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.

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.

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.

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.

Ketosis and cardiac failure: common signs of a single condition.

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.