Cell Population Data and Serum Polyclonal Immunoglobulin Free Light Chains in the Assessment of COVID-19 Severity.

Distinguishing between severe and nonsevere COVID-19 to ensure adequate healthcare quality and efficiency is a challenge for the healthcare system. The aim of this study was to assess the usefulness of CBC parameters together with analysis of FLC serum concentration in risk stratification of COVID-19. MATERIALS AND METHODS: CBC was analyzed in 735 COVID ICU, COVID non-ICU, and non-COVID ICU cases. FLC concentration was analyzed in 133 of them. RESULTS: COVID ICU had neutrophils and lymphocytes with the greatest size, granularity, and nucleic acid content. Significant differences in concentrations of κ and λ FLCs were shown between COVID ICU and COVID non-ICU. However, no difference was found in the κ/λ ratio between these groups, and the ratio stayed within the reference value, which indicates the presence of polyclonal FLCs. FLC κ measurement has significant power to distinguish between severe COVID-19 and nonsevere COVID-19 (AUC = 0.7669), with a sensitivity of 86.67% and specificity of 93.33%. The κ coefficients' odds ratio of 3.0401 was estimated. CONCLUSION: It can be concluded that the results obtained from the measure of free light immunoglobulin concentration in serum are useful in distinguishing between severe and nonsevere COVID-19.

Comparison of Sebia Free Light Chain Assay With Freelite Assay for the Clinical Management of Diagnosis, Response, and Relapse Assessment in Multiple Myeloma.

BACKGROUND: Serum free light chain (FLC) measurement has become an important marker for the management of multiple myeloma (MM). However, several analytical challenges remain unresolved. We compared the clinical performances of the Sebia FLC assay in MM to the Freelite assay. PATIENTS AND METHODS: A total of 177 patients from the IFM DFCI 2009 trial were enrolled onto this study, with a total of 368 samples analyzed. At baseline, concordance of the involved to noninvolved FLC ratio (iFLC/niFLC) was evaluated. During therapy, comparison of the disease response assessments according to International Myeloma Working Group criteria was performed. RESULTS: Compared to Freelite, the Sebia FLC assay demonstrated lower results, with a proportional bias with increased values. We demonstrated that the Sebia equivalent of the iFLC/niFLC ratio of 100 was 16. During follow-up, agreement in response assessment was moderate (for light chains MM) to good (for intact immunoglobulin MM). In the context of relapse, the concordance was moderate, but longitudinal follow-up showed a similar kinetics. CONCLUSION: The Sebia FLC assay provides inequivalent absolute results from the Freelite assay. Despite lower absolute FLC values, the kinetics of response and relapse is exactly the same. As with other FLC assays available, follow-up of MM with the same method is advisable.

Circulating free light chain measurement in the diagnosis, prognostic assessment and evaluation of response of AL amyloidosis: comparison of Freelite and N latex FLC assays.

BACKGROUND: The measurement of circulating free light chain (FLC) is essential in the diagnosis, prognostic stratification and evaluation of response to therapy in light chain (AL) amyloidosis. For more than 10 years, this has been done with an immunonephelometric assay based on polyclonal antibodies (Freelite), and cutoffs for staging and response assessment have been validated with this method. Recently, a new assay based on monoclonal antibodies (N latex FLC) has been marketed in Europe. METHODS: We evaluated and compared the clinical performance of the two assays in 426 patients with newly diagnosed AL amyloidosis. RESULTS: We found suboptimal agreement between the two methods, with differences between values obtained with the Freelite and N latex FLC assays increasing with the concentration of clonal FLC. The diagnostic sensitivity of the Freelite (82%) and N latex FLC (84%) assays was similar, and both improved to 98% in combination with serum and urine immunofixation. The concentration of FLC measured with both methods had prognostic significance. Less pronounced decreases in FLC best predicted improved survival with the N latex FLC assay (33% vs. 50%), and there was poor concordance (84%) in discrimination of responders. CONCLUSIONS: The two assays have similar diagnostic and prognostic performance. However, they are not interchangeable, and follow-up should be done with either one. New response criteria are needed for the N latex FLC assay.

Comprehensive Assessment of M-Proteins Using Nanobody Enrichment Coupled to MALDI-TOF Mass Spectrometry.

BACKGROUND: Electrophoretic separation of serum and urine proteins has played a central role in diagnosing and monitoring plasma cell disorders. Despite limitations in resolution and analytical sensitivity, plus the necessity for adjunct methods, protein gel electrophoresis and immunofixation electrophoresis (IFE) remain front-line tests. METHODS: We developed a MALDI mass spectrometry-based assay that was simple to perform, automatable, analytically sensitive, and applicable to analyzing the wide variety of monoclonal proteins (M-proteins) encountered clinically. This assay, called MASS-FIX, used the unique molecular mass signatures of the different Ig isotypes in combination with nanobody immunoenrichment to generate information-rich mass spectra from which M-proteins could be identified, isotyped, and quantified. The performance of MASS-FIX was compared to current gel-based electrophoresis assays. RESULTS: MASS-FIX detected all M-proteins that were detectable by urine or serum protein electrophoresis. In serial dilution studies, MASS-FIX was more analytically sensitive than IFE. For patient samples, MASS-FIX provided the same primary isotype information for 98% of serum M-proteins (n = 152) and 95% of urine M-proteins (n = 55). MASS-FIX accurately quantified M-protein to <1 g/dL, with reduced bias as compared to protein electrophoresis. Intraassay and interassay CVs were <20% across all samples having M-protein concentrations >0.045 g/dL, with the ability to detect M-proteins <0.01 g/dL. In addition, MASS-FIX could simultaneously measure κ:λ light chain ratios for IgG, IgA, and IgM. Retrospective serial monitoring of patients with myeloma posttreatment demonstrated that MASS-FIX provided equivalent quantitative information to either protein electrophoresis or the Hevylite(™) assay. CONCLUSIONS: MASS-FIX can advance how plasma cell disorders are screened, diagnosed, and monitored.

Regional myocardial microvascular dysfunction in cardiac amyloid light-chain amyloidosis: assessment with 3T cardiovascular magnetic resonance.

BACKGROUND: Coronary microvascular dysfunction is highly prevalent in patients with amyloid light-chain (AL) cardiac amyloidosis (AL-CA). The aim of this study was to clarify the feasibility of first-pass perfusion imaging using 3 T cardiovascular magnetic resonance (CMR) for evaluating the difference in left ventricular (LV) regional myocardial microvascular function among normal subjects and in patients with AL-CA. The amyloidosis patients were classified into those with impaired systolic function [LV ejection fraction (LVEF) < 50 %] and those with preserved systolic function. METHODS: In total, 32 patients with biopsy-proven AL-CA, including 11 AL-CA patients with systolic dysfunction, 21 AL-CA patients with preserved systolic function, and 25 healthy subjects, underwent CMR examination. LV regional myocardial perfusion parameters included upslope, time to maximum signal intensity (TTM) and max signal intensity (MaxSI) were compared among the three patient groups. Receiver operating characteristic analysis was performed to determine whether perfusion parameters could be used in discriminating regional myocardial microvascularity between AL-CA patients and normal subjects. RESULTS: The patients with AL-CA had significantly reduced first-pass perfusion upslope and MaxSI, and increased TTM compared with the normal subjects (all P < 0.01). Compared with the patients with AL-CA and preserved LVEF, the patients with AL-CA and impaired systolic function had a longer TTM in the basal (47.05 ± 16.59 vs. 39.68 ± 19.11; P = 0.002) and mid-ventricular (44.61 ± 16.34 vs. 37.74 ± 18.25; P = 0.002) segments; lower upslope in the basal (2.41 ± 1.32 vs. 3.60 ± 1.68; P < 0.0001), mid-ventricular (2.82 ± 1.34 vs. 4.15 ± 2.02; P < 0.0001), and apical (3.71 ± 1.38 vs. 4.97 ± 2.55; P = 0.004) segments; and lower MaxSI (31.67 ± 15.23 vs. 37.96 ± 11.15; P < 0.0001) in the basal segment. The ROC curve analysis revealed that the first-pass upslope, TTM, and MaxSI may be used as indicators for differentiating microcirculation between AL-CA patients with preserved or impaired systolic function and normal subjects. CONCLUSIONS: The differences in LV regional myocardial microvascular function among normal subjects, AL-CA patients with systolic dysfunction, and AL-CA patients with preserved systolic function can be monitored using first-pass perfusion CMR.

Development of scoring functions for antibody sequence assessment and optimization.

Antibody development is still associated with substantial risks and difficulties as single mutations can radically change molecule properties like thermodynamic stability, solubility or viscosity. Since antibody generation methodologies cannot select and optimize for molecule properties which are important for biotechnological applications, careful sequence analysis and optimization is necessary to develop antibodies that fulfil the ambitious requirements of future drugs. While efforts to grab the physical principles of undesired molecule properties from the very bottom are becoming increasingly powerful, the wealth of publically available antibody sequences provides an alternative way to develop early assessment strategies for antibodies using a statistical approach which is the objective of this paper. Here, publically available sequences were used to develop heuristic potentials for the framework regions of heavy and light chains of antibodies of human and murine origin. The potentials take into account position dependent probabilities of individual amino acids but also conditional probabilities which are inevitable for sequence assessment and optimization. It is shown that the potentials derived from human sequences clearly distinguish between human sequences and sequences from mice and, hence, can be used as a measure of humaness which compares a given sequence with the phenotypic pool of human sequences instead of comparing sequence identities to germline genes. Following this line, it is demonstrated that, using the developed potentials, humanization of an antibody can be described as a simple mathematical optimization problem and that the in-silico generated framework variants closely resemble native sequences in terms of predicted immunogenicity.

Appearance of neoantigen in mouse IgG1 upon reduction of interchain disulfide bridges: assessment of local and general conformational rearrangements using monoclonal antibody and small-angle X-ray scattering.

Mouse hybridoma antibody E5D2 reacting with murine mono- and polyclonal IgG1 has been produced. MonAb E5D2 recognizes the antigenic determinant (epitope) buried in intact IgG1 and expressed upon mild reduction of interchain S-S bridges. Neither H nor L chains alone maintain epitope E5D2. Reassociation of gamma 1 chains (H chains of IgG1) with L chains results in complete restoration of this antigenic determinant. The data strongly suggest that epitope E5D2 depends on the quaternary structure of IgG1. The epitope is also expressed by reduced F(ab)2 fragment of IgG1 but is not connected with its antigen binding site. The likely localization of the epitope E5D2 is the interface between CH and CL domains. The second produced monAb F6C2 reacts with CH1-CL region of reduced mouse IgG2. Small-angle X-ray scattering experiments have demonstrated pronounced decrease of the radius of gyration of reduced IgG1 as compared to the intact one. This indicates general conformational changes of IgG1 molecule following mild reduction of Fab region S-S groups. Epitope E5D2 is the first quaternary antigenic subclass specific determinant described for C the region of mouse IgG. Thus, serologic expression of epitope E5D2 reveals precise conformational perturbations of small area near reduced S-S bridges while small-angle scattering demonstrates accompanying general transformation of IgG structure.