September 1997, Volume 47, Issue 9

Original Article

Fluorescent Antinuclear Antibody and Anti-Double-Stranded DNA Antibody Testing: A Four Years Experience

Farzal Anwer  ( Department of Laboratory Medicine, Al-Hada Armed Forces Hospital, Taif, Saudi Arabia. )
Abdel-Moneim Abou-Azama  ( Department of Laboratory Medicine, Al-Hada Armed Forces Hospital, Taif, Saudi Arabia. )
Abdullah Mandourah  ( Department of Laboratory Medicine, Al-Hada Armed Forces Hospital, Taif, Saudi Arabia. )
Margarita Borja  ( Department of Laboratory Medicine, Al-Hada Armed Forces Hospital, Taif, Saudi Arabia. )
Sonja Grace Mercado  ( Department of Laboratory Medicine, Al-Hada Armed Forces Hospital, Taif, Saudi Arabia. )
Rodrigo Soriano  ( Department of Laboratory Medicine, Al-Hada Armed Forces Hospital, Taif, Saudi Arabia. )

Abstract

Fluorescent antinuclear antibody test (FANA) and anti-double stranded deoxytribonucleic acid (dsDNA) antibody testing is an integral part of the evaluation of the patients who are suspected of having connective tissue disease. We tested 2,140 serum samples for FANA and 1,460 serum samples for anti-dsDNA antibodies. Of 2,140 serum samples tested for FANA, 492 (23%) yielded a positive result (titre of 1:80 or greater) and of 1,460 serum samples tested for anti-ds DNA, 69 (4.7%) yielded positive results. Highest number (n=27) of serum samples positive for anti-dsDNA antibodies were found in serum samples that were positive for FANA test at a titre of 1:1280 or greater. In conclusion, FANA test can be used as an initial screening test for connective tissue/autoimmune disorders (JPMA 47:226,1997).

Introduction

Indirect Immunofluorescent Antinuclear Antibody (FANA) test is used to screen for patients with suspected connective tissue diseases like systemic lupus eiythematosus (SLE), scieroderma, Sjogren’s syndrome, mixed connective tissue disease (MCTD). The FANA results are positive in over 90% patients with connective tissue disease, suggesting it as a sensitive test and supporting its role as the laboratozy “gold standard” for assistance in the diagnosis of connective tissue disorders1. DNA was the first specific autoantigen identified in SLE2,3. These antibodies am rarely found in diseases other than SLE, and their titres often fluctuate in accord with the changes in disease activity4-6. Thus, analysis of anti-dsDNA antibodies has become an important laboratoiy test for the evaluation of patients with SLE. FANA, together with anti-dsDNA antibody test also helps in differentiating drug-induced lupus from other forms of lupus7,8. The present study reports our experience of FANA and anti-ds DNA antibody testing for over four years and general aspects of these parameters are discussed.

Material and Methods

A total of 2,140 serum samples were analyzed for FANA test and anti-dsDNA antibodies from Januaiy, 1992 to March, 1996. Of 2,140 serum samples, 1,460 were tested for both FANA and anti-dsDNA antibodies, whereas 680 serum samples were analyzed for FANA test alone.
FANA Testing
The FANA test for antinuclear antibodies was performed using ANAJ Hep-2 Test System (Zeus Scientific Inc., USA). Zeus, ANA/Hep- 2 Test System use ZORBA-NS sample diluent formulated to reduce non-specific staining. Polyclonal goat anti-human fluorescein- conjugated immunoglobulin was used as second antibody. These assays were performed according to the manufacturer’s instructions. Initial screening of serum samples was done at 1:40 dilution. Test slides were read by using fluorescence microscope x10 for positivity and x25 for pattern identification. A titre of 1:40 was considered insignificant because of the frequency of such low titres in the presumed normal population.
Anti-dsDNA Antibody Testing
For most part of our study, we used the indirect immunofluorescence assay to detect anti-dsDNA antibodies. Cnthidia lucilae substrate and polyclonal goat anti-human fluoresccin-conj ugated immunoglobulin as a second antibody (Zeus Scientific Inc., USA). Manufacturer’s instructions were followed for the procedure. Serum samples were screened at 1:10 dilution. Results were recorded as negative or positive with titres of 1:10 to greater than or equal to 1:160. No fuaher dilutions were performed for samples positive at 1:160 dilution. Recently, we have started radioimmunoassay for the detection of anti-dsDNA antibodies (DPC, Diagnostic Products Corporation, CA, USA). 1251- labelled recombinant DNA binds to anti-dsDNA in the patient sample. The bound fraction is then counted on a gamma counter (Gamma 4000, Beckman, USA) and patient sample concentrations are read from a calibration curve.

Results

FANA Testing
Of 2,140 serum samples tested for FANA, 492 (23%) yielded a positive result (titre of 1:80 or greater), while in 1,648 (77%) no immunofluorescence was detected (Table I).

A titre of 1:40 was detected in 119(5.56%). Of the total positive for immunofluorescence (n=492), majority (128) of serum samples showed a litre of 1:640 with a highest titre of 1:10,240 (Table II).

The most common patterns of fluorescence detected were speckled and homogenous.
Anti-dsDNA Testing
Of 1,460 serum samples tested for anti-dsDNA antibodies, 69 (4.7%) yielded positive results and 1,391 (95.2%) were negative (Table I).
Comparison of FANA Titre and Anti-dsDNA Antibody results Of the total 69 serum samples positive for anti-dsDNA antibody, 6 (8.7%) were positive with negative FANA test (Table III),

5(7.2%) were positive with FANA test at titre 1:40.
A total of 27 (3 9.1%) anti-dsDNA antibodies were detected at a FANA litre of 1:1,280 or greater.

Discussion

Antinuclear antibodies (ANAs) are found in different individuals, including healthy blood donors and patients with a variety of connective tissue disease. The study of autoantibodies is important in patient care and research in rheumatic/connective tissue diseases. The first understanding of ANAs began in 1948 when Hargraves and colleagues9, demonstrated that the concentrated bone marrow of patients with SLE contained “L.E.” cells. Later, Holman and KunkeI10 demonstrated that the basis for LE cells was antibodies to deoxyribonucleoprotein, which acted as opsonins for directed phagocylosis by polyrnorphonuclear leukocytes of antibody-sensitized nuclei. LE ecli phenomenon was widely used as a diagnostic tool until the middle of the 1970s when its use began to be supported by more specific test for individual ANAs (like antibodies to extractable nuclear antigens). The second major development in the study of ANAs occurred in 1957, when Friou used indirect immunofluorescence to the study of ANSs popularly known as FANA (fluorescence antinuclear antibody test). This technique is much more sensitive than the LE cell test, and since it detects ANAs in a broad variety of other disease and has become the method of choice for initial screening purposes in systemic connective tissue disorders. With the advent of new techniques and our understanding of the molecular biology of ANAs and their antigens, it is now possible to define the exact specification of these ANAs and their antigens, it is now possible to define the exact specification of these ANAs and help in the diagnosis of underlying rheumatic/connective tissue disorder. With all these developments, FANA test still holds to be the important screening tool for the initial evaluation of patients who are suspected of having connective tissue disease. Like all other laboratory parameters, FANA test has its own limitations and pitfalls’
DNA was the first specific autoantigen identified in SLE14. Antibodies to dsDNA are quite specific forSLE. These antibodies recognized the native, double-stranded DNA and bind the deoxyribose phosphate back-bone of DNA. A number of assay systems are available for the detection of these antibodies. One of the common method is the indirect immunOfluorescence technique using Crithidia lucilae substrate15-16. However, them am radioimmunoassay (RIA)17 and ELISA18 also available for this purpose.
It is concluded that FANA and anti-dsDNA antibody testing is useful for the evaluation of patients who are suspected of having connective tissue disease. We found 23% serum samples positive for FANA test. This test may be used as an initial screening tool when a connective tissue disease is suspected. Further investigations like anti-dsDNA antibody or antibodies to extractable nuclear antigens (ENA) may then follow. Whether to evaluate further the FANA test negative specimen for antibodies to other extractable nuclear antigens is slightly controversial. Rare cases of SLE patients have negative FANA test19,20. FANA test is unable to detect anti-Ro antibodies, antibodies to exclusively single-stranded DNA or cytoplasmic nbonucleic protein (RNPs) such as the Jo-I antigen21,22. Some of the published data indicate that further evaluation of FANA test negative serum samples is generally unjustifiable23,24. We however, believe that clinical findings remain the cornerstones for the diagnosis of rheumatic/connective tissue disease and selected tests must be used to refine the pretest assessments of disease probability.

Acknowledgement

The authors thank Ms Sarah Cortes for her assistance in the preparation of this manuscript.

References

1. Tan, EM., Robinson, CA., Nakamura, R.M. ANAs in systemic rheumatic disease. Postgrad. Med., 1985;78: 141.148.
2. Robins, W.C., Holman, HR. and Kunkel, H.G. Complement fixation with cell nuclei and DNA in lupus erythanatosus. Proc. Soc. Exp. Biol. Med., 1957,96:575-580.
3. Ceppallini, R., Rolli, E. and Celada, F. A DNA reacting factor in serum of a patient with lupus erythematosus diffuse. Proc. Soc. Exp. Biol. Med., 1957;96:572-575.
4. Pincus, T., Schur, PH.. Rose, J.A. et al. Measuremen is of serum DNA binding activity inSLE. N. Engi. J.Med.. 1969;281:701-705.
5. Schur, PH. and Sandson, J. Immunological factors and clinical activity in systemic lupus ecythcmatosus. N. Engi. J. Med., 1968;278:533-538.
6. Miniter, M.F., Stolla, B.D. and Angello, V. Reassessment of the clinical significance of native DNA antibodies in systemic lupus eiythematosus. Arthritis Rheum., 1979,22:959-968.
7. Weinstein, A. Laboratory diagnosis of drug-induced lupus. In: Lahita, R.G. Editor. Systemic lupus Erythamatosus. New York, John Wiley. 1987. pp.881-886.
8. Burlingame, RW. and Rubin, R.L. Drug-induced antihisionc autoantibodies display two patterns ofactivity with substratè of chromatin. Clin. Invest, 1991 ;88:680-690.
9. Hargraves, MM., Richmond. H. and Morton, R. Presentation of two bone marrow elements: The Tart* Cell and ‘LE’ Cell. Proc. Staff Meet. Mayo Clinic, l948;23:25-28.
10. Ilolman, HR. and Kunkel, HG. Reports: Affinity between the lupus erythematosus serum factor and cell nuclei and nucleoprotein. Science. 1957;126:162-165.
11. Cleymaet, J.E. and Nakamura, R.M. Indirect immunofluorescent antinuclear antibody tests; Comparison of sensitivity and specificity ofdiffercnt substrates, Am. J. Clin. Pathol., 1 972;58 :388-393.
12. Cheryll, M. and Linda, MM. ANA substrate comparison study.Lab. Med., I 994;25:5 15-519.
13. Molden, D.P, Nakamura, RM and Tan, EM. Standardization of the immunofiuo­rescent teat for auto-antibody to nuclear antigens (ANA): use of reference sera of defined antibody specificity. Am. J. Clin. Pathol., 1984;82:57-66.
14. Bardana, Li., Harbeck, R. J. Hoffman, A.A. ci al. The prognostic and thaapeutic implications of DNA: anti-DNA immune-complexes in systemic lupus erythe­matosus(SLE). Am. J. Med., 1975;59:515-522.
15. Jansen, EM., Deng, J.S., Beutner, E.H. ctal. Comparison ofcommcrcial kits for the detection of anti-DNA antibodies using Crithidia lucilae. Am. J. Clin Pathol., 1987;87:461-469.
16. Ballou, S.P. and Kushner, I. Anti-native DNA detection by the cnthidia lucilae method. An improved guide to the diagnosis and clinical managanent of systemic lupus erythematosus. Arthritis Rheum., 1 979;22:321 -327.
17. Rubin, R.L., Joslin, F.J. and Tan, EM. A solid-phase radioimmunoassay for antihistone antibodies in human sera: Comparison with an immunof luoreacence assay. Scand. J. Immunol., 1982;15:63-70.
18. Rubin, R.L., Joslin, F.J. and Tan, E. M. An improved ELISA for anti-native DNA by elimination of interference by antiihistone antibodies. J. Irnmunol. Methods, 1983;63 :359-366.
19. Fritzeer, M.J. Antinuclear antibodies in the investigation of rheumatic disease. Bull. Rheum., 1985;35:1-10.
20. Urowitz, M.B. and Gladman, D. Antinuclear antibody-negative systemic lupus erythematosus. In: Lahita, R.G. editor. Systemic lupus erythernatosus. New York, John Wiley, 1987. pp. 555-563.
21. Reichlin, M. and Wasicek, C.A. Clinical biological significance ofantibodies to Ro/SSA.Hum. Pathol., 1983; 14:401-405.
22. Tan, EM. Antinuclear antibodies. Diagnostic markers for autoimmune disease and probes for cell biology. Adv. Immunol., 1 989;44:93- 151.
23. Manoussaki, MW., Garalea, K.L., Tzioufas, AG. et al. Testing for antibodies to ENA and to dsDNA is not indicated in FANA- negative sera. Clin. Rheumatol., 1 988;7:465-469.
24. James, K. and Meek, 0. Evaluation of commercial enzyme Immunoassays compared for antibodies associated with autoimmune diseases. Am. J. Clin. Pathol., l992;97:559-565.

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