Background/Purpose:

Due to the poor specificity of the anti-nuclear antibodies (ANA) indirect immunofluorescence (IIF) assay, enhanced by the huge increase in ANA requests by non-rheumatologists, second line identification of specific anti-double stranded DNA (dsDNA) and anti- extractable nuclear antigen (ENA) is necessary.

Our study aims to objectify a cost-effective diagnostic ANA algorithm, standardizing the work-out of positive ANA IIF tests in a routine, secondary care setting.

Methods:

The ANA test results reported in our laboratory were retrospectively reviewed over a 9-month period. Positive ANA IIF test results on NOVA View® (Inova, USA) at 1:80 screening dilution (cut off = 49 Light Intensity Units (LIU)) were further analysed by dsDNA- (dsDNA-NcX IgG ELISA, Euroimmun, Germany) and ENA-screen (ANA screen 11 IgG ELISA, Euroimmun). For positive ENA-screen samples, ANA identification was performed with EUROLINE ANA profile 3 (Euroimmun). Based on ROC-curve analysis of LIU versus ENA/dsDNA identification, the LIU cut off at 95% sensitivity was determined.

Results:

3276 samples of 2916 patients were tested for ANA, of which 49,8% were from the rheumatology ward. 279 (9,6%) patients had repeated ANA requests. None of the repeated ANA IIF, dsDNA/ENA screen tests had a clinically significant result. 45,9% patients tested ANA IIF positive, with identification of a specific Ab (dsDNA/ENA) in 11,6% of the patients. ROC analysis of LIU in function of ENA/dsDNA identification revealed a specificity of 3,0% (2,1-4,3) for the 49 LIU cut off. A global ANA IIF sensitivity of 94.8% for ENA/dsDNA identification was obtained at a cut off of 88 LIU, with a specificity of 36,1% (33,6-39,5%) (Table 1). ROC curve analysis for isolated homogeneous, speckled, centromere and speckled metaphase positive IIF patterns confirmed an acceptable analytical performance and LR’s of ENA/dsDNA positivity at LIU of 88 (Table 2), in concordance with earlier published LR’s for ANA associated rheumatic disease positivity. Using this 88 LIU cut off for second line testing, can result in a yearly cost reduction of 5.060€. Introducing a limitation of the ANA workout to once yearly, if ANA IIF titer and pattern are stable, implies a supplementary cost reduction of 4.290€.

Conclusion:

Analytical performance analysis reveals a clinical and cost effective cut off for ANA IIF of 88 LIU for the initiation of second-level testing. If the patient is clinically stable and ANA IIF pattern and titer has not significantly changed, a yearly repetition of the diagnostic ANA workout is more than clinically sufficient.