Biomarkers of B cell activation in autoimmune connective tissue diseases: More than markers of disease activity.

B cells play a central role in the pathogenesis of many autoimmune diseases, acting as antigen-presenting cells, producing inflammatory cytokines, and acting as a source of autoantibodies after differentiating into plasma cells. In this review, we aim to summarize and synthesize the literature for the utility of biomarkers of B cell activation (plasma immunoglobulins (Ig), free light chains (FLCs), and beta-2 microglobulin (ß2M)) in monitoring inflammatory rheumatic connective tissue diseases, such as Sjogren's syndrome (SS), systemic lupus erythematosus (SLE), dermatomyositis (DM), and systemic sclerosis (SSc). Clinically, it is quite difficult to gauge prognosis in these conditions as there, historically, have not been many quantitative markers of disease activity available. From our extensive literature review, Ig, FLC, and ß2M may function as invaluable prognostic markers of ongoing disease activity, and potentially as biomarkers for response to therapy or disease relapse. They are inexpensive and unsophisticated tests that are vastly underused in the setting of autoimmune disease. However, clinicians still need to be aware of the potential of false positives in times of infection or plasma cell dyscrasia, as these disease states can artificially increase these biomarkers. Ultimately, the utility of serum Ig, FLCs, and ß2M is clearly delineated in SS and SLE, and least investigated in DM, and additional prospective studies utilizing these biomarkers, and specific B cell targeted therapies are still needed.

The effect of vitamin D3 and thyroid hormones on the capillaroscopy-confirmed microangiopathy in pediatric patients with a suspicion of systemic connective tissue disease-a single-center experience with Raynaud phenomenon.

Raynaud phenomenon (RP) may be the first manifestation of a systemic connective tissue disease (SCTD). Early detection of dysfunction of small vessels called microangiopathy is essential for the diagnostic process. The focus of this single-center, retrospective study was to investigate the potential dependencies between microvascular image and laboratory markers measured in children with RP. The study analyzed the nail-fold video-capillaroscopy (NVC) findings and laboratory results of 81 children between the ages 6 and 17 who were referred to pediatric rheumatologist with a suspicion of SCTD. Out of 52 patients presenting with RP at the time of evaluation, abnormalities in capillary microscopic imaging were found in 34. NVC findings were then compared to levels of specific biomarkers in serum. Vitamin D3 serum levels have been significantly decreased in patients with RP (23.4 ng/mL ± 8.76 vs. 30.0 ng/mL ± 12.66, P = 0.0148). There were positive significant correlations between levels of vitamin D3 and acute-phase reactants in serum, such as C-reactive protein (P = 0.0292). Furthermore, free thyroxine levels (fT4) in patients with both RP (P = 0.0126) and micro-angiopathy (P = 0.05496) persisted in the lower range of the normal limit (< 1.0 ng/dL). Regular oral supplementation of vitamin D3 should be always considered in children with RP if deficiency is found. Additionally, low fT4 level (< 1.0 ng/dL) should be considered as an indication to perform NVC in patients suspected of SCTD even when they do not present RP.

Interstitial Lung Disease in Connective Tissue Disease: A Common Lesion With Heterogeneous Mechanisms and Treatment Considerations.

Connective tissue disease (CTD) related interstitial lung disease (CTD-ILD) is one of the leading causes of morbidity and mortality of CTD. Clinically, CTD-ILD is highly heterogenous and involves rheumatic immunity and multiple manifestations of respiratory complications affecting the airways, vessels, lung parenchyma, pleura, and respiratory muscles. The major pathological features of CTD are chronic inflammation of blood vessels and connective tissues, which can affect any organ leading to multi-system damage. The human lung is particularly vulnerable to such damage because anatomically it is abundant with collagen and blood vessels. The complex etiology of CTD-ILD includes genetic risks, epigenetic changes, and dysregulated immunity, which interact leading to disease under various ill-defined environmental triggers. CTD-ILD exhibits a broad spectra of clinical manifestations: from asymptomatic to severe dyspnea; from single-organ respiratory system involvement to multi-organ involvement. The disease course is also featured by remissions and relapses. It can range from stability or slow progression over several years to rapid deterioration. It can also present clinically as highly progressive from the initial onset of disease. Currently, the diagnosis of CTD-ILD is primarily based on distinct pathology subtype(s), imaging, as well as related CTD and autoantibodies profiles. Meticulous comprehensive clinical and laboratory assessment to improve the diagnostic process and management strategies are much needed. In this review, we focus on examining the pathogenesis of CTD-ILD with respect to genetics, environmental factors, and immunological factors. We also discuss the current state of knowledge and elaborate on the clinical characteristics of CTD-ILD, distinct pathohistological subtypes, imaging features, and related autoantibodies. Furthermore, we comment on the identification of high-risk patients and address how to stratify patients for precision medicine management approaches.

Clinical utility of ANA-ELISA vs ANA-immunofluorescence in connective tissue diseases.

We investigated the performance of ANA-ELISA for CTDs screening and diagnosis and comparing it to the conventional ANA-IIF. ANA-ELISA is a solid-phase immune assay includes 17 ANA-targeted recombinant antigens; dsDNA, Sm-D, Rib-P, PCNA, U1-RNP (70, A, C), SS-A/Ro (52 and 60), SS-B/La, Centromere B, Scl-70, Fibrillarin, RNA Polymerase III, Jo-1, Mi-2, and PM-Scl. During the period between March till December 2016 all requests for ANA from primary, secondary, and tertiary care centers were processed with both techniques; ANA-IIF and ANA-ELISA. The electronic medical record of these patients was reviewed looking for CTD diagnosis documented by the Senior rheumatologist. SPSS 22 is used for analysis. Between March and December 2016, a total of 12,439 ANA tests were requested. 1457 patients were assessed by the rheumatologist and included in the analysis. At a cut-off ratio ≥ 1.0 for ANA-ELISA and a dilutional titre ≥ 1:80 for ANA-IIF, the sensitivity of ANA-IIF and ANA-ELISA for all CTDs were 63.3% vs 74.8% respectively. For the SLE it was 64.3% vs 76.9%, Sjogren's Syndrome was 50% vs 76.9% respectively. The overall specificity of ANA-ELISA was 89.05%, which was slightly better than ANA-IIF 86.72%. The clinical performance of ANA-ELISA for CTDs screening showed better sensitivity and specificity as compared to the conventional ANA-IIF in our cohort.

Understanding and interpreting antinuclear antibody tests in systemic rheumatic diseases.

Antinuclear antibodies (ANAs) are valuable laboratory markers to screen for and support the diagnosis of various rheumatic diseases (known as ANA-associated rheumatic diseases). The importance of ANA testing has been reinforced by the inclusion of ANA positivity as an entry criterion in the 2019 systemic lupus erythematosus classification criteria. In addition, specific ANAs (such as antibodies to Sm, double-stranded DNA (dsDNA), SSA/Ro60, U1RNP, topoisomerase I, centromere protein B (CENPB), RNA polymerase III and Jo1) are included in classification criteria for other rheumatic diseases. A number of techniques are available for detecting antibodies to a selection of clinically relevant antigens (such as indirect immunofluorescence and solid phase assays). In this Review, we discuss the advantages and limitations of these techniques, as well as the clinical relevance of the differences between the techniques, to provide guidance in understanding and interpreting ANA test results. Such understanding not only necessitates insight into the sensitivity and specificity of each assay, but also into the importance of the disease context and antibody level. We also highlight the value of titre-specific information (such as likelihood ratios).

Comparative diagnostic efficacy of serum Krebs von den Lungen-6 and surfactant D for connective tissue disease-associated interstitial lung diseases: A meta-analysis.

PURPOSE: The aim of the study was to estimate and compare the diagnostic accuracy of serum Krebs von den Lungen-6 (KL-6) and surfactant protein D (SP-D) for identifying interstitial lung disease (ILD) from non-ILD among connective tissue disease (CTD) patients. MATERIALS AND METHODS: Original articles on the diagnostic accuracy of serum KL-6 and SP-D in differentiating CTD-ILD from CTD-nonILD were identified from three public databases. The overall quality of evidence and methodologic quality of each eligible study were assessed by the Grading of Recommendations, Assessment, Development and Evaluation approach and Quality Assessment of Diagnostic Accuracy Studies, respectively. We used the bivariate model to calculate random-effect sensitivity, specificity, likelihood ratios, and area under curve. Furthermore, trial sequential analysis (TSA) was used to determine whether sample sizes incorporated in the meta-analysis were powerful for evaluating the diagnostic utility. Bayesian network analysis was performed to compare the diagnostic accuracy of 2 serum biomarkers in differentiating ILD among CTD patients and various subgroups. RESULTS: Twenty-nine studies were included in the quantitative synthesis. No threshold effects were observed (all P values >.05). For diagnosis of ILD among CTD patients, overall sensitivity and specificity of serum KL-6 were 0.76 (95% confidence interval [CI]: 0.68-0.82) and 0.89 (95% CI: 0.83-0.93), whereas those for serum SP-D were 0.65 (95% CI: 0.45-0.80) and 0.88 (95% CI: 0.80-0.93). Comprehensive comparison of 2 circulating biomarkers using back-calculated likelihood ratio (LR) demonstrated that serum KL-6 corresponded to a higher LR+ and a lower LR- in comparison to serum SP-D, as well as in SSc-ILD. TSA indicated that evidence for serum KL-6 and SP-D in identifying CTD-ILD is powerful; nonetheless, more trials were needed for validation of serum KL-6 and SP-D in differentiating CTD-ILD subtypes, including different CTD and ethnicities. CONCLUSIONS: This meta-analysis suggested that serum KL-6 had superior diagnostic accuracy to SP-D for differentiating ILD from non-ILD among CTD patients, providing a convenient and non-invasive approach for screening and management of ILD among CTD patients.

[The clinical features and prognosis of interstitial lung disease patients with positive anti-neutrophil cytoplasmic antibody].

Objective: To investigate the clinical features and prognosis of interstitial lung disease patients with positive anti-neutrophil cytoplasmic antibody. Methods: The patients with interstitial lung disease who visited Peking Union Medical College Hospital from March 2006 to March 2016 were divided into three groups: interstitial lung disease with ANCA-positive(ANCA-ILD), connective tissue disease associated interstitial lung disease and interstitial pneumonia with autoimmune features (CTD-ILD/IPAF) and idiopathic interstitial pneumonia (IIP). The three groups were analyzed in terms of clinical manifestations, serology, lung function, imaging, survival and recurrence. Results: Two hundred and seventy four patients were enrolled and 38 (14%) were ANCA-positive of whom 16 were male and 22 were female. The age of 38 ANCA-positive patients was (59±10) years and the follow-up time was (52±31) months. Seven among the 38 ANCA-positive patients died and the death rate is 18.42%. The ANCA-positive patients with interstitial lung disease have higher onset age (ANCA-ILD:59±10,CTD-ILD/IPAF:52±10,IIP:53±11,H=19.29, P<0.001), lower hemoglobin (ANCA-ILD: 129±21, CTD-ILD/IPAF: 138±15, IIP: 140±19, H=8.17, P=0.017), higher erythrocyte sedimentation rate (ANCA-ILD:45±35, CTD-ILD/IPAF:26±24,IIP:19±22,H=19.73, P<0.001), lower lung function improvement rate after treatment (ANCA-ILD:31%,CTD-ILD/IPAF:59%,IIP: 39%,χ(2)=11.74,P=0.003), lower absorption rate of CT lesion (ANCA-ILD:61%,CTD-ILD/IPAF:82%,IIP:67%, χ(2)=9.23,P=0.010) and higher death rate(ANCA-ILD:18%,CTD-ILD/IPAF:6%,IIP:12%, χ(2)=7.16,P=0.028). Conclusions: There are significant differences in clinical characteristics between ANCA-positive patients and other types of pulmonary interstitial disease. And both the treatment effect and the prognosis is poor for the ANCA-positive patients.

Utility of the AVISE Connective Tissue Disease test in predicting lupus diagnosis and progression.

Background: The AVISE Connective Tissue Disease (CTD) test uses autoantibody, erythrocyte-bound C4d (EC4d) and B-cell-bound C4d (BC4d) levels to aid in diagnoses of SLE, other CTDs and fibromyalgia. We evaluated the utility of the AVISE CTD test in predicting SLE disease development and damage progression. Methods: Patients who had undergone AVISE CTD testing were assessed for SLE diagnosis by the Systemic Lupus International Collaborating Clinics (SLICC) and American College of Rheumatology criteria and for SLE damage by the Systemic Lupus International Collaborating Clinics Damage Index (SDI) at the time of AVISE testing (t=0) and 2 years later (t=2). Results: Among 117 patients without a previous diagnosis of SLE, 65% of patients who tested positive developed SLE at t=2, compared with 10.3% of patients who tested non-positive (p<0.0001). AVISE-positive patients fulfilled significantly more SLICC diagnostic criteria than AVISE-non-positive patients at both t=0 (3.8±2.1 vs 1.9±1.1, p=0.001) and t=2 (4.5±2.2 vs 2.1±1.2, p<0.0001). AVISE-positive patients also had had significantly higher SDI at t=2 (1.9±1.3 vs 1.03±1.3, p=0.01). BC4d levels correlated with the number of SLICC criteria at t=0 (r=0.33, p<0.0001) and t=2 (r=0.34, p<0.0001), as well as SDI at t=0 (r=0.25, p=0.003) and t=2 (r=0.26, p=0.002). Conclusions: The AVISE CTD test can aid in SLE evaluation by predicting SLE disease development and future damage progression.

Vitamin D levels are prognostic factors for connective tissue disease associated interstitial lung disease (CTD-ILD).

OBJECTIVE: Vitamin D deficiency was associated with CTD-ILD and reduced lung function. We sought to confirm that lower Vitamin D level would be related to shorter survival times. RESULTS: The CTD-ILD patients had lower Vitamin D level(P<0.05). Among patients with CTD-ILD who have improved lung function after treatment, elevation of Vitamin D level was positively associated with ΔFVC (%), ΔFEV1(%) and ΔDLCO-SB (%). The median survival time of patients with high serum 25(OH)D level was significantly longer than the patients with low 25(OH)D level group (16.5 months vs14.0 months, P=0.007). The Vitamin D was identified as an independent prognostic factor with a hazard ratio of 0.869 (95% CI 0.772-0.977, P =0.019). CONCLUSIONS: Vitamin D level was lower in patients with CTD-ILD and associated with poor prognosis. Continuous levels of Vitamin D may be an important serum biomarker of prognosis. METHODS: 85 CTD-ILD patients, 71 Idiopathic pulmonary fibrosis (IPF) patients and 78 healthy control patients were included in the study. In the subgroup analysis, the CTD-ILD patients were divided into anti-MDA5 antibody-positive group and anti-MDA5 antibody-negative group according to the serum autoantibodies results. The survival analysis evaluated effect of Vitamin D level on disease prognosis.

The value of serum Krebs von den lungen-6 as a diagnostic marker in connective tissue disease associated with interstitial lung disease.

OBJECTIVES: The purpose of this study was to evaluate the value of serum krebs von den lungen-6 (KL-6) level as a diagnostic indicator for connective tissue disease associated with interstitial lung disease (CTD-ILD). METHODS: One hundred fifty five patients with newly diagnosed CTD in our hospital were enrolled and divided into two groups by their ILD manifestations, the CTD-ILD group and the CTD group. In parallel, 61 patients with pulmonary infection and 60 cases of healthy subjects were also enrolled into the study. The difference of serum KL-6 level among the four groups were compared. In CTD-ILD group, carbon monoxide diffusing capacity (DLCo) and high-resolution computed tomography (HRCT) of lung were also tested. The serum KL-6 level of 32 patients from the CTD-ILD group who received cyclophosphamide (CTX) pulse therapy were sampled and measured, by enzyme linked immunosorbent assay (ELISA), at three time points: before treatment, 3 months after treatment and 6 months after treatment. RESULTS: The serum KL-6 level in the CTD-ILD group (1004.9 (676.41738.1) IU/ml) is significantly higher than three other groups (χ2 = 72.29, P < 0.001). In the CTD-ILD group the level of serum KL-6 was positively correlated with disease severity on HRCT (r = 0.75, P <  0.001), while was negatively correlated with DLCo (r = - 0.50, P <  0.001). In 32 patients who received CTX pulse therapy, the level of serum KL-6 was gradually decreased in 20 cases whose lesions were absorbed within 6 months (F = 13.67, P <  0.001), whereas it remained unchanged in the rest of 12 patients (Z = -1.328, P = 0.198). CONCLUSIONS: Serum KL-6 level can potentially serve as a diagnostic marker for CTD-ILD and be utilized to evaluate the effectiveness of CTX pulse therapy.

Serum KL-6 level as a biomarker of interstitial lung disease in childhood connective tissue diseases: a pilot study.

Krebs von den Lungen-6 (KL-6) has been described as a promising biomarker in the diagnosis and determining the severity of interstitial lung disease in adults with connective tissue disease. This study was performed to determine whether the serum KL-6 level is useful as a biomarker for detecting the interstitial lung disease (ILD) in pediatric cases of connective tissue disease (CTD). In total, 88 patients [36 patients with systemic juvenile idiopathic arthritis (sJIA), 27 patients with juvenile systemic lupus erythematosus (JSLE), 14 patients with juvenile dermatomyositis (JDM), and 11 patients with juvenile systemic sclerosis (JSSc)] and 68 healthy controls were included in this study. Age, sex, and duration of CTD and ILD (if any) were recorded. Blood samples from all the patients and controls were examined by ELISA. Eleven of the 88 patients with CTD (12.5%) had ILD and all of them were symptomatic. Subgroup analysis indicated that eight patients had JSSc, two had JSLE, and one had systemic JIA. The median serum KL-6 level was 1450.5 U/mL (interquartile range (IQR) 742.9-2603.2) in the CTD with ILD group, 415.9 U/mL (IQR 233.4-748.4) in the CTD without ILD group, and 465.9 U/mL (IQR 273.6-1036) in the control group. KL-6 levels were significantly higher in the CTD with ILD group than the CTD without ILD group and the control group (p = 0.003). At a cut-off of 712.5 U/ml identified by ROC curve, serum KL-6 yielded a sensitivity of 81% and specificity of 72% for CTD with ILD group. There was no significant difference in serum KL-6 level among the disease subgroups (sJIA, JSLE, JSSc, JDM), in either the CTD with ILD group or the CTD without ILD group (p > 0.05). In conclusion, KL-6 is a useful biomarker of CTD with ILD in pediatric patients.

Comparative analysis of connective tissue disease-associated interstitial lung disease and interstitial pneumonia with autoimmune features.

OBJECTIVE: This retrospective clinical study aimed to examine the similarities and differences between connective tissue disease-associated interstitial lung disease (CTD-ILD) and interstitial pneumonia with autoimmune features (IPAF) and to identify the influencing factors of CTD-ILD, with a goal of early detection and active treatment of the disease. METHODS: We conducted a retrospective study of 480 patients: 412 with CTD-ILD and 68 with IPAF. Demographic features, clinical characteristics, laboratory indicators, and chest high-resolution computed tomography (HRCT) imaging data were analyzed. RESULTS: Compared with the IPAF group, the CTD-ILD group contained more women, and the incidences of joint pain, dry mouth/dry eyes, and Raynaud's phenomenon were higher; erythrocyte sedimentation rate (ESR) and D-dimer levels were higher; red blood cell (RBC) and hemoglobin (Hb) levels were lower; a high rheumatoid factor (RF) titer (> 2 times the normal upper limit) was observed, and anti-cyclic citrullinated peptide antibody (anti-CCP), anti-keratin antibody (AKA), antinuclear antibody (ANA), and anti-melanoma differentiation-associated gene 5 antibody (anti-MDA5) levels were higher. Compared with CTD-ILD patients, IPAF patients were more likely to present initially with respiratory symptoms, with higher rates of fever, cough and expectoration, dyspnea, and Velcro crackles; anti-Ro52 titers were higher; incidences of honeycombing opacity, reticulate opacity, patchy opacity, and pleural thickening were greater. Female sex, a high RF titer (> 2 times the normal upper limit), anti-CCP positivity, ANA positivity, and anti-MDA5 positivity were risk factors for CTD-ILD when the odds ratios were adjusted. CONCLUSION: CTD-ILD and IPAF patients differed in demographic features, clinical characteristics, laboratory indicators, and chest HRCT imaging data. Female sex, a high RF titer (> 2 times the normal upper limit), anti-CCP positivity, ANA positivity, and anti-MDA5 positivity were risk factors for CTD-ILD.Key Points• This retrospective clinical study comprehensively compared the demographic features, clinical characteristics, laboratory indicators, and chest HRCT imaging data of CTD-ILD and IPAF patients.• The evidence suggested that female sex, a high RF titer, anti-CCP positivity, ANA positivity, and anti-MDA5 positivity were risk factors for CTD-ILD.

[Clinical value of myositis antibodies in patients with connective tissue disease-associated interstitial lung diseases].

Objective: To investigate the clinical significance of detection of myositis-specific antibodies (MSAs) and myositis-associated antibodies (MAAs) in patients with connective tissue disease-associated interstitial lung diseases (CTD-ILD). Methods: Serum samples of 120 patients with CTD-ILD admitted to the Department of Respiratory, Affiliated Drum Tower Hospital of Nanjing University Medical College from December 2016 to April 2018 were collected for analysis. The patients included 45 with polymyositis/dermatomyositis (PM/DM), 36 with Sjogren's syndrome (SS) and 39 with undifferentiated connective tissue disease (UCTD). There were 37 males and 83 females with an average age of (56±11) years. Thirty-two patients with non-CTD-ILD, 10 males and 22 females with an average age of (42±17) years, were enrolled as the control group. Euroline Autoimmune Inflammatory Myopathies 16 Ag kit was used for detecting MSAs and MAAs, and the positive rates of serum MSAs and MAAs were calculated. The antibody distribution and clinical characteristics of different groups were analyzed and compared. Results: Eighty-nine of the 120 patients with CTD-ILD were positive for MSA and/or MAA (74.2%), and the detection rates of MSAs and MAAs were 52.5% (63/120) and 61.7% (74/120) respectively. No myositis antibody was detected in the non-CTD-ILD group. The detection rates of MSAs in PM/DM-ILD group, SS-ILD group and UCTD-ILD group were 75.6% (34/45), 33.3%(12/36) and 43.6%(17/39) respectively. The total detection rate of MSAs in PM/DM group was significantly higher than that in SS group and UCTD group (χ(2)=14.53, 8.95, 0.01). The anti-ARS was the most frequent (50/120, 41.7%). The positive rates of MAAs in the three groups were 64.4%(29/45), 77.8%(28/36), 43.6%(17/39) respectively, and anti-Ro-52 accounted for 60%(72/120), and were highly correlated with MSAs such as anti-Jo-1 antibodies. Conclusion: Myositis antibody profiling should be performed in patients with ILD who were negative for conventional autoimmune antibody testing and had no CTD. In patients with SS-ILD and UCTD-ILD, the myositis antibody spectrum could detect the presence of myositis-specific antibodies and myositis-related antibodies in some patients, and its role in clinical diagnosis and treatment needed further observation.

Prevalence of other connective tissue diseases in idiopathic inflammatory myopathies.

We sought to determine the prevalence of additional connective tissue diseases (CTDs) in patients with idiopathic inflammatory myopathies (IIM), and to study the muscle biopsy patterns in various clinico-serologic subsets of myositis. We undertook a retrospective cohort study of 648 patients with a histological diagnosis of IIM. The following was determined from the South Australian Myositis Database: presence of associated CTDs, histological details and presence of myositis-specific (MSA) or myositis-associated (MAA) antibodies. Among patients with IIM, a significantly greater proportion had systemic sclerosis 32/648 (4.9%) than mixed connective tissue disease (12/648, p = 0.003), primary Sjogren's syndrome (12/648, p = 0.003), systemic lupus erythematosus (10/648, p < 0.001) or rheumatoid arthritis (6/648, p = 0.0001). Polymyositis was the most common IIM diagnosis regardless of the presence or absence of CTD. MSA/MAA was more commonly detected in those with systemic sclerosis than those with IIM alone (OR 5.35, p < 0.005). The higher prevalence of SSc (compared with other CTDs) in IIM, together with the more frequent detection of autoantibodies in this group, suggests that these conditions may be linked.

Red Cell Distribution Width and Platelet Count as Biomarkers of Pulmonary Arterial Hypertension in Patients with Connective Tissue Disorders.

INTRODUCTION/OBJECTIVE: In the present paper, we aimed to test the value of the red cell distribution width (RDW) coefficient of variation as a candidate biomarker for pulmonary arterial hypertension (PAH) in patients with connective tissue disorders (CTD), correlating it with the degree of cardiopulmonary impairment in these patients. METHODS: The study population included N = 141 patients with CTD and N = 59 patients affected by pulmonary hypertension of other etiologies, all referred to the Pulmonary Hypertension Clinic of the Cardiology Division of an Academic Hospital in Northern Italy for evaluation (including right catheterization). Clinical, instrumental, and laboratory data were collected and related to RDW and other full blood count indexes. RESULTS: Twenty out of 141 CTD patients (14%) received a diagnosis of PAH. In comparison to those without PAH, CTD patients with PAH displayed a larger RDW (14.9% (13.5-17.2) vs. 13.8% (13.1-15.0); p = 0.02) and a lower platelet count (205 (177-240) × 109/l vs. 244 (197.5-304.2) × 109/l; p = 0.005). Moreover, with respect to CTD patients without PAH, RDW was significantly larger also in PH of other etiologies. In contrast, the platelet count was significantly lower only in CTD-related PAH, with a value > 276 × 109/l being 100% sensitive in ruling out PAH. Finally, RDW, but not the platelet count, was related directly to systolic pulmonary arterial pressure (r = 0.381; p = 0.0008) and right ventricle diameter (r = 0.283; p = 0.015) and inversely to diffusing capacity of the lung for carbon monoxide (r = -0.325; p = 0.014). CONCLUSION: RDW is a promising candidate biomarker for the screening and the prognostic stratification of PAH in CTD patients.

Clinical use of anti-DFS70 autoantibodies.

The dense fine speckled (DFS) nuclear pattern is one of the most common indirect immunofluorescence (IIF) patterns detected during routine anti-nuclear antibody (ANA) screening. There is a negative association between anti-DFS70 status and systemic autoimmune rheumatic disease (SARD), especially in the absence of concomitant SARD-specific autoantibodies. The purpose of this study was to determine the need for confirming anti-DFS70 status when a DFS pattern is observed in IIF-ANA. The frequency of anti-DFS70 detection on Western blot and the positive rate of connective tissue disease (CTD)-related autoantibody screening with a fluorescence-based enzyme immunoassay was evaluated in DFS (n = 182) and non-DFS (n = 359) groups. Specific autoantibodies against 15 autoantigens were identified by line immunoassay. We evaluated the frequency of cases of DFS mistaken for non-DFS and non-DFS cases mistaken for DFS, as well as the clinical impacts of these misinterpretations. Among cases of IIF-ANA with an observable DFS pattern, 68.1% had only anti-DFS70 without CTD-related autoantibodies, 20.3% were false positive for IIF-ANA, and the remaining 11.5% had CTD-related autoantibodies independent of anti-DFS70 status. These results indicated that CTD-related autoantibodies may be present with or without anti-DFS70 even if a DFS pattern is observed in IIF-ANA. Among patients who are ANA negative or have a low probability of SARD, an anti-DFS70 confirmation test has no clinical benefit and cannot replace specific tests for detecting CTD-related autoantibodies. Specific tests to detect CTD-related autoantibodies should be performed instead of anti-DFS70 confirmation tests when a DFS pattern is observed in IIF-ANA.

Serum KL-6 levels reflect the severity of interstitial lung disease associated with connective tissue disease.

BACKGROUND: Biomarkers have been actively investigated to supplement functional and imaging modalities to predict the severity, therapeutic responsiveness, and progression of connective tissue disease-associated interstitial lung disease (CTD-ILD). This study aimed to evaluate Krebs von den Lungen 6 (KL-6) as a potential biomarker reflecting the severity of CTD-ILD as assessed through computed tomography (CT) and pulmonary function test (PFT) parameters. METHODS: This retrospective study included 549 Korean patients with rheumatoid arthritis, systemic sclerosis, inflammatory myositis, and other CTDs with or without concurrent ILD. Serum KL-6 concentration (U/mL) was measured using the latex-enhanced immunoturbidimetric assay method. CT and PFT results were collected within 1 year of serum collection. A semiquantitative grade of ILD extent was evaluated through CT scan (grade 1, 0-25%; grade 2, 26-50%; grade 3, 51-75%; grade 4, 76-100%). RESULTS: CTD-ILD patients (n = 165) had elevated serum KL-6 levels compared to CTD patients without ILD (n = 384) (p < 0.001), and those findings were preserved after adjusting for age, sex, and CTD type. The semiquantitative grade of ILD on CT scan was significantly proportional to the KL-6 level, and the optimal cut-off KL-6 value effectively differentiated each ILD grade. The percent diffusing capacity of the lung for carbon monoxide (DLCO) (p < 0.001) and forced vital capacity (FVC) (p < 0.001) parameters had a moderate, negative correlation with the KL-6 level. CONCLUSION: Serum KL-6 levels were increased in CTD-ILD patients and had a positive correlation with CT grade and a negative correlation with FVC and DLCO. Serum KL-6 levels may reflect CTD-ILD severity.

Antinuclear antibodies in interstitial lung disease: Prevalence and clinical significance.

INTRODUCTION: The diagnosis of interstitial lung disease (ILD) requires elimination of underlying connective tissue disease. Consequently, antinuclear antibodies (ANA) are routinely screened in patients with idiopathic interstitial pneumonia. However the clinical usefulness of this practice is not well clear. AIM: In this study, we evaluated the frequency of ANA in ILD's patients and investigated the clinical significance of the ANA's presence in these patients. METHODS: We conducted a retrospective study of hospitalized patients diagnosed ILD at pulmonary department and for which ANA was performed in the immunology laboratory of our institution. Demographic features, clinical symptoms, biological and radiologic findings and CTD-ILD diagnoses were compared between patients with positive ANA versus negative ANA. RESULTS: We enrolled 73 patients. The ANA's prevalence was 32%. There were no significant differences in demographics, pulmonary function test values and radiologic findings between patients with and without ANA. Patients with positive ANA had more cutaneous manifestations (p꞊0.011) and Raynaud's phenomenon (p꞊0.029). The diagnosis of connective tissue disease was made in 42% of patients with positive ANA versus 8% with negative ANA (p꞊ 0.001). ANA's titer higher than 1/320 was predictive of CTD diagnosis (OR꞊14.4) (p<0.001). CONCLUSIONS: The research of ANA in PID's patients is an important tool of CTD diagnosis specially in those with suggestive symptoms of autoimmune disease.

A better definition of the anti-DFS70 antibody screening by IIF methods.

BACKGROUND: Anti-DFS70 antibodies have been recently included in a new testing algorithm for patients with suspicion of connective tissue diseases (CTDs). This algorithm enables to assess the probability of having a CTD in patients with a positive antinuclear antibodies (ANA) result. The aim of the study was to analyze the the inter-method agreement between three different HEp-2 cell substrates for anti-DFS70 detection, focusing on two novel IIF methods that assess the presence of monospecific anti-DFS70 antibodies. METHODS: Immunological and clinical records of 29 patients who were double positive for anti-DFS70 autoantibodies using chemiluminescence assay (CIA) and Immunoblot (IB) were studied. The IIF on HEp-2 cells were determined using slides from Inova Diagnostics, Euroimmun and Immco. The capability to detect isolated anti-DFS70 antibodies was compared using immunoadsorption on NOVA Lite HEp-2 Select (Inova Diagnostics) and the HEp-2 ELITE/DFS70 knockout test (Immco). RESULTS: The three substrates had very good sensitivity for detecting patients with anti-DFS staining pattern (93.1%, 79.3% and 72.4% for Euroimmun, Immco and Inova respectively). Most of the patients had full inhibition of DFS pattern (65.5%) by immunoabsorption test. Also, the 55.2% of the subjects were positive for monospecific DFS pattern using HEp-2 ELITE/DFS70 knockout test. However, the correlation between the full inhibition by immunoadsorption and the monospecific DFS pattern in knockout cells was very low (kappa: 0.22). CONCLUSION: The evaluation of monospecific anti-DFS70 antibodies is clinically fundamental and challenging using traditional HEp-2 IIF. Results obtained in this study support the hypothesis that the lack of standardization across IIF kits along with the subjectivity of user interpretation among other factors contribute to the overall reduction in the agreement.