The occurrence of autoimmune disease (AID) has increased 3–5% and has had a significant impact on human health. The detection of serum antinuclear antibody (ANA) is beginning to receive more attention from physicians for the diagnosis of AIDS.
Due to its ability to simply, specifically, and simultaneously detect multiantigens, indirect immunofluorescence assay (IIF) with HEP-2 cells is widely used in screening AID using HEp-2 cells.1 As the confirmatory test of ANA, the Euroline assay (LIA) (Euroimmun, Lübeck, Germany) for detecting the antinuclear antibody spectrum (ANAS) is often used in clinical laboratories because of these characteristics, i.e., simplicity, specificity, and multiantibody detection.2 In many laboratories worldwide, IIF is used as the screening test for ANA; if the result is positive, the LIA is utilized to confirm the specific antibodies. In some cases, ANA/ANAS is detected by only one of the methods.3
A total of 6659 sera samples were collected in the authors’ laboratory from 2011 to 2013 from in- and outpatients thought to be autoimmune or diagnosed as autoimmune. Serum ANA was determined by IIF using a commercialized ANA kit (Euroimmun) with HEp-2 cell lines as substrate at an initial serum dilution of 1:100 according to the manufacturer’s instructions. The serum was further diluted to 1:320, 1:1000, and 1:3200, in turn, when a positive result was obtained with the lower dilution. In order to reduce the deviation caused by different laboratorians, the results were determined by two workers simultaneously according to the double-blind principle. ANAs were detected with LIA, the results were read with EUROLineScan software (Euroimmun), and the positive result of each antibody was divided into four classes: 1) equivocal (+), 2) weak positive (+), 3) moderate/strong positive (++), and 4) very strong positive (+++). All the assay kits were purchased from Euroimmun.
Figure 1 ‒ Distribution of negative ANAS in different ANA fluorescence patterns.
The total consistent rate between single ANA and ANAS was 86.7%, and the positive and negative consistent rates were 61.1% and 95.2%, respectively. The positive rate was 83.8% in the nuclear speckle pattern, 73.4% in the nuclear homogeneous pattern, and 68.0% in the cytoplasmic speckle pattern. For the single ANA pattern, SSA and Ro-52 had a comparative high positive ratio, while there was no proliferating cell nuclear antigen (PCNA) plus centromere Protein B (CENPB), and Sjögren’s syndrome B SSB in the nucleolus and centromere patterns, respectively. In ANA positive and ANAS negative cases, the percentages of nuclear speckle and homogeneous patterns were highest (30.0% and 14.6%, respectively); while in ANAS positive and ANA negative samples, an average 62.5% of antibodies were weakly positive; CENPB was even 100%. Data are given in Tables 1‒6 and Figure 1.
Table 1 ‒ Coincidence rate of ANA and ANAS
Table 2 ‒ ANA fluorescence patterns and ANAS positive ratio
Table 3 ‒ Analysis of key single ANA fluorescence patterns and specific antibodies
Table 4 ‒ Consistency analysis: ANA fluorescence patterns and specific antibodies
Table 5‒ Consistency analysis: ANA fluorescence patterns and specific antibodies
Table 6 ‒ Consistency analysis: ANA fluorescence patterns and specific antibodies
Of the 6659 specimens, the positive and negative coincidence rates were not completely consistent between ANA and ANAS. For the negative IIF test, it is possible that the LIA was positive; otherwise, for the negative ANAS cases, the test for ANA may be positive.
This inconsistency is due to the deficiencies of the different methodologies. For IIF, the low concentration of autoantigens and the destruction of antigens during the preparation of the HEp-2 substrate possibly made the result negative.4 For LIA, according to the current reports, only 15 specific antigens can be detected in the clinical laboratory5 and, if the AID was caused by the antigen, not including the 15 specific antigens, the LIA results were possibly negative.6,7 Based on this, ANA-IIF is perceived as the “gold standard” or screening test and ANAS-LIA as the confirmatory test.8 The combination of ANA and IIF and ANAS and LIA is a scientific and effective method for the diagnosis of diseases related to autoimmunity, and may reduce the incidences of missed diagnoses and enhance the diagnostic capability of laboratory clinicians.
For each of the ANA fluorescence patterns, there was variation between the different patterns. The centromere pattern had a high coincidence rate with ANAS, followed by the nuclear speckle, cytoplasmic speckle, and nuclear homogeneous patterns, respectively. This shows that, except for the known antigens, there were unknown specific antigens or other reasons that caused the ANA to be positive; further research is required in this regard.
In the 280 ANA positive and ANAS negative samples, there were certain negative ratios of ANAS in different ANA patterns. The ratios were consistent with the percentage of every pattern in the ANA profiles. Further analysis of the titer of immunofluorescence test results demonstrated that most of the ANA titer was lower than 1:320, indicating that the low concentration of the specific antibodies was the reason for the negative ANAs. Of course, the existence of unknown antigens cannot be precluded.
As shown in Table 4, the correlation of each antibody with every single ANA pattern was analyzed. It can be seen that SSA and Ro-52 had a high positive rate in each of the patterns, indicating that the specificities of both SSA and Ro-52 were poor, especially the latter. Several researchers thought that Ro-52 had no specificity to disease,9‒11 and its role in the diagnosis of AID needs further study.
Vandenbroucke reported that Ro-52 has a cross-reaction with Jo-1 and RNP;12 however, this was not the finding of the authors of the current paper. Comparatively, CENPB had a high specificity to the centromere pattern with a consistency rate of 93.24%. However, in the nuclear speckle patterns, there was a certain percentage of CENPB, probably because the CENPB concentration was so low in the HEp-2 cells that could not be detected, and the main strong positive pattern (nuclear speckle) covered the weak centromere pattern. Therefore, the single ANA pattern is almost absent in some sense, but demonstrates the limitations of using ANA-IIF.
In addition, there was an interesting finding. All PCNA and CENPB were negative in the nucleolus pattern, as was SSB in the centromere pattern. If this phenomenon really exists, further investigation is warranted. In the ANA negative and ANAS positive samples, an average 64.5% of ANAs were weak positive. The highest were Pcl-70 and PCNA, and the lowest was CENPB. These data proved that because of the low titer of antoantibodies, the ANA pattern could not be identified by IIF at a titer of 1:100, and if the diagnosis standard was decreased from 1:100 to 1:80, the inconsistency rate between ANA and ANAS was likely reduced to some extent.
Whether for the detection of ANA with IIF or ANAS with LIA, opportunities exist for misdiagnosis. The combination of the two methods may avoid this and prove beneficial for the clinical laboratory.13 Although some new antoantigens have been found,14 a comparison of more than 100 in the ANAS profile demonstrates the need for further research.
- Kumar, Y.; Bhatia, A. et al. Antinuclear antibodies and their detection methods in diagnosis of connective tissue diseases: a journey revisited. Diagn. Pathol. 2009, 4(2), 1.
- Van den Bergh, K.; Hooijkaas, H. et al. Heterogeneous nuclear ribonuceoprotein h1, a novel nuclear autoantigen. Clin. Chem. 2009, 55(5), 946.
- Mariz, H.A.; Sato, E.I. et al. Pattern on the antinuclear antibody-HEp-2 test is critical parameter for discriminating antinuclear antibody-positive healthy individuals and patients with autoimmune rheumatic diseases. Arthrit. Rheum. 2011, 63(1). 191.
- Tanaka, N.; Muro, Y. et al. Anti-ss-a/ro antibody determination by indirect immunofluorescence and comparison of different methods of anti-nuclear antibody screening. Mod. Rheum. 2008, 18(6), 585.
- Lee, S.A.; Kahng, J. et al. Comparative study of immunofluorescent antinuclear antibody test and line immunoassay detecting 15 specific autotibodies in patients with sysytemic rheumatic disease. J. Clin. Lab. Anal. 2012, 26(4), 307.
- Kroshimsky, D.; Stoen, J.H. et al. Case records of the Massachusetts General Hospital. Case 5-2009. A 47-year-old woman with a rash and numbness and pain in the legs. N. Engl. J. Med. 2009, 360(7). 711.
- Hansson-Hamlin, H.; Rönnelid J. Detection of antinuclear antibodies by the Inno-Lia ANA update test in canine systemic rheumatic disease. Vet. Clin. Pathol. 2010, 39(2), 215.
- Meroni, P.L.; Schur, P.H. ANA screening: an old test with new recommendations. Ann. Rheum. Dis. 2010, 69(8), 1420.
- Dugar, M.; Cox, S. et al. Diagnositc utility of anti-Ro52 detection in systemic autoimmunity. Postgr. Med. J.2010, 86(1012), 79.
- Menéndez, A.; Gómez, J. et al. Clinical associations of anti-SSA/Ro60 and anti-Ro52/TRIM21 antibodies: diagnositic utility of their separate detection. Autoimmunity 2013, 46, 32.
- Ghillani, P.; André, C. et al. Clinical significance of anti-Ro52 (TRIM21) antibodies non-asscociated with anti-SSA 60 kDa antibodies: results of a multicentric study. Autoimmune Rev. 2011, 10(9), 509.
- Vandenbroucke, E.; Grutters, J.C. et al. Rituximab in life threatening antisynthetase syndrome. Rheumatol. Int. 2009, 29, 1499.
- Sebastian, W.; Roy, A. et al. Correlation of antinuclear antibody immunofluorescence patterns with immune profile using line immunoassay in the Indian scenario. Ind. J. Pathol. Microbiol. 2010, 53(3). 427.
- Van den Bergh, K.; Hooijkaas, H. et al. Heterogeneous nuclear ribnucleoprotein h1, a novel nuclear autoantigen. Clin. Chem. 2009, 55(5), 946.
Cui Kong is with the Department of Cardiology; Jianwei Zhou, Aihua Shen, Xinke Chen, and Li Li are with the Clinical Laboratory; Jinsong Sun is with the Rheumatoid Immunology Clinic; and Qin Song is with Pediatric Surgery, Affiliated Hospital of Jining Medical College, Jining 272029, Shandong Province, China; tel.: +86 537 2903223/+86 537 2903218; fax: +86 537 2903223; e-mail: firstname.lastname@example.org . This article was supported by the Provincial Science and Technology Development Project (Grant 2012YD18054), Provincial Nature Science Foundation (Grant ZR2012HL29), High School Science and Technology Plan Project (Grant J11LF18), Population and Family Planning Commission (Grant 13), Development Plan Project of Jining Science and Technology Bureau of Shandong Province (Grant  57), Youth Foundation of Jining Medical College (Grant ), and Miaopu Program of Affiliated Hospital of Jining Medical College (Grant JYFY-MP-2013-09).