Budsakorn Darawankul, MD. Maharat Nakhon Ratchasima Hospital
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1 Budsakorn Darawankul, MD. Maharat Nakhon Ratchasima Hospital
2 Outline What is ANA? How to detect ANA? Clinical application Common autoantibody in ANA diseases
3 Outline What is ANA? How to detect ANA? Clinical application Common autoantibody in ANA diseases
4 Autoantibody : antibody against the individual's own proteins.
5
6 Autonuclear antibody (ANA) : autoantibodies directed against intracellular antigen Nuclear Chromatin associated antigen : DNA, Histone etc. Small Nuclear Ribonucleoproteins (snrnp) Other Ribonucleoproteins Nucleolar (Cytoplasmic)
7
8
9 Outline What is ANA? How to detect ANA? Clinical application Common autoantibody in ANA diseases
10
11 Method for the Detection of Autoantibodies
12 IF-ANA : Gold standard for ANA testing American College of Rheumatology Slide collection
13 Hep-2 cells are grown on a microscope slide, fixed/permeabilized, and then incubated with a patient s diluted serum. After washing off unbound serum, the cells are incubated with fluorescently labeled goat anti-human IgG antibodies before viewing in an epifluorescence microscope. (b) ANA titer. Patient serum is diluted 1:40, and then serial twofold dilutions are performed and used to stain Hep-2 cells. In the example there is a weak fluorescent signal at 1:320 dilution and no staining at 1:640 (a 1:320 titer).
14 Homogeneous Peripheral Speckled Nucleolar
15
16
17 High sensitivity & specificity but technician dependent Previously used Ag substrate : rodent organ; liver, stomach In 1975, Human Epithelium cell line (HEp2 cells) was introduced to improve sensitivity.
18 American College of Rheumatology Slide collection
19
20 Outline What is ANA? How to detect ANA? Clinical application Common autoantibody in ANA diseases
21
22
23 Chronic infectious diseases, such as mononucleosis, HCV, IE, TB, and HIV Lymphoproliferative diseases The presence of ANAs is rarely associated with malignancy, with the exception of dermatomyositis. Drugs : procainamide, hydralazine, chlorpromazine, quinidine, penicillamine, isoniazid, anti-tnf agents
24 Usefulness of testing for ANAs To help establish a diagnosis To exclude in patients with few or uncertain clinical findings To subclassify a patient To monitor disease activity Evaluated : pattern & titers
25 Anti-nuclear Antibodies (Patterns, Autoantigens, & Disease association) Homogenous Rim Speckled Nucleolar Histone ds-dna Sm U1-RNP Ro L a Scl-70 + High titer DIL SLE MCTD SS PSS
26 Anti-centromere : Can be seen during meiosis and mitotic phase Correlate with limited sclerosis, CREST syndrome
27 Outline What is ANA? How to detect ANA? Clinical application Common autoantibody in ANA diseases
28 ANA diseases High prevalence of ANAs, often screened for by the fluorescent antinuclear antibody (FANA) test: SLE Systemic sclerosis (SSc) Mixed connective tissue disease (MCTD). The prevalence of ANAs in polymyositis (PM), dermatomyositis (DM), and Sjo gren s syndrome (SS) has been reported to be lower, but they are often grouped together.
29 Robbins Basic Pathology. 8 th edition
30 Common autoantibody in SLE Autoantibody Frequency IFA pattern Clinical correlation Anti-dsDNA 70-80% Homo, Rim Specific to SLE, correlate with disease activity (LN), bad prognostic serology Anti-ssDNA 90% Negative Non specific to SLE Anti-Histone 50-70% Homo Associated with drug induced Lupus, myositis, SSc, chronic infection (EBV) Anti-Sm 20-30% Speckle Specific to SLE Anti-U1 RNP 30-40% Speckle Good prognosis for SLE, MCTD like Anti-Ro 40% Speckle or Negative Neonatal LE, cutaneous lupus, sicca, lung involvement, Anti-La 10-15% Speckle Neonatal LE, late onset SLE, sicca, protect from anti-ro asso. nephritis Anti-Ribosomal P protein (anti-p) 10-20% Cytoplasm Neuropsychiatric LE, lupus hepatitis
31 Common autoantibody in Systemic sclerosis Autoantibody Frequency IFA pattern Clinical correlation Anti-Scl70 (Anti-topoisomearse1) Anti-Kinetochore (Anti-centromere) 22-40% Nucleolar Diffuse form, sclerodermal renal crisis, lung fibrosis 22-36% Centromeric Limited form, CREST syndrome, PHT Anti-RNAP III Nucleolar Diffuse form, sclerodermal renal crisis, Anti-U3RNP (Anti-fibrillalin) Nucleolar Diffuse form, PHT, internal organ involvement, myositis, lung involvement Anti-Th/T0 Nucleolar sclerodermal renal crisis, PHT Anti-PM-Scl Speckle Myositis-SSc overlap Anti-Ku Speckle Myositis, also found in SLE
32 Mixed Connective Tissue Disease (MCTD)
33 Common autoantibody in myositis Myositis-specific autoantibodies (MSAs) Myositis associated autoantibodies (MAAs) (myositis overlap autoantibodies) : Anti-snRNP, anti-pm-scl
34 Myositis-specific autoantibodies (MSAs) Autoantibody IFA pattern Clinical correlation Anti-Jo1 Cytoplasmic Anti-synthetase syndrome (Fever, arthritis, ILD, Raynaud s phenomenon, mechanic hand) Anti-Mi2 Speckle Dermatomyositis, good prognosis, good response to steroid Anti-CADM Amyopathic dermatomyositis Anti-TIF1γ (Anti-P155/140) Anti-SRP Anti-HMG-CoA Reductase Paraneoplastic syndrome Severe necrotizing myopathy, +/- cardiac involvement Statin induced necrotizing autoimmune myopathy (NAM)
35 Sjogren s syndrome Anti-Ro antibodies appear in approximately 40% to 95% of SS patients. Anti-La antibodies appear in as many as 87% of SS patients. Both antibodies correlated with extraglandular manifestations.
36
37 Red Flags for connective tissue disease Constitutional symptoms Arthralgia arthritis Photosensitive rash Urine abnormality Leukopenia Serositis Raynaud s phenomenon
38 Case 1 Female 25 years old Present with arthritis at small joints of hand with morning stiffness, rash at malar area, generalized edema. CBC WBC 3400, PMN 80%, L 15%, Hct 32%, Plt 120,000 Cr 0.5, Albumin 2.4 UA Protein 3+, RBC 3-5/HPF UPCI 4 gm/day
39 Case 1.1 ANA 1:320 Homogeneous pattern 1:640 Speckle pattern
40 ANA : negative Case 1.2
41 ANA negative SLE Protein losing condition Inappropriate substrate Prozone phenomenon Anti-Ro, Anti-ssDNA Complement deficiency True ANA negative SLE (less than 5%)
42 Case 2 Female 65 years old Present with constitutional symptoms, weight loss 6 kg/2 months, mild arthritis both hands, no rash. Mild splenomegaly, LN not palpable. CBC WBC 22,000, PMN 25%, L 65%, Hct 22%, Plt 90,000 PBS : microspherocyte, polychromasia, Lymphocyte predominate, platelet decrease DCT positive
43 Case 2 ANA 1:160 Speckle pattern Anti-dsDNA, Anti-Sm : negative
44 Case 3 Female 30 years old Present with prolonged fever 2 months with weight loss 10 kg, arthritis both hands, LCV both legs, Raynaud s phenomenon + Fine crepitation both lungs
45 Case 3 ANA 1:160 Speckle pattern Anti-dsDNA, Anti-Sm : negative Anti-HIV positive, Anti-HCV positive
46
47 Clinical features Idiopathic SLE Drug-induced Lupus Age / Sex Young / Female Older / Male (Children) Onset Insidious Abrupt Severity Severe Mild Systemic symptoms Common Common Musculoskeletal symptoms Common Common Polyserositis Common Common Multi-organ involvement Common Unusual Classical skin lesions Common Unusual CNS, renal involvement Common Rare Complements Decreased Normal ANA Anti Sm, Anti-DNA Anti-(H2A-H2B)-DNA Therapeutic trail No response Improvement
Clinical Laboratory. [None
Clinical Laboratory Procedure Result Units Ref Interval Accession Collected Received Double-Stranded DNA (dsdna) Ab IgG ELISA Detected * [None 18-289-900151 Detected] Double-Stranded DNA (dsdna) Ab IgG
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