Antinuclear Antibodies in Non-Rheumatic Diseases.

Eighty-one patients with sera positive for antinuclear antibodies were studied to determine the clinical significance of serum antinuclear antibody titers and immunofluorescent nuclear staining patterns. Attempts were made to (1) determine clinically significant serum ANA titers for various connective tissue diseases; (2) note the specificity of immunofluorescent nuclear staining patterns among various connective tissue diseases; (3) observe the spectrum of serum ANA titers and immunofluorescent nuclear staining patterns among various non-rheumatic diseases; (4) correlate serum ANA titers and immunofluorescent nuclear patterns with the clinical courses of connective tissue diseases. No clinically significant serum ANA titer could be determined either for specific connective tissue diseases or for connective tissue diseases in general, although patients with titers of 1:160 or above more often than not had clinically evident connective-tissue disease. Furthermore, no pattern of nuclear fluorescence proved to be specific for any given clinical syndrome, with a variety of nuclear fluorescent patterns being observed in specific connective-tissue disorders and other non-rheumatic diseases. Only a few correlations could be made between serum ANA titer, nuclear fluorescent pattern, and the clinical courses of connective-tissue disorders. These correlations occurred most frequently with lupus nephritis.

An enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of antinuclear antibodies (ANAs) previously established as diagnostic and/or prognostic marker ANAs for various connective tissue diseases. The antigen used in ELISA is a mixture of purified recombinant or natural antigens including single-and double-stranded DNA, RNP, Sm, SS-A/Ro, SS-B/La, centromere, topoisomerase I and Jo-1 antigens. Thirty hundred and fifty nine patients sera from a variety of connective tissue diseases and 113 normal human sera (NHS) were examined. ELISA ANAs were positive in 3.5% of NHS and 80.2% of patients sera at cut off index 11.5, whereas indirect immunofluorescent antinuclear antibodies (FANAs) using HEp-2 cells were positive in 9.7% of NHS and 92.5% of patients sera at 1:160 serum dilution. More than 80% of sera from systemic lupus erythematosus, mixed connective tissue disease and primary Sjögrens disease were ELISA ANAs positive. Mean value of ELISA ANAs was highest in sera of patients with MCTD. ELISA ANAs were positive in 92.5% of sera with marker ANAs for connective tissue diseases. Mean value of ELISA ANAs was higher in sera with more than two marker ANAs than in sera with a single ANA or in sera without marker ANAs. In contrast incidence and mean value of ELISA ANAs were low in sera positive for anti topoisomerase I antibody or anti Jo-1 antibody. Sensitivity, specificity and agreement (accuracy) for connective tissue diseases with marker ANAs were as follows: ELISA ANAs (at index 11.5): 92.5%, 88.3% and 90.9%: FANAs (at 1:160 serum dilution): 99.0%, 70.4% and 88.1%, respectively. ELISA ANAs, thus, are specific for connective tissue diseases when compared to FANAs and previous ELISA for the detection of total ANAs. Moreover, ELISA ANAs are able to measure precise ANAs titers and are much less labor intensive when screening a large number of clinical specimens.

Patients with autoimmune connective tissue diseases often have antibodies that are directed against multiple nuclear antigens called as antinuclear antibodies (ANA). Two testing methods i.e. ELISA and indirect immunofluorescence (IF) techniques are used to detect these antibodies. Though ELISA is a cheaper method, IF is a preferred method for detection of ANA. In our study we have compared these two techniques for their diagnostic performance. Both the testing methods were applied on 155 samples. Of these, 135 samples were from test group and 20 samples were controls (negative and positive controls). Of the 135 test samples, IF yield positive results in 25(18.51%) cases and was found negative in 110(81.49 %) cases. Positive results were found by ELISA in 20(14.81%) cases and negative in 115(85.19 %) cases. Samples showing positive results with both methods were 18(13.33%) and samples showing negative results with both methods were 108(80.0%). 07(5.18%) cases that gave negative results by ELISA were found to be positive by IF. 02(1.48%) samples that were found to be positive by ELISA were negative by IF. Sensitivity & specificity of ELISA was compared with IF and was found to be 90.0% and 93.9% respectively. From this study it can be concluded that for testing ANA, IF is better than ELISA. Keywords: IF (Immunofluorescence), ELISA (Enzyme Linked Immunosorbent Assay), ANA (Antinuclear Antibodies).

Event Abstract Back to Event Diagnostic value of Immunoblotting assay for determination of anti nuclear antibody (ANA) concentration in rheumatologic diseases Mohammad Naghavi-Behzad1* 1 Tabriz University of Medical Sciences, Students' Research Committee, Iran Abstract Background: Antinuclear antibodies (ANAs) are common features of autoimmune connective tissue diseases. Various detection methods are used and there are newer techniques that are continuously put forward to facilitate diagnosis and therapeutic monitoring in connective tissue disease (CTD) patients. Immunofluorescence (IF) or Fluorescent Antibody Technique the most used and “gold standard” test for diagnosis. Enzyme-Linked Immunosorbent Assay (ELISA) is another routine test. For Immunoblotting (IB) assay, autoimmune ANA profiles are used which provide a qualitative in vitro assay for human autoantibodies to 15 different antigens. This study was conducted to compare three techniques (IF, IB, ELISA) in detection of ANA. If the sensitivity and specificity of IB superior to other methods, we can replace IF and ELISA with IB. Materials and Methods: An analytical cross-sectional study of 85 sera from patients with Systemic lupus erythematous (SLE), Systemic sclerosis (SSc) and Dermatomyosities(DM) was undertaken at rheumatology and nephrology department and clinic of Emam Reza hospital from 89/11/1 to 90/10/30. Sera collected and stored at -80˚c. Then they were used to detection of ANA with three techniques. Results: Of all sera 63 (74.1%) were ELISA positive and 22 (25.9%) had negative ELISA.74 (87.1 %) IF and IB positive and 11 (12.9%) IF and IB negative were observed. The sensitivity and specificity of IB in comparison with IF was 98.65% and 90.91% respectively. In comparison with ELISA we found 93.65% and 31.82% of sensitivity and specificity. Conclusion: Immunoblotting, has high sensitivity and specificity, can be used in screening of ANA.

To evaluate the value of immunofluorescent and ELISA techniques in early diagnosis of systemic lupus erythematosus (SLE) and to find out whether there is a correlation between Antinuclear antibodies (ANA) pattern and prognosis by observing clinical score changes using British Isles Lupus Assessment Group score. The study included 75 SLE patients, 11 disease control group, and 18 healthy control group. ANA and ds-DNA antibodies detection were done by ELISA and Immunofluorescence for all groups. Immunofluorescence technique is more sensitive for ANA and ds-DNA detection than ELISA technique (100% versus 90.7%,and 93% versus 89.3% respectively); ELISA showed 89.7 % specificity for ANA detection compared to 86.2% for Immunofluorescence, and both have 100% specificity for ds-DNA detection ;homogenous ANA pattern, showed statistically significant higher BILAG score compared to speckled pattern either at the start of the study or after the follow up period (p = 0.000); and ds-DNA titer showed statistically significant decrease in titer after therapy (p = 0.01). ANA detection by Immunofluorescence is more sensitive and effective screening assay in patients with clinical features of SLE and both ds-DNA titer by ELISA and BILAG score for severity index are considered the best markers for follow-up.

Background Testing for antinuclear antibodies (ANA) is useful for the diagnosis of autoimmune connective tissue diseases (CTD). Solid-phase assay such as the enzyme-linked immunosorbent (ELISA) assay has replaced the use of indirect immunofluorescence assay (IIF) for the detection of ANA. Patients and methods In this study, ELISA which is based on a qualitative screening of IgG class autoantibodies using commercially available kits from Orgetec Diagnostika GmbH was compared with IIF for the detection of ANA in patients with different connective tissue diseases. The study involved 73 patients with confirmed diagnosis (38 patients diagnosed as systemic lupus erythematosus (SLE), 27 patients with rheumatoid arthritis (RA), and eight patients with other connective tissue diseases: systemic sclerosis, mixed connective tissue diseases, and Sjogren’s syndrome). They were recruited from the outpatient clinic of the Rheumatology and Rehabilitation Department for follow-up and others were admitted patients of the Rheumatology Department at Assuit University Hospitals. Twenty-five apparently healthy participants served as the control group. Results ANA results by IIF: of the 73 patients, 39 (53.4%) had positive ANA results and 34 (46.6%) patients had negative ANA results. All healthy control group ANA results by IIF were negative (100%). ANA results by ELISA: from a total of 73 patients, 42 (57.5%) had positive ANA results, 27 (37%) patients had negative ANA results, and four (5.5%) patients had borderline ANA results. All control group ANA results by ELISA had negative ANA results. ELISA sensitivity was 86.8% compared with 84.2% by IIF in the SLE. In other CTD both tests had the same sensitivity (87.5%). The ELISA and IIF had the same high specificity (100%) in SLE and other CTD. Conclusion There is a comparable result between sensitivity, specificity, PPV, NPV, and accuracy of both ANA tests. So we can rely on the results of ELISA in our laboratories in Assuit University Hospitals as they can deal with large numbers of patients and it saves time. IIF is partly subjective, and therefore there is considerable variation in the interpretation of results between different observers, so we can avoid it. So ANA by ELISA tests can be used as a screening test and if we want to identify the ANA pattern we can use IIF on HEp-2 cells.

For detection of anti-nuclear antibodies (ANAs) and antibodies to extractable nuclear antigens (ENAs), samples frequently are screened with indirect immunofluorescence (IIF); further determination of anti-ENA antibodies is performed only when the result is positive. However, because anti-ENA reactivities are found in samples with low fluorescence intensities, we determined anti-ENA antibodies in samples with negative IIF and thus calculated the sensitivity of IIF for specific ANAs. We collected 494 samples consecutively referred by rheumatologists for routine ANA testing. IIF on HEp-2 and HEp-2000 (HEp-2 cells transfected with Ro60 cDNA) and line immunoassay (LIA) for the detection of specific ANAs were performed on all samples. Fluorescence intensities and patterns on HEp-2 were strongly correlated with those on HEp-2000 [Spearman rho = 0.852 (P <0.001) and 0.838 (P <0.001), respectively]. Sixty-eight of 494 samples were positive on LIA, of which only 72% (confidence interval, 68-76%) were detected with HEp-2 and 75% (confidence interval, 70-78%) with HEp-2000. Of 291 samples negative on both substrates, 12 were positive on LIA. Connective tissue diseases were diagnosed in four of these patients and suspected in at least three others. The HEp-2 and HEp-2000 substrates perform comparably for fluorescence intensities and patterns and for detecting specific ANAs, but some patients with negative IIF show reactivity on LIA. We recommend testing for fine reactivities, regardless of the IIF result, when the clinical suspicion for rheumatic connective tissue disease is high.

Introduction: Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease. Detection of antinuclear antibodies (ANAs) aids in the diagnosis of SLE. The indirect immunofluorescence (IIF) assay is often used a routine screening test for the detection of ANA. The pathogenic role and significance of various patterns produced in IIF is yet to be explored.Aim: This study aimed to detect ANA patterns generated by IIF and correlate these patterns with specific antibodies detected by line immunoassay. We also investigated the significance of each ANA pattern and its association with specific serological SLE markers, such as complement molecules, anti-dsDNA, antiphospholipid antibody, and C-reactive protein (CRP), along with associations with direct Coombs test (DCT).Materials and methods: We conducted a retrospective study that included 204 patients newly diagnosed with SLE according to the European Alliance of Associations for Rheumatology/American College of Rheumatology (EULAR/ACR) criteria. The detection and pattern determination of ANA was performed by IIF using HEp-20-10. Furthermore, line immunoassay was performed, and the antibody profile of each sample was obtained. Other immunodiagnostic markers were analyzed, including C3, C4, anti-dsDNA, antiphospholipid antibodies (anti-cardiolipin antibodies, anti-beta-2-glycoprotein I, and lupus anticoagulant), CRP, and DCT.Results: Of the 204 samples, the most frequent ANA pattern observed was nucleus speckled (52.9%), followed by nucleus homogenous (27.5%), mixed (13.7%), and cytoplasm speckled (5.9%). The nucleus homogenous pattern showed the most pathogenic immune profile due to its close association with markers of disease activity, namely, high anti-dsDNA titer, low C3 level, and DCT positivity. Conclusion: This study showed that the most common pattern associated with SLE is nucleus speckled, followed by the nucleus homogenous pattern. Based on associations with specific serological markers, the nucleus homogenous pattern may be linked to a high disease activity in SLE.

Antinuclear Antibodies (ANA) are present in many autoimmune disorders and these disorders are collectively called as Connective Tissue Diseases (CTD). There are various CTDs which includes Systemic Lupus Erythematosus (SLE), Sjogren’s syndrome, Systemic Sclerosis (SS), Inflammatory Myositis (IM), Mixed Connective Tissue Disorder (MCTD) and Rheumatoid Arthritis (RA). Detection of ANAs in these CTDs is highly sensitive and is of utmost importance. The ANAs specific to SLE includes anti-double stranded Deoxyribonucleic Acid (anti-dsDNA), single stranded DNA (ssDNA). Scleroderma or Systemic Sclerosis (SS) is an immune mediated rheumatic disease where autoantibodies like topoisomerase 1, Ribonucleic Acid (RNA) polymerase 1 and fibrillarin are useful in diagnosis. Idiopathic Inflammatory Myositis (IIM) such as polymyositis and dermatomyositis are characterised by the presence of autoantibodies like PM-scl (Polymyositis-Scleromyositis), Mi-1 (Myositis specific autoantibody found in idiopathic inflammatory myositis), Mi-2 and Ku (DNA Binding Protein in dermatomyositis). Antibody titres against polypeptides on the U1 Ribonucleoprotein (U1RNP) is useful in the detection mixed CTD. Sjogren’s syndrome is characterised by the presence of serum autoantibodies against two ribonucleoproteinic complexes like Ro/SSA (Extractable nuclear antigen found in Sjogren's syndrome related antigen A auto antibodies) and La/SSB (Extractable nuclear antigen found in Sjogren's syndrome or lupus erythematous). ANA analysis can be done by techniques like indirect immunofluorescence method, Enzyme Linked Immuno Sorbent Assay (ELISA), Immunoprecipitation in agar and western blotting. All these diagnostic methods give precise identification of these antibodies with high accuracy. Farr assay, Multiplex Immunoassay (MIA), Flow cytometry, antigen microarray is also gaining importance in the diagnosis of ANAs. The objective of this study was to discuss various methods of ANA testing so that the clinicians know its relevance in diagnosis. Certain advancement in the diagnosis of ANA is also included in this review.

Antinuclear antibodies (ANA) are detected in approximately a quarter of COVID-19 patients when assessed by indirect immunofluorescence. Since there is no information, our study investigated the presence of ANA detected by Enzyme-Linked Immunosorbent Assay (ELISA) and its clinical and laboratory associations. A longitudinal study was conducted on 92 patients with severe COVID-19, 20 patients with acute myocardial infarction, and 25 healthy subjects. Blood samples were obtained at hospital admission. Commercial ELISA was used to detect ANA, while flow cytometry was used to measure serum interferons. ANAs were positive in 8.6% of COVID-19 patients, 10% of myocardial infarction patients, and 4% in healthy individuals (p=0.676). COVID-19 patients with ANA+ had less ferritin, troponin, and neutrophils but more albumin and lymphocytes than ANA- patients. Serum levels of type I, II, and III interferons were similar between groups. At follow-up, all ANA+ patients survived, while mortality was significant in ANA- patients (0 vs. 36%; p=0.048). ANA detection is not increased in severe cases of COVID-19 when assessed by ELISA. However, its presence appears to be associated with a less aggressive disease phenotype, regardless of circulating levels of interferons.

Background: Detection of antinuclear antibodies (ANAs) supports the clinical diagnosis of ANA-associated rheumatic diseases, such as systemic sclerosis (SSc), systemic lupus erythematosus (SLE), primary sjogren’s syndrome (SjS) and mixed connective tissue disease (MCTD) [1-3]. Throughout history, a number of autoantibody detection methods have emerged, for instance, indirect immunofluorescence (IIF), radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and line immunoassay (LIA) [4]. With the development of detection technology, new methods to detect ANAs were continuously developed by numerous manufacturers, for example, particle-enhanced turbidimetric immunoassay (PETIA). Therefore, in the current study, we evaluated for the first time the performance of PETIA in the detection of anti-nuclear antibody and compared it with commercial LIA. Objectives: To evaluate the clinical performance of PETIA for the detection of ANAs in comparison to the currently commonly used LIA. Methods: Total 606 serum samples from diseased and healthy controls were assayed to simultaneously determine SSA, Sm/RNP, SSB, Sm and U1-SnRNP antibodies by the PETIA and LIA. The sensitivity, specificity, consistency and area under curve (AUC) were analyzed for each antibody between PETIA and LIA. Results: The positive rate and specificity of PETIA and LIA for ANA specific target antibodies were comparable. Compared with LIA, the sensitivity of SSA, SSB and Sm were 100.00%, 88.89% and 90.00%, while Sm/RNP and U1-SnRNP were 75.00%, 70.59%, respectively; Sm/RNP, SSB, Sm and U1-SnRNP have high specificity, respectively 97.87%, 98.90%, 97.80% and 94.68%, while SSA specificity is general, 81.52%. Under manufacturer’s cut-off values, the consistent rates of SSA, Sm/RNP, SSB, Sm, and U1-SnRNP between PETIA and LIA were 87.22% (116/133), 92.06% (116/126), 96.61% (114/118), 97.03% (98/101) and 88.28 (113/128), respectively. Excellent consistencies were found between PETIA and LIA for the detection of Sm/RNP, SSB and Sm antibodies (kappa>0.75), and kappa coefficients were 0.776 (p Conclusion: The performance of PETIA for the detection of antibodies to nuclear specific antigen was satisfying to correlate with that of LIA. With the additional benefits of short detection time, quantitative output and high universality. PETIA can better meet the requirements of quantitative detection of specific target antibodies.

Objectives: Whereas antinuclear antibodies (ANAs) detected by indirect immunofluorescence (IIF) have diagnostic significance, the dense fine speckled (DFS) pattern on HEp-2 cells may be an exclusionary marker for ANA-associated rheumatic disease (AARD). The aim of this study was to evaluate a new algorithm considering anti-DFS70 antibodies for routine ANA testing.Method: From ANA requested sequential 10 528 sera, 181 sera samples showing the DFS pattern were additionally tested for anti-DFS70 antibodies by an enzyme-linked immunosorbent assay (ELISA-DFS70) and for specific-ANAs. Specific-ANAs(+)/IIF-DFS(–) control sera samples (n = 50) were also tested.Results: Of the 181 IIF-DFS-positive sera samples, 82.9% (n = 150) were from non-AARD patients and 112 (61.9%) patients had non-rheumatic diseases (NRD), including the most common clinical feature of dermatitis (18.2%). The ELISA-DFS70 was positive in 109 (60.2%) sera and was negative in all control sera. Specific-ANAs were similarly detected as 25.7% (28/109) and 22.2% (16/72) of ELISA-DFS70(+) and ELISA-DFS70(+) patients, respectively (p > 0.05). The prevalence of non-AARD was 95.1% and 25.1% in the ELISA-DFS70(+)/specific-ANAs(–) and ELISA-DFS70(–)/specific-ANAs (+) groups, respectively.Conclusions: In patients with a HEp-2 DFS pattern, the additional ELISA-DFS70 and specific-ANAs test could improve the efficiency of diagnosing AARD. The detection of anti-DFS70 antibodies should be included in test algorithms for ANA testing.

Objective To evaluate the performance of antinuclear antibody(ANA) detection in clinical laboratories. Methods There were 2 external quality assessments(EQA) scheme for nuclear antibody detection.The panel consisting of 5 samples was distributed.Each participant laborotory of the EQA program was required to report the ANA qualitative results, patterns, titers and anti-double strain DNA(dsDNA) antibody, anti-extractable nuclear antigen(ENA) antibody, the percent agreements of which were calculated respectively. Results The number of laboratories performing ANA test with IIF increased from 77.6%（149/192）in 2006 to 82.2%（342/416） in 2011, while the number of laboratories performing ANA test with ELISA was in the range of 14.5%（53/365） and 16.0%（52/326）.The positive percent agreements of IIF was over 98%.The positive percent agreement of ELISA were all over 90%.IIF showed more satisfying positive percent agreements than ELISA every year.Over 90% of the laboratories reported correct results for samples with granular ANA pattern except 0613 and 0624.Over 95% of the laboratories reported correct results for samples with homogeneous ANA pattern.Two samples with centromere pattern were correctly detected by 88.5%（161/182），79.0%（147/186）of the laboratories in 2007, while the sample with centromere pattern was correctly detected by 98.4%（299/304）, which indicated an improvement in the detection of centromere pattern. In ANA positive results, the lowest percentage of the laboratories reporting the median result was 36%（94/261）, while the highest percentage was only 85.5%（224/262）.The satisfied results of anti-ENA antibody were over 90%.And those of anti-dsDNA antibody was over 85%. Conclusions IIF is the most common method for ANA screening in clinical laboratories.ELISA is also used in some laboratories.The two methods reported satisfying results in ANA test.The detection of anticentromere antibodies is improved.But the results of ANA titer reported are unsatisfactory. ANA detection in routine practice needs to be improved by standardization.（Chin J Lab Med, 2012,35:271-276） Key words: Autoantibodies; Autoimmune diseases; Quality control; Clinical laboratory techniques

Antinuclear antibodies

BackgroundAnti-SSA autoantibodies can be differentiated according to their antigenic target proteins as anti-Ro60 (60 kDa) or anti-Ro52 (52 kDa). Anti-SSA(Ro60) are clearly associated with Connective Tissue Diseases (CTD), but the...