Anti-DFS70 autoantibodies in undifferentiated connective tissue diseases subjects: what’s on the horizon?

Anti-DFS70 autoantibodies in undifferentiated connective tissue diseases subjects: what’s on... Abstract Objective The main objective was to determine the prevalence of anti-dense fine speckled (DFS70) antibodies in a stable population of undifferentiated connective tissue disease (UCTD) to better define their potential role. Methods Immunological and clinical records of 91 long-standing UCTD patients were studied. DFS pattern was determined using the IIF ANA test on HEp-2 cells and anti-DFS70 antibodies were tested by chemiluminescence assay and by DFS70 line immunoassay. Results Twelve (13.2%) of 91 serum samples were positive for anti-DFS70 antibodies by chemiluminescence assay and line immunoassay. There was no statistical significance between the prevalence of anti-ENA and anti-DNA autoantibodies in patients with and without anti-DFS70 antibodies. No differences were found in the clinical characteristics of both groups. The presence of the anti-DFS70 antibodies was related to the younger age class. Conclusion The high prevalence of anti-DFS70 antibodies in the UCTD patients suggested the potential role of these autoantibodies as a marker in the evolution of UCTD to CTD. anti-DFS70 antibodies, undifferentiated connective tissue disease, antinuclear antibodies Rheumatology key messages The prevalence of anti-DFS70 autoantibodies in UCTD population is 13.3%. The presence of anti-DFS70 antibodies is associated with younger age in UCTD subjects. The anti-DFS70 autoantibodies could be a good biomarker in the evolution of UCTD to CTD. Introduction The clinical phenotype of connective tissue disease (CTD) is widely heterogeneous and can be undifferentiated when the presence of signs and symptoms suggestive of autoimmune disease do not fulfill the classification criteria for a defined CTD [1–8]. It has been shown that two-thirds of undifferentiated CTD (UCTD) patients eventually develop a classical CTD and typically within the first 5 years of suspected diagnosis [9–23]. UCTD may evolve into SLE, SSc, SS, mixed CTD, systemic vasculitis, PM/DM and more rarely, RA [10, 11, 13, 24–28]. UCTD patients were historically described as having undifferentiated or lanthanic collagen disease, since they clinically were characterized by an inflammatory tissue injury [29]. Later, in 1980, Le Roy introduced the new term UCTD to better define the early stage of CTD alluding to the concept that the condition could also remain indefinitely as an undifferentiated form of disease [30]. Although the pathogenesis of UCTD is still unknown, nowadays clinical and serological features are defined in the UCTD condition.The most common symptoms are arthralgia (37–80%), non-erosive arthritis (14–70%), RP (45–60%), leukopenia (11–42%), xerostomia (7–40%), xerophtalmia (8–36%), cutaneous rash and oral aphtosis [31]. The serological profile is positive for ANA (58–100%), anti-Ro/SSA (8–30%), anti-RNP (10–30%), anti-dsDNA and aPL [9, 13, 15, 19, 32, 33]. Moreover, 80% of UCTD patients commonly present a single autoantibody specificity [19]. Recently we reported two UCTD patients positive for ANA and anti-dense fine speckled 70 (DFS70) antibodies in which a monospecific finding of anti-DFS70 antibodies helped us to exclude a misdiagnosis of CTD [34]. These autoantibodies were traditionally detected by IIF assay on HEp-2 cells [35]. Recent studies have confirmed that monospecific anti-DFS70 autoantibodies are very uncommon in patients with autoimmune diseases. In many cases, the presence of these autoantibodies excludes the diagnosis of autoimmune diseases or the future development of autoimmunity in healthy subjects [36–41]. Previous studies found that anti-DFS70 antibodies are more prevalent in individuals that do not have a well-defined CTD and in most cases can be detected in apparently healthy individuals [39, 42]. From these observations and from the preliminary data on the two case reports described, we enlarged the UCTD cohort studied, aiming to determine the prevalence of anti-DFS70 antibodies in a stable UCTD population to better define their potential role. Furthermore, we investigated the best laboratory strategy to identify these autoantibodies in the UCTD cohort, considering two different tests to detect anti-DFS70 antibodies. Methods Patients Serum samples were obtained from 91 patients who were diagnosed as UCTD [87 females and 4 males; mean age 55 (15) years; mean disease duration 10 (3.3) years] who attended the Rheumatology Unit of San Giovanni di Dio Hospital, Florence (Italy), between June 2015 and September 2016. The inclusion criterion was diagnosis of UCTD with disease duration as assessed from the onset of the first symptom/sign ⩾5years [14]. Dense fine speckled (DFS) pattern was determined using the IIF ANA test on HEp-2 cells and anti-DFS70 antibodies were tested by chemiluminescence immunoassay (CIA) and by DFS70 line immunoassay (LIA) [38, 40, 43]. All samples were tested for anti-dsDNA and anti-ENA by BioPlex 2200 multiplex flow immunoassay (MFI). Serological analyses were performed blindly, and patient records and information remained anonymous. All patients gave their informed consent to this retrospective study according to the Declaration of Helsinki and to Italian legislation (Authorization of the Privacy Guarantor No. 9, 12 December 2013). The institutional review board, the Health Director of San Giovanni di Dio Hospital in Florence, reviewed and approved this study and the use of clinical and laboratory data of common clinical practice, with respect to privacy law, for clinical and scientific studies and publications. IIF assay on HEp-2 cells The DFS samples were identified by IIF-ANA test on HEp-2 cells using the NOVA Lite DAPI ANA kit processed on the QUANTA Lyser Instrument (Inova Diagnostics, San Diego, CA, USA). The results were interpreted by manual microscope reading and using the NOVA View Instrument (Inova Diagnostics), an automated fluorescence microscope that provides IIF pattern recognition for confirmation. The NOVA View software also outputs light intensity units where each sample was interpreted as negative or positive based on a pre-set cut-off (light intensity unit ⩾48 is positive). The samples were diluted with phosphate-buffered saline at 1: 80, according to the manufacturer’s instructions. In brief: 30 µl of each diluted serum was incubated on one well with fixed HEp-2 cells. After incubation and rinsing off, the cells were incubated with FITC-IgG-conjugated antibody. Anti-DFS70 antibodies detection by CIA Determination of anti-DFS70 antibodies was performed by CIA using QUANTA Flash DFS70 on the BIO-FLASH instrument (Inova Diagnostics). The antigen used in this test consisted of a recombinant DFS70 fragment expressed in Escherichia coli spanning amino acids 349–435, coated onto paramagnetic beads [44]. In brief, the diluted sera were incubated with paramagnetic beads. After incubation and rinsing off, isoluminol-conjugated secondary antibodies were added. After additional washing the results were expressed in chemiluminescence units proportional to the amount of anti-DFS70 antibodies in the serum samples. The assay cut-off for reactivity was 20 chemiluminescence units. Anti-DFS70 antibodies determined by LIA All the 91 samples were tested by the EUROLINE ANA Profile 3 lineblot (Euroimmun AG, Lübeck, Germany; Ro60, Ro52, La, RNP, Sm, Scl70, CENP-B, PM-Scl, Jo-1, PCNA, dsDNA, nucleosomes, histones, ribosomal P-proteins, AMA M2, DFS70) using a full-length DFS70 antigen [45]. Sera were incubated in accordance with the manufacturer’s standard protocol (30 min in serum, 30 min in anti-human immunoglobulin G/alkaline phosphatase and 10 min in 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium substrate). Reaction intensities expressed in greyscale units were automatically evaluated using commercially available EUROLineScan software (Euroimmun AG) and line intensity readings of >11 arbitrary units were considered specific. Anti-dsDNA and anti-ENA analysed with BioPlex2200 ANA screen (MFI) The BioPlex 2200 (Bio-Rad, Hercules, CA, USA) system is an automated analyser that uses multiplex bead technology (Luminex, Austin, TX, USA) to simultaneously detect antibodies to several antigens in a single tube. The BioPlex 2200 ANA Screen kit is intended for the qualitative screening of ANAs, the quantitative detection of antibodies to dsDNA and the semi-quantitative detection of 10 separate antibodies (chromatin, ribosomal P, SS-A, SS-B, Sm, SmRNP, RNP, Scl-70, Jo-1 and centromere B) in human serum and/or EDTA or heparinized plasma. Magnetic particles (8 μm diameter, carboxyl surface) are dyed with two fluorophores (classification dyes, CL1 and CL2) which emit at distinct wavelengths and adsorb significantly at 635 nm. The reporter fluorophore, B-phycoerythrin (PE) was chosen for its high molar extinction coefficient, quantum yield, resistance to photobleaching, lack of self-quenching and stability. The detector simultaneously measures light at four wavelengths; the two classification dyes, the reporter dye and the scatter of incident light. Following removal of excess conjugate, the magnetic beads are passed through the detector. Analyte concentration is proportional to the fluorescence intensity. Results are expressed in IU/ml for anti-dsDNA and Antibody Index for ANA antibodies and the results’ interpretation established by the manufacturer are 5–9 IU/ml (indeterminate) and ⩾10 IU/ml (positive) for dsDNA and ⩾1.0 Antibody Index (positive) for ANA antibodies. Statistical analysis The data were processed and analysed using the statistical program MedCalc for Windows version 15.2 (MedCalc Software, Ostend, Belgium). Data are presented as mean (s.e.m.). Significance of differences was analysed by Student’s t-test. For all statistical tests, P < 0.05 was considered significant. Results Twelve (13.3%) out of 91 subjects were positive for anti-DFS70 antibodies by CIA and LIA (Table 1). There were no significant differences between anti-ENA and anti-DNA antibody frequencies between patients with and without anti-DFS70 (P = 0.54 and P = 0.27, respectively). Considering the IIF-ANA testing, eight out of the 12 patients (75%) with anti-DFS70 autoantibodies showed a DFS pattern, and two of which had a second ANA pattern added. Moreover, two patients had a different pattern from the DFS pattern, and one was negative (Table 1). There was no difference on ANA titre between patients with and without anti-DFS70 antibodies (P = 0.4). The clinical characteristics of UCTD patients with and without anti-DFS70 autoantibodies were analysed by retrospective chart review and are summarized in Table 2. There were no statistical differences in the clinical characteristics of the two groups of patients. The mean age of patients with anti-DFS70 was significantly lower than subjects without anti-DFS70 [47 (13) vs 56 (16), P = 0.05]. There were no significant differences in the months of UCTD duration between patients with and without anti-DFS70 [9 (2) vs 10.4 (3.4), P = 0.13]. Table 1 Serological characterization of anti-DFS70 autoantibody-positive patients Patient No. Anti-DFS70 (CIA), CU Anti-DFS70 (LIA), AU IIF-ANA, LIU ANA pattern Anti-ENAa (MFI) Anti-dsDNAb (MFI) CLIFTb 1 450.8 130 556 Dense fine speckled and homogeneous N N — 2 410.4 113 293 Dense fine speckled N N — 3 393.7 142 788 Speckled and homogeneous N P N 4 248.0 110 352 Dense fine speckled N N — 5 246.0 116 1194 Dense fine speckled N N — 6 205.0 132 672 Speckled and dense fine speckled N N — 7 82.5 27 47 Dense fine speckled Anti-Ro N — 8 74.2 19 911 Dense fine speckled Anti-Ro Anti-La P P 1:20 9 38.8 72 781 Centromeric Anti-CENP B P N 10 37.6 80 217 Dense fine speckled Anti-RNP A Anti-RNP68 P N 11 28.4 64 52 Dense fine speckled N N — 12 23.1 47 85 Negative Anti-RNP A N — Patient No. Anti-DFS70 (CIA), CU Anti-DFS70 (LIA), AU IIF-ANA, LIU ANA pattern Anti-ENAa (MFI) Anti-dsDNAb (MFI) CLIFTb 1 450.8 130 556 Dense fine speckled and homogeneous N N — 2 410.4 113 293 Dense fine speckled N N — 3 393.7 142 788 Speckled and homogeneous N P N 4 248.0 110 352 Dense fine speckled N N — 5 246.0 116 1194 Dense fine speckled N N — 6 205.0 132 672 Speckled and dense fine speckled N N — 7 82.5 27 47 Dense fine speckled Anti-Ro N — 8 74.2 19 911 Dense fine speckled Anti-Ro Anti-La P P 1:20 9 38.8 72 781 Centromeric Anti-CENP B P N 10 37.6 80 217 Dense fine speckled Anti-RNP A Anti-RNP68 P N 11 28.4 64 52 Dense fine speckled N N — 12 23.1 47 85 Negative Anti-RNP A N — a Detection of autoantibodies against: chromatin, ribosomal P, Ro, La, Sm, SmRNP, RNP, Scl-70, Jo-1 and centromere B. b Anti-dsDNA positive samples by Multiplex (BioPlex) were analysed by Crithidia luciliae immunofluorescence test (CLIFT) (screening dilution 1:10). AU: arbitrary units; CIA: chemiluminescence assay; CU: chemiluminiscence units; DFS70: dense fine spleckled 70 kDa; IIF: indirect immunofluorescence (screening diluition 1:80); LIA: line immunoassay; LIU: light intensity units; MFI: multiplex flow immunoassay; N: negative; P: positive. Table 1 Serological characterization of anti-DFS70 autoantibody-positive patients Patient No. Anti-DFS70 (CIA), CU Anti-DFS70 (LIA), AU IIF-ANA, LIU ANA pattern Anti-ENAa (MFI) Anti-dsDNAb (MFI) CLIFTb 1 450.8 130 556 Dense fine speckled and homogeneous N N — 2 410.4 113 293 Dense fine speckled N N — 3 393.7 142 788 Speckled and homogeneous N P N 4 248.0 110 352 Dense fine speckled N N — 5 246.0 116 1194 Dense fine speckled N N — 6 205.0 132 672 Speckled and dense fine speckled N N — 7 82.5 27 47 Dense fine speckled Anti-Ro N — 8 74.2 19 911 Dense fine speckled Anti-Ro Anti-La P P 1:20 9 38.8 72 781 Centromeric Anti-CENP B P N 10 37.6 80 217 Dense fine speckled Anti-RNP A Anti-RNP68 P N 11 28.4 64 52 Dense fine speckled N N — 12 23.1 47 85 Negative Anti-RNP A N — Patient No. Anti-DFS70 (CIA), CU Anti-DFS70 (LIA), AU IIF-ANA, LIU ANA pattern Anti-ENAa (MFI) Anti-dsDNAb (MFI) CLIFTb 1 450.8 130 556 Dense fine speckled and homogeneous N N — 2 410.4 113 293 Dense fine speckled N N — 3 393.7 142 788 Speckled and homogeneous N P N 4 248.0 110 352 Dense fine speckled N N — 5 246.0 116 1194 Dense fine speckled N N — 6 205.0 132 672 Speckled and dense fine speckled N N — 7 82.5 27 47 Dense fine speckled Anti-Ro N — 8 74.2 19 911 Dense fine speckled Anti-Ro Anti-La P P 1:20 9 38.8 72 781 Centromeric Anti-CENP B P N 10 37.6 80 217 Dense fine speckled Anti-RNP A Anti-RNP68 P N 11 28.4 64 52 Dense fine speckled N N — 12 23.1 47 85 Negative Anti-RNP A N — a Detection of autoantibodies against: chromatin, ribosomal P, Ro, La, Sm, SmRNP, RNP, Scl-70, Jo-1 and centromere B. b Anti-dsDNA positive samples by Multiplex (BioPlex) were analysed by Crithidia luciliae immunofluorescence test (CLIFT) (screening dilution 1:10). AU: arbitrary units; CIA: chemiluminescence assay; CU: chemiluminiscence units; DFS70: dense fine spleckled 70 kDa; IIF: indirect immunofluorescence (screening diluition 1:80); LIA: line immunoassay; LIU: light intensity units; MFI: multiplex flow immunoassay; N: negative; P: positive. Table 2 Clinical characteristics of UCTD patients with (n = 12) and without (n = 79) anti-DFS70 antibodies Clinical manifestation Anti-DFS70+ (n = 12), n (%) Anti-DFS70− (n = 79), n (%) Arthralgia 4 (33.3) 26 (32.9) Arthritis 3 (25.0) 14 (17.7) Raynaud syndrome 3 (25.0) 10 (12.6) Photosensitivity 1 (8.3) 3 (3.8) Recurrent oral aphthosis 1 (8.3) 3 (3.8) Skin purpura 0 (0.0) 2 (2.5) Serositis 0 (0.0) 0 (0.0) Xerostomia 3 (25.0) 21 (26.6) Xerophtalmia 4 (33.3) 23 (29.1) Intestinal lung disease 0 (0.0) 0 (0.0) Leukopenia 4 (33.3) 26 (32.9) Haemolytic anaemia 1 (8.3) 3 (3.8) Thrombocytopenia 0 (0.0) 2 (2.5) Lymphopenia 0 (0.0) 0 (0.0) Thrombophlebitis 0 (0.0) 1 (1.3) Mononeuritis 0 (0.0) 1 (1.3) Clinical manifestation Anti-DFS70+ (n = 12), n (%) Anti-DFS70− (n = 79), n (%) Arthralgia 4 (33.3) 26 (32.9) Arthritis 3 (25.0) 14 (17.7) Raynaud syndrome 3 (25.0) 10 (12.6) Photosensitivity 1 (8.3) 3 (3.8) Recurrent oral aphthosis 1 (8.3) 3 (3.8) Skin purpura 0 (0.0) 2 (2.5) Serositis 0 (0.0) 0 (0.0) Xerostomia 3 (25.0) 21 (26.6) Xerophtalmia 4 (33.3) 23 (29.1) Intestinal lung disease 0 (0.0) 0 (0.0) Leukopenia 4 (33.3) 26 (32.9) Haemolytic anaemia 1 (8.3) 3 (3.8) Thrombocytopenia 0 (0.0) 2 (2.5) Lymphopenia 0 (0.0) 0 (0.0) Thrombophlebitis 0 (0.0) 1 (1.3) Mononeuritis 0 (0.0) 1 (1.3) DFS70: dense fine speckled 70 kDa. Table 2 Clinical characteristics of UCTD patients with (n = 12) and without (n = 79) anti-DFS70 antibodies Clinical manifestation Anti-DFS70+ (n = 12), n (%) Anti-DFS70− (n = 79), n (%) Arthralgia 4 (33.3) 26 (32.9) Arthritis 3 (25.0) 14 (17.7) Raynaud syndrome 3 (25.0) 10 (12.6) Photosensitivity 1 (8.3) 3 (3.8) Recurrent oral aphthosis 1 (8.3) 3 (3.8) Skin purpura 0 (0.0) 2 (2.5) Serositis 0 (0.0) 0 (0.0) Xerostomia 3 (25.0) 21 (26.6) Xerophtalmia 4 (33.3) 23 (29.1) Intestinal lung disease 0 (0.0) 0 (0.0) Leukopenia 4 (33.3) 26 (32.9) Haemolytic anaemia 1 (8.3) 3 (3.8) Thrombocytopenia 0 (0.0) 2 (2.5) Lymphopenia 0 (0.0) 0 (0.0) Thrombophlebitis 0 (0.0) 1 (1.3) Mononeuritis 0 (0.0) 1 (1.3) Clinical manifestation Anti-DFS70+ (n = 12), n (%) Anti-DFS70− (n = 79), n (%) Arthralgia 4 (33.3) 26 (32.9) Arthritis 3 (25.0) 14 (17.7) Raynaud syndrome 3 (25.0) 10 (12.6) Photosensitivity 1 (8.3) 3 (3.8) Recurrent oral aphthosis 1 (8.3) 3 (3.8) Skin purpura 0 (0.0) 2 (2.5) Serositis 0 (0.0) 0 (0.0) Xerostomia 3 (25.0) 21 (26.6) Xerophtalmia 4 (33.3) 23 (29.1) Intestinal lung disease 0 (0.0) 0 (0.0) Leukopenia 4 (33.3) 26 (32.9) Haemolytic anaemia 1 (8.3) 3 (3.8) Thrombocytopenia 0 (0.0) 2 (2.5) Lymphopenia 0 (0.0) 0 (0.0) Thrombophlebitis 0 (0.0) 1 (1.3) Mononeuritis 0 (0.0) 1 (1.3) DFS70: dense fine speckled 70 kDa. Discussion In recent years, better knowledge about the UCTD condition as a distinct clinical entity has underlined the importance of identifying patients at high risk of developing a defined CTD. Up to 50% of CTDs do not have a clearly defined profile at disease onset [46] and are referred to as UCTD. Three years after diagnosis 33–50% of UCTD cases will evolve into defined CTD, while ∼15–20% of all CTD will remain UCTD [21, 34, 47]. Certain combinations of clinical and laboratory features may predict the development of a defined CTD. For example, patients with fever, serositis and/or anti-Sm or anti-dsDNA antibodies are prone to developing SLE, while patients with Raynaud’s syndrome, abnormal nail fold capillaries and nucleolar ANA usually develop scleroderma [9–23]. However, since other patients do not develop specific clinical features or disease specific antibodies, reliable markers that may predict the evolution of UCTD to a defined form of CTD are important. The recently revised CTD classification and diagnostic criteria partially solved this critical point though there are still only a few biomarkers that predict UCTD evolution into defined CTD [14, 17]. Considering the therapeutic and prognostic impact of the disease, there is a great need for new serological markers that may help the clinician to differentiate stable and unstable UCTD. Relative to the serological profile, global ANA prevalence in UCTD is high, ranging from 58 to 100%. The IIF-ANA pattern can be considered an indicator of both high risk (i.e. homogeneous) and low risk (i.e. DFS), depending on the specific pattern considered [39, 40, 48]. Regarding the DFS IIF-ANA pattern, it can be difficult sometimes to accurately recognize it, resulting in misinterpretation. This is especially relevant when discriminating between DFS and mixed homogeneous and speckled or quasi-homogeneous patterns [40, 49, 50]. Additionally, the IIF-ANA test is not a reliable method to identify anti-DFS70 autoantibodies, because of its inconsistency [45]. The DFS pattern may be associated with the presence of anti-DFS70 antibodies but specific ANAs, including anti-dsDNA and anti-ENA, might lead to similar pattern recognition [51, 52]. In light of this, samples with DFS staining pattern identified by IIF should be tested for anti-DFS70 antibodies by a specific assay in order to avoid the misinterpretation of the ANA. Therefore the specific assays (CIA and LIA) are considered nowadays a better tool to identify the anti-DFS70 autoantibodies [45]. Our data (κ = 0.95) reinforced the excellent agreement between the two methods reported in literature and for this reason they can both be used interchangeably in the detection of the anti-DFS70 antibodies [38, 51, 53–56]. Additionally, consistent with the previous findings, we confirmed the remarkable prevalence in the UCTD individuals and the highest prevalence of anti-DFS70 antibodies in young females [54]. However, this finding has not been found in a recent paper on a large cohort of SLE patients [57]. The existing literature showed a high but heterogeneous prevalence of anti-DFS70 antibodies in UCTD patients ranging from 8.3 to 40% [36, 55], depending on the method used. We felt these data were worthy of further consideration and insights, and so we focused on a longstanding UCTD population to better define the role of antibodies as well as the best laboratory strategy, considering the currently available methods. Because our study confirmed the marker’s high frequency in the UCTD population studied (13.3%) and because it is uncommon in CTD patients, it follows that nowadays anti-DFS70 antibodies could represent a serological marker for UCTD and a reliable tool for excluding the evolution of UCTD to CTD. Taking into consideration the high prevalence of this condition in the rheumatology field and that a great percentage of UCTD patients will remain undifferentiated during follow-up, the cost-saving impact in developing predictive factors of evolution is increasingly clear. Since the 66.7% of the anti-DFS70 positive patients (8/12) were negative for anti-ENA and/or anti-dsDNA, the ANA testing could be interpreted as an ANA negative result in such situations, without any overestimation of the CTD evaluation diagnosis due to the ANA report. Consequently, detection of anti-DFS70 antibodies should be included in ANA testing algorithms to aid in interpreting ANA positivity in UCTD patients. The DFS pattern can be detected in ANA testing and it may act as an exclusion marker, especially when monospecific anti-DFS70 specificity is found. In fact, over the past decade monospecific anti-DFS70 antibodies have proved to be a crucial element in a reverse diagnostic flowchart [51] and therefore, similarly, in the UCTD population they could stratify patients at low risk for the CTD condition. The main limitation of the study was that we could not include a control group of a retrospective UCTD cohort developing a CTD in their next follow-up. However, it will be interesting to closely monitor the patients studied in the current paper for any changes in the clinical and serological phenotype of CTD over the years. Larger and prospective studies are needed to support these preliminary data and to better define the predictive role of anti-DFS70 antibodies in UCTD patients. Funding: No specific funding was received from any bodies in the public, commercial or not-for-profit sectors to carry out the work described in this manuscript. Disclosure statement: The authors have declared no conflicts of interest. References 1 Tan EM , Cohen AS , Fries JF et al. The 1982 revised criteria for the classification of systemic lupus erythematosus . Arthritis Rheum 1982 ; 25 : 1271 – 7 . Google Scholar CrossRef Search ADS PubMed 2 Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus . Arthritis Rheum 1997 ; 40 : 1725 . Google Scholar CrossRef Search ADS PubMed 3 Subcommittee for scleroderma criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Preliminary criteria for the classification of systemic sclerosis (scleroderma) . Arthritis Rheum 1980 ; 23 : 581 – 90 . CrossRef Search ADS PubMed 4 Bohan A , Peter JB. Polymyositis and dermatomyositis (first of two parts) . N Engl J Med 1975 ; 292 : 344 – 7 . Google Scholar CrossRef Search ADS PubMed 5 Bohan A , Peter JB. Polymyositis and dermatomyositis (second of two parts) . N Engl J Med 1975 ; 292 : 403 – 7 . Google Scholar CrossRef Search ADS PubMed 6 Arnett FC , Edworthy SM , Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis . Arthritis Rheum 1988 ; 31 : 315 – 24 . Google Scholar CrossRef Search ADS PubMed 7 Vitali C , Bombardieri S , Moutsopoulos HM et al. Preliminary criteria for the classification of Sjogren's syndrome. Results of a prospective concerted action supported by the European Community . Arthritis Rheum 1993 ; 36 : 340 – 7 . Google Scholar CrossRef Search ADS PubMed 8 Asherson RA , Khamashta MA , Ordi-Ros J et al. The “primary” antiphospholipid syndrome: major clinical and serological features . Medicine 1989 ; 68 : 366 – 74 . Google Scholar CrossRef Search ADS PubMed 9 Danieli MG , Fraticelli P , Salvi A , Gabrielli A , Danieli G. Undifferentiated connective tissue disease: natural history and evolution into definite CTD assessed in 84 patients initially diagnosed as early UCTD . Clin Rheumatol 1998 ; 17 : 195 – 201 . Google Scholar CrossRef Search ADS PubMed 10 Danieli MG , Fraticelli P , Franceschini F et al. Five-year follow-up of 165 Italian patients with undifferentiated connective tissue diseases . Clin Exp Rheumatol 1999 ; 17 : 585 – 91 . Google Scholar PubMed 11 Vila LM , Mayor AM , Valentin AH , Garcia SM , Vila S. Clinical outcome and predictors of disease evolution in patients with incomplete lupus erythematosus . Lupus 2000 ; 9 : 110 – 5 . Google Scholar CrossRef Search ADS PubMed 12 Swaak AJ , van de Brink H , Smeenk RJ et al. Incomplete lupus erythematosus: results of a multicentre study under the supervision of the EULAR Standing Committee on International Clinical Studies Including Therapeutic Trials (ESCISIT) . Rheumatology 2001 ; 40 : 89 – 94 . Google Scholar CrossRef Search ADS PubMed 13 Bodolay E , Csiki Z , Szekanecz Z et al. Five-year follow-up of 665 Hungarian patients with undifferentiated connective tissue disease (UCTD) . Clin Exp Rheumatol 2003 ; 21 : 313 – 20 . Google Scholar PubMed 14 Mosca M , Neri R , Bombardieri S. Undifferentiated connective tissue diseases (UCTD): a review of the literature and a proposal for preliminary classification criteria . Clin Exp Rheumatol 1999 ; 17 : 615 – 20 . Google Scholar PubMed 15 Mosca M , Neri R , Bencivelli W , Tavoni A , Bombardieri S. Undifferentiated connective tissue disease: analysis of 83 patients with a minimum followup of 5 years . J Rheumatol 2002 ; 29 : 2345 – 9 . Google Scholar PubMed 16 Mosca M , Neri R , Strigini F et al. Pregnancy outcome in patients with undifferentiated connective tissue disease: a preliminary study on 25 pregnancies . Lupus 2002 ; 11 : 304 – 7 . Google Scholar CrossRef Search ADS PubMed 17 Mosca M , Tani C , Bombardieri S. Undifferentiated connective tissue diseases (UCTD): a new frontier for rheumatology . Best Pract Res Clin Rheumatol 2007 ; 21 : 1011 – 23 . Google Scholar CrossRef Search ADS PubMed 18 Mosca M , Baldini C , Bombardieri S. Undifferentiated connective tissue diseases in 2004 . Clin Exp Rheumatol 2004 ; 22 : S14 – 8 . Google Scholar PubMed 19 Mosca M , Tani C , Neri C , Baldini C , Bombardieri S. Undifferentiated connective tissue diseases (UCTD) . Autoimmun Rev 2006 ; 6 : 1 – 4 . Google Scholar CrossRef Search ADS PubMed 20 Mosca M , Tani C , Bombardieri S. Defining undifferentiated connective tissue diseases: a challenge for rheumatologists . Lupus 2008 ; 17 : 278 – 80 . Google Scholar CrossRef Search ADS PubMed 21 Mosca M , Tani C , Carli L et al. Analysis of the evolution of UCTD to defined CTD after a long term follow-up . Clin Exp Rheumatol 2013 ; 31 : 471 . Google Scholar PubMed 22 Mosca M , Tani C , Talarico R , Bombardieri S. Undifferentiated connective tissue diseases (UCTD): simplified systemic autoimmune diseases . Autoimmun Rev 2011 ; 10 : 256 – 8 . Google Scholar CrossRef Search ADS PubMed 23 Vaz CC , Couto M , Medeiros D et al. Undifferentiated connective tissue disease: a seven-center cross-sectional study of 184 patients . Clin Rheumatol 2009 ; 28 : 915 – 21 . Google Scholar CrossRef Search ADS PubMed 24 Ganczarczyk L , Urowitz MB , Gladman DD. “Latent lupus”. J Rheumatol 1989 ; 16 : 475 – 8 . PubMed 25 Greer JM , Panush RS. Incomplete lupus erythematosus . Arch Intern Med 1989 ; 149 : 2473 – 6 . Google Scholar CrossRef Search ADS PubMed 26 Alarcon GS , Williams GV , Singer JZ et al. Early undifferentiated connective tissue disease. I. Early clinical manifestation in a large cohort of patients with undifferentiated connective tissue diseases compared with cohorts of well established connective tissue disease . J Rheumatol 1991 ; 18 : 1332 – 9 . Google Scholar PubMed 27 Dijkstra S , Nieuwenhuys EJ , Swaak AJ. The prognosis and outcome of patients referred to an outpatient clinic for rheumatic diseases characterized by the presence of antinuclear antibodies (ANA) . Scand J Rheumatol 1999 ; 28 : 33 – 7 . Google Scholar CrossRef Search ADS PubMed 28 Williams HJ , Alarcon GS , Joks R et al. Early undifferentiated connective tissue disease (CTD). VI. An inception cohort after 10 years: disease remissions and changes in diagnoses in well established and undifferentiated CTD . J Rheumatol 1999 ; 26 : 816 – 25 . Google Scholar PubMed 29 Sabo I. The lanthanic or undifferentiated collagen disease . Hiroshima J Med Sci 1969 ; 18 : 259 – 64 . Google Scholar PubMed 30 LeRoy EC , Maricq HR , Kahaleh MB. Undifferentiated connective tissue syndromes . Arthritis Rheum 1980 ; 23 : 341 – 3 . Google Scholar CrossRef Search ADS PubMed 31 Mosca M , Tani C , Carli L , Bombardieri S. Undifferentiated CTD: a wide spectrum of autoimmune diseases . Best Pract Res Clin Rheumatol 2012 ; 26 : 73 – 7 . Google Scholar CrossRef Search ADS PubMed 32 Mosca M , Tavoni A , Neri R , Bencivelli W , Bombardieri S. Undifferentiated connective tissue diseases: the clinical and serological profiles of 91 patients followed for at least 1 year . Lupus 1998 ; 7 : 95 – 100 . Google Scholar CrossRef Search ADS PubMed 33 Cavazzana I , Franceschini F , Belfiore N et al. Undifferentiated connective tissue disease with antibodies to Ro/SSa: clinical features and follow-up of 148 patients . Clin Exp Rheumatol 2001 ; 19 : 403 – 9 . Google Scholar PubMed 34 Infantino M , Meacci F , Grossi V et al. The clinical impact of Anti-DFS70 antibodies in undifferentiated connective tissue disease: case reports and a review of the literature . Immunol Res 2017 ; 65 : 293 – 5 . Google Scholar CrossRef Search ADS PubMed 35 Meroni PL , Schur PH. ANA screening: an old test with new recommendations . Ann Rheum Dis 2010 ; 69 : 1420 – 2 . Google Scholar CrossRef Search ADS PubMed 36 Muro Y , Sugiura K , Morita Y , Tomita Y. High concomitance of disease marker autoantibodies in anti-DFS70/LEDGF autoantibody-positive patients with autoimmune rheumatic disease . Lupus 2008 ; 17 : 171 – 6 . Google Scholar CrossRef Search ADS PubMed 37 Muro Y , Sugiura K , Nakashima R , Mimori T , Akiyama M. Low prevalence of anti-DFS70/LEDGF antibodies in patients with dermatomyositis and other systemic autoimmune rheumatic diseases . J Rheumatol 2013 ; 40 : 92 – 3 . Google Scholar CrossRef Search ADS PubMed 38 Mahler M , Hanly JG , Fritzler MJ. Importance of the dense fine speckled pattern on HEp-2 cells and anti-DFS70 antibodies for the diagnosis of systemic autoimmune diseases . Autoimmun Rev 2012 ; 11 : 642 – 5 . Google Scholar CrossRef Search ADS PubMed 39 Mahler M , Parker T , Peebles CL et al. Anti-DFS70/LEDGF antibodies are more prevalent in healthy individuals compared to patients with systemic autoimmune rheumatic diseases . J Rheumatol 2012 ; 39 : 2104 – 10 . Google Scholar CrossRef Search ADS PubMed 40 Mariz HA , Sato EI , Barbosa SH et al. Pattern on the antinuclear antibody-HEp-2 test is a critical parameter for discriminating antinuclear antibody-positive healthy individuals and patients with autoimmune rheumatic diseases . Arthritis Rheum 2011 ; 63 : 191 – 200 . Google Scholar CrossRef Search ADS PubMed 41 Schmeling H , Mahler M , Levy DM et al. Autoantibodies to dense fine speckles in pediatric diseases and controls . J Rheumatol 2015 ; 42 : 2419 – 26 . Google Scholar CrossRef Search ADS PubMed 42 Watanabe A , Kodera M , Sugiura K et al. Anti-DFS70 antibodies in 597 healthy hospital workers . Arthritis Rheum 2004 ; 50 : 892 – 900 . Google Scholar CrossRef Search ADS PubMed 43 Miyara M , Albesa R , Charuel JL et al. Clinical phenotypes of patients with anti-DFS70/LEDGF antibodies in a routine ANA referral cohort . Clin Dev Immunol 2013 ; 2013 : 703759 . Google Scholar CrossRef Search ADS PubMed 44 Bentow C , Lakos G , Rosenblum R et al. Clinical performance evaluation of a novel, automated chemiluminescent immunoassay, QUANTA Flash CTD Screen Plus . Immunol Res 2015 ; 61 : 110 – 6 . Google Scholar CrossRef Search ADS PubMed 45 Bizzaro N , Pesente F , Cucchiaro F et al. Anti-DFS70 antibodies detected by immunoblot methods: a reliable tool to confirm the dense fine speckles ANA pattern . J Immunol Methods 2016 ; 436 : 50 – 3 . Google Scholar CrossRef Search ADS PubMed 46 Doria A , Mosca M , Gambari PF , Bombardieri S. Defining unclassifiable connective tissue diseases: incomplete, undifferentiated, or both? J Rheumatol 2005 ; 32 : 213 – 5 . Google Scholar PubMed 47 Mosca M , Tani C , Neri C et al. [Analysis of the evolution to defined connective tissue diseases of patients with “early unidifferentiated connective tissue diseases (UCTD)”] . Reumatismo 2008 ; 60 : 35 – 40 . Google Scholar PubMed 48 Sherer Y , Gorstein A , Fritzler MJ , Shoenfeld Y. Autoantibody explosion in systemic lupus erythematosus: more than 100 different antibodies found in SLE patients . Semin Arthritis Rheum 2004 ; 34 : 501 – 37 . Google Scholar CrossRef Search ADS PubMed 49 Bizzaro N , Tonutti E , Villalta D. Recognizing the dense fine speckled/lens epithelium-derived growth factor/p75 pattern on HEP-2 cells: not an easy task! Comment on the article by Mariz et al . Arthritis Rheum 2011 ; 63 : 4036 – 7 . author reply 7–8. Google Scholar CrossRef Search ADS PubMed 50 Bentow C , Fritzler MJ , Mummert E , Mahler M. Recognition of the dense fine speckled (DFS) pattern remains challenging: results from an international internet-based survey . Auto Immun Highlights 2016 ; 7 : 8 . Google Scholar CrossRef Search ADS PubMed 51 Lee H , Kim Y , Han K , Oh EJ. Application of anti-DFS70 antibody and specific autoantibody test algorithms to patients with the dense fine speckled pattern on HEp-2 cells . Scand J Rheumatol 2016 ; 45 : 122 – 8 . Google Scholar CrossRef Search ADS PubMed 52 Basu A , Woods-Burnham L , Ortiz G et al. Specificity of antinuclear autoantibodies recognizing the dense fine speckled nuclear pattern: preferential targeting of DFS70/LEDGFp75 over its interacting partner MeCP2 . Clin Immunol 2015 ; 161 : 241 – 50 . Google Scholar CrossRef Search ADS PubMed 53 Mahler M , Fritzler MJ. The clinical significance of the dense fine speckled immunofluorescence pattern on HEp-2 cells for the diagnosis of systemic autoimmune diseases . Clin Dev Immunol 2012 ; 2012 : 494356 . Google Scholar CrossRef Search ADS PubMed 54 Conrad K , Rober N , Andrade LE , Mahler M. The clinical relevance of anti-DFS70 autoantibodies . Clin Rev Allergy Immunol 2017 ; 52 : 202 – 16 . Google Scholar CrossRef Search ADS PubMed 55 Bizzaro N , Tonutti E , Tampoia M et al. Specific chemoluminescence and immunoasdorption tests for anti-DFS70 antibodies avoid false positive results by indirect immunofluorescence . Clin Chim Acta 2015 ; 451(Pt B) : 271 – 7 . Google Scholar CrossRef Search ADS 56 Shovman O , Gilburd B , Chayat C et al. Prevalence of anti-DFS70 antibodies in patients with and without systemic autoimmune rheumatic diseases . Clin Exp Rheumatol 2018 ; 36 ( 1 ): 121 – 6 . Google Scholar PubMed 57 Choi MY , Clarke AE , St Pierre Y et al. The prevalence and determinants of anti-DFS70 autoantibodies in an international inception cohort of systemic lupus erythematosus patients . Lupus 2017 ; 26 : 1051 – 9 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Rheumatology Oxford University Press

Anti-DFS70 autoantibodies in undifferentiated connective tissue diseases subjects: what’s on the horizon?

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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10.1093/rheumatology/key012
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Abstract

Abstract Objective The main objective was to determine the prevalence of anti-dense fine speckled (DFS70) antibodies in a stable population of undifferentiated connective tissue disease (UCTD) to better define their potential role. Methods Immunological and clinical records of 91 long-standing UCTD patients were studied. DFS pattern was determined using the IIF ANA test on HEp-2 cells and anti-DFS70 antibodies were tested by chemiluminescence assay and by DFS70 line immunoassay. Results Twelve (13.2%) of 91 serum samples were positive for anti-DFS70 antibodies by chemiluminescence assay and line immunoassay. There was no statistical significance between the prevalence of anti-ENA and anti-DNA autoantibodies in patients with and without anti-DFS70 antibodies. No differences were found in the clinical characteristics of both groups. The presence of the anti-DFS70 antibodies was related to the younger age class. Conclusion The high prevalence of anti-DFS70 antibodies in the UCTD patients suggested the potential role of these autoantibodies as a marker in the evolution of UCTD to CTD. anti-DFS70 antibodies, undifferentiated connective tissue disease, antinuclear antibodies Rheumatology key messages The prevalence of anti-DFS70 autoantibodies in UCTD population is 13.3%. The presence of anti-DFS70 antibodies is associated with younger age in UCTD subjects. The anti-DFS70 autoantibodies could be a good biomarker in the evolution of UCTD to CTD. Introduction The clinical phenotype of connective tissue disease (CTD) is widely heterogeneous and can be undifferentiated when the presence of signs and symptoms suggestive of autoimmune disease do not fulfill the classification criteria for a defined CTD [1–8]. It has been shown that two-thirds of undifferentiated CTD (UCTD) patients eventually develop a classical CTD and typically within the first 5 years of suspected diagnosis [9–23]. UCTD may evolve into SLE, SSc, SS, mixed CTD, systemic vasculitis, PM/DM and more rarely, RA [10, 11, 13, 24–28]. UCTD patients were historically described as having undifferentiated or lanthanic collagen disease, since they clinically were characterized by an inflammatory tissue injury [29]. Later, in 1980, Le Roy introduced the new term UCTD to better define the early stage of CTD alluding to the concept that the condition could also remain indefinitely as an undifferentiated form of disease [30]. Although the pathogenesis of UCTD is still unknown, nowadays clinical and serological features are defined in the UCTD condition.The most common symptoms are arthralgia (37–80%), non-erosive arthritis (14–70%), RP (45–60%), leukopenia (11–42%), xerostomia (7–40%), xerophtalmia (8–36%), cutaneous rash and oral aphtosis [31]. The serological profile is positive for ANA (58–100%), anti-Ro/SSA (8–30%), anti-RNP (10–30%), anti-dsDNA and aPL [9, 13, 15, 19, 32, 33]. Moreover, 80% of UCTD patients commonly present a single autoantibody specificity [19]. Recently we reported two UCTD patients positive for ANA and anti-dense fine speckled 70 (DFS70) antibodies in which a monospecific finding of anti-DFS70 antibodies helped us to exclude a misdiagnosis of CTD [34]. These autoantibodies were traditionally detected by IIF assay on HEp-2 cells [35]. Recent studies have confirmed that monospecific anti-DFS70 autoantibodies are very uncommon in patients with autoimmune diseases. In many cases, the presence of these autoantibodies excludes the diagnosis of autoimmune diseases or the future development of autoimmunity in healthy subjects [36–41]. Previous studies found that anti-DFS70 antibodies are more prevalent in individuals that do not have a well-defined CTD and in most cases can be detected in apparently healthy individuals [39, 42]. From these observations and from the preliminary data on the two case reports described, we enlarged the UCTD cohort studied, aiming to determine the prevalence of anti-DFS70 antibodies in a stable UCTD population to better define their potential role. Furthermore, we investigated the best laboratory strategy to identify these autoantibodies in the UCTD cohort, considering two different tests to detect anti-DFS70 antibodies. Methods Patients Serum samples were obtained from 91 patients who were diagnosed as UCTD [87 females and 4 males; mean age 55 (15) years; mean disease duration 10 (3.3) years] who attended the Rheumatology Unit of San Giovanni di Dio Hospital, Florence (Italy), between June 2015 and September 2016. The inclusion criterion was diagnosis of UCTD with disease duration as assessed from the onset of the first symptom/sign ⩾5years [14]. Dense fine speckled (DFS) pattern was determined using the IIF ANA test on HEp-2 cells and anti-DFS70 antibodies were tested by chemiluminescence immunoassay (CIA) and by DFS70 line immunoassay (LIA) [38, 40, 43]. All samples were tested for anti-dsDNA and anti-ENA by BioPlex 2200 multiplex flow immunoassay (MFI). Serological analyses were performed blindly, and patient records and information remained anonymous. All patients gave their informed consent to this retrospective study according to the Declaration of Helsinki and to Italian legislation (Authorization of the Privacy Guarantor No. 9, 12 December 2013). The institutional review board, the Health Director of San Giovanni di Dio Hospital in Florence, reviewed and approved this study and the use of clinical and laboratory data of common clinical practice, with respect to privacy law, for clinical and scientific studies and publications. IIF assay on HEp-2 cells The DFS samples were identified by IIF-ANA test on HEp-2 cells using the NOVA Lite DAPI ANA kit processed on the QUANTA Lyser Instrument (Inova Diagnostics, San Diego, CA, USA). The results were interpreted by manual microscope reading and using the NOVA View Instrument (Inova Diagnostics), an automated fluorescence microscope that provides IIF pattern recognition for confirmation. The NOVA View software also outputs light intensity units where each sample was interpreted as negative or positive based on a pre-set cut-off (light intensity unit ⩾48 is positive). The samples were diluted with phosphate-buffered saline at 1: 80, according to the manufacturer’s instructions. In brief: 30 µl of each diluted serum was incubated on one well with fixed HEp-2 cells. After incubation and rinsing off, the cells were incubated with FITC-IgG-conjugated antibody. Anti-DFS70 antibodies detection by CIA Determination of anti-DFS70 antibodies was performed by CIA using QUANTA Flash DFS70 on the BIO-FLASH instrument (Inova Diagnostics). The antigen used in this test consisted of a recombinant DFS70 fragment expressed in Escherichia coli spanning amino acids 349–435, coated onto paramagnetic beads [44]. In brief, the diluted sera were incubated with paramagnetic beads. After incubation and rinsing off, isoluminol-conjugated secondary antibodies were added. After additional washing the results were expressed in chemiluminescence units proportional to the amount of anti-DFS70 antibodies in the serum samples. The assay cut-off for reactivity was 20 chemiluminescence units. Anti-DFS70 antibodies determined by LIA All the 91 samples were tested by the EUROLINE ANA Profile 3 lineblot (Euroimmun AG, Lübeck, Germany; Ro60, Ro52, La, RNP, Sm, Scl70, CENP-B, PM-Scl, Jo-1, PCNA, dsDNA, nucleosomes, histones, ribosomal P-proteins, AMA M2, DFS70) using a full-length DFS70 antigen [45]. Sera were incubated in accordance with the manufacturer’s standard protocol (30 min in serum, 30 min in anti-human immunoglobulin G/alkaline phosphatase and 10 min in 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium substrate). Reaction intensities expressed in greyscale units were automatically evaluated using commercially available EUROLineScan software (Euroimmun AG) and line intensity readings of >11 arbitrary units were considered specific. Anti-dsDNA and anti-ENA analysed with BioPlex2200 ANA screen (MFI) The BioPlex 2200 (Bio-Rad, Hercules, CA, USA) system is an automated analyser that uses multiplex bead technology (Luminex, Austin, TX, USA) to simultaneously detect antibodies to several antigens in a single tube. The BioPlex 2200 ANA Screen kit is intended for the qualitative screening of ANAs, the quantitative detection of antibodies to dsDNA and the semi-quantitative detection of 10 separate antibodies (chromatin, ribosomal P, SS-A, SS-B, Sm, SmRNP, RNP, Scl-70, Jo-1 and centromere B) in human serum and/or EDTA or heparinized plasma. Magnetic particles (8 μm diameter, carboxyl surface) are dyed with two fluorophores (classification dyes, CL1 and CL2) which emit at distinct wavelengths and adsorb significantly at 635 nm. The reporter fluorophore, B-phycoerythrin (PE) was chosen for its high molar extinction coefficient, quantum yield, resistance to photobleaching, lack of self-quenching and stability. The detector simultaneously measures light at four wavelengths; the two classification dyes, the reporter dye and the scatter of incident light. Following removal of excess conjugate, the magnetic beads are passed through the detector. Analyte concentration is proportional to the fluorescence intensity. Results are expressed in IU/ml for anti-dsDNA and Antibody Index for ANA antibodies and the results’ interpretation established by the manufacturer are 5–9 IU/ml (indeterminate) and ⩾10 IU/ml (positive) for dsDNA and ⩾1.0 Antibody Index (positive) for ANA antibodies. Statistical analysis The data were processed and analysed using the statistical program MedCalc for Windows version 15.2 (MedCalc Software, Ostend, Belgium). Data are presented as mean (s.e.m.). Significance of differences was analysed by Student’s t-test. For all statistical tests, P < 0.05 was considered significant. Results Twelve (13.3%) out of 91 subjects were positive for anti-DFS70 antibodies by CIA and LIA (Table 1). There were no significant differences between anti-ENA and anti-DNA antibody frequencies between patients with and without anti-DFS70 (P = 0.54 and P = 0.27, respectively). Considering the IIF-ANA testing, eight out of the 12 patients (75%) with anti-DFS70 autoantibodies showed a DFS pattern, and two of which had a second ANA pattern added. Moreover, two patients had a different pattern from the DFS pattern, and one was negative (Table 1). There was no difference on ANA titre between patients with and without anti-DFS70 antibodies (P = 0.4). The clinical characteristics of UCTD patients with and without anti-DFS70 autoantibodies were analysed by retrospective chart review and are summarized in Table 2. There were no statistical differences in the clinical characteristics of the two groups of patients. The mean age of patients with anti-DFS70 was significantly lower than subjects without anti-DFS70 [47 (13) vs 56 (16), P = 0.05]. There were no significant differences in the months of UCTD duration between patients with and without anti-DFS70 [9 (2) vs 10.4 (3.4), P = 0.13]. Table 1 Serological characterization of anti-DFS70 autoantibody-positive patients Patient No. Anti-DFS70 (CIA), CU Anti-DFS70 (LIA), AU IIF-ANA, LIU ANA pattern Anti-ENAa (MFI) Anti-dsDNAb (MFI) CLIFTb 1 450.8 130 556 Dense fine speckled and homogeneous N N — 2 410.4 113 293 Dense fine speckled N N — 3 393.7 142 788 Speckled and homogeneous N P N 4 248.0 110 352 Dense fine speckled N N — 5 246.0 116 1194 Dense fine speckled N N — 6 205.0 132 672 Speckled and dense fine speckled N N — 7 82.5 27 47 Dense fine speckled Anti-Ro N — 8 74.2 19 911 Dense fine speckled Anti-Ro Anti-La P P 1:20 9 38.8 72 781 Centromeric Anti-CENP B P N 10 37.6 80 217 Dense fine speckled Anti-RNP A Anti-RNP68 P N 11 28.4 64 52 Dense fine speckled N N — 12 23.1 47 85 Negative Anti-RNP A N — Patient No. Anti-DFS70 (CIA), CU Anti-DFS70 (LIA), AU IIF-ANA, LIU ANA pattern Anti-ENAa (MFI) Anti-dsDNAb (MFI) CLIFTb 1 450.8 130 556 Dense fine speckled and homogeneous N N — 2 410.4 113 293 Dense fine speckled N N — 3 393.7 142 788 Speckled and homogeneous N P N 4 248.0 110 352 Dense fine speckled N N — 5 246.0 116 1194 Dense fine speckled N N — 6 205.0 132 672 Speckled and dense fine speckled N N — 7 82.5 27 47 Dense fine speckled Anti-Ro N — 8 74.2 19 911 Dense fine speckled Anti-Ro Anti-La P P 1:20 9 38.8 72 781 Centromeric Anti-CENP B P N 10 37.6 80 217 Dense fine speckled Anti-RNP A Anti-RNP68 P N 11 28.4 64 52 Dense fine speckled N N — 12 23.1 47 85 Negative Anti-RNP A N — a Detection of autoantibodies against: chromatin, ribosomal P, Ro, La, Sm, SmRNP, RNP, Scl-70, Jo-1 and centromere B. b Anti-dsDNA positive samples by Multiplex (BioPlex) were analysed by Crithidia luciliae immunofluorescence test (CLIFT) (screening dilution 1:10). AU: arbitrary units; CIA: chemiluminescence assay; CU: chemiluminiscence units; DFS70: dense fine spleckled 70 kDa; IIF: indirect immunofluorescence (screening diluition 1:80); LIA: line immunoassay; LIU: light intensity units; MFI: multiplex flow immunoassay; N: negative; P: positive. Table 1 Serological characterization of anti-DFS70 autoantibody-positive patients Patient No. Anti-DFS70 (CIA), CU Anti-DFS70 (LIA), AU IIF-ANA, LIU ANA pattern Anti-ENAa (MFI) Anti-dsDNAb (MFI) CLIFTb 1 450.8 130 556 Dense fine speckled and homogeneous N N — 2 410.4 113 293 Dense fine speckled N N — 3 393.7 142 788 Speckled and homogeneous N P N 4 248.0 110 352 Dense fine speckled N N — 5 246.0 116 1194 Dense fine speckled N N — 6 205.0 132 672 Speckled and dense fine speckled N N — 7 82.5 27 47 Dense fine speckled Anti-Ro N — 8 74.2 19 911 Dense fine speckled Anti-Ro Anti-La P P 1:20 9 38.8 72 781 Centromeric Anti-CENP B P N 10 37.6 80 217 Dense fine speckled Anti-RNP A Anti-RNP68 P N 11 28.4 64 52 Dense fine speckled N N — 12 23.1 47 85 Negative Anti-RNP A N — Patient No. Anti-DFS70 (CIA), CU Anti-DFS70 (LIA), AU IIF-ANA, LIU ANA pattern Anti-ENAa (MFI) Anti-dsDNAb (MFI) CLIFTb 1 450.8 130 556 Dense fine speckled and homogeneous N N — 2 410.4 113 293 Dense fine speckled N N — 3 393.7 142 788 Speckled and homogeneous N P N 4 248.0 110 352 Dense fine speckled N N — 5 246.0 116 1194 Dense fine speckled N N — 6 205.0 132 672 Speckled and dense fine speckled N N — 7 82.5 27 47 Dense fine speckled Anti-Ro N — 8 74.2 19 911 Dense fine speckled Anti-Ro Anti-La P P 1:20 9 38.8 72 781 Centromeric Anti-CENP B P N 10 37.6 80 217 Dense fine speckled Anti-RNP A Anti-RNP68 P N 11 28.4 64 52 Dense fine speckled N N — 12 23.1 47 85 Negative Anti-RNP A N — a Detection of autoantibodies against: chromatin, ribosomal P, Ro, La, Sm, SmRNP, RNP, Scl-70, Jo-1 and centromere B. b Anti-dsDNA positive samples by Multiplex (BioPlex) were analysed by Crithidia luciliae immunofluorescence test (CLIFT) (screening dilution 1:10). AU: arbitrary units; CIA: chemiluminescence assay; CU: chemiluminiscence units; DFS70: dense fine spleckled 70 kDa; IIF: indirect immunofluorescence (screening diluition 1:80); LIA: line immunoassay; LIU: light intensity units; MFI: multiplex flow immunoassay; N: negative; P: positive. Table 2 Clinical characteristics of UCTD patients with (n = 12) and without (n = 79) anti-DFS70 antibodies Clinical manifestation Anti-DFS70+ (n = 12), n (%) Anti-DFS70− (n = 79), n (%) Arthralgia 4 (33.3) 26 (32.9) Arthritis 3 (25.0) 14 (17.7) Raynaud syndrome 3 (25.0) 10 (12.6) Photosensitivity 1 (8.3) 3 (3.8) Recurrent oral aphthosis 1 (8.3) 3 (3.8) Skin purpura 0 (0.0) 2 (2.5) Serositis 0 (0.0) 0 (0.0) Xerostomia 3 (25.0) 21 (26.6) Xerophtalmia 4 (33.3) 23 (29.1) Intestinal lung disease 0 (0.0) 0 (0.0) Leukopenia 4 (33.3) 26 (32.9) Haemolytic anaemia 1 (8.3) 3 (3.8) Thrombocytopenia 0 (0.0) 2 (2.5) Lymphopenia 0 (0.0) 0 (0.0) Thrombophlebitis 0 (0.0) 1 (1.3) Mononeuritis 0 (0.0) 1 (1.3) Clinical manifestation Anti-DFS70+ (n = 12), n (%) Anti-DFS70− (n = 79), n (%) Arthralgia 4 (33.3) 26 (32.9) Arthritis 3 (25.0) 14 (17.7) Raynaud syndrome 3 (25.0) 10 (12.6) Photosensitivity 1 (8.3) 3 (3.8) Recurrent oral aphthosis 1 (8.3) 3 (3.8) Skin purpura 0 (0.0) 2 (2.5) Serositis 0 (0.0) 0 (0.0) Xerostomia 3 (25.0) 21 (26.6) Xerophtalmia 4 (33.3) 23 (29.1) Intestinal lung disease 0 (0.0) 0 (0.0) Leukopenia 4 (33.3) 26 (32.9) Haemolytic anaemia 1 (8.3) 3 (3.8) Thrombocytopenia 0 (0.0) 2 (2.5) Lymphopenia 0 (0.0) 0 (0.0) Thrombophlebitis 0 (0.0) 1 (1.3) Mononeuritis 0 (0.0) 1 (1.3) DFS70: dense fine speckled 70 kDa. Table 2 Clinical characteristics of UCTD patients with (n = 12) and without (n = 79) anti-DFS70 antibodies Clinical manifestation Anti-DFS70+ (n = 12), n (%) Anti-DFS70− (n = 79), n (%) Arthralgia 4 (33.3) 26 (32.9) Arthritis 3 (25.0) 14 (17.7) Raynaud syndrome 3 (25.0) 10 (12.6) Photosensitivity 1 (8.3) 3 (3.8) Recurrent oral aphthosis 1 (8.3) 3 (3.8) Skin purpura 0 (0.0) 2 (2.5) Serositis 0 (0.0) 0 (0.0) Xerostomia 3 (25.0) 21 (26.6) Xerophtalmia 4 (33.3) 23 (29.1) Intestinal lung disease 0 (0.0) 0 (0.0) Leukopenia 4 (33.3) 26 (32.9) Haemolytic anaemia 1 (8.3) 3 (3.8) Thrombocytopenia 0 (0.0) 2 (2.5) Lymphopenia 0 (0.0) 0 (0.0) Thrombophlebitis 0 (0.0) 1 (1.3) Mononeuritis 0 (0.0) 1 (1.3) Clinical manifestation Anti-DFS70+ (n = 12), n (%) Anti-DFS70− (n = 79), n (%) Arthralgia 4 (33.3) 26 (32.9) Arthritis 3 (25.0) 14 (17.7) Raynaud syndrome 3 (25.0) 10 (12.6) Photosensitivity 1 (8.3) 3 (3.8) Recurrent oral aphthosis 1 (8.3) 3 (3.8) Skin purpura 0 (0.0) 2 (2.5) Serositis 0 (0.0) 0 (0.0) Xerostomia 3 (25.0) 21 (26.6) Xerophtalmia 4 (33.3) 23 (29.1) Intestinal lung disease 0 (0.0) 0 (0.0) Leukopenia 4 (33.3) 26 (32.9) Haemolytic anaemia 1 (8.3) 3 (3.8) Thrombocytopenia 0 (0.0) 2 (2.5) Lymphopenia 0 (0.0) 0 (0.0) Thrombophlebitis 0 (0.0) 1 (1.3) Mononeuritis 0 (0.0) 1 (1.3) DFS70: dense fine speckled 70 kDa. Discussion In recent years, better knowledge about the UCTD condition as a distinct clinical entity has underlined the importance of identifying patients at high risk of developing a defined CTD. Up to 50% of CTDs do not have a clearly defined profile at disease onset [46] and are referred to as UCTD. Three years after diagnosis 33–50% of UCTD cases will evolve into defined CTD, while ∼15–20% of all CTD will remain UCTD [21, 34, 47]. Certain combinations of clinical and laboratory features may predict the development of a defined CTD. For example, patients with fever, serositis and/or anti-Sm or anti-dsDNA antibodies are prone to developing SLE, while patients with Raynaud’s syndrome, abnormal nail fold capillaries and nucleolar ANA usually develop scleroderma [9–23]. However, since other patients do not develop specific clinical features or disease specific antibodies, reliable markers that may predict the evolution of UCTD to a defined form of CTD are important. The recently revised CTD classification and diagnostic criteria partially solved this critical point though there are still only a few biomarkers that predict UCTD evolution into defined CTD [14, 17]. Considering the therapeutic and prognostic impact of the disease, there is a great need for new serological markers that may help the clinician to differentiate stable and unstable UCTD. Relative to the serological profile, global ANA prevalence in UCTD is high, ranging from 58 to 100%. The IIF-ANA pattern can be considered an indicator of both high risk (i.e. homogeneous) and low risk (i.e. DFS), depending on the specific pattern considered [39, 40, 48]. Regarding the DFS IIF-ANA pattern, it can be difficult sometimes to accurately recognize it, resulting in misinterpretation. This is especially relevant when discriminating between DFS and mixed homogeneous and speckled or quasi-homogeneous patterns [40, 49, 50]. Additionally, the IIF-ANA test is not a reliable method to identify anti-DFS70 autoantibodies, because of its inconsistency [45]. The DFS pattern may be associated with the presence of anti-DFS70 antibodies but specific ANAs, including anti-dsDNA and anti-ENA, might lead to similar pattern recognition [51, 52]. In light of this, samples with DFS staining pattern identified by IIF should be tested for anti-DFS70 antibodies by a specific assay in order to avoid the misinterpretation of the ANA. Therefore the specific assays (CIA and LIA) are considered nowadays a better tool to identify the anti-DFS70 autoantibodies [45]. Our data (κ = 0.95) reinforced the excellent agreement between the two methods reported in literature and for this reason they can both be used interchangeably in the detection of the anti-DFS70 antibodies [38, 51, 53–56]. Additionally, consistent with the previous findings, we confirmed the remarkable prevalence in the UCTD individuals and the highest prevalence of anti-DFS70 antibodies in young females [54]. However, this finding has not been found in a recent paper on a large cohort of SLE patients [57]. The existing literature showed a high but heterogeneous prevalence of anti-DFS70 antibodies in UCTD patients ranging from 8.3 to 40% [36, 55], depending on the method used. We felt these data were worthy of further consideration and insights, and so we focused on a longstanding UCTD population to better define the role of antibodies as well as the best laboratory strategy, considering the currently available methods. Because our study confirmed the marker’s high frequency in the UCTD population studied (13.3%) and because it is uncommon in CTD patients, it follows that nowadays anti-DFS70 antibodies could represent a serological marker for UCTD and a reliable tool for excluding the evolution of UCTD to CTD. Taking into consideration the high prevalence of this condition in the rheumatology field and that a great percentage of UCTD patients will remain undifferentiated during follow-up, the cost-saving impact in developing predictive factors of evolution is increasingly clear. Since the 66.7% of the anti-DFS70 positive patients (8/12) were negative for anti-ENA and/or anti-dsDNA, the ANA testing could be interpreted as an ANA negative result in such situations, without any overestimation of the CTD evaluation diagnosis due to the ANA report. Consequently, detection of anti-DFS70 antibodies should be included in ANA testing algorithms to aid in interpreting ANA positivity in UCTD patients. The DFS pattern can be detected in ANA testing and it may act as an exclusion marker, especially when monospecific anti-DFS70 specificity is found. In fact, over the past decade monospecific anti-DFS70 antibodies have proved to be a crucial element in a reverse diagnostic flowchart [51] and therefore, similarly, in the UCTD population they could stratify patients at low risk for the CTD condition. The main limitation of the study was that we could not include a control group of a retrospective UCTD cohort developing a CTD in their next follow-up. However, it will be interesting to closely monitor the patients studied in the current paper for any changes in the clinical and serological phenotype of CTD over the years. Larger and prospective studies are needed to support these preliminary data and to better define the predictive role of anti-DFS70 antibodies in UCTD patients. Funding: No specific funding was received from any bodies in the public, commercial or not-for-profit sectors to carry out the work described in this manuscript. Disclosure statement: The authors have declared no conflicts of interest. References 1 Tan EM , Cohen AS , Fries JF et al. The 1982 revised criteria for the classification of systemic lupus erythematosus . Arthritis Rheum 1982 ; 25 : 1271 – 7 . Google Scholar CrossRef Search ADS PubMed 2 Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus . Arthritis Rheum 1997 ; 40 : 1725 . Google Scholar CrossRef Search ADS PubMed 3 Subcommittee for scleroderma criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Preliminary criteria for the classification of systemic sclerosis (scleroderma) . Arthritis Rheum 1980 ; 23 : 581 – 90 . CrossRef Search ADS PubMed 4 Bohan A , Peter JB. Polymyositis and dermatomyositis (first of two parts) . N Engl J Med 1975 ; 292 : 344 – 7 . Google Scholar CrossRef Search ADS PubMed 5 Bohan A , Peter JB. Polymyositis and dermatomyositis (second of two parts) . N Engl J Med 1975 ; 292 : 403 – 7 . Google Scholar CrossRef Search ADS PubMed 6 Arnett FC , Edworthy SM , Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis . Arthritis Rheum 1988 ; 31 : 315 – 24 . Google Scholar CrossRef Search ADS PubMed 7 Vitali C , Bombardieri S , Moutsopoulos HM et al. Preliminary criteria for the classification of Sjogren's syndrome. Results of a prospective concerted action supported by the European Community . Arthritis Rheum 1993 ; 36 : 340 – 7 . Google Scholar CrossRef Search ADS PubMed 8 Asherson RA , Khamashta MA , Ordi-Ros J et al. The “primary” antiphospholipid syndrome: major clinical and serological features . Medicine 1989 ; 68 : 366 – 74 . Google Scholar CrossRef Search ADS PubMed 9 Danieli MG , Fraticelli P , Salvi A , Gabrielli A , Danieli G. Undifferentiated connective tissue disease: natural history and evolution into definite CTD assessed in 84 patients initially diagnosed as early UCTD . Clin Rheumatol 1998 ; 17 : 195 – 201 . Google Scholar CrossRef Search ADS PubMed 10 Danieli MG , Fraticelli P , Franceschini F et al. Five-year follow-up of 165 Italian patients with undifferentiated connective tissue diseases . Clin Exp Rheumatol 1999 ; 17 : 585 – 91 . Google Scholar PubMed 11 Vila LM , Mayor AM , Valentin AH , Garcia SM , Vila S. Clinical outcome and predictors of disease evolution in patients with incomplete lupus erythematosus . Lupus 2000 ; 9 : 110 – 5 . Google Scholar CrossRef Search ADS PubMed 12 Swaak AJ , van de Brink H , Smeenk RJ et al. Incomplete lupus erythematosus: results of a multicentre study under the supervision of the EULAR Standing Committee on International Clinical Studies Including Therapeutic Trials (ESCISIT) . Rheumatology 2001 ; 40 : 89 – 94 . Google Scholar CrossRef Search ADS PubMed 13 Bodolay E , Csiki Z , Szekanecz Z et al. Five-year follow-up of 665 Hungarian patients with undifferentiated connective tissue disease (UCTD) . Clin Exp Rheumatol 2003 ; 21 : 313 – 20 . Google Scholar PubMed 14 Mosca M , Neri R , Bombardieri S. Undifferentiated connective tissue diseases (UCTD): a review of the literature and a proposal for preliminary classification criteria . Clin Exp Rheumatol 1999 ; 17 : 615 – 20 . Google Scholar PubMed 15 Mosca M , Neri R , Bencivelli W , Tavoni A , Bombardieri S. Undifferentiated connective tissue disease: analysis of 83 patients with a minimum followup of 5 years . J Rheumatol 2002 ; 29 : 2345 – 9 . Google Scholar PubMed 16 Mosca M , Neri R , Strigini F et al. Pregnancy outcome in patients with undifferentiated connective tissue disease: a preliminary study on 25 pregnancies . Lupus 2002 ; 11 : 304 – 7 . Google Scholar CrossRef Search ADS PubMed 17 Mosca M , Tani C , Bombardieri S. Undifferentiated connective tissue diseases (UCTD): a new frontier for rheumatology . Best Pract Res Clin Rheumatol 2007 ; 21 : 1011 – 23 . Google Scholar CrossRef Search ADS PubMed 18 Mosca M , Baldini C , Bombardieri S. Undifferentiated connective tissue diseases in 2004 . Clin Exp Rheumatol 2004 ; 22 : S14 – 8 . Google Scholar PubMed 19 Mosca M , Tani C , Neri C , Baldini C , Bombardieri S. Undifferentiated connective tissue diseases (UCTD) . Autoimmun Rev 2006 ; 6 : 1 – 4 . Google Scholar CrossRef Search ADS PubMed 20 Mosca M , Tani C , Bombardieri S. Defining undifferentiated connective tissue diseases: a challenge for rheumatologists . Lupus 2008 ; 17 : 278 – 80 . Google Scholar CrossRef Search ADS PubMed 21 Mosca M , Tani C , Carli L et al. Analysis of the evolution of UCTD to defined CTD after a long term follow-up . Clin Exp Rheumatol 2013 ; 31 : 471 . Google Scholar PubMed 22 Mosca M , Tani C , Talarico R , Bombardieri S. Undifferentiated connective tissue diseases (UCTD): simplified systemic autoimmune diseases . Autoimmun Rev 2011 ; 10 : 256 – 8 . Google Scholar CrossRef Search ADS PubMed 23 Vaz CC , Couto M , Medeiros D et al. Undifferentiated connective tissue disease: a seven-center cross-sectional study of 184 patients . Clin Rheumatol 2009 ; 28 : 915 – 21 . Google Scholar CrossRef Search ADS PubMed 24 Ganczarczyk L , Urowitz MB , Gladman DD. “Latent lupus”. J Rheumatol 1989 ; 16 : 475 – 8 . PubMed 25 Greer JM , Panush RS. Incomplete lupus erythematosus . Arch Intern Med 1989 ; 149 : 2473 – 6 . Google Scholar CrossRef Search ADS PubMed 26 Alarcon GS , Williams GV , Singer JZ et al. Early undifferentiated connective tissue disease. I. Early clinical manifestation in a large cohort of patients with undifferentiated connective tissue diseases compared with cohorts of well established connective tissue disease . J Rheumatol 1991 ; 18 : 1332 – 9 . Google Scholar PubMed 27 Dijkstra S , Nieuwenhuys EJ , Swaak AJ. The prognosis and outcome of patients referred to an outpatient clinic for rheumatic diseases characterized by the presence of antinuclear antibodies (ANA) . Scand J Rheumatol 1999 ; 28 : 33 – 7 . Google Scholar CrossRef Search ADS PubMed 28 Williams HJ , Alarcon GS , Joks R et al. Early undifferentiated connective tissue disease (CTD). VI. An inception cohort after 10 years: disease remissions and changes in diagnoses in well established and undifferentiated CTD . J Rheumatol 1999 ; 26 : 816 – 25 . Google Scholar PubMed 29 Sabo I. The lanthanic or undifferentiated collagen disease . Hiroshima J Med Sci 1969 ; 18 : 259 – 64 . Google Scholar PubMed 30 LeRoy EC , Maricq HR , Kahaleh MB. Undifferentiated connective tissue syndromes . Arthritis Rheum 1980 ; 23 : 341 – 3 . Google Scholar CrossRef Search ADS PubMed 31 Mosca M , Tani C , Carli L , Bombardieri S. Undifferentiated CTD: a wide spectrum of autoimmune diseases . Best Pract Res Clin Rheumatol 2012 ; 26 : 73 – 7 . Google Scholar CrossRef Search ADS PubMed 32 Mosca M , Tavoni A , Neri R , Bencivelli W , Bombardieri S. Undifferentiated connective tissue diseases: the clinical and serological profiles of 91 patients followed for at least 1 year . Lupus 1998 ; 7 : 95 – 100 . Google Scholar CrossRef Search ADS PubMed 33 Cavazzana I , Franceschini F , Belfiore N et al. Undifferentiated connective tissue disease with antibodies to Ro/SSa: clinical features and follow-up of 148 patients . Clin Exp Rheumatol 2001 ; 19 : 403 – 9 . Google Scholar PubMed 34 Infantino M , Meacci F , Grossi V et al. The clinical impact of Anti-DFS70 antibodies in undifferentiated connective tissue disease: case reports and a review of the literature . Immunol Res 2017 ; 65 : 293 – 5 . Google Scholar CrossRef Search ADS PubMed 35 Meroni PL , Schur PH. ANA screening: an old test with new recommendations . Ann Rheum Dis 2010 ; 69 : 1420 – 2 . Google Scholar CrossRef Search ADS PubMed 36 Muro Y , Sugiura K , Morita Y , Tomita Y. High concomitance of disease marker autoantibodies in anti-DFS70/LEDGF autoantibody-positive patients with autoimmune rheumatic disease . Lupus 2008 ; 17 : 171 – 6 . Google Scholar CrossRef Search ADS PubMed 37 Muro Y , Sugiura K , Nakashima R , Mimori T , Akiyama M. Low prevalence of anti-DFS70/LEDGF antibodies in patients with dermatomyositis and other systemic autoimmune rheumatic diseases . J Rheumatol 2013 ; 40 : 92 – 3 . Google Scholar CrossRef Search ADS PubMed 38 Mahler M , Hanly JG , Fritzler MJ. Importance of the dense fine speckled pattern on HEp-2 cells and anti-DFS70 antibodies for the diagnosis of systemic autoimmune diseases . Autoimmun Rev 2012 ; 11 : 642 – 5 . Google Scholar CrossRef Search ADS PubMed 39 Mahler M , Parker T , Peebles CL et al. Anti-DFS70/LEDGF antibodies are more prevalent in healthy individuals compared to patients with systemic autoimmune rheumatic diseases . J Rheumatol 2012 ; 39 : 2104 – 10 . Google Scholar CrossRef Search ADS PubMed 40 Mariz HA , Sato EI , Barbosa SH et al. Pattern on the antinuclear antibody-HEp-2 test is a critical parameter for discriminating antinuclear antibody-positive healthy individuals and patients with autoimmune rheumatic diseases . Arthritis Rheum 2011 ; 63 : 191 – 200 . Google Scholar CrossRef Search ADS PubMed 41 Schmeling H , Mahler M , Levy DM et al. Autoantibodies to dense fine speckles in pediatric diseases and controls . J Rheumatol 2015 ; 42 : 2419 – 26 . Google Scholar CrossRef Search ADS PubMed 42 Watanabe A , Kodera M , Sugiura K et al. Anti-DFS70 antibodies in 597 healthy hospital workers . Arthritis Rheum 2004 ; 50 : 892 – 900 . Google Scholar CrossRef Search ADS PubMed 43 Miyara M , Albesa R , Charuel JL et al. Clinical phenotypes of patients with anti-DFS70/LEDGF antibodies in a routine ANA referral cohort . Clin Dev Immunol 2013 ; 2013 : 703759 . Google Scholar CrossRef Search ADS PubMed 44 Bentow C , Lakos G , Rosenblum R et al. Clinical performance evaluation of a novel, automated chemiluminescent immunoassay, QUANTA Flash CTD Screen Plus . Immunol Res 2015 ; 61 : 110 – 6 . Google Scholar CrossRef Search ADS PubMed 45 Bizzaro N , Pesente F , Cucchiaro F et al. Anti-DFS70 antibodies detected by immunoblot methods: a reliable tool to confirm the dense fine speckles ANA pattern . J Immunol Methods 2016 ; 436 : 50 – 3 . Google Scholar CrossRef Search ADS PubMed 46 Doria A , Mosca M , Gambari PF , Bombardieri S. Defining unclassifiable connective tissue diseases: incomplete, undifferentiated, or both? J Rheumatol 2005 ; 32 : 213 – 5 . Google Scholar PubMed 47 Mosca M , Tani C , Neri C et al. [Analysis of the evolution to defined connective tissue diseases of patients with “early unidifferentiated connective tissue diseases (UCTD)”] . Reumatismo 2008 ; 60 : 35 – 40 . Google Scholar PubMed 48 Sherer Y , Gorstein A , Fritzler MJ , Shoenfeld Y. Autoantibody explosion in systemic lupus erythematosus: more than 100 different antibodies found in SLE patients . Semin Arthritis Rheum 2004 ; 34 : 501 – 37 . Google Scholar CrossRef Search ADS PubMed 49 Bizzaro N , Tonutti E , Villalta D. Recognizing the dense fine speckled/lens epithelium-derived growth factor/p75 pattern on HEP-2 cells: not an easy task! Comment on the article by Mariz et al . Arthritis Rheum 2011 ; 63 : 4036 – 7 . author reply 7–8. Google Scholar CrossRef Search ADS PubMed 50 Bentow C , Fritzler MJ , Mummert E , Mahler M. Recognition of the dense fine speckled (DFS) pattern remains challenging: results from an international internet-based survey . Auto Immun Highlights 2016 ; 7 : 8 . Google Scholar CrossRef Search ADS PubMed 51 Lee H , Kim Y , Han K , Oh EJ. Application of anti-DFS70 antibody and specific autoantibody test algorithms to patients with the dense fine speckled pattern on HEp-2 cells . Scand J Rheumatol 2016 ; 45 : 122 – 8 . Google Scholar CrossRef Search ADS PubMed 52 Basu A , Woods-Burnham L , Ortiz G et al. Specificity of antinuclear autoantibodies recognizing the dense fine speckled nuclear pattern: preferential targeting of DFS70/LEDGFp75 over its interacting partner MeCP2 . Clin Immunol 2015 ; 161 : 241 – 50 . Google Scholar CrossRef Search ADS PubMed 53 Mahler M , Fritzler MJ. The clinical significance of the dense fine speckled immunofluorescence pattern on HEp-2 cells for the diagnosis of systemic autoimmune diseases . Clin Dev Immunol 2012 ; 2012 : 494356 . Google Scholar CrossRef Search ADS PubMed 54 Conrad K , Rober N , Andrade LE , Mahler M. The clinical relevance of anti-DFS70 autoantibodies . Clin Rev Allergy Immunol 2017 ; 52 : 202 – 16 . Google Scholar CrossRef Search ADS PubMed 55 Bizzaro N , Tonutti E , Tampoia M et al. Specific chemoluminescence and immunoasdorption tests for anti-DFS70 antibodies avoid false positive results by indirect immunofluorescence . Clin Chim Acta 2015 ; 451(Pt B) : 271 – 7 . Google Scholar CrossRef Search ADS 56 Shovman O , Gilburd B , Chayat C et al. Prevalence of anti-DFS70 antibodies in patients with and without systemic autoimmune rheumatic diseases . Clin Exp Rheumatol 2018 ; 36 ( 1 ): 121 – 6 . Google Scholar PubMed 57 Choi MY , Clarke AE , St Pierre Y et al. The prevalence and determinants of anti-DFS70 autoantibodies in an international inception cohort of systemic lupus erythematosus patients . Lupus 2017 ; 26 : 1051 – 9 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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RheumatologyOxford University Press

Published: Apr 3, 2018

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