Suggested Rational Considerations for ANA-IF and ENA-Profile Test Requisition: Clinical Manifestation, Gender, Pattern, and Titer of ANA-IF

Yenny Surjawan, Umi Solekhah Intansari


BACKGROUND: The anti-nuclear antibody immunofluorescence (ANA-IF) test is used for screening of autoantibody presence in patients with suspected autoimmune disease. Positive ANA-IF should be followed-up with extractable nuclear antigens profile (ENA-profile). High ANA-IF sensitivity combined with low ENA-profile sensitivity, and the evolution of ANA-IF requests may result in a higher number of positive ANA-IF but negative ENA-profile. It is necessary to make an objective assessment in determining the conditions in which rational ANA-IF and ENA-profile should be suggested.

METHODS: Data were retrieved retrospectively from the medical records of subjects who performed both ANA-IF and ENA-profile. ANA-IF were examined using immunofluorescence principle with cut-off 1:100. ENA-profile which contained sixteen purified antigens was performed using line-immunoblot principle. Data was analyzed descriptively and analytically using SPSS, and significant result was indicated if p<0.05.

RESULTS: The ANA-IF result was dominated by negative (44.9%) and positive-speckled, titer 1:100 (32.9%). Of 923 subjects with positive ANA-IF, 45.4% had a negative ENA-profile. Of 751 subjects with negative ANA-IF, 10.2% had positive ENA-profile. In subjects whose specific clinical entity, the ANA-IF sensitivity and negative predictive value (NPV) in detecting ENA-profile were 93.8% and 93.3%, respectively, but the positive predictive value (PPV) was 63.2%. Women with specific autoimmune manifestation accompanied by ANA-IF homogeneous ≥1:100, or centromeres ≥1:100, or speckled ≥1:320 might have been predicted as subsequent positive ENA-profile with area under curve (AUC) of 77.2%, 76.9%, 79.2%, respectively.

CONCLUSION: ANA-IF should only be indicated for those with specific clinical symptoms. For woman with typical symptoms, the presence of positive ANA-IF with homogeneous ≥1:100, or centromeres ≥1:100, or speckled ≥1:320 should be further followed-up by ENA-profile.

KEYWORDS: ANA-IF, ENA-profile, autoimmune, autoantibody

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Andrade LEC, Damoiseaux J, Vergani D, Fritzler MJ. Antinuclear antibody (ANA) as a criterion for classification and diagnosis of systemic autoimmune diseases. J Transl Autoimmun 2022; 5: 100145, CrossRef.

UpToDate [Internet]. Clinical significance of antinuclear antibody staining patterns and associated autoantibodies [update 2022 Sept 28; cited 2023 Jan 17]. Available from:

Chan EKL, von Muhlen CA, Fritzler MJ, Damoiseaux J, Infantino M, Klotz W, et al. The International Consensus on ANA Patterns (ICAP) in 2021 – The 6th Workshop and Current Perspectives. J Appl Lab Med 2022; 7(1): 322-30, CrossRef.

Didier K, Bolko L, Giusti D, Toquet S, Robbins A, Antonicelli F, et al. Autoantibodies associated with connective tissue disease: what meaning for clinicians? Front Immunol 2018; 9: 541, CrossRef.

Suurmond J, Diamond B. Autoantibodies in systemic autoimmune diseases: specificity and pathogenicity. J Clin Invest 2015; 125(6): 2194-202, CrossRef.

Ventura JI, Lopez-Hoyos M. The past, present, and future in antinuclear antibodies (ANA). Diagnostics. 2022; 12: 647, CrossRef.

Almagro RM, Gutierrez JF, Martin-Martinez MA, Valls MJR, Valera CA, de la Iglesia Salgado JL. Association between antinuclear antibodies titers and connective tissue diseases in a rheumatology department. Rheumatol Clin. 2017; 13(3): 150-5, CrossRef.

Jang J, Kim S, Kim HS, Lee KA, Park J, Park Y. Comparison of antinuclear antibody profiles obtained using line immunoassay and fluorescence enzyme immunoassay. J Int Med Res. 2021: 49(6): 3000605211014390, CrossRef.

Angum F, Khan T, Kaler J, Siddiqui L, Hussain A. The prevalence of autoimmune disorders in women: A narrative review. Cureus. 2020; 12(5): e8094, CrossRef.

Euroimmun. Package Insert IIFT: ANA Mosaic 1A EUROPattern, Test Instruction for the Indirect Immunofluorescence Test from Euroimmun. Lubeck: Euroimmun; 2019, article.

International Consensus on ANA Patterns (ICAP) [Internet]. ANA Patterns - Nuclear Patterns [cited 2023 Jan 18]. Available from:

Euroimmun. Package Insert EUROLINE ANA Profile 3 plus DFS70 (IgG). Test Instruction from Euroimmun. Lubeck: Euroimmun; 2019.

Kaur J, Sing A. Comparative analysis of line immunoassay with immunofluorescence assay for the identification of autoantibodies in patients with suspected systemic autoimmune disorders: A cross-sectional study. J Clin Diagnostic Res. 2022; 16(6): 17-22, CrossRef.

Garg S, Srivastava A, Prasad S. Correlation of line immuno assay with indirect immunofluorescence assay for the detection of anti-nuclear antibodies in various autoimmune disorders. J Autoimmune Disord. 2017; 3(37): 1-7, CrossRef.

Chauhan R, Jain D, Dorwal P, Roy G, Raina V, Nandi SP. The incidence of immunofluorescence patterns and specific autoantibodies observed in autoimmune patients in a tertiary care centre. Eur Ann Allergy Clin Immunol 2019: 51(4): 165-73, CrossRef.

Petchiappan V, Guhan A, Selvam S, Nagaprabu VN. ANA immunofluorescence versus profile – how well they perform in autoimmune diseases: An analysis of their clinical utility in a tertiary care centre. Int J Res Med Sci. 2018; 6(9): 3140-6, CrossRef.

Imran K, Loya A, Hameed M, Siddiqui IA, Sheikh UN. The frequency of immunofluorescence antinuclear antibody patterns and extractable nuclear antigen: Experience from a large laboratory in Pakistan. Cureus. 2023; 15(1): e33343, CrossRef.

Sharmin S, Ahmed S, Abu Saleh A, Rahman F, Choudhury MR, Hassan MM. Association of immunofluorescence pattern of antinuclear antibody with specific autoantibodies in the Bangladesh population. Bangladesh Med Res Counc Bull. 2014; 40(2): 74-8, CrossRef.

Alsubki R, Tabassum H, Alfawaz H, Alaqil R, Aljaser F, Ansar S, et al. Association between antinuclear antibodies (ANA) patterns and extractable nuclear antigens (ENA) in Hep-2 cells in patients with autoimmune disease in Riyadh, Saudi Arabia. Intractable Rare Dis Res. 2020; 9(2):89-94, CrossRef.

Grygiel-Gomiak B, Rogacka N, Puszczewicz. Antinuclear antibodies in healthy people and non-rhemautic diseases – diagnostic and clinical implications. Rheumatologia. 2018: 56(4): 243-8, CrossRef.

Hamijoyo L, Candrianita S, Rini IA, Sutedja E, Setiabudiawan B, Sahiratmadja R. N-Acetyltrasnferase 2 (NAT-2) acetylator status among systemic lupus erythematosus patients from a tuberculosis endemic area in Bandung, Indonesia. Indones Biomed J 2019; 11(2): 175-81, CrossRef.

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, CrossRef.

Jury EC, Morrow WJW. Autoantibodies and overlap syndromes in autoimmune rheumatic disease. J Clin Path 2001; 54(5): 340-7, CrossRef.

Marin GG, Cardiel MH, Cornejo H, Viveros ME. Prevalence of antinuclear antibodies in 3 groups of healthy individuals: blood donors, hospital personnel, and relatives of patients with autoimmune diseases. J Clin Rheumatol. 2009; 15(7): 325-9, CrossRef.

Ludwig RJ, Vanhoorelbeke K, Leypoldt F, Kaya Z, Bieber K, McLachlan SM, et al. Mechanisms of autoantibody-induced pathology. Front Immunol. 2017; 8: 603, CrossRef.

Rodsaward P, Chottawornsak N, Suwanchote S, Rachayon M, Deekajorndech T, Wright HL, et al. The clinical significance of antinuclear antibodies and specific autoantibodies in juvenile and adult systemic lupus erythematosus patients. Asian Pac J Allergy Immunol. 2021; 39: 4, CrossRef.


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