%, and these alterations frequently occurred together. Hypertension in 77%, disturbances of carbohydrate metabolism in 41.7%, increased BMI in 43.2% were found. Patients were smokers in 26.5%, given up smoking in 13.2%. They had cardiovascular problems in 56.5%, cerebrovascular problems in 18.6% and peripheral vascular ones in 20.3%. Patients were treated with lipid lowering drugs in 87.7%, statins were used in 81.7%, fibrates in 29.6% and both of them in 11.5%. The main results of continuing care were: in the whole group frequency of cholesterol level > 5.2 mmol/l decreased by 13%, frequency of triglyceride level >1.7 mmol/l decreased by 4%; cholesterol target values were reached in 3% of patients with high cardiovascular risk, in 22.2% of patients with medium risk, and in 66.7% of patients with mild risk. CONCLUSIONS: These results suggest that there is a need for more intensive drug treatment of dyslipidemic patients to reach the target lipid levels, and for regular assessment of continuing care of these patients.     

Our experiences with nephrectomy with special emphasis on using biocompatible materials

We performed 43 open partial nephrectomies with different indications between 1996 and 2004, most of them (29) for renal cancer. We used different types of biocompatible materials for haemostasis. We describe our own recommendations and what is described in literature.     

Experience with radiofrequency ablation in the treatment of unresectable pelvic recurrence of rectal cancer

More than half of colorectal cancers are located in the rectum, and the number of such cancers is increasing. In Hungary colorectal cancers are diagnosed predominantly in advanced stages. In the last five years 736 patients with colorectal cancer were operated on at our Department, with the following stage distribution: Dukes A 10%, BI 10%, B2 31%, C 36% and D 13%. The local recurrence rate is decreasing since the introduction of total mesorectal excision and preoperative radiation. Effective treatment options are however poor for unresectable pelvic recurrences. Chemo- and radiotherapy have severe limitations in this advanced stage cancer. In recent years there are a few publications on the minimal-invasive radiofrequency tumour ablation (RFTA) technique, which is an effective treatment for primary and metastatic liver carcinomas and is a new palliative for the local treatment of pelvic recurrence. The aim of this study was to assess the response to treatment using ultrasound-guided radiofrequency ablation in two patients with unresectable pelvic recurrent rectal cancer.     

Effect of herpesvirus infection on pancreatic duct cell secretion

AIM: To examine the effect of acute infection caused by herpesvirus (pseudorabies virus, PRV) on pancreatic ductal secretion. METHODS: The virulent Ba-DupGreen (BDG) and nonvirulent Ka-RREpolacgfp (KEG) genetically modified strains of PRV were used in this study and both of them contain the gene for green fluorescent protein (GFP). Small intra/ interlobular ducts were infected with BDG virus (107 PFU/mL for 6 h) or with KEG virus (1010 PFU/mL for 6 h), while non-infected ducts were incubated only with the culture media. The ducts were then cultured for a further 18 h. The rate of HCO3- secretion [base efflux -J(B-)] was determined from the buffering capacity of the cells and the initial rate of intracellular acidification (1) after sudden blockage of basolateral base loaders with dihydro-4,4,-diisothiocyanatostilbene-2,2,-disulfonic acid (500 μmol/L) and amiloride (200 μmol/L), and (2) after alkali loading the ducts by exposure to NH4CI. All the experiments were performed in HCO3--buffered Ringer solution at 37℃ (n = 5 ducts for each experimental condition). Viral structural proteins were visualized by immunohistochemistry. Virallyencoded GFP and immunofluorescence signals were recorded by a confocal laser scanning microscope. RESULTS: The BDG virus infected the majority of accessible cells of the duct as judged by the appearance of GFP and viral antigens in the ductal cells. KEG virus caused a similarly high efficiency of infection. After blockage of basolateral base loaders, BDG infection significantly elevated -J(B-) 24 h after the infection, compared to the non-infected group. However, KEG infection did not modify -J(B-). After alkali loading the ducts, -J(B-) was significantly elevated in the BDG group compared to the control group 24 h after the infection. As we found with the inhibitor stop method, no change was observed in the group KEG compared to the non-infected group. CONCLUSION: Incubation with the BDG or KEG strains of PRV results in an effective infection of ductal epithelial cells. The BDG strain of PRV, which is able to initiate a lytic viral cycle, stimulates HCO3- secretion in guinea pig pancreatic duct by about four- to fivefold, 24 h after the infection. However, the KEG strain of PRV, which can infect, but fails to replicate, has no effect on HCO3- secretion. We suggest that this response of pancreatic ducts to virulent PRV infection may represent a defense mechanism against invasive pathogens to avoid pancreatic injury.     

Sandwich type ELISA and a fluorescent cytometric microbead assay for quantitative determination of hepatitis B virus X antigen level in human sera

The hepatitis B virus X protein (HBxAg) is responsible for severe complications of HBV infections including primary hepatocellular carcinoma. A sandwich type ELISA and a flow cytometric microbead assay for quantitative determination of serum levels of Hbx-Ag are introduced. We have previously developed monoclonal antibody families against well-conserved epitopes on HbxAg, characterized by different immunohistochemical and immunoserological techniques. Special selection of the antibody pairs provided highly sensitive and highly specific tools for quantitative immunoassay development. The resulting assays were tested on human sera (208 samples) collected from patients suffering from different clinical forms of HBV infection. The sensitivity range of the sandwich type ELISA was between 4 and 2000 ng/ml as measured on both the recombinant antigen and the sera of chronic hepatitis patients. A further flow cytometric microbead assay was established and tested in parallel with the ELISA. The quantitative results of these two immunoserological techniques were in strong correlation and they were found to be highly specific and sensitive on clinical samples. The HBxAg ELISA technique is applicable for routine clinical laboratory measurements, and our HBxAg microbead technique is recommended for complex multiparametric measurements combined with other markers.