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Human Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) infection activates a complex interaction host/virus, leading to the reprogramming of the host metabolism aimed at the energy supply for viral replication. Alterations of the host metabolic homeostasis strongly influence the immune response to SARS-CoV-2, forming the basis of a wide range of outcomes, from the asymptomatic infection to the onset of COVID-19 and up to life-threatening acute respiratory distress syndrome, vascular dysfunction, multiple organ failure, and death. Deciphering the molecular mechanisms associated with the individual susceptibility to SARS-CoV-2 infection calls for a system biology approach; this strategy can address multiple goals, including which patients will respond effectively to the therapeutic treatment. The power of metabolomics lies in the ability to recognize endogenous and exogenous metabolites within a biological sample, measuring their concentration, and identifying perturbations of biochemical pathways associated with qualitative and quantitative metabolic changes. Over the last year, a limited number of metabolomics- and lipidomics-based clinical studies in COVID-19 patients have been published and are discussed in this review. Remarkable alterations in the lipid and amino acid metabolism depict the molecular phenotype of subjects infected by SARS-CoV-2; notably, structural and functional data on the lipids-virus interaction may open new perspectives on targeted therapeutic interventions. Several limitations affect most metabolomics-based studies, slowing the routine application of metabolomics. However, moving metabolomics from bench to bedside cannot imply the mere determination of a given metabolite panel; rather, slotting metabolomics into clinical practice requires the conversion of metabolic patient-specific data into actionable clinical applications.

Objectives There is increasing interest regarding the relationship between serum levels of free triiodothyronine (fT3) and outcomes of COronaVIrus Disease-19 (COVID-19) patients. As several recent reports have described a worse prognosis in patients with low fT3 levels, we performed a meta-analysis to assess the prognostic role of fT3 serum levels in patients with COVID-19 as this information could be clinically relevant for the management of these patients. Methods The methodology was registered in the International prospective register of systematic reviews (PROSPERO) database under the protocol number CRD42021260952. A systematic search was carried out on PubMed, Embase, Web of Science, and Scopus from May to June 2021 without time and language restrictions. The literature search strategy was based on the following keywords: (T3 OR fT3 OR triiodothyronine) AND (COVID-19) AND (prognosis OR survival). Results The literature search identified 163 studies. Seven retrospective studies met the inclusion and exclusion criteria and were included in the meta-analysis. The included studies had a total of 1,183 patients. From the analysis of the included studies, lower fT3 serum levels were consistently observed in intensive care unit (ICU) than in non-ICU patients and in non-survivors than survivors, respectively. Conclusions Serum fT3 concentrations are significantly lower in patients with severe COVID-19 than in non-severely ill patients and predict all-cause mortality of patients with severe COVID-19. Accordingly, fT3 may become a simple tool for stratified management of patients with severe COVID-19.

Although being the recommended laboratory test to diagnose acute pancreatitis, serum pancreatic lipase (LIP) is among the poorly standardized laboratory tests, and laboratory stakeholders often appear to not take enough care of the quality of its measurements. Here we discuss some important issues that, if not correctly managed and solved, make misdiagnosis of acute pancreatitis by using serum LIP a real possibility. First, the current unavailability of a suitable higher-order reference material to be used as common calibrator should be filled up to definitively improve the inter-method bias. Second, knowledge of the analytical characteristics that may explain the defective performance of LIP assays should be deepened. IVD manufacturers should be more explicit in providing this information, including description of their internal protocol for transferring LIP values from internal references to commercial calibrators. Third, recommended models for accurately estimating measurement uncertainty and reliably defining analytical performance specifications for LIP measurements should be applied. Finally, investments considering alternative options for measuring LIP (e.g., targeted to the development of automated LIP immunoassays) should be warranted. All involved stakeholders (standardization bodies, higher-order reference providers, in vitro diagnostics manufacturers, and laboratory professionals) should contribute to fill the existing gap.

Objectives Multicentre international trials relying on diagnoses derived from biochemical results may overlook the importance of assay standardisation from the participating laboratories. Here we describe a study protocol aimed at harmonising results from total bile acid determinations within the context of an international randomised controlled Trial of two treatments, URsodeoxycholic acid and RIFampicin, for women with severe early onset Intrahepatic Cholestasis of pregnancy (TURRIFIC), referred to as the Bile Acid Comparison and Harmonisation (BACH) study, with the aims of reducing inter-laboratory heterogeneity in total bile acid assays. Methods We have simulated laboratory data to determine the feasibility of total bile acid recalibration using a reference set of patient samples with a consensus value approach and subsequently used regression-based techniques to transform the data. Results From these simulations, we have demonstrated that mathematical recalibration of total bile acid results is plausible, with a high probability of successfully harmonising results across participating laboratories. Conclusions Standardisation of bile acid results facilitates the commutability of laboratory results and collation for statistical analysis. It may provide the momentum for broader application of the described techniques in the setting of large-scale multinational clinical trials dependent on results from non-standardised assays.

Objectives Low-density lipoprotein cholesterol (LDL-C) estimation is critical for risk classification, prevention and treatment of atherosclerotic cardiovascular disease (ASCVD). Predictive equations and direct LDL-C are used. We investigated the comparability between the Martin/Hopkins, Sampson, Friedewald and eight other predictive equations on two analysers, to determine whether the equation or analyser influences predicted LDL-C result. Methods In two unpaired datasets, 9,995 lipid profiles were analysed by the Abbott Architect and 4,782 by the Roche Cobas analysers. Non-parametric statistics and Bland Altman plots were used to compare LDL-C. Results On the Abbott analyser; the Martin/Hopkins, Sampson and Friedewald LDL-C were comparable (median bias ≤1.8%) over a range of 1–4.9 mmol/L. On the Roche platform, Martin/Hopkins LDL-C was comparable to Friedewald (median bias 0.3%) but not to Sampson LDL-C (median bias 25%). In patients with LDL-C <1.8 mmol/L and triglycerides (TG) ≤1.7 mmol/L, predicted LDL-C using Abbott reagents was similar between Martin/Hopkins, Sampson and Friedewald equations but not comparable using Roche reagents. Abbott reagents classified 10–20% of patients in the 1.0–1.8 mmol/L range (Martin/Hopkins 13.4%; Sampson 14.5%; Friedewald 16%; direct LDL-C 13.2%). Roche reagents classified 11–30% in the 1.0–1.8 mmol/L range (Martin/Hopkins 23%; Sampson 11%; Friedewald 25%; direct LDL-C 17%). Conclusions Performance of predictive equations is influenced by the choice of analyser for total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and TG. Replacement of the Friedewald equation with Martin/Hopkins estimation to improve quality of LDL-C results can be safely implemented across analysers, whereas caution is advised regarding the Sampson equation.

Objectives In the clinical setting, the analysis and quantification of vitamin C (ascorbic acid) poses several challenges including analyte instability and poor retention by reverse phase HPLC systems. In this article we describe a rapid hydrophilic interaction chromatography ultraviolet method for the measurement of total vitamin C in plasma which overcomes these issues. Methods Ascorbic acid and the internal standard were separated under isocratic conditions using a Waters BEH-Amide column and a mobile phase containing 0.005 M potassium phosphate in 80% acetonitrile. Results The proposed method was validated and showed good precision (coefficient of variation <5%), accuracy (>99%), and analyte stability after extraction (>24 h). Conclusions The simple sample preparation allows full automation and rapid analytical run times of the assay and is therefore suitable for a high-throughput clinical chromatography laboratory.

Objectives Anti phosphatidylserine/prothrombin antibodies (aPS/PT) are often present in patients with antiphospholipid syndrome (APS) and might be relevant in the pathogenesis of this condition. They are major determinant of lupus anticoagulant (LA) in triple-positive antiphospholipid (aPL) profile. Whether they are present and pathogenic in patients with isolated LA [negative anticardiolipin (aCL) and anti β2-glycoprotein I (aβ2GPI) antibodies] is a matter of debate. Methods We measured aPS/PT in a large number of isolated LA with the aim to ascertain whether there is a link between the way isolated LA is assessed and the presence of these antibodies. APS/PT were measured in 86 patients with isolated LA (aCL- and abeta2GPI-). LA was assessed by two test systems, the dilute Russell Viper Venom Time (dRVVT) and the Silica Clotting Time (SCT). Results Sixty-six (77%) individuals with isolated LA were positive for aPS/PT (IgM 44, IgG and IgM 15, IgG in 7). Diagnosis of LA was made based on positive results in both dRVVT and SCT in 40 patients (Group 1) and based on only one positive test in the remaining 46 patients (Group 2). The rate of positive aPS/PT antibodies was significantly higher in Group 1 (OR=7.2, 95% CI 1.9–27.0, p<0.002). Moreover, the titre of IgM aPS/PT was significantly increased in Group 1 as compared to Group 2 (137 U, IQR 64–179 vs. 43 U, IQR 11–120, p=0.008). Conclusions These data indicate an association between LA based on two positive coagulation tests and the presence of aPS/PT antibodies, especially of IgM isotype.

Objectives To evaluate the impact of different biologic, histopathologic and lifestyle factors on serum levels of human epididymis protein 4 (HE4) and Cancer antigen 125 (CA125) in the diagnostic work up of women with an ovarian cyst or pelvic tumor. Methods The statistical evaluation was performed on a population of 445 women diagnosed with a benign ovarian disease, included in a large Swedish multicenter trial (ClinicalTrials.gov NCT03193671). Multivariable logistic regression analyses were performed to distinguish between the true negatives and false positives through adjusting for biologic, histopathologic and lifestyle factors on serum samples of CA125 and HE4 separately. The likelihood ratio test was used to determine statistical significance and Benjamini-Hochberg correction to adjust for multiple testing. Results A total of 31% of the women had false positive CA125 but only 9% had false positive results of HE4. Smoking (OR 6.62 95% CI 2.93–15.12) and impaired renal function, measured by eGFR (OR 0.18 95% CI 0.08–0.39), were independently predictive of falsely elevated serum levels of HE4. Endometriosis was the only variable predictive of falsely elevated serum levels of CA125 (OR 7.96 95% CI 4.53–14.39). Age correlated with increased serum levels of HE4. Conclusions Smoking, renal failure, age and endometriosis are factors that independently should be considered when assessing serum levels of HE4 and CA125 in women with an ovarian cyst or pelvic mass to avoid false indications of malignant disease.

Objectives The therapeutic monoclonal antibody (t-mAb) daratumumab, used to treat multiple myeloma (MM) patients, interferes with routine, electrophoretic based M-protein diagnostics. Electrophoretic response assessment becomes increasingly difficult when multiple t-mAbs are combined for use in a single patient. This is the first study to address the analytical challenges of M-protein monitoring when multiple t-mAbs are combined. Methods In this proof-of-principle study we evaluate two different methods to monitor M-protein responses in three MM patients, who receive both daratumumab and nivolumab. The double hydrashift assay aims to resolve t-mAb interference on immunofixation. The MS-MRD (mass spectrometry minimal residual disease) assay measures clonotypic peptides to quantitate both M-protein and t-mAb concentrations. Results After exposure to daratumumab and nivolumab, both t-mAbs become visible on immunofixation electrophoresis (IFE) as two IgG-kappa bands that migrate close to each other at the cathodal end of the γ-region. In case the M-protein co-migrates with these t-mAbs, the observed interference was completely abolished with the double IFE hydrashift assay. In all three patients the MS-MRD assay was also able to distinguish the M-protein from the t-mAbs. Additional advantage of the MS-MRD assay is that this multiplex assay is more sensitive and allows quantitative M-protein-, daratumumab- and nivolumab-monitoring. Conclusions Daratumumab and nivolumab interfere with electrophoretic M-protein diagnostics. However, the M-protein can be distinguished from both t-mAbs by use of a double hydrashift assay. The MS-MRD assay provides an alternative method that allows sensitive and simultaneous quantitative monitoring of both the M-protein and t-mAbs.

Objectives Macrotroponin is due to cardiac troponin (cTn) binding to endogenous cTn autoantibodies. While previous studies showed a high incidence of macrotroponin affecting cTnI assays, reports of macrotroponin T, particularly without cTnI reactivity, have been rare. Although the clinical significance of macrotroponin is not fully understood, macroenzymes and complexes are recognised to cause confusion in interpretation of laboratory results. The potential for adverse clinical consequences due to misinterpretation of affected results is very high. Methods We describe four cases of macrotroponin T with persistently low high sensitivity cTnT (hs-cTnT) by the 9 min compared to the 18 min variant of the assay. Three cases were serendipitously identified due to the use of a lot number of Roche hs-cTnT affected by non-reproducible results, necessitating measurement of cTnT in duplicate. We identified and characterised these macrotroponin specimens by immunoglobulin depletion (Protein A and PEG precipitation), mixing studies with EDTA and recombinant cTnT. Results In cases of macro-cTnT, a lower result occurred on the hs-cTnT using the 9 min compared to 18 min variant assay (ratio of 9–18 min hs-cTnT <0.80). Mixing studies with recombinant cTnT or EDTA demonstrated a difference in recovery vs. controls. One of these patients demonstrated a high molecular weight complex for cTnI and cTnT demonstrating a macrocomplex involving both cTn. This patient demonstrated a rise and fall in cTn when measured by several commercial assays consistent with genuine acute cardiac injury. Conclusions We identified several cases of macro-cTnT and described associated clinical and biochemical features.

Objectives Pre-analytical plasma glucose (PG) sampling methodology may significantly affect gestational diabetes mellitus (GDM) incidence, but no studies directly examined the impact on perinatal outcomes. We compared the effect on oral glucose tolerance test (OGTT) results of using for blood sampling the traditional sodium fluoride (NaF) tubes, batched at controlled temperature, and the more effective citrate-buffered tubes, in terms of GDM diagnosis and related outcomes. Methods We evaluated 578 pregnant women performing OGTT between 24- and 28-weeks’ gestation. Paired NaF and citrate blood samples were drawn and analyzed for PG. GDM diagnosis was made by applying the ‘one-step’ American Diabetes Association strategy. Data on perinatal outcomes were collected in a subset of 330 women who delivered in our hospital network. Results Using the standard NaF approach, 69 (11.9%) GDM women were detected. Using citrate PG values, 90 women were additionally identified as GDM, increasing the GDM prevalence to 27.5%. Perinatal outcomes were analyzed according to the different diagnostic allocation (NaF-diagnosed GDM, additional citrate-diagnosed GDM, and no GDM). NaF-diagnosed GDM showed a higher incidence of large for gestational age (LGA) (p=0.034), and of cesarean and preterm delivery (p<0.01) vs. no GDM. The only outcome remaining more frequent in the additional citrate diagnosed GDM when compared with no GDM group was LGA (17.2 vs. 6.8%, p=0.025). Conclusions If a health care system plans to use citrate tubes for GDM diagnosis, considerations about clinical implications are mandatory by balancing higher sensitivity in detecting a poor glycemic control with effects on outcomes to avoid “overdiagnosis”.

Objectives We hypothesized that the amount of antigen produced in the body during a COVID-19 infection might differ between patients, and that maximum concentrations would predict the degree of both inflammation and outcome for patients. Methods Eighty-four hospitalized and SARS-CoV-2 PCR swab-positive patients, were followed with blood sampling every day until discharge or death. A total of 444 serial EDTA plasma samples were analyzed for a range of biomarkers: SARS-CoV-2 nuclear antigen and RNA concentration, complement activation as well as several inflammatory markers, and KL-6 as a lung marker. The patients were divided into outcome groups depending on need of respiratory support and death/survival. Results Circulating SARS-CoV-2 nuclear antigen levels were above the detection limit in blood in 65 out of 84 COVID-19 PCR swab-positive patients on day one of hospitalization, as was viral RNA in plasma in 30 out of 84. In all patients, complete antigen clearance was observed within 24 days. There were definite statistically significant differences between the groups depending on their biomarkers, showing that the concentrations of virus RNA and antigen were correlated to the inflammatory biomarker levels, respiratory treatment and death. Conclusions Viral antigen is cleared in parallel with the virus RNA levels. The levels of antigens and SARS-CoV-2 RNA in the blood correlates with the level of IL-6, inflammation, respiratory failure and death. We propose that the antigens levels together with RNA in blood can be used to predict the severity of disease, outcome, and the clearance of the virus from the body.

Objectives Accurate detection of SARS-CoV-2 RNA is essential to stopping the spread of SARS-CoV-2. The aim of this study was to evaluate the performance of the recently introduced MassARRAY ® SARS-CoV-2 Panel and to compare it to the cobas ® SARS-CoV-2 Test. Methods The MassARRAY ® SARS-CoV-2 Panel consists of five assays targeting different sequences of the SARS-CoV-2 genome. Accuracy was determined using national and international proficiency panels including 27 samples. For clinical evaluation, 101 residual clinical samples were analyzed and results compared. Samples had been tested for SARS-CoV-2 RNA with the cobas ® SARS-CoV-2 Test. Results When accuracy was tested with the MassARRAY ® SARS-CoV-2 Panel, 25 of 27 (92.6%) samples revealed correct results. When clinical samples were analyzed with the MassARRAY ® SARS-CoV-2 Panel and compared to the cobas ® SARS-CoV-2 Test, 100 samples showed concordant results. One sample was found to be inconclusive with the MassARRAY ® SARS-CoV-2 Panel. When time-to-results were compared, the new assay showed longer total and hands-on times. Conclusions The MassARRAY ® SARS-CoV-2 Panel showed a good performance and proved to be suitable for use in the routine diagnostic laboratory. Especially during phases of shortage of reagents and/or disposables, the new test system appears as beneficial alternative to standard assays used for detection of SARS-CoV-2 RNA.

Objectives The detection of SARS-CoV-2 in infected people is a key tool to help in controlling COVID-19 pandemic. Like rapid antigenic tests, automated antigen tests, that present the advantage of a higher throughput flow, may be of interest. The LIAISON ® SARS-CoV-2 Ag test was evaluated for the quantification of SARS-CoV-2 nucleocapsid antigen in nasopharyngeal swabs by comparison to RT-PCR. Methods The study involved 378 nasopharyngeal samples (UTM ® and FLOQSwab™, Copan Diagnostics), including 46 swabs positive for SARS-CoV-2 by RT-PCR. These samples came from asymptomatic (n=99, 26.2%) or symptomatic people (n=279, 73.8%), at different times from symptom onset. The samples were analyzed on LIAISON ® XL. Results The overall specificity was 99.4% (CI95% [98.6–100]). The negative predictive value reached 100% in asymptomatic people. Among the 46 positive samples, the overall sensitivity was 84.8% (CI95% [74.4–95.2]), reached 91.9% (CI95% [83.1–100]) in the first fourth days after symptoms onset and was 100% for Cq values ≤25. Antigen was not detected in samples with Cq values >25. Similar results were observed on nasopharyngeal swabs coming from patients infected with the 20I/501Y.V1 variant or the 20H/501Y.V2 variant. Conclusions According to technical performances, the LIAISON ® SARS-CoV-2 Ag test may be a useful tool for COVID-19 diagnosis, especially during the first four days of symptoms.

Objectives Simple and standardized methods to establish correlates to vaccine-elicited SARS-CoV-2 protection are needed. Methods An observational study on antibody response to a mRNA vaccine (Comirnaty) was performed on health care workers (V, n=120). Recovered COVID-19 patients (N, n=94) were used for comparison. Antibody response was evaluated by a quantitative anti-receptor binding domain IgG (anti-RBD) commercial assay and by virus microneutralization test (MNT), in order to establish a threshold of anti-RBD binding antibody units (BAU) able to predict a robust (≥1:80) MNT titer. Results Significant correlation between BAU and MNT titers was found in both V and N, being stronger in V (rs=0.91 and 0.57 respectively, p<0.001); a higher incremental trend starting from MNT titer 1:80 was observed in the V group. The 99% probability of high MNT titer (≥1:80) was reached at 1,814 and 3,564 BAU/mL, and the area under the receiver operating characteristic (ROC) curve was 0.99 (CI: 0.99–1.00) and 0.78 (CI: 0.67–0.86) in V and N, respectively. Conclusions A threshold of 2,000 BAU/mL is highly predictive of strong MNT response in vaccinated individuals and may represent a good surrogate marker of protective response. It remains to be established whether the present results can be extended to BAU titers obtained with other assays.

Objectives After exceptional research efforts, several vaccines were developed against SARS-CoV-2 which sustains the pandemic COVID-19. The Comirnaty vaccine showed high efficacy in clinical trials and was the first to be approved for its distribution to the general population. We evaluated the immune response induced by the first vaccine dose in different sex/age groups and subjects with or without naturally present anti-SARS-CoV-2 antibodies. Methods As part of an Italian multicenter project ( Covidiagnostix ), serum samples from 4,290 health-professionals were serologically tested the day of the first vaccination dose, and 21 days later, using two different instrumentations (Siemens-Healthineers and Roche). Results In total, 97% of samples showed the presence of specific antibodies 21 days after the vaccination dose; the percentage of non-responders increased with age in both genders. Remarkably, naturally seropositive individuals showed antibody persistence up to 11 months and an exceptionally higher vaccination response compared to subjects never infected by SARS-CoV-2. Conclusions This study highlighted the importance of the serological test i) to identify naturally SARS-CoV-2 seropositive individuals and ii) to evaluate the antibody level elicited by the first vaccination dose. Both tests, highlighted differences in the immune response, when subjects were stratified by sex and age, and between naturally seropositive and seronegative subjects. The data obtained show how serological tests could play a crucial role in the triage of the population subjected to the vaccination campaign for COVID-19. The definition of suitable instrumentation-specific thresholds is needed to correctly follow eventually acquired post-vaccination immunity in the general population.