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Antibodies to extractable nuclear antigens in antinuclear antibody-negative samples.

To the Editor:

Antibodies to extractable nuclear antigens (ENAs)--SSA, SSB, U1RNP, Sm, Scl-70, and Jo-1--are clinically important in patients with systemic rheumatic diseases. Indirect immunofluorescence (IIF) with HEp-2 cells is a common initial screening test for detection of antinuclear antibodies (ANAs) and antibodies to ENAs. IIF-positive samples are further screened with more specific assays, but few studies have addressed the value of this cascade testing (1, 2). Although screening with conventional HEp-2 cells may miss some antigens, such as SSA (3) and Jo-1 (4), false-negative ANA results are infrequent (1, 2). SSA-transfected cells in particular, which overexpress SSA (60 kDa), are considered highly sensitive for detection of anti-SSA antibodies (5). Some antibodies to ENAs can be missed by IIF, however (6). Hoffman et al. (6) found that of 291 ANA-negative samples, 12 were positive for antibodies to ENAs, including antibodies to SSA (Ro52 and Ro60), SSB, RNP-A, RNP-C, RNP-70, SmD, Scl-70, and Jo-1.

We performed a prospective study to evaluate whether and to what extent IIF performed with SSA-transfected HEp-2000[TM] cells (hninunoconcepts) failed to detect antibodies to ENAs. Most samples (>95%) were referred for ANA testing by in-house specialists (University Hospital, Leuven, Belgium) in general internal medicine (18.6%), rheumatology (17.2%), gastroenterology (6%), hepatology (5.5%), pneumology (4.6%), allergy (4.5%), neurology (4.3%), and nephrology (4%). Evaluations were performed by 4 experienced technologists with an Eclipse E400 microscope (Nikon Corporation) fitted with an XBO 100 OFR Xenon bulb (100 W) and a 60x Nikon Plan Fluor objective for screening. All readings were done in duplicate (by 2 technologists).

A total of 1840 consecutive ANA-negative samples (i.e., ANA titers <1:80 and anti-cytoplasmic antibody titers <1:40) (5) collected over 15 weeks (starting December 2004) were tested for the presence of antibodies to ENAs by a screening ELISA (Bio-Rad). The ELISA revealed 42 (borderline) positive samples. These samples were further tested for the presence of antibodies to the individual antigens by dot-blot analysis (Biomedical Diagnostics) (7) and EliA[TM] fluorescent enzyme immunoassay (Pharmacia Diagnostics). In the dot-blot analysis, the nitrocellulose membrane was coated with purified antigens (SSA, SSB, Sm/U1RNP, Sm, Scl-70, and Jo-1) from bovine and rabbit thymus or calf spleen. In the EliA system, the wells were coated with human recombinant SSA (60 and 52 kDa), SSB, U1RNP (70 kDa; A and C proteins), U1RNP-70, Scl-70, Jo-1, and native Sm purified from bovine tissue. The readers of the ELISA, dot-blot analysis, and EliA results were not blinded to the IIF analysis results. All assays were done on the same serum sample in a short time interval (<5 days).

Twenty-one samples (from 16 different patients) were positive in at least 1 of the 2 assays. The clinical finding results are summarized in Table 1. Clinical diagnoses of systemic rheumatic diseases were established as described previously (5). In 6 patients, dot-blot analysis and EliA results were similar. In 7 patients, dot-blot analysis was negative, but EliA revealed the presence of anti-SSA. Because EliA detects antibodies to SSA-52 and 55-60 and dot-blot analysis detects only antibodies to SSA-60, we also analyzed these samples for the presence of antibodies to SSA-52 (Split ANA profile; Pharmacia Diagnostics). A sample from a rheumatoid arthritis patient contained anti-SSA-52 but not anti-SSA-60, and a sample from a neonatal lupus patient contained anti-SSA-60 and anti-SSA-52.

During the collection period of the 1840 ANA-negative samples, 565 ANA-positive samples were identified. Anti-ENA antibodies were found in 102 of these samples. The prevalence of anti-ENA antibodies was 16-fold higher in ANA-positive samples (18%)than in ANA-negative samples (1.1%). The sensitivity of ANA testing on HEp-2000 for detection of anti-ENA antibodies was calculated to be 82.9% (95% confidence interval, 75.1%-89%;calculated with Analyze-it in Microsoft[R] Office Excel). Hoffman et al. (6) reported a sensitivity of 72%, which varies from earlier studies in which the HEp-2000 assay was compared with counterimmunoelectrophoresis and immunodiffusion (8, 9). The differences between the reported frequencies of specific autoantibodies in IIF-negative samples may be related to the sensitivity of the technique used for detection of specific autoantibodies and to the definition of ANAs (some laboratories do not consider cytoplasmic staining as indicative of ANAs). For example, in our study, the dot-blot system was less sensitive than the EliA system. Dot-blot analysis and ELISA are also known to be more sensitive than counterimmuno-electrophoresis and immunodiffusion.

In conclusion, we confirmed that anti-ENA antibodies (especially anti-Jo-1 and anti-SSA) may be over-looked by IIF on HEp-2000 cells. When there is a high clinical suspicion, irrespective of the ANA result, focused testing for specific autoantibodies should be performed.

We are indebted to Bio-Rad for providing the ENA screening reagent set. We thank L. Meurs, G. Godefridis, and J. L'Heureux for expert technical assistance.


(1.) Solomon DH, Kavanaugh AJ, Schur P, and the American College of Rheumatology and Ad Hoc Committee on Immunologic Testing Guidelines. Evidence-based guidelines for the use of immunologic tests: antinuclear antibody testing. Arthritis Rheum 2002;4:434-4.

(2.) Kavanaugh A, Tomar R, Reveille J, Solomon DH, Homburger HA. Guidelines for clinical use of the nuclear antibody test and tests for specific autoantibodies to nuclear antigens. Arch Pathol Lab Med 2000;124:71-81.

(3.) Dahle C, Skogh T, Aberg AK, Jalal A, Olcen P. Methods of choice for diagnostic antinuclear antibody (ANA) screening. Benefit of adding antigen-specific assays to immunofluorescence microscopy. J Autoimmun 2004; 22: 241-8.

(4.) Bahrt KM, McCarty FA. A negative fluorescent antinuclear antibody test in a patient with Jo-1 antibody. J Rheumatol 1985;12:624-5.

(5.) Bossuyt X, Frans J, Hendrickx A, Godefridis G, Westhovens R, Marien G. Detection of anti-SSA antibodies by indirect immunofluorescence. Clin Chem 2004;50:2361-9.

(6.) Hoffman IEA, Peene I, Veys E, De Keyser F. Detection of specific antinuclear reactivities in patients with negative anti-nuclear antibody immunofluorescence screening test. Clin Chem 2002;48:2171-6.

(7.) Mewis A, Marien G, Blanckaert N, Bossuyt X. Evaluation of a dot-blot method for identification of antibodies against extractable nuclear antigens and cytoplasmic antibodies. Clin Chem 1999;45:311-2.

(8.) Bossuyt X, Meurs L, Mewis A, Marien G, Blanckaert N. Screening for autoantibodies to SSA/Ro by indirect immunofluorescence using HEp-2000 cells. Ann Clin Biochem 2000;37: 216-9.

(9.) Fritzler M, Hanson C, Miller J, Eystathioy T. Specificity of autoantibodies to SSA/Ro on a transfected and overexpressed human 60 kDa Ro autoantigen substrate. J Clin Lab Anal 2002;16:103-8.

Xavier Bossuyt *

Ariane Luyckx

Laboratory Medicine, Immunology

University Hospital Leuven

Leuven, Belgium

* Address correspondence to this author at: Department of Laboratory Medicine, Immunology, University Hospital Leuven, Herestraat 49, B-3000 Leuven, Belgium. Fax 32-13-347042; e-mail

DOI: 10.1373/clinchem.2005.058552
Table 1. Clinical findings and test results for patients with negative
IIF on HEp-2000 cells but with antibodies to ENAs. (a)

Patient Dot-blot (EliA
no. analysis units/mL) Clinical findings

 1 SSA SSA (13.6) Rheumatoid arthritis
 2 SSA SSA (29.7) Systemic lupus erythematosus
 3 SSA SSA (37.2) No data available
 4 SSA SSA (20.7) Sjogren syndrome, cutaneous lupus,
 5 Jo-1 Jo-1 (119) Polymyositis
 6 Sm/RNP [U.sub.1] Drug-induced lupus (2)
 + Sm RNP (51) +
 RNP 70 (19)
 + Sm (6)
 7 Scl-70 Negative Reactive arthritis
 8 Negative SSA (11.2) B-CLL (c) (bone marrow
 9 Negative SSA (38.7) Neonatal lupus erythematosus
 (skin rash)
10 Negative SSA (18.4) Psoriatic spondylarthropathy
11 Negative SSA (23.5) Chronic urticaria
12 Negative SSA (20.3) Pregnancy and thrombocytopenia
13 Negative SSA (27.1) Subacute cutaneous lupus
14 Negative SSA (19.5)4 Rheumatoid arthritis
15 Negative Sm (12.5) Subacute sclerosing panencephalitis
16 Negative [U.sub.1]RNP Cervical tension

(a) Dot-blot analysis detects antibodies to SSA-60, whereas EliA
detects antibodies to SSA-60 and SSA-52. Range of expected (cutoff)
values (Pharmacia Diagnostics) for EliA are as follows: negative,
<5 EliA units/mL for anti-Sm and <7 EliA units/mL for the other
antibodies; equivocal, 5-10 EliA units/mL for anti-Sm and 7-10 EliA
units/mL for the other antibodies; positive, >10 EliA units/mL for all

(b) The drug was peginterferon. In follow-up samples from the same
patient, the antibodies disappeared (on dot-blot analysis and by EliA).

(c) B-CLL, B-cell chronic lymphocytic leukemia.

(d) Further analysis (Split ANA profile; Pharmacia Diagnostics)
revealed that this sample contained antibodies to SSA-52 but not
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Title Annotation:Letters
Author:Bossuyt, Xavier; Luyckx, Ariane
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Dec 1, 2005
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