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

2. What are all those letters? 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

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 1.Positive anti-cardiolipin antibodies (aCL) IgG – associated with thrombosis IgM – associated with thrombocytopenia only 2.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 3.Dilute Russell Viper Venom Test (DRVVT) 4.β 2 glycoprotein-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