Negative ANA-IIF in SLE patients: what is beyond?

A prospective cross-sectional study included 393 newly diagnosed patients as SLE according to the 2012 Systemic Lupus International Collaborating Clinics (SLICC) classification criteria [17]. These patients had visited the Sohag University Hospital Department of Rheumatology and Rehabilitation in Upper Egypt. Drug-induced lupus and discoid lupus without systemic manifestations were excluded. A complete medical history, a general exam, and evaluations of the skin, cardiovascular, chest, abdomen, neurological, and locomotor systems were obtained. In addition, the age at disease onset (defined as the age at which the first SLE-related symptoms occurred) and disease duration (from the time the first SLE-related symptoms arose until the date of the visit) were recorded; regular laboratory parameters and autoimmune markers were evaluated.

The local ethics committee accepted the study protocol with the number IBR#S20-139, and it adheres to the principles stated in the Declaration of Helsinki. Before participating in this study, all patients provided written informed consent.

Five milliliters of venous blood was withdrawn in a sterile vacutainers tube from each subject under aseptic conditions. Serum was separated using the conventional procedure. The following laboratory investigations were done:

Approximately 20–30 µl of diluted serum (1/40) in phosphate buffered saline (PBS) was placed on fixed HEp-2 cell (Anafluor, DiaSorin, USA) for 30 min at room temperature in a covered damp chamber. Positive and negative controls were run with each test. Slides were rinsed carefully with PBS, and then slides were placed into a Coplin jar filled with PBS for about 10 min. Then, slides were removed from the wash buffer and the excess PBS was drained and the excess PBS was removed using blotting strip.

Approximately 20–30 µl of antihuman immunoglobulin conjugated with fluorescein isothiocyanate (FITC) was dispensed to each well and incubated for an additional 30 min in a dark room to be protected from excessive light. After washing with PBS as before, small drop of mounting media was applied and then a coverslip was placed gently over the slide, and the slides were read using a fluorescence microscope at × 40 power by a technical expert.

ANA by IIF titer of at least 1/80 was regarded as positive. A serum was considered positive if the nuclei of the cell fluoresce with greater or equal intensity than the endpoint reference control well and there is a clearly discernible pattern of fluorescence. Any positive patient specimen was confirmed by repeating the test with twofold dilutions of serum.

The endpoint titer is the serial dilution which best approximates the fluorescent intensity of the endpoint reference control reaction. All positive ANA patterns should be tittered to endpoint dilution to detect possible mixed antinuclear reactions that may not be apparent when interpreting a single screening dilution.

A commercially available dot immunoassay was performed by using Blue Diver Quantrix ANA19 IgG kit (code ANA19Q-24) and Blue Diver Instrument (BDI) (D-tek, Belgium), for the detection of IgG autoantibodies in human sera against nineteen antigens: Nucleosome, dsDNA, Histones, Sm, RNP (68kD/A/C), Sm/RNP, SSA/Ro 60kD, SSA/Ro 52kD, SSB, Scl-70, RNA polymerase III, Ku, PM-Scl 100, Mi-2, Jo-1, CENP-A/B, PCNA, Ribosome P0, and DFS70.

The test is based on a conventional enzyme immunoassay (EIA). Antigens were coated in a microdot-shaped format on a nitrocellulose membrane adhered to a specific plastic support for the test strip. The antigens and controls were all spotted in triplicate.

During the automated testing procedure, the BDI sequentially incubates the strips in the wells of cartridges containing ready-to-use reagents. Initially, the strips were incubated with diluted patient sera. Human antibodies, if present, bind to the specific antigens dotted on the membrane and unbound or excess antibodies are eliminated by washing. The enzyme conjugate binds to antigen–antibody complexes after incubation with goat antibodies conjugated with alkaline phosphatase against human IgG. After rinsing away any excess conjugate, the strips were finally incubated in a substrate solution. The presence of enzyme activity would result in the formation of purple dots. The color intensity was directly related to the concentration of antibody in the sample.

All measured values were totally quantitative when compared to a 6-point, built-in calibration curve, which included a blank control. Multiple types of controls (sample, conjugate, and substrate) were also coated on the strip, and their presence validated the entire testing technique.

Strips were visually examined for discoloration and scanned with a Blue Scan scanner (D-tek, Belgium). Dr. DOT Software was used for the evaluation of the results (D-tek, Belgium). Each strip includes an integrated 6-point standard (calibration) curve with the values 0 (blank), 6, 12, 25, 50, and 100 U/ml; the Dr DOT software measured the mean color intensity of each antigen triplicate and the corresponding quantitative value was calculated from the calibration curve and compared to the predetermined cut-off value to evaluate the result.

The manufacturer recommends a cutoff value of 6 U/ml for the Blue Diver Quantrix ANA19 IgG, while values between 6 and 12 U/ml considered equivocal. A sample was considered positive for a particular antibody if the associated antigen dot value was greater than 12 U/ml.

The research involved 393 newly diagnosed SLE patients according to the SLICC 2012 classification criteria, 358 (91.1%) are females and 35 (8.9%) are males; the mean age of the patients is 37 ± 12.28, with median 35 and range 26–42 years, mean age at diagnosis 36 ± 11.28 years, and mean disease duration 9 ± 3.6 months.

There were 324 patients (82.4%) positive for ANA and 69 (17.55%) negative; the ANA patterns showed 181 (46.1%) speckled, 167 (42.4%) homogenous, mixed 7 (1.8%), nucleolar 1 (0.3%), mixed pattern included (homogenous and centromere 1 (0.3%), homogenous and nucleolar 2 (0.5%), speckled and homogenous 2 (0.5%), speckled and peripheral 1 (0.3%), and speckled and cytoplasmic ribosomal 1 (0.3%).

The most frequent ANA titer was 1/80 (19.3%) and 1/160 (13.7%), and the maximum titer was 1/10240 (0.3%).

ANA-IIF-positive patients showed 13 cases negative for immunoblot assay autoantibodies, 7 of them were homogenous and the other 6 were speckled. Comparison between the ANA +ve patients with the ANA −ve patients has shown significant differences in the frequency of autoantibodies, and the score of the SLICC 2012 criteria (Table 1).

The most common findings were for dsDNA in 194 patients (49.4%), nucleosome in 149 (37.9%), and SS-A/Ro 60 in 169 (43%). Twelve autoantibodies were detected in the patients with negative ANA (IIF). Moreover, the incidence of specific autoantibodies varied significantly between SLE patients with negative and positive ANA-IIF (Table 2).

Negative ANA-IIF showed 34.7% with dsDNA, 30.4% with SS-A/Ro 52, 18.8% with nucleosome, 13.04% with SS-A/Ro 60, and 4.3% with SS-B/La. Homogenous pattern showed 80.8% with ds DNA, 65.9% with nucleosome, 27.5% SS-A/Ro60, 25.1% with histones, 18% with Sm/RNP, and 15% with anti-Sm. Speckled pattern showed 63% with SS-A/Ro 60, 48.6% with SS-A/Ro52, 26% with Sm/RNP, 25.4% with SS-B/La, 24.3% with ds DNA, 20.4% with RNP/68 kD/A/C), and 18.8%with anti-Sm (more details in Table 3).