Autoimmunity Highlights

, Volume 3, Issue 2, pp 59–65

Automation in indirect immunofluorescence testing: a new step in the evolution of the autoimmunology laboratory

  • Renato Tozzoli
  • Antonio Antico
  • Brunetta Porcelli
  • Danila Bassetti
Review Article


Indirect immunofluorescence (IIF) plays an important role in immunological and immunometric assays for detecting and measuring autoantibodies. This technology was the first multiplex method used to detect cardinal autoantibodies for the diagnosis of autoimmune diseases. Over the last 20 years, research has enabled the progressive identification of cell and tissue autoantigens which are the target of autoantibodies originally detected by IIF. Accordingly, newer immunometric methods, capable of measuring concentrations of specific autoantibodies directed against these autoantigens, allowed for a gradual replacement of the IIF method in the autoimmunology laboratory. Currently, IIF remains the method of choice only in selected fields of autoimmune diagnostics. Following the recent statement by the American College of Rheumatology that the IIF technique should be considered as the standard screening method for the detection of ANA, the biomedical industry has developed technological solutions which significantly improve automation of the procedure, not only in the preparation of substrates and slides, but also in microscope reading. This review summarizes the general and specific features of new available commercial systems (Aklides, Medipan; Nova View, Inova; Zenit G Sight, A. Menarini Diagnostics; Europattern, Euroimmun; Helios, Aesku.Diagnostics; Image Navigator, Immuno Concepts; Cytospot, Autoimmun Diagnostika) for automation of the IIF method. The expected advantages of automated IIF are the reduction in frequency of false negative and false positive results, the reduction of intra- and inter-laboratory variability, the improvement of correlation of staining patterns with corresponding autoantibody reactivities, and higher throughput in the laboratory workflow.


Autoimmune diseases Indirect immunofluorescence Automation Standardization 


  1. 1.
    Tozzoli R, Bonaguri C, Melegari A, Antico A, Bassetti D, Bizzaro N (2012) Current state of diagnostic technologies in the autoimmunology laboratory. Clin Chem Lab Med (in press)Google Scholar
  2. 2.
    Friou GJ (1957) Clinical application of lupus serum-nucleoprotein reaction using fluorescent antibody technique. J Clin Invest 86:890–896Google Scholar
  3. 3.
    Nairn RC, Ghose T, Porteous IB, Urquhart JA (1962) A routine immunofluorescence method for detecting autoantibodies to thyroid colloid. J Clin Pathol 15:594–595PubMedPubMedCentralCrossRefGoogle Scholar
  4. 4.
    Taylor KB, Roitt IM, Doniach D, Couchman KG, Shapland C (1962) Autoimmune phenomena in pernicious anaemia: gastric antibodies. Brit Med J 2:1347–1352PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Beutner EH, Jordon RE (1964) Demonstration of skin antibodies in sera of pemphigus vulgaris patients by immunofluorescent staining. Proc Soc Exp Biol Med 117:505–510PubMedCrossRefGoogle Scholar
  6. 6.
    Walker JG, Doniach D, Roitt IM, Sherlock S (1965) Serological tests in diagnosis of primary biliary cyrrhosis. Lancet 1:827–831PubMedCrossRefGoogle Scholar
  7. 7.
    Johnson GD, Holborow EJ, Glynn LE (1965) Antibody to smooth muscle in patients with liver disease. Lancet 2:878–879PubMedCrossRefGoogle Scholar
  8. 8.
    Irvine WJ, Stewart AG, Scarth L (1967) A clinical and immunological study of adrenal insufficiency (Addison’s disease). Clin Exp Immunol 2:31–70PubMedPubMedCentralGoogle Scholar
  9. 9.
    Anderson JR, Goudie RB, Gray K, Stuart-Smith DA (1968) Immunological features of idiopathic Addison’s disease: an antibody to cells producing steroid hormones. Clin Exp Immunol 3:119–131PubMedPubMedCentralGoogle Scholar
  10. 10.
    Seah PP, Fry L, Rossiter MA, Hoffbrand AV, Holborow EJ (1971) Anti-reticulin antibodies in childhood celiac disease. Lancet 7726:681–682CrossRefGoogle Scholar
  11. 11.
    Rizzetto M, Swana G, Doniach D (1973) Microsomal antibodies in active chronic hepatitis and other disorders. Clin Exp Immunol 15:331–344PubMedPubMedCentralGoogle Scholar
  12. 12.
    Bottazzo GF, Florin-Christiansen A, Doniach D (1974) Islet-cell antibodies in diabetes mellitus with autoimmune polyendocrine deficiencies. Lancet 7892:1279–1283CrossRefGoogle Scholar
  13. 13.
    Bottazzo GF, Pouplard A, Fiorin-Christiansen A, Doniach D (1975) Autoantibodies to prolactin-secreting cells oh human pituitary. Lancet 7925:97–101CrossRefGoogle Scholar
  14. 14.
    Unsworth DJ, Leonard JN, McMinn RM, Swain AF, Holborow EJ, Fry L (1981) Anti-gliadin antibodies and small intestinal mucosal damage in dermatitis herpetiformis. Br J Dermatol 105:653–658PubMedCrossRefGoogle Scholar
  15. 15.
    Chorzelski TP, Beutner EH, Sulej J, Tchorzewska H, Jablonska S, Kumar V et al (1984) IgA anti-endomysium antibody. A new immunological marker of dermatitis herpetiformis and celiac disease. Br J Dermatol 111:395–402PubMedCrossRefGoogle Scholar
  16. 16.
    Wiik A, Hoier-Madsen M, Forslid J, Charles P, Meyrowitsch J (2010) Antinuclear antibodies: a contemporary nomenclature using HEp-2 cells. J Autoimmun 35:276–290PubMedCrossRefGoogle Scholar
  17. 17.
    Tozzoli R (2007) Recent advances in diagnostic technologies and their impact in autoimmune diseases. Autoimmun Rev 6:334–340PubMedCrossRefGoogle Scholar
  18. 18.
    Tozzoli R, Bizzaro N (2012) The clinical autoimmunologost and the laboratory autoimmunologist: the two sides of the coin. Autoimmun Rev (in press)Google Scholar
  19. 19.
    Solomon DH, Kavanaugh AJ, Schur PH (2002) Evidence-based guidelines for the use of immunologic tests: antinuclear antibody testing. Arthritis Rheum 47:434–444PubMedCrossRefGoogle Scholar
  20. 20.
    Tozzoli R, Bizzaro N, Tonutti E, Villalta D, Bassetti D, Manoni F et al (2002) Guidelines for the laboratory use of autoantibody tests in the diagnosis and monitoring of autoimmune rheumatic diseases. Am J Clin Pathol 117:316–324PubMedCrossRefGoogle Scholar
  21. 21.
    Meroni PL, Schur PH (2010) ANA screening: an old test with new recommendations. Ann Rheum Dis 69:1420–1422PubMedCrossRefGoogle Scholar
  22. 22.
    Savige JF, Gillis DF, Benson E, Davies D, Esnault V, Falk RJ et al (1999) International Consensus Statement on testing and reporting of antineutrophil cytoplasmic antibodies (ANCA). Am J Clin Pathol 111:507–513PubMedGoogle Scholar
  23. 23.
    Rigon A, Soda P, Zennaro D, Iannello G, Afeltra A (2007) Indirect immunofluorescence in autoimmune diseases: assessment of digital images for diagnostic purpose. Cytometry B 72B:472–477CrossRefGoogle Scholar
  24. 24.
    Fritzler MJ (2011) The antinuclear antibody test: last or lasting gasp? Arthritis Rheum 63:19–22PubMedCrossRefGoogle Scholar
  25. 25.
    Jaskowski TD, Schroder C, Martins TB, Mouritsen CL, Litwin CM, Hill HR (1996) Screening of antinuclear antibodies by enzyme immunoassay. Am J Clin Pathol 105:468–473PubMedGoogle Scholar
  26. 26.
    Emlen W, O’Neill L (1997) Clinical significance of antinuclear antibodies. Arthritis Rheum 40:1612–1618PubMedCrossRefGoogle Scholar
  27. 27.
    Gniewek RA, Stites DP, McHugh TM, Hilton JF, Nakagawa M (1997) Comparison of antinuclear antibody testing: immunofluorescence assay versus enzyme immunoassay. Clin Diagn Lab Immunol 4:185–188PubMedPubMedCentralGoogle Scholar
  28. 28.
    Homburger HA, Cahen YD, Griffiths J, Jacob GL (1998) Detection of antinuclear antibodies: comparative evaluation of enzyme immunoassay and indirect immunofluorescence methods. Arch Pathol Lab Med 122:993–999PubMedGoogle Scholar
  29. 29.
    Rondeel JM, van Gelder W, van der Leeden H, Dinkelaar RB (1999) Different strategies in the laboratory diagnosis of autoimmune disease: immunofluorescence, enzyme-linked immunosorbent assay or both? Ann Clin Biochem 36:189–195PubMedCrossRefGoogle Scholar
  30. 30.
    Olaussen E, Rekvig OP (1999) Screening tests for antinuclear antibodies: selective use of central nuclear antigens as a rational basis for screening by ELISA. J Autoimmun 13:95–102PubMedCrossRefGoogle Scholar
  31. 31.
    Ulvestad E (2001) Performance characteristics and clinical utility of a hybrid ELISA for detection of ANA. APMIS 109:217–222PubMedCrossRefGoogle Scholar
  32. 32.
    Bernardini S, Infantino M, Bellicampi L, Nuccetelli M, Afeltra A, Lori R et al (2004) Screening of antinuclear antibodies: comparison between enzyme immunoassay based on nuclear homogenates, purified or recombinant antigens and immunofluorescence assay. Clin Chem Lab Med 42:1155–1160PubMedCrossRefGoogle Scholar
  33. 33.
    Tonutti E, Bassetti D, Piazza A, Visentini D, Poletto M, Bassetto F et al (2004) 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 37:171–176CrossRefGoogle Scholar
  34. 34.
    Fenger M, Wiik A, Hoier-Madsen M, Lykkegaard JJ, Rozenfeld T, Hansen MS et al (2004) Detection of antinuclear antibodies by solid-phase immunoassays and immunofluorescence analysis. Clin Chem 50:2141–2147PubMedCrossRefGoogle Scholar
  35. 35.
    Sinclair D, Saas M, Williams D, Hart M, Goswami R (2007) Can an ELISA replace immunofluorescence for the detection of anti-nuclear antibodies? The routine use of anti-nuclear antibody screening ELISAs. Clin Lab 53:183–191PubMedGoogle Scholar
  36. 36.
    Lopez-Hoyos M, Rodriguez-Valverde V, Martinez-Taboada V (2007) Performance of antinuclear antibody connective tissue disease screen. Ann NY Acad Sci 1109:322–329PubMedCrossRefGoogle Scholar
  37. 37.
    Hayashi N, Kawamoto T, Mukai M, Morinobu A, Koshiba M, Kondo S et al (2001) Detection of antinuclear antibodies by use of an enzyme immunoassay with nuclear HEp-2 cell extract and recombinant antigens: comparison with immunofluorescence assay in 307 patients. Clin Chem 47:1649–1659PubMedGoogle Scholar
  38. 38.
    Gonzalez C, Garcia-Berrocal B, Perez J, Navajo JA, Herraez O, González-Buitrago JM (2005) Laboratory screening of connective tissue diseases by a new automated ENA screening assay (EliA Symphony) in clinically defined patients. Clin Chim Acta 359:109–114PubMedCrossRefGoogle Scholar
  39. 39.
    Ghillani P, Rouquette AM, Desgruelles C, Hauguel N, Le Pendeven C, Piette JC et al (2007) Evaluation of the LIAISON ANA screen assay for antinuclear antibody testing in autoimmune diseases. Ann NY Acad Sci 1109:407–413PubMedCrossRefGoogle Scholar
  40. 40.
    Op De Beeck K, Vermeesch P, Verscheueren P, Westhovens R, Marien G, Blockmans D et al (2011) Detection of antinuclear antibodies by indirect immunofluorescence and by solid phase assay. Autoimmun Rev 10:801–808PubMedCrossRefGoogle Scholar
  41. 41.
    Nifli A-P, Notas G, Mamoulaki M, Niniraki M, Ampartzaki V, Theodoropoulos PA et al (2006) Comparison of a multiplex, bead-based fluorescent assay and immunofluorescence methods for the detection of ANA and ANCA autoantibodies in human serum. J Immunol Methods 311:189–197PubMedCrossRefGoogle Scholar
  42. 42.
    Bonilla E, Francis L, Allam F, Ogrinc M, Neupane H, Phillips PE et al (2007) Immunofluorescence microscopy is superior to fluorescent beads for detection of antinuclear antibody reactivity in systemic lupus erythematosus patients. Clin Immunol 124:18–21PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Salamunic I, Paukovic-Sekulic B, Galetovic A (2008) Comparative analysis of multiplex AtheNA multi-lyte ANA test system and conventional laboratory methods to detect autoantibodies. Biochemia Medica 18:88–98CrossRefGoogle Scholar
  44. 44.
    Hanly JG, Thompson K, McCurdy G, Fougere L, Theriault C, Wilton K (2010) Measurement of autoantibodies using multiplex methodology in patients with systemic lupus erythematosus. J Immunol Methods 352:147–152PubMedCrossRefGoogle Scholar
  45. 45.
    Hu Y, Murphy RF (2004) Automated interpretation of subcellular patterns from immunofluorescence microscopy. J Immunol Methods 290:93–105PubMedCrossRefGoogle Scholar
  46. 46.
    Glory EM, Murphy RF (2007) Automated subcellular location determination and high throughput microscopy. Dev Cell 12:7–16PubMedCrossRefGoogle Scholar
  47. 47.
    Rigon A, Buzzulini F, Soda P, Onofri L, Arcarese L, Iannello G et al (2011) Novel opportunities in automated classification of antinuclear antibodies on HEp-2 cells. Autoimmun Rev 10:647–652PubMedCrossRefGoogle Scholar
  48. 48.
    Hiemann R, Buttner T, Krieger T, Roggenbuck D, Sack U, Conrad K (2009) Challenges of automated screening and differentiation of non-organ specific autoantibodies on HEp-2 cells. Autoimmun Rev 9:17–22PubMedCrossRefGoogle Scholar
  49. 49.
    Egerer K, Roggenbuck D, Hiemann R, Weyer MG, Büttner T, Radau B et al (2010) Automated evaluation of autoantibodies on human epithelial-2 cells as an approach to standardize cell-based immunofluorescence tests. Arthritis Res Ther 12:R40PubMedPubMedCentralCrossRefGoogle Scholar
  50. 50.
    Kivity S, Gilburd B, Agmon-Levin N, Garcia Carrasco M, Tzafrir Y, Sofer Y et al (2012) A novel automated indirect immunofluorescence autoantibody evaluation. Clin Rheumatol 31:503–509PubMedCrossRefGoogle Scholar
  51. 51.
    Melegari A, Bonaguri C, Russo A, Luisita B, Trenti T, Lippi G (2012) A comparative study on the reliability of an automated system for the evaluation of cell-based indirect immunofluorescence. Autoimmun Rev (Epub ahead of print)Google Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Renato Tozzoli
    • 1
  • Antonio Antico
    • 2
  • Brunetta Porcelli
    • 3
  • Danila Bassetti
    • 4
  1. 1.Laboratory of Clinical Pathology, Department of Laboratory MedicineS. Maria degli Angeli HospitalPordenoneItaly
  2. 2.Laboratory of Clinical PathologyCity HospitalCittadellaItaly
  3. 3.Laboratory of Clinical Pathology, Department of Internal MedicineUniversity HospitalSienaItaly
  4. 4.Laboratory of Microbiology and Virology, S. Chiara HospitalTrentoItaly

Personalised recommendations