Overview of Rheumatology Labs: Alphabet soup?

1. Overview of Rheumatology Labs:Alphabet soup? Pediatric Rheumatology Red Team Resident Teaching Series

2. ESR ANA CRP dsDNA ENA RNP Smith SS-a/SS-b Cardiolipin Jo-1 CPK p-ANCA/ c-ANCA RF C3/C4 CH50 Histone Centromere Scl-70 VDRL/RPR DRVVT Beta 2 glycoprotein 1 LDH Aldolase What are all those letters?

3. Background • Rheumatology spans a group of diseases that have auto-immune components • Proposed mechanisms to auto-immunity including cross reactive antigens, molecular mimicry, and autoantibody amplification • We can identify auto-antibodies and therefore characterize clinical diseases • However, presence of auto-antibodies does not always predict disease

4. Diagnostic vs. Evaluative Tests • Need to distinguish to determine which test is appropriate • Diagnostic tests accurately distinguish a group of patients with a specific disease from a non-disease group • Evaluative tests monitor disease activity over time

5. Erythrocyte Sedimentation Rate (ESR) • Mainly used as a disease activity indicator • Method:Westergren method (most common) which measures the rate of settling of RBCs in anticoagulated whole blood • Nonspecific test of inflammation • Elevated in infection, IBD, cancer, pregnancy, trauma, and stress • Can be falsely low in conditions that don’t let RBCs undergo rouleaux formation (sickle cell anemia, Hereditary Spherocytosis, CHF, polycythemia)

6. Anti-nuclear Antibodies (ANA) • Immunoglobulins directed against structures within the cell (i.e. DNA, ribonuclear proteins, histones, and centromere) • Titer is important; pattern not important • Found in a variety of autoimmune diseases such as SLE, MCTD, JRA, scleroderma, Sjogren’s syndrome in high titers (>1:320) • Almost always present in SLE (95-98%) • Low titers (<= 1:160) found in: • Infections (EBV, CMV, Hepatitis B, bacterial endocarditis, HIV) • Drugs (hydralazine, INH, dilantin, tegretol, ETX, PCN, and sulfas) • Neoplasias (lymphoma) • It is sensitive but not that specific • Consider using as a screening test in only symptomaticpatients • Approximately 10% of the population has a positive low titer ANA and can be asymptomatic • ANAs do not correlate with disease activity ( i.e. diagnostic test) • If positive, should subtype (anti-dsDNA, ENA panel) • Must measure ANAs in patients with JIA to assess risk of uveitis

7. ANA Subtype: Double Stranded DNA (anti-dsDNA) • Anti-dsDNA antibody is an ANA subtype • In higher titers, highly specific for SLE • Seen in >80% of SLE patients at some time during their course • Associated with the presence of active lupus nephritis • Can detect flare up before clinically significant • Check anti-dsDNA levels if you suspect SLE in a child with positive ANA

8. ANA Subtype: Anti-Extractable Nuclear Antibodies (anti-ENA) • anti-Smith (Sm) and anti-ribonucleoprotein (RNP) • Both are directed against RNA proteins and are readily soluble in neutral buffers • Anti-RNP is specific for MCTD (high titer) • Anti-Sm ab is highly specific for SLE • 10-20% in Caucasians, 30% in Asians, 40% in African Americans • One of the lupus criteria

9. ANA Subtype: Anti Ro/SS-A and Anti La/SS-B • Associated with Sjogren’s, SLE, and neonatal lupus • Anti Ro/SS-A antibodies seen in: • 5-15% of normals • 50% of Sjogren’s patients • 30% of SLE patients (many have negative ANA or subacute cutaneous lupus) • Correlates with active nephritis and cytopenias • Crosses the placenta and is associated with neonatal SLE and heart block • Anti La/SS-B antibodies seen in: • 5% of normals • 15-85% of Sjogren’s patients • 10-15% of SLE patients • Also associated with neonatal SLE but do not see the cardiac manifestations

10. ANA Subtype: Anticentromere, Anti-Scl-70, and AntihistoneAntibodies • Anticentromere Antibodies seen in limited cutaneous systemic sclerosis • Anti-Scl-70 Antibodies (also known as anti-topoisomerase I) are assoicated with increased risk of pulmonary fibrosis in both limited and diffuse cutaneous systemic sclerosis • Anti-histone antibodies are found in 95% of patients with drug-induced lupus syndrome • Seen with: • Procainamide • Quinidine • Hydralazine • Phenytoin or other anti-epileptics

11. Antineutrophil Cytoplasmic Antibodies (ANCA) • Associated with vasculitides • Used as diagnostic test and possibly an evaluative test (still questionable) • 2 main staining categories: c-ANCA and p-ANCA: • Cytoplasmic ANCA (c-ANCA) – coarse granular staining of the cytoplasm. The main antigen is proteinase-3 (PR3). Seen in 90% of Wegener’s granulomatosis. • Perinuclear ANCA (p-ANCA) – staining of the nucleus and perinuclear area leaving cytoplasm clear. Main antigen is myloperoxidase (MPO). Associated with microscopic polyarteritisnodosa, Churg-Strauss, and Ulcerative Colitis.

12. Rheumatoid Factor (RF) • Uncommon in children • Should NOT be used as a screening test for rheumatic disease in children • Only indication is for polyarticular JIA patients to classify and offer prognostic information • Low titers seen in healthy children (<5%), infections (viral, SBE), malignancy, SLE • High titers seen in JIA (<20%) and predictive of erosive joint disease (follows adult RA course more) and MCTD

13. HLA-B27 • One of the histocompatibility genes • Associated with seronegativespondyloarthropathies: • ankylosing spondylitis (AS) (up to 90%) • IBD (25-50%) • psoriatic arthritis (<25%) • reactive arthritis (50-75%) • Less than 20% patients with HLA-B27 develop AS

14. Complements • Used to document complement consumption and diagnose rare complement deficiencies • CH50 (overall complement level) is decreased in SLE, MCTD, and immune complex vasculitis • Most common congenital complement deficiency is C2 • Low C3 and C4 levels seen in active lupus • Complement levels help follow disease activity and response to treatment in SLE • Considered a diagnostic and evaluative test

15. Antiphospholipid Antibodies (aPL antibodies) • Group of antibodies against a variety of phospholipids and phospholipid binding plasma proteins • Associated with syndrome of coagulopathy, thrombocytopenia, recurrent spontaneous abortions, livedoreticularis, migraines, TTP, chorea, myelitis, and avascular necrosis of bone • Can occur with SLE (30-40%), or seen without other autoimmune disease (antiphospholipid antibody syndrome) • Positive anti-cardiolipin antibodies (aCL) • IgG – associated with thrombosis • IgM – associated with thrombocytopenia only • Positive lupus anticoagulant: misnomer; antibody on the phospholipid of the prothrombin activator complex that causes in vitro anticoagulation (elevated aPTT and positive DRVVT) but in vivo paradoxical thrombosis • Dilute Russell Viper Venom Test (DRVVT) • β2glycoprotein-1 IgG and IgM

16. Practice Question #1a • A 3 year old girl with a two month history of a swollen and painful knee and eye findings

17. Practice Question #1b • A 15 year old girl with multiple joint pains and joint swelling

18. Practice Question #1c • A 8 yo boy with persistent fevers, intermittent rashes, and joint pain

19. Answer for Question 1a-c: Juvenile Idiopathic Arthritis • CBC-D • ESR • ANA to determine uveitis risk • If polyarticular course, add RF • If older onset with sacroiliac tenderness and tendon insertion site tenderness, add HLA-B27 • If systemic course, add LFTs, Ferritin, DDimer

20. Practice Question #2 • A 13 year old Hispanic girl with a facial rash, joint pain, mouth sores, fatigue, and blood in her urine

21. Answer for Question #2: Systemic Lupus Erythematosus • CBC-D • ESR • ANA • dsDNA • C3/C4 levels • Urinalysis and Urine protein/creatinine ratio • Antiphospholipid Antibodies (Anti-cardiolipin ab, PTT, DRVVT, and LAC, B2glycoprotein)

22. Practice Question #3 • A 6 year old boy with muscle weakness, leg pains, and rash over his eyelids and on his elbows

23. Answer for Question #3: Dermatomyositis • CBC-D • ESR • ANA • CPK, Aldolase, LDH (muscle enzymes) • AST/ALT (in this case, as a muscle enzyme)