Antinuclear Antibody Screening Research Articles

Antinuclear Antibodies in Systemic Lupus Erythematous – Their Complex Role and Clinical Significance in Diagnosis

Antinuclear antibodies (ANA) are a wide group of proteins directed against autologous cellular components, primarily nucleic acids and histones. Their levels are assessed using immunofluorescence on Hep-2 cells or a solid-phase ANA screening immunoassay to subsequently obtain titer value with positive cut-point of ≥1:80. Studies show that ANA can be found in 13% of general population, but typically they are associated with autoimmune conditions and inflammatory connective tissue diseases for example: systemic lupus erythematosus (SLE), sjögren's syndrome, rheumatoid arthritis, mixed connective tissue disease, juvenile idiopathic arthritis, systemic sclerosis, inflammatory myopathies. ANA detection is especially important for SLE, where 2 antibody types - anti-Sm and anti-dsDNA - serve as an entry diagnostic criterion. With increasing patient screening for those antibodies, it is important to determine that alone, positive ANA assays cannot confirm nor deny any disease and to classify their presence as a marker of a disease it is required to satisfy additional additive criteria approved in 2019 by European League Against Rheumatism/American College of Rheumatology. SLE diagnosis can be made when patient collects 10 points from clinical or immunologic domains described by EULAR/ACR, which makes SLE diagnosis challenging and therefore, describing the guidelines is vital to remain cautious about overestimation of the position positive ANA values hold in clinical practice.In conclusion the positive ANA test may be a basis for diagnosis, when additional symptoms occur, but alone does not hold any diagnostic significance and may lead to unnecessary stress for patient.

The Relative Timing, Outcomes, and Economic Impact of Anti-Nuclear Antibody (ANA) and Extractable Nuclear Antigen (ENA) Laboratory Ordering.

Objective: To determine the rates of simultaneous antinuclear antibodies (ANA) screening and extractable nuclear antigen (ENA) testing that do not follow recommendations.Design, Setting, and Participants: Retrospective cohort study of adult patients (≥18 years) with a HEp-2 ANA or ENA ordered in the Marshfield Clinic Health System.Main Outcome(s) and Measure(s): Counts of patients having simultaneous ANA and ENA laboratory testing or ENA testing without ANA screening. Relevant ENA positivity in ANA negative patients. Secondary measures included relative timing of ANA and ENA ordering, potential cost savings of unnecessary testing, and provider ordering characteristics including specialty and provider type.Results: Of 58,627 cohort patients, 39,155 (66.8%) were women, and the mean (SD) age at first laboratory testing was 48.7 (19.0) years. The negative ANA with positive ENA rate was 2%. Further stratification identified only 23 diagnosed autoimmune connective tissue diseases (AI-CTDs) in this 2%, with a resulting negative ANA with relevant positive ENA rate of 0.37%. Simultaneous ANA and ENA testing occurred in 8.3% of patients, and an ENA only was ordered in 24.2% of patients. The simultaneous or non-sequential ordering of ANA and ENA testing resulted in significant health care costs of $2,293,251.80 over 20,112 unique patients.Conclusions and Relevance: A significant percentage of providers do not follow recommendations to sequentially order ANA and ENA testing on patients with suspected AI-CTDs. Significant saving in health care spending without failure to diagnose AI-CTDs can be achieved if ANA testing is performed first, followed by ENA testing when suspecting AI-CTDs in patients.

Evaluation of the Accuracy of Estimated Endpoint Titer of NOVA View in Indirect Immunofluorescent Antinuclear Antibody Testing.

For antinuclear antibody (ANA) screening, the gold standard method is an indirect immunofluorescence assay (IIFA) using HEp-2 cells, and a serial dilution test is needed to determine the endpoint titer. We aimed to evaluate the accuracy of the estimated endpoint titer (eEPT) by the NOVA View system, by comparing it with the EPT by the serial dilution method (dEPT). The endpoint titers of a total of 1518 ANA positive cases with five major patterns including speckled, homogeneous, centromere, nucleolar, and nuclear dots patterns were determined using both the estimation function and the serial dilution method by the NOVA View system. A significant correlation between the light intensity unit (LIU) values and dEPTs was identified in all five patterns with high ρ values, ranging from 0.666 to 0.832. However, the overall exact match rate between dEPT and eEPT was 22.1% (336/1518), with the ±one-titer match rate being highest in the centromere pattern (62.8%, 81/129), and lowest in the homogeneous pattern (37.6%, 200/532). This suggests that while LIU values correlate well with dEPT, there are discrepancies in numerical agreement. Most cases that did not show an exact match, showed one-to-three-titer overestimations by eEPT. Therefore, adjusting eEPT downward significantly improved the concordance rates with dEPTs. Further investigation for an appropriate cutoff of LIU values for determining eEPT should be performed for clinical application and contribution to the standardization of the ANA titer.

O-201 Antinuclear antibodies in women with recurrent early pregnancy loss: prevalence and impact on subsequent pregnancy outcome

Abstract Study question What is the prevalence of antinuclear antibodies (ANAs) in women with recurrent early pregnancy loss (REPL) and do they affect subsequent pregnancy outcome? Summary answer The prevalence of ANAs in women with REPL was 13.0%, and their presence did not negatively impact subsequent pregnancy outcomes, including EPL and livebirth rates. What is known already An etiological factor cannot be identified in more than half of the couples experiencing REPL. Immunologic mechanisms could be involved, with the presence of autoimmune disease (AD) associated to lower reproductive outcomes. For the mere presence of ANAs, in the absence of AD, the evidence on the association with REPL is controversial, with most studies having a small sample size with important clinical and methodological heterogeneity. Study design, size, duration This is a single-centre, retrospective cohort study from a tertiary referral hospital including 960 women referred for REPL between January 2014 and December 2019. During the entire study period, maternal serum ANA detection was part of the routine diagnostic REPL work-up, defined as two or more pregnancy losses before the 10th week of gestation, including previous spontaneous conceptions and pregnancies following autologous IVF/ICSI treatment. Participants/materials, setting, methods Women under the age of 40 at ANA analysis were eligible for inclusion. Confirmed AD, uterine anomalies, parental chromosomal abnormalities and uncontrolled endocrine disorders were exclusion criteria. The prevalence of positive ANA titers and the impact of ANA positivity on the outcome of a subsequent pregnancy were investigated with EPL rate as the main outcome parameter and biochemical pregnancy rate, clinical miscarriage rate and live birth rate as secondary outcome measures. Main results and the role of chance Positive ANA screening at a titer of ≥ 1/80 was observed in 125/940 women (13.0%), a prevalence in line with previously published data in the general population not experiencing REPL. There were no statistically significant differences in maternal age, body mass index, ethnicity, or conception method between ANA-positive and -negative women. No higher number of previous EPLs was observed in ANA-positive compared to ANA-negative women (respectively 2.7 versus 2.8, p = 0.06). Women with ANAs had significantly more thyroid antibodies (21.6% versus 10.2%, p < 0.001). For a subsequent pregnancy, no association was observed between ANA positivity and reproductive outcome with an unadjusted EPL rate of 30/86 (34.9%, ANA-positive) versus 206/509 (40.5%, ANA-negative, p = 0.32). Logistic regression analysis did not identify ANAs as an independent predictive risk factor for additional EPL (OR 0.73, 95%CI 0.45-1.20, p = 0.22). Similarly, there was no significant difference in live birth rates between both groups (p = 0.5), with 54/86 (62.8%) in ANA-positive and 300/509 (58.9%) in ANA-negative patients. Limitations, reasons for caution The current data are limited by the absence of a formal control group without REPL, the retrospective nature of the study and potential bias due to unmeasured confounders. Wider implications of the findings These findings further questions systematic ANA measurement in the diagnostic work-up of REPL without AD. Furthermore, when ANAs are detected, patient counselling and treatment adaptations should be considered with caution, given our inability to eventually detect any significant differences in the pregnancy outcomes between ANA-positive and -negative REPL patients. Trial registration number not applicable

The Onset of Antinuclear Antibodies (ANAs) as a Potential Risk Factor for Mortality and Morbidity in COVID-19 Patients: A Single-Center Retrospective Study.

The immune system's amplified response to SARS-CoV-2 may lead to the production of autoantibodies, but their specific impact on disease severity and outcome remains unclear. This study aims to assess if hospitalized COVID-19 patients face a worse prognosis based on ANA presence, even without autoimmune diseases. We performed a retrospective, single-center, observational cohort study, enrolling 638 COVID-19 patients hospitalized from April 2020 to March 2021 at Hospital "Policlinico Riuniti" of Foggia (Italy). COVID-19 patients with a positive ANA test exhibited a significantly lower 30-day survival rate (64.4% vs. 83.0%) and a higher likelihood of severe respiratory complications during hospitalization than those with negative ANA screening (35.4% vs. 17.0%) (p < 0.001). The association between poor prognosis and ANA status was identified by calculating the HALP score (Hemoglobin-Albumin-Lymphocyte-Platelet), which was lower in COVID-19 patients with a positive ANA test compared to ANA-negative patients (108.1 ± 7.4 vs. 218.6 ± 11.2 AU; p < 0.011). In detail, COVID-19 patients with a low HALP showed a lower 30-day survival rate (99.1% vs. 83.6% vs. 55.2% for high, medium, and low HALP, respectively; p < 0.001) and a higher incidence of adverse respiratory events compared to those with high and medium HALP (13.1% vs. 35.2% vs. 64.6% for high, medium, and low HALP, respectively; p < 0.001). In summary, ANA positivity in COVID-19 patients appears to be linked to a more aggressive disease phenotype with a reduced survival rate. Furthermore, we propose that the HALP score could serve as a valuable parameter to assess prognosis for COVID-19 patients.

Combination of cytoplasmic and nuclear patterns on Hep-2 antinuclear antibody is useful as a screening test for anti-synthetase syndrome.

This study aimed to establish a screening model for differentiating anti-synthetase syndrome (ASS) from other ANA-associated rheumatic diseases (AARDs) using a combination of cytoplasmic and non-cytoplasmic ANA (ncANA) patterns. : This retrospective observational study included patients with AARDs such as SLE, SSc, SS, MCTD and PM/DM who underwent ANA screening between April 2012 and December 2021. Variables included age, sex, ANA patterns (Cytoplasmic and ncANA) and titres. Logistic regression analysis of Cytoplasmic and ncANA patterns was performed to differentiate ASS from other AARDs. : The 981 diagnosed cases of AARDs consisted of SS (n = 451), SSc (n = 264), SLE (n = 201), PM/DM (n = 104), MCTD (n = 52) and ASS, including PM/DM (n = 64). Of these, 155 patients had ≥2 overlapping diseases; however, there was no overlap between AARDs and ASS. ASS is more likely to occur when the cytoplasmic titre is positive and the ncANA <320. Receiver operating characteristic analysis of the Cytoplasmic and ncANA range revealed an area under the receiver operating characteristic curve of 0.885 (95% CI: 0.844-0.927). : It is important to detect cytoplasmic patterns as an ANA screening test for ASS diagnosis, even if the titre is low. Additionally, combining the cytoplasmic and ncANA patterns yields more accurate ASS screening results.

Phototherapy for vitiligo: A narrative review on the clinical and molecular aspects, and recent literature.

Vitiligo is characterized by depigmented patches resulting from loss of melanocytes. Phototherapy has emerged as a prominent treatment option for vitiligo, utilizing various light modalities to induce disease stability and repigmentation. This narrative review aims to explore the clinical applications and molecular mechanisms of phototherapy in vitiligo. The review evaluates existing literature on phototherapy for vitiligo, analyzing studies on hospital-based and home-based phototherapy, as well as outcomes related to stabilization and repigmentation. Narrowband ultra-violet B, that is, NBUVB remains the most commonly employed, studied and effective phototherapy modality for vitiligo. Special attention is given to assessing different types of lamps, dosimetry, published guidelines, and the utilization of targeted phototherapy modalities. Additionally, the integration of phototherapy with other treatment modalities, including its use as a depigmenting therapy in generalized/universal vitiligo, is discussed. Screening for anti-nuclear antibodies and tailoring approaches for non-photo-adapters are also examined. In conclusion, this review provides a comprehensive overview of phototherapy for vitiligo treatment. It underscores the evolving landscape of phototherapy and offers insights into optimizing therapeutic outcomes and addressing the challenges ahead. By integrating clinical evidence with molecular understanding, phototherapy emerges as a valuable therapeutic option for managing vitiligo, with potential for further advancements in the field.

Recognition of rare antinuclear antibody patterns based on a novel attention-based enhancement framework.

Rare antinuclear antibody (ANA) pattern recognition has been a widely applied technology for routine ANA screening in clinical laboratories. In recent years, the application of deep learning methods in recognizing ANA patterns has witnessed remarkable advancements. However, the majority of studies in this field have primarily focused on the classification of the most common ANA patterns, while another subset has concentrated on the detection of mitotic metaphase cells. To date, no prior research has been specifically dedicated to the identification of rare ANA patterns. In the present paper, we introduce a novel attention-based enhancement framework, which was designed for the recognition of rare ANA patterns in ANA-indirect immunofluorescence images. More specifically, we selected the algorithm with the best performance as our target detection network by conducting comparative experiments. We then further developed and enhanced the chosen algorithm through a series of optimizations. Then, attention mechanism was introduced to facilitate neural networks in expediting the learning process, extracting more essential and distinctive features for the target features that belong to the specific patterns. The proposed approach has helped to obtained high precision rate of 86.40%, 82.75% recall, 84.24% F1 score and 84.64% mean average precision for a 9-category rare ANA pattern detection task on our dataset. Finally, we evaluated the potential of the model as medical technologist assistant and observed that the technologist's performance improved after referring to the results of the model prediction. These promising results highlighted its potential as an efficient and reliable tool to assist medical technologists in their clinical practice.

Antinuclear antibody–associated autoimmune cytopenia in childhood is a risk factor for systemic lupus erythematosus

AbstractAutoimmune cytopenia (AIC) in children may be associated with positive antinuclear antibodies (ANA) and may progress to systemic lupus erythematosus (SLE). We evaluated the risk of progression to SLE of childhood-onset ANA-associated AIC. In the French national prospective OBS’CEREVANCE cohort, the long-term outcome of children with ANA-associated AIC (ANA titer ≥1/160) and a subgroup of children who developed SLE were described. ANA were positive in 355 of 1803 (20%) children with AIC. With a median follow-up of 5.8 (range, 0.1-29.6) years, 79 of 355 (22%) patients developed SLE at a median age of 14.5 (1.1-21.4) years; 20% of chronic immune thrombocytopenic purpura, 19% of autoimmune hemolytic anemia, and 45% of Evans syndrome. None of the patients with ANA-negative test developed SLE. Severe manifestations of SLE were observed in 21 patients, and 2 patients died. In multivariate analysis including patients with positive ANA within the first 3 months after AIC diagnosis, age >10 years at AIC diagnosis (relative risk [RR], 3.67; 95% confidence interval [CI], 1.18-11.4; P = .024) and ANA titer >1/160 (RR, 5.28; 95% CI, 1.20-23.17; P = .027) were associated with the occurrence of SLE after AIC diagnosis. ANA-associated AIC is a risk factor for progression to SLE, especially in children with an initial ANA titer >1/160 and an age >10 years at AIC diagnosis. ANA screening should be recommended in children with AIC, and patients with ANA should be monitored long-term for SLE, with special attention to the transition period. This trial was registered at www.ClinicalTrials.gov as #NCT05937828.

The Role of Anti-DFS70 in the Diagnosis of Systemic Autoimmune Rheumatic Diseases

The diagnosis of systemic autoimmune rheumatic disease (SARD) or its exclusion is carried out taking into account the results of immunological studies, primarily antinuclear antibodies (ANA) and specific autoantibodies. Often, during ANA analysis via indirect immunofluorescence reaction on cellular and tissue substrates, a dense fine speckled 70 (DFS70) fluorescence pattern is observed. Studies on the diagnostic significance of antibodies to anti-DFS70 allow for optimizing the stepwise diagnosis of SARD. Currently, a two-step strategy for laboratory diagnostic investigation is recommended: in the first step, ANA screening is performed, and in the second step, patients with positive results undergo confirmatory tests to detect specific antibodies against individual nuclear antigens. The detection of anti-DFS70 in ANA-seropositive patients without clinical and/or other specific serological markers characteristic of a particular disease within the SARD group may be considered a negative prognostic marker. Also, in the process of decision making in clinical practice, we should remember that anti-DFS70 can be found in the blood of patients with a different, non-SARD pathology and that most people showing anti-DFS70 are healthy individuals.

Prevalence and sociodemographic correlates of antinuclear antibody testing by indirect immunofluorescence or solid-phase assays in a Spanish population: the Camargo Cohort

Autoantibodies are the hallmark of autoimmunity, and specifically, antinuclear antibodies (ANA) are one of the most relevant antibodies present in systemic autoimmune diseases (AID). In the present study, we evaluate the relationship between ANA and sociodemographic and biobehavioral factors in a population with a low pre-test probability for systemic AID. ANA were determined in serum samples at baseline visit from 2997 participants from the Camargo Cohort using indirect immunofluorescence assay, and two solid phase assays (SPA), addressable laser bead immunoassay, and fluorescence enzyme immunoassay. Sociodemographic and biobehavioral features of the subjects were obtained at baseline visit using a structured questionnaire. The prevalence of ANA positive results was significantly higher when indirect immunofluorescence assay was used as screening method in comparison with SPAs, being higher in females, older subjects, and those with higher C-reactive protein levels. Considering biobehavioral features, the prevalence was higher in those individuals with a sedentary lifestyle, and in ex- and non-alcohol users. Moreover, considering the relevance of the antibody load using ANA Screen, the prevalence of the antibody load also increased with age, especially in females. In conclusion, the prevalence of ANA varies depending on sociodemographic and biobehavioral features of the subjects, which could be relevant specifically in a population with a low pre-test probability for systemic AIDs.

The Role of Indirect Immunofluorescence (IIF) and Line Immunoassay (LIA) in the Diagnosis of Autoimmune Diseases and Their Clinical Correlation: An Observational Study From a Tertiary Care Center in Bihar.

Background and aim The presence of distinct sets of autoantigens and autoantibodies bestow these autoimmune diseases (ADs) with specific immune profiles or fingerprints, which has cleared the diagnostic dilemma associated with these ADs. This study was planned to collate and compare the reporting of indirect immunofluorescence (IIF) with line immunoassay (LIA) and their clinical correlations.This study was conducted to investigate the association between the reporting of anti-nuclear antibody (ANA) screening by IIF and ANA profile reporting by LIA. Additionally, it aimed to explore the association of ANA pattern detection by IIF with the detection of autoantibodies against nuclear antigens by LIA and the association of autoantibody detection by LIA with clinical diagnosis. Methodology A total of 98 samples from patients suspected of having ADs were subjected to both IIF and LIA, and results were correlated with clinical diagnosis. Results In the homogenous pattern noted by IIF, the clustered antigens identified by LIA included dsDNA, Nucleosome, Histone, and Mi-2. In the speckled pattern, the identified antigens were SS-A/Ro52, P0, SS-A/Ro60, SS-B/La, and U1-snRNP. On the other hand, the nucleolar pattern revealed antigens AMA M2, PCNA, and CENP-B. The centromere pattern was mostly associated with CENP-B. The speckled pattern was found to be most commonly associated with systemic lupus erythematosus (SLE). The most common autoantibody found in total ANA profile-positive samples was anti-U1-snRNP followed by anti-SS-A/Ro60 and anti-SS-B/La, and all three were found to be associated with SLE. Conclusions SLE was the most common AD identified in our study samples, with the speckled pattern being the most common pattern on IIF and anti-U1-snRNP being the most common ANA identified by LIA. The fluorescence pattern of IIF predicts the presence of specific antibodies. LIA should be reserved for IIF-positive but dubious casesand whose signs and symptoms are nebulous and do not match the disease dictated by IIF.

B-131 The Prevalence and Clinical Relevance of the DFS Immunofluorescent Staining Pattern in a Large ANA-positive Cohort

Abstract Background The dense fine-speckled (DFS) nuclear pattern is one of the most common indirect immunofluorescence (IIF) patterns detected during routine antinuclear antibody (ANA) screening. Although various researchers have studied the DFS pattern in recent years, it presents a clinical puzzle since its significance remains elusive. Anti-DFS-70 antibodies occur in healthy individuals with different medical conditions. Unlike the other ANA patterns, they are not associated with systemic autoimmune disease. The present study examined the association of a DFS ANA pattern with the medical conditions of patients who presented to a tertiary care medical center. Methods Retrospective chart review of all laboratory values (January 2020 through June 2022) with ANA- and DFS-positive patterns in patients with ANA tests ordered by various clinicians and compared to the patient diagnosis reported. UMass Human IRB approves this study. Results Of 8885 ANA samples analyzed, 67.8% (6022) were reported negative. Of total positive ANA (2863) samples, 78.6% (2257) had nuclear patterns, 9.1% (260) had mitotic patterns, and 12.1% (346) had cytoplasmic patterns. Of the positive ANA samples, 14.6% (417) had DFS patterns. For samples with DFS pattern, titers ranged from 1:40 to 1:1280; 62.5% were between1:40, and 1:80. Of samples positive for ANA DFS patterns, 76% (317) were from female patients, and 90% (375) were from patients &amp;gt;21 years of age. Among patients with DFS patterns, the most commonly reported diseases were neurological, followed by respiratory, digestive, circulatory, and musculoskeletal (Table 1). Conclusion Clinicians should use a positive ANA with a DFS pattern to indicate pathological phenomena other than autoimmune diseases. Only 3.1% of the patients with DFS patterns have a systemic rheumatic disorder. Clinicians should pay close attention to ANA Patterns, especially DFS staining when using ANA testing.

BH12 Granulomatous alopecia areata

Abstract A 46-year-old woman with Fitzpatrick skin type V presented with sudden-onset accelerated diffuse hair loss. Her other relevant medical history included Graves disease. Trichoscopy showed black dots, broken hairs with exclamation mark-like hairs and yellow dots. She was provisionally diagnosed with diffuse alopecia areata (AA). Laboratory work-up, including complete blood count, liver/renal parameters, antinuclear antibody and complement screen did not reveal any abnormality. Thyroid function and adjusted calcium level were all within the normal range. She had slight iron, folate and vitamin D deficiencies, which were treated with the appropriate supplements. A biopsy was taken from the scalp, and histological examination showed a nonscarring process with miniaturization, significant telogen shift and peribulbar granulomatous inflammation. Subsequent syphilis serology was negative, and serum angiotensin-converting enzyme was within normal limits. No clinical features of sarcoidosis or syphilis were observed. After careful clinicopathological correlation, she was diagnosed with granulomatous AA. We planned to treat her with diphencyprone; however, her hair grew back spontaneously after 2 months. AA is a nonscarring hair loss that can commonly affect the scalp and beard, as well as any hair-bearing surface on the body. It is a T-cell-mediated disease with characteristic histological features, including peribulbar lymphocytic inflammation with or without eosinophils, follicular miniaturization and an increase in catagen/telogen hairs. Peribulbar granulomatous inflammation is a rare histopathological finding in AA. In the context of alopecia, other conditions associated with histopathological granulomatous findings include sarcoidosis, syphilis and various forms of inflammatory and cicatrizing alopecia. To our knowledge, only five other cases of granulomatous AA have been reported. Notably, we describe the first case from the UK and only the second case internationally to show spontaneous resolution, suggesting that the prognosis of granulomatous AA is not dissimilar to histologically typical AA. To summarize, we have described a clinically typical AA with atypical histopathological findings. Granulomatous inflammation should not preclude a diagnosis of AA. This demonstrates the importance of clinicopathological correlation as the histopathology of alopecia could potentially be misleading.

A novel magnetic bead-based immunoprecipitation method for anti-dense fine speckled 70 antibodies

The indirect immunofluorescence assay (IIFA) on HEp-2 cells has been widely used for screening anti-nuclear antibodies (ANA) that are associated with systemic autoimmune rheumatic diseases (SARD). Sera containing ANA display multiple distinct fluorescence patterns on HEp-2 cells. Among them, a dense fine speckled (DFS) pattern caused by anti-DFS70 antibodies has been reported to have higher prevalence in healthy individuals than in patients with SARD. This DFS pattern is often difficult to distinguish amongst other SARD-associated ANA patterns, in particular a mixed homogeneous and speckled pattern. Furthermore, a strong DFS pattern can mask other SARD-associated patterns. Hence, we developed a novel immunoprecipitation method using magnetic beads to remove anti-DFS70 antibodies in serum prior to running IIFA. We also aimed to confirm the presence of anti-DFS70 and to uncover any SARD-associated ANA patterns masked by a strong DFS pattern. The sera used in our study were from 70 individuals who had routine ANA screen, of which 35 sera had an isolated DFS pattern with monospecific anti-DFS70 antibodies confirmed by a complementary assay, and 35 were control sera without a DFS pattern. An immunoprecipitation method using magnetic beads coated with recombinant human full length DFS70 protein was developed. The performance of this new method was evaluated in comparison to an immunoadsorption method using the same DFS70 protein. Our newly developed immunoprecipitation method demonstrated excellent sensitivity (91.4%) and specificity (100%) in confirming the DFS pattern associated with anti-DFS70 in sera. Additionally, our method was able to remove anti-DFS70 and uncover SARD-associated ANA patterns masked by a strong DFS pattern. It also showed a clearer background on IIFA than that of the immunoadsorption method. The novel magnetic bead-based immunoprecipitation method demonstrated satisfactory performance in removing anti-DFS70 without interfering with the detection of other antibodies. It can be easily integrated with IIFA to confirm anti-DFS70 associated DFS pattern. Additionally, it can simultaneously unmask other ANA patterns, which cannot be achieved by a conventional protocol that requires a complementary anti-DFS70 specific assay to be performed. Therefore, the novel method provides a more effective and accurate solution for ANA screening.

An Investigation of the Relationship Between Isolated Anti-dense Fine-Speckled 70 Autoantibodies and Allergen-Specific Immunoglobulin E.

Background Although the mechanisms of the formation of anti-dense fine-speckled 70 (anti-DFS70) antibodies are not fully known, there is evidence in the literature that allergic reactions may play a role in their formation. Immunoglobulin E (IgE)-mediated immunopathological mechanisms are increasingly being elucidated in diseases such as atopic dermatitis and urticaria-related diseases. We aimed to reveal its relationship with anti-DFS70 in allergen-sensitive patients with positive specific IgE (sIgE) levels. Methodology The study included samples of 758 patients who underwent antinuclear antibody (ANA) screening and allergen-sIgE testing between January 2019 and January 2022. Patients' clinical diagnoses were retrospectively obtained from the hospital information management system. ANA was tested according to the instructions of the manufacturer by the indirect immunofluorescent antibody method using HEp-2 cell substrates (Euroimmun Luebeck, Germany). Allergen-sIgE was determined by chemiluminescence on the Immulite 2000 XPI system (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany) according to the instructions of the manufacturer. Results ANA pattern was detected in 74 samples included in the study. ANA-positive patients were divided into DFS70 (+) and DFS70 (-) groups. A statistically significant increase in the DFS70 pattern was observed in patients with a positive allergen-sIgE test (p < 0.0001). Both allergen-sIgE and DFS70 positivity were statistically significant in younger age groups (p < 0.05). The most common diagnosis was urticaria-related conditions in 23 (31%) patients with a positive allergy test. Conclusions Our study shows that the positivity of the DFS70 pattern is increased in allergen-sensitive patients. Therefore, the allergen-sIgE-mediated allergic disease should be considered in patients with isolated anti-DSF70. Studies with related disease groups are needed to determine whether there is a relationship between anti-DFS70 and allergy-related disease in these patients. If an immunopathological mechanism is not found, these false-positive results can be considered clinically insignificant, and unnecessary consultations can be avoided.

Evaluation of Antinuclear Antibody and Subserology Reflex Testing for the Diagnosis of Systemic Autoimmune Rheumatic Disorders in an Academic Teaching Hospital.

The aim of this study was to examine appropriate utilization of antinuclear antibody (ANA) screening tests with follow-up subserology tests (reflex testing) for diagnosing systemic autoimmune rheumatic disorder (SARD). We conducted a retrospective chart review of 3003 SARD-test orders at an academic teaching hospital from January to December 2019. Testing patterns were categorized as American College of Rheumatology (ACR)-recommended reflex testing, panel testing, or single subserology testing. We described testing patterns, assessed their diagnostic accuracy, and explored factors associated with reflex testing. Reflex testing accounted for 79.7% of SARD test-ordering, whereas improper testing (panel or single subserology) accounted for the other 20.3%. Reflex testing was associated with significantly more SARD diagnoses than improper testing (P = .004). Testing patterns were significantly associated with race/ethnicity (P = .008), with reflex testing being less frequent than improper testing in Hispanics and Whites. In summary, one-fifth (20.3%) of testing patterns for suspected SARD did not follow the ACR-recommended guidelines for using reflex testing. Use of reflex testing was associated with an increased frequency of SARD diagnosis.

Antinuclear antibodies in children: indirect immunofluorescence patterns, antigen targets and associated diagnoses

Antinuclear antibodies tests are of a paramount importance in the diagnosis, classification, prognostic evaluation and management of autoimmune diseases in children. The present study aimed to describe the immuno-clinical profile of antinuclear antibodies tests in a pediatric population in order to guide the clinical practice of biologists and clinicians. Our study enrolled 268 children. Antinuclear antibodies screening was performed using the indirect immunofluorescence assay on HEp-2 cells. Identification of target antigens was conducted using separately or at one timepoint the following techniques: enzyme-linked immunosorbent assay, Immunodot and Chemiluminescence. The average age of patients was 9.6 ± 4.3 years, with a female predominance (sex-ratio = 1.9). Antinuclear antibodies screening was positive in 40.67% of cases. The most frequently observed antinuclear antibodies patterns were speckled (52.3%), homogeneous (13.8%) and mixed homogeneous-speckled (13.8%). Autoantibodies were detected in 4 patients (2.51%) for whom ANA testing using the indirect immunofluorescence assay was negative. Positive antinuclear antibodies specificities were detected in connective tissue diseases (44.03%; n = 48), organ-specific autoimmune diseases (10.09%; n = 11), and in non-autoimmune conditions (inflammatory diseases, infections, hematological diseases, vasculitis and Wilson’s disease) (32.08%; n = 35). Our study revealed a high rate of positive antinuclear antibodies tests in the pediatric population, mainly related to autoimmune diseases (54.12%) besides non-autoimmune conditions (32.08%). Therefore, screening and interpretation of antinuclear antibodies testing in children require the consideration of clinical data and a close collaboration between clinicians and biologists.

Prevalence of Anti-Dense Fine Speckled 70 Antibodies in Systemic Autoimmune Rheumatic Diseases and Healthy Controls: A Cross-sectional Study

Introduction: Recognition of Dense Fine Speckled (DFS) Antinuclear Antibody (ANA) pattern is challenging as it is frequently confused with speckled or homogeneous patterns. Identification and confirmation of DFS pattern is important as it is more often seen in healthy individuals, routine laboratory referrals with relatively lesser prevalence in Systemic Autoimmune Rheumatic Diseases (SARD). As there is limited data on the prevalence of DFS pattern in Indian population, present study determined the prevalence and clinical significance of DFS pattern followed by anti-DFS 70 Enzyme Linked Immunosorbent Assay (ELISA) for confirmation. Aim: To estimate the prevalence of anti-DFS 70 antibodies in ANA screening tests, SARD, and healthy individuals by Indirect Immunofluorescence (IIF) screening and confirmation by anti- DFS 70 ELISA. Materials and Methods: A cross-sectional study was conducted over a period of 15 months from January 2017 to April 2018 at Nizams Institute of Medical Sciences, a tertiary care institute in Hyderabad, Telangana, India. A total of 7,550 serum samples were tested by IIF during the study duration. Sera displaying DFS pattern was tested for anti-DFS antibodies and anti-Extractable Nuclear Antigen (ENA) antibodies by ELISA. A total of 49 patients from rheumatology department satisfying SARD criteria and 184 healthy patients were also tested for anti-DFS antibodies. Results: Anti-DFS 70 antibodies were seen in 0.47%, 16.3%, 39.6% among routine ANA screening referrals, SARD patients and healthy individuals, respectively. DFS 70 antibodies were positive in 60% of DFS IIF positive samples with none of them showing anti-ENA positivity or clinical evidence of SARD. Anti- DFS 70 positivity was more commonly seen in younger patients. Anti-DFS antibodies were significantly more common in healthy individuals compared to SARD patients (p -value=0.0022). Conclusion: It is important to confirm DFS IIF pattern with a specific ELISA as isolated DFS antibodies are frequently associated with non SARD conditions.

Rare Antinuclear Antibody Patterns: Relevance in Routine Laboratory Reporting

Introduction: Uncommon patterns on Human Epithelial 2 (Hep 2) substrate during Antinuclear Antibody (ANA) screening by Indirect Immunofluorescence (IIF) microscopy are not routinely reported by many laboratories since their clinical relevance is not well established. Aim: To elucidate rare ANA patterns on Hep 2 and their possible association with clinical presentation. Materials and Methods: A retrospective study was conducted on ANA reports in the duration January 2021 to March 2022 at the Department of Laboratory Medicine, Dr. Sulaiman Al Habib Medical Group, Olaya Medical Complex, Riyadh, Saudi Arabia, to recognise rare ANA patterns. Gold standard method of IIF on Hep 2 was used for screening. Statistical evaluation was done to obtain frequencies of various ANA patterns. Those with frequency of less than 1% were classified as rare patterns. Results: Overall, 4207 consecutive ANA reports were evaluated out of which 1388 were positive and 210 (4.99%) demonstrated rare ANA patterns including nuclear, cytoplasmic and mitotic subtypes. Most commonly encountered among the rare ANA patterns was intercellular bridge (AC 27) with frequency of 0.78% (n=33). Systemic Lupus Erythematosus (SLE) (10/210) was the most often observed clinical association with rare cytoplasmic and mitotic patterns at titer ≥1:160. Conclusion: Uncommon ANA patterns may be useful in initial work-up of autoimmune illness hence, should be routinely reported. Further studies to enlighten the significance of these patterns, analogous antibodies could be of diagnostic relevance in autoimmune and other diseases.