Diagnostica e Ricerca San Raffaele SpA

Milano, Italy

Diagnostica e Ricerca San Raffaele SpA

Milano, Italy
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PubMed | Diagnostica e Ricerca San Raffaele S.p.A.
Type: Journal Article | Journal: EJIFCC | Year: 2016

This paper looks at the topic of reference intervals from the point of view of the patient or the clinician. The differences between the concepts of reference intervals (biological characteristic of a well defined population) and the various types of decision limits are illustrated and discussed. Decision limits can be defined in different ways: based on a Bayesian approach, on epidemiological studies or on clinical experience, but differ from reference intervals because, while the latter deals with physiology, decision limits are related to some kind of disease or risk of developing it.

Pietra D.,University of Pavia | Brisci A.,San Raffaele Scientific Institute | Rumi E.,University of Pavia | Boggi S.,University of Pavia | And 10 more authors.
Haematologica | Year: 2011

Somatic mutations of MPL exon 10, mainly involving a W515 substitution, have been described in JAK2 (V617F)-negative patients with essential thrombocythemia and primary myelofibrosis. We used direct sequencing and high-resolution melt analysis to identify mutations of MPL exon 10 in 570 patients with myeloproliferative neoplasms, and allele specific PCR and deep sequencing to further characterize a subset of mutated patients. Somatic mutations were detected in 33 of 221 patients (15%) with JAK2 (V617F)-negative essential thrombocythemia or primary myelofibrosis. Only one patient with essential thrombocythemia carried both JAK2 (V617F) and MPL (W515L). High-resolution melt analysis identified abnormal patterns in all the MPL mutated cases, while direct sequencing did not detect the mutant MPL in one fifth of them. In 3 cases carrying double MPL mutations, deep sequencing analysis showed identical load and location in cis of the paired lesions, indicating their simultaneous occurrence on the same chromosome. © 2011 Ferrata Storti Foundation.

Pighin S.,University of Trento | Savadori L.,University of Trento | Barilli E.,University of Trento | Rumiati R.,University of Padua | And 5 more authors.
Medical Decision Making | Year: 2013

The present research provides empirical evidence of whether communicating the prenatal risk of chromosomal anomalies using comparison scenarios influences women's ability to distinguish between different risk levels. In 2 experiments, participants read a description of a hypothetical woman who was learning of the risk of chromosomal anomaly as a result of a prenatal screening test. Both experiments used a 3 (risk level) × 3 (scenario) full between-subjects design. In accordance with the experimental condition, participants were presented with a low (e.g., 1 in 5390), a medium (e.g., 1 in 770), or a high risk value (e.g., 1 in 110). Such risk values were presented either on their own or along with additional information illustrating a comparison scenario that provided 2 numerical comparison points. Participants were asked to evaluate the risk of chromosomal anomaly. In Experiment 2, participants' numeracy skills were also assessed. Results showed that the use of comparison scenarios results in significant differences in perceived risk across risk levels whereas such differences are not significant without the comparison scenario, but such a technique has differential effects according to participants' capacity to deal with numbers. Although the technique is beneficial for high-numerate participants, it has no effect on low-numerate participants.

Galbiati S.,San Raffaele Scientific Institute | Damin F.,CNR Institute of Chemistry of Molecular Recognition | Pinzani P.,University of Florence | Mancini I.,University of Florence | And 7 more authors.
PLoS ONE | Year: 2013

Molecular diagnostics of human cancers may increase accuracy in prognosis, facilitate the selection of the optimal therapeutic regimen, improve patient outcome, reduce costs of treatment and favour development of personalized approaches to patient care. Moreover sensitivity and specificity are fundamental characteristics of any diagnostic method. We developed a highly sensitive microarray for the detection of common KRAS and BRAF oncogenic mutations. In colorectal cancer, KRAS and BRAF mutations have been shown to identify a cluster of patients that does not respond to anti-EGFR therapies; the identification of these mutations is therefore clinically extremely important. To verify the technical characteristics of the microarray system for the correct identification of the KRAS mutational status at the two hotspot codons 12 and 13 and of the BRAFV600E mutation in colorectal tumor, we selected 75 samples previously characterized by conventional and CO-amplification at Lower Denaturation temperature-PCR (COLD-PCR) followed by High Resolution Melting analysis and direct sequencing. Among these samples, 60 were collected during surgery and immediately steeped in RNAlater while the 15 remainders were formalin-fixed and paraffin-embedded (FFPE) tissues. The detection limit of the proposed method was different for the 7 KRAS mutations tested and for the V600E BRAF mutation. In particular, the microarray system has been able to detect a minimum of about 0.01% of mutated alleles in a background of wild-type DNA. A blind validation displayed complete concordance of results. The excellent agreement of the results showed that the new microarray substrate is highly specific in assigning the correct genotype without any enrichment strategy. © 2013 Galbiati et al.

Brisci A.,San Raffaele Scientific Institute | Damin F.,CNR Institute of Chemistry of Molecular Recognition | Pietra D.,University of Pavia | Galbiati S.,San Raffaele Scientific Institute | And 9 more authors.
Clinical Chemistry | Year: 2012

BACKGROUND: Myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Somatic mutations in exon 10 of the MPL (myeloproliferative leukemia virus oncogene) gene, mainly substitutions encoding W515 variants, have recently been described in a minority of patients with ET or PMF. We optimized analytically sensitive methods for detecting and genotyping MPL variants. METHODS: We used DNA previously isolated from circulating granulocytes of 60 patients withMPNthat had previously been analyzed by high-resolution melting (HRM), direct sequencing, and the TaqMan allelicdiscrimination assay. We developed conditions for enriching tumor mutant alleles with COLD-PCR (coamplification at lower denaturation temperature PCR) and coupled it with direct sequencing. Assays were designed for identifying MPL W515 substitutions with full COLD-PCR protocols. In parallel, we used innovative microarray substrates to develop assays for evaluating the mutant burden in granulocyte cells. RESULTS: Mutations that were present at very low levels in patients who had previously been scored as having an MPL variant by HRM and as wild type by direct sequencing were successfully identified in granulocyte DNA. Notably, the microarray approach displayed analytical sensitivities of 0.1% to 5% mutant allele, depending on the particular mutation. This analytical sensitivity is similar to that obtained with COLD-PCR. The assay requires no enrichment strategy and allows both the characterization of each variant allele and the evaluation of its proportion in every patient. CONCLUSIONS: These procedures, which are transferable to clinical diagnostic laboratories, can be used for detecting very low proportions of minority mutant alleles that cannot be identified by other, conventional methods. © 2012 American Association for Clinical Chemistry.

Castellanos-Rizaldos E.,Dana-Farber Cancer Institute | Liu P.,Dana-Farber Cancer Institute | Milbury C.A.,Dana-Farber Cancer Institute | Guha M.,Dana-Farber Cancer Institute | And 8 more authors.
Clinical Chemistry | Year: 2012

BACKGROUND: Low-level mutations in clinical tumor samples often reside below mutation detection limits, thus leading to false negatives that may impact clinical diagnosis and patient management. COLD-PCR (co-amplification at lower denaturation temperature PCR) is a technology that magnifies unknown mutations during PCR, thus enabling downstream mutation detection. However, a practical difficulty in applying COLD-PCR has been the requirement for strict control of the denaturation temperature for a given sequence, to within ±0.3°C. This requirement precludes simultaneous mutation enrichment in sequences of substantially different melting temperature (Tm) and limits the technique to a single sequence at a time. We present a temperature-tolerant (TT) approach (TT-COLD-PCR) that reduces this obstacle. METHODS: We describe thermocycling programs featuring a gradual increase of the denaturation temperature during COLD-PCR. This approach enabled enrichment of mutations when the cycling achieves the appropriate critical denaturation temperature of each DNA amplicon that is being amplified. Validation was provided for KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) and TP53 (tumor protein p53) exons 6-9 by use of dilutions of mutated DNA, clinical cancer samples, and plasma-circulating DNA. RESULTS: A single thermocycling program with a denaturation-temperature window of 2.5-3.0°C enriches mutations in all DNA amplicons simultaneously, despite their different Tms. Mutation enrichments of 6-9-fold were obtained with TT-full-COLD-PCR. Higher mutation enrichments were obtained for the other 2 forms of COLD-PCR, fast-COLD-PCR, and ice-COLD-PCR. CONCLUSIONS: Low-level mutations in diverse amplicons with different Tms can be mutation enriched via TT-COLD-PCR provided that their Tms fall within the denaturation-temperature window applied during amplification. This approach enables simultaneous enrichment of mutations in several amplicons and increases significantly the versatility of COLD-PCR. © 2012 American Association for Clinical Chemistry.

Foglieni B.,San Raffaele Scientific Institute | Brisci A.,San Raffaele Scientific Institute | Biagio F.S.,CPG Group | Pietro P.D.,CPG Group | And 6 more authors.
Clinical Chemistry and Laboratory Medicine | Year: 2010

Background: Several microdevices have been developed to perform only a single step of a genotyping process, such as PCR or detection by probe hybridization. Here, we describe a Lab-on-Chip (LoC) platform integrating a PCR amplification microreactor with a customable microarray for the detection of sequence variations on human genomic DNA. Methods: Preliminary work was focused on developing the single analytical steps including PCR and labeling strategies of the amplified product by conventional reference systems. The optimized protocols included a 1:4 forward:reverse primer ratio for asymmetric PCR, and Cy5-dCTP multiple incorporation for the generation of a labeled PCR product to be hybridized to complementary probes bound to the chip surface. Results: Final conditions were applied to the fully integrated LoC platform for the detection of the IVSI-110 G>A mutation in the human β-globin (HBB) gene associated with β-thalassemia, used as a model of genetic application, allowing for correct genotyping of 25 samples that were heterozygous, homozygous or wild-type for this mutation. Conclusions: The overall results show that the present platform is very promising for rapid identification of DNA sequence variations in an integrated, cost effective and convenient silicon chip format. © 2010 by Walter de Gruyter.

Carobene A.,Diagnostica e Ricerca San Raffaele S.p.A. | Carobene A.,Working Group Biological Variation | Braga F.,Working Group Biological Variation | Braga F.,University of Milan | And 5 more authors.
Clinical Chemistry and Laboratory Medicine | Year: 2013

Background: Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyl transferase (GGT) are enzymes measured in serum or plasma to investigate liver disease. The aim of this work is to assess the validity of published biological variation (BV) data currently available for these enzymes. Methods: Publications containing BV data for ALT, AST and GGT were identified by searching PubMed using the following keywords: biological varia*, RCV, CVw, CVi, CVb, and CV g. The 95% confidence intervals for the within- and between-subject coefficients of variation were calculated using the analytical imprecision, the number of subjects, samples and replicates. Results: The searches identified 10 publications with ALT, 14 with AST and nine with GGT data. The protocols presented in those publications as used were varied. The ranges of within-subject variation reported were: ALT: 11.1%-58.1%, AST: 3.0%-32.3% and for GGT: 3.9%-14.5%. The median values (ALT: 18.0%, AST: 11.9% and GGT: 13.8%) were similar to those listed in a BV database commonly used as a reference source. Conclusions: Published BV data for ALT, AST and GGT demonstrate a wide range of values derived from inconsistent protocols. The quality of the presentations of the data is variable. These findings raise concerns around the utility of the data currently available and highlight the need for critical appraisal of such publications. The working group on BV of the European Federation of Clinical Chemistry and Laboratory Medicine is undertaking work to develop a critical appraisal checklist for the production and publication of reliable BV data.

Infusino I.,University of Milan | Ceriotti F.,Diagnostica e Ricerca San Raffaele S.p.A. | Panteghini M.,University of Milan
Biochimica Clinica | Year: 2010

The goal of standardization for measurements of catalytic concentrations of enzymes is to achieve comparable results in human samples, independent of the reagent kits, instruments, and laboratory where the assay is carried out. To pursue this objective, the IFCC has established reference measurement systems for the most important clinical enzymes. These systems are based on the following requirements: a) reference methods, well described in procedures that are extensively evaluated; b) suitable reference materials; and c) reference laboratories operating in a highly controlled manner. Using these reference systems appropriately, the diagnostic industry can assign traceable values to commercial calibrators. Clinical laboratories, which use routine procedures with validated calibrators to measure human specimens, can finally obtain values which are traceable to higher-order reference procedures. These reference systems constitute the structure of the traceability chain to which the routine methods can be linked via an appropriate calibration process, provided that they have a comparable specificity (i.e., they are measuring the same catalytic quantity).

Passerini G.,Diagnostica e Ricerca San Raffaele S.p.A. | Basile U.,Catholic University of the Sacred Heart
Biochimica Clinica | Year: 2010

Cryoglobulins are immunoglobulins that precipitate out of serum at temperature <37 °C and resolubilize on warming. The clinical syndrome for cryoglobulinemia tipically includes purpura, weakness and arthralgias, but also underlying disease may contribute to symptoms. Cryoglobulin testing is carried out on blood sample collected, transported, clotted and spun at 37 °C, before the precipitate is allowed to form in serum stored at 4°C in a Wintrobe tube for at least 7 days. The most important confounding factor affecting cryoglobulin test is the preanalytical phase not fully done at 37 °C. The easiest way to quantify cryoglobulins is the cryocrit estimate, directly measured in the Wintrobe tube. However, this approach shows low accuracy and sensitivity. Furthermore, the precipitate should be resolubilized by warming to confirm that it is truly formed by cryoglobulins. Characterization of cryoglobulins requires several washing of the precipitate before immunofixation, technique by which cryoglobulins can be classified according to Brouet into three types depending from the characteristics (monoclonal vs. polyclonal) of the detected immunoglobulins. We suggest a wider classification of cryoglobulins undertaking also the microheterogeneous and biclonal patterns detected by high resolution immunoelectrophoretic techniques.

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