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Biochimica Clinica: VOL.40 N.4

La “whole genome amplification” su singola cellula
Whole genome amplification on single cell

The whole genome amplification (WGA) is a method for an entire genome amplification, starting with low amounts of DNA. Particularly, it allows downstream analysis, such as genomic screening [i.e., comparative genomic hybridization (CGH) array, next generation sequencing] and single gene mutation detection in single cells. Because WGA could introduce few bias, dependent on different methods, their selection should be related to the application. The first WGA method was based on amplification reaction and differently from a regular polymerase chain reaction (PCR), in which a single genetic locus is amplified, different locus were amplified simultaneously. Nowadays, several methods have been developed for WGA: degenerate oligonucleotide PCR and primer extension preamplification based on PCR, and multiple displacement amplification achieved with isothermal reaction setup. Each WGA approach has limitations, such as the genome coverage, chimeric DNA molecules, preferential allele amplification or allele drop-out and the guanine-cytosine (GC) richness (GC%). In this review, we detailed different WGA methods for single cell and their most important applications, such as cancer diagnosis and reproductive medicine.

Biochimica Clinica 2016; 40(4) 293-301
DOI: 10.19186/BC_2016.034
Pubblicato online il: 17.11.2016
Rassegne - Reviews
Ruolo del saggio Hevylite® nella diagnosi e nel monitoraggio delle gammopatie monoclonali
Role of Hevylite® assay in the diagnosis and monitoring of monoclonal gammopathies

Clinical tests for detection and characterization of monoclonal immunoglobulins include serum and urine protein electrophoresis, and serum and urine immunofixation. The quantification of monoclonal components provides a surrogate for monitoring the size of malignant cell population in patients affected by plasma cell dyscrasia. As complementary test, immunoglobulin quantification is useful in patients with high concentrations of monoclonal IgG and in patients with monoclonal IgA whose electrophoretic migration is in the -fraction. Serum free light chain / ratio and the concentration of free light chains can also be used in different conditions. To overcome the limitations of traditional methods, e.g., for the quantification of monoclonal components that are indistinguishable from other proteins at electrophoresis, a new nephelometric immunoassay, called Hevylite assay (HLC), was developed. HLC separately measures in pairs light chain types of each intact immunoglobulin class, generating ratios of monoclonal immunoglobulin/uninvolved polyclonal immunoglobulin concentrations. Studies have shown that HLC and immunofixation are complementary methods. In this review, we summarize the role of HLC in the identification of clonality, prediction of prognosis in patients with multiple myeloma and in the evaluation of response to treatment. HLC ratio may also reveal immunoparesis and serve as a new marker for validating remission depth and relapse probability.

Biochimica Clinica 2016; 40(4) 302-306
DOI: 10.19186/BC_2016.040
Pubblicato online il: 21.11.2016
Rassegne - Reviews
Analytical and clinical evaluation of the chemiluminescent microparticle immunoassay for galectin-3 determination

This study tested if the chemiluminescent microparticle immunoassay (CMIA) method on the Architect platform meets the analytical and clinical quality goals required for the galectin-3 (GAL-3) use in clinical practice. We evaluated results obtained from 121 apparently healthy adults and 382 patients with heart failure (HF). All healthy subjects and patients showed GAL-3 concentrations in plasma above the limit of detection (1.9 μg/L) and the limit of quantitation (2.4 μg/L). GAL-3 in healthy subjects ranged between 6.4 and 40.6 μg/L (median, 13.0 μg/L, interquartile range, 11.2-15.2 μg/L, 97.5th percentile, 33.7 μg/L). GAL-3 values were found significantly increased in patients with chronic HF (median, 15.1 μg/L, interquartile range, 11.7-19.4 μg/L) compared to healthy subjects (P <0.0001). HF patients with cardiac fibrosis, confirmed by magnetic resonance, showed significantly higher GAL-3 values (median, 15.3 μg/L, interquartile range, 11.2-21.9 μg/L) than those without cardiac fibrosis (median, 12.9 μg/L, interquartile range, 11.2-15.0 μg/L) (P=0.03). ROC analysis showed that GAL-3 discriminates the presence of cardiac fibrosis with an area under the curve of 0.635 (0.526-0.744), with a specificity of 76.7% and a sensitivity of 54.1% at the cut-off of 14.6 μg/L. Using multivariable models cardiac fibrosis was significantly associated with the logGAL-3.

Biochimica Clinica 2016; 40(4) 307-315
DOI: 10.19186/BC_2016.041
Pubblicato online il: 18.11.2016
Contributi scientifici - Scientific Papers
Valutazione di un sistema esperto per la validazione di tracciati elettroforetici delle proteine del siero
Evaluation of an expert system for computer-supported evaluation of serum protein electrophoresis patterns

Serum proteins electrophoresis (SPE) is mainly ordered for screening and follow-up of monoclonal gammopathies (MG). Several softwares have been developed to assist the process, but none has been able to replace human validation. In this paper we describe the performance of an expert system (ES), integrated by Sebia into Phoresis software, for validation of negative SPE patterns obtained on Capillarys 2, combining qualitative analysis of SPE patterns by neural networks with a semi-quantitative evaluation of SPE fractions and providing classification in real time. 10,055 patterns, evaluated by supervisor scientists without knowing ES classification, subsequently underwent ES analysis. Sensitivity, specificity and predictive values (PV) of ES classification were calculated. Agreement with human operator for classifying patterns was calculated by Cohen’s kappa, obtaining a value of 0.31 indicating fair agreement. Sensitivity and specificity of software alerts for MG detection were 100% and 49.3%, respectively, with 23.7% positive PV and 100% negative PV. Sensitivity and negative PV for a1-antitrypsin deficit and hypogammaglobulinemia were 100%. The combination of morphologic analysis by neural networks and quantitative assessment of every SPE fraction allowed a very good performance in detection of SPE patterns that did not display abnormalities. In conclusion, the evaluated ES is a safe and efficient method for validating normal SPE patterns, allowing ~40% time saving and a better standardization of inter-observer evaluations.

Biochimica Clinica 2016; 40(4) 316-321
DOI: 10.19186/BC_2016.039
Pubblicato online il: 8.11.2016
Contributi scientifici - Scientific Papers
Confronto tra l’applicazione “body fluid” di due analizzatori ematologici e il metodo in microscopia ottica per l’analisi cellulare del liquido cefalorachidiano
Comparison between “body fluid” mode of two hematology analyzers and the microscopy method for cellular analysis of the cerebrospinal fluid
M. Seghezzi  |  T. Mecca  |  P. Dominoni  |  E. Lochis  |  C. Saleri  |  B. Manenti  |  A. Crippa  |  S. Buoro  | 

In the cerebrospinal fluid (CSF), the white blood cell count (WBC) and cell differentiation in mononuclear (MN) and polymorphonuclear (PMN) cells are useful tools in the diagnosis of central nervous system disease. We evaluated the analytical performance of the “body fluid” module of the hematology analyzers Mindray BC-6800 and Sysmex XN-9000 in the cytometric analysis of the CSF. 110 CSF samples were collected and analyzed with BC-6800, XN-9000 and evaluated by optical microscopy (OM). The diagnostic concordance between each analyzer and OM, as well as between the two analyzers were evaluated.The comparison between each analyzer and OM showed correlation coefficients ranging from 0.65 to 0.99 and bias ranging from 0.06x106 cell/L to 19.9x106 cell/L. The comparison between BC-6800 and XN-9000 showed a good correlation for total cell counts, WBC, MN and PMN (both in absolute and percentage values) with a correlation coefficient ranging from 0.83 to 0.98 and bias ranging from -0.2 to -43.8x106 cell/L. The introduction of automated methods for CSF analysis could be very useful in the routine work of a laboratory analysis.

Biochimica Clinica 2016; 40(4) 322-327
DOI: 10.19186/BC_2016.042
Pubblicato online il: 23.11.2016
Contributi scientifici - Scientific Papers
miR-199a and miR-125b expression levels in serum of women affected by epithelial ovarian cancer

Recent studies show that microRNA (miRNAs) are involved in cancer by regulating cell proliferation, apoptosis and angiogenesis. Accordingly, their deregulation could contribute to cancer development and progression. It has been demonstrated that in ovarian tissue the over-expression of miR-199a and miR-125b inhibits tumor angiogenesis, a fundamental process for cancer development and growth. Aims of our study were to investigate the expression levels of miR-199a and miR-125b in serum of patients with ovarian cancer (OC) and to evaluate the correlation between miRNAs expression and traditional biomarkers [CA125 and human epididymis protein 4 (HE4)]. 32 patients with epithelial OC (54±14 years old) and 31 healthy controls (55±17 years old) were enrolled. Serum samples were collected prior to definitive surgical treatment and RNA extraction was performed by using the miRNeasy Serum/Plasma kit (Qiagen GmbH). miR-199a and miR-125b expression was determined by quantitative real timepolymerase chain reaction (TaqMan MicroRNA Assay, Applied Biosystems). The expression levels of miRNAs were normalized to miR-16 and calculated utilizing the 2-ΔCt method. Serum levels of miR-199a and miR-125b were significantly higher in OC patients compared to controls (P=0.007 and P=0.002, respectively). A marginally statistically significant correlation was found between miR-199a and miR-125b expression levels (r=0.38, P=0.03). The ROC curve analysis of the diagnostic performance between healthy controls and OC patients revealed that HE4 had a significantly higher area under the curve (AUC=0.90) when compared to CA125 (AUC=0.85), miR-199a (AUC=0.70) and miR-125b (AUC=0.67). Anyway, the determination of circulating miRNAs may be relevant, since their expression is known to be aberrant in cancer, having potential ability to monitor tumor dynamics.

Biochimica Clinica 2016; 40(4) 328-333
DOI: 10.19186/BC_2016.035
Pubblicato online il: 16.11.2016
Contributi scientifici - Scientific Papers
Varianti rare dell’emoglobina: la nostra esperienza con gli analizzatori Sysmex di ematologia
Rare hemoglobin variants: our experience with Sysmex hematology analyzers

The consolidation of laboratory activities optimizes resources and improves the expertise of the staff that encounters more frequently rare and exceptional cases. In the recent months, the Romagna Greater Area Laboratory, which provides a diagnostic service for over a million inhabitants in North Italy, detected an unusual instrumental alarm in a few routine blood counts. Further investigations demonstrated the presence in the samples of hemoglobin (Hb) variants, characterized with subsequent molecular analysis as Hb Leiden and Hb G-Ferrara. These Hb variants reduce the fluorescence signal hampering the differential leukocyte count carried out by the Sysmex XE2100 hematology analyzer. In all the investigated cases, irregular contracted red blood cells (RBC) and target RBC were present. HPLC analysis of the two Hb variants shows Hb Leiden and Hb G-Ferrara peaks at 4.28 min and 3.23 min of elution time, respectively. It is clinically important to identify carriers of these variants in specific clinical setting, since heterozygotes for these Hb variants are asymptomatic, but complications could be induced by drug therapies, viral infection or heterozygosity for b-thalassemia or other Hb variants. Our findings demonstrate the importance to investigate samples with low fluorescence when assayed using Sysmex analyzers. Finally, the reported cases confirm the effectiveness of tight integration between huge routine workload and specialized competence for the detection of unusual clinical conditions.

Biochimica Clinica 2016; 40(4) 334-337
DOI: 10.19186BC_2016.043
Pubblicato online il: 18.11.2016
Contributi scientifici - Scientific Papers
La diagnostica di laboratorio delle dislipidemie
The laboratory diagnosis of dyslipidemia

Dyslipidemias represent a major contributor to cardiovascular risk in Western countries, including Italy, that can be modified. After examining familial dyslipidemias and describing the essential issues for clinical and laboratory diagnostics, the paper considers the laboratory testing in detail. The preanalytical sources of variability (biological, sample collection and handling) are reviewed and essential indications to reduce them are given. About the analytical variability, the paper examines the methods routinely used for measuring the basic lipid parameters (total, LDL and HDL cholesterol, triglycerides and apolipoproteins A-I and B) and describes the state of art of the standardization of these analytes. The last section of the document deals with the reporting of laboratory results. The main indications of the document are the following: a) report the desirable values established by the European guidelines besides the measured concentrations; b) make always clear that the reported values are decisional cut points and not reference limits; c) add a note stating that the reported desirable values refer to individuals at low risk; d) report as critical values lipid concentrations deserving rapid clinical attention, i.e., total cholesterol, ≥8,00 mmol/L (310 mg/dL); LDL cholesterol, ≥4,90 mmol/L (190 mg/dL); triglycerides, ≥10,0 mmol/L (880 mg/dL).

Biochimica Clinica 2016; 40(4) 338-346
DOI: 10.19186/BC_2016.031
Pubblicato online il: 24.10.2016
Documenti SIBioC - SIBioC Documents
Verifica locale dei sistemi di prelievo nei laboratori clinici: adattamento delle linee guida EFLM
Blood collection systems in clinical laboratories: local adaptation of the EFLM guidelines

The importance of the process of purchasing or changing blood collection devices is often overlooked. This is likely attributable to many factors such as the limited knowledge that policymakers, healthcare administrators and also laboratory managers have on the significance of preanalytical quality, but also to the lack of validated criteria for analyzing the quality of blood collection devices. Since a gap remains to be filled between companies’ and laboratory’s validation, the EFLM Working Group on Preanalytical Phase (WG-PRE) has published a comprehensive document, which contains essential prerequisites and technical issues (e.g., physical imperfections, defects of functioning, safety deficiencies) to support local clinical laboratories for the development of tenders for blood tubes and for the validation of new materials ahead of local routine use. This consensus document is a national adaptation of these guidelines.

TAG: Errori   Variabilità Preanalitica   Raccolta di sangue  
Biochimica Clinica 2016; 40(4) 347-352
DOI: 10.19186/BC_2016.032
Pubblicato online il: 26.10.2016
Documenti SIBioC - SIBioC Documents
Esame fisico, chimico e morfologico delle urine: proposta di linee guida per la fase analitica del Gruppo Intersocietario Analisi delle Urine (GIAU)
Physical, chemical and morphological urine examination: proposed guidelines for the analytical phase by the Intersociety Urinalysis Group

With these guidelines, the Intersociety Urinalysis Group (GIAU) aims to stimulate the following aspects: a) improvement and standardization of the analytical approach to physical, chemical and morphological urine examination (ECMU); b) to emphasize the value added to ECMU by automated analyzers for the study of the morphology of the corpuscular fraction urine; c) improvement of the chemical analysis of urine with particular regard to the reconsideration of the diagnostic significance of parameters that are traditionally evaluated in dipstick analysis, together with an increasing awareness of the limits of sensitivity and specificity of this analytical method; d) to increase the awareness of the importance of professional skills in the field of urinary morphology and of the relationships with clinicians; e) implementation of a policy for the evaluation of the analytical quality by using, in addition to traditional IQC and EQA, a program for the evaluation of morphological competence; f) to stimulate the diagnostic industry to focus research efforts and development methodology and instrumental catering to the needs of clinical diagnosis. The hope is to revalue the enormous diagnostic potential of ECMU, by implementing an urinalysis based on personalized diagnostic needs.

Biochimica Clinica 2016; 40(4) 353-382
DOI: 10.19186/BC_2016.037
Pubblicato online il: 17.11.2016
Documenti SIBioC - SIBioC Documents
La funzionalità tiroidea in gravidanza: quali sono i valori fisiologici?
Thyroid function in pregnancy: what is normal?

Gestational thyroid dysfunction is common and associated with maternal and child morbidity and mortality. During pregnancy, profound changes in thyroid physiology occur, resulting in different thyroid-stimulating hormone (TSH) and free thyroxine (FT4) reference intervals compared to the nonpregnant state. Therefore, international guidelines recommend calculating trimester- and assay-specific reference intervals per center. If these reference intervals are unavailable, TSH reference intervals of 0.1–2.5 mU/L for the first trimester and 0.2–3.0 mU/L for the second trimester are recommended. In daily practice, most institutions do not calculate institution-specific reference intervals but rely on these fixed reference intervals for the diagnosis and treatment of thyroid disorders during pregnancy. However, the calculated reference intervals for several additional pregnancy cohorts have been published in the last few years and show substantial variation. We provide a detailed overview of the available studies on thyroid function reference intervals during pregnancy, different factors that contribute to these reference intervals, and the maternal and child complications associated with only minor variations in thyroid function. There are large differences in thyroid function reference intervals between different populations of pregnant women. These differences can be explained by variations in assays as well as population-specific factors, such as ethnicity and body mass index. The importance of using correct reference intervals is underlined by the fact that even small subclinical variations in thyroid function have been associated with detrimental pregnancy outcomes, including low birth weight and pregnancy loss. It is therefore crucial that institutions do not rely on fixed universal cut-off concentrations, but calculate their own pregnancy-specific reference intervals.

Biochimica Clinica 2016; 40(4) 383-392
DOI: 10.19186/BC_2016.036
Pubblicato online il: 17.11.2016
Il meglio di Clinical Chemistry - Clinical Chemistry highlights
In ricordo di Luigi Spandrio
In memory of Luigi Spandrio
Biochimica Clinica 2016; 40(4) 393
Pubblicato online il: 29.10.2016
Notizie SIBioC - SIBioC News
In ricordo di Massimo Luzzana
In memory of Massimo Luzzana
Biochimica Clinica 2016; 40(4) 394
Pubblicato online il: 28.10.2016
Notizie SIBioC - SIBioC News
Indice dei contenuti 2016
Index of contents 2016
Biochimica Clinica 2016; 40(4) 397-400
Pubblicato online il: 25.11.2016
Indice dei contenuti e degli autori - Index of contents and authors
Indice degli autori 2016
Index of authors 2016
Biochimica Clinica 2016; 40(4) 401-404
Pubblicato online il: 25.11.2016
Indice dei contenuti e degli autori - Index of contents and authors
Echinocitosi associata a diminuita espressione della banda 3 eritrocitaria in un bambino con epilessia idiopatica
Echinocytosis and decreased expression of erythrocyte band 3 in a child with idiopathic epilepsy: a case report

Echinocytosis (EC) is a morphologic change of the erythrocytes usually linked to electrolyte exchange abnormalities, energy depletion and cell dehydration. Herein, we report a case of a child presenting with complex partial epilepsy, consistent peripheral EC, mild unexplained microcitemia and a significantly decreased expression of band 3. No pathogenic mutations were found on the band 3 encoding gene, i.e., solute carrier family 4 (anion exchanger), member 1 (SLC4A1). The observed changes in band 3 expression likely originated at the transcriptional and/or post-transcriptional level. To date, band 3 is considered as a key protein in several neurodevelopmental diseases. The described modifications probably explain the observed clinical phenotype. The likelihood that an alteration in band 3 function could contribute to an erythrocyte morphological abnormality and neurological symptoms represents a fascinating and intriguing hypothesis.

Biochimica Clinica 2016; 40(4) e27-e30
DOI: DOI: 10.19186/BC_2016.038
Pubblicato online il: 18.11.2016
Casi clinici - Case report
Sensibilità al glutine: il test di attivazione dei basofili (BAT) può aiutare nella diagnosi differenziale?
Gluten hypersensitivity: basophil activation test (BAT) may help in the differential diagnosis

Gluten hypersensitivity: basophil activation test (BAT) may help in the differential diagnosis? The main forms of gluten-related disorders are wheat allergy (WA), celiac disease (CD) and possibly immune-mediated disease (gluten sensitivity). S-IgE play an essential role in WA. For CD tests available are anti-tissue transglutaminase (tTG) IgA, anti-endomysium (EMA) and deamidated gliadin peptides (DGP) antibodies IgG particular. For other immunemediated diseases there is currently no test available. The usefulness of basophil activation test (BAT) in anaphylactic adverse reactions, late-onset allergy has been demonstrated. We report the case of a woman of 46 years old with disorders of wheat tied overt clinical signs, intestinal and extra-intestinal symptoms, whose tests above were all negative. Only the BAT showed a stimulation of basophils exposed (52.9%) to extract wheat. We diagnosed gluten sensitivity (GS) on the basis of an algorithm for the differential diagnosis of gluten-related disorders, including CD, GS and WA. We believe that the BAT confirms a hypersensitivity reaction to wheat not-IgE-mediated, not CD.

Biochimica Clinica 2016; 40(4) e31-e34
DOI: 10.19186/BC_2016.033
Pubblicato online il: 14.11.2016
Casi clinici - Case report