Abstract
Objectives
Currently available computer-aided diagnosis (CAD) systems for the detection of anti-nuclear antibodies (ANA) by indirect immunofluorescence (IIF) assay enable a standardized measurement of system-specific fluorescent intensity (FI) measures. We aimed to evaluate an internal quality control (iQC) program that controls the total ANA IIF process in routine practice.
Methods
In addition to the kit iQC materials, supplemental quality indicators were integrated in a total quality assurance (QA) program: patient-derived iQC’s samples (negative, 1/160 fine speckled and 1/160 homogeneous), median sample FI per run and percentage of ANA IIF positive samples per run. Analytical rejection criteria were based on the imprecision of the positivity index (PI) measure of the Zenit PRO system (Menarini). Clinical rejection criteria were based on changes in FI that correspond to a change in ANA IIF titer of ≥2. To evaluate the QA program, different artificial errors were introduced during the ANA IIF process. After every run, quality indicators were evaluated and compared to the pre-set target values.
Results
Rescanning the ANA IIF slides five times, using an old conjugate and a needle obstruction resulted in analytically and even clinically relevant errors in ANA IIF results. All errors were correctly detected by the different defined quality indicators. Traditional Westgard rules, including analytically (and clinically) defined rejection limits were useful in monitoring quality indicators.
Conclusions
The integration of a total process iQC program in CAD systems, based on the specific FI measurands and performance criteria of the system, adds value to QA.
Acknowledgments
We gratefully acknowledge lab technicians of OLV Hospital Aalst for their practical support and the colleagues of ASZ Aalst for the preliminary testing of the ANA IIF samples on their Zenit PRO instrument.
Research funding: None declared.
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Competing interests: Heidi de Baere, Daria Franceschi and Emiliano Tosi are employees of A. Menarini Diagnostics; Marco Meoni is an employee of Visia Lab Srl. All other authors state no conflict of interest.
Ethical approval: The study was performed according to the Declaration of Helsinki and approved by the local ethical committee of OLV Hospital Aalst (study registration number B126201942365).
References
1. Meroni, PL, Schur, PH. ANA screening: an old test with new recommendations. Ann Rheum Dis 2010;69:1420–2. https://doi.org/10.1136/ard.2009.127100.Search in Google Scholar PubMed
2. Damoiseaux, J, von Mühlen, CA, Garcia-De La Torre, I, Carballo, OG, de Melo Cruvinel, W, Francescantonio, PL, et al.. International consensus on ANA patterns (ICAP): the bumpy road towards a consensus on reporting ANA results. Auto Immun Highlights 2016;7:1. https://doi.org/10.1007/s13317-016-0075-0.Search in Google Scholar PubMed PubMed Central
3. Damoiseaux, J, Andrade, LEC, Carballo, OG, Conrad, K, Francescantonio, PLC, Fritzler, MJ, et al.. Clinical relevance of HEp-2 indirect immunofluorescent patterns: the International Consensus on ANA patterns (ICAP) perspective. Ann Rheum Dis 2019;78:879–89. https://doi.org/10.1136/annrheumdis-2018-214436.Search in Google Scholar PubMed PubMed Central
4. Andrade, LEC, Klotz, W, Herold, M, Conrad, K, Rönnelid, J, Fritzler, MJ, et al.. International consensus on antinuclear antibody patterns: definition of the AC-29 pattern associated with antibodies to DNA topoisomerase I. Clin Chem Lab Med 2018;56:1783–8. https://doi.org/10.1515/cclm-2018-0188.Search in Google Scholar PubMed
5. Oyaert, M, Bossuyt, X, Ravelingien, I, Van Hoovels, L. Added value of indirect immunofluorescence intensity of automated antinuclear antibody testing in a secondary hospital setting. Clin Chem Lab Med 2016;54:e63–6. https://doi.org/10.1515/cclm-2015-0887.Search in Google Scholar PubMed
6. Schouwers, S, Bonnet, M, Verschueren, P, Westhovens, R, Blockmans, D, Mariën, G, et al.. Value-added reporting of antinuclear antibody testing by automated indirect immunofluorescence analysis. Clin Chem Lab Med 2014;52:547–51. https://doi.org/10.1515/cclm-2013-0610.Search in Google Scholar PubMed
7. Bossuyt, X, Cooreman, S, De Baere, H, Verschueren, P, Westhovens, R, Blockmans, D, et al.. Detection of antinuclear antibodies by automated indirect immunofluorescence analysis. Clin Chim Acta 2013;415:101–6. https://doi.org/10.1016/j.cca.2012.09.021.Search in Google Scholar PubMed
8. Vulsteke, JB, Van Hoovels, L, Willems, P, Vander Cruyssen, B, Vanderschueren, S, Westhovens, R, et al.. Titre-specific positive predictive value of antinuclear antibody patterns. Ann Rheum Dis 2019. https://doi.org/10.1136/annrheumdis-2019-216245.Search in Google Scholar PubMed
9. Tozzoli, R, Bonaguri, C, Melegari, A, Antico, A, Bassetti, D, Bizzaro, N. Current state of diagnostic technologies in the autoimmunology laboratory. Clin Chem Lab Med 2013;51:129–38. https://doi.org/10.1515/cclm-2012-0191.Search in Google Scholar PubMed
10. Bizzaro, N, Antico, A, Platzgummer, S, Tonutti, E, Bassetti, D, Pesente, F, et al.. Automated antinuclear immunofluorescence antibody screening: a comparative study of six computer-aided diagnostic systems. Autoimmun Rev 2014;13:292–8. https://doi.org/10.1016/j.autrev.2013.10.015.Search in Google Scholar PubMed
11. Rigon, A, Infantino, M, Merone, M, Iannello, G, Tincani, A, Cavazzana, I, et al.. The inter-observer reading variability in anti-nuclear antibodies indirect (ANA) immunofluorescence test: a multicenter evaluation and a review of the literature. Autoimmun Rev 2017;16:1224–9. https://doi.org/10.1016/j.autrev.2017.10.006.Search in Google Scholar PubMed
12. Copple, SS, Giles, SR, Jaskowski, TD, Gardiner, AE, Wilson, AM, Hill, HR. Screening for IgG antinuclear autoantibodies by HEp-2 indirect fluorescent antibody assays and the need for standardization. Am J Clin Pathol 2012;137:825–30. https://doi.org/10.1309/ajcpicnfg7uces1s.Search in Google Scholar PubMed
13. Infantino, M, Meacci, F, Grossi, V, Manfredi, M, Benucci, M, Merone, M, et al.. The burden of the variability introduced by the HEp-2 assay kit and the CAD system in ANA indirect immunofluorescence test. Immunol Res 2017;65:345–54. https://doi.org/10.1007/s12026-016-8845-3.Search in Google Scholar PubMed
14. Mahler, M, Meroni, PL, Bossuyt, X, Fritzler, MJ. Current concepts and future directions for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies. J Immunol Res 2014;2014:315179. https://doi.org/10.1155/2014/315179.Search in Google Scholar PubMed PubMed Central
15. Tozzoli, R, Villalta, D, Bizzaro, N. Challenges in the standardization of autoantibody testing: a comprehensive review. Clin Rev Allergy Immunol 2017;53:68–77. https://doi.org/10.1007/s12016-016-8579-y.Search in Google Scholar PubMed
16. Tozzoli, R, Bizzaro, N. The clinical and the laboratory autoimmunologist: where do we stand? Auto Immun Highlights 2020;11:10. https://doi.org/10.1186/s13317-020-00133-1.Search in Google Scholar PubMed PubMed Central
17. Krause, C, Ens, K, Fechner, K, Voigt, J, Fraune, J, Rohwäder, E, et al.. EUROPattern Suite technology for computer-aided immunofluorescence microscopy in autoantibody diagnostics. Lupus 2015;24:516–29. https://doi.org/10.1177/0961203314559635.Search in Google Scholar PubMed
18. van Beers JJBC, Hahn, M, Fraune, J, Mallet, K, Krause, C, Hormann, W, et al.. Performance analysis of automated evaluation of antinuclear antibody indirect immunofluorescent tests in a routine setting. Auto Immun Highlights 2018;9:8. https://doi.org/10.1007/s13317-018-0108-y.Search in Google Scholar PubMed PubMed Central
19. Copple, SS, Jaskowski, TD, Giles, R, Hill, HR. Interpretation of ANA indirect immunofluorescence test outside the darkroom using NOVA view compared to manual microscopy. J Immunol Res 2014;2014:149316. https://doi.org/10.1155/2014/149316.Search in Google Scholar PubMed PubMed Central
20. Tozzoli, R, Antico, A, Porcelli, B, Bassetti, D. Automation in indirect immunofluorescence testing: a new step in the evolution of the autoimmunology laboratory. Auto Immun Highlights 2012;3:59–65. https://doi.org/10.1007/s13317-012-0035-2.Search in Google Scholar PubMed PubMed Central
21. Picchioni, D, Nencini, F, Franceschi, D. Zenit PRO: towards total automation and IIF standardization. Autoimmun Close Up 2017;4:6–14.Search in Google Scholar
22. Van Hoovels, L, Schouwers, S, Van den Bremt, S, Bogaert, L, Vandeputte, N, Vercammen, M, et al.. Analytical performance of the single well titer function of NOVA View®: good enough to omit ANA IIF titer analysis? Clin Chem Lab Med 2018;56:258–61. https://doi.org/10.1515/cclm-2018-0338.Search in Google Scholar
23. Sack, U, Knoechner, S, Warschkau, H, Pigla, U, Emmrich, F, Kamprad, M. Computer-assisted classification of HEp-2 immunofluorescence patterns in autoimmune diagnostics. Autoimmun Rev 2003;2:298–304. https://doi.org/10.1016/s1568-9972(03)00067-3.Search in Google Scholar
24. Rigon, A, Soda, P, Zennaro, D, Iannello, G, Afeltra, A. Indirect immunofluorescence in autoimmune diseases: assessment of digital images for diagnostic purpose. Cytometry B Clin Cytometry 2007;72:472–7. https://doi.org/10.1002/cyto.b.20356.Search in Google Scholar PubMed
25. Rigon, A, Buzzulini, F, Soda, P, Onofri, L, Arcarese, L, Iannello, G, et al.. Novel opportunities in automated classification of antinuclear antibodies on HEp-2 cells. Autoimmun Rev 2011;10:647–52. https://doi.org/10.1016/j.autrev.2011.04.022.Search in Google Scholar PubMed
26. Van den Bremt, S, Schouwers, S, Van Blerk, M, Van Hoovels, L. ANA IIF automation: moving towards harmonization? Results of a multicenter study. J Immunol Res 2017;2017:6038137. https://doi.org/10.1155/2017/6038137.Search in Google Scholar PubMed PubMed Central
27. Van Hoovels, L, Schouwers, S, Van den Bremt, S, Bossuyt, X. Variation in antinuclear antibody detection by automated indirect immunofluorescence analysis. Ann Rheum Dis 2019;78:e48. https://doi.org/10.1136/annrheumdis-2018-213543.Search in Google Scholar PubMed
28. Infantino, M, Manfredi, M, Soda, P, Merone, M, Afeltra, A, Rigon, A. ANA testing in ’real life’. Ann Rheum Dis 2020;79:e3. https://doi.org/10.1136/annrheumdis-2018-214615.Search in Google Scholar PubMed
29. Meroni, PL, Chan, EK, Damoiseaux, J, Andrade, LEC, Bossuyt, X, Conrad, K, et al.. Unending story of the indirect immunofluorescence assay on HEp-2 cells: old problems and new solutions? Ann Rheum Dis 2019;78:e46. https://doi.org/10.1136/annrheumdis-2018-213440.Search in Google Scholar PubMed
30. Mahler, M. Lack of standardisation of ANA and implications for drug development and precision medicine. Ann Rheum Dis 2019;78:e33. https://doi.org/10.1136/annrheumdis-2018-213374.Search in Google Scholar PubMed
31. Bogaert, L, Van den Bremt, S, Schouwers, S, Bossuyt, X, Van Hoovels, L. Harmonizing by reducing inter-run variability: performance evaluation of a quality assurance program for antinuclear antibody detection by indirect immunofluorescence. Clin Chem Lab Med 2019;57:990–8. https://doi.org/10.1515/cclm-2018-0933.Search in Google Scholar PubMed
32. Maenhout, TM, Bonroy, C, Verfaillie, C, Stove, V, Devreese, K. Automated indirect immunofluorescence microscopy enables the implementation of a quantitative internal quality control system for anti-nuclear antibody analysis. Clin Chem Lab Med 2014;52:989–98. https://doi.org/10.1515/cclm-2013-0912.Search in Google Scholar PubMed
33. Mulliez, SM, Maenhout, TM, Bonroy, C. Impact of the routine implementation of automated indirect immunofluorescence antinuclear antibody analysis: 1 year of experience. Clin Chem Lab Med 2016;54:e183–6. https://doi.org/10.1515/cclm-2015-0900.Search in Google Scholar PubMed
34. Bizzaro, N, Bossuyt, X, Haapala, AM, Shoenfeld, Y, Sack, U. Accreditation in autoimmune diagnostic laboratories. A position paper of the European Autoimmunity Standardisation Initiative (EASI). Autoimmun Rev 2017;16:81–6. https://doi.org/10.1016/j.autrev.2016.09.021.Search in Google Scholar PubMed
35. ISO 15189:2012. Medical laboratories — requirements for quality and competence. Geneva: ISO; 2012.Search in Google Scholar
36. Sack, U, Bossuyt, X, Andreeva, H, Antal-Szalmás, P, Bizzaro, N, Bogdanos, D, et al.. European Autoimmunity Standardisation Initiative. Quality and best practice in medical laboratories: specific requests for autoimmunity testing. Auto Immun Highlights 2020;11:12. https://doi.org/10.1186/s13317-020-00134-0.Search in Google Scholar PubMed PubMed Central
37. Galli, C, Plebani, M. Quality controls for serology: an unfinished agenda. Clin Chem Lab Med 2020;58:1169–70. https://doi.org/10.1515/cclm-2020-0304.Search in Google Scholar PubMed
38. Van Hoovels, L, Bossuyt, X. Harmonisation of laboratory tests for rheumatic diseases: still a long way to go. Ann Rheum Dis 2020;79:e5. https://doi.org/10.1136/annrheumdis-2018-214696.Search in Google Scholar PubMed
39. Rahman, S, Wang, L, Sun, C, Zhou, L. Deep learning based HEp-2 image classification: a comprehensive review. Med Image Anal 2020;65:101764. https://doi.org/10.1016/j.media.2020.101764.Search in Google Scholar PubMed
Supplementary Material
The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2020-1669).
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