Detection and identification of significant ANAs in previously determined ANA negative samples

Background: An antinuclear antibody (ANA) substrate transfected with the cDNA to hyperexpress the 60 kD SS-A/Ro antigen (HEp-2000®) has been shown to detect anti-SS-A/Ro antibodies missed by standard HEp-2 and other immunoassays. Despite this evidence, many laboratories remain convinced that with experienced technicians, standard HEp-2 is acceptable for ANA detection. Aim: To challenge the ability of HEp-2000® to detect anti-SS-A/Ro antibodies in samples previously determined to be ANA negative using standard HEp-2. Methods: Three hundred and seventy-one pre-screened "negative" ANA samples were provided by a university hospital laboratory in Germany. These samples were tested on the HEp-2000® substrate at a dilution of 1:40 by indirect immunofluorescence (IIF). Samples that screened positive for a nuclear pattern were titered (range of 1:40-1:640) and all ANA-positive patterns were identified. Samples containing at least one positive ANA pattern at a dilution greater than or equal to 1:160 were further tested. Samples that produced a speckled pattern were tested for antibodies to the extractable nuclear antigens (ENA) and samples that showed homogeneous staining were tested for antibodies to dsDNA, and if negative, were then tested for anti-histone antibodies. Results: Ninety-one patient samples were positive with titers ≥1:160. Speckled patterns were the most common finding (30 samples) followed by speckled/homogeneous mixed patterns (19 samples) and samples demonstrating the SS-A/Ro pattern (16 samples) either alone or in combination with other ANA patterns. The remaining 26 positive samples consisted of various other ANA patterns. The most commonly identified ENAs were SS-A/Ro (14 samples), Scl-70 (11 samples) and SSB (6 samples). No antibodies to dsDNA were identified in 23 positive samples with homogeneous staining patterns, though 17 of these samples tested positive for antibodies to histone. Conclusions: HEp-2000® detected anti-SS-A/Ro antibodies in 16 (4%) of the "ANA negative" samples. In addition to improved sensitivity for anti-SS-A antibodies, HEp-2000® demonstrated improved sensitivity over standard HEp-2 substrate for other significant ANAs including anti-Scl-70, anti-histone, and anti-SS-B antibodies.