Biochimica Clinica: VOL.46 N.3

The use of Cannabis and its derivatives is increasingly at the center of intense political debates related to regulation or prohibition. Beyond the political or ideological debates, a scientifically based information policy is however necessary to understand the differences between Cannabis used for recreational purposes, light Cannabis that can be legally sold, and Cannabis administered for therapeutic purposes, with a special focus to the analytical methods that can be utilized to differentiate the different types of Cannabis. Regarding the different properties, considered that the differentiation between the various types of Cannabis, (recreational, light or medical) is essential for legal purposes, the role played by the analytical toxicology laboratories is crucial. A number of studies with light and medical Cannabis have illustrated the possible use of Cannabidiol (CBD) as a possible biomarker to identify the type of Cannabis, since CBD is predominant in light Cannabis; the ratio of CBD to Δ9-tetrahydrocannabinol (THC), the only cannabinoid with psychotropic effects, has been suggested to be of particular relevance. This review aims to analyze the current legislative, medical and laboratory medicine aspects connected to Cannabis in Italy, by focusing on the legislation currently in force and on the role of the toxicology laboratory in differentiating the different types of Cannabis.

Parkinson’s disease (PD) is a neurodegenerative condition characterized by a series of symptoms that anticipate overt disease. In particular, rapid eye movement (REM) sleep behavior disorders, intestinal problems and olfactory impairment are the most frequent prodromal signs of the disease. Compelling evidence suggests that, among other factors, the microbiota might play an important role in the onset and progression of PD. While several findings confirmed that there might be an important link between intestinal microbiota alterations and PD onset, little is known about the potential contribution of the nasal microbiota. Here, we will describe the latest findings on this topic by considering that more than 80% of patients with PD develop remarkable olfactory deficits in their prodromal disease stage. Therefore, the nasal microbiota might contribute to PD, eventually boosting the intestinal microbiota in promoting disease onset.

Background: we report here data on humoral immune response post-BNT162b2 primary vaccination and booster in pre-vaccination baseline severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seronegative and seropositive subjects. Methods: the study population consisted in 51 baseline SARS-CoV-2 seronegative and 11 baseline SARS-CoV-2 seropositive subjects, who underwent primary mRNA-based BNT162b2 vaccination (two doses) followed by homologous booster administration (third dose). Venous blood was sequentially collected up to 1 months after vaccine booster administration, and humoral response was monitored by measuring anti-SARS-CoV-2 spike trimeric IgG antibodies. Results: the humoral response after the three doses of BNT162b2 displayed an overlapping trend in the two groups, although the baseline and post-primary vaccination concentration of anti-SARS-CoV-2 spike trimeric IgG were constantly higher in baseline SARS-CoV-2 seropositive than in baseline SARS-CoV-2 seronegative subjects (all p<0.001). Unlike before vaccine booster administration, the levels of anti-SARS-CoV-2 spike trimeric IgG, 1 month after receiving the third BNT162b2 dose were not significantly different between pre-vaccination baseline SARS-CoV-2 seropositive and seronegative subjects (7 430 versus 9 020 kBAU/L; p=0.232). In both cohorts, all recipients of vaccine booster displayed antibodies levels >264 kBAU/L. Conclusion: the results of this study demonstrate that although baseline SARS-CoV-2 seropositive subjects have magnified humoral response to primary BNT162b2 vaccination, vaccine booster generates anti-SARS-CoV-2 spike trimeric IgG values not different from those found in baseline SARS-CoV-2 seronegative subjects. Thus, this study provides evidence that a prior SARS-CoV-2 infection does not mitigate the need for additional vaccine boosters.

Introduction: the health emergency caused by the COVID-19 pandemic has forced health providers to rethink and reformulate patients’ care management, particularly during lockdown, in view of the need of stronger interactions between hospitals and local medical care with the aim of reducing the number of patients’ accesses to the hospitals. Patient under oral anticoagulant therapy, particularly those treated with antivitamin K drugs (AVK), need continuous therapy monitoring and drug dose adjustment, thus increasing their risk of possible SARS-CoV-2 exposure during the visits to the hospitals. The aim of this study is to illustrate, using appropriate indicators, the outcomes of an alternative clinical management of anticoagulated patients, adopting a new organizational model and an extended use of digital tools. Methods: a dedicated telephone number and a digital platform for therapy issuing were activated to maintain continuous patient surveillance and counselling activity. Specific indicators were: number of telephone calls received; number of digital therapy prescriptions; time in range (TTR) of AVK patients; number of major adverse events. Results: during the pandemic phase 10 215 telephone calls were received (with an increase of 200% compared to the pre-pandemic era). When compared to the values observed in the pre-pandemic period, the values of the indicators remained stable during the pandemic phase: TTR of AVK patients was 74% versus 74.6%; 22 minor and 2 major bleeding events were recorded versus 21 and 2 respectively. Discussion: the results obtained show, from the patient side, an optimal acceptance of the model proposed and, from the clinical side, the maintenance of the pre-pandemic quality standards of care

Introduction: the Homologous Recombination Repair (HRR) system is essential for DNA repair and genomic stability. Homologous Recombination Deficiency (HRD) arises upon the inactivation of several genes involved in HRR, and it is commonly observed in breast and ovarian cancer. The detection of HRD is a valuable tool of clinical relevance, indicative of sensitivity to agnostic therapy and DNA damaging reagents. 
Methods: shallow Whole Genome Sequencing (sWGS) was performed on 26 ovarian cancer (OC) samples divided into training (n=13) and test group (n=13). The training set included 7 somatic and 6 germline carrying BRCA+ve (n=8) and BRCA-ve (n=5) status. In particular, the BRCA+ve were 3/7 somatic and 5/6 germline samples; the BRCA-ve were 4/7somatic and 1/6 germline. All samples were prepared using the KAPA HyperPlus Library Preparation kit (Roche), pooled and sequenced on NextSeq550 Dx (Illumina). Large-Scale Transitions (LST) profiles were calculated using WGS data at low coverage (<1x) using different sliding window sizes spanning 10-1 000 Kbases. The HRD score was estimated using our bioinformatics pipeline. 
Results: the BRCA status was assessed in 22/26 OC samples, and the HRD score was evaluated in all somatic samples. In the test group, 7 BRCA+ve samples were classified as HRD+ve, 2 BRCA-ve were classified as HRD-ve, and 4 BRCA unknown samples were predicted as HRD+ve in two cases and HRD-ve in the other half. 
Discussion: germline BRCA1/2 mutation status is currently the main genetic biomarker of HRD, but it has its drawbacks: HRD can be driven purely by somatic events. A bioinformatics pipeline to evaluate the HRR system status in breast and ovarian cancer has been completed; it is based on sWGS to support therapeutics and follow up strategies.

Introduction: the term “gut dysbiosis” refers to an alteration in the composition and functions of the microbiota that causes the loss of beneficial microorganisms and species diversity with the potential growth of harmful bacteria. This condition has been related to many pathologies, both intestinals and extraintestinals. One method for evaluating the dysbiosis is the determination of urinary metabolites related to intestinal fermentative processes. Among these different molecules, indoxyl-sulfate (IS) is well studied. In this paper, the IS measurement was performed in a group of subjects in order to establish a reference interval.
Methods: the measurement was carried out by HPLC method using a  fluorescence detector on early morning urine void. The results were expressed for urine creatinine concentration.
Results: in a group of 68 subjects (35 males, 33 females), the mean IS concentration was 22.3 µmol/mmol creatinine with a standard deviation of 6.8 and a range of 8.2-36.6. The reference interval at 95%, calculated with the “robust method”, was 9.0-36.3 µmol/mmol creatinine. The 90% confidence intervals, calculated with the “boostrap method”, for the lower and upper limit of the reference interval were 6.8-11.5 and 34.0-38.6 respectively.
Conclusion: this study is  one of the first contributions to the definition of a reference interval for this metabolite in the first morning urine void.

Introduction: alcohol and cocaine are frequently used together; this co-abuse compromises the physical and mental requirements included in the assessment of fitness to drive. The assessment for the issuing of the certificate of fitness to drive is released by Local Medical Commissions after clinical examination and toxicological analysis. The aim of this paper is to estimate the prevalence of cocaine use among drunk-drivers during a specific driving regranting protocol, in order to assess the usefulness of the inclusion of cocaine metabolites in hair analysis in this category of subjects. 
Methods: according to the toxicological protocol at the Regional Reference toxicological laboratory of the “Liguria Levante” in Sarzana (La Spezia) at San Bartolomeo Hospital, in addition to the quantification of carbohydrate-deficient transferrin (CDT) in serum by HPLC and Ethylglucuronide (ETG) in keratin matrix, also cocaine and its metabolites, especially Coca-ethylene (CE) has been tested in hair by LC-MS/MS. The protocol has been applied on biological samples collected from 2 215 drunk-drivers.
Results: the 6.6% of the 2 215 hair samples analyzed were classified as non-moderate alcohol users (ETG >30 pg/mg). Among these 0.1% were cocaine users and 0.9% consumed together alcohol and cocaine, as highlighted by the presence of CE. Out of the 94.4% of the 2 215 hair samples with ETG <30 pg/mg, 3.9% and 6.1% were positive for cocaine, and CE respectively.
Conclusion: This study demonstrates the importance of toxicological analysis of drugs of abuse in hair and the need to include the analysis in specific protocols for driving regranting, such as the one here adopted.

The Italian National Recovery and Resilience Plan (NRRP) provides resources for the activation for over a thousand Community Houses that will serve as permanent points of reference for the population with the aim to guarantee both the prevention activity and the  care of patients. These structures need to be provided with Information Technology  (essential for the implementation of telemedecine), an adequate multi-specialty instrumental equipment (radiology, clinical laboratory) and a multidisciplinary team of medical specialists, paediatricians, nurses and other health professionals. In this context, the clinical laboratory activity takes on a strategic role especially in the diagnosis of the acute miocardial damage (AMI) which is fundamentally based on the determination of Troponin I o T and no longer on instrumental investigations, such as ECG. High quality POCT devices are currently available on the market for the determination of Troponin I (and T) with high sensitivity methods characterized by adequate costs for the diagnostic efficacy they should guarantee. Recently, the POCT system QuidelTriage High Sensitivity Troponin I test has been evaluated  using 75 whole blood samples from patients. Overall, the outcome of the trial was satisfactory and corresponded to the quality and performance specifications recommended by AACC and IFCC for a method that should assure  an elevated level of diagnostic accuracy. The tested POCT device was effective in the management of patients with suspected AMI using either single cut-off value or 0/1 hour algorithm. This demonstrates the important contribution that POCT devices can offer for of early diagnosis and monitoring of chronic disease

Cardiovascular risk evaluation in patients undergoing major non-cardiac surgery: role of cardiac-specific biomarkers.

Joint document of the Italian Societies of Clinical Biochemistry: European Ligand Assay Society, Sezione Italiana (ELAS), Società Italiana di Biochimica Clinica e Biologia Molecolare Clinica (SIBioC), Società Italiana di Patologia Clinica e Medicina di Laboratorio (SIPMeL) Patients undergoing major surgery have a substantial risk of cardiovascular events during the perioperative period.

Despite the introduction of several risk scores based on medical history, classical risk factors and non-invasive cardiac tests, the possibility to predict cardiovascular events in patients undergoing non-cardiac surgery remains limited. The cardiac-specific biomarkers natriuretic peptides (NPs) and cardiac troponins (cTn) have been proposed as additional tools for risk prediction in the peri-operative period. This Consensus Document aims to discuss the value of preoperative levels and perioperative changes in cardiac-specific biomarkers to predict adverse outcomes in patients undergoing major non-cardiac surgery. Based on several prospective observational studies and 6 meta-analyses, some guidelines recommended the measurement of NPs to refine perioperative cardiac risk estimation in patients undergoing noncardiac surgery. More recently, several studies reported a higher mortality in surgical patients presenting an elevation in high-sensitivity cTnT and cTnI, especially in elderly patients or those with comorbidities. This evidence should be considered in future international guidelines on the evaluation of perioperative risk in patients undergoing major noncardiac surgery.

HbA1c is a major hemoglobin characterized by nonenzymatic binding of glucose to the N-terminal valine residue of the hemoglobin β-chain, which reflects average glucose levels during the erythrocyte lifespan. This test has been recommended for diabetes monitoring and even for diagnosis, as well as in assessing the risk for chronic complications in diabetic patients. Therefore, an accurate measurement of HbA1c is extremely important. However, the reliability of HbA1c is impaired in certain clinical conditions, such as hemolytic anemia, blood transfusion, renal disease, and pregnancy, that increase the erythrocyte turnover or reduce its lifespan. We report the case of a 38-years-old woman with previous history of high fasting plasma glucose level who underwent routine laboratory assessment. The analysis of HbA1c by capillary electrophoresis (CE) showed an atypical profile with a clear presence of abnormal hemoglobin that did not allow to obtain a reliable result for HbA1c. The same sample analyzed by HPLC showed, the presence of an abnormal Hb and obtained a different result for HbA1c. Subsequently, the analysis of hemoglobin fractions in CE (using Hemoglobin kit- Sebia) confirmed an atypical profile with the presence of an abnormal hemoglobin peak (27.3%) in the “zone Z15” and low HbA2 (0.5%). The molecular investigation of the globin genes highlighted the presence of three mutations of the α-genes compatible with HbH disease. The HbH disease is responsible for a hemolytic condition that is associated with reduced erythrocyte survival, making it impossible to use HbA1c for diagnosis and monitoring the glycemic status in this patient. The use of separative technologies, such as CE and HPLC, has been useful to detect a thalassemic defect, which must be reported to allow correct diagnostic conclusions. In this condition, the introduction of alternative biomarkers like glycated albumin (GA) is thought to be more reliable than HbA1c, since GA values are not influenced by the modifications of the erythrocyte lifespan.

Diabetes mellitus is a worldwide disease and glycated hemoglobin (HbA1c) is the gold standard for the diagnosis and monitoring. Hemoglobin variants can interfere with HbA1c measurement in both preanalytical and analytical phases, and their incidental detection is continuously increasing, due to the recent migration waves. In the Roma 1 HUB Laboratory, HbA1c is measured by capillary electrophoresis (CE). A 51-year-old man from Bangladesh underwent HbA1c analysis and a variant was evident in the electrophoretic pattern. After informed consent for further analysis was given, a hemoglobin electrophoresis by CE was performed. A suspected HbE was found and then confirmed by molecular testing. The patient showed mild microcytosis and hypochromia, but no anemia. The presence of the variant HbE was without influence on HbA1c measurement by CE. This technique offers the advantage to detect hemoglobin variants as well as a reliable measurement of hemoglobin A2 (HbA2). These features allow a correct diagnosis of thalassemia and hemoglobinopathies without interfering with HbA1c measurement.

A 63-year-old man, without previously known cancer disease, arrived at the Emergency room because of severe dyspnea. Electrocardiogram revealed right branch block and low ventricular repolarization voltages. Chest X ray and echocardiogram, instead, showed right pleural effusion and cardiac massive effusion. The patient underwent pericardiocentesis. The morphological study of pericardial fluid revealed the presence of numerous nonhematopoietic cells and the patient was admitted to the hosptital Thoracentesis was performed during hospitalization. The analysis of pleural fluid showed similar results to those obtained in pericardial fluid. A subsequent paracentesis was performed. Accordingly, this fluid showed similar atypical cells. Adenopathy in the supraclavicular area was found at the TC scan. Lymphnode biopsy confirmed a poorly differentiated proliferation, consistent with squamous cell carcinoma. In conclusion, morphological analysis of the pericardial fluid is a simple examination that, in a short time, may alert about the presence of unexpected atypical cells.