Comparison of the effect of reporting cytoplasmic patterns as anti-nuclear antibody positive and anti-nuclear antibody negative on reflex test ordering

Abstract

Objective Anti-nuclear antibody (ANA) patterns are classified as nuclear, cytoplasmic or mitotic. The International Consensus on ANA patterns (ICAP) suggests three parameters for ANA reporting – assay type, results and advice for reflex testing – but has not yet reached a consensus on the reporting of cytoplasmic/mitotic patterns as ANA-negative or positive. We investigated the effect of ICAP’s two proposals for reporting cytoplasmic patterns as ANA-positive and negative with a view to the recommendation for reflex testing in a country that has no national reimbursement policies for automatic reflex testing.

Methods This non-interventional descriptive study included 1241 patients with positive cytoplasmic ANA patterns. 442 patients were reported as ANA-negative and 799 as ANA-positive. Patients were followed up for a two-year period to determine testing recommendations based upon nuclear and cytoplasmic patterns. For statistical analysis, the t-test was used, with a significance threshold of p-value<0.05.

Results Appropriate reflex orders were seen more commonly with cytoplasmic patterns reported as ANA-positive (27.30%) than with those reported as ANA-negative (5.51%, p-value<0.05). However, ANA-positive reports led to higher ordering of nuclear pattern reflex tests (12.97%) compared with ANA-negative reports (1.10%, p-value<0.05). A large group of patients (59.73% ANA-positive, 93.39% ANA-negative) did not receive reflex testing.

Conclusion Reporting cytoplasmic patterns as ANA-positive was considered more significant, but reading the result report without considering the pattern and recommendation notes could lead to inappropriate reflex testing. Besides reaching a consensus for reporting cytoplasmic patterns as ANA-negative or positive, it is important to consider solutions to reimbursement policies for automatic reflex testing to decrease the impediments in reporting cytoplasmic ANA patterns.

Zusammenfassung

Hintergrund Antinukleäre Antikörper (ANA) werden als nukleäre, zytoplasmatische oder mitotische Muster klassifiziert. Der internationale Konsens zur ANA-Bestimmung (ICAP) schlägt drei Parameter für die ANA-Berichterstattung vor – die Art des Tests, dessen Ergebnisse und Empfehlungen für Reflextests. Für die Meldung zytoplasmatischer/mitotischer Muster als ANA-negativ oder -positiv wurde jedoch noch kein Konsens erzielt. Wir untersuchten die Auswirkungen der beiden Vorschläge des ICAP für die Meldung von zytoplasmatischen Mustern als ANA-positiv und -negativ auf die Empfehlung für einen Reflextest in einem Land, das über keine nationalen Erstattungsrichtlinien für automatische Reflextests verfügt.

Methoden Diese nicht-interventionelle deskriptive Studie umfasste 1241 Patienten mit positiven zytoplasmatischen ANA-Mustern. 442 Patienten wurden als ANA-negativ eingestuft und 799 als ANA-positiv. Zur Ermittlung der Testempfehlungen auf Grundlage der nukleären und zytoplasmatischen Muster wurden die Patienten über einen Zeitraum von zwei Jahren nachbeobachtet. Für die statistische Analyse wurde der t-Test mit einer Signifikanzschwelle von p-Wert<0,05 angewendet.

Ergebnisse Ordnungsgemäße Anordnungen von Reflextests wurden häufiger bei zytoplasmatischen Mustern gesehen, die als ANA-positiv angezeigt wurden (27,30%) als bei negativen (5,51%, p-Wert<0,05). Bei ANA-positiven Befunden wurde jedoch häufiger ein Kernmuster-Reflextest angeordnet (12,97%) als bei ANA-negativen Befunden (1,10%, p-Wert<0,05). Bei einer großen Gruppe von Patienten (59,73% ANA-positiv, 93,39% ANA-negativ) wurden keine Reflextests angeordnet.

Schlussfolgerung Die Meldung von zytoplasmatischen Mustern als ANA-positiv wurde als aussagekräftiger erachtet. Das Lesen des Ergebnisberichts ohne Berücksichtigung des Musters und der Empfehlungshinweise könnte jedoch zu unangebrachten Reflextests führen. Neben der Erzielung eines Konsenses zur Meldung zytoplasmatischer Muster als ANA-negativ oder -positiv ist es wichtig, Lösungen für die Erstattungsrichtlinien für automatische Reflextests zu prüfen, um die Hindernisse bei der Meldung zytoplasmatischer ANA-Muster zu verringern.

Key words

cytoplasmic ANA pattern - reflex testing - anti-mitochondrial antibody - autoimmunity - anti-nuclear antibody

Schlüsselwörter

Antinukleäre Antikörper - zytoplasmatisches ANA-Muster - Reflextest - antimitochondrialer Antikörper - Autoimmunität

Publication History

Article published online:
19 September 2022

• References

• 1 Agmon-Levin N, Damoiseaux J, Kallenberg C. et al. International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies. Ann Rheum Dis 2014; 73: 17-23
• 2 Damoiseaux J, Mühlen C, Torre I. et al. International consensus on ANA patterns (ICAP): the bumpy road towards a consensus on reporting ANA results. Autoimmune Highlights 2016; 7: 1-8
• 3 Solomon DH, Kavanaugh AJ, Schur PH. Evidence-based guidelines for the use of immunologic tests: antinuclear antibody testing. Arthritis Rheum 2002; 47: 434-444
• 4 Chan E.K.L, Damoiseaux J. de Melo Cruvinel, et al. Report on the second International Consensus on ANA Pattern (ICAP) workshop in Dresden 2015. Lupus. 2016; 25: 797-804
• 5 Meroni PL, Schur PH. ANA screening: an old test with new recommendations. Ann Rheum Dis 2010; 69: 1420-1422
• 6 Scholz J, Grosmann K, Knütter I. et al. Second generation analysis of antinuclearantibody (ANA) by combination of screening and confirmatory testing. Clin Chem Lab Med 2015; 53: 1991-2002
• 7 Bonaguri C, Melegari A, Ballabio A. et al. Italian multicentre study for application of a diagnostic algorithm in autoantibody testing for autoimmune rheumatic disease: conclusive results. Autoimmun Rev 2011; 11: 1-5
• 8 Rigon A, Infantino M, Merone M. et al The inter-observer reading variability in anti-nuclear antibodies indirect (ANA) immunofluorescence test: A multicenter evaluation and a review of the literature. Clin Chem Lab Med 2015; 53: 1991-2002
• 9 Bizzaro N, Tozzoli R, Tonutti E. et al. Variability between methods to determine ANA, anti-dsDNA and anti-ENA autoantibodies: a collaborative study with the biomedical industry. J Immunol Methods 1998; 219: 99-107
• 10 Song L, Hennink E, Young IT. et al. Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy. Biophys J 1995; 68: 2588-2600
• 11 Kavanaugh A, Tomar R, Reveille J. et al. American College of Pathologists. Guidelines for clinical use of the antinuclear antibody test and tests for specific autoantibodies to nuclear antigens. Arch Pathol Lab Med 2000; 124: 71-81
• 12 Copple SS, Giles SR, Jaskowski TD. et al. Screening for IgG antinuclear autoantibodies by HEp-2 indirect fluorescent antibody assays and the need for standardization. Am J Clin Pathol 2012; 137: 825-830
• 13 Tonutti E, Bassetti D, Piazza A. et al. Diagnostic accuracy of ELISA methods as an alternative screening test to indirect immunofluorescence for the detection of antinuclear antibodies. Evaluation of Five Commercial Kits. Autoimmunity. 2004; 37: 171-176
• 14 Tan EM, Smolen JS, McDougal JS. et al. A critical evaluation of enzyme immunoassays for detection of antinuclear autoantibodies of defined specificities. I. Precision, sensitivity, and specificity. Arthritis Rheum 1999; 42: 455-464
• 15 Satoh M, Chan EK, Ho LA. et al. Prevalence and sociodemographic correlates of antinuclear antibodies in the United States. Arthritis Rheum 2012; 64: 2319-2327
• 16 Meroni PL, Chan EK, Damoiseaux J. et al. Unending story of the indirect immunofluorescence assay on HEp-2 cells: old problems and new solutions? Ann Rheum Dis. published online April 17, 2018
• 17 Tonutti E, Bizzaro N, Morozzi G. et al. The ANA-reflex test as a model for improving clinical appropriateness in autoimmune diagnostics. Autoimmun Highlights. 2016; 7: 9
• 18 Bizzaro N, Morozzi G. A proposed model for effective collaboration between rheumatologists and clinical pathologists for the diagnosis of autoimmune rheumatic diseases. Rheumatol Int 2009; 29: 849-851
• 19 Damoiseaux J, Agmon-Levin N, Van BM. et al. From ANA-screening to antigen-specificity: an EASI-survey on the daily practice in European countries. Clin Exp Rheumatol 2014; 32: 539-546
• 20 Mariz HA, Sato EI, Barbosa SH. et al. Pattern on the antinuclear antibody-HEp-2 test is a critical parameter for discriminating antinuclear antibody-positive healthy individuals and patients with autoimmune rheumatic diseases. Arthritis Rheum 2011; 63: 191-200
• 21 Fabris M, Zago S, Tosolini R. et al. Anti-DFS70 antibodies: a useful biomarker in a pediatric case with suspected autoimmune disease. Pediatrics. 2014; 134: 706-708
• 22 Infantino M, Meacci F, Grossi V. et al. The burden of the variability introduced by the HEp-2 assay kit and the CAD system in ANA indirect immunofluorescent test. Immunol Res 2017; 65: 345-354
• 23 Petri M, Orbai AM, Alarcón GS. et al. Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum 2012; 64: 2677-2686
• 24 Shiboski SC, Shiboski CH, Criswell L. et al. American College of Rheumatology classification criteria for Sjogren’s syndrome: a data-driven, expert consensus approach in the Sjögren’s International Collaborative Clinical Alliance cohort. Arthritis Care Res 2012; 64: 475-487
• 25 Amigues JM, Cantagrel A, Abbal M. et al. Comparativestudy of 4 diagnosis criteria sets for mixed connectivetissue disease in patients with anti-RNP antibodies. AutoimmunityGroup of the Hospitals of Toulouse. J Rheumatol 1996; 23: 2055-2062
• 26 Van den Hoogen F, Khanna D, Fransen J. et al. 2013 classification criteria for systemic sclerosis: an American College of Rheumatology/European League against Rheumatism collaborative initiative. Arthritis Rheum 2013; 65: 2737-2747
• 27 Meroni PL, Biggioggero M, Pierangeli SS. et al. Standardization of autoantibody testing: aparadigm for serology in rheumatic diseases. Nat Rev Rheumatol 2014; 10: 35-43
• 28 Alvarez F, Berg PA, Bianchi FB. et al. International Autoimmune HepatitisGroup Report: review of criteria for diagnosis of autoimmunehepatitis. J Hepatol 1999; 31: 929-938
• 29 Hennes EM, Zeniya M, Czaja AJ. et al. Simplified criteria for the diagnosis of autoimmune hepatitis. Hepatology. 2008; 48: 169-176
• 30 Liberal R, Grant CR, Longhi MS. et al. Diagnostic criteria of autoimmune hepatitis. Autoimmun Rev 2014; 13: 435-440
• 31 Van Hoovels L, Broeders S, Chan EL. et al. Current laboratory and clinical practices in reporting and interpreting anti-nuclear antibody indirect immunofuorescence (ANA IIF) patterns: results of an international survey. Autoimmun Highlights 2020; 11: 17
• 32 Von Mühlen CA, La Torre IG, Infantino M. How to report the antinuclear antibodies (anti-cell antibodies) test on HEp-2 cells: guidelines from the ICAP initiative. Immunologic Research 2021; 69: 594-608