How standardization of the pre-analytical phase of both research and diagnostic biomaterials can increase reproducibility of biomedical research and diagnostics.

Comparison of published biomedical studies shows that a large proportion are irreproducible, causing severe damage to society and creating an image of wasted investments. These observations are of course damaging to the biomedical research field, which is currently full of future promise. Precision medicine and disease prevention are successful, but are progressing slowly due to irreproducible study results. Although standardization is mentioned as a possible solution, it is not always clear how this could decrease or prevent irreproducible results in biomedical studies. In this article more insight is given into what quality, norms, standardization, certification, accreditation and optimized infrastructure can accomplish to reveal causes of irreproducibility and increase reproducibility when collecting biomaterials. CEN and ISO standards for the sample pre-analytical phase are currently being developed with the support of the SPIDIA4P project, and their role in increasing reproducibility in both biomedical research and diagnostics is demonstrated. In particular, it is described how standardized methods and quality assurance documentation can be exploited as tools for: 1) recognition and rejection of 'not fit for purpose' samples on the basis of detailed sample metadata, and 2) identification of methods that contribute to irreproducibility which can be adapted or replaced.

Influence of pre-analytical procedures on genomic DNA integrity in blood samples: the SPIDIA experience.

BACKGROUND: DNA integrity is a critical part of the definition of genomic DNA (gDNA) quality and can influence downstream molecular applications. Pre-analytical variables as sample storage and DNA extraction methods can influence the quality and quantity of isolated DNA and affect molecular test performances. The aim of this paper is to investigate the role of blood sample storage and DNA extraction procedures on gDNA integrity and gDNA fragmentation impact on a molecular test. METHODS: 157 DNA samples deriving from the Pan European 1st SPIDIA DNA External Quality Assessment (EQA), aimed to investigate the influence of blood storage on gDNA quality and quantity, have been analyzed by Pulsed Field Gel Electrophoresis and ImageJ imaging software. 157 DNA samples derived from the Pan European 1st SPIDIA DNA External Quality Assessment (EQA), which aimed to investigate the influence of blood storage on gDNA quality and quantity, have been analyzed by Pulsed Field Gel Electrophoresis and ImageJ imaging software. RESULTS/CONCLUSIONS: Our results demonstrate that blood sample storage and DNA extraction procedures influence gDNA integrity and that the same blood sample which underwent a long range multiplex PCR based analytical test can provide different results if the adopted pre-analytical procedures are not standardized.

Circulating BRAFV600E in the diagnosis and follow-up of differentiated papillary thyroid carcinoma.

CONTEXT: Cell-free nucleic acids circulating in plasma are considered a promising noninvasive tool for cancer monitoring. BRAF(V600E) mutation in cell-free DNA (cfDNA) could represent an appropriate marker for papillary thyroid carcinoma (PTC). OBJECTIVE: Our aim is to investigate the role of BRAF(V600E)-mutated allele in cfDNA as a marker for the diagnosis and follow-up of PTC. STUDY DESIGN: BRAF(V600E) allele was detected and quantified by an allele-specific real-time quantitative PCR assay in plasma from 103 patients affected by nodular goiter. As control populations, we enrolled 49 healthy subjects and 16 patients with non-nodular thyroid diseases. RESULTS: The percentage of circulating BRAF(V600E) was significantly different between patients and controls and throughout different cytological categories of ultrasound-assisted fine-needle aspiration. Patients with a histopathological diagnosis of PTC showed a higher percentage of circulating BRAF(V600E) (P = .035) compared to those with benign histology. In 19 patients, a second blood draw, taken 3-6 months after surgery, showed a lower percentage of BRAF(V600E) in cfDNA than the presurgical sample (P < .001). The diagnostic performance of circulating BRAF(V600E) was assessed by receiver operating characteristic curve analysis resulting in an area under the curve of 0.797. A cutoff value was chosen corresponding to maximum specificity (65%) and sensitivity (80%). On this basis, we evaluated the predictive value of BRAF(V600E) in Thy 3 patients with a resulting positive predictive value of 33% and a negative predictive value of 80%. CONCLUSIONS: The results of the present study provide encouraging data supporting the possibility to take advantage of circulating BRAF(V600E) in the management of PTC.

SPIDIA-DNA: an External Quality Assessment for the pre-analytical phase of blood samples used for DNA-based analyses.

BACKGROUND: The EC-funded project SPIDIA is aimed to develop evidence-based quality guidelines for the pre-analytical phase of blood samples used for DNA molecular testing. To this purpose, a survey and a pan-European External Quality Assessment (EQA) were implemented. METHODS: SPIDIA facility sent to all the participants the same blood sample to be processed without time or temperature limitation. DNA quality parameters performed at SPIDIA facility included: UV spectrophotometric analysis of DNA purity and yield, PCR interferences study by Kineret software and DNA integrity analysis by pulsed field gel electrophoresis. RESULTS: 197 applications have been collected from 30 European countries. A high variability of DNA fragmentation was observed whereas purity, yield and PCR interferences had a narrow distribution within laboratories. A significant difference between the RNase P single copy gene quantity obtained in the DNA samples extracted with the precipitation-based method respect to those obtained with beads and column-based methods was observed. CONCLUSIONS: The results of this study will be the basis for implementing a second pan-European EQA and the results of both EQAs will be pooled and will provide the basis for the implementation of evidence-based guidelines for the pre-analytical phase of DNA analysis of blood samples.

SPIDIA-RNA: first external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses.

The diagnostic use of in vitro molecular assays can be limited by the lack of guidelines for collection, handling, stabilization and storage of patient specimens. One of the major goals of the EC funded project SPIDIA (www.spidia.eu) is to develop evidence-based quality guidelines for the pre-analytical phase of blood samples used for molecular testing which requires intracellular RNA analytes. To this end, a survey and a pan-European external quality assessment (EQA) were implemented. This report is the summary of the results of that trial. With the European Federation of Laboratory Medicine (EFLM) support, 124 applications for participation in the trial were received from 27 different European countries, and 102 laboratories actually participated in the trial. Each participating laboratory described their respective laboratory policies and practices as well as blood collection tubes typically used in performing this type of testing. The participating laboratories received two identical blood specimens: in an EDTA tubes (unstabilized blood; n=67) or in tubes designed specifically for the stabilization of intracellular RNA in blood (PAXgene® Blood RNA tubes; n=35). Laboratories were requested to perform RNA extraction according to the laboratory's own procedure as soon as possible upon receipt of the tubes for one tube and 24h after the first extraction for the second tube. Participants (n=93) returned the two extracted RNAs to SPIDIA facility for analysis, and provided details about the reagents and protocols they used for the extraction. At the SPIDIA facility responsible for coordinating the study, the survey data were classified, and the extracted RNA samples were evaluated for purity, yield, integrity, stability, and the presence of interfering substances affecting RT-qPCR assays. All participants received a report comparing the performance of the RNA they submitted to that of the other participants. All the results obtained by participants for each RNA quality parameter were classified as "in control", "warning", "out of control" and "missing" by consensus mean analysis. From the survey data, the most variable parameters were the volume of blood collected and the time and storage temperature between blood collection and RNA extraction. Analyzing the results of quality testing of submitted RNA samples we observed a data distribution of purity, yield, and presence of assay interference in agreement with expected values. The RNA Integrity Number (RIN) values distribution was, on the other hand, much wider than the optimal expected value, which led to an "in control" classification, even for partly degraded RNA samples. On the other hand, RIN values below 5 significantly correlated with a reduction of GAPDH expression levels. Furthermore, the distribution of the values of the four transcripts investigated (c-fos, IL-1ß, IL-8, and GAPDH) was wide and the RNA instability between samples separated by 24h were similar. Assuming the presence of at least two quality parameters "out of control" as an indication of a critical performance of the laboratory, 33% of the laboratories were included in this group. The results of this study will be the basis for implementing a second pan-European EQA and the results of both EQAs will be pooled and will provide the basis for the implementation of evidence-based guidelines for the pre-analytical phase of RNA analysis of blood samples.

Red or white wine assumption and serum antioxidant capacity.

The biochemistry of reactive oxygen species is an important field with wide implications. Both preventive and chain breaking antioxidants have a role in the limitation of oxidative stress that accompanies aging and diseases. The potent antioxidant activity of phenolic substances of red wine, in particular, have been proposed as an explanation for the "French Paradox" (the apparent incompatibility of a high fat diet with a low incidence of coronary heart diseases). A lot of researchers emphasize beneficial effects of red wine and insist on lower or no antioxidant effect of white wine. We have been studying the effect of both white and red wine on blood antioxidant capacity in humans. The white wine we have been testing was produced by an ancient Tuscany procedure (the same used for red winemaking) which includes fermentation with grapes juice together with peelings and seeds. A statistically significant increase in total antioxidant capacity (TAC) levels was observed after 2h from red or white wine ingestion. White wine effect appears to be faster than that of the red one, since a significant difference can also be reported after 1h. We can conclude that the big difference in the results of serum TAC due to white wine reported by us, in comparison to those reported by others relatively to white wine produced using the French method, can be explained by the difference in the winemaking procedure we adopted.

Centenarians in Tuscany: The role of the environmental factors.

Statistical data of Italy, issued on 1st January 2005, show that Tuscany has a high percentage of elderly subjects, and that to reach 100 years is a common possibility, mainly for women. Comparable values appear for longevity index (LI%), either calculated on the basis of the various resorts above sea level, or on the basis of the number of inhabitants. However, some differences are observed considering the various provinces: Grosseto Province has the second higher value of >65-year-old subjects, while its LI% is the lowest. Centenarity index (CI%) considerably differs from LI%: if ranking Tuscany according to the various heights of territorial positions, the first displays a negative, while the second one shows a positive correlation with the local height. If comparing for inhabitants number, CI% is maximal in provincial cities, while LI% is higher in smaller communities. All these data indicate that residents in small communities of the mountains reach easily 90 years, while 100 years are more frequently reached in larger cities in the plane areas. In conclusion, the highest province longevity (Lucca) coincides with the highest centenarian sex ratio, and it suggests that in this province the older women may enjoy particular forms of environmental protection, which helps them for reaching 100 years of age. Differences between the results of this study and others performed in Sardinia are discussed.

Longevity index (LI%) and centenarity index (CI%): new indicators to evaluate the characteristics of aging process in the Italian population.

Centenarians represent a group of population displaying the most rapid expansion. This progressive increase of the presence of centenarians is a multi-factorial phenomenon, which is due to the improvements of the environmental conditions and the life style and also to the progress of the medical science. In order to obtain a more reliable estimate of the longevity per gender and territorial entities, we propose two new indicators. (i) The ratio between the ultranonagenarians and the total population above 65 years old (called longevity index: LI%); (ii) the ratio between the centenarians and the total population above 90 years old (called centenarity index: CI%). An analysis of the data of the Italian National Office of Statistics (ISTAT, ) using these two indicators demonstrates that the subjects above the age of 90 are more frequent in the regions of Central and Northern Italy, which are more developed regarding the economic conditions and technological progress. Nevertheless, the Southern and Insular regions of Italy have a higher occurrence of centenarians among the ultranonagenarian population, and also a higher prevalence of male centenarians, as compared to the northern regions. This demonstrates that achievement of the threshold of 100 years old does not require only particular socio-economic conditions, but also an adequate climate and environment, as well as a favorable genetic composition.

Early detection of Biscogniauxia nummularia in symptomless European beech (Fagus sylvatica L.) by TaqMan quantitative real-time PCR.

AIMS: To develop a quantitative real-time PCR (Rt PCR) assay for the early detection of Biscogniauxia nummularia, a xylariaceous fungus that causes strip-canker and wood decay on European beech (Fagus sylvatica L.). METHODS AND RESULTS: The molecular assay was based on TaqMan chemistry using species-specific primers and a fluorogenic probe designed on the ITS1 sequence of rRNA gene clusters. The specificity of the oligonucleotides and the probe were tested using the DNA of B. nummularia isolates from different geographic areas, of phylogenetically related species, and of some fungi commonly colonizing European beech bark and wood. A total of 31 symptomless and symptomatic shoots of European beech were collected from three forest sites in the Apennine Mountains of Italy. The percentage of positive detections of B. nummularia with the TaqMan assay was 78.6%, compared with only 14.3% of positive isolations on growth media for two sites. CONCLUSIONS: In shoots, the quantitative Rt PCR assay detected down to 8.0-fg fungal DNA per microgram of total DNA extracted. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay developed in quantitative Rt PCR, by using TaqMan chemistry, revealed a rapid and sensitive method useful for the early detection of B. nummularia in symptomless European beech twigs.

Laser-assisted microdissection for real-time PCR sample preparation.

Laser-assisted microdissection (LMD) has been developed to procure precisely the cells of interest in a tissue specimen, in a rapid and practical manner. Together with real-time PCR and RT-PCR techniques, it is now feasible to study genetic alterations, gene expression features and proteins in defined cell populations from complex normal and diseased tissues. The process that brings from sample collection to the final quantitative results is articulated in several steps, each of which requires optimal choices in order to end up with high-quality nucleic acid or protein that allows successful application of the final quantitative assays. This review will describe shortly the development of LMD technologies and the principles they are based on. Trying to highlight the advantages and disadvantages of LMD, the main problems related to specimens collection and processing, section preparation and extraction of bio-molecules from microdissected tissue samples have been analysed.

Inhibition of acetylcholine-induced activation of extracellular regulated protein kinase prevents the encoding of an inhibitory avoidance response in the rat.

It has been demonstrated that the forebrain cholinergic system and the extracellular regulated kinase signal transduction pathway are involved in the mechanisms of learning, encoding, and storage of information. We investigated the involvement of the cholinergic and glutamatergic systems projecting to the medial prefrontal cortex and ventral hippocampus and of the extracellular regulated kinase signal transduction pathway in the acquisition and recall of the step-down inhibitory avoidance response in the rat, a relatively simple behavioral test acquired in a one-trial session. To this aim we studied by microdialysis the release of acetylcholine and glutamate, and by immunohistochemistry the activation of extracellular regulated kinase during acquisition, encoding and recall of the behavior. Cholinergic, but not glutamatergic, neurons projecting to the medial prefrontal cortex and ventral hippocampus were activated during acquisition of the task, as shown by increase in cortical and hippocampal acetylcholine release. Released acetylcholine in turn activated extracellular regulated kinase in neurons located in the target structures, since the muscarinic receptor antagonist scopolamine blocked extracellular regulated kinase activation. Both increased acetylcholine release and extracellular regulated kinase activation were necessary for memory formation, as administration of scopolamine and of extracellular regulated kinase inhibitors was followed by blockade of extracellular regulated kinase activation and amnesia. Our data indicate that a critical function of the learning-associated increase in acetylcholine release is to promote the activation of the extracellular regulated kinase signal transduction pathway and help understanding the role of these systems in the encoding of an inhibitory avoidance memory.

Effect of diet and red wine consumption on serum total antioxidant capacity (TAC), dehydroepiandrosterone-sulphate (DHEAS) and insulin-like growth factor-1 (IGF-1) in Italian centenarians.

The traditional mediterranean diet is associated with a hope for longer survival. It has also been shown that the red wine possesses a protective effect against the oxidative stress. We studied TAC, the DHEAS and the IGF-1 in a group of 26 healthy centenarians, 17 women and 9 men, of the age range of 100--105 years. Furthermore, we analyzed also serum urate and bilirubin levels between drinkers and abstainers. Most of centenarian subjects have been moderate wine consumers (<500 ml/day of red wine). These subjects were subdivided as follows: (i) Group A: those who had maintained the style of their dietary habits as compared to the previous years (n=3 males, 10 females); (ii) Group B: those who actually consumed a diet being deficient compared to that of the previous years, but remained moderate drinkers of red wine (n=3 males, 4 females); and (iii) Group C: those who actually consumed a diet being deficient compared to that of the previous years, and at the same time, were abstainers in wine consumption (n=3 males, 3 females). The results show that in men three of the studied parameters decreased from Group A to C to considerable extents, as follows (mean+/-S.D.). TAC: 302.4+/-32.3; 142.0+/-24.1 and 96.4+/-20.1 micromol/l; DHEAS: 3.35+/-0.81; 2.52+/-0.18 and 1.34+/-0.14 micromol/l; IGF-1: 85.7+/-6.7; 76.6+/-6.7 and 65.6+/-2.6 ng/ml, respectively. For the same parameters, the results in the women were: TAC: 258.4+/-12.2; 182.1+/-14.0 and 107.6+/-10.0 micromol/l; DHEAS: 3.85+/-0.16; 2.34+/-0.19 and 2.05+/-0.04 micromol/l; IGF-1: 89.7+/-6.7; 76.6+/-4.7 and 64.2+/-2.7 ng/ml, respectively. We did not find any significant difference in the other serum parameters between drinkers (n=14) and abstainers (n=3) (urate: 267.6+/-52.9, and 289.5+/-80.1; bilirubin: 9.81+/-4.29 and 7.18+/-2.89 micromol/l, respectively). Our data suggest that the deteriorated diet caused a reduction of TAC, DHEAS and IGF-1 in the centenarians. However, red vine consumption exerted a protective effect against this trend, even if this protection is not reaching statistical significance in some cases (in men), which is due most probably to the lower number of male subjects in the study.

Real-time PCR detection of Biscogniauxia mediterranea in symptomless oak tissue.

AIMS: Real-time PCR, based on TaqMan chemistry, was used to detect Biscogniauxia mediterranea, a fungal pathogen that after a long endophytic phase may cause charcoal disease in oak trees. METHODS AND RESULTS: Specific primers and probe were designed and tested on axenic cultures of B. mediterranea and other fungi commonly colonizing oaks. Twig samples were collected in Tuscany from apparently healthy oaks (Quercus cerris, Quercus ilex and Quercus pubescens) growing near trees infected with the fungus. Twigs were divided into two groups: one for isolation in agar plates, and one for real-time PCR after DNA extraction. The detection limit of the assay was 0.01 pg/DNA, whereas the amounts of fungal DNA detected in asymptomatic tissue were >0.5 pg microg(-1) total DNA extracted. In the apparently healthy twigs the frequency of isolation found on agar was 25.0%, much lower than that with real-time PCR (96.4%). CONCLUSIONS: Real-time PCR is a sensitive and fast technique able to specifically detect and quantify the DNA of B. mediterranea in oak tissue. SIGNIFICANCE AND IMPACT OF THE STUDY: This diagnostic method is a precise tool to localize fungi in symptomless plant tissues and promises to advance our understanding of fungal infection during their latent phase.

Healthy centenarian subjects: the effect of red wine consumption on liver function tests.

Liver function is well maintained with increasing age. The aim of our study was to investigate if long-term moderate (

Statistical diagnostics emerging from external quality control of real-time PCR.

Besides the application of conventional qualitative PCR as a valuable tool to enrich or identify specific sequences of nucleic acids, a new revolutionary technique for quantitative PCR determination has been introduced recently. It is based on real-time detection of PCR products revealed as a homogeneous accumulating signal generated by specific dyes. However, as far as we know, the influence of the variability of this technique on the reliability of the quantitative assay has not been thoroughly investigated. A national program of external quality assurance (EQA) for real-time PCR determination involving 42 Italian laboratories has been developed to assess the analytical performance of real-time PCR procedures. Participants were asked to perform a conventional experiment based on the use of an external reference curve (standard curve) for real-time detection of three cDNA samples with different concentrations of a specific target. In this paper the main analytical features of the standard curve have been investigated in an attempt to produce statistical diagnostics emerging from external quality control. Specific control charts were drawn to help biochemists take technical decisions aimed at improving the performance of their laboratories. Overall, our results indicated a subset of seven laboratories whose performance appeared to be markedly outside the limits for at least one of the standard curve features investigated. Our findings suggest the usefulness of the approach presented here for monitoring the heterogeneity of results produced by different laboratories and for selecting those laboratories that need technical advice on their performance.

Measurement of somatostatin receptor subtype 2 mRNA in breast cancer and corresponding normal tissue.

Somatostatin analogs are effective in inhibiting growth of human breast cancer cell lines. These antiproliferative effects are mediated by specific receptors located on cell membranes. The somatostatin receptor subtype 2 (sst2) is the principal mediator of somatostatin effects in normal and cancer cells, and its presence has already been demonstrated in breast cancer. The purpose of our study was to evaluate the clinical relevance of the expression of sst2 by quantifying its mRNA in a large group of infiltrating breast cancers and their corresponding normal tissues. The expression of sst2 mRNA was measured with quantitative real time RT-PCR in 169 breast cancers and in their corresponding unaffected tissues. We evaluated the association of sst2 expression with the commonest clinical-pathologic features of breast cancer. The correlation with a marker of cell proliferation (Ki-67) and with receptor concentration was also evaluated. In cancer tissues, we found that the absolute concentrations of sst2 mRNA were significantly higher in estrogen receptor (ER)-positive samples (P=0.002) as well as in lymph-node-negative cancers (P=0.04) (Student's t-test or one-way ANOVA). In addition, sst2 mRNA was significantly higher in breast cancers than in corresponding unaffected tissues (P=0.0002). However, when the clinical-pathologic parameters were considered, this gradient maintained its statistical significance only in tumors expressing positive prognostic markers, such as the presence of ER (P=0.0005) and progesterone receptors (PgR) (P=0005), and the lack of lymph-node involvement (P=0.0003). The same difference was also significant in postmenopausal women (P=0.001) and in T1 patients (P=0.001). In addition, sst2 mRNA expression was significantly higher (P=0.008) in low-proliferating breast cancers. Finally, we found that the quantitative expression of sst2 mRNA was directly related to the PgR concentration in breast cancer tissues (P<0.001). Our data seem to indicate that an upregulation of sst2 gene expression is a common feature of breast cancers which, on the basis of conventional predictive parameters, are expected to have a better prognosis. Featuring a possible role of somatostatin analogs in combined endocrine therapies for breast cancer, our results seem to confirm that the sst2 status of the tumor should be previously investigated.

Rapid detection of Oenococcus oeni in wine by real-time quantitative PCR.

AIMS: To develop a real-time polymerase chain reaction (PCR) method for rapid detection and quantification of Oenococcus oeni in wine samples for monitoring malolactic fermentation. METHODS AND RESULTS: Specific primers and fluorogenic probe targeted to the gene encoding the malolactic enzyme of O. oeni were developed and used in real-time PCR assays in order to quantify genomic DNA either from bacterial pure cultures or wine samples. Conventional CFU countings were also performed. The PCR assay confirmed to be specific for O. oeni species and significantly correlated to the conventional plating method both in pure cultures and wine samples (r = 0.902 and 0.96, respectively). CONCLUSIONS: The DNA extraction from wine and the real-time PCR quantification assay, being performed in ca 6 h and allowing several samples to be concurrently processed, provide useful tools for the rapid and direct detection of O. oeni in wine without the necessity for sample plating. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid quantification of O. oeni by a real-time PCR assay can improve the control of malolactic fermentation in wines allowing prompt corrective measures to regulate the bacterial growth.

Multidrug resistance in ovarian cancer: comparing an immunocytochemical study and ATP-tumor chemosensitivity assay.

The aim of our study was to evaluate the possible prognostic and predictive significance of the expression of P-glycoprotein, a transmembrane transport protein related to multidrug resistance, in previously untreated patients with FIGO stage III ovarian cancer; to compare the results of immunocytochemical analysis of tissue sections of tumors to the in vitro chemosensitivity to cytotoxic drug of fresh samples of the same tumors; and to evaluate survival in women who underwent the same surgical treatment and the same adjuvant chemotherapy.