Laboratory Testing in Rheumatology: Take the

High Value RoadWilliam E Davis, MD, FACP

Markers of inflammation◦ ESR◦ CRP

Rheumatoid factor and anti-CCP antibodies Anti-nuclear antibodies

Acute phase response◦ Cytokine production◦ Hepatic plasma

proteins ↑ 25% CRP SAA Complement Ceruloplasmin Haptoglobin Fibrinogen

◦ Negative acute phase proteins albumin, prealbumin,

transferrin

Transcription factors◦ Signal transducer

and activator of transcription 3 (STAT3)

◦ Janus activated kinase (JAK)

◦ Nuclear factor κB

Inflammation

Edmund Biernacki

Robert Sanno Fåhræus

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

ESR: Erythrocyte Sedimentation Rate

Simple Inexpensive Strong evidence base

ESR: Advantages

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

C-Reactive Protein

Advantages◦ Modest cost◦ Automated nephelometry◦ Serum test◦ Evidence data base solid

Limitations◦ Obese, elderly, ethnicity

CRP

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?

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?

1. Evaluate the extent or severity of inflammation

2. Monitor disease activity over time and with treatment

3. Assess prognosis

Utility of ESR / CRP

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

Rheumatoid Factor

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Anti-perinuclear factor (APF) Anti-keratin antibodies (AKA) Citrullinated filaggrin Cyclic citrullinated peptide (CCP)

Anti-Cyclic Citrullinated Peptide (CCP) Antibodies

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

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 ?

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

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 ?

RF and Anti-CCP

1948 LE Cell 1957 FANA test

Anti-nuclear antibodiesΥ

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ΥΥΥΥFITC

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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-tRNA sythetases (Jo-1)

Anti-nuclear antibodies

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

Titer Positive?

1:40

1:80

1:160

1:320

1:640

1:1280

1:2560

>1:5120

Coat beads or microtiter plates with multiple antigens

Incubate patient plasma; measure reactivity Any reactivity - positive

-SSA/Ro

-dsDNA

-Sm

RNP-

SSB/La-

Histone-

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

Systemic Lupus Erythematosus

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

Drug induced SLE

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

Sjogren’s Syndrome

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

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

Inflammatory muscle disease

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

Overlap syndromes

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)

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

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

Summary