Common Rheumatologic Tests: Evaluation And Interpretation

Common Rheumatologic Tests: Evaluation and Interpretation Beth Valashinas, D.O. Chief Rheumatology Fellow University of North Texas Health Science Center/ Plaza Medical Center

Disclosures • Nothing to disclose

Introduction • Immunologic laboratory testing in

rheumatology is useful for supporting or refuting a clinically suspected diagnosis • “Shotgun approaches” or “screening tests” often lead to false positives, and further unnecessary workups/referrals

Background • Defining attributes of a test – – – – – –

Sensitivity Specificity Positive predictive value Negative predictive value Likelihood ratios Pretest and posttest probabilities

Attributes of a test • Sensitivity

– Proportion of patients with a disease who have a positive test result

• Specificity

– Proportion of patients without a disease who have a negative test result

• Both sensitivity and specificity are independent of disease prevalence

Attributes of a test • Predictive value – likelihood of disease or lack thereof based on a positive or negative test result – Negative predictive value (NPV) • True negative/(true negative + false negative) – Positive predictive value (PPV) • True positive/(true positive + false positive)

Predictive value • Predictive value is significantly affected by disease prevalence

– Predictive value of a positive rheumatologic test in patient with polyarthralgias is likely to be higher in a rheumatology practice than in a family physician’s office – As pretest probability increases, so does the clinical utility of a specific test Lane, SK and Gravel, JR. American Family Physician. 65;6,1073,2002.

Attributes of a test • Likelihood ratio – LR for a negative test result:

(1-sensitivity)/specificity – LR for a positive test result:

sensitivity/(1-specificity)

Likelihood ratio • Provides additional measure by allowing

calculation of posttest probability based on pretest probability and test result • Decision to use a test should be based upon whether posttest probability will be significantly different from the pretest probability given a positive or negative test result

ACR AD HOC Committee. Arthritis Care and Research. 47:429, 2002.

Attributes of a test • If a test has a high positive likelihood

ratio (e.g., 10), and the test result is positive, then the posttest probability of the test will be greatly increased • If the likelihood ratio is only 1, then no difference would be expected between pretest and posttest probabilities

Performance characteristics of specific ANAs

Antigen

Condition

Sensitivity Specificity + LR - LR

Anti-dsDNA SLE Ab

57%

97%

16.3

0.49

Anti-Sm Ab

25-30%

high

*

*

Anti-Ro/SSA Sjogren’s, Ab SCLE

8-70%

87%

*

*

Anti-La/SSB Sjogren’s, Ab SCLE

16-40%

94%

*

*

Scl-70

20%

100%

>25

0.8

Anticentrom CREST e

65%

99.9%

650

0.4

Anti-U-3 RNP Scleroderma

12%

96%

3

0.92

SLE

Scleroderma

Colglazier, CL et al. Southern Medical Journal.2005

Acute phase reactants • Heterogeneous group of proteins

synthesized in liver in response to inflammation – – – –

Fibrinogen Haptoglobin C-reactive protein Alpha-1-antitrypsin

Acute phase protein response

Adapted from Gitlin JD, Colten HR in Pick E, Landy M [eds]: Lymphokines.14;123,1987.

Common markers of inflammation • ESR – Measures distance (in mm) that RBCs fall within specified tube (Westergren or Wintrobe) over 1 hour – Indirect measure of changes in acutephase reactants and quantitative Igs – Decreases by ~50% in 1 week after inflammation resolves

Mechanism of elevated ESR • If higher concentration of

asymmetrically charged acute-phase protein or hypergammaglobulinemia occurs, dielectric constant of plasma increases and dissipates inter-RBC repulsive forces, leads to closer aggregation of RBCs, so they fall faster, and cause ESR elevation

Hobbs, K in West, S. Rheumatology Secrets,2002

Noninflammatory conditions with elevated ESR • Aging • Female sex • Obesity • Pregnancy

Rule of thumb • Age-adjusted upper limit normal for ESR

– Male: age/2 – Female: (age + 10)/2

Causes of markedly elevated ESR • ESR >100 – – –

Infection, bacterial (35%) CTD (GCA, PMR, SLE, vasculitides (25%) Malignancy: lymphomas, myeloma, etc (15%) – Other causes (25%) Hobbs, K in West, S. Rheumatology Secrets,2002.

Causes of extremely low ESR • ESR ~ 0mm/hr – – –

Agammaglobulinemia Afibrinogenemia/dysfibrinogemia Extreme polycythemia (Hematocrit >65%) – Increased plasma viscosity

Approach to elevated ESR • Complete H&P • Routine labs (CBC, CMP, UA) • Up-to-date cancer screening/health

maintenance • Repeat ESR • If still elevated without other association

– Consider SPEP, CRP – Recheck in 1-3 months (up to 80% normalize)

C-reactive protein (CRP) • Pentameric protein – Trace concentrations in human plasma – Highly conserved over hundreds of millions of years of evolution – Properties of recognition and activation • Activates classic complement pathway • Modulates behavior of phagocytic cells (both inflammatory and non-inflammatory influence)

CRP • Acute phase reactant produced by liver – Response to IL-6, other cytokines

• Rises and falls quickly – – –

Elevation within 4 hr of tissue injury Peak at 24-72 hr Half-life ~18 hr

Rule of thumb • CRP <0.2 mg/dL: normal • CRP 0.2-1.0 mg/dL: indeterminate (may be seen in smoking, DM) • CRP >1.0 mg/dL: inflammatory • Levels > 10mg/dL suggest bacterial infection (up to 85%), or possibly systemic vasculitis, metastatic cancer Morely JJ, et al. Ann N Y Acad Sci;389,1982.

Serum protein electrophoresis (SPEP) • Quantifies the acute-phase response – Increase in alpha-1 and -2 zones (alpha1 antitrypsin and haptoglobin) – Increase in beta-gamma area (fibrinogen and CRP) – Decrease in pre-albumin, albumin, and the beta zone (transferrin)

Normal SPEP

erl.pathology.iupui.edu/LABMED/IMAGES/SPE_16A.JPG

SPEP- acute inflammation

erl.pathology.iupui.edu/LABMED/IMAGES/SPE_16 A.JPG

SPEP- Polyclonal gammopathy

erl.pathology.iupui.edu/LABMED/IMAGES/SPE_16A.JPG

Antinuclear antibodies (ANA) • Initial LE test in 1940s – Incubate bare nucleus with pt’s serum, allowing ANAs to bind to nucleus – Then add normal PMNs and if sufficient Ab have bound to nucleus, nucleus is opsonized and PMNs engulf the material – LE cell is PMN containing phagocytosed nucleus

LE Cell

Current ANA measurement • Fluorescence microscopy

– HEp-2 cells (derived from human epithelial tumor cell line) incubated with pt’s serum – Fluoresceinated Ab added, binds to pt’s Abs bound to nucleus – Amount of ANA determined by dilution of the pt’s serum - the greater the dilution (titer) at which nuclear fluorescence detected, the higher the ANA concentration

ANA • Arbitrary definition of positive ANA is the level that exceeds that seen in 95% of the population • Titers usually “positive” at 1:40 to 1:80 • Clinically significant titers (with HEp2 cells) ~1:160

ANA • High sensitivity in SLE, but poor

specificity • Positive ANA has predictive value of only 11% (positive LR =2.2) • ANA found in 5-10% of pts without CTD – Healthy pts, chronic infections (e.g., Hep C), multiple meds, etc.

ANA • Condition – – – –

SLE Drug induced lupus MCTD Autoimmune liver dz – Sjogren’s syndrome – Polymyositis – RA

• % ANA-positive – – – – – – –

99% 95-100% 95-100% 60-100% 75-90% 30-80% 30-50%

Adapted from Hobbs, K in West, S Rheumatology Secrets, 2002.

ANA • Condition – Multiple sclerosis – Pts with silicone breast implants – Healthy relatives of pts with SLE – Neoplasms – Normal elderly (>70 yrs)

• % ANA-positive – 25%

– 15-25%

– 20%

Adapted from Hobbs, K in West, S. Rheumatology Secrets, 2002

ANA • Is the ANA a good screening test for SLE?

– If >5% of normal U.S. population has positive ANA, then over 12.5 million “normal” people in U.S. are ANA positive – Prevalence of SLE is only ~1/1000, so only 250,000 individuals with SLE and positive ANA – If entire population was screened, more normal individuals would be detected with positive ANA than SLE pts. by ~50:1

Hobbs, K. in West, S Rheumatology Secrets. 2002..

ANA • Clinical value of ordering an ANA test can be dramatically enhanced when there is a reasonable pre-test probability of an autoimmune disease

ANA patterns • Homogeneous (diffuse) – SLE, drug-induced SLE, other diseases

ANA patterns • Rim (peripheral) – SLE, autoimmune hepatitis

ANA patterns • Speckled – SLE, MCTD, Sjogren’s, Scleroderma, other dz

ANA patterns • Nucleolar – Scleroderma, hepatocellular carcinoma

ANA patterns • Centromere – Limited scleroderma (CREST)

Drug-induced ANAs • Common drugs that cause positive ANAs – – – – – –

Procainamide Hydralazine Phenothiazines Diphenylhydantoin Isoniazid Quinidine

Lupus or ANA profile • If screening ANA is positive and

additional info needed to further delineate type of autoimmune disease • In extremely rare instances, ANA may be negative but SS-A antibodies may be detected in pts. with an SS-A associated disease

Lupus Profile dsDNA RNP

SM

SS-A

SS-B

CENTROMERE

SLE

60%

30%

30%

30%

15%

Rare

RA

(-)

(-)

(-)

Rare

Rare

(-)

MCTD

(-)

>95%

(-)

Rare

Rare

Rare

Scleroderm (-) a

Low titer

(-)

Rare

Rare

10-15%

CREST

(-)

(-)

(-)

(-)

(-)

60-90%

Sjogren’s

(-)

Rare

(-)

70%

60%

(-)

Hobbs, K. in West, S Rheumatology Secrets. 2002.

Lupus Profile • Antibodies to dsDNA are associated with lupus

nephritis, and often parallel disease activity • Antibodies to SS-A/Ro and SS-B/La are commonly associated with Sjogren’s syndrome • Anti-Ro/SSA antibodies increase risk for neonatal lupus/congenital heart block (CHB), especially when in conjunction with anti-La/SSB Ab – Overall risk is ~5%

Antibodies to ribonuclear protein (RNP) • Target is spliceosomal snRNPs in

nucleoplasm • Seen in SLE, scleroderma, mixed connective tissue disease (MCTD) • High levels very suggestive of MCTD – MCTD is overlap disease with features of SLE, scleroderma, and polymyositis

Anticentromere and SCL-70 Ab • Anticentromere Ab – up to 98% pts with limited scleroderma (CREST) – 22-36% pts with diffuse scleroderma

• Anti-SLC70 (anti-topoisomerase I) – 22-40% pts with diffuse scleroderma • longer disease duration, association with cancer, pulmonary fibrosis, digital pitting scars, cardiac manifestations

Anti-dsDNA Ab prior to Dx of SLE • Serum from 130 SLE patients

– 55% had anti-dsDNA Ab prior to SLE Dx – Mean onset of Ab 2.7 years prior to Dx

(range <1mo-9.3 years) – 58% of cases with at least 2 positive samples had significant rise in anti-dsDNA within 6 months of Dx

M. R. Arbuckle, et al. Scandinavian Journal of Immunology 54 (1-2) , 211–219.

Evaluation of pt with positive ANA and generalized arthralgias • H & P - any signs of CTD? • If ANA titer > 1:160, consider lupus

profile • Other possible tests: CBC, CMP, C3, C4, SPEP, RF, ESR, UA, lupus anticoagulant, anticardiolipin antibody

Antiphospholipid antibodies • Heterogeneous group of Ab that bind to plasma proteins, have affinity for phospholipid surfaces – – –

Anticardiolipin Ab (ACL) Lupus anticoagulant (LAC) Beta 2-glycoprotein I

Antiphospholipid antibodies • ACL measured by ELISA assay for IgG,

IgM, and IgA isotypes • LAC measured by phospholipiddependent screening test, if prolonged, add 1:1 mix with normal plasma - if no correction, LAC present • Beta 2-glycoprotein I measured by ELISA

Antiphospholipid antibodies • Conditions with positive aPL

– ~8% normal population – chronic infections e.g., HIV, Hep C – Medications e.g., phenothiazines, hydralazine, phenytoin, procainamide, quinidine – ~20% pts. with systemic vasculitis – ~15% pts. with recurrent miscarriage – ~50% pts. with SLE

Hansen, KE. in West, S Rheumatology Secrets, 2002.

Antiphospholipid antibodies • ~50% pts. with SLE and aPL will develop a thrombotic event • ~3-7% pts. per year who have aPL will experience a new thrombotic event • Overall positive predictive value of an aPL for future CVA, venous thrombosis, or recurrent MC is between 10-25% Hansen, KE. in West, S. Rheumatology Secrets, 2002.

Cryoglobulins • Immunoglobulins that precipitate in cold

temperatures • May cause hyperviscosity or vasculitis • Symptoms include fatigue, arthralgias/arthritis, cutaneous vasculitis or purpura, neuropathies, visceral organ involvement, and digital ischemia

Cryoglobulins • Type I- Monoclonal Ig (IgG or IgM) – Lymphoproliferative disorders

• Type II- Monoclonal IgM directed against polyclonal IgG

– Majority associated with Hepatitis C

• Type III- Mixed polyclonal IgG and IgM – Connective tissue diseases, chronic infections

Anticytoplasmic Antibodies • Often more helpful in diagnosis than antibodies against nuclear antigens • Seen with multiple autoimmune diseases and several forms of vasculitis

Anticytoplasmic antibodies Disease

Cytoplasmic Antigen

Frequency

Polymyositis

tRNA synthetase (anti-Jo-1, etc)

20-30%

SLE

Ribosomal P

5-10%

Wegener’s granulomatosis

Serine proteinase- 90% 3 (in neutrophils)

Microscopic polyarteritis

Myeloperoxidase (in neutrophils)

70%

Primary biliary cirrhosis

Mitochondria

80%

Hobbs, K. in West, S. Rheumatology Secrets. 2002.

Anti-neutrophil cytoplasmic Antibodies (ANCA) • C-ANCA – Most commonly seen in Wegener’s granulomatosis, microscopic polyarteritis, rarely Churg-Strauss vasculitis

ANCA • P-ANCA – seen in multiple diseases as well as vasculitis

P-ANCA • MPO positive – Microscopic polyarteritis – Pauci-immune GN – Churg-Strauss vasculitis – Drug-induced syndromes

• MPO negative – Ulcerative colitis – Autoimmune disease – HIV – Chronic infections or neoplasms (rare)

ANCA • If pt. tests positive to ANCA,

evaluation of specific antigen testing for MPO and PR3 should be undertaken • If C-ANCA is not against PR3 or PANCA is not against MPO, must consider causes other than vasculitis

Rheumatoid factor • Autoantibody directed against the Fc (constant) region of an IgG molecule

– Multiple isotypes, including IgM, IgG, IgA, and IgE – IgM RF is routinely measured using latex agglutination titers, nephelometry, and ELISA

Rheumatoid factor • Very low levels normal, but higher

production secondary to chronic immune stimulation • RF positive in ~80% of patients with RA • Multiple other causes of positive RF

Conditions associated with a positive rheumatoid factor • Rheumatologic diseases – – – – – –

RA (80-85%) Sjogren’s (75-95%) MCTD (50-60%) Scleroderma (20-30%) Sarcoidosis (15%) Polymyositis (5-10%)

• Non-rheumatologic conditions – – – – –

Chronic hepatitis Pulmonary disease Neoplasms Aging Cryoglobulinemia (40-100%) – Infections

• AIDS, Mono, TB, syphilis, parasites, endocarditis

Adapted from Kathryn Hobbs, from Rheumatology Secrets, 2002, p.60.

Frequency of RF positivity in normal population • AGE – 20-60 years – 60-70 years – >70 years

• Frequency of +RF – 2-4% – 5% – 10-25%

Adapted from Kathryn Hobbs in West, S. Rheumatology Secrets, 2002.

Anti-CCP antibodies • ELISA assay based on filaggrin from

human skin or synthetic citrullinated peptides • Target amino acid in filaggrin is citrulline, a post-translationally modified arginine residue • High specificity and moderate sensitivity for RA

Anti-CCP antibodies • Sensitivity 68% for RA • Specificity 98% for RA • Can be seen in active TB, other CTD • Clinical implications – Predictive of more aggressive disease with more progressive joint damage

Early antibody production as indicator of future disease? • Longitudinal study of 79 RA patients – ~50% produced anti-CCP Ab and/or IgM-RF prior to onset of disease – Positive results occurred median of 4.5 years (range 0.1-13.8) before symptom onset – Elevated levels of either IgM-RF or anti-CCP may imply high risk for development of RA M. J. Nielen, et al. Arthritis Rheum 50:380, 2004.

Complement • Cascade of proteins activated by many agents, including immune or antigenantibody complexes • May be decreased due to

– Increased consumption (proteolysis) • Increased levels of circulating immune complexes activate classical pathway

– Decreased production • Hereditary deficiency or liver disease

Hereditary complement deficiencies • May see SLE-like disease with

deficiencies in C1-C4 • Terminal complement (C5-9) deficiencies lead to recurrent infections • Deficiency in C1 INH leads to angioedema (hereditary or acquired)

Diseases associated with low complement levels • Rheumatic diseases

– SLE, systemic vasculitis, cryoglobulinemia,

RA (rare) • Glomerulonephritis

– Post streptococcal and membranoproliferative

• Infectious diseases

– Bacterial sepsis, SBE, Hepatitis B, other viremias, parasitemias

Complement level assessment • C3 and C4 generally decreased with increased disease activity in SLE • Decreased levels may predict impending disease flares

– C4 lowers before C3 and remains lower longer

• CH50 not useful as disease activity marker

Serum uric acid levels • Age- and sex-dependent • Concentration levels rise with puberty in males and menopause in females • Age of onset – Peak for males: 40-50 years – Peak for females: >60 years

Serum uric acid levels • Hyperuricemia – –

> 7.0 mg/dL in males >6.0 mg/dL in females

• 24 hour urine collection – Urate >800 mg/24 hrs suggests overproduction – Urate <800 mg/24 hrs suggests underexcretion

Serum uric acid levels • Important considerations

– Only 15% of pts. with hyperuricemia develop gout – If uric acid level>10mg/dL, risk increases to 30-50% – In ~10% of patients with acute gout, serum uric acid levels are normal • Need joint aspiration and polarized light microscopy to diagnose with certainty

Asymptomatic hyperuricemia • Treatment indications – Acute overproduction e.g., tumor lysis syndrome – Severe hyperuricemia e.g., uric acid levels >12mg/dL • Risk of uric acid nephrolithiasis is ~50%

HLA-B27 • Sensitivity – – – – –

~95% for AS ~80% for Reactive Arthritis ~70% for SpA associated with psoriasis ~50% for SpA associated with IBD ~70-84% for uSpA

Shmerling RH. Geriatrics;51:22, 1996.

HLA-B27 • Specificity – Low given prevalence is ~8% in Caucasian population

• In patients with inflammatory back pain, HLA-B27 positivity yields

– 20-fold increased risk of SpA – 15-fold higher risk of radiological sacroiliitis Braun J, et al. Arthritis Rheum;41:58, 1998.

Synovial fluid analysis • Studies to perform – – –

Gram stain and culture Total leukocyte count with differential Polarized microscopy

Synovial fluid analysis Fluid type

Appearanc Total WBC %PMNs e Count/mm 3

Normal

Clear, 0-200 <10% viscous NonClear to sl. 200-2000 <20% inflammator turbid y Inflammator Slightly 2000y turbid 50,000 Adapted from Spencer, RT in West, S. Rheumatology Secrets, 2002

20-70%

Synovial fluid analysis • Noninflammatory joint effusions – OA, joint trauma, mechanical derangement, AVN

• Inflammatory synovial fluid – Multiple rheumatic disorders – Infectious arthritis

• Pyarthrosis – Joint sepsis – Pseudosepsis in gout, reactive arthritis or RA

Polarized light microscopy Crystal

Gout Monosodium urate (MSU)

Pseudogout Calcium pyrophosphate dihydrate (CPPD)

Shape

Needle

Rhomboid or rectangular Positive Blue

Birefringence Negative Crystal color Yellow parallel to axis Adapted from Spencer, RT in West, S. Rheumatology Secrets, 2002

CPPD and MSU crystals

Conclusions • Immunologic laboratory tests facilitate

diagnosis and provide information regarding specific disease manifestations, disease activity and prognosis • Clinical utility of laboratory evaluation can be enhanced by the employment of evidence-based guidelines • A thorough history and physical examination remain the best screening and diagnostic tools

References 1. 2. 3. 4. 5. 6. 7. 8. 9.

ACR AD HOC Committee on Immunologic testing in the rheumatic diseases: an introduction. Arthritis Care and Research. August 15, 2002, Vol. 47, No. 4 pp.429-433. Gitlin JD, Colten HR: Molecular biology of the acute phase plasma proteins. In Pick E, Landy M [eds]: Lymphokines. Vol. 14. San Diego, Academic Press, 1987, pp 123-153.) Morley JJ, Kushner I: Serum C-reactive protein levels in disease. Ann N Y Acad Sci 389:406-418, 1982. Macy EM, Hayes TE, Tracy RP: Variability in the measurement of C-reactive protein in healthy subjects: implications for reference intervals and epidemiological applications. Clin Chem 43:52-58, 1997. Morely JJ, et al. Serum C-reactive protein levels in disease. Ann N Y Acad Sci 1982;389:406-418. M. R. Arbuckle, J. A. James, K. F. Kohlhase, M. V. Rubertone, G. J. Dennis, J. B. Harley (2001) Development of Anti-dsDNA Autoantibodies Prior to Clinical Diagnosis of Systemic Lupus Erythematosus. Scandinavian Journal of Immunology 54 (1-2) , 211–219. M. J. Nielen, et al. Specific Autoantibodies Precede the Symptoms of Rheumatoid Arthritis. A Study of Serial Measurements in Blood Arthritis Rheum 2004,50:380-386. Shmerling RH. Rheumatic disease: choosing the most useful diagnostic tests. Geriatrics 1996;51:226,29-30,32. Braun J, Bollow M, Remlinger G et al. Prevalence of spondylarthropathiesin HLA-B27 positive and negative blood donors. .Arthritis Rheum 1998;41:58–67.

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Sheldon, J. Laboratory testing in autoimmune diseases. Best Pract Res Clin Rheum. 2004; 18;3; 249-69. Dorner, T and Hansen, A. Autoantibodies in normals- the value of predicting rheumatoid arthritis. Arthritis Res and Therapy.2004;6,5. Lane, SK and Gravel, JW. Clinical utility of common serum rheumatologic tests. American Family Physician. 2002; 65,6. Harris, E et al. Kelley’s textbook of rheumatology, Ed. 7th Ed., 2006. Kavanaugh, A and ACR AD HOC committee. Guidelines for immunologic laboratory testing in the rheumatic diseases: anti-DNA antibody testing. Arthritis Care and Res. 2002;47;5;546-55. Shojania, K. Rheumatology:2. What laboratory tests are needed? CMAJ 2000;162 (8):115763. Zochling, J et al. The current concept of spondyloarthropathies with special emphasis on undifferentiated spondyloarthropathies. Rheumatology (Oxford) 2005;44:1483. Schellekens GA, et al. The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrillunated peptide. Arthritis and Rheumatism 2000;42:155-163.