CORRELATION OF ANA IMMUNOFLUOROSCENT TITRE AND PATTERN DISTRIBUTION WITH CLINICAL FEATURES : JOURNEY BEYOND SLE

This is a Retrospective study conducted at Pathology department Bombay Hospital Indore. 300 patients were tested for presence of ANA antibody using indirect immunofluorescent test (IMMUNOSHOP AESKU SLIDES) over the period of one year. ANA testing by IIF is a highly valuable and time tested technique for diagnosis of autoimmune disorder. Results should be interpretated in the light of clinical and biochemical findings as normal individuals have positive results on traditional ANA testing. The most definitive result from ANA testing is a negative test. This result, especially when coupled with negative tests on an ANA profile, suggests strongly that ANA associated diseases are unlikely to be present. This imparts a high NPV to ANA IIF tests. Apart from the usually described clinical features this study highlights few of the uncommon isolated clinical features like cytopenias, myopathies and Pyrexia of unknown origin and utility of ANA IIF in establishing diagnosis. We at our centre perform ANA profile of patients to further classify the disease which is beyond the scope of this article.


Introduction:
Anti nuclear antibodies are directed against intracellular antigens (ANAs), are important in the diagnosis of systemic autoimmune rheumatic diseases (SARDs) like systemic lupus erythematosus (SLE), Sjögren's syndrome (SjS) mixed connective tissue disease (MCTD),systemic sclerosis (SSc) , idiopathic inflammatory myopathies (IIMs).ANA may connote a form of systemic autoimmunity that is expressed as a wide variety of complaints, even in the absence of a definite diagnosis of CTD.Common clinical features of autoimmune destruction include fever of unknown origin, nephropathy, and dryness of mouth, paraesthesias, muscle weakness, Raynauds phenomenon, pleural effusions and other multisystem involvement.
Detection of ANA by indirect immunofluoroscent method is a reliable technique for diagnosing SARD.Apart from specific diagnosis there are certain uncommonly described features which we highlighted in our study.These symptoms along with ANA testing can help in diagnosis of the patient.Such uncommon features are cytopenias of unexplained origin, high grade fever with raised TLC etc.
This study aims at defining the correlation of ANA immunofluoroscent staining pattern with clinical features; both common and uncommon in a tertiary care setting

Material and Methods
This is a Retrospective study conducted at Pathology department Bombay Hospital Indore.300 patients were tested for presence of ANA antibody using indirect immunofluorescent test (IMMUNOSHOP AESKU SLIDES) over the period of one year.Immunofluorescence testing involves incubating dilutions of patien sera with a monolayer of fixed, permeabilized cells [F1].Antibodies adherent to the cell monolayer are visualized with an anti-human immunoglobulin reagent that has been conjugated to a fluorescent tag.
Presence or absence of nuclear staining and the pattern of nuclear staining is assessed by fluorescence microscopy.
Patients clinical details were recorded and then correlated with ANA staining, pattern and titre.

Results
ANA was found to be positive in about 43 % of the total patients tested, more in females as compared to males.The most frequent pattern in descending order were Speckled followed by Homogenous, Nuclear memberane Nucleolar, Centromeric and one rare pattern also encountered that is mixed nucleolar speckled also called as SS PATTERN.Cytoplasmic pattern that were seen and described in our study were Golgi pattern and Ribosomal P pattern, in patient of myopathy.
For the patterns associated with cytoplasmic staining (AC-15 to AC-23), the ICAP classification tree indicates the fibrillar, speckled, reticular/antimitochondrial antibody (AMA), and polar/Golgi-like patterns, as well as rods and rings, as belonging to the competent level.All of the patterns associated with the mitotic category were assigned expert level recognition.In our study we found following patterns and their associations as shown in table.

Figure 3:
In the homogeneous staining pattern, the entire nucleus is diffusely stained, (Titre: 1:1280)  First and foremost fact to keep in mind with ANA testing is that it is a diagnostic test.Once the diagnosis is established reassesment of the patient's ANA status is not required.ANA positivity does not mean that patient has autoimmune disease as ANA positivity is quiet common in general population in low titres.This imparts a low positive predictive value to the test.This can be overcome by increasing serial dilutions.ANA titre in general population is usually 1: 80 and as dilutions are increased to 1:160 or 320 the chances of false positivity decreases,as low as 5% 5,6 Furthermore , ANA pattern or titre do not provide any information regarding disease activity so repeat testing is not indicated. 7In patients with established diagnoses of autoimmune diseases, however ANA positivity can subdivide patients with regard to prognosis and response to therapy.In juvenile chronic arthritis, where ANA positivity is associated with an increased risk of uveitis, autoimmune hepatitis where ANA positivity defines a disease subtype 8 .In patients with scleroderma, the presence of a centromere pattern of staining may suggest the CREST syndrome, while a diffuse or nucleolar pattern of staining would be more consistent with diffuse cutaneous scleroderma.This distinction may be important, since the incidence of major end organ complications such as interstitial lung disease is lower in CREST syndrome.
In contrast, scleroderma patients with antitopoisomerase (Scl-70) antibodies (one of the causes of a diffuse pattern of ANA staining) have increased scleroderma lung disease.

Figure 7 :Figure 8 :
Figure 7: testing by IIF is a highly valuable and time tested technique for diagnosis of autoimmune disorder.Results should be interpretated in the light of clinical and biochemical findings, as normal individuals have positive results on traditional ANA testing.The most definitive result from ANA testing is a negative test.This result, especially when coupled with negative tests on an ANA profile, suggests strongly that ANA associated diseases are unlikely to be present.This imparts a high NPV to ANA IIF tests.Apart from the usually described clinical features this study highlights few of the uncommon isolated clinical features like cytopenias, myopathies and Pyrexia of unknown origin and utility of ANA IIF in establishing diagnosis.