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Articoli in pubblicazione

Identificazione di geni di normalizzazione per studi trascrizionali con PCR quantitativa: revisione della letteratura
Identification of genes for normalization of RT-qPCR gene expression data: a review of published literature

Reverse-transcriptase quantitative Polymerase Chain Reaction (RT-qPCR) is a well-established technique to quantifygene expression levels and critically depends on reference genes for data normalization.
We performed a review of biomedical literature to analyse the usage of RT-qPCR in relation to other techniques fortranscriptional analyses and to describe practices for the identification of suitable reference genes for RT-qPCR.
In the 81 analysed studies, 3 genes (GAPD, ACTB, B2M) were included in ≥70% of cases, but ranked among themost stable genes in ≤1/3 of cases. The most frequently used normalizing algorithm was geNorm(83%), followed byNormFinder(73%) and BestKeeper(32%).
We also analysed transparency and good laboratory practices based on adherence to the Minimum Information forPublication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, using selected validated evaluationcriteria. Overall, key MIQE criteria were satisfied in ≥50% of analyzed studies, but only four criteria (details ofemployed kit/enzyme for reverse transcription, priming method, primers/probes and DNA polymerase) were satisfiedin ≥90% of cases. Data on assay repeatability were reported only in 15% of studies. The presence of pseudogenesas a potential confounder of assay specificity was evaluated only in 13% of studies. Finally, as few as 6% of studiesaccounted for the presence of known mutations of singly nucleotide polymorphisms when designing assayprimers/probes.
Better adherence to the MIQE guidelines should be encouraged. Publicly available transcriptomic and genomic datasets could be employed to refine the identification of suitable normalizing genes and to assist assay design.

Biochimica Clinica
DOI: 10.19186/BC_2019.051
Pubblicato online il: 05.09.2019
Rassegne - Reviews
Vitamin D plasma level can affect nivolumab drug exposure in a cohort of patients with non-small-cell lung cancer

Introduction: immune-checkpoint inhibition using programmed cell death-1 and its ligand drug inhibitors haveimproved survival, among patients with advanced non-small-cell lung cancer (NSCLC): nivolumab is one of the lastapproved. Vitamin D deficiency (<20 ng/mL) is frequent in lung cancer patients and studies showed this pre-hormonemodulates the expression of genes involved in drug pathway and in the immune system regulation. Furthermore, notmany biomarkers related to nivolumab therapy are present in literature. The aim of this study was to understand whichfactors were able to predict nivolumab concentrations and its anti-antibody levels in patients’ plasma at 15, 45 and 60days of therapy.
Methods: forty-five patients with advanced NSCLC were enrolled to receive nivolumab. Enzyme-linkedimmunosorbent assay was used for drug and vitamin D quantification.
Results: Median nivolumab plasma levels were 12.5, 22.3 and 27.1 μg/mL respectively at 15, 45 and 60 days(p<0.001). No anti-nivolumab antibodies have been detected. 25-hydroxyvitamin D median concentrations were 12.8ng/mL at baseline, 13.6 ng/mL at 15 days, 11.8 ng/mL at 45 days and 12.9 ng/mL at 60 days. Gender significantlyaffected nivolumab concentrations (p=0.010 at 15 days, p=0.033 at 45 days and p=0.006 at 60 days). In linearregression analyses, 25-hydroxyvitamin D <20 ng/mL before starting therapy, gender and 25-hydroxyvitamin D <20ng/mL at 15 days were able to predict nivolumab concentrations respectively at 15, 45 and 60 days of treatment.
Conclusions: for the first time, this study shows factors able to predict nivolumab exposure at different timings, butfurther, studies in bigger and different cohorts are needed to clarify these data.

TAG: immune-therapy   monoclonal antibody   NSCLC   
Biochimica Clinica
DOI: 10.19186/BC_2019.046
Pubblicato online il: 31.07.2019
Contributi Scientifici - Scientific Papers
Pazienti diabetici di tipo 2, non in terapia insulinica e albumina glicata: una valutazione multidimensionale
Insuline-Naive type 2 diabetic patients: a multidimensional evaluation on the role of glycated albumin

Insuline-Naaïve type 2 diabetic patients: a multidimensional evaluation on the role of glycated albuminIntroduction: glycated Albumin (GA) is an innovative glycemic marker, that could be used in the clinical practice, asan add-on strategy, to the traditional glycemic monitoring systems, such as glycated haemoglobin (Hb1Ac) and fastingplasma glucose (FPG). The study aims at presenting the results of a multidimensional analysis conducted in Italy,exploring the main clinical, economic, ethical, social and organizational implications, related to the introduction of GA.Methods: an Health Technology Assessment (HTA) approach was implemented. The analysis considered the ItalianNational Healthcare Service (NHS) perspective, and assumed a 12-month time horizon, focusing on type 2 diabetespatients insulin-naïve, assuming oral therapy. The 9 HTA dimensions (derived from the Core Model developed by theEuropean Network of HTA – EUnetHTA) were deployed, considering scientific evidence, health economics tools andqualitative approaches, through the administration of specific questionnaires to 15 diabetes experts.Results: literature reported better GA safety and efficacy profiles, thus being a predictor of the relative risk for diabetescomplications development, and increasing the therapeutic success after 3 months of therapy (97.0% versus71.6%).From an economic point of view, GA introduction resulted in an economic advantage of 1.06% and in a better trade-off between costs sustained and efficacy gained. Considering a 7-item Likert Scale (ranging from -3 to +3), negativeperceptions emerged with regard to equity aspects (0.13 versus0.72) due to GA limited accessibility, whereas it wouldimprove both patients (2.17 versus1.33) and care givers (1.50 versus0.83) quality of life. In the short term, GArequired training courses and equipment update, whereas, in the long term, it could be considered the preferablesolution from an organizational perspective (0.30 versus0.01).Conclusions: the results of this study demonstrated GA strategic relevance, its economic sustainability and feasibility,as well as the potential clinical pathway improvement.

TAG: Diabete mellito   albumina   insulina  
Biochimica Clinica
DOI: 10.19186/BC_2019.049
Pubblicato online il: 26.07.2019
Contributi Scientifici - Scientific Papers
Studio preliminare del microbiota intestinale, cutaneo e della mucosa orale in pazienti affetti da Pemfigo Volgare e Pemfigoide Bolloso
Preliminary study of the microbiome in the gut, skin and oral mucosa of patients affected by Pemphigus Vulgaris and Bullous Pemphigoid

Introduction: the study of the human microbiome is one of the most dynamic current topics in biomedical research. Asignificantchallenge in this field is the development of cost effective method for a robust interrogation of microbiotacomposition.Advances in next-generation sequencing (NGS) technologies have allowed for efficient and molecular-based analysisof microbial communities. Association between diseases and imbalance of the microbial populationsare today wellinvestigated.
Methods: Pemphigus Vulgaris (PV) and Bullous Pemphigoid (BP) are two rare autoantibody-mediated blisteringskindiseases. In this pilot study, we characterized the intestinal, cutaneous and oral mucosal microbiota compositionsinPV/BP patients, in order to evaluate the potential role of the bacterial composition in these dermatologicaldisorders.Particularly, we performed a high-throughput sequencing analysis of the V3-V4 hypervariable regions of16S rRNA forthe evaluation of bacterial composition of stool, skin and oral mucosa samples in PV (n=12) and BP(n=8) patients.
Results: a similar composition of the intestinal microbiota was observed in PV and BP patients. The evaluationof skinlesions revealed a prevalence of Firmicutes phylum in both patients’ groups. In the cutaneous microbiota, weidentifieda significant decreased abundance of Bacteroidetes phylum compared to healthy controls.
Conclusions: the results obtained from our standardized NGS pipeline, reinforced by correlation with other clinical andbiochemicalparameters, will contribute to clarify the mechanisms of these rare diseases.

TAG: Microbiota   sequenziamento   pemfigo  
Biochimica Clinica
DOI: 10.19186/BC_2019.041
Pubblicato online il: 26.07.2019
Contributi Scientifici - Scientific Papers
Valutazione delle prestazioni analitiche dei conteggi cellulari in automazione su liquidi biologici mediante un programma di controllo di qualità dedicato
Evaluation of the analytical performances of the cell counting on biological fluids using a dedicated program of quality control

Introduction: the development of new technologies in the era of information technology and digitization has certainlyinfluenced the clinical laboratory and quality management system. The aim of this work is to verify the utility of adedicated software (SNCS - Sysmex, Inc. Kobe, Japan) for the improvement of quality control managementprocedures of quantitative parameters obtained by the automated cell counting in biological liquids.
Methods: the measurements were performed on the XN "Body Fluid Mode" (XN-BF) analytical system (Sysmex, Inc.Kobe, Japan) according to the manufacturer's specifications. The parameters that can be included in the report usingthis mode are the total nucleated elements count, the leukocyte count, the differentiation of mononuclear frompolymorphonuclear cells and the erythrocyte count.
Results: our study included: a comparative evaluation between the laboratory CVs and the CV of an internationalhomogeneous group; a retroactive evaluation from the SNCS software using the standard deviation index and theprecision index used as accuracy and precision measurements. Finally, a daily comparison was made between theLevi-Jennings cards of the instrument and the SNCS intra-day report, to improve the timely evaluation of the randomerror.
Conclusion: using the SNCS software, the performance of the analyzers can be precisely monitored and comparedon an internaional basis. Its utility lies in the possibility of comparing internal performances with those of a group oflaboratories using the same instruments and the same controls, allowing the quantification of the analytical bias.

TAG: Conta dei leucociti   differenziazione cellulare   eritrociti  
Biochimica Clinica
DOI: 10.19186/BC_2019.042
Pubblicato online il: 08.07.2019
Contributi Scientifici - Scientific Papers
Attività fisica e biomarcatori dello stress ossidativo nell’invecchiamento
Physical exercise and oxidative stress biomarkers in the elderly

Introduction: aging is regulated by a number of aging genes including sirtuins, linked to redox status andepigenetically controlled. Physical exercise, depending on intensity, type of training and duration, seems to modulatepositively the redox state in aging. It was demonstrated an increase in susceptibility to oxidative stress by thedepletion of intracellular glutathione (GSH) and total glutathione (tGSH) levels in the muscles of aged animals.Moreover, physical activity, especially if moderate and regular, seems to activate adaptive mechanisms, reducingbody's vulnerability to oxidative stress and improving systemic metabolic activities.
Methods: this study is aimed to evaluate the effects of regular exercise (3 times/week for 2 months) on the levels ofradical species (d-ROM test), thiol groups (SHp test), plasma antioxidant capacity (BAPtest), 8-OH-guanosine(COMET test) and some redox-related proteins (HSP27, HSP70, Sirt1 and 2) in 40 healthy elderly subjects, agedfrom 65 to 74 years. In addition, plasma enzymatic activities of superoxide dismutase (SOD), glutathione peroxidase(GPx) and glutathione reductase (GR) were evaluated.
Results: the enrolled subjects showed oxidative stress before exercise with the presence of radical species, oxidationof purine bases and reduced antioxidant capacity. After the scheduled training, a global improvement of oxidativestress markers was observed with an upregulation of HSP27, HSP70, Sirt1 and Sirt2 expression, as adaptiveresponse.
Conclusion: our results underline the importance of these biochemical parameters to monitor oxidative stress in agingand the adaptive redox response to physical exercise in elderly.Therefore, these biomarkers may be routinely usedin order to identify individuals at risk for developing oxidative stress related pathologies as well as in personalizedphysical therapy.

TAG: Sirtuine   glutatione   superossido dismutasi  
Biochimica Clinica
DOI: 10.19186/BC_2019.044
Pubblicato online il: 08.07.2019
Contributi Scientifici - Scientific Papers
Comparative study of galectin-3, brain natriuretic peptide, cystantine C and high sensitivity troponin determinations in the diagnosis of heart failure in elderly italian males

Introduction: heart failure (HF) has been defined a modern pandemic. The complex array of physiologic,psychological, social and health care issues makes it a challenging chronic disease to diagnose and manage. Ourstudy is aimed to evaluate a multi-markers approach in diagnosis of HF in aged males.
Methods: 68 Italian males aged >65 years have been enrolled; 25 were patients with heart failure (HF) and 43 werehealthy controls. In all the subjects, measurements of high sensitivity troponin I (TNI), galectin-3 (GAL), cystatin C(CYS) and brain natriuretic peptide (BNP) were performed using routine methods.
Results: in patients with HF, mean concentrations of TNI, GAL, CYS and BNP were significantly higher (p<0.001) thanvalues observed in controls subjects. Only BNP and GAL showed a correlation with NHYA stage of HF. In this study,GAL and BNP demonstrated better diagnostic perfomamces to differentiate of HF patients from controls subjects.
Conclusions: our study showed the usefulness of a strategy involving multiple biomarkers determination in laboratorydiagnosis of HF in elderly males.

TAG: Biomarkers   Heart failure   Elderly  
Biochimica Clinica
DOI: 10.19186/BC_2019.040
Pubblicato online il: 04.07.2019
Contributi Scientifici - Scientific Papers
Evaluation of a multiplex immunochromatographic assay for the rapid detection of carbapenemase-producing Enterobacteriaceaefrom culture colonies

Introduction: the increasing worldwide spread of multidrug resistant bacteria, in particular of carbapenemase-producing Enterobacteriaceae(CPE), represents a serious clinical and public health concern. An accurate and fastdetection of infected patients or colonized carriers is thus mandatory.
Aim of this study was to assess the performance of a multiplex immunochromatographic assay (NG-Test CARBA 5,NG Biotech, Guipry, France) for the rapid detection of carbapenemases directly from pure bacterial colonies.
Methods: seventy-five non-replicated Enterobacteralesisolates with decreased susceptibility to carbapenems,including 71 Klebsiella pneumoniae, 3Escherichia coli and1Enterobacter cloacae, were analysed with NG-TestCARBA 5. At the same time the combination disk test (CDT) was performed according to the European Committeeon Antimicrobial Susceptibility Testing (EUCAST) indications, while confirmation of carbapenemase production wasachieved by polymerase chain reaction (PCR).
Results: PCR assay could find 66 CPE strains, including 64 Klebsiella pneumoniae[53 producing Klebsiellapneumoniaecarbapenemase (KPC), 5 New Delhi metallo-β-lactamase (NDM), 2 class D oxacillinases (OXA-48), 1Verona integron-encoded metallo-β-lactamase (VIM) and 3 co-producing NDM and OXA-48] and 2 Escherichia coli(2 NDM+OXA-48) while 9 isolates were found as non-carbapenemase producing: 7 Klebsiella pneumoniae, 1Escherichia coli, 1Enterobacter cloacae. CDT allowed us to consider those 9 strains as extended spectrum β-lactamase (ESBL) or AmpC β-lactamase producers. NG-Test CARBA 5 successfully identified 66/66 CPE showing100% sensitivity and 100% specificity. Unlike NG-Test CARBA 5, CDT was not able to correctly identify 5 strains co-producing NDM and OXA-48 carbapenemases.
Conclusion: NG-Test CARBA 5 is a reliable assay that can be useful in settings requiring a rapid identification of CPEdirectly from culture colonies. Moreover, this test is an easy-to-use option that could avoid misidentification ofcarbapenemases co-producers strains.

TAG: carbapenemase   Escherichia coli   β-lactamase  
Biochimica Clinica
DOI: 10.19186/BC_2019.033
Pubblicato online il: 04.07.2019
Contributi Scientifici - Scientific Papers
Variabilità biologica dei parametri dell’esame emocromocitometrico in soggetti sani
Biological variation estimates of complete blood count parameters in healthy subjects

Background: the complete blood count (CBC) is the test more frequently requested in clinical practice. Therefore,estimating the biological variation (BV) of CBC parameters is essential for assessing the analytical performance ofhematological analyzers and for enabling accurate data interpretation and appropriate clinical management. Thisstudy was aimed to define BV estimates and reference change value (RCV) of CBC parameters.
Methods: the study population consisted of 21 healthy volunteers, who had BV of CBC parameters assessed withSysmex XN. The study protocol, the analytical measurements and the statistical analysis were carried out accordingto current recommendations of the European Federation for Clinical Chemistry and Laboratory Medicine (EFLM).
Results: Within-subject BV ranged between 0,3% for mean cell hemoglobin (MCH) and 19,7% for immaturegranulocytes (IG), whilst between-subjects BVs ranged between 0,9% for mean corpuscolar haemoglobinconcentration (MCHC) and 66,6% for microcytic red blood cells (Micro-R). The RCV ranged between 2,3% for MCHand 73,5% for IG.
Conclusion: This study has allowed the estimation of BV of many CBC parameters, some of which have not beencurrently explored, thus leading the way to use RCV calculated according to time of monitoring and/or differentiatedby sex.

TAG: Valori di riferimento   indici eritrocitari   variabilità biologica  
Biochimica Clinica
DOI: 10.19186/BC_2019.031
Pubblicato online il: 10.06.2019
Contributi Scientifici - Scientific Papers
Patologie cardiovascolari e troponine cardiache: la storia di un legame indissolubile
Cardiovascular diseases and cardiac troponins: the history of a lasting connection

The evolution of thebiochemical diagnosis of cardiac diseases, represents a paradigm of the laboratory medicine evolution in the recent years.
Starting from the use of poor specific and sensitive biomarkers, the “so-called” cardiac enzymes (aspartateaminotransferase; lactate dehydrogenase; creatine kinase) recommended by World Health Organization for the acutemyocardial infarction (AMI) diagnosis, a fundamental development in biochemical knowledge has been obtained,providing new biomarkers (CK-MB, myoglobin) for a more specific and early diagnosis according to the clinical andtherapeutic needs. However, the revolutionary biochemical issue has been represented by the discovery of cardiactroponins and by the implementation of methods allowing their measurement in emergency setting in patients withacute chest pain. Cardiac troponins, are characterized by an absolute cardiac specificity and by a high sensitivity thatallow to carry out a timely and safe diagnosis of AMI, being recognized as “gold standard” in all clinical andbiochemical guidelines. In patients with acute chest pain and in ischemic clinical setting, a typical kinetic release ofbiomarker concentration may be suggestive of AMI even if ECG typical patterns are lacking. The actual improvementin analytical performance of troponins methods, particularly in the analytical sensitivity, allows to extend themeasurement also in diagnosis of minor myocardial damage in patients suffering from different cardiac disease, tomonitor the efficacy of therapy, the progression of the disease and to provide prognostic information and risk-stratification in addition to the clinical pathway.

TAG: Infarto acuto del miocardio   mioglobina   biomarcatori  
Biochimica Clinica
DOI: 10.19186/BC_2019.021
Pubblicato online il: 10.06.2019
Opinioni - Opinions
La Medicina di Laboratorio: gli specialisti di domani
Laboratory Medicine: specialists of tomorrow

Laboratory Medicine rides the wave of technological progress, themetamorphosis of information systems and data management. The Young Specialist is not a mere observer, butrather takes a leading role in this change, taking advantage of the opportunities offered by “omics” technologies,capturing new ideas and innovative stimuli that lead to a new concept of work and research oriented to health andprevention. Thanks to the support of international web platforms, training and exchange programs supported by theInternational Scientific Societies and Federations that favor professional and scientific growth, Young Scientists workin a global context. In this scenario, the SIBioC Young Scientists Study Group, with the auspices of SIBioC, EFLMand IFCC, organized a meeting on "Laboratory Medicine: Specialists of tomorrow" with the aim of discussing andhighlighting some of the most important challenges, such as technological progress, training and internationalizationof young people. Finally, the future of laboratory medicine looks at a multidisciplinary approach that leads tointegrated diagnosis, identification of the frail patient, the use of the Point of Care Testing as an indispensable tool incrisis areas, making the dialogue between physician and laboratory specialist a fundamental step for the diagnosisand treatment with the final aim of a better outcome for the patient.

TAG: Società scientifiche   studi interdisciplinari   ricerca scientifica  
Biochimica Clinica
DOI: 10.19186/BC_2019.039
Pubblicato online il: 04.07.2019
Documenti - Documents
Documento di consenso Gruppo di Studio di Farmacotossicologia Clinica e Doping SIBioC e Gruppo Tossicologi Forensi (GTFI). Modalità per lo svolgimento di indagini di laboratorio per la determinazione delle sostanzed’abuso
Consensus document of the Study Group on Clinical Pharmacotoxicology and Doping of Italian Society ofClinical Biochemistry and Italian Forensic Toxicologists Group (GTFI) – for laboratories involved in thedetermination of substances of abuse

The greatest workload for the laboratories performingpharmacotoxicological tests remains the routine activity for detection and measurement, in different biologicalmatrices, of psychotropic substances such as opiates, cocaine, cannabinoids, amphetamines, methadone,buprenorphine and ethyl alcohol. In addition to the investigations requested for clinical reasons, the requests to thepharmacotoxicological laboratories may also include medico-legal investigations, whose variety and complexitycontributed to the adoption of personalized and extremely diverse operating modalities implemented in the Italianlaboratories. The purpose of this document is to provide the Laboratories of the National Health Service in Italy thatare planning to carry out or that already perform determination of drugs of abuse for medico-legal purposes, withrecommendations at national level that take into account the “good laboratory practices” recognized at theinternational level in order to perform accurate and precise analytical tests so that they can meet the requirementsnecessary to provide a high quality and legally unassailable service.

TAG: Cannabinoidi   farmaci psicotropi   programmi sanitari nazionali  
Biochimica Clinica
DOI: DOI: 10.19186/BC_2019.037
Pubblicato online il: 17.04.2019
Documenti SIBioC - SIBioC Documents