Consensus Group Determines Clinical Relevance of HEp-2 Indirect Immunofluorescent Patterns

The International Consensus on Antinuclear Antibody Patterns (ICAP) initiative has reached consensus regarding the clinical relevance of 29 distinct patterns of indirect immunofluorescence assay (IIFA) on HEp-2 cells. The consensus paper has been published in Annals of the Rheumatic Diseases.¹

HEp-2 IIFAs are commonly used for antinuclear antibody (ANA) detection, the outcomes of which are integrated in diagnostic and classification criteria for many systemic autoimmune diseases. The HEp-2 IIFA test can also provide fluorescence patterns that may be clinically relevant for disease classification.

To organize the names and descriptions of HEp-2 IIFA patterns, an ordered classification taxonomy was proposed. The ICAP initiative has previously reached consensus on the definitions of these patterns.²

A summary of the clinical relevance for the HEp-2 IIFA patterns is as follows:

Nuclear HEp-2 IIFA Patterns

• AC-1: Found in patients with systemic lupus erythematosus (SLE), chronic autoimmune hepatitis, or juvenile idiopathic arthritis (JIA). Follow-up testing for anti-dsDNA antibodies is suggested if SLE is suspected; it is not recommended if hepatitis or JIA is suspected because the respective autoantigens revealing the AC-1 pattern are not clearly defined.
• AC-2: Found as high titer HEp-2 IIFA-positive in healthy individuals or in those who do not have a systemic autoimmune rheumatic disease. The negative association with a rheumatic disease is valid only if autoreactivity is confirmed as being directed to DFS70 and if no other common extractable nuclear antigen (ENA) is recognized. This pattern may be caused by autoantibodies to other antigens than DFS70.
• AC-3: Found in patients with limited cutaneous systemic sclerosis (SSc), and, in combination with Raynaud phenomenon, is prognostic for onset of limited cutaneous SSc. AC-3 is associated with CENP-B antibodies. Confirmation by an antigen-specific immunoassay is recommended to support the association with limited cutaneous SSc.
• AC-4: Found in various rheumatic diseases, including Sjogren syndrome, SLE, subacute cutaneous lupus erythematosus, neonatal lupus, congenital heart block, SSc, and SSc-autoimmune myopathy (AIM) overlap syndrome, and dermatomyositis.
  • Follow-up tests for anti-SS-A/Ro and anti-SS-B/La antibodies are recommended for Sjogren syndrome, SLE, subacute lupus erythematosus, neonatal lupus, or congenital heart block.
  • Follow-up tests for Mi-2, TIF1-y, and Ku antibodies are recommended for SSc-AIM overlap syndrome.
• AC-5: Found in various rheumatic diseases, including SLE, SSc, SSc-autoimmune myopathy overlap syndrome, mixed or undifferentiated connective tissue disease (CTD).
  • Follow-up tests for anti-Sm and anti-U1RNP antibodies are recommended for SLE.
  • Follow-up tests for anti-RNApol III antibodies are recommended for SSc.
  • Follow-up tests for anti-U1RNP antibodies are recommended for mixed CTD.
  • Follow-up tests for anti-U1RNP and anti-Ku antibodies are recommended for SSc-AIM overlap syndrome.
• AC-6: Found in many rheumatic diseases, including primary biliary cholangitis, autoimmune myopathy, and other inflammatory conditions. Follow-up tests for anti-Sp100 are recommended if primary biliary cholangitis is clinically suspected, and follow-up for anti-MJ/NXP-2 antibodies is recommended if dermatomyositis is clinically suspected.
• AC-7: Has low predictive value for any disease. Antigens that are primary localized in the dots include p80-coilin and SMN complex.
• AC-8: Found in patients with SSc, SSc-AIM overlap syndrome, or clinical manifestations of other rheumatic diseases. Follow-up tests for anti-Th/To antibodies are recommended if limited cutaneous SSc is clinically suspected, and testing for anti-PM/Scl antibody reactivity is recommended if SSc-AIM overlap syndrome is suspected. Other identified antigens include B23/nucleophosmin, No55/SC65, and C23/nucleolin, but the significance of these autoantibodies is not clearly defined.
• AC-9: Found in patients with SSc. A follow-up test for anti-U3RNP/fibrillarin antibodies is recommended if SSc is recommended.
• AC-10: Found in various conditions, including SSc, Raynaud phenomenon, Sjogren syndrome, and cancer. If the pattern is observed in the serum of patients with these conditions, follow-up testing for anti-NOR90(hUBF) antibodies is recommended.
  • AC-10 is associated with anti-RNApol I antibodies, but these antibodies almost always coexist with anti-RNApol III antibodies, which reveal the AC-5 pattern. If SSc is clinically suspected, it is recommended to perform a follow-up test for anti-RNApol III antibodies.
• AC-11: Infrequently found in routine autoantibody testing, but described in autoimmune-cytopenias, autoimmune liver diseases, linear scleroderma, antiphospholipid syndrome, and systemic autoimmune rheumatic diseases. Recognized antigens include lamins (A, B, C) and LAP2.
• AC-12: Found in PBC and other autoimmune liver disease and systemic autoimmune rheumatic diseases. A follow-up test for anti-gp210 antibodies is recommended if PBC is suspected. Other recognized antigens include p62 nucleoporin, LBR, and Tpr.
• AC-13: Formerly considered highly specific for SLE, but the specificity has been debated. A follow-up test for anti-PCNA antibodies is recommended is SLE is suspected. Studies with antigen-specific immunoassays have also shown clinical associations with SSc, AIM, rheumatoid arthritis, and hepatitis C.
• AC-14: The majority of sera originate from patients with diverse neoplastic conditions of the breast, lung, colon, lymphoma, ovary, or brain; however, the frequency of this pattern in patients with these malignancies is low. The pattern is also noted in inflammatory conditions, including Crohn disease, autoimmune liver disease, and Sjogren syndrome. Associations are only confirmed if CENP-F reactivity is confirmed in an antigen-specific immunoassay.
• AC-29: This pattern is highly specific for SSc, particularly with diffuse cutaneous SSc and with more aggressive forms of the condition. A follow-up test for anti-Topoisomerase I antibodies is recommended if SSc is suspected.

Cytoplasmic HEp-2 IIFA Patterns

• AC-15: Found in autoimmune hepatitis type 1, chronic hepatitis C, and celiac disease, but rarely found in rheumatic diseases. Clinicians should confirm reactivity with smooth muscle antibodies if autoimmune hepatitis type 1 is suspected.
  
• AC-16: Not typically found in rheumatic diseases. Recognized antigens include cytokeratins 8, 18, & 19, tubulin, and vimentin.
• AC-17: Found very infrequently in a serology diagnostic setting. Recognized antigens include α-actinin and vinculin.
  
• AC-18: Autoantibodies of this pattern have been reported in distinct systemic autoimmune rheumatic diseases, but their prevalence has not been thoroughly studied. Recognized antigens include GW-body antigens and endosomal antigens.
• AC-19: Found in patients with SLE and anti-synthetase syndrome, interstitial lung disease, polyarthritis, and Raynaud phenomenon.
  • Follow-up tests for antibodies to ribosomal P phosphoproteins (P0, P1, P2, C22 RibP peptide) are recommended if SLE is suspected.
  • Follow-up tests for antibodies to tRNA synthetases is recommended if AIM, in particular the anti-synthetase syndrome, is suspected.
  • Follow-up tests for anti-SRP antibodies is recommended if AIM, in particular necrotizing myopathy, is suspected.
• AC-20: Found in anti-synthetase syndrome, interstitial lung disease, polyarthritis, and Raynaud phenomenon. Autoantibodies associated with this pattern are mostly reported for the anti-Jo-1 antibody. However, because AC-20 is not specific for Jo-1, a follow-up test for anti-Jo-1 antibodies is recommended.
• AC-21: Often found in PBC, but also found in SSc, PBC-SSc overlap syndrome, and PBC-Sjogren syndrome. A follow-up test for anti-mitochondrial antibodies is recommended if PBC is suspected. Other recognized antigens include E1α and E1β subunits of PDH, the E3-binding protein of PDH, and the 2-OGDC.
• AC-22: Found in few patients with various conditions. Recognized antigens include giantin/macrogolgin and distinct golgin molecules.
• AC-23: Primarily found in patients with hepatitis C receiving pegylated interferon-α/ribavirin combination therapy; autoantibodies revealing this pattern were undetected prior to treatment.

Mitotic HEp-2 IIFA Patterns

• AC-24: Has low positive predictive value for any disease. This pattern is found in Raynaud phenomenon, localized scleroderma, SSc, SLE, and RA. Recognized antigens include α-enolase, γ-enolase, ninein, Cep-250, Mob1, PCM-1/2, and pericentrin.
• AC-25: Has low positive predictive value for any disease and found very infrequently in a routine serology diagnostic setting. A recognized antigen includes HsEg5.
• AC-26: About half of patients with this pattern have clinical features of a rheumatic disease, including Sjogren syndrome, SLE, undifferentiated CTD, limited SSc, or RA. This pattern has also been reported in patients with organ-specific autoimmune diseases and was reported less frequently in non-autoimmune conditions.
• AC-27: Has low positive predictive value for any disease and found very infrequently in a routine serology diagnostic setting. Recognized antigens include DCA, MCA1, and MCA5.
• AC-28: Has low positive predictive value for any disease and found very infrequently in a routine serology diagnostic setting. Recognized antigens include CENP-E, CENP-F, TD60, MSA36, KIF-14, MKLP-1, MPP1/KIF20B, and INCENP.

References

1. Damoiseaux J, Andrade LEC, Carballo OG, et al. Clinical relevance of HEp-2 indirect immunofluorescent patterns: the International Consensus on ANA patterns (ICAP) perspective [published online March 12, 2019]. Ann Rheum Dis. doi:10.1136/annrheumdis-2018-2144362. Chan EKL,
2. Damoiseaux J, Carballo OG, et al. Report of the first international consensus on standardized nomenclature of antinuclear antibody HEp-2 cell patterns 2014–2015. Front Immunol. 2015;6:412.