Maria Infantino, Italy
Immunology and Allergy laboratory San Giovanni di Dio HospitalPresenter of 3 Presentations
NEW TECHNOLOGIES FOR ANTI-DSDNA ANTIBODIES DETECTION: THERE IS STILL NEED OF HARMONIZATION
Abstract
Background and Aims
Anti-double stranded DNA antibodies (anti-dsDNA) are considered a specific marker for SLE. Although the Farr technique has been considered the “gold standard” for their detection, currently it has been replaced by numerous newly developed assays. However, there is still no solid evidence of the equivalence of these methods in terms of diagnostic accuracy and of their correlation with the Crithidia luciliae immunofluorescence test (CLIFT).
Methods
Anti-dsDNA antibody levels were measured in 60 patients suffering from different connective tissue diseases, and in 20 diseased controls using four different immunoassays (fluorescence enzyme immunoassay, microdot array, chemiluminescent immunoassay, multiplex flow immunoassay) and by two CLIFT methods.
Results
Average agreement among methods for positive/negative results assessed by the Cohen’s Kappa was 0.71, ranging from moderate to almost perfect (0.53-0.92) (Table1).
Conclusions
Despite good individual performance, new technologies for anti-dsDNA antibody detection are not fully comparable. In particular, their different correlation with CLIFT influences their positioning in the diagnostic algorithm for SLE (either in association or sequentially). Considering the high inter-method variability, harmonization of anti-dsDNA antibody testing remains an unachieved goal.
PRE-ANALYTICAL EVALUATION OF CIRCULATING CALPROTECTIN IN SERUM AND PLASMA FROM HEALTHY DONORS
Abstract
Background and Aims
Calprotectin (S100A8/A9) is a heterodimeric protein expressed in neutrophils and macrophages. It has recently generated interest in the rheumatology setting as an inflammation marker. However, its detection can be highly altered by pre-analytical processing of the specimen. This study aims to evaluate the impact of differences in sample pre-processing using serum as well as EDTA and citrate plasma samples processed at different time points.
Methods
Blood was drawn from five apparently healthy individuals into serum, EDTA plasma and citrate plasma sample tubes. Samples were kept at room temperature (1, 5, 10 and 24 hours) and subsequently clarified by centrifugation for 10 minutes at 3000 g at selected time points. Sample supernatants were collected and stored frozen for later testing. Calprotectin levels were measured using the novel QUANTA Flash Circulating Calprotectin chemiluminescent immunoassay (Inova Diagnostics). Percent recovery results were calculated at each time point using the sample processed after 1 hour as reference.
Results
All three sample matrices exhibited significant sensitivity to the pre-analytical phase (delay of centrifugation). In serum, calprotectin levels were significantly more elevated than in plasma, but the results were fluctuating over time and therefore difficult to interpret. For EDTA plasma a significant and continuous increase of measured calprotectin was observed reaching 2693% after 24 hours. Citrate plasma was the matrix showing very similar percent recovery values over time (p=0.170). (Table 1).
Conclusions
The pre-analytic phase needs to be considered when evaluating levels of circulating calprotectin. Of the three matrices evaluated, citrate plasma provided more consistent results for measuring circulating calprotectin.
A NEW DIAGNOSTIC ALGORITHM FOR PATTERN-ORIENTED AUTOANTIBODY TESTING ACCORDING TO THE ICAP NOMENCLATURE: A PILOT STUDY
Abstract
Background and Aims
The commercial tests currently available as second-level tests to detect ANA sub-specificities are generally used independently from the ANA immunofluorescence (IIF) pattern. The aim of this study was to evaluate the efficacy of the use of a customizable pattern-oriented antigenic panel by immunoblot (IB) using the International Consensus on ANA Patterns (ICAP) classification scheme, in order to introduce a novel and updated autoimmune diagnostic flowchart.
Methods
710 sera referred for routine ANA testing were selected on the basis of the ANA pattern according to the ICAP nomenclature (nuclear speckled AC-2,4,5; nucleolar AC-8,9,10,29; cytoplasmic speckled AC 18,19,20) and on an IIF titer ≥1:320. They were then assayed by three experimental IB assays using a panel of selected antigens.
Results
ICAP-oriented IB detected 515 antibody reactivities vs. 457 of traditional anti-ENA in the nuclear speckled pattern group, 108 vs. 28 in the nucleolar pattern group, and 43 vs. 34 in the cytoplasmic speckled pattern.
Conclusions
This pilot study may lead the way for a new approach introducing an ICAP pattern-oriented follow up testing as a valid alternative to the existing standard panels, thus enabling more patients with autoimmune rheumatic disease to be accurately diagnosed.