Defect propagation in NiTi rotary instruments: a noncontact optical profilometry analysis.

AIM: To evaluate the presence and propagation of defects and their effects on surfaces of nickel-titanium (NiTi) instruments using noncontact, three-dimensional optical profilometry, and to assess the accuracy of this method of investigation. METHODOLOGY: The flute surface areas of instruments from two commercial instrumentation systems, namely Reciproc R25 (n = 5) and WaveOne Primary (n = 5), were assessed and compared before and after performing two instrumentation cycles in simulated root canals in clear resin blocks. All the analyses were conducted on areas measuring 211 × 211 µm, located 3 mm from the tips of the instruments. A quantitative analysis was conducted before and after the first and second instrumentation cycles, using the Sa (average roughness over the measurement field), Sq (root mean square roughness) and Sz (average height over the measurement field) amplitude parameters. All the data were submitted to statistical analysis at a 5% level of significance. RESULTS: There was a significant increase (P = 0.007) in wear in both groups, especially between baseline and the second instrumentation cycle, with significantly higher wear values being observed on WaveOne instruments (Sz median values = 33.68 and 2.89 µm, respectively, for WO and RP groups). A significant increase in surface roughness (P = 0.016 and P = 0.008, respectively, for Sa and Sq) was observed in both groups from the first to the second instrumentation cycle, mostly in WaveOne specimens. Qualitative analysis revealed a greater number of defects on the flute topography of all the instruments after use. CONCLUSIONS: More defects were identified in WaveOne Primary instruments compared to Reciproc R25, irrespective of the evaluation stage. The investigation method provided an accurate, repeatable and reproducible assessment of NiTi instruments at different time-points.

Reference miRNAs for colorectal cancer: analysis and verification of current data.

MicroRNAs (miRNAs) hold great promise in cancer research. The use of appropriate reference miRNAs for normalization of qPCR data is crucial for accurate expression analysis. We present here analysis and verification of current data, proposing a workflow strategy for identification of reference miRNAs in colorectal cancer (CRC). We performed a systematic review of studies aimed to identify stable reference miRNAs in CRC through high-throughput screening. Among the candidate miRNAs selected from the literature we excluded those predicted to target oncogenes or tumor suppressor gene. We then assessed the expression levels of the remaining candidates in exosomes, plasma and tissue samples from CRC patients and healthy controls. The expression stability was evaluated by box-plot, ∆Cq analysis, NormFinder and BestKeeper statistical algorithms. The effects of normalisers on the relative quantification of the oncogenic miR-1290 was also assessed. Our results consistently showed that different combinations of miR-520d, miR-1228 and miR-345 provided the most stably expressed reference miRNAs in the three biological matrices. We identified suitable reference miRNAs for future miRNA expression studies in exosomes plasma and tissues CRC samples. We also provided a novel conceptual framework that overcome the need of performing ex novo identification of suitable reference genes in single experimental systems.

A new method for the assessment of the surface topography of NiTi rotary instruments.

AIM: To describe a new method for the assessment of nanoscale alterations in the surface topography of nickel-titanium endodontic instruments using a high-resolution optical method and to verify the accuracy of the technique. METHODOLOGY: Noncontact three-dimensional optical profilometry was used to evaluate defects on a size 25, .08 taper reciprocating instrument (WaveOne® ), which was subjected to a cyclic fatigue test in a simulated root canal in a clear resin block. For the investigation, an original procedure was established for the analysis of similar areas located 3 mm from the tip of the instrument before and after canal preparation to enable the repeatability and reproducibility of the measurements with precision. All observations and analysis were taken in areas measuring 210 × 210 µm provided by the software of the equipment. RESULTS: The three-dimensional high-resolution image analysis showed clear alterations in the surface topography of the examined cutting blade and flute of the instrument, before and after use, with the presence of surface irregularities such as deformations, debris, grooves, cracks, steps and microcavities. CONCLUSIONS: Optical profilometry provided accurate qualitative nanoscale evaluation of similar surfaces before and after the fatigue test. The stability and repeatability of the technique enables a more comprehensive understanding of the effects of wear on the surface of endodontic instruments.

Effects of magnesium supplements on blood pressure, endothelial function and metabolic parameters in healthy young men with a family history of metabolic syndrome.

BACKGROUND AND AIMS: Magnesium plays an important role in the modulation of vascular tone and endothelial function and can regulate glucose and lipid metabolism. Patients with hypertension, metabolic syndrome (MetS) and diabetes mellitus (T2DM) have low body magnesium content; indeed, magnesium supplementation has been shown to have a positive effect on blood pressure (BP) and gluco-metabolic parameters. The aim of our study was to evaluate the effect of magnesium supplements on hemodynamic and metabolic parameters in healthy men with a positive family history of MetS or T2DM. METHODS AND RESULTS: In a randomized, double-blind, placebo-controlled 8-week crossover trial with a 4 week wash-out period, oral supplements of 8.1 mmol of magnesium-pidolate or placebo were administered twice a day to 14 healthy normomagnesemic participants, aged 23-33 years. The primary endpoint was office BP, measured with a semiautomatic oscillometric device. Secondary endpoints included characteristics of the MetS, namely endothelial function, arterial stiffness and inflammation. Plasma and urinary magnesium were measured in all participants while free intracellular magnesium was measured only in a subsample. There was no significant difference in either systolic and diastolic BP in participants post-magnesium supplementation and post-placebo treatment when compared to baseline BP measurements. Further, the metabolic, inflammatory and hemodynamic parameters did not vary significantly during the study. CONCLUSIONS: Our study showed no beneficial effect of magnesium supplements on BP, vascular function and glycolipid profile in young men with a family history of MetS/T2DM (trial registration at clinicaltrial.gov ID: NCT01181830; 12th of Aug 2010).

Epigenetic alteration: new insights moving from tissue to plasma - the example of PCDH10 promoter methylation in colorectal cancer.

BACKGROUND: Tumour-released DNA in blood represents a promising biomarker for cancer detection. Although epigenetic alterations such as aberrant promoter methylation represent an appealing perspective, the discordance existing between frequencies of alterations found in DNA extracted from tumour tissue and cell-free DNA (cfDNA) has challenged their practical clinical application. With the aim to explain this bias of agreement, we investigated whether protocadherin 10 (PCDH10) promoter methylation in tissue was associated with methylation pattern in matched cfDNA isolated from plasma of patients with colorectal cancer (CRC), and whether the strength of concordance may depend on levels of cfDNA, integrity index, as well as on different clinical-pathological features. METHODS: A quantitative methylation-specific PCR was used to analyse a selected CpG site in the PCDH10 promoter of 67 tumour tissues, paired normal mucosae, and matched plasma samples. The cfDNA integrity index and cfDNA concentration were assessed using a real-time PCR assay. RESULTS: The PCDH10 promoter methylation was detected in 63 out of 67 (94.0%) surgically resected colorectal tumours and in 42 out of 67 (62.7%) plasma samples. The median methylation rate in tumour tissues and plasma samples was 43.5% (6.3-97.8%) and 5.9% (0-80.9%), respectively. There was a significant correlation between PCDH10 methylation in cfDNA and tumour tissue in patients with early CRC (P<0.0001). The ratio between plasma and tissue methylation rate increases with increasing cfDNA integrity index in early-stage cancers (P=0.0299) and with absolute cfDNA concentration in advanced cancers (P=0.0234). CONCLUSION: Our findings provide new insight into biological aspects modulating the concordance between tissues and plasma methylation profiles.

Brand of dipotassium EDTA vacuum tube as a new source of pre-analytical variability in routine haematology testing.

This study assesses the use of different dry K2 (dipotassium) EDTA vacuum tubes and whether or not they might represent a bias in haematological testing. Blood was collected in three dipotassium EDTA vacuum tubes from different manufacturers: Venosafe, Vacuette and Vacutainer. Samples were analysed on an Advia 2120i analyser. Significant differences among results and biases were compared with current quality specifications. Significant differences were found for haematocrit (HCT), mean corpuscular volume (MCV), white blood cell count (WBC) and platelet distribution width (PDW) when comparing Venosafe vs. Vacuette; for MCV, WBC and PDW when comparing Venosafe vs. Vacutainer; and for HCT and MCV when comparing Vacuette vs. Vacutainer. Clinically significant variations were observed for HCT and PDW in Venosafe vs. Vacuette; PDW in Venosafe vs. Vacutainer; and HCT and MCV in Vacuette vs. Vacutainer. The use of dipotassium EDTA vacuum tubes from different manufacturers represent a clinically relevant source of variation for HCT, MCV and PDW.

Vanin-1 T26I polymorphism, hypertension and cardiovascular events in two large urban-based prospective studies in Swedes.

BACKGROUND AND AIMS: Vanin-1 (gene name VNN1) is an enzyme with pantetheinase activity generating the amino-thiol cysteamine which is implicated in the regulation of red-ox status through its effect on glutathione. We tested the hypothesis that the rs2294757 VNN1 T26I polymorphism could affect blood pressure (BP) levels, hypertension prevalence, and risk of incident cardiovascular events. METHODS AND RESULTS: The VNN1 T26I polymorphism was genotyped in 5664 participants of the cardiovascular cohort of the "Malmö Diet and Cancer" (MDC-CVA) study and successively in 17874 participants of the "Malmö Preventive project"(MPP). The incidence of cardiovascular events was monitored for an average of nearly 12 years of follow-up in the MDC-CVA and for 25 years in the MPP. Both before and after adjustment for sex, age and BMI in the MDC-CVA the polymorphism had a mild lowering effect on diastolic BP and hypertension, especially in females. However in MPP no effect on BP phenotypes was detectable. Before and after adjustment for major cardiovascular risk factors, the hazard ratio for incident ischemic stroke and coronary events in the MDC-CVA was not significantly different in carriers of different genotypes. CONCLUSIONS: Our data do not support a major role for the VNN1 T26I variant in determining BP level and incident ischemic events.

Impact of the CYP4F2 p.V433M polymorphism on coumarin dose requirement: systematic review and meta-analysis.

A systematic review and a meta-analysis were performed to quantify the accumulated information from genetic association studies investigating the impact of the CYP4F2 rs2108622 (p.V433M) polymorphism on coumarin dose requirement. An additional aim was to explore the contribution of the CYP4F2 variant in comparison with, as well as after stratification for, the VKORC1 and CYP2C9 variants. Thirty studies involving 9,470 participants met prespecified inclusion criteria. As compared with CC-homozygotes, T-allele carriers required an 8.3% (95% confidence interval (CI): 5.6-11.1%; P < 0.0001) higher mean daily coumarin dose than CC homozygotes to reach a stable international normalized ratio (INR). There was no evidence of publication bias. Heterogeneity among studies was present (I(2) = 43%). Our results show that the CYP4F2 p.V433M polymorphism is associated with interindividual variability in response to coumarin drugs, but with a low effect size that is confirmed to be lower than those contributed by VKORC1 and CYP2C9 polymorphisms.

HE4 in ovarian cancer: from discovery to clinical application.

Despite the relatively low prevalence, ovarian cancer is the fifth leading cause of death from cancer among women. As such, an early diagnosis for establishing a timely surgical and/or chemotherapeutic treatment is essential for improving the outcome. The most reliable, but not always straightforward, approach to diagnose ovarian cancer relies on multiple, time-consuming and expensive investigative tools. These typically include clinical presentation (i.e., pelvic or abdominal pain, urinary frequency or urgency, increased abdominal size or bloating) with pelvic examination, transvaginal ultrasonography (US), and measurement of carbohydrate antigen 125 (CA125). Although the conventional pathway to develop and market a clinically useful biomarker is challenging, recent advances in genomic and proteomic technologies have led to the identification of previously unknown candidate markers of ovarian cancer. Some of these are currently under clinical validation. The human epididymis protein 4 (HE4) has recently been approved by the Food and Drug Administration for monitoring recurrence or progression of epithelial ovarian cancer. Nevertheless, reliable clinical evidence demonstrates that HE4, used alone or in combination with CA125, substantially improves the accuracy of screening and/or disease monitoring. This chapter will review the current knowledge on biologic and clinical applications of ovarian cancer biomarkers, with particular emphasis on the newly proposed marker, HE4.

Transillumination: a new tool to eliminate the impact of venous stasis during the procedure for the collection of diagnostic blood specimens for routine haematological testing.

INTRODUCTION: The collection of diagnostic blood specimens for routine haematological testing (RHT) is traditionally performed with tourniquet. However, the transillumination devices based on cold near-infrared LEDs have been formerly proposed as a valuable tool for identifying reliable venous accesses, especially in patients with difficult or small veins, such as children. This study was aimed to evaluate whether a transillumination device can advantageously replace the use of the tourniquet during the procedure for collection of blood specimens for RHT and thereby eliminating the discomfort and risk of spurious results caused by excessive or prolonged venous stasis. METHODS: Two hundred and fifty volunteers were divided into five groups (G1, G2, G3, G4 and G5) to compare the results of RHT between blood sample collected with transilluminator device (left arm) and with tourniquet application (right arm) for 30 s(G1), 60 s(G2), 90 s(G3), 120 s(G4) and 180 s(G5). RESULTS: No significant increases were observed in any of the haematological parameters tested in G1 when compared with blood collected by the transilluminator device. From G2 to G5, significant increases were observed for the platelet count, red blood cell count, haemoglobin, haematocrit, white blood cell count, neutrophils, monocytes and eosinophils. From G3-G5, further increases were observed for lymphocytes. Clinically significant variations were, however, observed for basophils in G2; red blood cell count, haemoglobin, haematocrit and basophils in G3 and eosinophils in G3 only. CONCLUSION: As such, considering that inappropriate use of the tourniquet is commonplace, we conclude that transillumination devices can represent a suitable tool to eliminate the venous stasis and to improve the quality of phlebotomy procedures.

Acute variation of leucocytes counts following a half-marathon run.

There is growing interest on haematological changes following elite and recreation sports. However, no information is available on leucocyte variations after an intermediate-distance run. Complete blood cell count was assessed on 17 trained, middle-aged males before a 21-km half-marathon, at the end and 3, 6, 24 h thereafter. Statistically significant variations by one-way analysis of variance were observed for all the parameters tested. Haematocrit, haemoglobin and red blood cells and platelet count increased significantly by the end of the run and returned to pre-run values 3 h thereafter. White blood cells, neutrophils, monocytes and basophils counts increased after the run, reached the peak at 3 h and returned to the baseline after 24 h. Conversely, the eosinophils count significantly decreased after the run, reached a nadir at 3 h and slowly returned to pre-run values at 24 h. The lymphocytes count significantly increased after the run, decreased 3 h thereafter and returned to pre-run values after 6 h. These results show that an intermediate-distance run, which is a popular form of recreational sports worldwide, is associated with transitory leucocytosis, neutrophilia, monocytosis and basophilia, but it also induces acute eosinopenia, all changes being completely reversed 24 h thereafter.

Influence of temperature and time before centrifugation of specimens for routine coagulation testing.

The accurate standardization of the preanalytical phase is of pivotal importance for achieving reliable results of coagulation tests. Because information on the suitable storage conditions for coagulation testing is controversial, we aimed at investigating the sample stability with regard to the temperature and time before centrifugation. The activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrinogen and D-dimer were assayed in specimens collected from 26 consecutive patients on antivitamin K therapy on the ACL TOP analyzer. Three primary 3.6-ml siliconized evacuated tubes containing 0.109 mol/l buffered trisodium citrate were sequentially collected from each patient. These three tubes were mixed, pooled and divided into seven identical aliquots. The first aliquot was immediately centrifuged according to the standard protocol [1500 g for 15 min at room temperature (RT)] and analyzed. The other aliquots were left for 3, 6 and 24 h, respectively, at RT or 4 degrees C, and then centrifuged and analyzed. Test results were compared with those obtained on the reference specimen. Statistically significant prolongations were observed for aPTT in all the samples. Such differences exceeded the analytical quality specifications for desirable bias in the samples stored for 24 h. A significant reduction, yet comprised within the desirable bias, was observed for PT and fibrinogen in uncentrifuged specimens stored at RT for 3 and 6 h. No significant biases could be recorded in D-dimer. In conclusion, a 6-h storage of uncentrifuged specimens at either RT or 4 degrees C may still be suitable to achieve results of routine coagulation testing comprised within the analytical quality specifications for desirable bias.

Separation of haemoglobin HbE and HbA by the fully automated, high-pressure liquid chromatography Tosoh HLC-723 G7 analyzer.

High-pressure liquid chromatography instruments specifically devised for separating haemoglobin (Hb) fractions have been increasingly employed by the hospital laboratories over the recent years since they allow easy and fast screening for several Hb variants. Although such instruments may be proposed as sensitive, specific and reliable alternatives to the classic electrophoretic techniques, a major drawback of this screening strategy is the almost identical retention time of several Hb variants. In particular, at least 18 Hb variants have been reported in the same retention window as HbA(2), including HbE, the second most common beta-chain variant in humans after sickle cell trait. Recently, we evaluated the performance characteristics of an improved buffer formulation originally conceived for Hb variants separation procedures on the fully automated high-pressure liquid chromatography instrument Tosoh G7. At variance with other fully automated high-pressure liquid chromatography analyzers, the elution pattern on the G7 in subjects heterozygous for HbE is characterized by the presence of four suggestive peaks (HbF, HbA, HbA(2) and HbE), confirming the effective separation of HbE from HbA(2). Because of its potential value in the diagnosis of the thalassaemia syndromes, the effective separation of HbA(2) from HbE can provide clinical laboratories with a valuable information for the diagnostic reasoning.

Influence of stable, long-term treatment with phenobarbital on the activity of serum alanine aminotransferase and gamma-glutamyltransferase.

Phenobarbital, a long-acting barbiturate, is generally considered to be a fairly safe and effective drug; however, hepatotoxicity is an infrequent but potentially fatal adverse effect and there is little information on the serum activity of liver enzymes in patients on stable, long-term monotherapy. The serum activity of alanine aminotransferase (ALT) and gamma-glutamyltransferase (GGT) are measured along with phenobarbital as part of the routine biochemical measurement in 128 consecutive adult out-patients on stable, long-term phenobarbital treatment. The control population consists of 2468 consecutive outpatients matched for age and gender. The patients on long-term phenobarbital therapy had significantly higher serum activities of ALT (27 IU/L versus 23 IU/L, P < 0.001) and GGT (79 IU/L versus 24 IU /L, P < 0.001). The prevalence of subjects with abnormal GGT values, but not ALT, was significantly higher than that in the control population. No significant differences were observed in either the mean activity or the prevalence of abnormal values of ALT or GGT between patients with suboptimal and therapeutic concentrations of the drug. These results suggest that chronic phenobarbital therapy may be associated with a clinically significant elevation of serum GGT activity. If confirmed, a specific GGT reference range should be adopted. Moreover, in those patients presenting with high serum GGT activity, it would not be necessary to reduce the dosage, discontinue the drug or change to a different anti-epileptic medication.