Biochimica Clinica: VOL.46 N.1

The introduction of vaccination programs aiming at inducing an active immune response against pathogens dates back to the first experimental approaches at the end of the 18th century and, since then, has represented a turning point in public health measures to contrast infections. The scientific improvements of the last few years in the field of molecular biology, immunology and genetic engineering have allowed to design new vaccines able to solve, at least in part, the hurdles of conventional vaccine platforms. From the first vaccines based on the inoculation of the whole microorganism, the scientific research has gone in the direction of platforms able to carry only a few or even a single antigenic component of the pathogen, ranging from subunit vaccines to those based on mRNA or DNA. This achievement has made vaccines easier and quicker to develop and, above all, much safer, and it has involved scientific fields that extend far beyond the attempt to fight infectious diseases, such as cancer research. The purpose of this review is to present an overview of the currently available vaccine platforms, their mechanism of action, and the advantages and pitfalls behind each approach.

The COVID-19 pandemic has prompted an unprecedented race to find the means to contrast the SARS-CoV-2 infection, resulting in a huge common effort to develop an efficacious vaccine as soon as possible and an exceptional acceleration of the review process to ensure its safety and efficacy. Many technological platforms are currently under investigation or have already been approved, including those based on the inactivated virus, mRNA- or DNA-based vaccines expressing viral antigens, recombinant SARS-CoV-2 proteins and vector-based vaccines exploiting chimeric adenoviruses. The emergence of new viral variants has represented ad additional challenge and has induced the entire scientific community to potentiate the monitoring process of the ongoing vaccination campaigns. In this scenario, laboratory medicine certainly plays a pivotal role not only in the diagnosis of the infection but also in monitoring the immune response to vaccines and in the detection and prevention of clinically significant adverse events, ultimately contributing to the determination of the biological and clinical efficacy of the available vaccines. This review offers an overview of the most recent and updated data on anti-SARS-CoV-2 vaccines and the technological principles behind them as well as on the resources that laboratory medicine can offer to support the vaccination campaigns. All these aspects represent a rapid step forward in the clinical field which transcends the COVID-19 outbreak and that will certainly pave the way for the future scientific research.

Introduction: biotinidase is required for biotin recycling; its deficiency results in loss of this coenzyme and, hence, the function of many critical enzymes. Delay in the diagnosis of biotinidase deficiency causes irreversible neurological damage; however, it is completely reversible and treatable if detected early. The aim of this study is to measure the activity of the biotinidase enzyme in dried blood specimens using a spectrophotometric technique.
Methods: dried blood specimens were collected from adults (n=81), neonates (n=50), as well as from a patient and his two siblings. A microplate-based enzyme kinetics assay was implemented to assess enzyme stability and activity using N-biotinyl-p-aminobenzoateas as a substrate.
Results: the enzyme was found to be stable for 28 days in dried blood spots stored at -20 ºC. The assay was effective in measuring similar activities of biotinidase among healthy adults and neonates. On the other hand, no biotinidase activity was detected in the patient and low activity was measured in his two siblings.
Conclusion: the assay provides a cost-effective method for the early detection of biotinidase deficiency.

Introduction: hair testing is an alternative matrix for diagnosis and monitoring of drug misuse. The aim of the present study was to evaluate the analytical performances of 7 semi-quantitative immunoassays using hair specimens according to the ISO 15189 standard, in order to introduce hair drug screening in our practice.
Methods: QuantILab DRI Cocaine Metabolite, Opiate, Cannabinoid, Methadone, Amphetamines, Ecstasy and Immunalysis Buprenorphine, were applied on Ilab Taurus (Instrumentation Laboratory SpA, a Werfen Company, Milano). The imprecision verification study consisted in calculating the within-laboratory imprecision (SWL) using positive quality control hair materials, followed by assessment of uniformity with manufacturer’s inter-assay claims and acceptability of test results. A total of 25 EQAS and 29 hair specimens were tested to verify the diagnostic sensitivity (SELAB) and specificity (SPLAB). To confirm the results, an UPLC-MS/MS method accredited according to ISO 15189 standard was used.
Results: SWL obtained for 3 assays were lower than those specified by the manufacturer, opposed to Cannabinoid, Amphetamines, Buprenorphine and Ecstasy assays; thus Upper Verification Limit (UVL) were calculated. SWL for Cannabinoid, Amphetamines and Buprenorphine assays were lower than UVL; Ecstasy assay SWL (8.40%) was greater than UVL (8.39%), but still lower than state-of-the-art imprecision (<15%). SELAB and SPLAB were 100% for all assays except SPLAB for Cannabinoid (95.2%) and Amphetamines (92.9%); 95% confidence intervals of manufacturer diagnostic specificity (SPPROD) were calculated. SPLAB for both assays were included in the 95% CI of SPPROD.
Discussion: for all immunoassays, the verifications were successful and exhibit good diagnostic efficiency for hair drug screening.

Introduction: incident reporting and risk analysis are pivotal activities in the clinical laboratory to ensure patient safety. This paper describes an adverse event that occurred in February 2021 in the clinical laboratory of the Istituto Fiorentino Cura e Assistenza (IFCA) relating to a SARS-CoV-2 testing. Briefly, patients need a negative molecular test on a sample taken 2 days pre-operatively, but in one occasion a number of tests were not done on time due to a failure of the laboratory organization, and the surgery was postponed.
Methods: the subsequent analysis of the event showed that a rethinking of the entire pre analytical phase was necessary and the Toyota Production System (TPS) was used to establish a completely revised work flow.
Results: with the involvement of the entire laboratory staff and the support of a TPS specialist, the pathway was entirely redesigned: tubes colour codes were defined, dedicated working areas were implemented in order to obtain a space where the staff members can verify, at a glance, the status and the results of each step of the workflow.
Discussion: the application of the Toyota system to the pre-analytical phase of the laboratory has created a tidy and clean environment, with dedicated spaces for all the samples. The involvement of the staff in the improvement process has guaranteed the compliance of the operators to the new organisation.

Introduction: recently, multi-analytes delta-check (MDC) has been proposed as a more effective tool in identification errors (IE) prevention. In this context, “Haematology” and “Clinical Risk” SIBioC working groups launched a project aiming to develop a cell blood count (CBC) MDC. This work is aimed to describe the project and some preliminary results.
Methods: the project consists of four phases: collection of CBC results from 15 Italian laboratories to create an original dataset (OD); pilot study on a smaller dataset (SD) i.e., creation of an artificial mix-up dataset-MD containing IE by casual resampling of the SD and identification of the best statistical model to create a MDC; identification of the most accurate MDC on OD; testing the MDC in involved labs and verification of its effectiveness.
Results: the SD included 2,367 pair of consecutive results for the same patient (patients’ age: 0-100 years; the majority of repetitions were within days). The SD casual resampling generated a MD with 2,000 pair of patient-mixed consecutive results. When one of the most frequent used delta-check alert (ΔMCV=7fL) was applied to detect IE in MD, the method accuracy was low (AUC=0.542). On the contrary, testing of a multivariate model, obtained by a stepwise logistic analysis, allowed to obtain a more accurate MDC in IE detection (AUC=0.931, sensitivity=91.6%, specificity=94%).
Conclusions: MDC may offer a practical strategy to identify IE prior to test reporting, improving patient safety. However a good planning of project workflow, selection of methodology, tools and staff competence are key elements to reach the objectives.

Introduction: electrophoresis of serum proteins (EF) is indicated for the identification and monitoring of monoclonal components (CM). It has been shown that interleukins play a role in the differentiation of B cells in plasma cells producing immunoglobulins; it has been also demonstrated that COVID-19 patients show a higher prevalence of CM in comparison to the general population. The aim of this work is to retrospectively evaluate the presence of CM in patients hospitalized with COVID-19, compared to a population of patients admitted in non-COVID-19 wards.
Methods: EF was performed in the two groups of patients (COVID positive and negative) using capillary electrophoresis. Patients with previous plasma cell dyscrasias have been excluded.
Results: the results show that in the COVID positive group, the incidence of CM is statistically higher compared to the COVID negative group (39.7% versus 13.3%). In one patient, the CM was no longer detectable when the swab became negative.
Conclusions: the study confirmed that the viral infection produces detectable CM, probably transitory as shown by a case index. The pathogenesis of the phenomenon could be explained by the cytokine stimulus on B cells and by the interaction of the virus with the lymphocyte ACE 2 receptor. Larger studies are needed to confirm the presented data.

Introduction: in this study we evaluated the analytical performance of the Trinity Biotech HbPremier Hb9210 (Bray, Ireland/Kansas city, US), a boronate affinity chromatography-based high performance liquid chromatography (HPLC) system for the measurement of glycated hemoglobin. The ADAMSTM A1c HA-8180V (ARKRAY, Inc., Kyoto, Japan) analyzer was used for the comparison.
Methods: three pool of blood (L1, L2, L3) and two control materials (B1, B2) were used to evaluate analytical precision of the HbPremier Hb9210, analyzing five replicates of each sample per day for five days. For the comparison, blood specimens were analyzed according to the Clinical and Laboratory Standard Institute (CLSI) guidelines. Both the available reference systems (IFCC, NGSP) were used.
Results: the total CVs of for low, medium and high values of the three pools (L1, L2, L3) were 0.68%, 1.72%, 1.31% respectively; and were 1.57%, 1.38% for low, and medium values (B1, B2) of the two controls when the values were expressed in mmol/mol (SI units) and 0.39%, 1.18%, 1.08% (L1, L2, L3), 0.95%, 1.07% (B1, B2) when the values were reported in % (NGSP units). Passing-Bablok regression used for the comparison of methods showed a small proportional error (slope 1.05, 95%CI: 1.02-1.07) using the IFCC reference system (mmol/mol); the error was not present with the NGSP (%). The bias found was -1.87 mmol/mol (95%CI: -4.33- 0.58) and -0.17% (95%CI: -0.39-0.05).
Discussion: the analytical performances of HbPremier Hb9210 have been successfully verified. The instrument performs well and provides results in good agreement with those obtained with the ADAMS analyzer.

Introduction: the new fully automated HPLC ion-exchange system ADAMS A1c HA-8190V analyzer, developed by ARKRAY Inc., running in two different modes (Variant and Fast) has been evaluated.
Methods: reproducibility was evaluated according to the EP-15A3 standard. Method comparison was performed on 122 fresh blood samples, according to the EP-9 standard. The system was compared to 3 other HPLC analyzers, based on ion-exchange (Tosoh G11 and Bio-Rad D-100) and boronate affinity chromatography (Trinity Biotech Premier Hb9210). Usability was evaluated by using to a score evaluation system.
Results: reproducibility proved to be very good at normal and high HbA1c concentration, with total CVs always <0.7 %, when HbA1c was expressed in mmol/mol as well as in % units. The HA-8190V system was well correlated to the other HPLC analyzers, with a mean bias not clinically relevant. Finally, the usability of the system was evaluated and proved to be well acceptable.
Conclusions: the ARKRAY HA-8190 V system was found to be a reliable and suitable method for routine HbA1c measurement in clinical chemistry laboratories.

Description and clinical use of autoantibody determination in autoimmune liver diseases – update 2021, on behalf of the Study Group “Autoimmune Diseases” of the Italian Society of Clinical Biochemistry. The autoantibody assessment in the field of autoimmune liver disease is crucial both for diagnosis and prognosis. Although these autoantibodies are sometime present even in the normal healthy individuals, their presence is a prerequisite to diagnose autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC); some of these autoantibodies are included in the diagnostic scoring system for these diseases. The laboratory diagnostics of autoimmune liver diseases, traditionally carried out using indirect immunofluorescence testing (IFT) on rodent tissue slices, has achieved substantial improvements due to innovative analytical opportunities, such as ELISA-based diagnostic assay and Multiple Immunodot Liver profile test based on recombinant or purified antigens. The aim of this document is to highlight the crucial role of the new antigen specific tests for a better diagnostic strategy in the field of the three major autoimmune liver diseases AIH, PBC and Primary sclerosing cholangitis (PSC).

The required blood to anti-coagulant ratio in the tubes for coagulation tests, is 9:1; any deviation from this ratio should be avoided as it may lead to erroneous analytical results. One of the variables influencing the ratio is an elevated hematocrit (>55%), because the elevated concentration of citrate in the plasma specimen can cause clotting times falsely increased. These erroneous results can lead to possible misdiagnosis, incorrect patient treatment and/or the performance of additional unnecessary investigations. We present the case of a 46-year-old man referred to the laboratory for a pre-surgery check. The presence of elevated values in his coagulation tests led to delayed surgery and to additional tests. Once the error was detected, a root cause analysis was performed. The main contributing factors to the pre-analytical error were identified using Ishikawa method; factors related to the organization, staff and environment were identified and analyzed. Preventive actions (safety barriers) were designed to prevent the error from recurring. The safety barriers implemented were: an automatic flag displayed in the information laboratory system (LIS) in cases of elevated hematocrit (>55%), to enhancement of the possible error detection plus additional laboratory staff training. At the best of our knowledge, since these safety barriers were applied three years ago, no coagulation test results interfered by high hematocrit have been released from our laboratory. This case demonstrates the importance of implementing safety barriers to prevent errors arising from situations that, although already described and well known, may go unnoticed by the laboratory staff