Martine Vercammen

Diagnostic accuracy of the FIDIS multiplex fluorescent microsphere immunodetection system for anti-extractable nuclear antigen (ENA) antibodies in connective tissue diseases

Abstract Background: Anti-extractable nuclear antigen antibodies (ENA) are markers of connective tissue diseases (CTDs). Methods: We compared FIDIS reagents in the multiplex fluorescent microsphere immunodetection system to INNO-LIA and immunodiffusion for 174 antinuclear antibody-positive patients, 102 with well-defined CTDs and 72 disease controls. Results: No significant differences were found in sensitivity or specificity between FIDIS and immunodiffusion, or between FIDIS and INNO-LIA for all anti-ENA in all CTD patients; nor were any differences found for individual anti-ENAs within distinct CTDs. The FIDIS sensitivity was 41% (anti-SSA) and 17% (anti-SSB) in lupus erythematosus (LE) or primary Sjögrens syndrome; 5% (anti-ribosome and anti-Sm) in LE; 17% (anti-RNP) in LE or mixed CTD; 21% (anti-Scl70) in systemic sclerosis; and 61% (anti-centromere) in limited systemic sclerosis. The specificity reached 88%–100%. Receiver operating characteristic curve areas did not differ between FIDIS and INNO-LIA. Agreement ranged from 91% (anti-SSB) to 99% (anti-Jo1) between FIDIS and INNO-LIA, and from 95% (anti-Scl70) to 100% (anti-Sm) between FIDIS and immunodiffusion. Samples scored positive with all techniques in 83% (anti-centromere), 70% (anti-RNP), 67% (anti-Jo1), 60% (anti-SSA), 40% (anti-SSB), 33% (anti-ribosome), 25% (anti-Sm) and 13% (anti-Scl70) of cases. Conclusions: The diagnostic performance of FIDIS anti-ENA reagents is comparable to immunodiffusion and INNO-LIA. Clin Chem Lab Med 2007;45:505–12.

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.

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.

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.

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.

Belgian recommendations on ANA, anti-dsDNA and anti-ENA antibody testing

Abstract Autoantibodies to nuclear antigens, i.e. antinuclear antibodies (ANA), antibodies to double-stranded DNA (dsDNA) and extractable nuclear antigens (ENA), are useful as diagnostic markers for a variety of autoimmune diseases. In March 2010, the Belgian national External Quality Assessment Scheme sent a questionnaire on ANA, anti-dsDNA and anti-ENA antibody testing designed by the Dutch EASI (European Autoimmunity Standardization Initiative) team, to all clinical laboratories performing ANA testing. Virtually all laboratories completed the questionnaire (97·7%, 127/130). This paper discusses the results of this questionnaire and provides valuable information on the state-of-the-art of ANA, anti-dsDNA and anti-ENA antibody testing as practiced in the Belgian laboratories. In addition, this work presents practical recommendations developed by the members of the advisory board of the scheme as a result of the outcome of this study.

Analytical performance of the single well titer function of NOVA View®: good enough to omit ANA IIF titer analysis?

Antinuclear antibodies (ANAs) are a diagnostic marker for ANA-associated rheumatic diseases [1]. The gold standard method for ANA detection is considered indirect immunofluorescence (IIF) on human epithelial (HEp-2) cells because of its high sensitivity [2]. Most recent ANA IIF guidelines recommend not only to report the presence of ANA as positive or negative but also to give a quantitative result [3, 4]. Nowadays, automated digital reading systems for ANAs by IIF testing have been integrated in routine immunodiagnostic laboratory practice [5, 6]. Several studies already showed a good correlation between fluorescence intensity by automated reading and end point titer (ET) obtained by manual reading [7, 8]. Furthermore, recent studies objectified that the likelihood for a systemic rheumatic disease increases with increasing ANA IIF fluorescence intensity [8, 9]. This illustrates that estimation of fluorescence intensity by automated IIF systems (without serial dilution) has clinical utility. NOVA View® (Inova, San Diego, CA, USA) is a digital IIF microscope that can be used for automated ANA detection. The system is able to assign five basic fluorescent ANA patterns (homogeneous, speckled, centromere, nucleolar and nuclear dots) and reports the measured average nuclear fluorescence intensity in nominal units, called Light-intensity units (LIUs). For positive ANA IIF samples, the instrument allows estimation of a “single well titer” (SWT) based on the LIUs measured at the 1/80 screening dilution and pattern-specific dilution curves [5, 7]. We evaluated the analytical performance (total imprecision and accuracy) of the SWT function on the NOVA View® for different ANA IIF nuclear patterns. Total imprecision of LIU measurement and of SWT function was evaluated by analyzing samples with a high and low level of a homogeneous, speckled, centromere and nucleolar pattern in 10 different ANA IIF runs. The target ET of the samples was determined by manual serial dilution. The total imprecision values of LIU measurement for the high-level and low-level samples were, respectively, 8% and 34% for a homogeneous pattern, 26% and 45% for a speckled pattern, 35% and 47% for a centromere pattern, and 38% and 39% for a nucleolar pattern ( Supplemental Data, Table 1). When results were expressed as SWT, the estimated SWT deviated from the target ET by maximum 1 SWT for all patterns except for the centromere pattern where the deviation was 2 titers in 30% of the sample with a low antibody level and in 10% of the sample with a high antibody level. Of note, for the sample with a high level of nucleolar antibodies, 60% of the determinations deviated by 1 titer from the target value. To evaluate the accuracy of the SWT function, samples with an isolated homogeneous (n = 389), speckled (n = 458), centromere (n = 101) and nucleolar (n = 69) ANA IIF pattern were analyzed in four hospitals: University Hospital Leuven, Gasthuiszusters Hospital Antwerp, OLV *Corresponding author: Lieve Van Hoovels, Department of Laboratory Medicine, OLV Hospital Aalst, Moorselbaan 164, 9300 Aalst, Belgium, Phone: +32 (0)53/72 42 91, Fax: +32 (0)53/72 45 88, E-mail: lieve.van.hoovels@olvz-aalst.be Sofie Schouwers and Laura Bogaert: Department of Laboratory Medicine, GZA Hospitals, Antwerp, Belgium Stefanie Van den Bremt: Department of Laboratory Medicine, OLV Hospital, Aalst, Belgium Nathalie Vandeputte: Department of Immunodiagnostics, Werfen N.V./S.A., Breda, Netherlands Martine Vercammen: Department of Laboratory Medicine, AZ SintJan Hospital Brugge, Brugge, Belgium Xavier Bossuyt: Department of Laboratory Medicine, University Hospital Leuven, Leuven, Belgium

Revised 2017 international consensus on ANCA testing in small vessel vasculitis: support from an external quality assessment

We read with great interest the multicentre study of Damoiseaux et al 1–3 on the detection of antineutrophil cytoplasmic antibodies (ANCA). ANCAs are important laboratory markers to support the diagnosis of ANCA-associated small vessel vasculitis (AAV), including granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). Traditionally, laboratories screen for ANCA by indirect immunofluorescence (IIF) and IIF positive samples are further evaluated for antibodies to proteinase 3 (PR3) or myeloperoxidase (MPO) by specific immunoassays. Such diagnostic algorithm is based on an international consensus statement on ANCA testing issued in 1999.4 Over the last two decades, the diagnostic performance of immunoassays has significantly improved. The recent multicentre study showed a …

Diagnostic thresholds for free light chains in multiple myeloma depend on the assay used

Leukemia accepted article preview online, 20 November 2017. doi:10.1038/leu.2017.335.