INTERPRITATION OF RESULT OF ANA

Interpretation of Antinuclear Antibodies (ANA) : Interpretation of Antinuclear Antibodies (ANA) Anupam Maity

What are Antinuclear Antibodies : : What are Antinuclear Antibodies : A group of antibodies that bind to various nuclear antigens, such as DNA, snRNPs, but with the advent of FANA(fluorescent antinuclear antibody test ) which detects autoantigens throughout the cells spectrum of ANA is expanded to both cytoplasmic & nuclear specificities.

Cell structure : : Cell structure :

Nuclear structure : : Nuclear structure :

Mitotic splindle apparatus : : Mitotic splindle apparatus :

History of ANA : : History of ANA : The "LE cell" was first used as an tool in the diagnosis of SLE and drug-induced lupus, as well as SS. LE cells were soon discovered to be due to a plasma factor, autoantibody against deoxyribonucleoprotein, which opsonized, induced apoptosis, or phagocytosed by PMNs indirect immunofluorescence which offered sensitive assay for autoimmunity in SLE as well as other diseases

ANA Positivity : : ANA Positivity : ANAs have remained important diagnostic and prognostic tools for these connective tissue diseases these autoantibodies appear in a variety of infectious, inflammatory, and neoplastic diseases, as well as in normal individuals.

THE ANA RELATED DISEASES AND CONDITIONS : : THE ANA RELATED DISEASES AND CONDITIONS :

Non-rheumatic conditions causing positive ANA : : Non-rheumatic conditions causing positive ANA : Normal individuals: females > males, increasing age, relatives of patients with rheumatic disease, pregnancy Hepatic diseases: eg. chronic active hepatitis Pulmonary diseases eg. idiopathic pulmonary fibrosis Chronic infections Malignancies: lymphoma, leukemia, melanoma, solid tumors (ovary, breast, lung, kidney) Hematologic disorders: idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia Drug- induced Miscellaneous: autoimmune thyroiditis, type 1 diabetes mellitus,

ANA positivity in normal individuals : : ANA positivity in normal individuals : ANA 1:40 Seen in almost 32% of normals ANA 1:80 Seen in almost 13% ANA 1:160 Seen in almost 5% ANA 1:320 Seen in almost 3% There is no set titer that can distinguish between those with and without SLE Most people with positive ANA don’t have disease However, ANA titre is not a marker of disease activity.

So, when should we advice an ANA? : So, when should we advice an ANA? When your pre-test probability for lupus is moderate Not recommended as a random screening test Not useful to diagnose other conditions but may support a clinical diagnosis

Methods of detection : : Methods of detection : indirect immunofluorescence ANA test (FANA) on HEp-2 cell culture. IMMUNODIFFUSION COUNTERIMMUNOELECTROPHORESIS ELISA IMMUNOPRECIPITATION IMMUNOBLOT ANTI-DNA ANTIBODY TESTS

Slide 13 : Anti-Nuclear Antibody : IFA

Cultered hep2 cell : : Cultered hep2 cell :

Slide 15 : Results typically are reported by two parameters: pattern and titer, with any pattern of reactivity at a titer of 1:40 or greater being considered positive. A positive ANA test will show a particular staining pattern that may have diagnostic value in differentiating between types of CTD, but only in conjunction with clinical evaluation. Positive specimens Apple-green fluorescence of nuclear region Patterns of nuclear fluorescence

Slide 16 : Homogeneous -Rim Speckled Nucleolar For higher titres, patterns of ANA may be given:

HomogenEous pattern : : HomogenEous pattern : This homogeneous pattern of diffuse bright green staining of nuclei seen by immunofluorescence microscopy with a Hep2 cell substrate is called homogenous, and is the most common pattern with autoimmune diseases overall. Common in anti DNA Antibodies.

Speckled pattern : : Speckled pattern : Speckled pattern Scl70, SSA, SSB, Sm Suggests Sjogren’s, scleroderma, or MCTD

Nucleolar pattern : : Nucleolar pattern : Nucleolar pattern Seen in Sceloderma, Poly Myositis.

Rim / peripheral pattern : : Rim / peripheral pattern : This rim (peripheral ) pattern of linear bright green staining around the peripheral of nuclei seen by immunofluorescence microscopy with a Hep2 cell substrate . Seen against dsDNA

Crithidia luciliae test : : Crithidia luciliae test : Its kinetoplast, a modified giant mitochondrion, contains a concentrated focus of stable, circularized ds-DNA, without contaminating RNA or nuclear proteins, providing a sensitive and specific fluorescence substrate by which to establish anti-ds-DNA activity

Diagnostic patterns of ana : : Diagnostic patterns of ana :

Contd. : Contd.

Contd.. : Contd..

Contd… : Contd…

Patterns of imF on HEp-2 CELL: : Patterns of imF on HEp-2 CELL:

Algorithm For diagnosis of connective tissue dis. With positive ana: : Algorithm For diagnosis of connective tissue dis. With positive ana:

Slide 28 :

Ana antibodies in sle : : Ana antibodies in sle :

Ana in sceloderma : : Ana in sceloderma :

Ana in inflammatory muscle diseases : : Ana in inflammatory muscle diseases :

MISCELLANEOUS DISEASES : : MISCELLANEOUS DISEASES : In contrast to the traditional ANA diseases, the presence of a positive ANA in Raynaud's phenomenon, juvenile rheumatoid arthritis (JRA), and antiphospholipid antibody syndrome (APS) In Raynaud's phenomenon, a positive result increases the likelihood, from 19 percent to 30 percent, of the development of a systemic rheumatic disease, including SLE, RA, and SSc. In JRA, ANA positivity may predict the development of uveitis.

TAKE HOME MESSAGE regarding ana: : TAKE HOME MESSAGE regarding ana: A negative excludes active Systemic Lupus Erythematosus (SLE) in >95% of cases. False-positive s occur in the following frequencies: - at 1:40: 32% - at 1:80: 13% - at 1:320: 3% - The number of false-positive Increases with age. Positive s lack specificity, and can occur in many autoimmune rheumatic diseases, chronic inflammatory and infectious diseases, malignancies, and can also be induced by certain drugs.

Slide 34 : Anti DsDNA Abs are essentially restricted to SLE and are seen infrequently in severe Rheumatoid Arthritis (RA). Increases in Anti-dsDNA Ab titers may predict flares in SLE. Anti Sm Abs. are 99% specific for SLE. High titer Anti RNP Abs. (>1:10,000) are characteristic of Mixed Connective Tissue Disease (MCTD), particularly if unaccompanied by other autoantibody specificities.

Once ANA is positive… : Once ANA is positive… Specific autoantibody tests possess diagnostic significance in the right clinical setting. ANA has no utility for disease monitoring. There is no need to repeat it.

Thank you everybody ……….. : Thank you everybody ………..

Slide 37 : Anti-Nuclear Antibody: ELISA

Slide 38 : Sensitivity 95% in patients with SLE Almost all patients with SLE have positive ANA The ANA test is not specific for SLE

Slide 39 : Homogeneous -Rim Speckled Nucleolar For higher titres, patterns of ANA may be given: Staining patterns are not specific and not reliable for diagnosing different diseases

Once ANA is positive… : Once ANA is positive… ANA has no utility for disease monitoring There is no need to repeat it