Maria Stella Graziani

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Davide Giavarina
Bruna Lo Sasso
Giampaolo Merlini
Martina Montagnana
Andrea Mosca
Paola Pezzati
Rossella Tomaiuolo
Matteo Vidali

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Biochimica Clinica: VOL.37 N.6

L’importanza della qualità analitica
The value of analytical quality
F. Ceriotti  | 
Biochimica Clinica 2013; 37(6) 452-453
Editoriale - Editorial
Emofilia, come si concluderà la storia?
Hemophilia, how will end the story?

Hemophilia A (HA) and B (HB) are the most frequent inherited bleeding disorders caused by defects in the F8C and F9 genes that encode coagulation factor VIII and factor IX, respectively. Both HA and HB are X-linked recessive diseases and have an incidence of 1:5000 and 1:30,000 males, respectively. The diagnosis is based on normal prothrombin time, altered activated partial thromboplastin time and reduced activity
of factor VIII or factor IX in plasma. Furthermore, laboratory contributes to identify the inhibitor (an immunoglobulin against the factor that some hemophilic patients develop during therapy) and to reveal acquired hemophilia. Carrier females of HA and HB are tipically asymptomatic and can be identified only by molecular analysis; their evaluation is important, as one third of cases of hemophilia is due to novel mutations and in these cases the mother (and consanguineous females) of the proband have no risk to be carrier. Both diseases are due to a myriad of different mutations (mostly private), so that the molecular diagnosis is based on scanning techniques or gene sequencing. Given the number of hemophilic patients that experience severe perinatal complications, high-risk couples usually require prenatal diagnosis. We revise here our experience on 50 prenatal diagnoses of hemophilia. The clinical heterogeneity of hemophilic patients prompted many groups to study prothrombotic gene variants in these subjects to investigate whether such variants modify the clinical expression of disease. Finally, therapy (using recombinant factors) and, in a near future, gene therapy will change the natural history of hemophilic patients.

Biochimica Clinica 2013; 37(6) 454-460
Rassegne - Reviews
Polimorfismo I/D del gene per l’enzima di conversione dell’angiotensina (ACE): gene della longevità o fattore di rischio nella patologia ipertensiva?
Polymorphism of the angiotensin converting enzyme gene: longevity gene or risk factor in hypertensive disease?

In recent decades, the increase in life expectancy stimulated the study of aging processes and the search for candidate genes involved in longevity. The angiotensin converting enzyme (ACE), present in all endothelial cells, plays an essential role in maintaining the homeostasis of blood flow by regulating the production of the vasoconstrictor angiotensin II and inactivating the bradykinin. Some studies reported a possible association between the polymorphism I/D of ACE gene and either hypertension and longevity. The present study was aimed to confirm these data. We studied two large cohorts of nonagenarians and centenarians. One was from Sardinia (200 subjects, 88 males, mean age: 96 years) and their data were compared to a group of 222 subjects (106 males, mean age: 44 years) from the general population of the same geographic area. The latter group of longeve subjects (161 subjects, 71 males, mean age: 97 years) was from Southern Italy. Furthermore, we studied 146 hypertensive patients (98 males, mean age: 51 years) and 172 normotensive subjects (86 males, mean age: 33 years) from Southern Italy. The ACE I/D polymorphism was typed by polymerase chain reaction; the amplified 490 bp (allele I) and 190 bp (allele D) were visualized on 2% agarose gel. Hypertensive subjects had a significantly different distribution of ACE genotypes as compared to normotensive ones (P=0.001) and a higher frequency of the D/D genotype. Long-lived subjects from Sardinia showed a significantly different distribution of ACE genotypes as compared to subjects from the general population of the same geographic area (P <0.001), to long-lived subjects from Southern Italy (P <0.001), to hypertensive patients (P=0.011) and to normotensive subjects from Southern Italy (P <0.001). Surprisingly, they had the highest frequency of the D/D genotype among the compared groups. Our study indicates that: i) centenarians of different ethnic origin have a different genetic background, ii) there is a possible association between longevity and allelic variants of ACE, even if only in specific ethnic groups (i.e., Sardinian) and iii) ACE polymorphisms are a predisposing factor to hypertension.

Biochimica Clinica 2013; 37(6) 461-464
Contributi scientifici - Scientific papers
Identificazione e caratterizzazione di mutazioni in regioni regolatorie del gene malattia della fibrosi cistica
Identification and characterization of mutations in regulatory regions of cystic fibrosis disease gene

Mutation epidemiology is crucial for cystic fibrosis (CF) diagnosis and counselling. ~6%-7% of alleles from CF patients do not bear mutations in the coding regions of the Cystic Fibrosis Transmembrane Regulator (CFTR) disease gene. In these patients, mutations may be present in non-coding, regulatory regions of the gene as i) intronic regions (particularly in high conserved sequences), ii) the promoter region or iii) the area at the 3’ of the gene, which is the target of microRNA regulation. We studied these regions by gene sequencing in a group of CF patients with one or both unidentified mutations after the analysis of CFTR coding regions, and in a group of CF patients with a different clinical expression of disease to evaluate if mutations in such regions may have a role in modulating CF clinical expression. Our analysis revealed: i) a dozen of mutations (most novel) in the large promoter area of 6000 bp at the 5’ of the gene; expression studies in four cell lines demonstrated that a half of such mutations may have a pathogenic effect; ii) a series of mutations in 52 conserved sequence tags (CSTs), i.e. intronic regions with a high homology between humans and mouse; expression studies revealed the pathogenic effect of one of these mutations; iii) finally, three mutations in the 1500 bp region at the 3’ of the gene; one of this has a pathogenic role, enhancing the interaction of CFTR with two inhibitory microRNAs. To the best of our knowledge, this is the first example of such pathogenic mechanism in a monogenic disorder. On the contrary, no mutations were identified in patients with different clinical expression in any of the three
non-coding regions. To conclude, the sequencing of non coding regions may improve the detection rate of molecular analysis in CF, but functional studies are required to define the pathogenic effect of novel mutations.

Biochimica Clinica 2013; 37(6) 465-469
Contributi scientifici - Scientific papers
Verifica della riferibilità metrologica dei dispositivi medico-diagnostici in vitro: responsabilità e strategie
Verification of in vitro medical diagnostics (IVD) metrological traceability: strategies and responsibilities

To be accurate and equivalent laboratory results should be traceable to higher-order references. Furthermore, their analytic quality should fulfil acceptable measurement uncertainty as defined to fit the intended clinical use. To this aim, IVD manufacturers should define a calibration hierarchy to assign traceable values to their system calibrators and to fulfil during this process uncertainty limits for calibrators that should represent a proportion of the uncertainty budget allowed for clinical laboratory results. It is important that end-users may know and verify how manufacturers have implemented the traceability of their calibrators and estimated the corresponding uncertainty. Currently, the full information about traceability and uncertainty of calibrator is not available as manufacturers only provide the name of higher-order reference material and/or procedure to which the assay calibration is traceable without any description of steps and their corresponding uncertainty of the implemented traceability chain. Important post-market tools for IVD traceability surveillance are related to the verification by clinical laboratories of the consistency of declared performance during daily operations performed in accordance with the manufacturer’s instructions and the organization of appropriately structured EQAS. The former activity should be accomplished by analyzing system control materials and confirming that current measurements are in the manufacturer’s established control range. With regard to EQAS, it is mandatory that target values to materials are assigned with reference procedures by an accredited reference laboratory, that materials are commutable and a clinically allowable inaccuracy for participant’s results is defined in order to prove the suitability of laboratory measurements in clinical setting.

Biochimica Clinica 2013; 37(6) 470-478
Opinioni - Opinions
Cancer biomarkers 2013: appunti a margine del convegno satellite EuroMedLab
Cancer biomarkers 2013: notes about the EuroMedLab satellite meeting

Like all other interventions and devices in health care, cancer biomarkers should undergo a careful validation process before they are introduced into clinical practice for diagnostic or prognostic purposes. Particularly, for those candidate diagnostic markers, validation implies the assessment of their diagnostic accuracy according to pre-specified and standardized criteria. For prognostic purposes, large, protocol-driven prospective studies should assure unbiased results on the effectiveness of the investigated biomarkers in predicting patient’s risk of a future outcome, in aiding individual treatment choice and in patient counselling. The improvement of the methodology of research should finally contribute evidence of high level and strength useful to recommend or not the appropriate application of specific biomarkers in different clinical settings (screening, primary/secondary care, treatment monitoring, follow-up). In this presentation, we summarized the major issues highlighted during the recent EuroMedLab satellite meeting held in Venice (IT) on May 2013, in which standardized processes from the evaluation of biomarkers’ diagnostic accuracy and prognostic value for guideline development were discussed. In addition, current efforts on the research of novel molecules and therapeutic targets (proteins, exosomes, circulating nucleic acids) candidate to clinical introduction were briefly reviewed.

Biochimica Clinica 2013; 37(6) 479-492
Opinioni - Opinions
I prodotti derivati dalla carbamilazione come potenziali biomarcatori nell’insufficienza renale cronica e nell’aterosclerosi
Carbamylation-derived products: bioactive compounds and potential biomarkers in chronic renal failure and atherosclerosis

Carbamylation is a posttranslational modification of proteins resulting from the nonenzymatic reaction between isocyanic acid and specific free functional groups. This reaction alters protein structural and functional properties and thus contributes to molecular ageing. Many studies have shown the involvement of carbamylated proteins in diseases, especially in chronic renal failure and atherosclerosis. In this review we describe the biochemical basis of the carbamylation process and its role in protein molecular ageing. We summarize the current evidence of protein carbamylation involvement in disease, identify available biomarkers of the carbamylation process and their related analytical methods, and discuss the practical relevance of these biomarkers. Carbamylation-induced protein alterations are involved in the progression of various diseases, because carbamylation-derived products (CDPs) are bioactive compounds that trigger specific and inappropriate cellular responses. For instance, carbamylation may promote hormone and enzyme inactivation, and carbamylated proteins, as diverse as collagen or LDLs, induce characteristic biochemical events of atherosclerosis progression. CDPs are potential biomarkers to monitor diseases characterized by an increased rate of carbamylation (e.g., chronic renal failure and atherosclerosis). Different methods (e.g., liquid chromatography–tandem mass spectrometry and immunoassays) to measure specific carbamylated proteins or general markers of carbamylation, such as protein-bound homocitrulline, have been described. Their use in clinical practice must still be validated by appropriate clinical studies.

Biochimica Clinica 2013; 37(6) 493-499
Il meglio di Clinical Chemistry - Clinical Chemistry highlights
Idiopatic acquired hemophilia A in two women in Chioggia

Acquired hemophilia A (AHA) is a rare, but often life-threatening hemorrhagic disorder characterized by antibodies directed against coagulation factor VIII. We report clinical and laboratory investigations of two cases with AHA observed in our hospital. These patients were two elderly women (73 and 62 years old), who presented with subcutaneous bleeding, intramuscular hematoma and a prolonged activated partial thromboplastin time (aPTT). On the basis of these findings as well as decreased factor VIII activities and the presence of factor VIII inhibitors, we made a diagnosis of AHA. Both patients were referred to a specialized hospital for treatment. The diagnosis of AHA should be considered in any elderly patient who presents with bleeding and prolonged aPTT. Moreover, the coexistence of a series of underlying diseases associated with AHA should be always searched for.

Biochimica Clinica 2013; 37(6) 500-503
Casi clinici - Case report
Utilità della misura dell’attività plasmatica di eparina-anti fattore Xa durante trattamento antitrombotico in gravidanza
Usefulness of heparin-anti factor Xa assay during antithrombotic treatment in pregnancy

A case of successful pregnancy in a woman with previous episodes of unprovoked deep vein thrombosis treated with vitamin K antagonist (warfarin) therapy is described. As the patient became pregnant, warfarin therapy was immediately stopped and lowmolecular-weight heparin (LMWH) prophylaxis was started. In the second and third trimesters of pregnancy we observed an increase in body weight (from 64 to 80 kg) and in body mass index (from 21 to 26.4). During the pregnancy, we used the heparin-anti factor Xa assay (Anti-FXa) to monitor the LMWH treatment and to adjust the dosage in a increasing way. The Anti-FXa can be very useful in improving safety and efficacy of LMWH treatment in pregnancy.

Biochimica Clinica 2013; 37(6) 504-507
Casi clinici - Case report
Report dal congresso satellite EuroMedLab sulle proteine plasmatiche
Report from the EuroMedLab Satellite Meeting on plasma proteins
F. Braga  | 
Biochimica Clinica 2013; 37(6) 508-509
Notizie SIBioC - SIBioC News
La qualità dei metodi molecolari nell’era della medicina personalizzata
The quality of molecular methods in the age of personalized medicine
Biochimica Clinica 2013; 37(6) 510-511
Notizie SIBioC - SIBioC News
Riferibilità metrologica e standardizzazione: report dal 7th CIRME International Scientific Meeting
Metrological traceability and standardization: report from the 7th CIRME International Scientific Meeting
D. Szőke  | 
Biochimica Clinica 2013; 37(6) 512-515
Notizie SIBioC - SIBioC News
Elezioni Consiglio Direttivo SIBioC - Medicina di Laboratorio
Biochimica Clinica 2013; 37(6) 516-522
Notizie SIBioC - SIBioC News
4° Congresso Interregionale SIBioC - Medicina di Laboratorio Le giornate mediterranee di Medicina di Laboratorio Sorrento (NA), 9-11 ottobre 2013
4th SIBioC - Laboratory Medicine Interregional Congress Sorrento (NA) 9-11 October 2013
Biochimica Clinica 2013; 37(6) 523-580
Atti congressuali - Meeting abstracts
Indice dei contenuti e degli autori - Volume 37, 2013
Index of contents and authors - Volume 37, 2013
Biochimica Clinica 2013; 37(6) 583-590
Indice dei contenuti e degli autori - Index of contents and authors