Digital immunofluorescence enables automated detection of antinuclear antibody endpoint titers avoiding serial dilution

Abstract

We noticed with great interest the study by Bonroy et al. reporting the evaluation of antinuclear antibody (ANA) detection by an automated interpretation system for indirect immunofluorescence (IIF) images [1]. We would like to comment on their intriguing data regarding the quantification of ANA titer levels based on our experience with the first commercially available automated IIF system AKLIDES ® . We have a long lasting interest in the development of quantification methods for autoantibody assessment by cell-based IIF tests and we generated a software module for the determination of ANA endpoint titers [2,3].With the development of digital IIF reading and novel pattern recognition algorithms, a new era in autoantibody diagnostics by IIF has been ushered in, overcoming such shortcomings like human bias in IIF reading and laborious data management [2,4]. Quantification of fluorescence signals equals IIF testing with other quantitative methods used in autoimmune diagnostics such as enzyme immunoassays. Thus, automated interpretation of cellbased fluorescence tests has become available addressing the rising demand for the standardized assessment of autoantibodies by IIF in routine laboratories [5][6][7][8]. Indirect IIF on human epidermoid laryngeal carcinoma (HEp-2) cells is the preferred method for the screening of ANA within the reco mmended two-stage diagnostic strategy for systemic autoimmune rheumatic diseases (SARD) [9]. Due to the unsurpassed sensitivity of ANA IIF, this method is the ideal diagnostic tool for the screening stage followed by confirmatory testing with different immunological assay technologies [10].Despite the controversial discussion on the need of quantitative ANA results in clinical practice, the determination of endpoint titers is offered by the majority of routine autoimmune laboratories for the serological diagnosis of SARD. In general, endpoint titers higher than 160 are scored positive and respective sera are recommended to be further diluted for their endpoint titer quantification. Endpoint titers should not to be reported higher than 5120 [9].For the first time in ANA IIF reading, digital IIF offers the opportunity to quantify ANA titers by using just one sample dilution. This decreases the hands-on time for an autoantibody-positive sample dramatically avoiding further dilution steps. Bonroy et al. demonstrated the usefulness of this new approach with a novel commercially available interpretation system for the detection of a probability index (PI) [1]. The authors report a significant correlation of the PI with the classical endpoint titer. However, there appears to be no statistical difference between the PI of positive samples with a classical titer of 320 and those with 640. The same phenomenon was reported for samples with classical titers of 1280 compared to samples with titers higher than 1280 hinting to a non-linear dependency.We fully concur with the author's view that quantification of ANA testing can be achieved with automated IIF reading. According to our experience, ...

Automated antinuclear immunofluorescence antibody analysis is a reliable approach in routine clinical laboratories

“…Our results showed a significant association between the LIU value and the end-point titer at a dilution of 1:80, which was consistent with the report of S. Schouwers et al [17,25]. However, we further proved that the custom cutoff was superior to the preset cutoff in NOVA View (local vs. preset cutoff: 57.4% vs. 43.7%, respectively, p < 0.01) in our laboratory.…”

“…Recently, many studies focused on the comparisons between automatic and manual microscope reading or discrepancies between various computer-aided ANA pattern diagnostic systems [11][12][13][14][15][16]. However, few studies evaluated the reliability of the autotiter evaluation system [17]. Could it really provide a quantitative reading, which was correlated with the end-point titer?…”

Second generation analysis of antinuclear antibody (ANA) by combination of screening and confirmatory testing

“…Indeed, the possibility of quantitative ANA screening and multiplex confirmatory testing has been reported in independent assays, respectively. Our group has pioneered the use of digital fluorescence microscopy in autoAb testing by developing an automated interpretation technique providing standardization of autoAb interpretation by IIF including ANA pattern reading [18][19][20]. Furthermore, we set up a multiplex IIF assay for the simultaneous detection of autoAbs employing fluorescent microbeads with chemically activated surfaces for autoantigen immobilization [21].…”

Anti-Nuclear antibodies: Current concepts and future direction for diagnosing connective tissue disease

“…[21][22][23] The systems differ from each other with respect to the use of DNA-binding counterstains, such as DAPI, the cell substrate used, the throughput, the number of patterns that can be identified, and user friendly Generally, these automated systems are based on a microscope fitted with an automated stage, a CCD digital camera, a LED light source, and software that controls the moving parts and directs image acquisition. All systems perform some kind of fluorescent light intensity measurement and use the results for preliminarily categorization of the samples as positive or negative and for pattern analysis.…”