|Year : 2019 | Volume
| Issue : 1 | Page : 29-33
Demystifying antinuclear antibodies and other serological tests in dermatology practice
Anupam Berwal1, Indira Bairy2, Akshita Gupta3
1 Department of Microbiology, Kasturba Medical College, Manipal Academy of Higher Education, Manipal, Karnataka, India
2 Department of Microbiology, Melaka Manipal Medical College, Manipal Academy of Higher Education, Manipal, Karnataka, India
3 Department of Mcrobiology, KMC Manipal, Manipal, Karnataka, India
|Date of Web Publication||14-Feb-2019|
Department of Microbiology, Melaka Manipal Medical College, Manipal Academy of Higher Education, Manipal - 576 104, Karnataka
Source of Support: None, Conflict of Interest: None
The presence of various autoantibodies in patients' sera is the most common marker of autoimmune diseases, and detection of these antinuclear antibodies (ANAs) aid the diagnosis of several autoimmune disorders in clinical practice; this review article is on ANA assays and other serological tests, specifically the ANA and their interpretation, correlation with its applicability in the dermatology practice.
Keywords: Antinuclear antibodies, autoimmunity, enzyme-linked immunosorbent assay
|How to cite this article:|
Berwal A, Bairy I, Gupta A. Demystifying antinuclear antibodies and other serological tests in dermatology practice. Clin Dermatol Rev 2019;3:29-33
|How to cite this URL:|
Berwal A, Bairy I, Gupta A. Demystifying antinuclear antibodies and other serological tests in dermatology practice. Clin Dermatol Rev [serial online] 2019 [cited 2019 Mar 25];3:29-33. Available from: http://www.cdriadvlkn.org/text.asp?2019/3/1/29/252314
| Introduction|| |
The antinuclear antibody (ANA) assay is very significant tool for diagnosis in cases of clinical suspicion of autoimmune diseases, mainly those belonging to the group of collagen diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), scleroderma, dermatomyositis (DM), Sjogren's syndrome, mixed and overlap syndromes. ANAs and rheumatoid factors (RFs) were the first autoantibodies to be described as serum factors that could bind nuclear antigens and immunoglobulins (Igs), respectively.,
ANA and RF were recognized as diagnostic features of SLE and RA, respectively. Nowadays, the significance of ANA assays is increasing, and it is the method of choice for diagnostic screening, classifying, and monitoring autoimmune diseases. Even though ANA is a collective terminology for nuclear antibodies, their positivity is interpreted as the presence of antibodies not only against elements of the cell nucleus but it also signifies the presence of autoantibodies against elements of the cytoplasm,,, and its interpretation should always be correlated clinically.
The detection of circulating autoantibodies can be made by different immunoassays such as indirect immunofluorescence (IIF), immunoblot, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). ANA global test detects more than hundred ANAs and is employed as a screening test. Currently, the gold standard method for ANA detection is IIF on human epithelial (HEp-2) cells. The diagnosis and prognosis of systemic autoimmune diseases is determined by detection of antibodies to extractable nuclear antigens (ENAs) and dsDNA. The diagnostic tests such as desmoglein (Dsg) 1 and 3 ELISA are available for the diagnosis of pemphigus, BP180, and BP230 ELISA for bullous pemphigoid (BP) and Type VII collagen ELISA for epidermolysis bullosa acquisita. In this review, we describe the practical aspects of different serological tests and their interpretation in dermatology.
| Background|| |
Finding of lupus erythematosus (LE) cells in the sera of lupus patients in 1948 led to investigations of the presence of autoantibodies. The pathogenic mechanism behind the formation of LE cells was found to be the result of antideoxyribonucleoprotein antibodies-induced apoptosis. ANA analysis by IF microscopy along with histopathology remains a diagnostic hallmark of SLE. ANAs are associated with a number of autoimmune conditions, and they are most useful in the diagnosis and classification of rheumatic diseases. Therefore, ANA screening is imperative in patients presenting with clinical symptoms or other laboratory results suggestive of rheumatic disease. However, the presence of ANA is nonspecific and can be associated with many nonautoimmune conditions such as carcinoma, infection, pharmaceuticals, and environmental factors. ANA-positive frequency in healthy individuals is found to be >20%. A study has reported that 31.7% of normal individuals were ANA positive at 1:40 dilution, 13.3% at 1:80, and 5.0% at 1:160 dilution. A titer of 1:100 is usually done to avoid false-positive result. It can be positive in up to 50% of patients with RA; 20% in scleroderma with no systemic manifestations; and over 80% in the systemic forms. Positivity can reach 40%–80% in DM/poliomyelitis.,
Genetic and familial factors as well as few drugs and viruses can also stimulate a positive ANA. Apart from genetic predisposition, several factors can trigger cell apoptosis (e.g., viral and bacterial infections, and drugs). Ultraviolet cytotoxic irradiation is responsible for inducing keratinocyte apoptosis., The exposure of both nuclear and cytoplasmic elements to predisposed immunocompetent cells initiates the formation of antibodies against certain nuclear or cytoplasmic proteins. These autoantibodies, when circulating, deposit in different organs and tissues, trigger fixation of the complement system, thus leading to inflammation and dysfunction of the target organ.
| Identification Methods|| |
Identification of autoantibodies includes different methods such as IIF of Hep-2 cells, ELISA, radioimmunoassay, immunodiffusion, counterimmunoelectrophoresis, immunoprecipitation, and immunoblot. All these methods are subject to false-positive results, mainly when the evaluated patient is above 60 years of age.
The use of Hep-2 substrate is preferred because of its high sensitivity, but certain patients with clinical features of SLE having negative tests for ANA have been reported. Technical factors or prozone effects may be the possible causes for ANA-negative SLE. A positive ANA should always be evaluated and correlated with clinical condition, deposit pattern, and dilution. Different possible deposit patterns correlate with likely cellular elements that behave as antigens. Thus, depending on ANA deposit pattern, the specificity of the ANA marker of the suspected disease is assessed.,,
| Autoantibody Characteristics and Specificity|| |
ANA are typically found in inflammatory rheumatic diseases, and it is described as nucleus homogenous, granular or speckled, nucleolar, cytoplasm granular, and mixed patterns. They can be associated with other autoimmune diseases in varied frequencies. ANA is detectable in up to 50% of patients with primary biliary cirrhosis (PBC). The preferred immunofluorescence pattern is multiple nuclear dots and perinuclear staining of the nuclear membrane. The ANA immunofluorescence patterns can be a sign of an AIH/PBC overlap syndrome. Certain ANA specificities can point to an associated systemic autoimmune disease, or an increased risk of developing a systemic autoimmune disease, e.g., anti-centromere antibodies in PBC may indicate associated systemic sclerosis.
Antidouble stranded DNA (ds-DNA) antibodies which can be detected by ENA line immunoassay (LIA) and the titer can be measured by ELISA are diagnostic and help in monitoring SLE in the correct clinical setting, but it does not alone always make a diagnosis as anti-dsDNA may be found in few other rheumatic diseases and may be negative in SLE with serositis cases. The disease cannot be excluded if anti-dsDNA is negative, as anti-dsDNA antibodies are found in 60% of SLE cases. Anti-dsDNA well correlates with overall activity of the disease, especially increasing titers points to disease flare, hence of diagnostic and prognostic importance.
Anti-centromere antibody is a type of ANA detected by IIF displaying a centromeric pattern (discrete speckled pattern). It is present in approximately 30% of patients who suffer from systemic scleroderma of the CREST type (calcinosis, Raynaud, esophageal disease, sclerodactyly, and telangiectasia). It can also be present in generalized cutaneous scleroderma in a proportion that varies between 30% and 40% of patients, in Hashimoto's thyroiditis, Raynaud's phenomenon, and PBC.,
Antiextractable nuclear antigen
The other ENAs such as Smith (Sm) antigen, ribonucleoprotein (RNP) or U1RNP, anti-SSA (Ro), and anti-SSB (La) can be detected by LIA or ELISA. The Sm antigen is highly specific for SLE, but it is found only in ~25% of SLE patients. The U1RNP antigen is seen in patients with SLE plus systemic sclerosis and in patients with mixed connective tissue disease. The SSA (Ro) and SSB (La) nuclear antigens are often found together in those patients with Sjögren's syndrome. Anti-SSA and anti-SSB are also seen in some subsets of SLE patients. This group includes those patients with subcutaneous lupus erythematosus (prominent photosensitive rashes and sometimes vasculitis), but without severe renal disease. The presence of anti-SSA and anti-SSB is associated with neonatal lupus, in which transplacental transfer of these antibodies (maternal IgG) can cause transient photosensitive rash and/or congenital heart block.
Anti-SRP, anti-JO-1, anti-Mi-2, and anti-PM/Scl are termed myositis-specific antibodies because of the high specificity to the autoimmune inflammatory myopathies.
| Protocol for Antinuclear Antibody Testing|| |
ANA global should be the initial investigation of choice whenever there is a suspicion of collagen disease. Clinical findings should always be correlated with the ANA results to obtain the best diagnosis. In a suspected rheumatic disease case if ANA immunofluorescence is positive, then ANA profile test is done to look for specific ENAs to diagnose the rheumatic disease [Table 1]. ANA profile is an immunoblotting test which detects more than 14 specific antibodies.
|Table 1: Autoantibody tests that are widely available and help diagnosis|
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| Clinical and Antinuclear Antibody Scenarios|| |
1. Negative antinuclear antibody + clinical suspicion
Request anti-Ro/SSA and/or anti-Jo-1 and/or antiphospholipid. Consider the possibility of negative-ANA LE or presence of plasmatic autoantibodies (antiphospholipid), in addition to cytoplasmatic autoantibodies with discontinuous fluorescence (anti-Jo-1).
2. Positive antinuclear antibody, low titer + nonspecific clinical findings
Request anti-Ro/SSA and/or antiJo-1 or antiphospholipid. Find out the specificity that best fits clinical hypothesis.
3. Positive antinuclear antibody, low titer + highly suspected clinical findings
Request as per suspected condition
- SLE – Anti-DNA, anti-Sm, anti-Ro/SSA
- Drug-induced SLE – Anti-DNA histone
- Scleroderma – Anti-Scl-70, anti-centromere, anti-PM-Scl
- DM – Anti-Jo-1, anti-PM-Scl
- Mixed syndrome – Anti-U1RNP, anti-RNA-synthetase
- Sjögren's syndrome – Anti-Ro/SSA, anti La/SSB
- Neonatal SLE – Anti-Ro/SSA, anti-La/SSB, antiU1RNP.
In Rheumatoid Arthritis, positivity rate is as follows: RF (66%), Histone (15%–20%), single-stranded DNA (8%), and U1 nRNP (3%). Other antibodies such as antikeratin, anticitrullinated peptides, anti-RA33, anti-Sa, and anti-p68 autoantibodies have been shown to have >90% specificity for RA.
Stills disease, a rare systemic inflammatory disorder of unknown etiology, has a heterogeneous presentation like fever, rash, and arthritis with multiorgan involvement. The RF and ANA tests are negative.
4. Positive antinuclear antibody, high titer antinuclear antibody + highly suspected clinical findings
Evaluation of location and deposit pattern can be sufficient to establish a correlation between ANA specificity and diagnostic conclusion or request specific autoantibodies for confirmation, in the same fashion as described above.
| Desmoglein 1 and Desmoglein 3|| |
These are the autoantibodies for pemphigus foliaceus (PF) and pemphigus vulgaris (PV), respectively. PV and PF is a disease that features blistering of the skin and mucous membranes caused by autoantibodies directed against Dsg3 and/or Dsg1. IIF and ELISA are the methods that are widely used for the diagnosis of pemphigus. ELISA test helps to measure the titers of antibody. Although the titers of these autoantibodies are generally correlated with disease activity, some patients with a high titer do not have severe symptoms.
PV and PF can be differentiated serologically by Dsg antibody profile measured by ELISA as shown in [Table 2].
ELISA score can be calculated by comparing titers measured by these ELISA with those from standard serum provided in the kit. ELISA score is very useful in evaluating the disease activity and monitoring the therapy; its decreased score can help in tapering the dose of steroid therapy.
BP180 and BP230 – Pemphigoid diseases are a heterogeneous group of subepidermal autoimmune blistering diseases, prototype being BP. Direct and Indirect IF tests are used for diagnosis using skin sections. However, ELISA is a sensitive tool for the detection of IgG anti-BP180 and anti-BP230 autoantibodies for diagnosis and monitoring. BP180 ELISA uses recombinant NC16a domain that is the proximal region of extracellular domain of BP180. BP230 ELISA uses the recombinant domain of the N- and C-terminus. It does not contain the middle part of BP230.
The target autoantibodies for this group are shown in [Table 3].
|Table 3: Subepidermal blistering diseases and their target autoantigens|
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| Antineutrophil Cytoplasmic Antibody|| |
Antineutrophil cytoplasmic antibody (ANCA) are group of autoantibodies to the antigens in neutrophil granulocytes and monocytes. Even though found in many autoimmune diseases, they are mainly associated with systemic vasculitis. The main antigens associated are proteinase – 3 (PR3) which produces cytoplasmic fluorescence (C ANCA) and myeloperoxidase which produces perinuclear pattern (P ANCA). Immunofluorescence is used as screening test, and ELISA tests detect antibodies to individual antigens and also the titer which is of prognostic importance. Physicians should always keep in mind different diseases that are associated with ANCA and recognize that certain patients with Wegener's granulomatosis, microscopic polyangiitis, Churg–Strauss syndrome, or pauci-immune glomerulonephritis may be ANCA negative. The value of an ANCA assay depends on its operational characteristics (sensitivity, specificity, positive, or negative predictive value).
| Conclusion|| |
ANA is the most important investigation of choice for the diagnosis of collagen diseases; but, it should always be correlated with clinical findings. Although it has a predictive value with high probability of an individual developing a collagen disease, its mere isolated presence is of no significance. Its negativity cannot be conclusive of the absence of collagen disease, either. The ANA assay, along with its interpretation, is dynamic. ANA test has contributed a lot in understanding connective tissue disorders, and it is proven to be a valuable tool for the clinician to diagnose and manage autoimmune diseases.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Neugebauer KM, Merrill JT, Wener MH, Lahita RG, Roth MB. SR proteins are autoantigens in patients with systemic lupus erythematosus. Importance of phosphoepitopes. Arthritis Rheum 2000;43:1768-78.
Vassileva S. Immunofluorescence in dermatology. Int J Dermatol 1993;32:153-61.
Chhabra S, Minz RW, Saikia B. Immunofluorescence in dermatology. Indian J Dermatol Venereol Leprol 2012;78:677-91.
] [Full text]
Mohan KH, Pai S, Rao R, Sripathi H, Prabhu S. Techniques of immunofluorescence and their significance. Indian J Dermatol Venereol Leprol 2008;74:415-9.
] [Full text]
Dellavance A, Gabriel A Jr., Cintra FU, Ximenes AC, Nuccitelli B, Taliberti BH, et al
. Brazilian consensus on Antinuclear antibodies in Hep-2 cells. Rev Bras Reumatol 2003;43:129-40.
Duarte AA, editor. Colagenoses and dermatology ASA Duarte - Sao Paulo: Ed. Do Autor, 2004. p. 12-38.
Holman HR, Kunkel HG. Affinity between the lupus erythematosus serum factor and cell nuclei and nucleoprotein. Science 1957;126:162-3.
Satoh M, Vázquez-Del Mercado M, Chan EK. Clinical interpretation of antinuclear antibody tests in systemic rheumatic diseases. Mod Rheumatol 2009;19:219-28.
Peng SL, Craft J. Antinuclear antibodies. In: Harris ED Jr., editor. Kelley's Textbook of Rheumatology. Philadelphia: Elsevier & Saunders; 2005. p. 311-31.
Lahita RG, Chiorazzi N, Reeves WH. Antinuclear antibodies. In: Harris ED Jr., editor. Textbook of the Autoimmune Diseases. Philadelphia: Williams & Wilkins; 2000. p. 87-101.
Schmidt-Acevedo S, Pérez-Romano B, Ruiz-Argüelles A. 'LE cells' result from phagocytosis of apoptotic bodies induced by antinuclear antibodies. J Autoimmun 2000;15:15-20.
Tan EM. The L.E. Cell and its legacy 1948. Clin Exp Rheumatol 1998;16:652-8.
Beck JS. Variations in the morphological patterns of “autoimmune” nuclear fluorescence. Lancet 1961;1:1203-5.
Solomon DH, Kavanaugh AJ, Schur PH; American College of Rheumatology Ad Hoc Committee on Immunologic Testing Guidelines. Evidence-based guidelines for the use of immunologic tests: Antinuclear antibody testing. Arthritis Rheum 2002;47:434-44.
Kim HA, Chung JW, Park HJ, Joe DY, Yim HE, Park HS, et al.
An antinuclear antibody-negative patient with lupus nephritis. Korean J Intern Med 2009;24:76-9.
Vaile JH, Dyke L, Kherani R, Johnston C, Higgins T, Russell AS, et al.
Is high titre ANA specific for connective tissue disease? Clin Exp Rheumatol 2000;18:433-8.
Conrad K. Textbook of – Autoantibodies in Organ Specific Autoimmune Diseases – A Diagnostic Reference. Vol. 8. Pabst science publishers lengerich, berlin, bremen, Miami, Riga, Viernheim, Wein, Zagreb; 2011. p. 27, 28.
Nakano M, Ohuchi Y, Hasegawa H, Kuroda T, Ito S, Gejyo F, et al.
Clinical significance of anticentromere antibodies in patients with systemic lupus erythematosus. J Rheumatol 2000;27:1403-7.
Weiner ES, Hildebrandt S, Senécal JL, Daniels L, Noell S, Joyal F, et al.
Prognostic significance of anticentromere antibodies and anti-topoisomerase I antibodies in Raynaud's disease. A prospective study. Arthritis Rheum 1991;34:68-77.
Castro C, Gourley M. Diagnostic testing and interpretation of tests for autoimmunity. J Allergy Clin Immunol 2010;125:S238-47.
Gopalarathinam R, Orlowsky E, Kesavalu R, Yelaminchili S. Adult onset still's disease: A review on diagnostic workup and treatment options. Case Rep Rheumatol 2016;2016:6502373.
Ishii K. Importance of serological tests in diagnosis of autoimmune blistering diseases. J Dermatol 2015;42:3-10.
Kobayashi M, Amagai M, Kuroda-Kinoshita K, Hashimoto T, Shirakata Y, Hashimoto K, et al.
BP180 ELISA using bacterial recombinant NC16a protein as a diagnostic and monitoring tool for bullous pemphigoid. J Dermatol Sci 2002;30:224-32.
Yoshida M, Hamada T, Amagai M, Hashimoto K, Uehara R, Yamaguchi K, et al.
Enzyme-linked immunosorbent assay using bacterial recombinant proteins of human BP230 as a diagnostic tool for bullous pemphigoid. J Dermatol Sci 2006;41:21-30.
Gross WL, Schmitt WH, Csernok E. ANCA and associated diseases: Immunodiagnostic and pathogenetic aspects. Clin Exp Immunol 1993;91:1-2.
Vassilopoulos D, Hoffman GS. Clinical utility of testing for antineutrophil cytoplasmic antibodies. Clin Diagn Lab Immunol 1999;6:645-51.
[Table 1], [Table 2], [Table 3]