Articoli in pubblicazione

Gamma-hydroxybutyric acid (GHB) can be considered both an endogenous metabolite and a precursor of the neurotransmitter gamma-aminobutyric acid (GABA) acting within the central nervous system as a neuromodulator.
Pharmacologically, GHB is classified as a central nervous system depressant and its mechanism of action involves interaction and binding with GABA-B receptors.
With the generic name of sodium oxybate, the sodium salt of GHB, is sold as a pharmaceutical product under the trade name of Xyrem® when prescribed for the treatment of people with narcolepsy and with the name of Alcover® when used to relieve alcohol withdrawal syndrome and treat alcohol dependence.
Between the end of the 90s of the twentieth century and the beginning of the twenty-first one, the abuse of GHB became increasingly widespread in the recreational field. The substance, often consumed in combination with alcohol, cannabis, ecstasy (3,4-Methylenedioxymethamphetamine, MDMA), ketamine, has been converted in one of the most used "club drugs", becoming one of the most serious health issues in the emergency department of many European and extra-European Countries because of the initial difficult clinical management of the growing number of cases of intoxication. At the same time, there was an increase of cases of sexual assaults of victims who were unaware they had been given GHB as odourless and colourless sedative substance.
In this narrative review, the three different aspects of GHB as endogenous neuromodulator, as prescription drug and as substance of abuse are illustrated. The main methods for qualitative and quantitative analysis in conventional and non-conventional biological matrices for clinical and forensic purposes are also described.

Background: the ozone autohemotherapy (O3-AHT) is an alternative medical practice where an aliquot of patient's blood is treated with an ozone-oxygen mixture (O3/O2), and reinfused to induce an hormetic effect, that is to obtain a mild oxidative stress that empowers the antioxidant response and improves the oxidative balance. The customization of the ratio O3/O2 and the amount of blood is a crucial aspect of this therapy. The measure of some biomarkers of oxidative balance may be useful to calibrate the treatment and avoid damages.
Methods: O3-AHT was performed on 9 volunteers healthy subjects, treating 150 g of blood with 30 μg/mL O3/mL O2/g. Aliquots of blood and urine were collected before therapy (t0), and 4, 8, 24 and 48 hours (t4-t48) after reinfusion to measure the derivates of reactive oxygen metabolites (dROMs), the biological antioxidant potential (BAP), the single components of antioxidant barrier coenzyme Q10, and glutathione in both total (GSH+GSSG) and reduced (GSH) forms, the oxidative biomarkers homocysteine (Hcy), 8-hydroxy-deoxyguanosine (8-OHdG) and 2-deoxyguanosine (2-dG) and the nitrative biomarker 3-nitrotyrosine (3-NT).
Results: within the first 4-8 hours after O3-AHT, as dROMs increased, the median of GSH had an initial considerable decrease (-20% at t8 versus t0) followed by a final net increase (+12% at t48). In a similar way, 3-NT had an initial decrease (-19% at t4) followed by a remarkable increase at t8 and t24 (+28% and +57% respectively) and a subsequent important decrease at t48 (-44%), which led to final nitration levels lower than the one observed at t0.
Conclusions: When used in correct concentrations, O3 induces a moderate oxidative stress which initially consumes antioxidant species and produces nitrative damages. Within 24-48 hours however, the beginning of a virtuous counter-regulatory response that increases the reducing power (particularly GSH levels) and decreases molecular damages, improving the redox balance, can be observed. Serial measurements of GSH and 3-NT, allow to monitor that the induced oxidative stress causes only moderate and transient damages, and help the physician to choose the more effective O3 therapeutic concentrations.

Introduction: the medical use of cannabis is increasingly being applied in the treatment and support of several diseases and syndromes. In the Liguria Region, olive oil galenic preparations are mainly prepared by hospital pharmacies, according to common standard procedures. The preparations must be analyzed in order to establish the concentration of the two main active compounds (delta-9 tetrahydrocannabinol, THC and cannabidiol, CBD) thus allowing the correct setting of the therapeutic prescription. Liguria Region is at the forefront in the use of medical cannabis with a high number of patients treated (>1000). The aim of this work is to describe the organization of the titration activity centralized at the two regional reference laboratories (Central Laboratory of Analyses of Giannina Gaslini Institute, Genova and Toxicology Laboratory of Sarzana, La Spezia), coordinated by the inter-hospital department (DIAR) of the Laboratories Area.
Methods: the phases of the analytical process (pre-analytical, analytical and post-analytical) have been identified and described. The analysis of the workflow has been carried out including the methods to prepare cannabis oil in the pharmacies, the intervals and production volumes, the medical-legal handling requirements and operational responsibilities. The definition of the pre-analytical phase foresees the methods of packaging, transport and recording of the samples and related responsibilities.
Results: the analytical phase included the development and validation of the analytical method Ultra High Performance Liquid Chromatography coupled to tandem mass spectrometry, (UHPLC-MS/MS) in the two laboratories, with common procedures and the comparison of results conducted both on reference material and real samples of olive oil galenic preparations. The definition of the post-analytical phase included the reporting procedures.
Discussion: the experimental phase has been concluded at the end of 2019 and the implementation phase of the project has started in march 2020.

Background: prostate cancer (Pca) is the second most common cancer among men and the sixth leading cause of death due to cancer among men worldwide. We aimed to verify if serum PSA levels and PCa risk/aggressiveness are modulated by polymorphisms of KLK3, RASA1 and NAALADL2 genes.
Methods: 1058 men have been studied; they consecutively underwent prostate biopsy for clinical suspicion of PCa. PCa was histologically diagnosed in 401 and ruled out in 657 men. Gleason score in PCa patients was ≤6 in 261, 7 in 83 and >7 in the remaining 57 PCa. tPSA and f/tPSA levels were determined. Four polymorphisms were studied: rs35148638 (RASA1), rs78943174 (NAALADL2), rs2735839 and rs17632542 (KLK3).
Results: PCa diagnosis was significantly predicted by the KLK3 rs17632542 polymorphism (p<0.001), tPSA (p<0.001) and f/tPSA (p<0.001). Carriers of the KLK3 rs17632542C rare allele had a significantly higher risk of PCa (p<0.001) (OR 2.1, 95% CI 1.40-3.19). Gleason score >7 was associated with increased tPSA (p<0.001), decreased f/tPSA (p=0.003) and the KLK3 rs2735839 A rare allele (p= 0.004). In controls, tPSA was significantly lower in subjects bearing NAALADL2 rs78943174T rare allele (p=0.029). f/tPSA was higher in subjects with the KLK3 rs17632542C rare allele (p<0.001) and with the RASA1 rs35148638 C/C genotypes (p=0.009). In PCa subjects, tPSA was not associated with the polymorphisms studied.
Conclusions: KLK3 rs17632542 and rs2735839 polymorphisms were significantly associated with the risk and aggressiveness of PCa respectively. NAALADL2, KLK3 rs17632542 and RASA1 polymorphisms were correlated with tPSA and f/tPSA serum levels, suggesting a genetically-based PSA expected values in absence of tumor. These results suggest a potential role of these polymorphisms as biomarkers for PCa in association with the diagnostic and prognostic indexes currently recognized.

Introduction: Laboratory Medicine is continuously changing because of the advent of new technologies and perspectives, such as automation, Big Data and omics sciences. Professionals’ profiles are changing concurrently, developing the new technological, clinical and management skills required nowadays. In order to assess training needs as well as education and working conditions, the SIBioC Young Scientists Working Group (YS-WG) promoted a questionnaire directed to professionals ≤40 years old.
Methods: the questionnaire was prepared using Survey Monkey and was sent to the 587 SIBioC members ≤40 years old; it was also diffused via the YS-WG social media pages, and through personal e-mails with the help of Specialty School Offices. The questionnaire included 54 questions examining different aspects: participation in SIBioC activities, scientific interests, working conditions, evaluations of training and education experiences, expectations for the future professional life.
Results: during three months, 282 responses have been collected. The most represented professionals are Biologists (PhD) (46%), followed by Medical Doctors (24%). 33% of participants has an open-ended contract, 15% temporary, 17% freelance and 17% has a scholarship/research grant; 46% of them do not receive any remuneration. Around 60% work in public institutions (Universities or Hospitals); 52% are involved in clinical area, 29% in research. Residents’ evaluation on educational quality of Specialty Schools is rather heterogeneous. Among the 193 SIBioC members, 35% is actively participating in at least one of the society’s Working Group. Most of the participants are regular readers of the SIBioC official journal (Biochimica Clinica), consult LabTestsOnline web site, and participate to SIBioC scientific events and/or to the Society e-learning courses.
Conclusions: the results of the survey are a key point for the Society, allowing to understand the young laboratory professionals needs, so that they can be accompanied and encouraged in a full development of their future professional life.

A recently published comment (Nature, 2019), proposing to give up the use of the P value in scientific literature, spurred several contributions on the topic. The main target was the need to avoid the dichotomization of P, with p<0.05 identifying the statistically significant results. A first proposal was to lower the threshold value to 0.005, labeling as “suggestive” results previously classified as significant but not meeting the new threshold. A more radical suggestion was to suppress the use of P, allowing its presence only in a descriptive sense. At the time of writing, only one journal took such a radical position, and this choice gave rise to problems in the interpretation of studies’ results. To avoid p-hacking and other inappropriate uses of P, the most sensible strategy would be to mandate the pre-publication of the study protocol, including the statistical analysis. The authors should then be required to adhere to their original published plan. This rule could be of great help for pragmatic trials, but does not apply to exploratory studies, which are more frequent in life sciences. It could also be imposed to report the P value only for sufficiently large sample sizes, reporting otherwise only descriptive statistics. Moreover, the term “statistical significance” could be replaced by “statistical accuracy,” in order to avoid the common confusion with “clinical significance”. This debate probably will not lead to the abandonment of P, but it may help to improve the quality of the statistical analysis of trials’ results.

Bence Jones protein (BJP) refers to urine monoclonal free immunoglobulin light chains produced by the clonal expansion of a plasma cell in the bone marrow. BJP is strongly associated with systemic amyloidosis AL, light chain deposition disease, and multiple myeloma; less frequently, BJP may be recognized either in patients with monoclonal gammopathies of uncertain significance (MGUS) and with other plasma cell dyscrasias or in patients with malignant non-Hodgkin's lymphomas and chronic lymphocytic leukemia. This paper contains updated recommendations for the detection and the measurement of BJP in clinical practice from the Working Group “Proteins” of the Italian Society of Clinical Biochemistry (SIBioC), with specific indications for improving all the steps of the preanalytical, analytical, and postanalytical phases. The first morning void is the urine sample recommended for BJP detection, while 24-hours urine collection is preferred for BJP quantification. Native urine cannot be used for samples with low or very low content in urine total protein; in these cases, samples should be concentrated by using specific disposables, such as ultrafiltration membranes retaining proteins with molecular weight around 10 kDa. The required degree of concentration may vary according to sensitivity of the electrophoretic method utilized and the protein content of the sample. The detection of BJP may be performed directly by the recommended method agarose gel immunofixation (IFE) with specific polyvalent immunoglobulin antisera IgG-IgA-IgM, total  and  light chains; alternatively, an electrophoretic screening may be acceptable to rule out negative test results. However, positive test results should be confirmed by IFE. Tests based on immunometric methods can be used neither as screening test, nor for the BJP quantification; however, it could be useful for monitoring purposes, provided that the renal function of the patient is preserved. BJP measurement should be performed by the densitometric scanning of the electrophoretic peak corresponding to BJP, and results should be expressed as ratio of the BJP peak percentage to the urine total protein. Test results should be always integrated by standardized interpretative comments included in the laboratory reports.

Vitamin D, and its metabolites, have key roles in the functioning of virtually all tissues. Indeed, besides the unquestioned role in bone mineral metabolism, evidences have pointed out roles in inflammation and autoimmunity, skeletal muscle and cardiac function, neuromuscular communication. Therefore, vitamin D deficiency associates with rickets in the child, osteomalacia and increased risk of fracture in the adulthood and possibly with the increased risk
of other pathological conditions. In order to regulate the prescription of vitamin D supplementation (cholecalciferol, cholecalciferol/calcium salts, calcifediol) to the adult population, and consequently to contain the costs sustained by the National Health System, the Italian Medicine Agency (AIFA) has recently drafted the “Nota 96” identifying the categories of patients, based on the laboratory measurement of serum levels of 25(OH)D, that can benefit from the reimbursement of vitamin D therapy. If the “Nota 96”, AIFA has the merit to define rules in a field only sligtly regulated, several considerations emerged from its analysis. From an analytical point of view, the “Nota 96” does not consider the issues of reliability and reproducibility of 25(OH)D measurement and standardization of the units of measurement and reporting. On the clinical side, the “Nota 96” does not consider the epidemic vitamin D insufficiency/deficiency that needs an adequate preventive approach and, furthermore, beyond its roles, the agency indicates the clinical conditions for whom vitamin D prescription is allowed. This document analyses the content of “Nota 96” highlighting the points that need further evaluations and by giving possible different solutions; the document provides also recommendations on laboratory reporting of vitamin D measurements.

The causative gene of Cystic Fibrosis (CF) is the Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Although monogenic, CF has a complex genotype – phenotype relationship mainly originated by the high number of CFTR variants, the challenging mutational analysis, the incomplete functional knowledge and the variable clinical outcome of most variants, the action of modifier genes. This complexity affects diagnosis, prognosis and precision therapy. A correct and complete genetic analysis is crucial for all these aspects. The Consensus Document 2019 on the genetic analysis of CF (1S) provides a guidance to practitioners for the appropriate request and proper use of genetic testing in CF, as well as for the correct interpretation of the test result. It is to be hoped that the broad sharing achieved between the scientific societies involved can standardize the approach at national level and can improve the usefulness of the information provided to users, as part of a continuous process of improvement.

A 13 year-old child whit headache, nausea and repeated episodes of vomiting was hospitalized at the Major Hospital of Charity of Novara. A lumbar puncture and biochemical investigations on cerebrospinal fluid (CSF) including oligoclonal bands (OCB) screen were performed. Kappa and Lambda free light chains (kFLC and λFLC) and IgG concentrations were measured in serum and CSF by nephelometry. OCB were evaluated by isoelectric focusing (IEF) followed by immunofixation (IMF). A negative IgG index, no OCB IgG (type I pattern), positive kFLC index with increased levels of kFLC (0.14 mg/dL) and λFLC (0.14 mg/dl in CSF) were found. The FLC data suggested an intrathecal synthesis. A CSF IMF was then performed using anti-IgM peroxidase together with the IgM index using Reiber’s hyperbolyc graphic. The presence of a monoclonal IgM band and 76% of intrathecal IgM synthesis was demonstrated. A subsequent molecular biology investigation was performed on CSF and revealed a clear positivity for enterovirus.

Light chain deposition disease (LCDD) is characterized by tissue deposition, mostly in the kidney, of monoclonal immunoglobulin light chains (LCs), causing renal dysfunction and end-stage renal disease. The main goal of therapy is the reduction of LCs concentration, that can be obtained using chemotherapy approaches. We report the case of a 28-year-old man with LCDD (IgGκ type) and underlying multiple myeloma who, after three ineffective lines of therapy, started a treatment with daratumumab, a monoclonal antibody (mAb, IgG1κ type) drug, recently introduced for multiple myeloma treatment. The drug seemed effective but a IgGκ spike remained visible at standard immunofixation. To discriminate the drug from the patient monoclonal component, immunofixation with Hydrashift system was used. This tool identified the visible IgGκ as mAb drug and complete response was documented. This case showed the utility of new clinical assays for the evaluation of response to therapy in patients treated with mAb drugs.

Isocitrate Dehydrogenase 2 (IDH2) mutations are reported in about 20% of Acute Myeloid Leukemia (AML) and they are promising Minimal/Measurable Residual Disease (MRD) molecular markers because of the possible therapy whit the inhibitor Enasidenib. A man with a diagnosis of AML, negative for the common molecular markers, resulted in complete remission (CR) only after three induction cycles. The patient underwent a transplant procedure but after 7 months a relapse was observed. We retrospectively studied the status of IDH2 mutation in this patient which was positive at diagnosis (29%), after two induction cycles and at the allogeneic transplantation (2%). In the next follow-up, both WT1 and IDH2 were normal. After 3 months, when the patient was in CR and WT1 was still normal, IDH2 resulted 0.12%. After further 2 months, the patient clinically relapsed; at that time, WT1 was very high and IDH2 was 7.8%. In this case, IDH2 monitoring by Droplet Digital PCR (ddPCR) predicted relapse two months before WT1 levels and clinical evidence.