Recognizing the dense fine speckled/lens epithelium–derived growth factor/p75 pattern on HEp-2 cells: Not an easy task! Comment on the article by Mariz et al
Article first published online: 29 NOV 2011
Copyright © 2011 by the American College of Rheumatology
Arthritis & Rheumatism
Volume 63, Issue 12, pages 4036–4037, December 2011
How to Cite
Bizzaro, N., Tonutti, E. and Villalta, D. (2011), Recognizing the dense fine speckled/lens epithelium–derived growth factor/p75 pattern on HEp-2 cells: Not an easy task! Comment on the article by Mariz et al. Arthritis & Rheumatism, 63: 4036–4037. doi: 10.1002/art.30621
- Issue published online: 29 NOV 2011
- Article first published online: 29 NOV 2011
- Accepted manuscript online: 30 AUG 2011 10:07AM EST
To the Editor:
We read with interest the recent article by Mariz et al (1) on the possibility of discriminating antinuclear antibody (ANA)–positive healthy subjects from patients with autoimmune rheumatic diseases (ARDs) based on the presence of the dense fine speckled pattern detected by indirect immunofluorescence (IIF) on HEp-2 cells.
Both those investigators and the author of an accompanying editorial (2) underline the necessity of confirming the dense fine speckled pattern observed by IIF, using a more objective method such as an enzyme-linked immunosorbent assay (ELISA) or Western blotting.
Our group studied 94 sera with dense fine speckled–like patterns by IIF, using an MBL ELISA that is specific for detecting anti–dense fine speckles 70 (DFS-70)/lens epithelium–derived growth factor (LEDGF) antibodies. All of the patients were referred in 2006 by rheumatologists or general practitioners for ANA testing for the presence of ARDs. The dense fine speckled–like pattern at titers from 1:80 to 1:1,280 was identified by expert examiners in 3 reference laboratories. Sera were then stored at −80°C until tested. To our surprise, the results with the DFS-70–specific ELISA revealed positive results for only 13 (14%) of the 94 sera tested. Several hypotheses might explain such poor concordance between IIF testing and the ELISA results.
First, it is possible that the antibodies that produce a dense fine speckled–like ANA pattern are very heterogeneous, and that the antigens coated on the microplates in the MBL ELISA contained only a few of the diverse epitopes against which the antibodies are directed. The antigen used in the MBL ELISA is a recombinant protein expressed on a baculovirus containing the C-terminal 338–530–aa region of LEDGF/p75 (3).
However, the most likely explanation is that samples were not selected properly, resulting in an overestimation of the dense fine speckled pattern. Although we recognize the probability of this error in identification of the pattern, the data imply that recognition of a dense fine speckled pattern by IIF testing, even by expert personnel, is very discretionary and subjective. In effect, other fine speckled patterns and, moreover, the pattern that Mariz and colleagues define as “quasi-homogeneous,” are not easily distinguishable from the dense fine speckled pattern, especially by nonexpert interpreters.
Another possible cause relates to differences arising from the type of cellular substrate used. To assess this possibility carefully, we analyzed 57 of the 94 samples, including the 13 with positive results by ELISA, using 4 different HEp-2 cell lines (from Inova, Bion, Bio-Rad, and Euroimmun, respectively). Some samples with low titers (1:80) had positive test results with only 1 or 2 of the cell lines and had negative results with the others; of the 13 ELISA-positive samples, only 3 had positive test results with all 4 cell lines, confirming that the use of different substrates contributes to different results. In addition, 2 high-titer sera that had positive test results with all 4 cell lines had negative results by ELISA. The conclusion that can be drawn from the results of our study is that the approach proposed by Mariz et al would be associated with enormous practical difficulties in any laboratory that might want to adopt it.
Another substantial concern (and maybe the most important one) relates to the clinical significance of recognizing a dense fine speckled pattern. Even if the prevalence of the dense fine speckled pattern in ARDs is low, one cannot say that it is always absent. If we rely on the prevalence data, the dense fine speckled pattern is present in ∼10% of healthy subjects but also is observed in 2–7% of patients with ARDs and in up to 11% of patients with Sjögren's syndrome (4–7). Because an ANA test is not usually requested for healthy subjects, a substantial part of the tests with positive results would be in patients with ARDs. This is why the proposal to use this pattern to exclude an ARD diagnosis is difficult to support.
Consistent with the data presented by Mariz and colleagues, a review of medical records of our 94 patients over 5 years from the discovery of the dense fine speckled pattern demonstrated that ARDs developed in 31 patients (33%) during followup, but none of these patients had shown anti–DFS-70 positivity by ELISA. However, 2 patients with lupus had positive IIF test results with 3 of the 4 cell lines. Among the 13 patients with anti–DFS-70 positivity by ELISA, the diagnoses during followup included rheumatic polymyalgia, colon cancer, Hashimoto's thyroiditis, type 1 diabetes mellitus, chronic lymphocytic leukemia, and infertility, while no definite diagnosis was made in the other 6 patients.
In summary, although the work by Mariz and colleagues is interesting from the speculative point of view, and certainly the dense fine speckled–like pattern is present only in a small number of patients with ARDs, we do not have sufficient evidence to be able to use it as a criterion for exclusion of a diagnosis of ARD. Our opinion is that special caution is warranted when attributing clinical significance to the dense fine speckled pattern, given that, as we have seen in our experience, the possibility of positive-false results is very high, and, consequently, the impact on patients could be great.
If multiplex methods for the simultaneous determination of several antibodies become available in the future, a possible solution is insertion of the DFS-70/LEDGF antigen in diagnostic panels. Only after many more data are gathered may we be able to state whether this antibody can be used to direct the diagnosis toward a specific disease (or group of diseases) or for excluding other diseases (e.g., ARDs), or whether it is merely an antibody without any particular diagnostic and clinical significance.
- 1Pattern on the antinuclear antibody–HEp-2 test is a critical parameter for discriminating antinuclear antibody–positive healthy individuals and patients with autoimmune rheumatic diseases. Arthritis Rheum 2011; 63: 191–200., , , , , .
- 2The antinuclear antibody test: last or lasting gasp? [editorial]. Arthritis Rheum 2011; 63: 19–22..
- 3Autoantigenicity of DFS70 is restricted to the conformational epitope of C-terminal α-helical domain. J Autoimmun 2004; 23: 221–31., , , , , , et al.
- 4Anti-DFS70 antibodies in 597 healthy hospital workers. Arthritis Rheum 2004; 503: 892–900., , , , , , et al.
- 5Autoantibodies to DFS70 kd/transcription coactivator p75 in atopic dermatitis and other conditions. J Allergy Clin Immunol 2000; 105: 1211–20., , , , , .
- 6Autoantibodies to lens epithelium-derived growth factor/transcription co-activator P75 (LEDGF/P75) in children with chronic nonspecific complaints and with positive antinuclear antibodies. Autoimmunity 2009; 42: 492–6., , , .
- 7High concomitance of disease marker autoantibodies in anti-DFS70/LEDGF autoantibody-positive patients with autoimmune rheumatic disease. Lupus 2008; 17: 171–6., , , .
Nicola Bizzaro MD*, Elio Tonutti MD, Danilo Villalta MD, * Santa Antonio Hospital, Tolmezzo, Italy, Santa Maria della Misericordia Hospital, Udine, Italy, Santa Maria degli Angeli Hospital, Pordenone, Italy.