Antinuclear Antibody Indirect Immunofluorescence Research Articles

Diagnostic value of screening enzyme immunoassays compared to indirect immunofluorescence for anti-nuclear antibodies in patients with systemic rheumatic diseases: A systematic review and meta-analysis.

This study aimed to review and compare the diagnostic accuracy of the screening enzyme immunoassay (SEIA) and indirect immunofluorescence (IIF) as anti-nuclear antibody (ANA) screening assays for patients with systemic rheumatic diseases (SRDs), including systemic lupus erythematosus (SLE), Sjögren's syndrome (SS), and systemic sclerosis (SSc). A systematic literature search was conducted in the Medline, Embase, Cochrane, Web of Science, and Scopus databases for articles published before August 2017. A bivariate random effects model was used to calculate pooled diagnostic values. Thirty-three studies including 3976 combined SRDs, 2839 SLE, 610 SS, and 1002 SSc patients and 11,716 non-healthy and 8408 healthy controls were available for the meta-analysis. The summary sensitivities of SEIA vs. IIF were 87.4% vs 88.4% for combined SRDs, 89.4% vs. 95.2% for SLE, 88.7% vs. 88.4% for SS, and 85.4% vs. 93.6% for SSc, respectively. Meanwhile, the summary specificities of SEIA vs. IIF were 79.7% vs.78.9% for combined SRDs, 89.1% vs. 83.3% for SLE, 89.9% vs. 86.8% for SS, and 92.8% vs. 84.2% for SSc, respectively. Although the differences in sensitivity and specificity between SEIA and IIF were not significant in most subgroups, the summary sensitivity of SLE presented statistically significant changes. Our systematic meta-analysis demonstrates that both SEIA and IIF are useful to detect ANAs for SRDs. Between the two assays, IIF is a more sensitive screening assay than SEIA, particularly in patients with SLE. SEIA is comparable to IIF, considering the specificity and standardization.

Evaluation of Diagnostic Significance and Cost Effectiveness of ELISA and IFA for the Diagnosis of Autoimmune Disorders

Background: The presence of antinuclear antibodies (ANA) is a hallmark of autoimmune diseases. As Clinicopathological classification of autoimmune diseases is difficult without laboratory support, laboratory testing is of helps in diagnosis, treatment, prognosis, and prediction of the pathological changes by disease activity. Although different tests are available for ANA detection enzyme linked immunosorbent assay (ELISA) is the mainstay of diagnosis in most routine laboratories. Indirect immunofluorescence antinuclear antibody test (IFA) though currently the “gold standard” it is not widely practiced. Most studies have used Hep2 cells for the detection of autoantibodies by IFA. However Hep 2000 Ro is superior compared to Hep 2 which lacks capability of detecting some autoantibodies like Ro antibodies. Hence, this study was undertaken to compare the diagnostic value and cost effectiveness of ANA pattern, ELISA with profile testing for patients suspected to have autoimmune disorders. Results: In the present study we observed high prevalence of autoimmune diseases in females (75.82%).ANA-ELISA in criteria matched cases with respect to ANA-IFA had a low sensitivity (59% versus 80%), higher specificity (84% versus 70%). Statistical analysis of ELISA and IFA with respect to ANA Profile showed a very less sensitivity by ELISA over IFA (51% vs. 78%) and equal specificity (70-72%) in 142 criteria matched cases. Conclusions: Statistically significant differences between ELISA and IFA infers IFA-ANA is a very appropriate method for screening purposes also IFA have capability of finding anti-mitochondrial and other cytoplasmic antibodies, which is not possible with ELISA.

Automated antinuclear immunofluorescence antibody analysis is a reliable approach in routine clinical laboratories.

Indirect immunofluorescence (IIF) assays are recommended as the gold standard method for the detection of antinuclear antibodies (ANAs). This study aimed to investigate the reliability of an automated system. We compared 3745 serum samples using NOVA View archived images with manual analysis via microscopy. A custom cutoff value was established to distinguish ANA titers and was validated in two clinical laboratories. The automatic ANA pattern recognition system was evaluated, and all ANA-positive sera were subjected to two commercial ANA IIF kits to compare the consistency of the pattern interpretation results. For inconsistent patterns, a third ANA IIF testing kit was utilized. Agreement of the interpretation of the ANA IIF test using the platform of NOVA View and manual microscopy was 96.9%. The local cutoff value to discriminate ANA titers in four main ANA patterns was calculated based on 1390 serum samples. In our laboratory, the titer prediction accuracy was superior to the preset cutoff in NOVA View (p<0.01); the performance was similar in another laboratory (p=0.11). The automatic pattern recognition accuracies of speckled, homogeneous, centromere, nucleolar and nuclear dot patterns were 62.7%, 57.4%, 92.6%, 30.5% and 27.3%, respectively. The consistency of the pattern interpretation results between INOVA and MBL kits was 95.3%. It is necessary to establish a custom value-added ANA report. However, confirmation of the digital immunofluorescence images by expert technicians was essential, and suspect results of an ANA pattern should be reconfirmed by another commercial ANA IIF kit to achieve more reliable results.

Use of panel testing for detection of antinuclear antibody in a resource-limited setting: an appraisal

ABSTRACTObjectives: Despite an increase in the incidence of systemic connective tissue diseases (CTD), panel testing for detection of antinuclear antibodies (ANA) is not a routine practice in many health centers of the Indian subcontinent. Consequently, the data on its significance is scanty.Methods: To evaluate utility of panel testing, line immunoassay (LIA) and indirect immunofluorescence antinuclear antibody test (IIF-ANA) were performed in 321 cases of CTD.Results: Out of 321 serum samples screened by the above tests, 227 were positive and 18 were negative by both LIA and IIF-ANA. Additional 11/321 (3.4%) cases were picked up by LIA. SSA was most common specificity in these cases followed by SSA/SSB, SSB, Ro-52, Jo-1, dsDNA and nRNP/Sm.Conclusion: Use of LIA along with IF-ANA and ELISA improves sensitivity of CTD screening.

The burden of the variability introduced by the HEp-2 assay kit and the CAD system in ANA indirect immunofluorescence test.

According to the recent recommendations of the American College of Rheumatology, ANA Task Force, IIF technique should be considered the gold standard in antinuclear antibodies (ANAs) testing. To overcome the lack of standardization, biomedical industries have developed several computer-aided diagnosis (CAD) systems. Two hundred and sixty-one consecutive samples with suspected autoimmune diseases were tested for ANA by means of IIF on routinely HEp-2 assay kit (Euroimmun AG). Assignment of result was made if consensus for positive/negative was reached by at least 2 out of 3 expert physicians. ANA-IIF was also carried out using 3 CAD systems: Zenit G-Sight (n=84), Helios (n=85) and NOVA View (n=92); human evaluation was repeated on the same substrate of each CAD system (Immco, Aesku and Inova HEp-2 cells, respectively). To anonymize the results, we randomly named these three systems as A, B and C. We ran a statistical analysis computing several measures of agreement between the ratings, and we also improved the evaluation by using the Wilcoxon's test for nonparametric data. Agreement between the human readings on routinely HEp-2 assay kit and human readings on CAD HEp-2 assay was substantial for A (k=0.82) and B (k=0.72), and almost perfect for C (k=0.89). Such readings were statistically different only in case A. Comparing experts' readings with the readings of CAD systems, when the samples were prepared using CAD HEp-2 assay kits, we found almost perfect agreement for B and C (k=0.86; k=0.82) and substantial agreement for A (k=0.73). Again, human and CAD readings were statistically different only in A. When we compared the readings of medical experts on routinely HEp-2 assay kit with the output of the CAD systems that worked using their own slides, we found substantial agreement for all the systems (A: k=0.62; B: k=0.65; C: k=0.71). Such readings were not statistically different. The change of the assay kit and/or the introduction of a CAD system affect the laboratory reporting, with an evident impact on the autoimmune laboratory workflow. The CAD systems may represent one of the most important novel elements of harmonization in the autoimmunity field, reducing intra- and inter-laboratory variability in a new vision of the diagnostic autoimmune platform.

Recognition of the dense fine speckled (DFS) pattern remains challenging: results from an international internet-based survey

PurposeThe dense fine speckled (DFS) pattern as detected by indirect immunofluorescence (IIF) on HEp-2 cells has been associated with several inflammatory diseases but is most commonly observed in individuals that do not have an antinuclear antibody (ANA)-associated rheumatic disease and even in apparently healthy individuals. Consequently, the accurate identification and correct reporting of this IIF pattern is of utmost importance and accordingly has been recognized by several international study groups for the detection of ANA. Furthermore, the DFS IIF pattern has recently been recommended as a competency level recognition pattern by the International Consensus on Antinuclear Antibody (ANA) Pattern (ICAP, http://www.anapatterns.org/) Committee. The objective of this study was to use an internet-based survey to assess how accurately the DFS IIF pattern was recognized by experienced technologists.MethodsHigh-resolution digital IIF images were captured using the automated IIF NOVA View instrument (Inova Diagnostics, San Diego, CA). Ten images were posted in an anonymous, international, internet-based interpretive survey. Two hundred and thirty IIF technologists were invited to participate. Four of the images in the survey were from previously characterized serum samples with classical ANA IIF patterns (nucleolar, centromere, homogeneous, and speckled) and two of the images were from samples with a DFS IIF ANA pattern and isolated anti-DFS70 antibodies as determined by a chemiluminescence immunoassay. The remaining four images were from sera with the classic IIF ANA patterns referred to above and mixed with a monospecific anti-DFS70-positive sample. The survey included multiple choice selections: homogeneous, DFS, centromere, nucleolar, speckled, other, or unrecognizable.Results125 of the 230 participants who completed the survey had diverse levels of experience in IIF pattern recognition on HEp-2 cells ranging from <1 year to >10 years of experience (average >10 years). Participants had a high concordance in correctly classifying the classical ANA IIF patterns: ranging from 95.2 % for centromere to 74.4 % for nucleolar patterns. The unmixed DFS pattern was recognized with significantly lower accuracy (~50 %; p < 0.05). However, less than 10 % correctly identified mixed patterns derived from the sera containing both clinically relevant ANA and anti-DFS70 antibodies.ConclusionsRecognizing the DFS ANA IIF pattern and mixed IIF patterns composed of DFS + clinically relevant ANA patterns poses a significant challenge. Consequently, it seems imperative that DFS-specific immunoassays should be used to confirm the presence of anti-DFS70 antibodies before definitive results are reported to physicians.

The comparison of antinuclear antibody positivity and thiol/disulfide levels / Antinükleer antikor pozitifliği ve tiyol/disülfid düzeylerinin karşılaştırılması

Abstract Objective: There is also a growing body of evidence showing that an abnormal thiol disulphide homeostasis state is involved in the pathogenesis of certain diseases. In the present study, it was aimed to investigate the relationship among ANA positivity and dynamic thiol/ disulphide homeostasis in serum samples Methods: Serum samples were collected from ANA-positive and ANA-negative individuals. The indirect immunofluorescence antinuclear antibody test (HEp 20-10, EUROIMMUN, Germany) was used. The serum thiol/disulfide levels were measured with the fully automated new method. Results: No statistically significant difference was detected between thiol/disülfide levels in individuals who were negative and those who were positive for ANA (p&gt;0.05). Besides, among the ANA patterns was not found statistically significant difference for thiol/disulfide levels (p&gt;0.05). However, serum native thiol, total thiol and disülfide levels were decreased with aging. The native thiol, total thiol levels and native thiol/disulfide, total thiol/disulfide, native thiol/total thiol ratio was found statistically significant difference among the different age groups (p&lt;0.05). Conclusion: Determination of thiol/disulfide homeostasis can not provide valuable information on normal or abnormal biochemical processes in ANA-positive individuals. However, determination of thiol/disulfide homeostasis which could be a contributing factor to the pathogenesis of some age-related diseases.

Impact of the routine implementation of automated indirect immunofluorescence antinuclear antibody analysis: 1 year of experience.

Impact of the routine implementation of automated indirect immunofluorescence antinuclear antibody analysis: 1 year of experience.

Frequency of dense fine speckled pattern in immunofluorescence screening test.

The presence of antinuclear antibodies (ANA), directed against intracellular antigens, is a distinctive feature of systemic autoimmune rheumatic diseases (SARDs). The standard test for antinuclear antibody screening is the indirect immunofluorescence (IIF). Anti-dense fine speckled 70 (anti-DFS70) antibodies were initially identified as an ANA IIF pattern from a patient with interstitial cystitis, but they were later associated with various other conditions. The objective of the study was to determine the frequency of anti-DFS70 antibodies in a cohort of patients undergoing routine ANA testing. From January 2011 to January 2012, a total of 5800 serum samples were screened for ANA by IIF (Euroimmune AG, Lübeck, Germany). DFS pattern was searched. ANA were present in 1302 (22.4%) of all patients. There were 16 (1.2%) anti-DFS70 antibody-positive patients. The number of females and males who have anti-DFS70 antibody was eleven and five, respectively. All of the samples presented a titer of ≥1/320. There was one patient with SARD from the rheumatology department. Another 15 patients were from gastroenterology, endocrinology, and general internal medicine. Although a distinctive clinical association has not been reported, anti-DFS70 have been proposed as a significant biomarker for the exclusion of SARD. The present study is a preliminary study. There is a need for a reliable assay to ensure reactivity to DFS70 and screening large populations.

A fully automated IIF system for the detection of antinuclear antibodies and antineutrophil cytoplasmic antibodies

Indirect immunofluorescence (IIF) is the main technique for the detection of antinuclear antibodies (ANA) and antineutrophil cytoplasmic antibodies (ANCA). The fully automated IIF processor HELIOS(®) is the first IIF processor that is able to automatically prepare slides and perform automatic reading. The objective of the present study was to determine the diagnostic performance of this system for ANA and ANCA IIF interpretation, in comparison with visual IIF. ANA detection by visual IIF or HELIOS(®) was performed on 425 sera samples including: 218 consecutive samples submitted to a reference laboratory for routine ANA testing, 137 samples from healthy subjects and 70 ANA/ENA positive samples. For ANCA determination, 170 sera samples were collected: 40 samples for routine testing, 90 samples from healthy blood donors and 40 anti-PR3/anti-MPO positive subjects. Good correlation was found for the visual and automated ANA IIF approach regarding positive/negative discrimination of these samples (kappa = 0.633 for ANA positive samples and kappa = 0.657 for ANA negative samples, respectively). Positive/negative IIF ANCA discrimination by HELIOS(®) and visual IIF revealed a complete agreement of 100% in sera from healthy patients and PR3/MPO positive samples (kappa = 1.00). There was 95% agreement between the ANCA IIF performed by automated and visual IIF on the investigation of routine samples. Based on these results, HELIOS(®) demonstrated a high diagnostic performance for the automated ANA and ANCA IIF interpretation that was similar to a visual reading in all groups of samples.

Detection of antinuclear antibodies by indirect immunofluorescence method and its comparison with line immunoassay in a tertiary care hospital: A laboratory based observational study

Antinuclear antibodies (ANA) are the hallmark of autoantibody production in autoimmune diseases and its testing is widely used as screening test in autoimmune diseases. ANA are directed against components of the cell nuclei such as DNA, histones, nucleoli and ribonucleoprotein. ANA are detected by indirect immunofluorescence (IIF) assay, which is among the most commonly used routine method for ANA detection as screening test, due to its ability to detect multiple antigens simultaneously. In this study, serum samples, referred to our laboratory for ANA testing were subjected for testing by IIF method and line immunoassay (LIA) during a study period of 20 months and the two were correlated with one another to establish any link between the two. A total of 279 serum samples were processed for ANA testing during the study period from June 2012 to January 2014. Of these 279 samples, 199(71.3%) were ANA IIF positive and 80(28.7%) were ANA IIF negative. The spectrum of various positive ANA IIF patterns are nucleus homogenous 52(26.1%), nucleus granular 50(25.1%), mixed pattern 57(28.6%), mitosis positive 15(7.5%), nucleus nucleolar 13(6.5%), nucleus dotted 8(4%), nuclear membrane 1(0.5%), cytoplasm positive 3(1.5%). All the samples tested by ANA IIF were subjected to LIA. Of these 159(56.9%) were both ANA IIF and LIA positive. In addition, 40(14.3%) samples were detected as IIF positive but LIA negative, whereas the rest 14(5%) samples were IIF negative but LIA positive. In the present study, a definite correlation was found in 201(71%) samples between ANA patterns and the LIA. Thus ANA IIF method using biochips can be used as a cost effective screening method for ANA testing and restricting LIA, which are expensive. This could economize on the cost of laboratory investigations in a developing country like India. Keywords: Antinuclear antibodies; Indirect immunofluorescence method; Immunoassay; Autoimmune diseases; Tertiary care hospital; Observational study

Automated indirect immunofluorescence antinuclear antibody analysis is a standardized alternative for visual microscope interpretation

Automated indirect immunofluorescence antinuclear antibody analysis is a standardized alternative for visual microscope interpretation

Anti-nuclear antibodies in daily clinical practice: prevalence in primary, secondary, and tertiary care.

For the diagnosis of systemic autoimmune rheumatic diseases (SARD), patients are screened for anti-nuclear antibodies (ANA). ANA, as assessed by indirect immunofluorescence (IIF), have a poor specificity. This hampers interpretation of positive results in clinical settings with low pretest probability of SARD. We hypothesized that the utility of positive ANA IIF results increases from primary to tertiary care. We retrospectively determined ANA, anti-ENA, and anti-dsDNA antibody prevalence in patient cohorts from primary (n = 1453), secondary (n = 1621), and tertiary (n = 1168) care settings. Results reveal that from primary care to tertiary care, ANA prevalence increases (6.2, 10.8, and 16.0%, resp.). Moreover, in primary care low titres (70% versus 51% and 52% in secondary and tertiary care, resp.) are more frequent and anti-ENA/dsDNA reactivities are less prevalent (21% versus 39% in secondary care). Typically, in tertiary care the prevalence of anti-ENA/dsDNA reactivities (21%) is lower than expected. From this descriptive study we conclude that positive ANA IIF results are more prone to false interpretation in clinical settings with low pretest probabilities for SARD, as in primary care. Whether alternative approaches, that is, immunoadsorption of anti-DFS70 antibodies or implementation of anti-ENA screen assays, perform better, needs to be determined.

AB1238 A comparison between automated and manual methods for detection antinuclear autoantibodies on human epithelial-2 cells

BackgroundA variety of antinuclear antibodies (ANAs) are found in serum samples obtained from patients with rheumatic autoimmune diseases1. Identification of the corresponding antigens and measurement of the antibody titer are...

Original Approach for Automated Quantification of Antinuclear Autoantibodies by Indirect Immunofluorescence

Introduction. Indirect immunofluorescence (IIF) is the gold standard method for the detection of antinuclear antibodies (ANA) which are essential markers for the diagnosis of systemic autoimmune rheumatic diseases. For the discrimination of positive and negative samples, we propose here an original approach named Immunofluorescence for Computed Antinuclear antibody Rational Evaluation (ICARE) based on the calculation of a fluorescence index (FI). Methods. We made comparison between FI and visual evaluations on 237 consecutive samples and on a cohort of 25 patients with SLE. Results. We obtained very good technical performance of FI (95% sensitivity, 98% specificity, and a kappa of 0.92), even in a subgroup of weakly positive samples. A significant correlation between quantification of FI and IIF ANA titers was found (Spearman's ρ = 0.80, P < 0.0001). Clinical performance of ICARE was validated on a cohort of patients with SLE corroborating the fact that FI could represent an attractive alternative for the evaluation of antibody titer. Conclusion. Our results represent a major step for automated quantification of IIF ANA, opening attractive perspectives such as rapid sample screening and laboratory standardization.

Automated indirect immunofluorescence antinuclear antibody analysis is a standardized alternative for visual microscope interpretation

Screening for antinuclear antibodies (ANA) is a basic tool in the serological work-up of systemic rheumatic disorders. Despite the emergence of alternative screening methods and the difficulties in standardization, indirect immunofluorescence (IIF) remains the recommended method for ANA detection. This study aimed to assess the reliability of automated ANA IIF analysis as a standardized alternative for the conventional manual approach. ANA testing on HEp-2000 cells was performed on 304 consecutive routine sera, 28 serumbank samples displaying rare staining patterns, 219 samples of well-defined disease cohorts [141 systemic sclerosis (SSc), 13 polymyalgia rheumatica, 22 osteoarthritis, 5 ANCA-associated vasculitis and 38 spondyloarthritis] and 100 healthy donors. All samples were analyzed by automated IIF (Zenit G-sight), by conventional visual IIF microscopy and two ANA screening enzyme immunoassays (EIA). Automated and conventional ANA IIF analysis were comparable for negative/positive interpretation as well as intensity assessment (>90% agreement). In contrast, the accuracy of pattern recognition (26%) was limited. Likelihood ratios (LR) for SSc on results intervals of both Zenit G-sight and EIA increased with increasing level of positivity. Sensitivity within the SSc-associated antibody subsets was higher for Zenit G-sight (97%-100%) than EIA (10%-96%). A significant correlation between the quantitative result obtained by Zenit G-sight and the conventional end-point titer was found. The use of Zenit G-sight for automated ANA IIF analysis offers opportunities to improve standardization. However, a complementary role of the expert technicians remains, especially for pattern recognition and classification of uncertain/negative samples.

Serum Autoantibodies: From Identification to Clinical Relevance

Serum Autoantibodies: From Identification to Clinical Relevance

Reconstructing a 3-dimensional image of the results of antinuclear antibody testing by indirect immunofluorescence

Indirect immunofluorescence anti-nuclear antibody testing (IIF-ANAT) is an essential screening tool in the diagnosis of various autoimmune disorders. ANA titer quantification and interpretation of immunofluorescence patterns are determined subjectively, which is problematic.First, we determined the examination conditions under which IIF-ANAT fluorescence intensities are quantified. Next, IIF-ANAT was performed using homogeneous, discrete speckled, and mixed serum samples. Images were obtained using Bio Zero BZ-8000, and 3-dimensional images were reconstructed using the BZ analyzer software. In the 2-dimensional analysis, homogeneous ANAs hid the discrete speckled pattern, resulting in a diagnosis of homogeneous immunofluorescence. However, 3-dimensional analysis of the same sample showed discrete speckled-type ANA in the homogeneous background.This study strengthened the current IIF-ANAT method by providing a new approach to quantify the fluorescence intensity and enhance the resolution of IIF-ANAT fluorescence patterns. Reconstructed 3-dimensional imaging of IIF-ANAT can be a powerful tool for routine laboratory examination.

Longitudinal Study of a Human Drug-Induced Model of Autoantibody to Cytoplasmic Rods/Rings following HCV Therapy with Ribavirin and Interferon-α

BackgroundA novel pattern in the indirect immunofluorescence antinuclear antibody assay on HEp-2 cells (IIF-HEp-2) characterized by cytoplasmic rods and rings (RR) was reported in HCV patients, but stringent disease specificity studies and longitudinal analysis are lacking. We investigated the clinical significance of anti-RR in an HCV cohort with up to a 12-month treatment follow up.Methodology/Results597 patients (342 HCV, 55 HCV/HIV, 200 non-HCV) were screened and titered for anti-RR. Serial samples were available from 78 of 176 treated and 27 of 166 untreated patients. Anti-RR was detected in 14.1% of 342 HCV patients, 9.1% of 55 HCV/HIV, 3.4% of 29 Hepatitis B, and none of 171 non-HCV (p<0.0001; HCV versus non-HCV). Anti-RR was present in 38% of 108 patients receiving interferon-α/ribavirin, but none in 26 receiving either interferon-α or ribavirin, or 166 untreated patients (p<0.0001). Other IIF-HEp-2 patterns were more frequently associated with interferon-α treatment alone (52.2%) as compared to interferon-α/ribavirin (25%), ribavirin alone (33.3%), and no therapy (26.5%). Anti-RR frequency was not associated with sex, age, ethnicity, HCV genotype or viral load. Anti-RR occurred only after initiation of treatment, beginning as early as 1 month (6%), but by the sixth month >47% tested positive for anti-RR. The anti-RR titer generally increased with sustained treatment and remained high in 53% of patients. After treatment, anti-RR titer was negative in 41%. Non-responders to HCV therapy were 77% in anti-RR-positive versus 64% in anti-RR-negative patients. Response to treatment was not associated with anti-RR titer or the dynamics of anti-RR reactivity during and after treatment.ConclusionsThe exquisite association of anti-RR reactivity with combined interferon-α/ribavirin therapy in HCV patients represents a unique model for drug-induced autoantibody generation in humans as demonstrated by the fact that a significant fraction of patients who have anti-RR during therapy becomes anti-RR-negative after completion of therapy.

The clinical significance of the dense fine speckled immunofluorescence pattern on HEp-2 cells for the diagnosis of systemic autoimmune diseases.

Antinuclear antibodies (ANAs) are a serological hallmark in the diagnosis of systemic autoimmune rheumatic diseases (SARD). The indirect immunofluorescence (IIF) assay on HEp-2 cells is a commonly used test for the detection of ANA and has been recently recommended as the screening test of choice by a task force of the American College of Rheumatology. However, up to 20% of apparently healthy individuals (HI) have been reported to have a positive IIF ANA test, primarily related to autoantibodies that target the dense fine speckles 70 (DFS70) antigen. Even more important, the DFS IIF pattern has been reported in up to 33% of ANA positive HI, but not in ANA positive SARD sera. Since the intended use of the ANA HEp-2 test is to aid in the diagnosis and classification of SARD, the detection and reporting of anti-DFS70 antibodies and their associated pattern (DFS) as a positive test significantly reduce the specificity and the positive likelihood of the ANA test. This has significant implications for medical management and diagnostic algorithms involving the detection of ANA. Recently, a novel immunoadsorption method has been developed that specifically blocks anti-DFS70 antibodies and, therefore, significantly increases the specificity of the ANA test for SARD. This immunoadsorption method has the potential to overcome a significant limitation of the ANA HEp-2 assay. The present paper summarizes the current knowledge about anti-DFS70 antibodies and their clinical impact on ANA testing.