Laboratory Testing in Rheumatology: Take the High Value Road

1. Laboratory Testing in Rheumatology: Take the High Value Road William E Davis, MD, FACP

3. Markers of inflammation • ESR • CRP • Rheumatoid factor and anti-CCP antibodies • Anti-nuclear antibodies

4. Inflammation • Transcription factors • Signal transducer and activator of transcription 3 (STAT3) • Janus activated kinase (JAK) • Nuclear factor κB • Acute phase response • Cytokine production • Hepatic plasma proteins ↑ 25% • CRP • SAA • Complement • Ceruloplasmin • Haptoglobin • Fibrinogen • Negative acute phase proteins • albumin, prealbumin, transferrin

5. Edmund Biernacki • Robert SannoFåhræus

6. ESR: Erythrocyte Sedimentation Rate • Electrostatic charges prevent rouleaux formation and sedimentation • Plasma proteins and fibrinogen ↑ • Microcytosis, polycythemia ↓ • Pregnancy, ESRD ↑ • Normal M <15mm, F < 20 mm • Elderly M = age/2 • Elderly F = age/2+10

7. ESR: Advantages • Simple • Inexpensive • Strong evidence base

8. C-Reactive Protein • Binds C-polysaccharide of streptococcus • Normal <1mg/dL (<10mg/L) • Binds apoptotic cells, Fcγ receptors, activates complement • >1000 fold increase in acute phase • Peak 2-3 days • T ½ = 19h • Persistently elevated in RA, tuberculosis, malignancy • > 15 mg/dL in bacterial infection

9. CRP • Advantages • Modest cost • Automated nephelometry • Serum test • Evidence data base solid • Limitations • Obese, elderly, ethnicity

10. 75 y/o caucasian male presents with new onset temporal headache x 2 weeks. • PMH: HTN, on ACE inhibitor • Normal vital signs and physical examination • ESR/CRP?

11. 47 y/o female with 10 year hx rheumatoid arthritis, on MTX and tnf-inhibitor (etanercept), presents with hx acute shaking chill, cough with brick red sputum, fever, physical examination and CXR c/w RML pneumonia • ESR/CRP?

12. Utility of ESR / CRP • Evaluate the extent or severity of inflammation • Monitor disease activity over time and with treatment • Assess prognosis

13. Rheumatoid Factor • Sheep cell agglutination test • IgM antibodies that recognize Fc of IgG • Normal: <15 I.U./L • 1% young healthy, up to 5% elderly • Present in RA, Sjogren’s syndrome, HCV-cryoglobulinemia • Prognostic Υ Υ Υ Υ Υ Υ Υ

14. Anti-Cyclic Citrullinated Peptide (CCP) Antibodies • Anti-perinuclear factor (APF) • Anti-keratin antibodies (AKA) • Citrullinatedfilaggrin • Cyclic citrullinated peptide (CCP)

15. Anti-CCP • Sensitivity 82.9% • Specificity 93-94% • Predicts development of RA in early arthritis • Associated with severe, destructive disease • Radiographic progression • Total joint prosthesis • Disability • May precede development of RA by years • 30-60% CCP+ up to 6 years before dx

16. RF and Anti-CCP • 48 y/o male with symmetric polyarthralgia progressive x 3-4 years • Hx HTN • PE: No joint swelling or deformity • Lab: normal CBC, mild increase AST, ALT <2x normal • RF + 55 IU • CCP negative • ?

17. RF and Anti-CCP • 48 y/o male with symmetric polyarthralgia progressive x 3-4 years • Hx HTN • PE: No joint swelling or deformity • Lab: normal CBC, mild increase AST, ALT <2x normal • RF + 55 IU • CCP negative • HCV – chronic HCV associated with RF and arthralgia

18. RF and Anti-CCP • 32 y/o female with symmetric polyarthralgia for 6 weeks; sx controlled with NSAID • PMHx: negative except G2P2 • PE: Slight joint swelling and tenderness MCP’s, wrists, ankles & MTP’s • Lab: normal CBC, CMP, slightly elevated ESR 30, CRP 2 mg/dL • RF + 55 IU • CCP >100 U/ml • ?

19. Anti-nuclear antibodies • 1948 LE Cell • 1957 FANA test Υ Υ Υ Υ Υ Υ FITC Υ Υ Υ Υ Υ Υ

20. Anti-nuclear antibodies • Chromatin associated antigens • DNA (dsDNA, ssDNA) • Histone • Kinetochore (centromere) • Ribonucleoproteins (snRNP) • Sm • U1 RNP • Anti-Ro/SSA and Anti-La/SSB • Ribosomal P protein • Nucleolar antigens • Kenetochore • Topoisomerase • RNA polymerase • PM-Scl-75 and PM-Scl-100 components of exoribonuclease • Aminoacyl-tRNAsythetases (Jo-1)

21. Fluorescent ANA test • Technician reads pattern and titer • Expensive • Subjective (1:160 or 1:320?) • Substrate • Rodent liver or kidney • Human cultured cell lines, e.g. Hep-2 • ELISA for specific antigen specificity • +ANA → ELISA testing

23. Coat beads or microtiter plates with multiple antigens • Incubate patient plasma; measure reactivity • Any reactivity - positive -SSA/Ro SSB/La- -dsDNA RNP- -Sm Histone-

24. Systemic Lupus Erythematosus • 31 y/o female presents with pericarditis • She reports intermittent joint swelling and pain, photosensitive dermatitis • WBC 3500, platelets 110,000 • ANA 95% sensitive • Anti-Sm specific • Anti-dsDNA specific and high levels predict renal disease

25. Drug induced SLE • 65 y/o F presents with several weeks inflammatory arthritis of hands. • PMH: HTN, CHF, multiple med’s • PE: swollen MCP joints • Lab: normal except WBC 4000, Platelets 125,000 • ANA: 1:320, homogenous • Negative DNA, Sm, SSA/Ro, SSB/La • Rheumatologist Rx’s hydroxychloroquine • Internist discontinues hydralazine • Anti-histone antibody positive

26. Sjogren’s Syndrome • 58 y/o female has symmetric joint swelling without deformity; she has dry eyes and dry mouth and swollen parotid glands • Lab normal except hypergammaglobulinemia • RF 150 IU • CCP negative • ANA 1:1280 • Anti-Sm, Anti-DNA neg • Anti-SSA, anti-SSB positive

27. 32 y/o female complains of fatigue, dyspnea, joint pain, and Raynaud’s phenomenon x 6 months • PE normal except Raynaud’s • Lab normal except ANA + 1:1280, nucleolar • Anti-topoisomerase (Scl70): diffuse systemic sclerosis • Anti-centromere : CREST syndrome • Pulmonary vascular hypertension • Raynaud’s with negative ANA: 7% risk of rheumatic disease • Raynaud’s with positive ANA: 19-30% risk of rheumatic disease

28. Inflammatory muscle disease • 62 yo male with joint pain, Raynaud’s, and symptoms of proximal muscle weakness • CPK 2000 • +ANA • 40-80% PM/DM patients have +ANA • Anti-Jo-1 associated with “anti-synthetase syndrome” and interstitial lung disease

29. Overlap syndromes • Myositis • Raynaud’s, arthritis, puffy fingers • Lupus or scleroderma overlap • “MCTD” • Anti-RNP, Anti-PM-Scl

30. Negative ANA: lupus unlikely • Positive ANA not helpful (%+): • Discoid lupus (5-25) • Fibromyalgia (15-25) • Rheumatoid arthritis (30-50) • Relatives of patients (5-25) • Multiple sclerosis (25) • Thyroid disease (30-50) • Silicone breast implants (15-25)

31. Summary • Symptomatic patient with Positive ANA: look for specificity • Lupus: DNA and Sm specific • Anti-DNA prognostic and an activity marker • Histone may indicate drug induced • SSA, SSB correlate with neonatal damage • Sjogren’s syndrome: SSA, SSB • Systemic sclerosis (SSc): 97% +ANA • Centromere: limited sclerosis and pulmonary hypertension (CREST) • Topoisomerase/Scl70: diffuse disease with poor prognosis • Inflammatory myositis: 40-80% + ANA, most specifics negative • Anti-Jo-1 : poor prognosis and risk of pulmonary hypertension • RNP , PM-Scl : associated with overlap syndromes (SLE, SSc) • Raynaud’s: ANA useful for prognosis

32. Summary • ESR/CRP • Identify extent or severity of inflammatory disease • Monitor disease activity (RA) • Assess prognosis in early arthritis • RF/CCP • Use anti-CCP test to improve the specificity for RA • +RF and +CCP predict worse prognosis • ANA • Very sensitive test for SLE but technically challenging • ANA specificities should be guided by clinical signs of autoimmune disease • Prevalence of ANA specificities may be very low