Table 1

Nuclear HEp-2 IIFA patterns

CodeAC pattern—clinical relevanceRefs
AC-1HOMOGENEOUS
  • Found in patients with SLE, chronic autoimmune hepatitis or juvenile idiopathic arthritis

  • If SLE is clinically suspected, it is recommended to perform a follow-up test for anti-dsDNA antibodies, alone or in combination with dsDNA/histone complexes (nucleosomes/chromatin); anti-dsDNA antibodies are included in the classification criteria for SLE

15, 16
  • If chronic autoimmune hepatitis or juvenile idiopathic arthritis is suspected, follow-up testing is not recommended because the respective autoantigens revealing the AC-1 pattern are not completely defined

17
Notes: Although autoantibodies to Topoisomerase I (formerly Scl-70) may be reported as nuclear homogeneous, they typically reveal a composite AC-29 HEp-2 IIFA pattern; as such, clinical suspicion of SSc may warrant follow-up testing for reactivity to this antigen. 14, 18
Although AC-1 is the most prevalent pattern in chronic autoimmune hepatitis, other HEp-2 IIFA patterns may occur, but also for these patterns the autoantigens are not completely defined. 19
AC-2DENSE FINE SPECKLED
  • Commonly found as high titer HEp-2 IIFA-positive in apparently healthy individuals or in patients who do not have a systemic autoimmune rheumatic disease (SARD)

9
  • The negative association with SARD is only valid if the autoreactivity is confirmed as being directed to DFS70 (also known as LEDGF/p75) and if no other common ENA is recognized

20, 21
  • Both in apparently healthy individuals as well as patients who do not have a SARD the AC-2 pattern may be caused by autoantibodies to other antigens than DFS70

22
Note: Confirmatory assays for anti-DFS70 antibodies may be available only in specialty clinical laboratories.
AC-3CENTROMERE (see online supplementary table S1 for further details
  • Commonly found in patients with limited cutaneous SSc, and as such included in the classification criteria for SSc

8, 15, 23
  • In combination with Raynaud phenomenon, the AC-3 pattern is prognostic for onset of limited cutaneous SSc

15, 23
  • Strongly associated with antibodies to CENP-B; especially in case of low titers, confirmation by an antigen-specific immunoassay is recommended to support the association with limited cutaneous SSc; the CENP-B antigen is included in many routine ENA profiles

15
  • The AC-3 pattern is also apparent in a subset of patients with PBC; these patients often have both SSc as well as PBC

15
AC-4FINE SPECKLED
  • Present to a varying degree in distinct SARD, in particular SjS, SLE, subacute cutaneous lupus erythematosus, neonatal lupus erythematosus, congenital heart block, DM, SSc, and SSc-AIM overlap syndrome

15
  • If SjS, SLE, subacute cutaneous lupus erythematosus, neonatal lupus erythymatosus, or congenital heart block is clinically suspected, it is recommended to perform follow-up tests for anti-SS-A/Ro (Ro60) and anti-SS-B/La antibodies; in most laboratories these antigens are included in the routine ENA profile

15
  • Autoantibodies to SS-A/Ro are part of the classification criteria for SjS (the criteria do not distinguish between Ro60 and Ro52/TRIM21)

25
  • If SSc, AIM, or to a lesser extend SLE, is clinically suspected, it is recommended to perform follow-up tests for detecting autoantibodies to Mi-2, TIF1γ, and Ku; these antigens are typically included in disease specific immunoassays (i.e., inflammatory myopathy profile*)

26
  • Autoantibodies to Mi-2 and TIF1γ are associated with DM; autoantibodies to TIF1γ in patients with DM, although rare in the overall AC-4 pattern, is strongly associated with malignancy in old patients

26, 27
  • Autoantibodies to Ku are associated with SSc-AIM and SLE-SSc-AIM overlap syndromes

26
Notes: Anti-SS-A/Ro (Ro60) and AIM-specific autoantibodies may be undetected in HEp-2 IIFA-screening. 28
AC-5LARGE/COARSE SPECKLED (see online supplementary table S1 for further details)
  • Present to a varying degree in distinct SARD, in particular SLE, SSc, MCTD, SSc-AIM overlap syndrome, and UCTD (i.e, patients with rheumatic symptoms without a definite SARD diagnosis)

29
  • If SLE is clinically suspected, it is recommended to perform follow-up tests for anti-Sm and anti-U1RNP antibodies; these antigens are commonly included in the routine ENA profile; anti-Sm antibodies are included in the classification criteria for SLE

16, 30, 31
  • If SSc is clinically suspected, it is recommended to perform a follow-up test for anti-RNApol III antibodies (e.g., SSc profile*); the anti-RNApol III antibodies are included in the classification criteria for SSc

8
  • If MCTD is clinically suspected, it is recommended to perform a follow-up test for anti-U1RNP antibodies; the antigen is commonly included in the routine ENA profile; anti-U1RNP antibodies are included in the diagnostic criteria for MCTD

32
  • If the SSc-AIM overlap syndrome is clinically suspected, it is recommended to perform follow-up tests for anti-U1RNP and anti-Ku antibodies; these antigens are included in the routine ENA profile (U1RNP), or in disease specific immunoassays (Ku, i.e., inflammatory myopathy profile* and SSc profile*)

26, 33
  • In non-SARD individuals in the general population, the presence of the AC-5 pattern is not associated with the autoantigens mentioned above and most often concerns low antibody titers

AC-6MULTIPLE NUCLEAR DOTS
  • Found in a broad spectrum of autoimmune diseases, including PBC, AIM (DM), as well as other inflammatory conditions

34
  • If PBC is clinically suspected, it is recommended to perform follow-up tests for anti-Sp100 (and PML/Sp140) antibodies; in particular anti-Sp100 antibodies have the best clinical association with PBC and have added value, especially when associated with AMA; the Sp100 (and PML-Sp140) antigen is included in disease specific immunoassays (ie, liver profile*)

17, 35, 36
  • If DM is clinically suspected, it is recommended to perform a follow-up test for anti-MJ/NXP-2 antibodies; these anti-MJ/NXP-2 antibodies are highly specific for AIM, are found in up to one third of patients with juvenile DM, and have been reported to be associated with malignancies in adult AIM patients; the antigen is included in disease specific immunoassays (i.e., inflammatory myopathy profile*)

37–39
AC-7FEW NUCLEAR DOTS (see online supplementary table S1 for further details)
  • The AC-7 pattern has low positive predictive value for any disease

40, 41
  • Antigens primarily localized in the dots include p80-coilin and SMN complex; specific immunoassays for these autoantibodies are currently not commercially available

42, 43
AC-8HOMOGENEOUS NUCLEOLAR (see online supplementary table S1 for further details)
  • Found in patients with SSc, SSc-AIM overlap syndrome, and patients with clinical manifestations of other SARD

44–46
  • If limited cutaneous SSc is clinically suspected, it is recommended to perform a follow-up test for anti-Th/To antibodies; the antigen is included in disease specific immunoassays (ie, SSc profile*)

44, 45
  • If SSc-AIM overlap syndrome is clinically suspected, it is recommended to perform a follow-up test for anti-PM/Scl antibody reactivity; the antigen may be included in the routine ENA profile and is included in disease specific immunoassays (i.e., inflammatory myopathy profile* and the SSc profile*); in general, anti-PM/Scl antibodies yield a diffuse nuclear fine speckled staining in addition to the AC-8 pattern

46
  • Other antigens recognized include B23/nucleophosmin, No55/SC65, and C23/nucleolin, but the clinical significance of these autoantibodies is not well established; specific immunoassays for these autoantibodies are currently not commercially available

Notes: Although some anti-Th/To antibody immunoassays are commercially available, technical issues relating to the limited sensitivity of these immunoassays should be taken in to consideration. 44, 47
AC-9CLUMPY NUCLEOLAR
  • Found in patients with SSc

48
  • If SSc is clinically suspected, it is recommended to perform a follow-up test for anti-U3RNP/fibrillarin antibodies; the antigen is included in disease specific immunoassays (i.e, SSc profile*)

48
  • If confirmed as anti-U3RNP/fibrillarin reactivity by immunoassay, the clinical association is with diffuse SSc, increased incidence of pulmonary arterial hypertension, skeletal muscle disease, severe cardiac involvement, and gastrointestinal dysmotility

23, 48–50
  • Among SSc patients, anti-U3RNP/fibrillarin antibodies are most commonly found in African American and Latin American patients

48, 49, 51
Notes: Although some anti-U3RNP/fibrillarin immunoassays are commercially available, technical issues relating to the limited sensitivity of these immunoassays should be taken into consideration. 24
AC-10PUNCTATE NUCLEOLAR
  • The AC-10 pattern can be seen in various conditions, including SSc, Raynaud’s phenomenon, SjS, and cancer

52–56
  • If the AC-10 pattern is observed in the serum of patients with conditions mentioned above, follow-up testing for anti-NOR90(hUBF) antibodies is to be considered; the antigen is included in disease specific immunoassays (i.e. SSc profile*)

54, 55
  • While AC-10 is associated with anti-RNApol I antibodies, these antibodies almost always coexist with anti-RNApol III antibodies which reveal the AC-5 pattern; therefore, if SSc is clinically suspected, it is recommended to perform a follow-up test for anti-RNApol III antibodies (See also AC-5); specific immunoassays for anti-RNApol I antibodies are currently not commercially available

52, 53, 57
AC-11SMOOTH NUCLEAR ENVELOPE
  • The AC-11 pattern is infrequently found in routine autoantibody testing and has been described in autoimmune-cytopenias, autoimmune liver diseases, linear scleroderma, APS, and SARD; current information on clinical associations is based mainly on case reports and small cohorts

58–60
  • Antigens recognized include lamins (A, B, C) and LAP-2; specific immunoassays for these autoantibodies are currently not commercially available

58–60
AC-12PUNCTATE NUCLEAR ENVELOPE (see online supplementary table S1 for further details)
  • Found in patients with PBC, as well as patients with other autoimmune liver diseases and SARD

61
  • If PBC is clinically suspected, it is recommended to perform a follow-up test for anti-gp210 antibodies; the antigen is included in disease specific immunoassays (ie, extended liver profile*)

62–64
  • Other antigens recognized include p62 nucleoporin, LBR, and Tpr; specific immunoassays for these autoantibodies are currently not commercially available

65–68
AC-13PCNA-like (see online supplementary table S1 for further details)
  • The AC-13 pattern has formerly been considered highly specific for SLE, but this specificity is debated

69, 70
  • If SLE is clinically suspected, it is recommended to perform a follow-up test for anti-PCNA antibodies; the antigen is included in several routine ENA profiles

69
  • Recent studies with antigen-specific immunoassays show clinical associations also with SSc, AIM, RA, HCV, and other conditions

70–73
AC-14CENP-F-like
  • The majority of sera exhibiting the AC-14 pattern are from patients with a diversity of neoplastic conditions (breast, lung, colon, lymphoma, ovary, brain); paradoxically, the frequency of the AC-14 pattern in patient cohorts with these malignancies is low

  • The AC-14 pattern is also seen in inflammatory conditions (Crohn’s disease, autoimmune liver disease, SjS, graft-versus-host disease); current information on clinical associations is based mainly on case reports and series of cases

  • Possible associations only hold if the reactivity to CENP-F is confirmed in an antigen-specific immunoassay; current information on clinical associations is based mainly on case reports and series of cases; specific immunoassays for this autoantibody are currently not commercially available

74–78
AC-29 TOPOI-like
  • The AC-29 pattern is highly specific for SSc, in particular with diffuse cutaneous SSc and more aggressive forms of SSc

14, 18, 23
  • If SSc is clinically suspected, it is recommended to perform a follow-up test for anti-Topoisomerase I (formerly Scl-70) antibodies; the anti-Topoisomerase I antibodies are included in the classification criteria for SSc and the antigen is included in routine ENA profiles

8, 23, 79
  • *Availability of the inflammatory myopathy profile, the SSc profile and the (extended) liver profile may be limited to specialty clinical laboratories.

  • AIM, autoimmune myopathy;AMA, antimitochondrial antibodies;APS, antiphospholipid syndrome;CENP, centromere-associated protein;DFS, dense fine speckled;DM, dermatomyositis;ENA, extractable nuclear antigens;HCV, hepatitis C virus;IIFA, indirect immunofluorescence assay;LAP, lamin-associated polypeptide;LBR, lamin B receptor;LEDGF, lens epithelial derived growth factor;NOR, nucleolus organiser region;NXP, nuclear matrix protein;PBC, primary biliary cholangitis;PCNA, proliferating cell nuclear antigen;PML, promyelocytic leukaemia;PM/Scl, polymyositis-scleroderma;RA, rheumatoid arthritis;RNApol, RNA polymerase;RNP, ribonucleoprotein;SARD, systemic autoimmune rheumatic diseases;SLE, systemic lupus erythematosus;SMN, survival of motor neuron;SSc, systemic sclerosis;SjS, Sjögren’s syndrome;TIF, transcription intermediary factor;TRIM, tripartite motif;Tpr, translocated promoter region; UCTD, undifferentiated connective tissue disease; dsDNA, double stranded DNA;hUBF, human upstream binding factor.