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FVG1691A (factor V Leiden, FVL) and FII G20210A (prothrombin G, GPRO) polymorphisms are the most common genetic causes of thrombophilia. For diagnosis of these polymorphisms, two years ago we adopted the GeneXpert instrument, after a preliminary study conducted on frozen plasma that had already been typed. We report our experience concerning the routine application of this analysis system. Between January 2011 and December 2012, 1106 patients were evaluated for detection of FVL and GPRO using the Cepheid GeneXpert system (Instrumentation Laboratory, Milan, Italy). The first 142 were also tested with the LightCycler system (Roche, Monza, MI, Italy). Results obtained with the GeneXpert system showed full agreement with those obtained with the LightCycler system. Full agreement was also observed with previously characterized frozen samples. Among the 1,106 subjects examined, 235 were FVL heterozygous, 20 homozygous FVL, 37 GPRO heterozygous, 3 homozygous GPRO, 15 double heterozygous FVL/GPRO, and 796 genetically normal. In the 2-year period we observed only 37 invalid results with the need to repeat the test. The GeneXpert system is a fully automated analytical system. In less than 35 minutes, it is possible to perform a combined determination of FVL and GPRO from a subject’s anticoagulated whole blood. The design of the kit, based on the use of individual disposable cartridges, in which are contained all the necessary reagents for the extraction of nucleic acids, their amplification and the detection of the amplicons, eliminates waste and allows determinations to be performed on demand. On the other hand, the extremely simple manual makes the test accessible to those laboratories not specializing in molecular biology techniques. In our experience, the GeneXpert system has proven reliable with total concordance with the results obtained with the system already in use in our laboratory. We also observed only a small percentage of invalid results with a satisfactory ratio (1.03) of the number of tests performed to the number producing a result.

Background. The aims of this retrospective study were to ascertain in large series of children and adults: the relationship of the infecting strain to gastric mucosal lesions; and the relationship of the infecting strain to its duodenal localization. Materials and Methods. We studied 307 and 604 consecutive children and adults. In gastric mucosal samples H. pylori was cultured, genotyped and histologically assessed, while inflammation, activity and intestinal metaplasia were graded. In a subset of 171 patients H. pylori ureaseA (ureA) and cagA genes were amplified (PCR) using mucosal biopsies from the duodenum. Results.H. pylori infection was diagnosed in 40 children and 308 adults. cagA was identified in 50% and 65.5% of infected children and adults. Antral activity was associated with the density of infecting bacteria (p

INTRODUCTION The transfer of drug-susceptible (suicide) genes to tumor cells by retroviral or adenoviral vectors is a novel approach to the treatment of human tumors. AIMS To ascertain the antitumor effect of retroviral transduction of the pancreatic cancer cell lines MIA PaCa 2, CAPAN-1, PANC1, and PSN1 with the herpes simplex virus thymidine kinase (HSV-TK) gene. METHODOLOGY The vector carried a neoselectable marker gene, the human interleukin-2 gene, an internal ribosome entry coding site, and the region coding HSV-TK. RESULTS Twenty micromoles or less of ganciclovir did not modify nontransduced TK- cell growth, whereas > or =100 micromol completely inhibited TK- cell growth, indicating that this dosage is cytotoxic per se. The 4 TK- and the 4 transduced cell lines were treated daily with 0.001, 0.01, 0.1, 1, 10, and 20 micromol of ganciclovir for 13 days. CAPAN-1 cell growth was completely inhibited by 0.1 micromol of ganciclovir; higher doses were required to kill PANC1 (10 micromol) and PSN1 (20 micromol). MIA PaCa 2 cell growth decreased following a 20-micromol ganciclovir dosing. The bystander effect was great in the CAPAN-1 cell line and moderate in PANC1; no bystander effect was recorded in MIA PaCa 2 and PSN1 cell lines. CONCLUSION Gene therapy with HSV-TK for pancreatic cancer seems effective in only a limited number of tumor-derived cell lines, and this limits its application in vivo.

The authors compare efficacy of two ELISA assays (one supplied by DIAMEDIX [Delta Biological s.r.l.], and the other by RADIM [RADIM I]) in detecting total anti‐H. pylori antibodies, and of two further ELISA methods (one supplied by EUROSPITAL [Helori CTX IgG] and the other by RADIM [RADIM 2]) in identifying anti‐CagA antibodies, using sera from 69 controls (20 adults and 49 children) and from 96 patients, obtained before endoscopy. Seventy‐three of the patients had H. pylori infection, while the remaining 23 were H. pylori negative (histology and polymerase chain reaction [PCR]). Fifty‐two of the H. pylori positive patients, had cagA‐positive strain infection, identified by PCR. The DIAMEDIX assay was found to be more sensitive (92%) than RADIM 1 (79%) in identifying H. pylori positive patients, irrespective of the infecting strain. On the other hand, the DIAMEDIX assay was less specific than RADIM 1 for H. pylori‐negative patients (43% vs. 83%). However, when patients already treated for H. pylori infection were excluded from the group of H. pylori‐negative patients, the DIAMEDIX assay had a specificity of 89%. In identifying anti‐CagA antibodies, the kit supplied by RADIM (RADIM 2) had a sensitivity of 90% and a specificity of 94%, whereas that supplied by EUROSPITAL had a sensitivity of 100% and a specificity of 76%. The performances of the two methods in the identification of anti‐CagA antibodies were found to be similar. The authors conclude that, in view of its high sensitivity, the DIAMEDIX assay may be useful in screening for H. pylori infection. J. Clin. Lab. Anal. 13:194–198, 1999. © 1999 Wiley‐Liss, Inc.

Transfusion-associated bacterial infections are a quite frequent collateral effect of administration of platelet concentrates (PC). We carried out a microbiological surveillance of bacterial contamination of apheresis platelet concentrates by studying microbial flora on donors' arms before and after skin disinfection, through blood cultures with the diversion volume and with the PC.Platelet aphereses were carried out using two Haemonetics MCS+ instruments. Cutaneous swabs were examined by the direct plate technique and blood cultures were performed using Bact/ALERT aerobic bottles. In the 5 years from January 2001 to December 2005 we tested 481 PC.Cutaneous swabs showed significant bacterial growth in 89% of cases before skin disinfection and in 44% after. None of the blood cultures performed on diversion blood was positive, one (0.2%) PC was positive on the fifth day after collection and the presence of a Staphylococcus epidermidis strain was demonstrated.Our results suggest that the skin disinfection protocol adopted in our structure is not fully satisfactory. The cultures performed on the PC showed a low prevalence of contamination, and the only positive sample was contaminated by a common skin contaminant (S. epidermidis). The culture became positive on the fifth day after collection, but on the second day the PC had been transfused to a patient, without any adverse reaction. In our experience a culture method using Bact/ALERT aerobic bottles was not able to prevent transfusion of the only contaminated PC identified in this study.

Pancreatic adenocarcinoma causes diabetes mellitus by releasing factors interfering with glucose metabolism.We verified in isolated rat hepatocytes the molecular weight (MW) of the fraction from pancreatic cancer cell conditioned media (CM) that altered glucose metabolism and ascertained, using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis, whether there is any common peptide in CM and in the sera of patients with pancreatic cancer.Sera was obtained from patients with pancreatic cancer ( n = 14) and chronic pancreatitis ( n = 9) and healthy control subjects ( n = 10). Conditioned medium (CM) was obtained from the following cell lines: MIA PaCa 2, PSK-1, PANC-1, and CAPAN-1. Two fractions (MW of less than 30,000 Da and less than 10,000 Da) were obtained from patients' sera, from CM, and from non-CM (NCM) after two-step ultrafiltration. Rat hepatocytes were incubated with CM and NCM. The peptide profile of patients' sera, CM, and NCM were analyzed using MALDI-MS.In rat hepatocytes, glucose metabolism was impaired by CM from all the pancreatic cancer cell lines and by CM with an MW of less than 10,000 Da. Two peptides (m/z 2030 and 2726) were found in CM and patients' sera. Only the peptide at m/z 2030 was found to be associated with the presence of diabetes.A peptide at m/z 2030 may be a putative pancreatic cancer-associated diabetogenic factor.

Our aim was to assess the clinical reliability of mutated K-ras detection in serum or bile for the diagnosis of pancreatic cancer using ME-PCR. DNA was extracted from 1 ml serum obtained from 29 patients with pancreatic cancer and 12 control subjects. ME-PCR was optimized using a mixture of normal DNA added with different amounts of mutated DNA. The analysis of sera obtained from the 29 patients and of bile obtained from 11 pancreatic cancer patients demonstrated the presence of mutated K-ras in two (6.9%) and four cases (36%). By contrast K-ras was not amplifiable in any of the 12 serum samples obtained from healthy controls. In conclusion the DNA obtained from pancreatic cancer patients' sera is suitable for K-ras amplification and for the identification of codon 12 point mutations. However ME-PCR alone has an unsatisfactory sensitivity for the detection of pancreatic cancer using serum DNA as starting template.

Interleukin 6 (IL-6), an autocrine growth factor for many tumors, seems to favour tumor spread to the liver. Our aims were first to evaluate the pattern of portal and systemic IL-6 levels in patients with pancreatic cancer (PC, n =18) and chronic pancreatitis (CP, n =22) compared with controls (CS, n =20); and second, to ascertain whether there was any relation between IL-6 levels and tumor spread or PC-associated Diabetes mellitus. For all subjects, a fasting serum sample was obtained from a cubital vein; a portal serum sample was obtained from nine PC and three CP patients. In cubital and portal sera we measured IL-6, interleukin 1 beta (IL-1b), CA 19-9, c-reactive protein (CRP) and amylase. Systemic IL-6 levels were significantly higher in PC patients than in CS. In PC, portal IL-6 levels were significantly higher than the corresponding systemic values. The same pattern was found in the three CP patients, whereas IL-1b, CA 19-9, CRP and amylase portal levels were the same as systemic values. No correlation was found between PC stage and systemic or portal IL-6 levels. Portal IL-6 levels were correlated with the corresponding fasting serum glucose values. A significant correlation was found between IL-6 values and CRP, ALT, total bilirubin, GGT and creatinine, but not amylase. In conclusion: (1) Portal IL-6, which is partly of pancreatic origin, is first metabolised in the liver; (2) Systemic IL-6 reflects hepatic and renal functions rather than local conditions in the pancreas; (3) IL-6 does not appear to influence PC spread; (4) IL-6, which is released in large amounts by the inflamed pancreas, may contribute to determining diabetes, thus interfering with the signal transducing pathways involved in glucose metabolism in liver cells.