Carolien Bonroy

Do worsening scleroderma capillaroscopic patterns predict future severe organ involvement? a pilot study

Objective Assessment of associations of nailfold videocapillaroscopy (NVC) scleroderma patterns (‘early’, ‘active’ and ‘late’) with future severe clinical involvement in a systemic sclerosis (SSc) population. Methods Sixty-six consecutive patients with SSc according to the LeRoy and Medsger criteria underwent NVC assessment at baseline. Videocapillaroscopic images were classified into ‘normal’, ‘early’, ‘active’ or ‘late’ NVC pattern. Clinical evaluation was performed for nine organ systems (general, peripheral vascular, skin, joint, muscle, gastrointestinal tract, lung, heart and kidney) according to the disease severity scale of Medsger (DSS) at 18–24 months of follow-up. Severe clinical involvement was defined as category 2–4 per organ of the DSS. Results NVC patterns were significantly associated with future severe, peripheral vascular/lung involvement at 18–24 months. The OR rose steadily throughout the patterns. The OR for future severe peripheral disease based on simple/multiple (correcting for disease duration, subset and medication) logistic regression was 2.49/2.52 (95% CI 1.33 to 5.43, p=0.003/1.11 to 7.07, p=0.026) for early, 6.18/6.37 for active and 15.35/16.07 for late NVC scleroderma patterns versus the normal NVC pattern. The OR for future severe lung involvement based on simple/multiple regression was 2.54/2.33 (95% CI 1.40 to 5.22, p=0.001/1.13 to 5.52, p=0.021) for early, 6.43/5.44 for active and 16.30/12.68 for late NVC patterns. Conclusions This pilot study is the first demonstrating an association between baseline NVC patterns and future severe, peripheral vascular and lung involvement with stronger odds according to worsening scleroderma patterns. This may indicate a putative role of capillaroscopy as a biomarker.

Nailfold Capillaroscopy for Prediction of Novel Future Severe Organ Involvement in Systemic Sclerosis

Objective. Assessment of associations of nailfold videocapillaroscopy (NVC) scleroderma (systemic sclerosis; SSc) (“early,” “active,” and “late”) with novel future severe clinical involvement in 2 independent cohorts. Methods. Sixty-six consecutive Belgian and 82 Italian patients with SSc underwent NVC at baseline. Images were blindly assessed and classified into normal, early, active, or late NVC pattern. Clinical evaluation was performed for 9 organ systems (general, peripheral vascular, skin, joint, muscle, gastrointestinal tract, lung, heart, and kidney) according to the Medsger disease severity scale (DSS) at baseline and in the future (18–24 months of followup). Severe clinical involvement was defined as category 2 to 4 per organ of the DSS. Logistic regression analysis (continuous NVC predictor variable) was performed. Results. The OR to develop novel future severe organ involvement was stronger according to more severe NVC patterns and similar in both cohorts. In simple logistic regression analysis the OR in the Belgian/Italian cohort was 2.16 (95% CI 1.19–4.47, p = 0.010)/2.33 (95% CI 1.36–4.22, p = 0.002) for the early NVC SSc pattern, 4.68/5.42 for the active pattern, and 10.14/12.63 for the late pattern versus the normal pattern. In multiple logistic regression analysis, adjusting for disease duration, subset, and vasoactive medication, the OR was 2.99 (95% CI 1.31–8.82, p = 0.007)/1.88 (95% CI 1.00–3.71, p = 0.050) for the early NVC SSc pattern, 8.93/3.54 for the active pattern, and 26.69/6.66 for the late pattern versus the normal pattern. Conclusion. Capillaroscopy may be predictive of novel future severe organ involvement in SSc, as attested by 2 independent cohorts.

Automated indirect immunofluorescence antinuclear antibody analysis is a standardized alternative for visual microscope interpretation

Abstract Background: Screening for antinuclear antibodies (ANA) is a basic tool in the serological work-up of systemic rheumatic disorders. Despite the emergence of alternative screening methods and the difficulties in standardization, indirect immunofluorescence (IIF) remains the recommended method for ANA detection. This study aimed to assess the reliability of automated ANA IIF analysis as a standardized alternative for the conventional manual approach. Methods: ANA testing on HEp-2000 cells was performed on 304 consecutive routine sera, 28 serumbank samples displaying rare staining patterns, 219 samples of well-defined disease cohorts [141 systemic sclerosis (SSc), 13 polymyalgia rheumatica, 22 osteoarthritis, 5 ANCA-associated vasculitis and 38 spondyloarthritis] and 100 healthy donors. All samples were analyzed by automated IIF (Zenit G-sight), by conventional visual IIF microscopy and two ANA screening enzyme immunoassays (EIA). Results: Automated and conventional ANA IIF analysis were comparable for negative/positive interpretation as well as intensity assessment (>90% agreement). In contrast, the accuracy of pattern recognition (26%) was limited. Likelihood ratios (LR) for SSc on results intervals of both Zenit G-sight and EIA increased with increasing level of positivity. Sensitivity within the SSc-associated antibody subsets was higher for Zenit G-sight (97%–100%) than EIA (10%–96%). A significant correlation between the quantitative result obtained by Zenit G-sight and the conventional end-point titer was found. Conclusions: The use of Zenit G-sight for automated ANA IIF analysis offers opportunities to improve standardization. However, a complementary role of the expert technicians remains, especially for pattern recognition and classification of uncertain/negative samples.

Optimization and diagnostic performance of a single multiparameter lineblot in the serological workup of systemic sclerosis

INTRODUCTION Detection of systemic sclerosis-associated antibodies (SSc-Ab) in routine clinical practice is mostly restricted to anti-centromere and anti-topoisomerase-I antibodies. However, also other SSc-Ab (e.g. anti-RNA-polymerase-III, anti-PM/Scl, anti-fibrillarin and anti-Th/To) have been shown to be valuable diagnostic and prognostic markers for the disease, but testing methodologies for their detection are laborious and time-consuming. This study aimed to optimize interpretational criteria of a multiparameter lineblot (LB) for the parallel detection of SSc-Ab. We also assessed its global diagnostic value as an alternative for combined conventional techniques (CCT) in the serological workup of systemic sclerosis (SSc) patients. METHODS The presence of SSc-Ab (anti-centromere, anti-topoisomerase-I, anti-RNA-polymerase-III, anti-PM/Scl, anti-fibrillarin and anti-Th/To) was identified by LB on 145 consecutive SSc patients and on 277 disease controls. Diagnostic sensitivity and specificity were calculated for both individual reactivities and the global LB. Cohens kappa coefficient was used to examine agreement between LB and CCT and guided the definition of final interpretational criteria for LB. RESULTS Applying the optimal cut-off values and interpretational criteria, LB identified SSc-Ab in 110 SSc patients (sensitivity=76%) and in 19 disease controls (specificity=93%). Globally, there was a substantial agreement between CCT and LB (κ=0.787, concordance 92.4%). LB and CCT showed a very good correlation (κ>0.800) for most SSc-Ab (anti-centromere, anti-topoisomerase-I, anti-RNA-polymerase-III and anti-PM/Scl). The best agreement for anti-RNA-polymerase-III and anti-PM/Scl was achieved when positivity for both components was taken as a criterion. CONCLUSIONS LB is a reliable alternative for the laborious and time-consuming conventional techniques in the diagnostic workup of SSc, especially for the detection of anti-centromere, anti-topoisomerase-I, anti-RNA-polymerase-III and anti-PM/Scl.

Screening for pulmonary arterial hypertension in an unselected prospective systemic sclerosis cohort

Screening for pulmonary arterial hypertension (PAH) in systemic sclerosis (SSc) improves outcomes. The DETECT screening algorithm is recommended in a high-risk SSc subgroup. This study aims to compare prospectively the positive predictive value of screening using the DETECT algorithm and the 2009 European Society of Cardiology/European Respiratory Society (ESC/ERS) guidelines, and to compare their cost-effectiveness in an unselected, day-to-day SSc population. Post hoc, screening according to the 2015 ESC/ERS guidelines using echocardiographic parameters alone (“2015 echo screening”) or combined with the DETECT algorithm (“2015 combined screening”) in high-risk subjects was analysed. 195 consecutive SSc patients included in the Ghent University Hospital SSc cohort were screened using different algorithms. The referral rate for right heart catheterisation was 32% (63 out of 195 patients) (46/4/13/34/40 patients using the DETECT algorithm/2009 guidelines/both/2015 echo screening/2015 combined screening). Right heart catheterisation was performed in 53 patients (84%) (36 (78%)/four (100%)/13 (100%)/28 (82%)/32 (80%) patients recommended by the DETECT algorithm/2009 guidelines/both/2015 echo screening/2015 combined screening). PAH was diagnosed in three patients (incidence 1.5%·year–1, 95% CI 0.5–4.4), in whom all algorithms recommended a right heart catheterisation. The positive predictive value was 6% (95% CI 2–17%; three out of 49 patients) for the DETECT algorithm, 18% (95% CI 6–41%; three out of 17 patients) for the 2009 guidelines, 23% (95% CI 8–50%; three out of 13 patients) for both, 11% (95% CI 4–27%; three out of 28 patients) for the 2015 echo screening and 9% (95% CI 3–24%; three out of 32 patients) for the 2015 combined screening. The cost was EUR224/80/90/112 per patient using the DETECT algorithm/2009 guidelines/2015 echo screening/2015 combined screening. Echocardiography may remain a candidate first step for PAH screening in SSc. Echocardiography remains a candidate first step in screening for PAH in an unselected systemic sclerosis population http://ow.ly/nuoh3096nRh

Specific anti-nuclear antibodies in systemic sclerosis patients with and without skin involvement: an extended methodological approach

OBJECTIVES To determine how sensitively SSc-associated ANAs are detected by a standard identification algorithm compared with an extensive panel of ANA identification assays, and to assess the distribution of SSc-associated ANAs and SSc organ system involvement in patients without skin involvement (limited SSc). METHODS Serum samples from 145 consecutive monocentric SSc patients fulfilling LeRoy and Medgsers criteria for early SSc were studied. ANAs were detected by IIF on HEp-2000 cells and identified by western blotting, protein radio-immunoprecipitation, RNA immunoprecipitation and line immunoassay (LIA). SSc organ involvement was assessed according to a modification of Medsgers disease severity scale. RESULTS At least one specific ANA reactivity was present in 88% of the patients. The standard algorithm (IIF and LIA) found at least one specific ANA in 74% of the patients. The main reactivities missed by this algorithm were anti-RNA polymerase III, anti-PM/Scl and anti-Th/To. Eighty-three percent of the patients with limited SSc had at least one ANA. ACAs and anti-Th/To antibodies were significantly associated with limited SSc, whereas anti-topoisomerase I and anti-RNA polymerase III were observed less frequently. SSc organ system involvement was present in 63% of the patients with limited SSc, most of whom had lung involvement. CONCLUSIONS Standard algorithms for ANA identification lack sensitivity for the detection of SSc-associated ANA and should be supplemented with additional assays, especially in a clinical environment that has particular interest in SSc. The spectrum of SSc-associated ANA differs according to the presence or absence of skin involvement.

Automated indirect immunofluorescence microscopy enables the implementation of a quantitative internal quality control system for anti-nuclear antibody (ANA) analysis.

Abstract Background: Screening for anti-nuclear antibodies by indirect immunofluorescence (ANA-IIF) remains mandatory in the serological work-up of connective tissue diseases. Recently, automated approaches were introduced that may improve harmonization. Here, we investigated whether the introduction of automated ANA-IIF and more specifically the use of its quantitative measure, could improve ANA-IIF internal quality control (IQC) management. Methods: We retrospectively reviewed results of two cohorts of routine samples and parallel IQC data collected from January 2010 to February 2013 and from February to mid October 2013. For the first cohort, data were collected using conventional microscopy. The second cohort was analyzed by an automated ANA-IIF microscope (Zenit G sight, A. Menarini). Retrospectively, we evaluated the applicability of the probability index (PI) of control material measurements and patient results for IQC management based on Westgard multirules. This approach was also compared with monthly monitoring of the %ANA-IIF positive samples. Results: In our historical data set, we showed that monitoring of %ANA positives identified systematic errors that were not detected by monitoring control material results. Data resulting from automated microscopy showed that PI measurements on control material remained stable within the observed period and that Westgard multirules can be used for IQC follow-up. Parallel monitoring of the daily median patient PI and the monthly %ANA positives, showed that the daily median was a sensitive and fast tool for detecting systematic errors. Conclusions: The introduction of the automated ANA-IIF microscope could enable objective IQC procedures and should be considered an important step forward in ANA-IIF harmonization.

Specific Antinuclear Antibody Level Changes after B Cell Depletion Therapy in Systemic Sclerosis Are Associated with Improvement of Skin Thickening

To the Editor: B cell depletion therapy using rituximab (RTX) has emerged as a promising therapy in systemic sclerosis (SSc). Our group reported a clinical benefit on skin thickening when RTX (2 × 1000 mg) in combination with methylprednisolone (100 mg) was administered at months 0 and 61. The rationale behind the use of B cell depletion in SSc treatment is based on the growing evidence that B cells play a role in the pathogenesis of the disease2,3. In addition, SSc-associated antinuclear antibody (ANA) are present in the majority of patients, but are generally accepted to be rather static markers, primarily important in diagnosis rather than in followup4. Moreover, conflicting results have been published5 on the change in disease-specific ANA titers in relation to clinical response in other connective diseases (e.g., systemic lupus erythematosus and rheumatoid arthritis) after RTX. To our knowledge, no studies in SSc are available documenting the longterm effect of RTX on these autoantibody levels. In this letter we report the 2-year serologic followup data (months 0, 3, 6, 12, 15, 18, and 24) on the same 8 patients with SSc with diffuse skin involvement (dcSSc) who were included in our initial RTX studies1,6. The idea was to evaluate the relationship between SSc-ANA level changes and clinical response. To document this latter relationship, we performed … Address correspondence to C. Bonroy, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital 2P8, De Pintelaan 185, B-9000, Ghent, Belgium. E-mail: carolien.bonroy{at}uzgent.be

Relevance of Different Results of Different Anti–Double-Stranded DNA Assays in Reporting Clinical Studies: Comment on the Article by Petri et al

Relevance of different results of different anti–doublestranded DNA assays in reporting clinical studies: comment on the article by Petri et al To the Editor: We read with interest the recent report by Petri and colleagues on baseline predictors of disease flares in patients with serologically active systemic lupus erythematosus (SLE) treated with standard therapy alone—a study that used the combined placebo group of the phase III belimumab trials (1). The definition of serologic activity included the presence of anti–double-stranded DNA (anti-dsDNA) antibodies in concentrations of at least 30 IU/ml. This inclusion criterion was based on a prior phase II placebo-controlled trial showing that belimumab reduced and stabilized disease activity within this subpopulation and was subsequently used in two phase III trials (2–4). In our opinion, however, it should be clearly anticipated that the serologic criterion for study inclusion used in the phase II and phase III studies (anti-dsDNA antibody levels 30 IU/ml) cannot be extrapolated directly for use in clinical practice to select patients eligible for belimumab therapy. From the same perspective, use of an anti-dsDNA level of 200 IU/ml to predict SLE flares, as reported by Petri et al (1), should be adopted with caution. Our arguments are as follows. First, assays assessing the presence and the level of dsDNA antibodies vary significantly (between techniques as well as between manufacturers) despite the use of international units (IU/ml) based on previous attempts to obtain standardization (5). Second, despite the emerging use of enzyme immunoassays (EIAs), it is generally accepted that the Farr assay remains the gold standard for determining SLE-specific DNA antibodies (6). Within this context, details are missing regarding the EIA and manufacturer used in the phase II and phase III belimumab studies, as well as their relationship with the results obtained using a Farr immunoassay (as was used in the phase I belimumab study) (1–4,7). To document this latter relationship as well as the variability between assays, we analyzed anti-dsDNA in a set of 44 patients with SLE, using a Farr assay (Anti-dsDNA Kit; Trinity Biotech) and a selection of commercially available EIAs (Figure 1). All assays included in the study were calibrated against the Wo/80 reference material (5). Using the cutoff values for positivity proposed by the manufacturers, moderate to substantial kappa value agreement between the EIAs was observed ( 0.462–0.732). For the comparison between EIAs and the Farr assay, only moderate agreement was observed ( 0.573–0.657) (results not shown). Never-

The integration of the detection of systemic sclerosis-associated antibodies in a routine laboratory setting: comparison of different strategies

Abstract Background: Detection of systemic sclerosis-associated autoantibodies (SSc-Ab) is mostly restricted to anti-centromere and anti-topoisomerase-I. However, anti-RNA-polymerase-III and anti-PM/Scl are also important diagnostic markers for the disease supporting their incorporation in the laboratory repertoire. The aim of this study was to compare different testing strategies integrating the identification of these extra SSc-Ab in a routine testing algorithm. Methods: Sera from 144 consecutive SSc-patients and 265 controls were screened for antinuclear antibodies (ANA) by indirect immunofluorescence (ANA IIF) and tested for anti-extractable nuclear antigen (ENA) using five different assays that differ in their ability to detect SSc-Ab [two screening enzyme immunossays (EIA) with antigen mixtures, one multi-parameter line-immunoassay and two EIA with individual antigens]. Results: The application of SSc-Ab testing in cascade with the routine ANA/anti-ENA tests improved diagnostic performance characteristics. Besides the type of algorithm, also the number of antigens included in the screening EIA as well as the expected patient/control ratio, influenced the average expected costs and the number of additional SSc-Ab tests to be performed. In laboratories with an expected patient/control ratio of 0.002, cascade testing was most exploited by the use of a screening EIA that included all SSc-Ab as a secondary test after ANA IIF. Conclusions: Restriction of the performance of additional SSc-Ab assays based on the results of prior ANA/anti-ENA tests is a cost-effective strategy allowing optimized use of laboratory resources with minimal loss in diagnostic capacity.