All patients admitted to the Rheumatology Unit of the Second University of Naples for a suspected secondary RP from 1 November 2000 to 31 October 2010 were considered eligible for the study if they fulfilled LeRoy and Medsger's criteria  for RP, that is, bilateral, episodic bi- or triphasic color changes of fingers (pallor followed by dusky blueness and/or redness) induced by a cold challenge. Patients who met the ACR criteria for the classification of SSc  or any other connective tissue disease were excluded from the study, as were patients displaying any feature consistent with definite SSc , that is, skin sclerosis, digital ulcers/scars, two or more teleangectasias, clinically visible nailfold capillaries, cutaneous calcinosis, X-ray bibasilar lung fibrosis, X-ray esophageal dysmotility, ECG signs of myocardial fibrosis (cardiac blocks, Q waves), blood tests (serum creatinine) indicative of previous scleroderma renal crisis.
After giving informed written consent, according to standard clinical practice and to ensure a correct classification, the selected RP patients underwent: a detailed history and physical examination, to identify any of the previously listed clinical signs that excluded enrolment in the study, puffy fingers, and present or previous arthritis; routine laboratory investigations, devoted to exclude comorbidities and not relevant to the definition of the disease subset (that is, early SSc, definite SSc, UCTD), including blood cell count, urinalysis, blood urea nitrogen (BUN), serum creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), erythrosedimentation rate (ESR), serum protein electrophoresis with the evaluation of gammaglobulin concentration, serum C3 and C4 concentration; nailfold videocapillaroscopy (NVC) using an optical probe videocapillaroscope equipped with a ×200 magnification contact lens and connected to image analysis software (Videocap, DS MediGroup, Milan, Italy). The nailfold of the second, third, fourth and fifth finger was examined bilaterally in each patient. Four consecutive fields extending over 1 mm in the middle of the nailfold were studied per finger. The procedure was carried out by a physician (MI) experienced in NVC [8–10]; an autoantibody screening and profiling of sera collected at the first visit, performed as previously described , including antinuclear antibodies (ANA), SSc and other connective tissue disease marker autoantibodies, namely anti-Scl-70, anticentromere (ACA), anti-RNA polymerase III, anti-fibrillarin, anti-PmScl, anti-Th/To, anti-SSA, anti-SSB, anti-Sm, anti-Jo1, anti-U1RNP and anti-dsDNA antibodies; chest X-ray, barium esophageal X-ray and ECG to identify patients not eligible for the study because of findings consistent with lung, esophageal or cardiac SSc involvement as assessed by routine examinations and current treatment. Thus, patients satisfying the Koenig et al. criteria for early SSc , that is, RP plus either SSc marker autoantibodies and/or megacapillaries or avascular areas and no manifestation other than puffy fingers and/or arthritis, were enrolled in the study. In addition, patients who met the criteria for undifferentiated connective tissue disease (UCTD) (ANA positivity, but no SSc marker or any other connective tissue disease autoantibody, no scleroderma videocapillaroscopic findings or any clinical manifestation pathognomonic of any other connective tissue disease) [12, 13], were also enrolled in the study.
At baseline, all patients underwent B-mode echocardiography, lung function tests, and esophageal manometry. The detection of diastolic abnormalities at B-mode echocardiography, indicated by an inverted ratio between early (E)/late (atrial = A) ventricular filling velocity (E/A ratio < 1), in the absence of arterial hypertension, coronary artery disease and other symptoms/signs of cardiac disease, was regarded as early scleroderma heart involvement . The detection of a diffusing lung capacity for carbon monoxide (DLCO) or a forced vital capacity (FVC) <80% of the predicted values in the absence of a smoking habit and/or obstructive lung disease at lung function study was regarded as SSc lung involvement [15, 16]. The detection of a basal low esophageal sphincter (LES) pressure <15 mmHg, with or without impaired peristalsis, at esophageal manometry was regarded as early SSc esophageal involvement .
Lastly, 45 out of the enrolled 76 patients (21 early SSc and 24 UCTD patients) and 25 controls, matched for sex and age and affected with osteoarthritis or primary fibromyalgia syndrome, were investigated for serum endothelial, T-cell and fibroblast activation markers, namely soluble E-selectin (sE-selectin); soluble IL-2 receptor alpha (sIL-2Rα); carboxyterminal telopeptide of type I collagen (ICTP), and aminoterminal propeptide of type III collagen (PIIINP). SIL-2Rα and sE-selectin concentrations were measured by a multiplex suspension immunoassay. The assay was based on the use of polystyrene spectrally encoded beads of 5 to 6 μm diameter as the solid support. Each bead was coupled with a capture antibody specific to the analyte of interest. A first incubation step allowed the binding of the analyte from the test sample to the solid support, then the complex bead-capture antibody-analyte was challenged with a biotinylated detection antibody. Finally, a streptavidin-phycoerythrin conjugate, the reporter molecule, was added to the system to complete the reaction on the surface of each bead, thus labeling the analytes (either sIL-2Rα or sE-selectin). The assay was read with a double laser-based instrument (Luminex 200, Luminex Corporation, Austin, Texas, USA), which identifies each bead by a distinct spectral region and quantifies the concentration of the bound analyte (pg/ml) by analyzing the fluorescence intensity of the streptavidin-phycoerythrin conjugate. Capture and detection antibodies directed against sIL-2Rα and sE-selectin were both originated in mice. All reagents for this assay were provided by Merk Millipore, Billerica, MA, USA. ICTP and PIIINP concentrations were measured by a conventional competitive radio-immunoassay (RIA) and expressed as μg/l, using the UniQ kits by Orion Diagnostica, Espoo, Finland. Again, none of the investigated activation markers contributed to the definition of the disease subset (that is, early SSc, definite SSc, UCTD).
Each patient was re-evaluated every six months for symptoms/signs of SSc or any other connective tissue disease, and underwent yearly ECG, chest and esophageal X-ray, and lung function tests. The follow-up status was assessed in December 2011.
The study protocol was reviewed and approved by the local Ethics Committee.
GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, California, U.S.A.) and MedCalc 11.3 (MedCalc Software bvba, Mariakerke, Belgium) for Windows software were used for statistical analyses. Continuous data were expressed as mean ± SD and median with range, and were compared by Student's t-test or Mann-Whitney U test as appropriate. Categorical data were analysed by Fisher's exact test. Kaplan-Meier curves were used to describe the cumulative rates of SSc manifestations over time in the subgroups of patients, and the log-rank test was applied to analyse differences. Risk prediction was assessed by univariate and multivariate logistic regression analysis. Receiver-operating characteristic (ROC) curve analysis was performed to identify the cut-off values of both activation markers predicting the evolution of early SSc to definite SSc and their respective sensitivity and specificity. Statistical significance was expressed by a P-value < 0.05.