Inter-assay diagnostic accuracy of cerebrospinal fluid kappa free light chains for the diagnosis of multiple sclerosis.

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, κIgG 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), κIgG 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 κIgG 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 κIgG 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 κIgG 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.

Detection of antinuclear antibodies: recommendations from EFLM, EASI and ICAP.

OBJECTIVES: 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). METHODS: 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). RESULTS: 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. CONCLUSIONS: These recommendations are an important step to achieve high quality ANA testing.

Analytical aspects of the antinuclear antibody test by HEp-2 indirect immunofluorescence: EFLM report on an international survey.

OBJECTIVES: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Serum free light chain analysis: persisting limitations with new kids on the block.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Laboratory evaluation of anti-dsDNA antibodies.

Antibodies to dsDNA are an important laboratory parameter for diagnosis, monitoring and classification of systemic lupus erythematosus (SLE). In clinical laboratories, several techniques are used to detect and quantify anti-dsDNA antibodies. Each technique has its advantages and disadvantages regarding sensitivity, specificity, avidity and assay procedure. Assays differ with respect to the antigen source (native versus synthetic versus molecular biological) used and the way the antigen is presented (e.g. in solution, covalently linked to a solid phase,…). Consequently, correlation between assays can be poor and standardization of anti-dsDNA antibody tests is challenging. We here provide an overview of the currently available anti-dsDNA tests frequently used in clinical laboratories [Crithidia luciliae immunofluorescence test (CLIFT), Enzyme linked immune sorbent assay (ELISA), fluoroenzyme immunoassay (FEIA), chemiluminisence immunoassay (CIA), multiplexed bead-based assays and Farr-RIA] and their performance characteristics. From this literature study, we concluded that performance characteristics differ between assays. Often, a combination of techniques is necessary for the best result interpretation.

Current laboratory and clinical practices in reporting and interpreting anti-nuclear antibody indirect immunofluorescence (ANA IIF) patterns: results of an international survey.

BACKGROUND: The International Consensus on Antinuclear Antibody (ANA) Patterns (ICAP) has recently proposed nomenclature in order to harmonize ANA indirect immunofluorescence (IIF) pattern reporting. ICAP distinguishes competent-level from expert-level patterns. A survey was organized to evaluate reporting, familiarity, and considered clinical value of ANA IIF patterns. METHODS: Two surveys were distributed by European Autoimmunity Standardization Initiative (EASI) working groups, the International Consensus on ANA Patterns (ICAP) and UK NEQAS to laboratory professionals and clinicians. RESULTS: 438 laboratory professionals and 248 clinicians from 67 countries responded. Except for dense fine speckled (DFS), the nuclear competent patterns were reported by > 85% of the laboratories. Except for rods and rings, the cytoplasmic competent patterns were reported by > 72% of laboratories. Cytoplasmic IIF staining was considered ANA positive by 55% of clinicians and 62% of laboratory professionals, with geographical and expertise-related differences. Quantification of fluorescence intensity was considered clinically relevant for nuclear patterns, but less so for cytoplasmic and mitotic patterns. Combining IIF with specific extractable nuclear antigens (ENA)/dsDNA antibody testing was considered most informative. Of the nuclear competent patterns, the centromere and homogeneous pattern obtained the highest scores for clinical relevance and the DFS pattern the lowest. Of the cytoplasmic patterns, the reticular/mitochondria-like pattern obtained the highest scores for clinical relevance and the polar/Golgi-like and rods and rings patterns the lowest. CONCLUSION: This survey confirms that the major nuclear and cytoplasmic ANA IIF patterns are considered clinically important. There is no unanimity on classifying DFS, rods and rings and polar/Golgi-like as a competent pattern and on reporting cytoplasmic patterns as ANA IIF positive.

Performance Evaluation of Serum Free Light Chain Analysis: Nephelometry vs Turbidimetry, Monoclonal vs Polyclonal Reagents.

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.

Overestimation of free light chain antigen excess rate.

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.

Betaine homocysteine methyl transferase 1, a novel auto-antigen associated with anti-Golgi immune reactivity.

Anti-Golgi antibodies are rare autoantibodies that have been described in systemic autoimmune diseases. Not all Golgi auto-antigens are known. The objective of this study was to identify a novel auto-antigen associated with anti-Golgi immune reactivity. Sera from a patient with Golgi immune reactivity and from a control individual were used for Western blotting after 2-dimensional gel separation of a rat Golgi-enriched extract. Betaine homocysteine S-methyltransferase 1 (BHMT1) was identified as an auto-antigen by MALDI-TOF/TOF mass spectrometry. Using human recombinant BHMT1, a strong positive blotting signal was obtained with serum from the patient but not from a control. Pre-absorption of the serum sample with reactivity to BHMT1 with recombinant human BHMT1 resulted in decreased reactivity on Western blotting and in disappearance of the Golgi-like pattern on indirect immunofluorescence. Using immunocytochemistry, we confirmed the subcellular localization of BHMT1 to the Golgi apparatus. Antibodies to BHMT1 were found in four of 80 samples with a Golgi-pattern on indirect immunofluorescence. The antibodies were not associated with a specific clinical condition. We identified BHMT1 as a novel auto-antigen associated with anti-Golgi immune reactivity.

Effect of sample dilution on serum free light chain concentration by immunonephelometric assay.

BACKGROUND: Free light chains (FLC) are useful biomarkers in the assessment of plasma cell disorders. Concerns have been raised about some technical aspects of the assay. This report examined the occurrence of dilution anomalies and/or antigen excess. METHODS: FLC were determined on a BNII instrument at at-least two dilutions (100- and 2000-fold) in 2088 consecutive samples from 865 different patients. In part of them, the 400-fold dilution was also available. RESULTS: Higher than 2-fold differences and inconsistencies ["<" or ">" result at one dilution not consistent with the result obtained at another dilution] between the 100- and 2000-fold dilutions were found in 12.7% of patients for κ FLC and in 3.1% of patients for λ FLC. More than 4-fold differences between results obtained at the 2000-fold and the 100-fold dilutions were observed in 5.4% of patients for κ FLC and in 1.2% of patients for λ FLC. CONCLUSIONS: The FLC assay on BNII suffers from sample dilution anomalies and/or failure of antigen excess detection in a substantial fraction of patients. Laboratory professionals and clinicians should be alert to such analytical difficulties.

HLA-G expression in human embryonic stem cells and preimplantation embryos.

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.

The importance of HLA-G expression in embryos, trophoblast cells, and embryonic stem cells.

The nonclassical HLA-G molecule is a trophoblast-specific molecule present in almost every pregnancy. It differs from classical HLA class I molecules by the low degree of allelic variants and the high diversity of protein structures. HLA-G is reported to be a tolerogenic molecule that acts on cells of both innate and adaptive immunity. At the maternal-fetal interface HLA-G seems to be responsible largely for the reprogramming of local maternal immune response. This review will focus on the HLA-G gene expression profile in pregnancy, in preimplantation embryos, and in human embryonic stem cells with emphasis on the structural diversity of the HLA-G protein and its potential functional and diagnostic implications.

Use of interval-specific likelihood ratios improves clinical interpretation of serum FLC results for the diagnosis of malignant plasma cell disorders.

BACKGROUND: We examined whether the use of test result interval-specific likelihood ratios (LR) could improve the clinical interpretation of serum FLC kappa/lambda ratio for the diagnosis of malignant plasma cell disorders. METHODS: We calculated LRs for different FLC kappa/lambda intervals using sera from patients diagnosed with intact multiple myeloma (MM), light chain MM (LCMM), non-secretory MM (NSMM) and light chain amyloidosis (AL-A). Consecutive patients with a clinical suspicion of a monoclonal B-cell disorder that were diagnosed with MGUS or no B-cell monoclonal disorder served as the disease control group. RESULTS: Using LRs for different test result intervals, a distinction can be made between FLC kappa/lambda ratios that are within the normal diagnostic range, ratios that are inconclusive (1.66-5.0, LR+/-1), ratios that indicate the possible presence of a malignant plasma cell disorder (0.05-0.25 and >5.0-10, LR+/-10) and ratios that were suggestive of a malignant plasma cell disorder (<0.05 or >10; LR+/-50). A FLC kappa/lambda ratio within the normal diagnostic range virtually excluded LCMM and AL-A, but not intact MM or NSMM. CONCLUSIONS: Interpreting serum FLC kappa/lambda ratios using LRs for different result intervals improves the clinical interpretation for the diagnosis of malignant plasma cell disorders excluding plasmacytoma.

Can soluble human leucocyte antigen-G predict successful pregnancy in assisted reproductive technology?

PURPOSE OF REVIEW: To review the predictive value of soluble human leucocyte antigen-G (HLA-G) in embryo culture fluid for implantation, considering origin, structure, function and detection method of soluble HLA-G. RECENT FINDINGS: Soluble HLA-G in embryo culture supernatant has been proposed as a noninvasive marker for the selection of embryos with implantation potential. Controversially, some centres detect soluble HLA-G in none of the culture supernatants of embryos that evolve towards pregnancy, whereas others report it to be mandatory. According to a recently published meta-analysis, the pooled diagnostic accuracy for predicting clinical pregnancy is low. Factors influencing soluble HLA-G results are numerous, for example, embryo culture (single or multiple, washing/denudation steps, medium, day 2 to 5 sample collection), sample preservation to prevent degradation and HLA-G immunoassays (standard, detection limit, reproducibility). Published studies are very heterogeneous. Moreover, the origin of soluble HLA-G in embryo culture fluid is unresolved. From the embryonic genome activation at day 3 onwards, the embryo should be able to produce HLA-G. Before, it is maternally derived, either from the oocyte, follicular fluid or follicular cells contaminating embryo culture. SUMMARY: At present, no studies have been published on the relationship between pregnancy and soluble HLA-G in supernatants from individually cultured and individually transferred embryos using standardized embryo culture and soluble HLA-G immunoassay, sensitive at the picogram level. As such, it remains undetermined whether the pregnancy is induced by an HLA-G-producing embryo. Therefore, the predictive value of soluble HLA-G in embryo culture supernatant for selection of embryos with good implantation potential remains unknown.