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Maria Stella Graziani

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Martina Zaninotto

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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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Kjell Grankvist Sweden
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☩Howard Morris Australia
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☩Jill Tate Australia
Tommaso Trenti Italy
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Biochimica Clinica: VOL.43 N.4

Scarica intero fascicolo
Le gammopatie monoclonali: una sfida continua per la Medicina di Laboratorio
G. Merlini  | 
Biochimica Clinica 2019; 43(4) 355-356
DOI: 10.19186/BC_2019.077
Pubblicato online il: 26.11.2019
Editoriale - Editorial
 
Identificazione di geni di normalizzazione per studi trascrizionali con Polymerase Chain Reaction
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 quantify gene 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 for transcriptional 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 the most stable genes in ≤1/3 of cases. The most frequently used normalizing algorithm was geNorm(83%), followed by NormFinder(73%) and BestKeeper(32%).
We also analysed transparency and good laboratory practices based on adherence to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, using selected validated evaluation criteria. Overall, key MIQE criteria were satisfied in ≥50% of analyzed studies, but only four criteria (details of employed kit/enzyme for reverse transcription, priming method, primers/probes and DNA polymerase) were satisfied in ≥90% of cases. Data on assay repeatability were reported only in 15% of studies. The presence of pseudogenes as a potential confounder of assay specificity was evaluated only in 13% of studies. Finally, as few as 6% of studies accounted for the presence of known mutations of singly nucleotide polymorphisms when designing assay primers/probes.
Better adherence to the MIQE guidelines should be encouraged. Publicly available transcriptomic and genomic data sets could be employed to refine the identification of suitable normalizing genes and to assist assay design.

TAG: trascrittoma   reazione a catena della polimerasi   DNA polimerasi  
Biochimica Clinica 2019; 43(4) 357-365
DOI: 10.19186/BC_2019.051
Pubblicato online il: 05.09.2019
Rassegne - Reviews
 
Gammopatie monoclonali: quadri clinici principali e ruolo del laboratorio
Monoclonal gammopathies: main clinical pictures and role of the clinical laboratory
F. Adami  |  T. Berno  |  M. Riva  |  F. Lessi  |  M.S. Graziani  | 

The finding of a monoclonal gammopathy both by chance and upon clinical suspicion is becoming more and more frequent and requires in-depth clinical, laboratory and instrumental analyses in order to establish the underlying disease, if any. In the last two decades the improvement of the diagnostic tools has led to the identification and precise definition of several clinical entities pathogenetically linked to the monoclonal gammopathy, thus shrinking the field of the "monoclonal gammopathy of undetermined significance". Such a striking improvement relies on the increasing sensitivity and accuracy of old analyses and availability of new ones (such as the serum Free Light Chain assay). Currently, data from the clinical biochemistry laboratory provide important clues to the diagnosis and prognosis, and are also crucial for monitoring the therapy. These new achievements, along with the availability of new therapeutic options, allowed a significant, sometimes dramatic improvement of the prognosis of many of the gammopathy-related diseases. In this review the main clinical pictures are described along with the contribution of the clinical biochemistry laboratory to the definition of the diagnosis, the risk profile and the monitoring of the specific diseases.

TAG: MGUS   mieloma multiplo   amiloidosi  
Biochimica Clinica 2019; 43(4) 366-383
DOI: 10.19186/BC_2019.070
Pubblicato online il: 15.10.2019
Rassegne - Reviews
 
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 of hematological analyzers and for enabling accurate data interpretation and appropriate clinical management. This study 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 with Sysmex XN. The study protocol, the analytical measurements and the statistical analysis were carried out according to 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 immature granulocytes (IG), whilst between-subjects BVs ranged between 0,9% for mean corpuscolar haemoglobin concentration (MCHC) and 66,6% for microcytic red blood cells (Micro-R). The RCV ranged between 2,3% for MCH and 73,5% for IG.
Conclusion: This study has allowed the estimation of BV of many CBC parameters, some of which have not been currently explored, thus leading the way to use RCV calculated according to time of monitoring and/or differentiated by sex.

TAG: valori di riferimento   indici eritrocitari   variabilità biologica  
Biochimica Clinica 2019; 43(4) 384-393
DOI: 10.19186/BC_2019.031
Pubblicato online il: 10.06.2019
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 fast detection 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-Test CARBA 5. At the same time the combination disk test (CDT) was performed according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) indications, while confirmation of carbapenemase production was achieved by polymerase chain reaction (PCR).
Results: PCR assay could find 66 CPE strains, including 64 Klebsiella pneumoniae[53 producing Klebsiella pneumoniae carbapenemase (KPC), 5 New Delhi metallo-β-lactamase (NDM), 2 class D oxacillinases (OXA-48), 1 Verona 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, 1 Escherichia coli, 1 Enterobacter 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 showing 100% 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 CPE directly from culture colonies. Moreover, this test is an easy-to-use option that could avoid misidentification of carbapenemases co-producers strains.

TAG: carbapenemase   escherichia coli   β-lactamase  
Biochimica Clinica 2019; 43(4) 394-398
DOI: 10.19186/BC_2019.033
Pubblicato online il: 04.07.2019
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. Our study 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 were healthy 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) than values 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 laboratory diagnosis of HF in elderly males.

TAG: biomarkers   heart failure   elderly  
Biochimica Clinica 2019; 43(4) 399-405
DOI: 10.19186/BC_2019.040
Pubblicato online il: 04.07.2019
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. A significant challenge in this field is the development of cost effective method for a robust interrogation of microbiota composition. 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 blisteringskin diseases. In this pilot study, we characterized the intestinal, cutaneous and oral mucosal microbiota compositionsin PV/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 for the 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 evaluation of skin lesions revealed a prevalence of Firmicutes phylum in both patients’ groups. In the cutaneous microbiota, we identified a 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 and biochemicalparameters, will contribute to clarify the mechanisms of these rare diseases.

TAG: microbiota   sequenziamento   pemfigo  
Biochimica Clinica 2019; 43(4) 406-412
DOI: 10.19186/BC_2019.041
Pubblicato online il: 26.07.2019
Contributi Scientifici - Scientific Papers
 
La metilazione del DNA nella diagnostica: stato dell’arte e prospettive
DNA methylation in diagnostics: state of the art and perspectives
A. Fuso  |  M. Lucarelli  | 

DNA methylation is the most known and studied among the epigenetic modifications; these are chemical modifications occurring on DNA or histone proteins, able at modifying the transcriptional efficiency of genes. DNA methylation consists in the binding of a methyl group (-CH3) on the carbon n. 5 of a cytosine moiety. Recently, it has been demonstrated that methylated cytosines can be further modified to hydroy-methyl-cytosines, but it is still unclear whether this transformation is just a demethylation intermediate or it canretain the functional role of an independent epigenetic modification. At first, DNA methylation has been studied for itsphysiological role in the regulation of one expression during the different stages of the cell life, particularly duringdifferential and embryogenesis. Then, during the last thirty year, it has been shown that the epigenetic modifications,particularly DNA methylation, are involved in the onset and progression processes of some pathologies. The role of DNA methylation in cancer processes is known since a long time, whereas only recently it becomes evident that this epigenetic modification is a component of some degenerative and aging-associated pathologies, particularly in neurodegenerative and inflammatory processes. Due to the incredible technical advances developed in the last years, it is now possible to study in detail the methylation pattern of a gene sequence with single cytosine resolution, rapidly and with high accuracy and precision. Besides allowing the rapid evolution of our knowledge of the physio-pathological states in which DNA methylation has a functional role, this favourable condition also allows us to consider the possible use of DNA methylation as diagnostic biomarker in different pathologies.

TAG: DNA methylation   biomarkers   epigenetics  
Biochimica Clinica 2019; 43(4) 413-420
DOI: 10.19186/BC_2019.064
Pubblicato online il: 02.10.2019
Opinioni - Opinions
 
Gammopatie monoclonali di significato clinico
Monoclonal gammopathies of clinical significance.
F. Adami  | 

This Opinion is aimed to comment and emphazise the main points raised in a seminal paper published in 2018 in Blood. The paper deals with a number of severe diseases caused by small lymphoid/plasma cell clones. These clones affect different organs by means of the biological properties of the clonal cells themselves and/or of their secretion products such as monoclonal immunoglobulins, cytokines and other molecules. More importantly, the paper also deals with the pathogenetic mechanisms involved in the organ damage and sheds light on most of them. The present comment aims to bring attention to these diseases, but it does not encompass the whole matter. We suggest that the readers involved in this matter refer to the paper in its original version.

TAG: MGUS   mieloma multiplo   amiloidosi  
Biochimica Clinica 2019; 43(4) 421-423
DOI: 10.19186/BC_2019.072
Pubblicato online il: 15.11.2019
Opinioni - Opinions
 
La Medicina di Laboratorio: gli specialisti di domani
Laboratory Medicine: specialists of tomorrow

Laboratory Medicine rides the wave of technological progress, the metamorphosis of information systems and data management. The Young Specialist is not a mere observer, but rather 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 and prevention. Thanks to the support of international web platforms, training and exchange programs supported by the International Scientific Societies and Federations that favor professional and scientific growth, Young Scientists work in a global context. In this scenario, the SIBioC Young Scientists Study Group, with the auspices of SIBioC, EFLM and IFCC, organized a meeting on "Laboratory Medicine: Specialists of tomorrow" with the aim of discussing and highlighting some of the most important challenges, such as technological progress, training and internationalization of young people. Finally, the future of laboratory medicine looks at a multidisciplinary approach that leads to integrated diagnosis, identification of the frail patient, the use of the Point of Care Testing as an indispensable tool in crisis areas, making the dialogue between physician and laboratory specialist a fundamental step for the diagnosis and treatment with the final aim of a better outcome for the patient.

TAG: società scientifiche   studi interdisciplinari   ricerca scientifica  
Biochimica Clinica 2019; 43(4) 424-434
DOI: 10.19186/BC_2019.039
Pubblicato online il: 04.07.2019
Documenti - Documents
 
Glossario di biologia molecolare e biologia molecolare clinica. Parte II: metodologie di biologia molecolare
Glossary of molecular biology and clinical molecular biology. Part II: laboratory methodologies

This document represents the second part of a glossary on molecular biology. In particular, the main laboratory techniques for molecular biology are be described. Indeed, recent technological advances made available a number of technologies featured by higher accuracy and sensitivity that are becoming commonly used in routine molecular diagnostics. Aiming to support less experienced readers, the terms related to the main molecular biology techniques are listed herein. For each term the corresponding English version is reported (see also the complete list, both in Italian and in English alphabetical order, reported in the Appendix). In addition, for some of the terms, a link to articles published in Biochimica Clinica, where they have been used, is reported.

TAG: metodologie di laboratorio   tecniche di biologia molecolare   avanzamenti tecnologici  
Biochimica Clinica 2019; 43(4) 435-448
DOI: 10.19186/BC_2019.052
Pubblicato online il: 02.10.2019
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 sostanze d’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 performing pharmacotoxicological tests remains the routine activity for detection and measurement, in different biological matrices, 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 the pharmacotoxicological laboratories may also include medico-legal investigations, whose variety and complexity contributed to the adoption of personalized and extremely diverse operating modalities implemented in the Italian laboratories. The purpose of this document is to provide the Laboratories of the National Health Service in Italy that are planning to carry out or that already perform determination of drugs of abuse for medico-legal purposes, with recommendations at national level that take into account the “good laboratory practices” recognized at the international level in order to perform accurate and precise analytical tests so that they can meet the requirements necessary to provide a high quality and legally unassailable service.

TAG: cannabinoidi   farmaci psicotropi   programmi sanitari nazionali  
Biochimica Clinica 2019; 43(4) 449-452
DOI: DOI: 10.19186/BC_2019.037
Pubblicato online il: 17.04.2019
Documenti SIBioC - SIBioC Documents
 
Una paziente con dolori ossei diffusi: il ruolo del laboratorio nel diagnosticarne la causa
A female patient with diffuse bone pain: the role of the clinical laboratory in the diagnostic process
D. Debbia  |  P. Natali  |  L. Ferrara  |  M. Varani  |  G. Longo  |  T. Trenti  | 

Multiple myeloma (MM) represents 10% of all hematologic malignancies; in 15% of MM the monoclonal component consists of only free light chains. A 53 year-old patient performs at the Corelab laboratory (AOU-AUSL Modena) blood tests for bone pain. Serum electrophoresis shows hypogammaglobulinemia (5,5 g/L). The laboratory professional decides to carry on further studies: a serum immunofixation that highlighted the presence of kappa free light chains not traceable to any heavy chain and the measure of the serum free light chains (sFLC) with the following results: FLC-κ 26 777 mg/L (i.r. 3.3-19.4); FLC-λ 6.15 mg/L (i.r. 5.7-26.3); ratio FLC (rFLC), 435.31 (i.r. 0.26-1.65). The light chain MM is a type of MM difficult to recognize. The laboratory professional's own initiative defines a procedure of "personalized medicine" oriented to to the patient's needs. The expertise of the laboratory professional is crucial in assuring the patient the best outcome when carried out on the basis of the available guidelines.

TAG: proteina di Bence Jones   catene leggere   immunoglobuline  
Biochimica Clinica 2019; 43(4) e35-e36
DOI: 10.19186/BC_2019.059
Pubblicato online il: 10.10.2019
Casi Clinici - Case Report
 
Un caso clinico di linfocitosi persistente
Persistent Lymphocytosis

Persistent Polyclonal Binucleated B-cell Lymphocytosis (PPBL) is a rare, benign hematological disorder characterized by a chronic polyclonal B-cell lymphocytosis with binucleated lymphocytes. We report the clinical case of a young woman with lymphocytosis who presented binucleate lymphocytes at the morphological examination of the peripheral venous blood smear. Flow cytometry immunophenotyping performed in peripheral blood showed a polyclonal lymphocyte B subpopulation. Serological tests were negative for viral infections. Because of its benign and indolent course, the diagnoses of PPBL is important in order to avoid unnecessary diagnostic procedures and therapies.

TAG: leucociti   malattie ematologiche   striscio di sangue periferico  
Biochimica Clinica 2019; 43(4) e38-e40
DOI: 10.19186/BC_2019.062
Pubblicato online il: 15.10.2019
Casi Clinici - Case Report
 
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