Testing for ANCA plays a pivotal role in the serological diagnosis of AAV [22
]. In accordance with international guidelines, IIF and independent detection techniques like ELISA should be used for the assessment of ANCA [4
]. Interestingly, IIF has been reported to be used as a screening method due to its high sensitivity followed by anti-PR3-ANCA and anti-MPO-ANCA ELISAs as confirmatory tests under routine laboratory conditions due to its high sensitivity and negative predictive value [24
]. In contrast to the ELISA technique, IIF is prone to human bias and ANCA IIF image interpretation has been not automated until recently [16
]. Thus, the recommended combination of IIF and confirmatory testing requires a high level of expert knowledge and lacks standardization [25
]. Several studies tried to demonstrate that ELISA technology is better adaptable for standardization of ANCA assessment and has a lower lab-to-lab variability compared to IIF [26
]. However, most commercially available ELISAs have been reported to be inferior to IIF in terms of sensitivity [29
]. Therefore, automated interpretation of ANCA IIF patterns as demonstrated for ANA detection by novel pattern recognition algorithms could provide a cost-efficient and standardized alternative approach for ANCA screening [13
]. In particular, regarding routine laboratories with large numbers of ANCA determinations, automated interpretation would overcome shortcomings of IIF such as high level of manual work and exceeding data management [13
Several studies have confirmed the usefulness of automated IIF systems for objective ANA pattern interpretations recently, providing the basis for the employment of IIF as gold standard for ANA testing as required by the American College of Rheumatology even under differing routine conditions [13
In contrast to ANA detection on HEp-2 cells, polymorphonuclear granulocytes are characterized by varying shapes of the nucleus, which is usually lobed into three segments. Therefore, algorithms for identification of granulocyte staining patterns proved to be more complex compared to those for HEp-2 cells. Furthermore, ethN and formN show different boundary characteristics with DAPI staining. Consequently, the type of fixation needs to be taken into account assessing signal intensity and pattern classification to adapt the algorithms to the changed morphology.
Due to polymorphic nucleus and cytoplasmic ANCA staining patterns, signal assessment and pattern classification cannot be performed simply inside or outside of the DAPI positive area as described for ANA pattern detection [12
]. Cytoplasmic and perinuclear ANCA patterns need to be detected and identified in border regions of the nucleus, rendering detection of ANCA more complex and challenging for automation.
As a fact, our study did not establish a significant difference between automated and visual ANCA IIF detection investigating patients with AAV and disease controls. Furthermore, the positive/negative discrimination and ANCA pattern assessment demonstrated a very good agreement for ethN and formN, confirming the usefulness of pattern recognition algorithms for the automated interpretation of IIF patterns. In contrast to the work of Melegari et al. reporting a very good consistency for positive/negative discrimination by automated and visual interpretation in a review, in this study ANCA patterns were also analyzed on neutrophils with two different fixation methods.
Moreover, this is the first study on ANCA IIF pattern reading, reporting quantitative data for interassay variability. As shown for anti-dsDNA and ANA detection by IIF recently, this approach allows determining a functional assay sensitivity giving the lowest fluorescence intensity with an interassay variability of equal or less than 20% [15
]. This creates the opportunity for a standardized cutoff determination improving the test accuracy for borderline samples and might provide a better reporting of ANCA IIF results to clinicians in order to support the progress in the treatment of AAV [33
]. Relying on quantitative data for ANCA IIF reading may even give the opportunity to report findings for different test-result intervals as proposed recently for ANCA testing by ELISA [34