Sequential Normothermic Regional Perfusion and End-ischemic Ex Situ Machine Perfusion Allow the Safe Use of Very Old DCD Donors in Liver Transplantation.

BACKGROUND: In Italy, 20 min of continuous, flat-line electrocardiogram are required for death declaration. Despite prolonged warm ischemia time, Italian centers reported good outcomes in controlled donation after circulatory death (cDCD) liver transplantation by combining normothermic regional and end-ischemic machine perfusion (MP). The aim of this study was to evaluate the safety and feasibility of the use of septuagenarian and octogenarian cDCD donors with this approach. METHODS: All cDCD older than 70 y were evaluated during normothermic regional perfusion and then randomly assigned to dual hypothermic or normothermic MP. RESULTS: In the period from April 2021 to December 2022, 17 cDCD older than 70 y were considered. In 6 cases (35%), the graft was not considered suitable for liver transplantation, whereas 11 (65%) were evaluated and eventually transplanted. The median donor age was 82 y, being 8 (73%) older than 80. Median functional warm ischemia and no-flow time were 36 and 28 min, respectively. Grafts were randomly assigned to ex situ dual hypothermic oxygenated MP in 6 cases (55%) and normothermic MP in 5 (45%). None was discarded during MP. There were no cases of primary nonfunction, 1 case of postreperfusion syndrome (9%) and 2 cases (18%) of early allograft dysfunction. At a median follow-up of 8 mo, no vascular complications or ischemic cholangiopathy were reported. No major differences were found in terms of postoperative hospitalization or complications based on the type of MP. CONCLUSIONS: The implementation of sequential normothermic regional and end-ischemic MP allows the safe use of very old donation after circulatory death donors.

Orientation of capture antibodies on gold nanoparticles to improve the sensitivity of ELISA-based medical devices.

The sensitivity of ELISA-based devices strongly depends on the right orientation of antibodies on the sensor surface. The aim of this work was to increase the analytical performance of a commercial ELISA-based medical device (VIDAS®), thanks to the specific orientation of antibodies on gold nanostructured disposables. For this purpose, fPSA VIDAS® assay was used as model and the disposable providing the antigen binding surface (SPR®) was functionalized with gold nanostructures coated with monovalent half-fragment antibodies (reduced IgG, rIgG). The functionalization of polystyrene SPRs® with gold nanostructures was achieved through a one-step incubation of gold dispersions in a mixture of non-toxic solvents. Five different concentrations of gold nanoparticles (NPs) were tested with a maximum fluorescence enhancement for NPs density around 3-8 *103 NPs/µm2 (752 ± 11 RFV vs 316 ± 5 RFV of bare SPRs®). The comparison of the dose-response curve obtained with commercial and gold coated-SPRs® revealed a significant improvement (p < 0.0001) of the analytical sensitivity of the VIDAS® system using nanostructured disposables. This improved version of SPRs® allows to distinguish small variations of fPSA concentrations opening the way to the application of this biomarker to other kinds of cancer as recently described in the literature.

A method to obtain purified free light chain monomers and dimers from urine samples of patients with multiple myeloma.

Antibody light chains are synthesized in excess by plasma cells, and this excess can be secreted into biological fluids as dimers or monomers in various proportions. Structural differences between monomers or dimers of free light chains (FLC) can affect their biological functions and possibly their pathogenicity. They also may exhibit differential immune reactivity, perhaps explaining discrepant quantifications when measured by different immunoreagents. Having purified FLC monomers and dimers available can be useful for studying their properties. Here we propose a simple preparatory procedure to purify FLC monomers and dimers from urine samples of patients with plasma cell disorders. Two representative urine samples containing lambda or kappa FLC were loaded into a nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gel strips containing separate monomers and dimers were excised, electroeluted, and the FLC recovered. The FLC were recovered from SDS-PAGE gel in sufficient amounts to be quantified by UV and two automated nephelometric assays immunochemical. The procedure was found to be simple, reproducible, and with a high yield, thus offering the opportunity to compare different assays. Not all urine samples are suitable for this procedure, but this approach allows for the purification of FLC monomers and dimers from many selected urine samples which maintain their oligomeric organization.

Old and New Systemic Immune-Inflammation Indexes Are Associated with Overall Survival of Glioblastoma Patients Treated with Radio-Chemotherapy.

Background. Systemic immunity and inflammation indexes (SI) derived from blood cells have gained increasing attention in clinical oncology as potential biomarkers that are associated with survival. Materials and methods. We tested 12 different SI using blood tests from patients with isocitrate dehydrogenase 1 and 2 wild-type glioblastomas, treated with radio-chemotherapy. The primary endpoint was their overall survival. Results. A total of 77 patients, comprising 43 males and 34 females, with a median age of 64 years (age range 26-84), who were treated between October 2010 and July 2020, were included in the present analysis (approved by a local ethics committee). In the univariate Cox regression analysis, all the indexes except two showed a statistically significant impact on OS. In the multivariate Cox regression analysis, neutrophil × platelet × leukocyte/(lymphocyte × monocyte) (NPW/LM) and neutrophil × platelet × monocyte/lymphocyte (NPM/L) maintained their statistically significant impact value. Conclusions. This univariate analysis confirms the potential of systemic inflammation indexes in patients with glioblastoma, while the multivariate analysis verifies the prognostic value of NPW/LM and NPM/L.

Big and Free Fractions of Gamma-Glutamyltransferase: New Diagnostic Biomarkers for Malignant Mesothelioma?

Malignant pleural mesothelioma (MPM) is a cancer mainly caused by asbestos fiber inhalation, characterized by an extremely long latency and poor prognosis. Recently, researchers have focused on testing the diagnostic ability of several biomarkers. Gamma-Glutamyltransferase (GGT) has been demonstrated to be the sum of several GGT sub-fractions activity, classified based on their molecular weight in big-GGT, medium-GGT, small-GGT, and free-GGT. This work aims to evaluate whether specific GGT fractional enzymatic activity patterns could be helpful in MPM diagnosis. We analyzed blood samples from 175 workers previously exposed to asbestos, 157 non-exposed healthy subjects, and 37 MPM patients through a molecular exclusion chromatographic method. We found a specific profile of GGT fractions activity, significantly associated with MPM, resulting in an increase in b-, m- activity, along with an evident, yet not significant, decrease in f-activity. Receiver-operating characteristic (ROC) analysis showed that the best Area Under Curve (AUC) value resulted from the combined index b/f (0.679, 95% CI: 0.582-0.777). Combining the b-/f-GGT activity with the levels of serum mesothelin-related protein (SMRP; another promising MPM biomarker) improved the diagnostic accuracy, increasing the AUC value to 0.875 (95% CI: 0.807-0.943, p = <0.0001). Since MPM has a specific pattern of GGT enzymatic activity, we could hypothesize that GGT fractions play different specific biochemical roles. The improvement in the diagnostic power given by the combination of these two biomarkers confirms that the strategy of biomarkers combination might be a better approach for MPM diagnosis.

A biomimetic enzyme-linked immunosorbent assay (BELISA) for the analysis of gonadorelin by using molecularly imprinted polymer-coated microplates.

An original biomimetic enzyme-linked immunoassay (BELISA) to target the small peptide hormone gonadorelin is presented. This peptide has been recently listed among the substances banned in sports by the World Antidoping Agency (WADA) since its misuse by male athletes triggers testosterone increase. Hence, in response to this emerging issue in anti-doping controls, we proposed BELISA which involves the growth of a polynorepinephrine (PNE)-based molecularly imprinted polymer (MIP) directly on microwells. PNE, a polydopamine (PDA) analog, has recently displayed impressive performances when it was exploited for MIP preparation, giving even better results than PDA. Gonadorelin quantification was accomplished via a colorimetric indirect competitive bioassay involving the competition between biotinylated gonadorelin linked to the signal reporter and the unlabeled analyte. These compete for the same MIP binding sites resulting in an inverse correlation between gonadorelin concentration and the output color signal (λ = 450 nm). A detection limit of 277 pmol L-1 was achieved with very good reproducibility in standard solutions (avCV% = 4.07%) and in urine samples (avCV% = 5.24%). The selectivity of the assay resulted adequate for biological specimens and non-specific control peptides. In addition, the analytical figures of merit were successfully validated by mass spectrometry, the reference anti-doping benchtop platform for the analyte. BELISA was aimed to open real perspectives for PNE-based MIPs as alternatives to antibodies, especially when the target analyte is a poorly or non-immunogenic small molecule, such as gonadorelin. Biomimetic enzyme-linked immunosorbent assay (BELISA).

MS-based targeted profiling of oxylipins in COVID-19: A new insight into inflammation regulation.

The key role of inflammation in COVID-19 induced many authors to study the cytokine storm, whereas the role of other inflammatory mediators such as oxylipins is still poorly understood. IMPRECOVID was a monocentric retrospective observational pilot study with COVID-19 related pneumonia patients (n = 52) admitted to Pisa University Hospital between March and April 2020. Our MS-based analytical platform permitted the simultaneous determination of sixty plasma oxylipins in a single run at ppt levels for a comprehensive characterisation of the inflammatory cascade in COVID-19 patients. The datasets containing oxylipin and cytokine plasma levels were analysed by principal component analysis (PCA), computation of Fisher's canonical variable, and a multivariate receiver operating characteristic (ROC) curve. Differently from cytokines, the panel of oxylipins clearly differentiated samples collected in COVID-19 wards (n = 43) and Intensive Care Units (ICUs) (n = 27), as shown by the PCA and the multivariate ROC curve with a resulting AUC equal to 0.92. ICU patients showed lower (down to two orders of magnitude) plasma concentrations of anti-inflammatory and pro-resolving lipid mediators, suggesting an impaired inflammation response as part of a prolonged and unsolvable pro-inflammatory status. In conclusion, our targeted oxylipidomics platform helped shedding new light in this field. Targeting the lipid mediator class switching is extremely important for a timely picture of a patient's ability to respond to the viral attack. A prediction model exploiting selected lipid mediators as biomarkers seems to have good chances to classify patients at risk of severe COVID-19.

Big gamma-glutamyltransferase is associated with epicardial fat volume and cardiovascular outcome in the general population.

AIMS: Gamma-glutamyltransferase (GGT) has been recognized as a cardiovascular risk factor, and its highest molecular weight fraction [big GGT (b-GGT)] is found in vulnerable atherosclerotic plaques. We explored the relationship between b-GGT, computed tomography findings, and long-term outcomes in the general population. METHODS AND RESULTS: Between May 2010 and October 2011, subjects aged 45-75 years living in a Tuscan city and without known cardiac disease were screened. The primary endpoint was a composite of cardiovascular death or acute coronary syndrome requiring urgent coronary revascularization. Gamma-glutamyltransferase fractions were available in 898 subjects [median age 65 years (25th-75th percentile 55-70), 46% men]. Median plasma GGT was 20 IU (15-29), and b-GGT was 2.28 (1.28-4.17). Coronary artery calcium (CAC) score values were 0 (0-60), and the volume of pro-atherogenic epicardial fat was 155 mL (114-204). In a model including age, sex, low-density lipoprotein (LDL) cholesterol, current or previous smoking status, hypertension, diabetes, obesity, b-GGT independently predicted epicardial fat volume (EFV) (r = 0.162, P < 0.001), but not CAC (P = 0.198). Over a 10.3-year follow-up (9.6-10.8), 27 subjects (3%) experienced the primary endpoint. We evaluated couples of variables including b-GGT and a cardiovascular risk factor, CAC or EFV. Big GGT yielded independent prognostic significance from age, LDL cholesterol, current or previous smoking status, hypertension, diabetes, obesity, but not CAC or EFV. Conversely, GGT predicted the primary endpoint even independently from CAC and EFV. CONCLUSION: Big GGT seemed at least as predictive as the commonly available GGT assay; therefore, the need for b-GGT rather than GGT measurement should be carefully examined.

Signal Enhancement in Oriented Immunosorbent Assays: A Balance between Accessibility of Antigen Binding Sites and Avidity.

The sensitivity of immunoassays was reported to be increased by the orientation of antibodies. We investigated how the size and valence of antigens and orientation and valence of antibodies contribute to the analytical sensitivity of ELISA. Antigens differing in size and number of epitopes were compared using oriented and non-oriented ELISAs: the orientation of antibodies was obtained coating half-fragment antibodies on maleimide microplates, while, in non-oriented ELISA, whole antibodies were randomly physisorbed. The oriented assay performed better than the non-oriented one at each concentration (0.4-3.3 ng/mL) of a small monomeric antigen (cardiac Troponin I, 24 kDa, Rh 3 nm). No significant differences were observed with a large monovalent antigen (prostate-specific antigen-alpha(1) antichymotrypsin, 90 kDa, Rh > 3 nm), since its steric hindrance overcame the increased availability of antigen binding sites given by orientation. Large multivalent antigens (ferritin, 280 kDa, Rh 6 nm; α-fetoprotein, >70 kDa, Rh > 3.3 nm) performed better in non-oriented assays. In this case, the repeated epitopes on the surface of the antigens favored the engagement of both antigen binding sites of the whole IgG, thus suggesting that avidity represented the leading force in this experimental setting. In conclusion, the design of high-sensitivity ELISAs should consider the dimension and valency of antigens in addition to the affinity and avidity of antibodies.

Determination and stability of N-terminal pro-brain natriuretic peptide in saliva samples for monitoring heart failure.

Heart failure (HF) is the main cause of mortality worldwide, particularly in the elderly. N-terminal pro-brain natriuretic peptide (NT-proBNP) is the gold standard biomarker for HF diagnosis and therapy monitoring. It is determined in blood samples by the immunochemical methods generally adopted by most laboratories. Saliva analysis is a powerful tool for clinical applications, mainly due to its non-invasive and less risky sampling. This study describes a validated analytical procedure for NT-proBNP determination in saliva samples using a commercial Enzyme-Linked Immuno-Sorbent Assay. Linearity, matrix effect, sensitivity, recovery and assay-precision were evaluated. The analytical approach showed a linear behaviour of the signal throughout the concentrations tested, with a minimum detectable dose of 1 pg/mL, a satisfactory NT-proBNP recovery (95-110%), and acceptable precision (coefficient of variation ≤ 10%). Short-term (3 weeks) and long-term (5 months) stability of NT-proBNP in saliva samples under the storage conditions most frequently used in clinical laboratories (4, - 20, and - 80 °C) was also investigated and showed that the optimal storage conditions were at - 20 °C for up to 2.5 months. Finally, the method was tested for the determination of NT-proBNP in saliva samples collected from ten hospitalized acute HF patients. Preliminary results indicate a decrease in NT-proBNP in saliva from admission to discharge, thus suggesting that this procedure is an effective saliva-based point-of-care device for HF monitoring.

C-C motive chemokine ligand 2 and thromboinflammation in COVID-19-associated pneumonia: A retrospective study.

PURPOSE: A derangement of the coagulation process and thromboinflammatory events has emerged as pathologic characteristics of severe COVID-19, characterized by severe respiratory failure. CC motive chemokine ligand 2 (CCL2), a chemokine originally described as a chemotactic agent for monocytes, is involved in inflammation, coagulation activation and neoangiogenesis. We investigated the association of CCL2 levels with coagulation derangement and respiratory impairment in patients with COVID-19. METHODS: We retrospectively evaluated 281 patients admitted to two hospitals in Italy with COVID-19. Among them, CCL2 values were compared in different groups (identified according to D-dimer levels and the lowest PaO2/FiO2 recorded during hospital stay, P/Fnadir) by Jonckheere-Terpstra tests; linear regression analysis was used to analyse the relationship between CCL2 and P/Fnadir. We performed Mann-Whitney test and Kaplan-Meier curves to investigate the role of CCL2 according to different clinical outcomes (survival and endotracheal intubation [ETI]). RESULTS: CCL2 levels were progressively higher in patients with increasing D-dimer levels and with worse gas exchange impairment; there was a statistically significant linear correlation between log CCL2 and log P/Fnadir. CCL2 levels were significantly higher in patients with unfavourable clinical outcomes; Kaplan-Meier curves for the composite outcome death and/or need for ETI showed a significantly worse prognosis for patients with higher (> median) CCL2 levels. CONCLUSIONS: CCL2 correlates with both indices of activation of the coagulation cascade and respiratory impairment severity, which are likely closely related in COVID-19 pathology, thus suggesting that CCL2 could be involved in the thromboinflammatory events characterizing this disease.

Data about performances of whole and monovalent half-fragments antibodies in immunosorbent assays.

The data here presented are related to the research article entitled "Sensitivity and reproducibility enhancement in enzyme immunosorbent assays based on half fragment antibodies" [1] aimed to compare the performance in ELISA of whole antibodies and their corresponding monovalent half-fragments obtained by reduction. Half-fragment antibodies represent an interesting method to orient antibodies in high-sensitive immunoassays taking advantage of the free sulfhydryl groups of the hinge region [2], [3], [4] that allow their oriented binding on maleimide functionalized microplates. Data here presented describe the contribution of both chemical reduction and orientation on the antigen binding capacity of whole and half-fragments antibodies. For this purpose, monoclonal anti-horseradish peroxidase (anti-HRP) or monoclonal anti-fPSA antibodies, and their respective half-fragments, were coated on polystyrene or maleimide functionalized microplates. The antigen binding capability was analyzed by in-house enzyme linked immunosorbent assays. These data would be used for further studies on the development of oriented immunoassays based on half fragment antibodies.

Sensitivity and reproducibility enhancement in enzyme immunosorbent assays based on half fragment antibodies.

The free sulfhydryl groups of the hinge region of monovalent antibody fragments (rIgG) allow the orientation of rIgG on functionalized surfaces in immunosensors. To evaluate the contribution of reduction and orientation on signal enhancement we compared the performance of whole antibodies and their rIgG in ELISA performed on polystyrene or maleimide-functionalized microplates. Monoclonal anti-horseradish peroxidase (anti-HRP) and monoclonal anti-fPSA antibodies (1 mg/mL) were reduced with 2-mercaptoethylamine (53 mM). Western blot confirmed the presence of rIgG as a band at 75 kDa, detectable only by anti-heavy chain but not by anti-light chain antibodies, suggesting a possible folding rearrangement. Using anti-HRP we confirmed the retention of the antigen binding capacity of rIgG. Moreover, we observed a signal enhancement for rIgG even if randomly absorbed on polystyrene [linear regression slope (95%CI): rIgG 0.524 (0.434-0.614), IgG 0.370 (0.430-0.399); P = 0.0016] suggesting that chemical reduction might affect the antigen binding capacity of antibodies. ELISA with anti-fPSA rIgG coated on polystyrene confirmed these observations. Oriented anti-fPSA rIgG on a maleimide surface showed comparable signals to the assay performed on polystyrene for each analyzed concentration of antigen (PANOVA = 0.1980), anyway, with a significant improvement of the repeatability likely providing a more homogeneous capturing surface (SD rIgGmaleimide-rIgGpolystirene: fPSA 0.725 ng/mL:0.74-2.89; 1.45 ng/mL:1.56-8.69; 3.625 ng/mL:3.52-15.03; 7.25 ng/mL:7.78-18.44).

Lack of neutralizing activity in nonconvalescent sera, regardless of ABO blood group and anti-A isoagglutinin titer.

Background: Several ABO blood groups have been associated with the likelihood of infection, severity, and/or outcome of COVID-19 in hospitalized cohorts, raising the hypothesis that anti-A isoagglutinins in non-A-group recipients could act as neutralizing antibodies against SARS-CoV-2. Materials and methods: We run live virus neutralization tests using sera from 58 SARS-CoV-2 seronegative blood donors (27 O-group and 31 A-group) negatives for SARS-CoV-2 IgG to investigate what degree of neutralizing activity could be detected in their sera and eventual correlation with anti-A isoagglutinin titers. Results: We could not find clinically relevant neutralizing activity in any blood group, regardless of anti-isoagglutinin titer. Discussion: Our findings suggest that mechanisms other than neutralization explain the differences in outcomes from COVID19 seen in different ABO blood groups.

Is There a Crucial Link Between Vitamin D Status and Inflammatory Response in Patients With COVID-19?

Background: Hypovitaminosis D has been suggested to play a possible role in coronavirus disease 2019 (COVID-19) infection. Methods: The aim of this study is to analyze the relationship between vitamin D status and a biochemical panel of inflammatory markers in a cohort of patients with COVID-19. A secondary endpoint was to evaluate the correlation between 25OHD levels and the severity of the disease. Ninety-three consecutive patients with COVID-19-related pneumonia were evaluated from March to May 2020 in two hospital units in Pisa, in whom biochemical inflammatory markers, 25OHD levels, P/F ratio at nadir during hospitalization, and complete clinical data were available. Results: Sixty-five percent of patients presented hypovitaminosis D (25OHD ≤ 20 ng/ml) and showed significantly higher IL-6 [20.8 (10.9-45.6) vs. 12.9 (8.7-21.1) pg/ml, p = 0.02], CRP [10.7 (4.2-19.2) vs. 5.9 (1.6-8.1) mg/dl, p = 0.003], TNF-α [8.9 (6.0-14.8) vs. 4.4 (1.5-10.6) pg/ml, p = 0.01], D-dimer [0.53 (0.25-0.72) vs. 0.22 (0.17-0.35) mg/l, p = 0.002], and IL-10 [3.7 (1.8-6.9) vs. 2.3 (0.5-5.8) pg/ml, p = 0.03]. A significant inverse correlation was found between 25OHD and all these markers, even adjusted for age and sex. Hypovitaminosis D was prevalent in patients with severe ARDS, compared with the other groups (75% vs. 68% vs. 55%, p < 0.001), and 25OHD levels were lower in non-survivor patients. Conclusions: The relationship between 25OHD levels and inflammatory markers suggests that vitamin D status needs to be taken into account in the management of these patients. If vitamin D is a marker of poor prognosis or a possible risk factor with beneficial effects from supplementation, this still needs to be elucidated.

Ruxolitinib Rapidly Reduces Acute Respiratory Distress Syndrome in COVID-19 Disease. Analysis of Data Collection From RESPIRE Protocol.

Background: The Coronavirus disease (COVID-19) pandemic is causing millions of infections and hundreds of thousands of deaths worldwide. Cumulative clinical and laboratory evidence suggest that a subset of patients with severe COVID-19 may develop a cytokine storm syndrome during the course of the disease, with severe respiratory impairment requiring ventilatory support. One field of research nowadays is to identify and treat viral-induced hyperinflammation with drugs used in other clinical conditions characterized by an hyperinflammation status. These drugs might help to reduce COVID19 mortality. Methods: Ruxolitinib, a JAK1 and JAK2 inhibitor, has been successfully used to treat severe immune-mediated diseases, such as graft vs. host disease and Hemophagocytic lymphohistiocytosis. We used ruxolitinib in 18 patients with clinically progressive COVID-19 related acute respiratory distress syndrome, with a primary endpoint to rapidly reduce the degree of respiratory impairment and as a secondary endpoint to rapidly restore the PaO2/FiO2 ratio, as an evaluation of clinical status, and monitoring of drug related Adverse Events. Parameters of inflammation responses and organ functions were assessed and monitored. The treatment plan was ruxolitinib 20 mg bid for the first 48 h and subsequent two-step de-escalation at 10 mg bid and 5 mg bid for a maximum of 14 days of treatment. Results: Our data collection shows a rapid clinical response with no evolution from non-invasive ventilation to mechanical ventilation in 16/18 patients and no response in two patients (overall response rate-ORR 89%). Already after 48 h of ruxolitinib treatment 16/18 patients showed evident clinical improvement, and after 7 days of treatment 11/18 patients showed fully recovered respiratory function (pO2 > 98% in spontaneous breathing), 4/18 patients had minimal oxygen requirement (2-4 L/m), 1/18 patient showed stable disease, and 2/18 patient showed progressive disease. After 14 days, 16/18 patients showed complete recovery of respiratory function (ORR 89%). Compliance to ruxolitinib planned treatment was 100% and no serious adverse event was recorded. In our case series of 18 critically ill patients with COVID-19 and ARDS, administration of ruxolitinib resulted in a clinical improvement that concurred to modify the standard course of disease. Ruxolitinib can be a therapeutic option for patients with respiratory insufficiency in COVID-19 related ARDS. RESPIRE Study (Ruxolitinib for the treatment of acute rESPIratory distREss syndrome, ClinicalTrials.gov Identifier: NCT04361903).

Sweat chloride assay by inductively coupled plasma mass spectrometry: a confirmation test for cystic fibrosis diagnosis.

The current guidelines for sweat chloride analysis identify the procedures for sweat collection, but not for chloride assay, which is usually performed by methods originally not aiming at the low concentrations of chloride found in sweat. To overcome this limitation, we set up, characterized, and adopted an original inductively coupled plasma mass spectrometry (ICP-MS) method for sweat chloride determination, which was designed for its easy use in a clinical laboratory. The method was linear in the range 8.5E-3 to 272.0E-3 mM, precision exhibited a relative standard deviation < 6%, and accuracy was in the range 99.7-103.8%. Limit of blank, limit of detection, and limit of quantitation were 2.1 mM, 3.2 mM, and 7.0 mM, respectively, which correspond to real concentrations injected into the mass spectrometer of 3.9E-3 mM for LOD and 8.5E-3 mM for LOQ. At first, the method was tested on 50 healthy volunteers who exhibited a mean chloride concentration of 15.7 mM (25-75th percentile 10.1-19.3 mM, range 2.8-37.4 mM); then, it was used to investigate two patients with suspected cystic fibrosis, who exhibited sweat chloride values of 65.6 mM and 81.2 mM, respectively. Moreover, the method was cross-validated by assaying 50 samples with chloride concentration values in the range 10-131 mM, by both ICP-MS and coulometric titration, which is the technology officially used in Tuscany for cystic fibrosis newborn screening. The reference analytical performances and the relatively low cost of ICP-MS, accompanied by the advantageous cost of a single sweat chloride assay, make this technology the best candidate to provide a top reference method for the quantification of chloride in sweat. The method that we propose was optimized and validated for sweat samples ≥ 75 mg, which is the minimum amount requested by the international protocols. However, the method sensitivity and, in addition, the possibility to reduce the sample dilution factor, make possible the quantification of chloride even in samples weighting < 75 mg that are discarded according to the current guidelines. Graphical abstract.

Free light chain UV quantification compared with immunochemical measurement: How dimers and monomers may influence the results.

Serum κ and λ free light chain (FLC) levels are important for the management of plasma cell disorders. Immunochemical measurements on automated platforms with different reagents occasionally return different results that make them not interchangeable. The reasons for this behaviour are not clear and it is not known which result is the most accurate. The aim of the study is to quantify naturally occurring FLCs with a reference method (UV absorbance) in a sample devoid of other sources of UV absorbance. This was possible on a particular urine sample containing only lambda FLC proteins, dialyzed to clear it from low molecular weight UV absorbing compounds. The sample was submitted to Fast Protein Liquid Chromatography separation with a size-exclusion column in order to separate the FLC monomers and dimers. FLCs were also measured with the Freelite and N Latex FLC methods and the results were compared. The results demonstrated that the amount of FLC calculated on the basis of UV absorbance was overestimated by both immunochemical methods, and that the amount measured by the two reagents was affected by the different proportions of dimers or monomers. The present findings may be useful for the comprehension of the immunochemical measurement of FLC.

Inter-assay variability in automated serum free light chain assays and their use in the clinical laboratory.

Serum κ and λ free light chain levels are markers of plasma cell proliferation, and their measurements have been included in recent guidelines by the International Myeloma Working Group for the management of patients with plasma cellular dyscrasias. Five in vitro diagnostic methods for the immunochemical quantification of serum free light chains (FLC) are available, three based on polyclonal antibodies (Freelite®, The Binding Site; FLC ELISA κ and λ, Sebia; human κ and λ FLC, Diazyme Laboratories) and two on monoclonal antibodies (N Latex FLC, Siemens Healthineers; Seralite®, Sebia). Several studies have shown that these methods cannot be used interchangeably for the follow-up of patients because measured κ and λ FLC concentrations may differ significantly, especially at high levels. Because no international reference material for the measurement of FLC is available, it is not possible to establish which method is the most accurate. For this reason, knowledge about the analytical and diagnostic performances of the assays used is important. The aim of this review is to describe the main analytical features of the κ and λ FLC assays and how they may influence the clinical use of these parameters.