Is it useful to test for antibodies to extractable nuclear antigens in the presence of a negative antinuclear antibody on Hep-2 cells?
- Department of Dermatology, Leeds Teaching Hospitals Trust, Leeds General Infirmary, Great George Street, Leeds LS1 3EX, UK
Antinuclear antibody (ANA) negative lupus has long been recognised as a distinct entity affecting a small number of patients with systemic lupus erythematosus (SLE).1 Initial estimates of the prevalence of this entity (5% of patients with lupus) were based upon studies using rodent tissues as substrate for antinuclear antibody testing. The increasing use of human epithelial cell lines (Hep-2 cells), which have greater sensitivity for extractable nuclear antibodies (ENA), has meant that new patients with true ANA negative lupus are now rarely encountered.
Many immunology laboratories are faced with a substantial number of requests for antibodies to ENA and double stranded DNA, even in patients with negative ANA, on the grounds that patients with ANA negative lupus might go undetected. Using Hep-2 cells, we have attempted to define the prevalence of ANA negative, anti-ENA positive disease in a series of consecutive, unselected serum samples.
Over a 12 month period, all laboratory requests for antibodies to ENA (antibodies to Sm, Ro, La, and ribonuclear protein) were scrutinised to determine the number of samples that had antibodies to ENA despite a negative ANA on Hep-2 cells. The notes of patients who were ANA negative, anti-ENA positive were examined to verify the clinical diagnosis.
During the 12 month study period, 7077 Hep-2 ANA samples were processed and 468 patients had an anti-ENA profile performed despite a negative ANA. Of these 468 patients, nine were identified who were ANA negative, anti-ENA positive. Review of their clinical notes indicated that six of these nine patients had previously been ANA positive and were known to have lupus but were receiving immunosuppressive treatment. Only three patients were persistently ANA negative despite positive anti-Ro antibodies before treatment. Thus, the prevalence of anti-ENA positivity combined with a negative ANA was three out of 468 (0.64%).
Because ANA negative lupus characteristically presents with cutaneous disease the clinical notes of 90 of the dermatology patients were reviewed. Twenty seven of these patients had confirmed lupus erythematosus. Only one patient from the dermatology group had ANA negative, anti-Ro positive lupus before the commencement of immunosuppressive treatment.
Our finding of a low prevalence of anti-ENA positivity in the presence of a negative ANA on Hep-2 cells is in keeping with other studies in the literature.2, 3 Manoussakis et al found that only 0.4% of 243 Hep-2 negative patients with systemic autoimmune disease had positive anti-ENA antibodies2 and Homburger,3 reporting on the experience of the Mayo Clinic immunopathology laboratory, stated that anti-ENA antibodies were unlikely to be positive in the presence of a negative ANA result on Hep-2 cells. However, neither of these studies included a clinical evaluation of the ANA negative, anti-ENA positive patients.
We recognise that our study is subject to potential sources of bias. The failure to scrutinise patients' notes on all ANA negative samples irrespective of anti-ENA antibody status might have resulted in some patients with strong clinical evidence of connective tissue disease being overlooked. We think it unlikely that this would have greatly changed our findings given the rarity of uniformly seronegative lupus (ANA negative, anti-ENA negative, and anti-DNA negative) and the general acceptance that a repeatedly negative ANA effectively excludes systemic lupus. Second, if clinicians failed to request ENA along with ANA, it is possible that some cases of ANA negative, ENA positive disease would be missed.
Based on these findings and others in the literature2–5 we have modified our testing strategy for antibodies to ENA. All requests for anti-ENA antibodies are “gated” by performing an initial ANA screen on Hep-2 cells. Samples that are ANA negative do not proceed to further testing unless there are compelling clinical reasons to suggest lupus. In conjunction with good clinical liaison this testing strategy allows streamlining in busy clinical laboratories.