Rheumatology Labs: Alphabet soup ?

1. 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 • Due to metabolic changes in liver causing increased plasma proteins • 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 issue important; pattern not important • Found in a variety of autoimmune diseases such as SLE, MCTD, JRA, scleroderma, Sjogren’s syndrome in high titres (>1:320) • Almost always present in SLE (95-98%)

7. Anti-nuclear Antibodies (ANA) • Low titres (<= 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 • Approximately 10% of the population has a positive low titer ANA and can be asymptomatic • As one ages, ANA titers increase (40% with low positive ANA by age > 60 yrs)

8. Anti-nuclear Antibodies (ANA) • ANAs do not correlate with disease activity ( i.e. diagnostic test) • Consider using as a screening test in only symptomatic patients (arthritis, rash, serositis, proteinuria) • Must measure ANAs in patients with JIA (esp. oligoarticular) to assess risk of uveitis

9. Anti-nuclear Antibodies (ANA) • Positive ANA is not in itself diagnostic and should only be viewed as a clue for possible underlying autoimmune processes. One must use other means (PE, hx, other labs) to make diagnosis • If the ANA is positive, it is helpful to further identify which antigen is being recognized by the ANA (ANA subtype)

10. ANA Subtype: Double Stranded DNA(anti-dsDNA) • 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

11. 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

12. ANA Subtype: Anti Ro/SS-A and Anti La/SS-b • Two sets of names assigned by two different groups; first seen in Sjogren’s patients and then seen in SLE patients • 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

13. ANA Subtype: Anti Ro/SS-A and Anti La/SS-b • 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

14. ANA Subtype: Anticentromere and Anti-Scl-70 Antibodies • Anticentromere Antibodies seen in limited cutaneous systemic sclerosis (CREST) • 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

15. ANA Subtype: Anti-histone antibodies • Anti-histone antibodies are found in 95% of patients with drug-induced lupus syndrome • Seen with: • Procainamide • Quinidine • Hydralazine • Phenytoin or other anti-epileptics

16. 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) • Perinuclear ANCA (p-ANCA) – staining of the nucleus and perinuclear area leaving cytoplasm clear. Main antigen is myloperoxidase (MPO).

17. ANCA • c-ANCA seen in 90% of Wegener’s granulomatosis • p-ANCA is associated with microscopic PAN, Churg-Strauss, and Ulcerative Colitis • Consider vasculitis (ANCA) if patient has: • Fever of unknown origin • Palpable purpura, vasculitis urticaria, or dermal necrosis • Mononeuritis multiplex • Unexplained arthritis, myositis, or serositis • Unexplained pulmonary, CV, or renal disease • Abnormal lab: ESR or CRP, WBC or eosinophils, low complements

18. 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

19. HLA-B27 • One of the histocompatibility genes important in transplantation • Associated with seronegative spondyloarthropathies: • 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

20. 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 an evaluative test and diagnostic test

21. Antiphospholipid Antibodies (aPL antibodies) • Group of heterogeneous antibodies against a variety of phospholipids (cardiolipin, phosphatidic acid, phosphatidyl-serine) and phospholipid binding plasma proteins (beta 2 GP-1, prothrombin, Factor X, protein C/S) • Associated with syndrome of coagulopathy, thrombocytopenia, recurrent spontaneous abortions, livedo reticularis, migraines, TTP, chorea, myelitis, and avascular necrosis of bone • Can occur with SLE (30-40%)

22. Antiphospholipid Antibodies: Screening • 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

23. What tests should be ordered? • A 3 year old girl with a two month history of a swollen and painful knee and eye findings

26. What tests should be ordered? • A 15 year old girl with multiple joint pains and joint swelling

28. What tests should be ordered? • A 8 yo boy with persistent fevers, intermittent rashes, and joint pain

29. Juvenile Idiopathic Arthritis • CBC-D • ESR • ANA • If polyarticular course, add RF • If older onset with sacroiliac tenderness and tendon insertion site tenderness, add HLA-B27 • If systemic course, add LFTs and coags (PT/PTT/d-dimers/fibrinogen)

30. What tests should be ordered? • A 13 year old Hispanic girl with a facial rash, joint pain, mouth sores, fatigue, and blood in her urine

32. 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)

33. What tests should be ordered? • A 6 year old boy with muscle weakness, leg pains, and rash over his eyelids and on his elbows

36. Dermatomyositis • CBC-D • ESR • ANA • CPK • Aldolase • LDH • AST/ALT

37. What tests should be ordered? • A 10 year old boy with difficulty swallowing, facial rash, joint pain, and shortness of breath, muscle pain

38. Mixed Connective Tissue Disease • CBC-D • ESR • ANA • ENA (anti-RNP and anti-Sm) • Consider Pulmonary Function Tests, ECHO, Swallowing studies

39. What tests should be ordered? • A 13 year old girl with recurrent sinusitis, rash, cough, joint pain, and hematuria

41. Vasculitis (Wegener’s Granulomatosis) • BUN/Cre • ANCA • ESR • CBC-D • Urinalysis, Uprot/cr • vWAg • (PFTs, Sinus and Chest CT)

42. What tests should be ordered? • A 17 year old woman with tightening of her skin, tapering of her fingertips, and cold blue hands and feet

44. Scleroderma • ANA • Anti-Scl 70 antibody • Anti-centromere antibody • Antiphospholipid antibody