Antinuclear Antibody Screening Research Articles

A step towards standardization: A method for end-point titer determination by fluorescence index of an automated microscope. End-point titer determination by fluorescence index

ObjectivesIndirect Immunofluorescence (IIF) is widely considered the Gold Standard for Antinuclear Antibody (ANA) screening. However, the high inter-reader variability remains the major disadvantage associated with ANA testing and the main reason for the increasing demand of the computer-aided immunofluorescence microscope. Previous studies proposed the quantification of the fluorescence intensity as an alternative for the classical end-point titer evaluation. However, the different distribution of bright/dark light linked to the nature of the self-antigen and its location in the cells result in different mean fluorescence intensities. The aim of the present study was to correlate Fluorescence Index (F.I.) with end-point titers for each well-defined ANA pattern. MethodsRoutine serum samples were screened for ANA testing on HEp-2000 cells using Immuno Concepts Image Navigator System, and positive samples were serially diluted to assign the end-point titer. A comparison between F.I. and end-point titers related to 10 different staining patterns was made. ResultsAccording to our analysis, good technical performance of F.I. (97% sensitivity and 94% specificity) was found. A significant correlation between quantitative reading of F.I. and end-point titer groups was observed using Spearman's test and regression analysis. A conversion scale of F.I. in end-point titers for each recognized ANA-pattern was obtained. ConclusionsThe Image Navigator offers the opportunity to improve worldwide harmonization of ANA test results. In particular, digital F.I. allows quantifying ANA titers by using just one sample dilution. It could represent a valuable support for the routine laboratory and an effective tool to reduce inter- and intra-laboratory variability.

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies available for ANA screening, indirect immunofluorescence (IIF) using Human epithelial cells-2 (HEp-2) substrate remains the primary and recommended method because of its superior sensitivity. HEp-2 substrates can detect a multitude of patterns resulting from autoantibody binding to various protein and nucleic acid autoantigens distributed throughout the nucleus and cytoplasm of the cells. The great diversity of monospecific and mixed patterns resulting from positive reactions on HEp-2 substrate also complicate the interpretation and accuracy of reporting. One specific example which received utmost attention recently is the dense fine speckled 70 (DFS70) pattern resulting from autoantibodies that specifically bind to a protein called lens epithelium derived growth factor (LEDGF). Lack of clear association with a specific systemic autoimmune disease and high prevalence in healthy populations have made accurate interpretation of DFS70 pattern important. Accurate distinction of DFS70 pattern from disease-associated patterns using conventional HEp-2 substrate is challenging. Moreover, frequent co-occurrence of DFS70 pattern along with disease-associated patterns such as homogeneous, speckled, and mixed homogeneous-speckled patterns complicate the IIF interpretation. The goal of this paper is to demonstrate the utility of a novel engineered HEp-2 IIF substrate that retains all advantages of conventional HEp-2 substrate while simultaneously providing the ability to distinguish DFS70 pattern with high confidence in both monospecific and mixed ANA positive examples. The new substrate is further able to unmask disease-associated ANA patterns previously concealed by DFS70 pattern.

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate.

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies available for ANA screening, indirect immunofluorescence (IIF) using Human epithelial cells-2 (HEp-2) substrate remains the primary and recommended method because of its superior sensitivity. HEp-2 substrates can detect a multitude of patterns resulting from autoantibody binding to various protein and nucleic acid autoantigens distributed throughout the nucleus and cytoplasm of the cells. The great diversity of monospecific and mixed patterns resulting from positive reactions on HEp-2 substrate also complicate the interpretation and accuracy of reporting. One specific example which received utmost attention recently is the dense fine speckled 70 (DFS70) pattern resulting from autoantibodies that specifically bind to a protein called lens epithelium derived growth factor (LEDGF). Lack of clear association with a specific systemic autoimmune disease and high prevalence in healthy populations have made accurate interpretation of DFS70 pattern important. Accurate distinction of DFS70 pattern from disease-associated patterns using conventional HEp-2 substrate is challenging. Moreover, frequent co-occurrence of DFS70 pattern along with disease-associated patterns such as homogeneous, speckled, and mixed homogeneous-speckled patterns complicate the IIF interpretation. The goal of this paper is to demonstrate the utility of a novel engineered HEp-2 IIF substrate that retains all advantages of conventional HEp-2 substrate while simultaneously providing the ability to distinguish DFS70 pattern with high confidence in both monospecific and mixed ANA positive examples. The new substrate is further able to unmask disease-associated ANA patterns previously concealed by DFS70 pattern.

A Multiplex Autoantibody Panel for Early Detection of Autoimmune Disease Activity

Background: Detection of autoantibodies has played a consolidate role in diagnosis of systemic autoimmune disorders. A cascade autoantibody testing is usually performed by employing antinuclear antibodies (ANA) test as a first screening test and the other tests as second level determinations. Here, we present that supplementing extractable nuclear antigens (ENA) tests to the ANA test may capture more autoimmunity and provide critical medical information at an early stage. In this study, we evaluated the clinical significance of a multiplex ANA + ENA panel. Methods: A cohort of 110 subjects, identified as ANA negative but ENA positive, were followed up for two years. The detection of their ANA and anti-ENA autoantibodies was assessed with a multiplex ANA + ENA panel at Vibrant America Clinical Laboratory. Results: During two years of multi-visit follow-up, 23 out of 110 subjects (20.9%) were found to become ANA positive within an average of 385 (±144) days. Histone (50/110, 45.5%) and Chromatin (25/110, 22.7%) antibodies were the most frequently found antibodies at their first visits. The subjects who were positive for RNP (5/8, 62.5%) and SSA (Ro) (10/22, 45.5%) have the highest ratio of conversion to positive ANA. No significant correlation was observed between the conversion frequency and the number of anti-ENA antibodies being carried. Conclusion: This study, which followed up on the subjects who had disparate ANA and ENA test results, showed that anti-ENA antibodies may exist years earlier than ANA. Combining ENA tests with ANA screening may reduce false negatives and improve sensitivity.

Carbimazole-induced agranulocytosis – A rare case report

A 30-year-old lady with hyperthyroidism for the past 1 year was initially on carbimazole 30 mg twice orally and then changed to 30 mg once daily. She presented with intermittent fever since 3 weeks, h/o oral ulcers, difficulty in swallowing, and palpitations. Routine investigations showed anemia, neutropenia, leucopenia, and C-reactive protein elevation. Peripheral smear showed normocytic normochromic anemia with leucopenia. Widal test, RA factor test, immunoglobulin M (Ig M) dengue, Ig M Lepto, antinuclear antibody screen, cytoplasmic antineutrophil cytoplasmic antibodies, and perinuclear anti-neutrophil cytoplasmic antibodies tests were negative. Viral markers are negative.

Ketosis-prone diabetes and SLE co-presenting in an African lady with previous gestational diabetes.

We describe the case of an African woman who was diagnosed with ketosis-prone diabetes with diabetes-associated autoantibodies, after being admitted for diabetic ketoacidosis (DKA) precipitated by her first presentation of systemic lupus erythematosus (SLE). She had a seven-year history of recurrent gestational diabetes (GDM) not requiring insulin therapy, with return to normoglycaemia after each pregnancy. This might have suggested that she had now developed type 2 diabetes (T2D). However, the diagnosis of SLE prompted testing for an autoimmune aetiology for the diabetes, and she was found to have a very high titre of GAD antibodies. Typical type 1 diabetes (T1D) was thought unlikely due to the long preceding history of GDM. Latent autoimmune diabetes of adults (LADA) was considered, but ruled out as she required insulin therapy from diagnosis. The challenge of identifying the type of diabetes when clinical features overlap the various diabetes categories is discussed. This is the first report of autoimmune ketosis-prone diabetes (KPD) presenting with new onset of SLE.Learning points:DKA may be the first presentation of a multi-system condition and a precipitating cause should always be sought, particularly in women with a history of GDM or suspected T2D.All women with GDM should undergo repeat glucose tolerance testing postpartum to exclude frank diabetes, even when post-delivery capillary blood glucose (CBG) tests are normal. They should also be advised to continue CBG monitoring during acute illness in case of new onset diabetes.KPD comprises a spectrum of diabetes syndromes that present with DKA, but subsequently have a variable course depending on the presence or absence of beta cell failure and/or diabetes autoantibodies.KPD should be considered in a patient with presumed T2D presenting with DKA, especially if there is a personal or family history of autoimmune diabetes.LADA should be suspected in adults presumed to have T2D, who do not require insulin therapy for at least six months after diagnosis and have anti-GAD antibodies.Patients with autoimmune diabetes have an increased risk of other autoimmune diseases and screening for thyroid, parietal cell, coeliac and antinuclear antibodies should be considered.

Predictive autoimmunity using autoantibodies: screening for anti-nuclear antibodies.

Abstract Background: Early detection of antinuclear antibodies (ANA) in asymptomatic subjects is useful to predict autoimmune diseases years before diagnosis. ANA have been determined by indirect immunofluorescence (IIF) using human epithelial type 2 (HEp-2) cells, which is considered the gold standard technique. Multiplex technology (BioPlex ANA Screen) has been introduced for ANA evaluation in recent years. Nevertheless, concordance between BioPlex and IIF is low and there is no harmonization between both methods for detection of autoantibodies. This study has aimed to clarify the clinical significance of autoantibodies detected by BioPlex ANA Screen in subjects with undiagnosed clinical suspicion of autoimmune disease and to determine the predictive value of autoantibodies detected by BioPlex ANA Screen. Methods: A 3-year follow-up study was performed of 411 subjects without a clear diagnosis of autoimmune diseases in whom autoantibodies were detected by BioPlex ANA Screen that were negative by IIF on HEp-2 cells. Results: At 3 years of follow-up, 312 (76%) subjects were positive for autoantibodies by IIF and 99 subjects continued to be negative. A diagnosis of autoimmune disease was found in most of the subjects (87%). Conclusions: BioPlex ANA Screen has greater sensitivity than IIF on HEp-2 cells for autoantibodies detection. Early detection of these antibodies by BioPlex can predict possible development of autoimmune diseases.

Quality Monitoring Approach for Optimizing Antinuclear Antibody Screening Cutoffs and Testing Work Flow.

An antinuclear antibody (ANA) testing strategy involving enzyme immunoassay (EIA) screening that reflexed to immunofluorescence assay (IFA) was implemented, monitored, and optimized for clinical utility. The clinical utility, test performance, and workload implications of various ANA testing strategies were compared during the following study phases: (a) Preimplementation (n = 469) when IFA was used for all ANA screening, (b) Verification (n = 58) when EIA performance was confirmed, (c) Implementation (n = 433) when a reflexive strategy (EIA screen/IFA confirmation) was implemented, and (d) Postimplementation (n = 528) after the reflexive strategy was optimized. Sequential samples were captured in the Preimplementation, Implementation, and Postimplementation phases for clinical performance evaluation. Clinical performance of the EIA screen, per ROC analysis yielded area under the curve (AUC) of 0.846 in the Implementation phase and increased to 0.934 Postimplementation (P < 0.01); AUC for IFA similarly increased, from 0.678 to 0.808 (P = 0.05). The reflexive testing strategy increased screening sensitivity from 61% Preimplementation (IFA) to 98% (EIA) at Implementation and was maintained after optimization (98%, Postimplementation). Optimization decreased the false-positive rates for both EIA (from 40% to 18%) and IFA (18% to 8%) and was associated with reductions in daily full-time equivalent (by 33%) and IFA slide use (by 50%). Continuous quality monitoring approaches that incorporate sequential data sets can be used to evaluate, deploy, and optimize sensitive EIA-based ANA screening methods that can reduce manual IFA work without sacrificing clinically utility.

Antinuclear Antibody Screening: A Delicate Balance for Clinical Laboratories.

In the article in this issue, “Quality monitoring approach for optimizing antinuclear antibody screening cutoffs and testing work flows,” Tacker and Perrotta address an issue faced by many laboratories—how to offer antinuclear antibody (ANA)3 testing that combines optimal clinical performance with realistic laboratory work flows (1). Specifically, Tacker and Perrotta evaluated an ANA enzyme immunoassay (EIA) as a way to decrease the number of manual immunofluorescence assays (IFAs) performed in their laboratory. Maintaining IFA testing is a significant challenge for many clinical laboratories (2, 3). It is usually a manual process with many steps that each have risk for error. In addition, the interpretation is subjective, relying on well-trained clinical technologists for accurate and consistent test results. For years, clinical laboratories have recognized the need for alternatives to IFA that are more automated and less subjective. These alternatives are available in the form of EIAs and multiplex immunoassays (MIAs). EIAs generally use a human epithelial type 2 (HEp-2) cell lysate as the capture antigen, which makes it fairly representative of the IFA method (4). The MIA is a bead-based method in which individual antigens are conjugated to different bead sets (5, 6). Collectively, these beads represent an …

Induction of Autoantibodies and Autoimmune Diseases in Patients with Psoriasis Receiving Tumor Necrosis Factor Inhibitors

BackgroundThe induction of antinuclear antibodies (ANA) and the onset of autoimmune diseases have been reported after treatment with tumor necrosis factor (TNF) inhibitors, though controversy persists. ObjectivesTo determine the frequency of onset of autoimmune diseases and of the appearance of autoantibodies in psoriasis patients administered TNF inhibitors (adalimumab and etanercept) subcutaneously and to correlate this with the effectiveness of treatment, adverse effects, and the order of use of TNF inhibitors. We also tried to identify any factors that might predict the appearance of ANA and autimmune diseases. MethodsWe performed a retrospective study of a cohort of 121 patients monitored over an 11-year period. ANA were measured at baseline and at 3, 6, and 12 months; positive results were followed up by study of antibodies to double-stranded DNA. Extractable nuclear antigen (ENA) antibodies were also studied at baseline and at 3, 6, and 12 months. Patients with a baseline assay of ANA and ENA at least one more assay during the first year were included in the study, and these antibodies were measured annually thereafter. Psoriasis area severity index was calculated and adverse effects were recorded at each visit. ResultsA significant increase in ANA positivity was observed during treatment of moderate-to-severe psoriasis with adalimumab and etanercept, but this was not associated with the onset of autoimmune diseases. No correlation was observed with treatment efficacy, the order of use of TNF inhibitors, or the appearance of adverse effects. No predictive factors for the appearance of ANA were identified, except for the body mass index. ConclusionsWe recommend ANA measurement and screening for autoimmune diseases prior to treatment with TNF inhibitors, but not routine serial measurements of ANA during follow-up except in patients with signs or symptoms suggestive of autoimmune disease.

Automated evaluation of ANA under real-life conditions

IntroductionVisual evaluation of indirect immunofluorescence (IIF) on human epithelial-2 cells is the routine method for screening for antinuclear antibodies (ANA) in connective tissue diseases. Since visual IIF is time-consuming and...

Analysis of DFS70 pattern and impact on ANA screening using a novel HEp-2 ELITE/DFS70 knockout substrate

Indirect immunofluorescence (IIF) using human epithelial cell (HEp-2) substrate is a widely used and the recommended method for screening of antinuclear antibodies (ANA). Dense fine speckled (DFS70) pattern on HEp-2 has been widely reported in various healthy and disease groups. Interpretation of DFS70 pattern can be challenging on a conventional HEp-2 substrate due to its similarity to some of the disease associated patterns. The high prevalence of DFS70 autoantibodies in normal population, lack of association with a particular disease group and a general negative association with systemic and ANA associated autoimmune rheumatic diseases (SARD/AARD) necessitates the confirmation of DFS70 pattern. Results using available commercial assays for confirmation of DFS70 autoantibodies do not always agree with IIF screening results further complicating the lab work flow and ANA algorithms. In this review, we discuss the prevalence of DFS70 antibodies and factors affecting the performance of IIF and DFS70 specific confirmatory assays. Factors that contribute to disagreement between DFS70 suspicion by IIF and confirmatory assays will also be discussed. In addition, we also describe a novel IIF HEp-2 substrate, and its positive impact on DFS70 reporting and ANA screening-confirmation algorithm.

Inducción de anticuerpos y enfermedades autoinmunes en pacientes con psoriasis tratados con fármacos anti-TNFα

BackgroundThe induction of antinuclear antibodies (ANA) and the onset of autoimmune diseases have been reported after treatment with tumor necrosis factor (TNF) inhibitors, though controversy persists. ObjectivesTo determine the frequency of onset of autoimmune diseases and of the appearance of autoantibodies in psoriasis patients administered TNF inhibitors (adalimumab and etanercept) subcutaneously and to correlate this with the effectiveness of treatment, adverse effects, and the order of use of TNF inhibitors. We also tried to identify any factors that might predict the appearance of ANA and autimmune diseases. MethodsWe performed a retrospective study of a cohort of 121 patients monitored over an 11-year period. ANA were measured at baseline and at 3, 6, and 12 months; positive results were followed up by study of antibodies to double-stranded DNA. Extractable nuclear antigen (ENA) antibodies were also studied at baseline and at 3, 6, and 12 months. Patients with a baseline assay of ANA and ENA at least one more assay during the first year were included in the study, and these antibodies were measured annually thereafter. Psoriasis area severity index was calculated and adverse effects were recorded at each visit. ResultsA significant increase in ANA positivity was observed during treatment of moderate-to-severe psoriasis with adalimumab and etanercept, but this was not associated with the onset of autoimmune diseases. No correlation was observed with treatment efficacy, the order of use of TNF inhibitors, or the appearance of adverse effects. No predictive factors for the appearance of ANA were identified, except for the body mass index. ConclusionsWe recommend ANA measurement and screening for autoimmune diseases prior to treatment with TNF inhibitors, but not routine serial measurements of ANA during follow-up except in patients with signs or symptoms suggestive of autoimmune disease.

Efficient evaluation of humoral immune responses by the use of serum pools

BackgroundCollection and testing of individual serum samples are often used in research to gain knowledge about e.g. the humoral response against bacteria or virus. This is a valid but time-consuming method and might be a waste of valuable serum samples for inefficient research. So far, no study has considered using serum pools as a quick and efficient screening method to confirm or deny hypotheses. MethodsWe created serum pools from four different patient groups (systemic lupus erythematosus n=85, rheumatoid arthritis n=77, Sjögren's syndrome n=91, systemic sclerosis n=66) and one healthy control group (n=67). Each serum pool was analyzed using three well-known immunoassays: enzyme-linked immunosorbent assay (ELISA), line blot, and immunofluorescence microscopy (anti-nuclear antibody (ANA) screening). The presence of Epstein-Barr virus (EBV) EA/D-, EBNA-1-, VCA p23-, and gp350-directed antibodies was used to validate serum pools as an efficient tool for further investigations by comparison to previous findings in this area. ResultsThe presence of EBV EA/D-, EBNA-1-, VCA p23-, and gp350-directed antibodies in each pool was consistent within the obtained ELISA and line blot results, as increased titers of IgG against the four antigens were found in all patient serum pools and also in individual sera regarding gp350. These results correspond to previous findings on individual samples from patients with these diseases. The presence of ANAs was observed in all four patient serum pools and not in the HC pool by both line blots and immunofluorescence microscopy, which corresponds with the expectations and further corroborate the application of serum pools for screenings. ConclusionWe developed and validated the use of serum pools that reliably and rapidly can confirm or deny hypotheses, which enables a more efficient research concentrating on the most evident factors.

In Response to: Utility of Antinuclear Antibody Screening by Various Methods in a Clinical Laboratory Patient Cohort-Differences in Sensitivity, Specificity, and Limitations.

Deng et al. (1) compared the clinical utility of three antinuclear antibody (ANA)1 screening assays, i.e., an enzyme immunoassay (EIA), an immunofluorescence assay (IFA) on human epithelial (HEp)-2 cells, and a multiplex immunoassay (MIA). Among the conclusions from their data were the following statements: “the overall diagnostic performance of the IFA, EIA, and MIA were not statistically different….” Our primary focus will be on the above quoted words, with additional attention given to the propensity of HEp-2 cell lines to miss anti–Sjogren's Syndrome …

In Reply to Geoffrey Baird on: Utility of Antinuclear Antibody Screening by Various Methods in a Clinical Laboratory Patient Cohort.

The authors would like to thank Dr. Baird for his constructive comments regarding the calculation of sensitivity and specificity of antinuclear antibody (ANA)1 testing in our recent publication (1). The goal of this study was to compare the diagnostic utility of various ANA methods in a representative testing population. Studies of this type may be performed using a case-control design, where cases (patients with the disease) and controls (individuals without the disease) are randomly selected, or by using a collection of consecutive …

In Response to: Utility of Antinuclear Antibody Screening by Various Methods in a Clinical Laboratory Patient Cohort.

In the inaugural issue of The Journal of Applied Laboratory Medicine , Deng et al. (1) reported the utility of antinuclear antibody screening by various methodologies. …

Association of anti-Ro52, anti-Ro60 and anti-La antibodies with diagnostic, clinical and laboratory features in a referral hospital in Jerez, Spain

ObjectiveSeveral antibodies have proven to be useful in autoimmune diseases, as markers for diagnosis, prognosis or clinical manifestations. Our objective was to evaluate the diagnosis and manifestations associated for antibodies anti-Ro52, anti-Ro60 and anti-La at a referral hospital in Spain. MethodsWe retrospectively analyzed the antigenic specificities of the consecutive samples submitted to the Immunology Unit for antinuclear antibody screening between 2002 and 2012. We included patients with more than one positive sample for some of the autoantibodies anti-Ro52, anti-Ro60 or anti-La. We also reviewed diagnosis, clinical and laboratory features. As dependent variable we evaluated possible combinations of anti-Ro52, anti-Ro60 and anti-La. Results322 patients, 91% females, were studied (age 44.3±15.51 years). The most frequent diagnosis was Sjögren's syndrome (40.06%) and systemic lupus erythematosus (SLE) (36.6%). The most prevalent pattern by indirect immunofluorescence was the fine speckled (69.9%). Anti-Ro52+/anti-Ro60+/anti-La+ combination was positively associated with fine speckled pattern (p: 0.001) and negatively with homogeneous (p: 0.016) and cytoplasmic pattern (p: 0.002). Isolated anti-Ro52+ was negatively associated with fine speckled pattern (p<0.001) and positively with the cytoplasmic one (p<0.001). The main positive associations with clinical symptoms were xerostomia and xerophthalmia with anti-Ro52+/anti-Ro60+/anti-La+ (p<0.001), oral ulcers with anti-Ro52+/anti-Ro60+/anti-La− (p: 0.002) and alopecia with anti-Ro52−/anti-Ro60+/anti-La− (p: 0.003). Negative associations were xerophthalmia and photosensitivity with anti-Ro52+/anti-Ro60−/anti-La− (p: 0.003). Laboratory positive associations were hypergammaglobulinemia with anti-Ro52+/anti-Ro60+/anti-La+ (p: 0.003), and hypocomplementemia with anti-Ro52−/anti-Ro60+/anti-La− (p: 0.003). Leucopenia was negatively associated with anti-Ro52+/anti-Ro60−/anti-La− (p: 0.003). ConclusionOur study found significant relationships between clinical and laboratory manifestations with different patterns of antibodies to anti-Ro52, anti-Ro60 and anti-La. The combination of antibodies might be clinically useful due to prognostic and therapeutic implications.

Utility of Antinuclear Antibody Screening by Various Methods in a Clinical Laboratory Patient Cohort.

Antinuclear antibody (ANA)5 testing is routinely performed during evaluation of patients with a suspected connective tissue disease (CTD), yet the question of which method is most appropriate remains controversial. The purpose of this study was to evaluate the clinical utility of ANA testing by an enzyme immunoassay (EIA), an immunofluorescence assay (IFA), and a multiplex immunoassay (MIA) in a routine laboratory population. Samples (n = 1000) were collected from specimens submitted for ANA testing by EIA (Bio-Rad). All samples were subsequently analyzed by IFA (Zeus) and MIA (Bio-Rad). The sample cohort was weighted to represent the routine testing population. Diagnostic information was obtained by chart review. For the diagnosis of a CTD, ROC curve analysis demonstrated no significant differences between IFA (area under the curve 0.81) and EIA (0.84) (P = 0.25), with overlay of a single point for the MIA. When normalized to a specificity of approximately 90%, the sensitivities of the MIA, EIA, and IFA were 67%, 67%, and 56%, respectively. By varying the clinical cutoff, the IFA could achieve the highest sensitivity of 94%; however, the corresponding specificity was only 43%. In contrast, a strongly positive EIA had a specificity of 97%, although, at this cutoff, the sensitivity was only 40%. Although the overall diagnostic performance of the IFA, EIA, and MIA were not statistically different, the clinical sensitivity and specificity varied dramatically based on the positive/negative cutoff. Knowledge about the performance characteristics of each method will significantly aid in the interpretation of ANA testing.

The Clinical Relevance of Anti-DFS70 Autoantibodies.

Despite all the progress in the establishment of specific autoantibody assays, screening for antinuclear antibodies (ANA) by indirect immunofluorescence on HEp-2 cells for quality-oriented laboratory diagnosis of ANA associated rheumatic diseases (AARD) remains indispensable but is not without limitations. Recent data on the relevance of the dense fine speckled (DFS) pattern and anti-DFS70 antibodies disclosed novel possibilities to optimize the serological stepwise diagnostics of AARD. The DFS pattern on HEp-2 cells is well differentiated from the classic "homogeneous" ANA pattern associated with dsDNA antibodies. This is the most frequent pattern in high titer ANA-positive healthy persons. The most characteristic ANA specificity associated with DFS pattern is the anti-DFS70 antibody (synonym LEDGF antibody). The prevalence of anti-DFS70 antibodies in AARD patients is significantly lower compared with the prevalence in ANA-positive healthy persons. There is a negative association between anti-DFS70 antibodies and AARD, especially if no concomitant AARD-specific autoantibodies are found. Isolated anti-DFS70 antibodies are detectable in less than 1% of AARD but are detectable in 2-22% of healthy persons. In the presence of an isolated anti-DFS70 antibody, the posttest probability for AARD is reduced significantly. The significance of anti-DFS70 antibodies as a criterion that helps to exclude AARD is also confirmed by follow-up studies on anti-DFS70 antibodies of positive, healthy individuals, who did not develop any AARD during a 4 year observation period. Consequently, anti-DFS70 antibodies are valuable novel biomarkers for better interpretation of positive ANA in cases of negative AARD-associated autoantibodies and should be integrated in modified test algorithms to avoid unnecessary referrals and examinations of ANA-positive persons.