Biochimica Clinica: VOL.47 N.1

Part I of this review was dedicated to the biological complexity of saliva and its relationship with the pathophysiology of many organs. In Part II, issues related to the pre-analytical and analytical phases are illustrated, focusing first the attention on the main sources of variability, starting from the few data available on biological variation to the different approaches for sample collection under basal or stimulated conditions. Up to now, published data, obtained on small groups of healthy or diseased individuals, seem to be indicative of wide pre-analytical errors and/or individual variability. The established clinical use of salivary tests is then reviewed and a series of possible new applications are presented. Methodologies employed in saliva are presented, ranging from commonly applied techniques, such as colorimetric enzymatic methods and ELISA, to more sophisticated technologies (gas chromatography–mass spectrometry, liquid chromatography-mass spectrometry and 1H NMR spectroscopy) to investigate proteomics and metabolomics. The aim of this review is to present the pre-analytical issues related to its collection and storage and to illustrate the knowledge gaps and the numerous opportunities that the analysis of this interesting biological fluid may offer.

An impressive number of studies published during the past years have demonstrated that low levels of high-density lipoprotein cholesterol (HDL-C) are associated with increased cardiovascular risk. Nevertheless, more recent data in both ischemic heart disease patients and general population revealed that also considerably increased HDL-C concentration could be associated with enhanced risk of all-cause and cardiovascular mortality, displaying an U-shaped association with adverse outcomes. These paradoxical findings prompted us to review the available information on this matter, concluding that the association between extremely elevated HDL-C and mortality may not be totally unexpected or unpredictable. Some unfavorable consequences of high HDL-C levels are confirmed by epidemiologic data in over-trained subjects, in whom a similar U-shaped curve is also observed between strenuous training and mortality. This review is hence aimed to summarize these evidences, proffering some possible mechanisms underlying this enigmatic association. If this association could be confirmed in further studies, it may be advisable that laboratory reporting of HDL-C should encompass a range of “desirable values” rather than indicating a single lower decision threshold, as has been suggested until presently. 

Introduction: Saliva is a promising biological fluid to be used for measuring a number of analytes. Aim of this ppaperis to verify if salivary anti-SARS-CoV-2 antibodies determination could be suitable for monitoring the viral spread andvaccination efficacy during the COVID-19 epidemic.Methods: a total of 69 subjects were enrolled at the Padova University Hospital: 39 COVID-19 patients and 30 health careworkers (HCW), who underwent a complete vaccination cycle with BNT162b2. All subjects collected a salivary sample,using Salivette, (SARSTEDT AG & Co, Nümbrecht, Germany). For 9 HCW, salivary samples were collected at threedifferent times within the same day. A serum sample was also obtained for all individuals. Salivary COVID-19 N/S1 RBD(sal-IgG) and serum anti-SARS-CoV-2 S-RBD IgG Ab (ser-IgG) were used for determining anti SARS-CoV-2 antibodies.Results: positive sal-IgG were found in 67/69 (97.1%) samples; in serum samples, the positivity for ser-IgG was foundin 68/69 (98.6%). The sal-IgG median levels differed from COVID-19 to vaccinated HCW, being 0,21 kAU/L in patientssamples and 0,8 kAU/L in vaccinated HCV samples (p =0.030). Median levels for ser-IgG in COVID-19 and patientsvaccinated HCW were 121 kBAU/L and 940 kBAU/L (p <0.001) respectively. A statistically significant correlation was foundbetween ser-IgG levels and time post-vaccination in HCW (rho =-0.6292, p <0.001). Sal-IgG levels were not influencedby the daytime of collection (rho =0.148, p=0.373). Passing-Bablok regressions showed that sal-IgG and ser-IgGcomparability was assessable only when ser-IgG values were divided by 1000, showing slope and intercept values of 0.016(95%CI: 0.016-0.078) and 0,221 (95%CI:-0.097 to 0.786), respectively.Conclusions: sal-IgG are detectable both in COVID-19 and in vaccinated individuals and the values are not influencedby the daytime of collection. As expected sal-IgG were much lower than ser-IgG.

Introduction: the increase in high-intensity fraction of training shifts the ox-inflammation balance towards a more oxidizing environment, overcoming the antioxidant barrier and causing molecular oxidative damage, impaired performance and greater risk of injury for athletes. Methods: a team of male cyclists and one of female basketball players were evaluated during a competitive season. A number of serum parameters usually employed in sports medicine together with some ox-inflammation biomarkers most recently suggested, were measured at definite intervals of time. Results: significant variations related to the extent of workload were observed for several biomarkers; glutathione (GSH), 3-nitrotyrosine (3NT) and 8-hydroxy-deoxy-guanosine (8OHdG) showed the greatest (exceeding the limits of the referenge ranges) and most significant changes (exceeding the reference change values, RCV). Discussion: the measurements of ox-inflammation biomarkers allowed to evaluate the extent of oxidative imbalance determined by training. In cyclists, which were subjected to the highest workloads and for a longer time, the increasing of 3NT levels suggested that the antioxidant systems was not completely able to neutralize the imbalance, causing a progressive oxidative damage on proteins. The lower levels of 3NT and 8OHdG in basketball players showed that minor workloads have lower impact on the imbalance. Since ox-inflammation biomarkers vary with training load with greater amplitude and significance compared to other parameters, their measurements are proposed for the evaluation of the effects of training programs in athletes.

Objectives: the Italian Society of Clinical Biochemistry and Clinical Molecular Biology (SIBioC) Big Data and Artificial Intelligence (BAI) Working Group promoted a survey to frame the knowledge, skills, and technological predisposition in Clinical Laboratories. Methods: questionnaire, focusing on digitization, IT infrastructures, data accessibility, and BAI projects underway was sent to 1351 SIBioC participants. The responses were evaluated using SurveyMonkey software and Google Sheets. Results: the 227 respondents (17%) from all over Italy (47% of 484 labs), mainly biologists, laboratory physicians and managers, mostly from laboratories of public hospitals, revealed lack of hardware, software and corporate Wi-Fi, and dearth of PCs. Only 25% work daily on clouds, while 65%-including Laboratory Directors-cannot acquire health data from sources other than laboratories. Only 50% of those with access can review a clinical patient’s health record, while the other access only to laboratory information. The integration of laboratory data with other health data is mostly incomplete, which limits BAI-type analysis. Many are unaware of integration platforms. Over 90% report pulling data from the LIS, with varying degrees of autonomy. Very few have already undertaken BAI projects, frequently relying on IT partnerships. The majority consider BAI as crucial in helping professional judgments, indicating a growing interest. Conclusions: the questionnaire received relevant feedback from SIBioC participants. It highlighted the level of expertise and interest in BAI applications. None of the obstacles stands out more than the others, emphasising the need to allaround work: IT infrastructures, data warehouses, BAI analysis software acquisition, data accessibility and training.

Forensic toxicology is the branch of toxicology that applies the principles and knowledge of the latter to issues and problems related to the field of law enforcement officers, lawyers, judges, and juries. There is a need to establish specific codes, currently lacking, for forensic toxicologists, to guide them in situations where ethical issues are linked to the job the toxicologists carry out. To fill this gap, two international scientific societies: The International Association of Forensic Toxicologists (TIAFT) and the Society of Forensic Toxicologists (SOFT) decreed the same Code of Ethics indicating personal and professional behaviours to be followed by societies' members. The code details what it is intended for: professionalism, competency, proficiency, and clear communication of forensic toxicologists, trying to answer the ethical dilemmas, not yet encoded in official norms. Concerning toxicological and forensic analyses, necessary to support medical evidences, the knowledge of laboratory data uncertainty is mandatory to ensure the reliability of analytical results to be used in medico-legal contexts. Gathered together, all the discussed recommendations constitute the authors’ vision of “special dose” of ethics in forensic toxicolog

It has now become clear that SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) will remain among us for long time, becoming a “harmingly” endemic infectious disease. Although virulence and pathogenicity have gradually decreased over time, the infectivity has instead increased after the emergence of new sublineages belonging to Omicron family, so that the volume of tests that need to be performed for diagnosing and monitoring coronavirus disease 2019 (COVID-19) remains paramount. The situation has become even more complicated after resurgence of other infectious respiratory diseases, namely Influenza and Respiratory Syncytial Virus (RSV), which are now responsible for an unprecedented “tripledemic”. In this catastrophic scenario, laboratory medicine needs to be proactive and timely adapt to the new challenge. Further development and implementation of SARS-CoV-2 antigen rapid diagnostic testing (Ag-RDT) will play a crucial role in the future, especially encompassing devices that will enable to confirm (or rule out) more infectious diseases at the same time (i.e., COVID-19, Influenza and RSV). Considering that the clinical phenotypes of the three viruses is overlapping, new diagnostic tests designed for simultaneous assessment of multiple viruses within a single Ag-RDT device represent a valuable option for achieving a rapid differential diagnosis between different respiratory infections, or even for diagnosing multiple concomitant infections. A novel strategy could hence be profiled, entailing the screening by means of combined Ag-RDT of all subjects whose infective status may jeopardize their own health as well as the health of other people.

In the last few years, artificial intelligence (AI) is gaining attention in several medical disciplines, including laboratory medicine (LM). The raised interest on AI has been fueled not only by the huge amounts of information daily generated, but also by the special natural context offered by laboratories, where digitalization have already occupied an important part of the routine workflow of patients’ data. Motivated by these topics and under the auspices of SIBioC, a conference on AI and big data was organized in May 2022 in Bologna, Italy. This conference covered several topics of AI and big data, including but not limited to the current and future perspectives, comprising ethical challenges and the role of laboratory specialists, including young professionals, the productive integration of AI with information technologies and with other digital infrastructure, such as the LOINC and the block chain. Furthermore, some examples of real application of AI in LM were reported, including diagnosis and monitoring of familiar hypercholesterolemia, management of insulin treatments for diabetes, reference intervals identification and verification by indirect methods, COVID-19 diagnosis and the monitoring of outpatients monoclonal gammopathy treatment by digital healthcare

Therapeutic monitoring (TDM) of antiepileptic drugs (AEDs), or antiseizure medicines (ASMs) represents a pragmatic approach to the management of patients with epilepsy to assess either compliance, efficacy and toxicity. Dose adjustments based on drug concentrations detected in biological fluids allow physicians to optimize and personalize the pharmacologic regimens for the single patient. TDM of AEDs has become increasingly popular since the 1970s because it is difficult to identify the optimal drug dose for each patient and because AEDs concentrations correlate better than dose with clinical effects. This document aims to suggest reference intervals to harmonize the pharmacokinetic activity among Italian Laboratories in the monitoring of AEDs plasma exposures according to recent international literature and guidelines. 

The EFLM recommends not to implement the race-free Chronic Kidney Disease Epidemiology Consortium (CKD-EPI) equation in European laboratories and to keep the 2009 version of the CKD-EPI equation, without applying a race correction factor. This recommendation is completely in line with a recent Editorial published by the European Renal Association who has also proposed to change to a novel equation only when it has considerably better performance, trying to reach global consensus before implementing such a new glomerular filtration rate (GFR) estimation equation. In Europe, this equation could be for instance the new European Kidney Function Consortium (EKFC) equation, which is population-specific, developed from European cohorts and accurate from in- fants to the older old. Beyond serum creatinine, the estimating equations based on cystatin C will probably gain in popularity, especially because cystatin C seems independent of race. Finally, we must keep in mind that all GFR equations remain an estimation of GFR, especially rough at the individual level. Measuring GFR with a reference method, such as iohexol clearance, remains indicated in specific patients and/or specific situations, and here also, the role of the clinical laboratories is central and should still evolve positively in the future.

In accordance with all the most recent international guidelines, the variation of circulating levels of cardiac troponins I and T, measured with high-sensitivity methods (hs-cTnI and hs-cTnT), should be used for the detection of acute myocardial injury. Recent experimental and clinical evidences have demonstrated that the evaluation of hs-cTnI an hs-cTnT variations is particularly relevant: for the differential diagnosis of Acute Coronary Syndromes in patients admitted to the Emergency Department (ED); for the evaluation of cardiovascular risk in patients undergoing major cardiac or non-cardiac surgery, and in asymptomatic subjects of the general population aged >55 years and with comorbidities; for the evaluation of cardiotoxicity caused by administration of some chemotherapy drugs in patients with malignant tumors. The aim of this document is to discuss the fundamental statistical and biological considerations on the intraindividual variability of hs-cTnI and hs-cTnT over time in the same individual. Firstly, it will be discussed in detail as the variations of circulating levels strictly depend not only on the analytical error of the method used but also on the intra-individual variability of the biomarker. Afterwards, the pathophysiological interpretation and the clinical relevance of the determination of the variability of the hs-cTnI and hs-cTnT values in patients with specific clinical conditions are discussed. Finally, the evaluation over time of the variation in circulating levels of hs-cTnI and hs-cTnT is proposed for a more accurate estimation of cardiovascular risk in asymptomatic subjects from the general population.

An 88-year old woman was admitted to the emergency room (ER) after a syncope and subsequent head trauma. A complete blood count and imaging studies showed cytopenia and bilateral cervical lymphadenopathy, respectively. After three months, the patient returned to ER with dyspnea and a large abdominal effusion. Cytopenia was progressed and protein electrophoresis revealed a monoclonal peak in the beta zone. Immunofixation showed the presence of gamma heavy-chains without associated light-chains. Capillary electrophoresis with immunosubtraction and immunophenotyping by flow cytometry returned the same results. Immunohistochemistry was apparently discordant, describing a monotypic kappa population. An important ascites with low total proteins in the ascitic fluid, a liver with lumpy margins and non-homogeneous structure, elevated GGT, and decreased albumin, suggested ascitogen liver cirrhosis in the context of a rare gamma heavy-chain disease.

This case report is about two frail women admitted to the Emergency Room. The first patient, with a history of alcohol dependence, accessed to First Aid because of an accidental fall; she presented anemia, and elevation of the coagulation tests (prothrombin time 3.02; activated partial thromboplastin time 2.45). After the clinicians’ prescription for the measurements of coagulation factors, the FII was found significantly reduced (6%). Suspecting previous assumption of dabigatran (not declared at the admission), the laboratory decided to measure the plasma concentration of dabigatran, which resulted rather elevated (658 ng/mL). The second patient, aged 94, accessed to the first aid for anuria. Laboratory tests revealed severe anemia, kidney impairment and elevated coagulation tests. The patient was on dabitagran and verapamil. The dabitagran plasma concentrations were particularly elevated (1 224 mg/L. The dabitagran was discontinued in both patients and its plasma concentrations decreased rapidly; the coagulation tests returned to normal. Stroke prevention in elderly people with atrial fibrillation is of great importance, as stroke risk rises greatly with age. The elderly and frail patients have more favourable outcomes when on direct oral anticoagulant (DOACs). The use of these drugs in daily clinical practice does not require monitoring of coagulation. However, the measurement of DOACs is advisable in certain situations, as demonstrated in these two case reports, since elevated drug concentrations. pose the patients at an increased bleeding risk. Anticoagulant activity can be measured easily measured in laboratory practice, using specific coagulation assays developed for the quantification of DOACs plasma levels