Use and Interpretation of Rheumatology Lab Tests

Thomas A. Griffin, MD, PhD Pediatric Rheumatology

Disclosure

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I have no relevant financial relationships with the manufacturer(s) of any commercial product(s) and/or provider(s) of commercial services discussed in this CME activity. I do not intend to discuss an unapproved/investigative use of a commercial product/device in our presentation.

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Purposes of Rheumatology Lab Tests

Assist in diagnosis – rarely establish diagnosis Provide evidence for inflammation Assess disease activity – response to treatment

Characteristics of Lab Tests

Sensitivity - % of diseased individuals with positive test Requires a gold standard ANA is highly sensitive for lupus “False negative” - % of diseased individuals with negative test ESR has a high rate of false negatives in JIA Specificity - % of normal individuals with a negative test Anti-CCP is highly specific for arthritis “False positive” - % of normal individuals with a positive test ANA has a high rate of false positives in children

Characteristics of Lab Tests

Positive & Negative Predictive Values Depend on disease prevalence, sensitivity & specificity 90% of patients with ankylosing spondylitis (AS) have HLA-B27 (highly sensitive) <20% patients with back pain and HLA-B27 have AS (low predictive value) – back pain is common, AS is not

Categories of Lab Tests

Autoantibodies Assist in diagnosis of autoimmune disease Markers of inflammation Assess disease activity Genetic tests Diagnostic, disease associations Miscellaneous tests

Rheumatoid Factor

Autoantibodies to the Fc portion of IgG RF present in 85% of adults with RA, but only 5-10% of children with JIA Predicts more severe disease and risk of joint damage PPV in JIA <0.5

Greater PPV at higher titers (>3x ULN) Seroconversion in JIA almost never occurs RF+ JIA diagnosis requires 2 positive tests >3 months apart RF with increased prevalence in other disorders SLE (15-35%), Sjogren ’s Syndrome (75-95%), MCTD (50-60%), Scleroderma (20-30%), SBE, tuberculosis, syphilis, viral infections, sarcoidosis, leukemia, leprosy

Anti-Cyclic Citrullinated Peptide

Autoantibodies to citrulline (post-translationally modified arginine) Presence greatly enhances specificity for JIA in the presence of RF positivity (>90%) Present in about 10% psoriatic arthritis without RF Presence of anti-CCP can predict future disease – can precede disease by several years

Anti-nuclear Antibodies (ANA)

“LE cells” 1948 – phagocytosis of antibody-sensitized nuclei ANA test Detection of antibodies to nuclear elements by indirect immunofluorescence of epithelial cells (Hep-2) incubated with serial dilutions of sera Report highest dilution where binding still present Pattern is not as helpful as specific ANA ’s Speckled Anti-double stranded DNA Anti-Smith

Anti-nuclear Antibodies (ANA)

Who should be tested?

ANA has a low positive predictive value in the absence of findings suggestive of autoimmune disease Fever, rash, photosensitivity, alopecia, mucosal lesions, serositis, nephritis, cerebritis, arthritis ANA in the absence of autoimmune disease does not predict the development of autoimmune disease Children with a positive ANA referred to a pediatric rheumatologist have <5% chance of developing an autoimmune disease if not diagnosed at the first visit

Anti-nuclear Antibodies (ANA)

Who should be tested?

Prevalence in healthy children 30% at 1:40 5% at 1:160 Prevalence of lupus in children 0.05% Only about 50% of children referred to a pediatric rheumatologist with an ANA have an autoimmune disease – vast majority of those have JIA

Anti-nuclear Antibodies (ANA)

Systemic Lupus Erythematosus (SLE) ANA is highly sensitive (nearly 100%), but low PPV (0.06) Anti-dsDNA and Anti-Smith are highly specific Diseases with >90% positivity for ANA Mixed Connective Tissue Disease (Anti-U1-RNP) Sjogren ’s Syndrome (Anti-SS-A and Anti-SS-B) Systemic Sclerosis (Anti-topoisomerase I (Scl-70)) Diseases with increased prevalence of ANA JIA (increased to about 75% with uveitis) Dermatomyositis (Anti-Jo-1)

Anti-nuclear Antibodies (ANA)

Other autoimmune diseases with increased prevalence of ANA Thyroiditis, hepatitis, ITP, psoriasis, diabetes, multiple sclerosis, drug-induced (minocycline) Other diseases with increased prevalence of ANA Post-viral, tuberculosis, SBE, chronic osteomyelitis, malaria, hepatitis C, parvovirus B19, lymphoma, leukemia

ENA Autoantibodies

Sm (Smith) Highly specific but relatively insensitive for SLE 20-40% of SLE, stable over time, poor prognosis U1-RNP 100% of MCTD (diagnostic criterion) – very high titer Most SLE with Smith also have RNP SS-A (Ro) 40% of SLE, 75% of Sjogren ’s Syndrome 100% of neonatal lupus, but only 2% SS-A+ mothers SS-B (La) 20% SLE, 60-70% Sjogren ’s Almost never without SS-A in SLE or SS

Anti-double stranded DNA

Highly specific for SLE, but only moderately sensitive (50-70%) Presence will predict SLE – 2/3 with positive test within 1 year Level of titer may correlate with disease activity (nephritis) Rise in titer is predictive of flare (PPV >90%) Can rise several weeks before clinical flare Responding to rise in anti-dsDNA with increased steroids can reduce flare rate Presence of both Anti-dsDNA and Anti-Smith Essentially 100% SLE

Anti-Phospholipid Antibodies

Target anionic phospholipids and proteins that bind these lipids Anti-phospholipid Antibody Syndrome Thrombosis, miscarriage, thrombocytopenia Primary (Sneddon Syndrome) or Secondary (SLE) Tests for APAs (Positive tests >3 months apart) Anti-cardiolipin (80% sensitive, but only 35% specific) Anti-beta-2 Glycoprotein (95% sensitive, 22% specific) Lupus anti-coagulant (greater risk of thrombosis) Other diseases with APAs SLE, stroke, mononucleosis, HIV, syphilis, RA, preeclampsia, gestational diabetes

Anti-neutrophil Cytoplasmic Antibodies

ANCAs target lysosomal enzymes in neutrophils and monocytes 3 Patterns Cytoplasmic (C-ANCA) – 90% anti-proteinase 3 Granulomatosis with Polyangiitis (Wegener ’s) Poor correlation with disease activity Tuberculosis, Hodgkin ’s, HIV, myeloma, gammopathies Perinuclear (P-ANCA) – 70% anti-myeloperoxidase Microscopic polyangiitis (60% without C-ANCA) Churg-Strauss Syndrome (<60% P-ANCA) IBD, PAN, Kawasaki ’s, SLE Atypical (X-ANCA) – mixture of C and P RA, SLE, IBD

Other Autoantibodies

Anti-SCL-70 (Topoisomerase I) 25% of systemic sclerosis, but highly specific if not SLE Presence with Raynaud ’s phenomenon predictive of SS Highly predictive of pulmonary fibrosis Anti-Centromere (an ANA) 20% of systemic sclerosis (almost never with anti-SCL-70) CREST Syndromes – calcinosis and telangiectasias Anti-Ribosomal P Highly specific for SLE, associated with cerebritis Anti-Jo-1 (an anti-tRNA synthetase) Specific for myositis, rare in children Associated with interstitial lung disease

Markers of Inflammation

Erythrocyte Sedimentation Rate (ESR) Distance that red blood cells sediment in one hour Anti-coagulated blood in Westergren Tube 2 ml in 200 mm column Largely a surrogate for fibrinogen (60-70%) Factors that increase ESR Age, female, pregnancy, anemia, cholesterol, elevated Ig ’s, tilting, heat Factors that lower ESR – Leukocytosis, polycythemia, abnormal RBC shape, low Ig ’s, coolness, hypofibrinogenemia, clotting, vibration, delay in processing

Markers of Inflammation

Erythrocyte Sedimentation Rate (ESR) Not a diagnostic test, but a screen for inflammation Can use to monitor disease activity/response to treatment Core outcome measure in clinical trials of JIA Prognostic factor – higher ESR at diagnosis of oligoarticular JIA correlates with worse outcome

Markers of Inflammation

C Reactive Protein (CRP) Reacts with C polysaccharide of S. pneunomiae Binds to pathogens & apoptotic/necrotic cells – activates complement and phagocytosis Responds more rapidly than ESR – can increase in 4 hours of stimulus Assay requires small amount of serum More consistent than ESR

Markers of Inflammation

C Reactive Protein (CRP) In SLE, rise in CRP may indicate infection ESR often already elevated due to elevated Ig ’s, etc.

Role in atherogenesis In JIA – CRP correlates better with acute flares and ESR correlates better with long-term outcomes

Markers of Inflammation

Anemia Impaired erythropoiesis due to pro-inflammatory cytokines May correlate with disease activity C3 and C4 Increased by inflammation Depressed in SLE, post-infectious GN, MPGN, liver disease, congenital deficiency Ferritin Elevated by inflammation even if iron deficient Particularly elevated with hemophagocytosis Ferritin >10,000 has 96% specificity for HLH May correlate with activity in systemic JIA

Genetic Tests

HLA Antigens MHC is most strongly associated region with autoimmunity B27 – 90% of ankylosing spondylitis, 8% of population PPV for AS 3 to 6% Reactive arthritis, IBD-associated arthritis, acute anterior uveitis DR4 (shared epitope) – 70% of RA, 30% population B51 – up to 80% Behcet’s Disease, 20% population Systemic Autoinflammatory Syndromes Familial Mediterranean Fever – Pyrin (MEFV) TRAPS –TNFRSA1A/TNFR1 – Treat with anti-TNF Hyper IgD Syndrome – Mevalonate Kinase Cryopyrin Associated Disorders Muckle-Wells/NOMID/FCAS Treat with anti-IL1

Miscellaneous Tests

Angiotensin Converting Enzyme (ACE) Elevated in 50-80% of sarcoidosis patients From epithelial cells in granulomas (non-specific) May correlate with disease activity in sarcoidosis Muscle Enzymes CPK, LDH, aldolase, AST, ALT Elevation of at least 1 is 90% sensitive for dermatomyositis Factor VIII-related Antigen (von Willebrand Factor Antigen) Marker of endothelial cell damage May correlate with vasculitis activity

Questions?