|Year : 2018 | Volume
| Issue : 3 | Page : 223-228
Detection of antinuclear antibody in autoimmune connective tissue diseases: a comparison between immunofluorescence and solid-phase assay
Neveen A Kamel, Ashraf A Hassaballa, Yomna M Hasan
Department of Clinical Pathology, Faculty of Medicine, Assiut University, Asyut, Egypt
|Date of Submission||15-Oct-2017|
|Date of Acceptance||14-Jan-2019|
|Date of Web Publication||15-Apr-2019|
Yomna M Hasan
Department of Clinical Pathology, Faculty of Medicine, Assiut University, Asyut
Source of Support: None, Conflict of Interest: None
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 Sjögren’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.
Keywords: antinuclear autoantibodies, connective tissue diseases, enzyme-linked immunosorbent, indirect immunofluorescence
|How to cite this article:|
Kamel NA, Hassaballa AA, Hasan YM. Detection of antinuclear antibody in autoimmune connective tissue diseases: a comparison between immunofluorescence and solid-phase assay. Al-Azhar Assiut Med J 2018;16:223-8
|How to cite this URL:|
Kamel NA, Hassaballa AA, Hasan YM. Detection of antinuclear antibody in autoimmune connective tissue diseases: a comparison between immunofluorescence and solid-phase assay. Al-Azhar Assiut Med J [serial online] 2018 [cited 2020 Aug 12];16:223-8. Available from: http://www.azmj.eg.net/text.asp?2018/16/3/223/255858
| Introduction|| |
Connective tissue diseases (CTD) are a group of autoimmune disorders which are characterized by the presence of antinuclear autoantibodies (ANA) in the blood of patients . CTD include rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, polymyositis-dermatomyositis, primary Sjögren’s syndrome, primary antiphospholipid syndrome, and mixed connective tissue disease. ANA are a specific class of autoantibodies that have the capability of binding and destroying certain structures within the nucleus of the cells. Not only these antibodies are involved in the disease pathogenesis but they also constitute the basis for the diagnosis and treatment of CTD . Indirect immunofluorescence (IIF) on HEp-2 cells is the gold standard technique for the detection of ANA. The presence of positive fluorescence staining indicates the presence of ANA. This method is inexpensive, but does not allow specific identification of these autoantibodies, semiquantitative, time consuming, and need trained and skilled persons. For these purposes, additional testing is required such as ELISA. ELISA assay that contains a mixture of known antinuclear antigens are increasingly being adopted for ANA detection as they are easily automated and useful in a large number of patient samples with high sensitivity and specificity .
| Aim|| |
Make a comparison between the two different methods ELISA-ANA and traditional IIF-ANA results in patients with different connective tissue diseases.
| Patients and methods|| |
The study included 73 patients with confirmed diagnosis made by the rheumatology department (38 patients diagnosed as systemic lupus erythematosus (SLE), 27 patients with rheumatoid arthritis (RA), eight patients with other connective tissue diseases such as systemic sclerosis, mixed connective tissue diseases, and Sjögren syndrome) and their ages ranged from 13 to 65 years. They were recruited from the outpatient clinic of the Rheumatology and Rehabilitation Department for follow-up and others were admitted patients in Rheumatology Department at Assuit University Hospitals. The study was conducted between December 2014 and June 2015. Twenty-five apparently healthy participants were chosen as the control group in the age range of 15–60 years. This study carried out after the approval of the Ethics and Research Committee of the Faculty of Medicine, Assuit University.
| Methods|| |
A measure of 4 ml of venous blood was collected by vein puncture into a clean Wassermann tube, rapidly separated after being clotted and the serum was stored at −20°C until the time of assay.
The samples used for ANA determination were performed with a serum specimen using IIF and ELISA.
IIF for ANA detection: ANA detection by HEp-2 Cell Line Substrate from DiaSorin (Alfred Nobel DriveHercules, California, USA).
ELISA: kits from Orgetec Diagnostika GmbH (Mainz, Germany) that contain 26 human recombinant and highly purified native antigens, including [SS-A-52 (Ro-52), SS-A-60 (Ro-60), SS-B (La), RNP/Sm, RNP-70, RNP-A, RNP-C, Sm-BB, Sm-D, Sm-E, Sm-F, Sm-G, Scl-70, Jo-1, double-stranded DNA (dsDNA), ssDNA, polynucleosomes, mononucleosomes, histone complex, histone H1, histone H2A, histone H2B, histone 3, histone H4, Pm-Scl-100, and centromere B].
Tests were performed according to the manufacturer’s instructions. For ANA by IIF s titer of at least 1/40 was regarded as positive. And then identify the pattern (the titer used was 1 : 40–1 : 80). If the index less than 1.0 is negative, 1.0–1.2 is borderline and an index more than 1.2 is positive.
| Results|| |
Patterns of antinuclear antibodies detected by indirect immunofluorescence
Eight (25%) cases had a coarse, speckled pattern, four (12.5%) cases had a fine speckled pattern, seven (21.9%) cases had a homogeneous pattern, three (9.4%) cases had a homogeneous+peripheral pattern, six (18.8%) cases had a peripheral pattern, two (6.2%) cases had a peripheral+coarse speckled pattern, and two (6.2%) cases had a peripheral+fine speckled pattern ([Table 1]).
|Table 1 The distribution of antinuclear antibodies pattern by indirect immunofluorescence method|
Click here to view
In other connective tissue diseases
Three (42.9%) cases had a coarse speckled pattern; two (28.5%) cases had a homogeneous pattern, and two (28.5%) cases had a fine speckled pattern.
Comparison between antinuclear antibodies results by enzyme-linked immunosorbent and antinuclear antibodies results by indirect immunofluorescence
Of the 73 patients, 39 (53.4%) had a positive ANA result by both methods, three (4.1%) patients had negative ANA by IIF and positive ANA by ELISA, 23 (37%) patients had ANA negative by both techniques, four (5.5%) patients were negative by IIF and borderline ANA result by ELISA ([Figure 1]).
All control group ANA results were negative by both methods (100%).
Among the 38 patients with SLE, 32 (43.8%) patients had ANA positive by both techniques, three (4.1%) patients had ANA negative by both techniques, one (1.4%) patient had negative ANA by IIF and positive ANA by ELISA, two (2.7%) patients were negative by IIF and borderline ANA result by ELISA.
Among the 27 patients with RA, 24 (32.9%) patients had ANA negative result by both techniques. Two (2.7%) patients were ANA negative by IIF and positive ANA by ELISA, one (1.4%) patient was negative by IIF, and borderline ANA result by ELISA.
Among the eight patients with other CTDs seven (9.6%) patients had ANA positive by both techniques and one (1.4%) case was negative ANA by IIF and borderline ANA result by ELISA ([Table 2]).
|Table 2 Comparison between antinuclear antibodies results by enzyme-linked immunosorbent and by indirect immunofluorescence|
Click here to view
There is no statistically significant difference between the two tests as the sensitivity of ELISA is 86.8% in the diagnosis of SLE and the sensitivity of IIF is 84.2% (P=1); in RA and other CTDs there is no statistically significant difference between the two tests.
Correlation between two tests results using the ROC curve
In ROC analysis of the ANA by ELISA the area under the curve was (0.963, indicating a highly accurate diagnosis. The cutoff index more than 1.2, sensitivity=100.0%, and the specificity=91.3% ([Figure 2]).
| Discussion|| |
Several studies have been published on the evaluation of ELISA method for ANA detection as a possible alternative screening test to the ANA-IIF method ,.
ANA detection has diagnostic roles in CTD and SLE particularly for which the presence of ANA is one of the classification criteria .
In our study, we found that the ANA results by ELISA had higher sensitivity (86.8%) in SLE patients than IIF (84.2%). Both methods had the same high specificity (100%) in ANA results. In RA and other CTD patients we found that there is no significant difference between the results of two methods used in sensitivity and specificity.
In 2009, Qin et al.  reported their comparison of HEp-2 IFA testing with ELISA for ANA and DNA testing. They concluded that ELISA prescreening combined with IIFA can obtain the information of the nuclear pattern and allow the observation of the titer alterations. The combination of two or more testing methods can greatly enhance the accuracy of the results.
Results from the present study are in agreement with what were reported by Gniewek et al. ; Homburger et al. ; Hayashi et al. ; Tonuttia et al. ; and Copple et al. , confirming that the ELISA technique has a higher sensitivity than IIF for the detection of ANA antibodies in SLE patients and other CTDs we worked on and they also found that the specificity is higher in ELISA, but in our study we found the specificity is equivalent in both methods.
However, the number of patients and control are different and the components of kits they used are not similar to our kits from Orgentec; this may explain the difference in sensitivity and specificity of our results.
The Gniewek et al.  study included 467 patients with different connective tissue diseases and 98 healthy blood donors; using the kits (Radias; Bio-Rad, USA) the plates are coated with a HEp-2 cell extract containing ANA antigens which include dsDNA, SS-A, SS-B Sm, RNP, Jo-1, and Scl-70. The sensitivity, specificity, positive predictive value, and negative predictive value for ANA-IFA were 87.2, 48.0, 29.1, and 93.9%, respectively. For ANA by ELISA, they were 90.7, 60.2, 35.8, and 96.4%, respectively.
The Homburger et al.  study included 313 patients and 102 healthy controls and different types of diseases as they work on systemic rheumatic diseases (SRDs) and patients with no SRDs. Among the patients diagnosed as SRDs the ELISA was positive for ANA in 100% and IIF was positive for ANA in 95.4% of cases. Among patients with no SRDs the ELISA and IIF were positive for ANA in 97.6 and 75.6% of cases, respectively. Among the healthy control group, both tests were positive in 15% of cases. They concluded that the ELISA is substantially equivalent to IIF for detecting ANA.
The Hayashi et al.  study included 492 healthy individuals and 307 patients with connective tissue diseases and they used Roche Diagnostics using seven recombinant proteins (60-kDa SSA/Ro, 52-kDa SSA/Ro, SS-B/La, Scl-70, Jo-1, dsDNA, and CENP-B). They found that the sensitivity and specificity of the IF method were 92% and 65%, respectively, and by EIA were 93 and 79% respectively.
Tonuttia et al.  used five commercially available five different EIA kits in the study different from the ones we use: Kit 1: (Sanofi Pasteur, Lyon, France), Kit 2: (Imtec Immunodiagnostika, Berlin, Germany), Kit 3: (Biochem Immuno-System, Bologna, Italy), Kit 4: (DiaSorin, Saluggia, Italy), and Kit 5: (Inova Diagnostics, San Diego, California, USA). The positive rate of the ANA by IIF test in participants with systemic autoimmune diseases was 92%, whereas in the five ANA-EIA kits there was broad diversity in response, with positive rates ranging from 74 to 94%. They concluded that the kits with higher levels of sensitivity and specificity than IIF could be used as an alternative screening test to IIF.
The Copple et al.  study included 30 patients with clinically defined SLE and 94 from patients with clinically defined rheumatoid arthritis and 100 samples from healthy donors. They sought to screen for ANAs by ELISA followed by selective use of IFA testing as a routine approach for screening and confirming the presence of ANAs. They used ANA ELISAs from four manufacturers that differ from the ones we use. Their results showed higher sensitivities, 90–97%, for the ELISAs evaluated compared with 80% sensitivity for the HEp-2 IFA test in the clinically defined SLE samples.
In contrast to our findings, other studies reported that the IF technique has a higher sensitivity and a lower specificity than ELISA for the detection of ANA antibodies ,,; this is explained by the difference in the number of patients and types of kits with different number of antigens.
The Vollset study included 471 patients (373 with different autoimmune diseases and 89 with nonautoimmune diseases) and 127 participants as control group, which was a much bigger number than our study; they also used different immunoassay kits with different number of antigens. As in the Varelisa ANA Rheuma Screen (Thermo Fisher Scientific Inc., Uppsala, Sweden) [one of ELISA kits they used] it contains a low number of antigens and the microtiter plates were coated with recombinant or purified nuclear antigens including only 10 (Sm, RNP, dsDNA, histones, centromere, Jo-1, SSA (Ro-60 and Ro-52), SS-B, PM-scl, and Scl-70). The sensitivity was 93.3% in SLE by IIF and ranged from 71.7 to 77.8% using different types of ELISA. They concluded that IIF using HEp-2 cells as substrate is the best assay when predicting SLE and SS.
The El-Chennawi and colleagues study included 75 patients with SLE and 18 healthy participants. They used different types of kits from the ones we used (Bio-Rad). The sensitivity by IIF was 100% and by ELISA was 90.7% in SLE patients. In the healthy control group, both techniques gave the same results for ANA. They explained that the specificity of the ELISA technique over the IIF technique may be due to the presence of/or absence of antibody to a specific antigen in a technique rather than the other and the overall low specificity of both techniques in ANA detection is due to the presence of ANA in different diseases and even in normal participants.
Although, Hira‐Kazal and colleagues use the same type of kits we used from Orgentec Diagnostika, the results are different. They reported that ELISA and HEp-2 screening assays yielded 45 and 81% positive test results, respectively, demonstrating lack of concordance between the test methods. They found that ELISA was less sensitive but a more specific screening tool for SLE and other ANA-associated connective tissue diseases. They reported ‘All these ANA that were not detected by ELISA testing proved nonreactive against dsDNA, but were not tested further for other antigenic reactivity’. They found that in patients with positive results of IIF against negative results of ELISA a sizeable proportion of them had an ANA-associated disorder, while a similar proportion had Raynaud’s phenomenon. They concluded that ELISA screening may fail to detect clinically relevant ANA that lack a defined specificity for antigen.
The contradiction in our results from this study can be attributed to: they used heterogeneous unselected population that were requested for ANA test; they used dilution 1/40 for HEp-2 testing which increases the sensitivity results of IIF; they admit in their discussion that other studies reported that some ELISAs had equivalent sensitivity  or were even more sensitive than HEp-2 testing for the detection of ANA ; and their results are far from other studies that reported that ELISA is less sensitive than IIF as the sensitivity of ELISA was 78–87% .
| Conclusion|| |
From this study we have concluded that there is a comparable result between sensitivity, specificity, PPV, NPV, and accuracy of both ANA tests. Therefore, ELISA can be used as an alternative to IIF testing.
The authors acknowledge the staff members, colleagues, and personnel of the Clinical Pathology, Faculty of Medicine, Assiut University, for their continuous help and encouragement.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Lernmark Å. Series Introduction: autoimmune diseases: are markers ready for prediction?. J Clin Invest 2001; 108:1091–1096.
Kumar Y, Bhatia A, Minz RW. Antinuclear antibodies and their detection methods in diagnosis of connective tissue diseases: a journey revisited. Diagn Pathol 2009; 4:1–10.
Lightfoote MM, Chirmule N, Homburger HA, Kavanaugh A, Nakamura RM et al.
Quality assurance of laboratory tests for autoantibodies to nuclear antigens: (1) indirect fluorescence assay for microscopy and (2) microtiter enzyme immunoassay methods; approved Guideline—Second Edition. Quality Assurance 2006; 26.13.
Keren DF. Antinuclear antibody testing. Clin Lab Med 2002; 22:447–474.
Lehmann HP, Fuehling I, Ott C, Huedepohl B, Haass M. HEp2 ANA EIA: a new fully automated assay for the screening of antinuclear antibodies. Isr Med Assoc J 2000; 2:646–648.
Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1997;40:1725–1725.
Qin X, Tao X, Chen ZJ, Jiang JQ, Xu MH, Li RL et al.
Comparison of indirect immunofluorescence assay and ELISA for detecting antinuclear antibodies and anti-double-stranded DNA antibodies. Nan Fang Yi Ke Da Xue Xue Bao 2009; 29:472–475.
Gniewek RA, Stites DP, Mchugh TM, Hilton JF, Nakagawa M. Comparison of antinuclear antibody testing methods: immunofluorescence assay versus enzyme immunoassay. Clin Diagn Lab Immunol 1997; 4:185–188.
Homburger H, Cahen Y, Griffiths J, Jacob G. Detection of antinuclear antibodies: comparative evaluation of enzyme immunoassay and indirect immunofluorescence methods. Arch Pathol Lab Med 1998;122:993.
Hayashi N, Kawamoto T, Mukai M, Morinobu A, Koshiba M, Kondo S et al.
Detection of antinuclear antibodies by use of an enzyme immunoassay with nuclear HEp-2 cell extract and recombinant antigens: comparison with immunofluorescence assay in 307 patients. Clin Chem 2001; 47:1649–1659.
Tonutti E, Bassetti D, Piazza A, Visentini D, Poletto M, Bassetto F et al.
Diagnostic accuracy of ELISA methods as an alternative screening test to indirect immunofluorescence for the detection of antinuclear antibodies. Evaluation of five commercial kits. Autoimmunity 2004; 37:171–176.
Copple SS, Sawitzke AD, Wilson AM, Tebo AE, Hill HR. Enzyme-linked immunosorbent assay screening then indirect immunofluorescence confirmation of antinuclear antibodies. Am J Clin Pathol 2011;135:678–684.
Vollset S. Evaluation of diagnostic tests for antinuclear antibodies in rheumatological practice. Scand J Immunol 2000; 52:309–315.
El-Chennawi FA, Mosaad YM, Habib HM, El-Degheidi T. Comparative study of antinuclear antibody detection by indirect immunofluorescence and enzyme immunoassay in lupus patients. Immunol Invest 2009; 38:839–850.
Hira‐Kazal R, Shea‐Simonds P, Peacock J, Maher J. How should a district general hospital immunology service screen for anti‐nuclear antibodies? an ‘in‐the‐field’ audit. Clin Exp Immunol 2015; 180:52–57.
Russell A, Johnston C. Relative value of commercial kits for ANA testing. Clin Exp Rheumatol 2003; 21:477–480.
Meroni P, Schur PH. ANA screening: an old test with new recommendations. Ann Rheum Dis 2010; 69:127100.
[Figure 1], [Figure 2]
[Table 1], [Table 2]