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Background: Cerebrospinal fluid (CSF) kappa free light chain (κFLC) measures gained increasing interest as diagnostic markers in multiple sclerosis (MS). However, the lack of studies comparing assay-dependent diagnostic cutoff values hinders their use in clinical practice. Additionally, the optimal κFLC parameter for identifying MS remains a subject of ongoing debate. Objectives: The aim of this study was to compare same-sample diagnostic accuracies of the κFLC index, kIgG index, CSF κFLC/IgG ratio, and isolated CSF κFLC (iCSF-κFLC) between two reference centers using different methods. Methods: Paired serum and CSF samples were analyzed for κFLC and albumin concentrations by Freelite®-Optilite (Sint-Jan Bruges hospital) and N Latex®-BNII (Ghent University hospital). Diagnostic performance to differentiate MS from controls was assessed using ROC curve analysis. Results: A total of 263 participants were included (MS, n = 80). Optimal diagnostic cutoff values for the κFLC index (Freelite®-Optilite: 7.7; N Latex®-BNII: 4.71), kIgG index (Freelite®-Optilite: 14.15, N Latex®-BNII: 12.19), and CSF κFLC/IgG ratio (Freelite®-Optilite: 2.27; N Latex®-BNII: 1.44) differed between the two methods. Sensitivities related to optimal cutoff values were 89.9% (Freelite®-Optilite) versus 94.6% (N Latex®-BNII) for the κFLC index, 91% (Freelite®-Optilite) versus 92.2% (N Latex®-BNII) for the kIgG index, and 81.3% (Freelite®-Optilite) versus 91.4% (N Latex®-BNII) for the CSF κFLC/IgG ratio. However, for iCSF-κFLC, optimal diagnostic cutoff values (0.36 mg/L) and related specificities (81.8%) were identical with a related diagnostic sensitivity of 89.9% for Freelite®-Optilite and 90.5% for N Latex®-BNII. The diagnostic performance of the κFLC index [area under the curve (AUC) Freelite®-Optilite: 0.924; N Latex®-BNII: 0.962] and kIgG index (AUC Freelite®-Optilite: 0.929;N Latex®-BNII: 0.961) was superior compared to CSF oligoclonal bands (AUC: 0.898, sensitivity: 83.8%, specificity: 95.9%). Conclusions: The κFLC index and the kIgG index seem to be excellent markers for identifying MS, irrespective of the method used for κFLC quantification. Based on the AUC, they appear to be the measures of choice. For all measures, optimal cutoff values differed between methods except for iCSF-κFLC. iCSF-κFLC might therefore serve as a method-independent, more cost-efficient, initial screening measure for MS. These findings are particularly relevant for clinical practice given the potential future implementation of intrathecal κFLC synthesis in MS diagnostic criteria and for future multicentre studies pooling data on κFLC measures. [ABSTRACT FROM AUTHOR]

Detection of antinuclear antibodies (ANA) by indirect immunofluorescence assay using HEp-2 cells (HEp-2 IFA) is used to screen for various autoimmune diseases. HEp-2 IFA suffers from variability, which hampers harmonization. A questionnaire was developed to collect information on HEp-2 IFA methodology, computer-assisted diagnosis (CAD) systems, training, inter-observer variability, quality assessment, reagent lot change control, and method verification. The questionnaire was distributed to laboratories by Sciensano (Belgium), national EASI groups (Italy, Croatia, Portugal, Estonia, Greece) and ICAP (worldwide). Answers were obtained by 414 laboratories. The results were analysed in the framework of the recent EFLM/EASI/ICAP ANA recommendations (companion paper). Laboratories used either HEp-2, HEp-2000, or HEp-20-10 cells and most laboratories (80%) applied the same screening dilution for children and adults. The conjugate used varied between laboratories [IgG-specific (in 57% of laboratories) vs. polyvalent]. Sixty-nine percent of CAD users reviewed the automatic nuclear pattern and 53% of CAD users did not fully exploit the fluorescence intensity for quality assurance. Internal quality control was performed by 96% of the laboratories, in 52% of the laboratories only with strongly positive samples. Interobserver variation was controlled by 79% of the laboratories. Limited lot-to-lot evaluation was performed by 68% of the laboratories. Method verification was done by 80% of the respondents. Even though many laboratories embrace high-quality HEp-2 IFA, substantial differences in how HEp-2 IFA is performed and controlled remain. Acting according to the EFLM/EASI/ICAP ANA recommendations can improve the global performance and quality of HEp-2 IFA and nurture harmonization. [ABSTRACT FROM AUTHOR]

Antinuclear antibodies (ANA) are important for the diagnosis of various autoimmune diseases. ANA are usually detected by indirect immunofluorescence assay (IFA) using HEp-2 cells (HEp-2 IFA). There are many variables influencing HEp-2 IFA results, such as subjective visual reading, serum screening dilution, substrate manufacturing, microscope components and conjugate. Newer developments on ANA testing that offer novel features adopted by some clinical laboratories include automated computer-assisted diagnosis (CAD) systems and solid phase assays (SPA). A group of experts reviewed current literature and established recommendations on methodological aspects of ANA testing. This process was supported by a two round Delphi exercise. International expert groups that participated in this initiative included (i) the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group "Autoimmunity Testing"; (ii) the European Autoimmune Standardization Initiative (EASI); and (iii) the International Consensus on ANA Patterns (ICAP). In total, 35 recommendations/statements related to (i) ANA testing and reporting by HEp-2 IFA; (ii) HEp-2 IFA methodological aspects including substrate/conjugate selection and the application of CAD systems; (iii) quality assurance; (iv) HEp-2 IFA validation/verification approaches and (v) SPA were formulated. Globally, 95% of all submitted scores in the final Delphi round were above 6 (moderately agree, agree or strongly agree) and 85% above 7 (agree and strongly agree), indicating strong international support for the proposed recommendations. These recommendations are an important step to achieve high quality ANA testing. [ABSTRACT FROM AUTHOR]

Serum free light chain (sFLC) measurements have inherent analytical limitations impacting sFLC clinical interpretation. We evaluated analytical and diagnostic performance of three polyclonal sFLC assays on four analytical platforms. sFLC concentration was measured using Diazyme FLC assays (Diazyme) on cobas c501/c503 analyzer (Roche); Freelite assays (The Binding Site) on Optilite analyzer (The Binding Site) and cobas c501 analyzer and Sebia FLC ELISA assays (Sebia) on AP22 ELITE analyzer (DAS). Imprecision, linearity, method comparison vs. Freelite/Optilite, antigen excess detection and reference value verification were assessed. Diagnostic performance was compared on 120 serum samples and on follow-up samples of five patients with κ and λ monoclonal gammopathy. Method comparison showed excellent correlation with Freelite/Optilite method for all assays. A large proportional negative bias was shown for both Sebia κ and λ ELISA and a significant positive proportional bias for λ in the low (<10 mg/L) Freelite/cobas c501 method. Clinically relevant underestimation of κ sFLC levels due to antigen excess was shown for 7% of each Diazyme/cobas application and for 11 and 32.1% of λ sFLC assay of respectively Diazyme/cobas and Sebia/AP22. sFLC reference values revealed application specific. Cohen's κ values were (very) good for κ sFLC but only moderate to good for λ sFLC. In 4/10 follow-up patients, significant differences in clinical interpretation between sFLC assays were noticed. Important analytical limitations remain for all sFLC applications. Differences in reference values and diagnostic performance hamper interchangeability of sFLC assays. Assay specific sFLC decision guidelines are warranted. [ABSTRACT FROM AUTHOR]

Keywords: antineutrophil cytoplasmic antibodies (ANCA); vasculitis; harmonization; SARS-CoV-2 antibody EN antineutrophil cytoplasmic antibodies (ANCA) vasculitis harmonization SARS-CoV-2 antibody e35 e39 5 01/21/21 20210201 NES 210201 To the Editor, Antineutrophil cytoplasmic antibodies (ANCA) are valuable laboratory markers to support the diagnosis of ANCAassociated vasculitis (AAV). Depending on the assay, 52-66% of AAV patients had a test result with an LR >30 and 14-27% of AAV patients had a test result with an LR between 10 and 30 (Figure 2, Panel B). [Extracted from the article]

To the Editor, Free light chains (FLCs) are valuable laboratory markers to support the diagnosis, prognosis and follow-up of patients with monoclonal gammopathies. / ratio by N-Latex and Freelite in MGUS and MM: The / ratio was determined in 525 samples from 255 MGUS patients (described in [[8]]) and the results were classified as normal or abnormal. When the minimum and maximum / ratio determined in the 282 controls in this study was used, then 39.4% of the samples from MGUS patients in this study had an abnormal / ratio with both assays, 9% with only Freelite, and 7.6% with only N-Latex. [Extracted from the article]

The article examines the analytical performance of NOVA View, a digital indirect immunofluorescence (IIF) microscope, for automated antinuclear antibodies (ANA) detection. Topics discussed include a pattern-specific association found between light-intensity units (LIU) and single well titer (SWT), the link between the LIU and the end point titer (ET), and data which show that SWT on isolated nuclear patterns tended to overestimate the titer in a substantial fraction of samples.

The article presents a study on antigen excess detection by automated assays for free light chains. Topics discussed include role of free light chains (FLCs) in diagnosis, prognosis and follow-up of patients with monoclonal gammopathy; information on antigen excess detection system; and the issues relating to the multiple myeloma, a cancer that forms in a type of white blood cell.

Background Free light chains (FLC) are useful biomarkers for diagnosis and follow-up of plasma cell disorders. FLC quantification is encumbered by non-linearity and antigen excess (> 4-fold difference between results obtained at the 2000- and 100-fold dilution). Methods FLC concentration was measured with Freelite® reagents on the BNII, using 100- and 2000-fold dilutions in 3645 samples. Samples displaying antigen excess were re-measured at the 2000- and/or 400-fold dilution. Carryover was evaluated by tracing samples to cuvettes and by measuring a normal sample in cuvettes that previously contained samples with a high FLC concentration. Results Antigen excess occurred in 0.93% of samples for κ and in 0.55% of samples for λ . In 81.5% of the cases, it could not be confirmed by a re-analysis of a 2000-fold and/or a 400-fold diluted sample. Real antigen excess was documented in 0.25% and 0.03% of the samples for κ and λ FLC, respectively. In the low concentration range (2000-fold dilution), imprecision was high. False antigen excess was reduced by batch analysis, introducing cleaning and rinsing procedures and using the 400-fold dilution. No antigen excess was detected in samples with normal FLC concentrations. Conclusion Falsely high results occur by imprecision in the low concentration range and/or by carryover in cuvettes. [ABSTRACT FROM AUTHOR]

We have recently shown that Toxoplasma gondii tachyzoites grown in in vitro culture can bind unspecific immunoglobulin (Ig) through their Fc moiety. We show now that Fc receptors are also present on T. gondii within the host animal, and that intraperitoneal parasites in immunocompetent mice are saturated with unspecific Ig. We have also investigated the effect of the parasite's Fc receptor on the interaction of tachyzoites with mammalian cells, using the Vero cell line as a model for nonphagocytic host cells and murine peritoneal macrophages in primary culture as a model for phagocytic cells. Coating of tachyzoites with parasite-unrelated Ig did not enhance their invasive capacity in either target cell type, but slightly decreased the parasite proliferation. Moreover, phagocytosis by macrophages was increased by approximately 50% when parasites were coated with unspecific Ig. These results indicate that the Fc receptor on T. gondii affects the balance between invasion and phagocytosis in a way that is detrimental to the parasites. [ABSTRACT FROM AUTHOR]

Human leukocyte Ag-G, a tolerogenic molecule that acts on cells of both innate and adaptive immunity, plays an important role in tumor progression, transplantation, placentation, as well as the protection of the allogeneic fetus from the maternal immune system. We investigated HLA-G mRNA and protein expression in human embryonic stem cells (hESC) derived from the inner cell mass (ICM) of blastocysts. hESC self-renew indefinitely in culture while maintaining pluripotency, providing an unlimited source of cells for therapy. HLA-G mRNA was present in early and late passage hESC, as assessed by real time RT-PCR. Protein expression was demonstrated by flow cytometry, immunocytochemistry, and ELISA on an hESC extract. Binding of HLA-G with its ILT2 receptor demonstrated the functional active status. To verify this finding in a physiologically relevant setting, HLA-G protein expression was investigated during preimplantation development. We demonstrated HLA-G protein expression in oocytes, cleavage stage embryos, and blastocysts, where we find it in trophectoderms but also in ICM cells. During blastocyst development, a downregulation of HLA-G in the ICM cells was present. This data might be important for cell therapy and transplantation because undifferentiated hESC can contaminate the transplant of differentiated stem cells and develop into malignant cancer cells. [ABSTRACT FROM AUTHOR]

Objectives: Free light chain (FLC) measurement gained a lot of interest for diagnostic workup of monoclonal gammopathy., Methods: We evaluated the performance of turbidimetric polyclonal Freelite (The Binding Site, Birmingham, UK) assays on Cobas 6000 (Roche Diagnostics, Rotkreuz, Switzerland) and nephelometric monoclonal N Latex (Siemens Healthcare Diagnostics, Marburg, Germany) assays on BN ProSpec (Dade Behring, Deerfield, IL) vs established nephelometric Freelite assays on BN ProSpec., Results: Analytical performance was acceptable. Method comparison (n = 118) showed significant proportional FLC differences for N Latex assays. However, good correlation and clinical concordance were shown. Recovery study in the low concentration range demonstrated consistent over- and underrecovery for Freelite reagents, hampering future research on prognostic value of suppressed noninvolved FLC. Antigen excess detection was successful for κ FLC in three-fourths of cases with Freelite reagents and in all cases with N Latex reagents. However, the latter resulted in underestimated κ FLC concentrations., Conclusions: FLC analysis requires continuous awareness of analytical limitations. Monitoring of disease response requires FLC analysis on the same platform using the same reagents., (© American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

The metabolic effects of chronic alcohol abuse can induce a broad spectrum of disorders. We describe the case of an initially unidentified alcoholic, poorly nourished woman who presented with ketoacidosis. She developed severe cardiac failure, which did not respond to classical treatment. The administration of intravenous thiamine resulted in an impressive recovery of cardiac function. Laboratory examinations confirmed the diagnosis of alcoholic ketoacidosis and cardiac beriberi. The clinical entity and treatment of these two uncommon disorders are discussed. If recognized early both diseases (and their combination) are fully reversible.