Laboratory Evaluation American College of Rheumatology

Transcription

1 Laboratory Evaluation

2 Objective 1 Describe laboratory tests which would be most useful in the evaluation, diagnosis, and prognosis of frequently occurring rheumatic diseases.

3 Laboratory Tests & Clinical Presentations of Rheumatic Disease Clinical Presentation Priority Tests Secondary Tests Acute monoarthritis Chronic monoarthritis Chronic polyarthritis Diffuse arthralgias or myalgias Vasculitis syndromes Thrombosis syndromes, recurrent miscarriage Synovial fluid exam: wbc, crystals, gram stain, culture Synovial fluid exam: wbc, crystals, gram stain, culture (include AFB and fungus), (joint imaging indicated, though not a lab test) ESR, C-reactive protein, rheumatoid factor, anticcp, ANA ESR, CRP, TSH, CBC Urinalysis, CBC, metabolic panel (creatinine, liver tests), ESR, CRP Lupus inhibitor, antiphospholipid panel, ANA, CBC with platelets Serum uric acid, serum calcium, consider CRP or ESR HLA-B27, chest x-ray ANA subsets if appropriate, creatine kinase, hepatitis antibodies, CBC, metabolic panel, urinalysis If indicative findings: ANA, CK ANCA panel, cryoglobulins, hepatitis serologies, ANA if SLE features Hematology thrombosis tests

4 Major Initial Questions for Diffuse Rheumatic Disease Presentation Is it arthritis (in the joints) or not (e.g. bursitis, fibromyalgia, etc.)? Answer by history and physical exam Is it inflammatory or not? Answer by history, physical exam, lab Lab tests: ESR and/or C-reactive protein (CBC) If arthritis, is it one of common inflammatory conditions? Answer by history, PE, lab Lab: rheumatoid factor, anti-citrullinated peptide, ANA panel.

5 Diagnostic Laboratory Evaluation of New Patient with Chronic Possibly Inflammatory Polyarthritis Inflammatory markers: ESR and/or C-reactive protein If elevated or inflammatory process suspected, add: For diagnosis/prognosis: Rheumatoid factor and/or antibody to cyclic citrullinated peptide (anticcp) Consider ANA panel If all normal, RA less likely, but patients with RA may have normal ESR & CRP, especially early in disease Follow-up depends on which tests are abnormal. Consider CBC, urinalysis, thyroid stimulating hormone (TSH) Consider Comprehensive Metabolic Panel (including creatinine, transaminases, alkaline phosphatase), HCV antibody (for diagnosis and as baseline for potentially hepatotoxic drugs)

6 Rheumatoid Factor (RF) RF is an autoantibody directed against IgG, the main immunoglobulin in normal serum The Fab (antigen-binding) portion of the RF binds to the Fc ( tail ) portion of the antigen, which is IgG Usually, the lab measures serum IgM RF, i.e. IgM anti-igg (see illustration) IgA RF and IgG RF tests rarely used in US Present in about 60-80% of rheumatoid arthritis patients, vs ~5% of normal Prognostic: high levels of RF in RA associated with more severe joint and extra-articular disease Present also in some other rheumatic diseases and chronic infections.

7 Rheumatoid Factor (RF) Antigen = normal IgG Y Antibody = IgM RF Y Y IgM RF binding to normal IgG

8 Utility of Rheumatoid Factor Assists in diagnosis Presence of RF in patient with compatible findings (i.e. symmetric, chronic polyarthritis) increases the certainty of a clinical diagnosis of rheumatoid arthritis, if other causes of RF excluded Assists in prognosis High titer RF (e.g. >50 international units) increases the likelihood that an untreated patient will have chronic and erosive arthritis. Assists in treatment decisions RA patient with high titer RF more likely to warrant early use of DMARDs

9 Clinical Associations of RF Rheumatoid arthritis (~80% of RA patients) Other rheumatic diseases Sjogren s syndrome (~90%) SLE (15-20%) Mixed cryoglobulinemia syndrome (95%) Sarcoidosis (~15%) Parvovirus arthropathy (~15%, transient) Chronic infection Chronic hepatitis C infection (~50% of HCV patients have serum RF) Chronic osteomyelitis Bacterial endocarditis Monoclonal IgM paraproteins Normal aging (RF present at low titers)

10 Problem with RF as Screening Test RA Prevalence of RA ~ % RF + rate in RA about 80% Chronic HCV Prevalence about 1-2% in USA RF + rate in HCV about 40-70% Given positive RF in random individual in US population, risk of HCV about same as RA Implications: Consider HCV arthritis in RF+ patients with non-erosive arthritis or arthralgias

11 Anti-CCP Diagnostic Test Similar role as RF, similar sensitivity but more specific Less common in Sjogren's or SLE- still some Not seen in chronic HCV or other infections Predicts severe and erosive disease Present in early synovitis Not perfectly correlated with RF, i.e. some patients discordant for RF and anticcp, though most RA patients positive for one are also positive for the other.

12 Antibodies to Cyclic Citrullinated Peptides (anticcp) Citrulline is a modified amino acid, derived by chemical change in the amino acid arginine. Autoantibodies to citrullinated peptides (citrulline contained within a protein or peptide) are highly specific for rheumatoid arthritis, rarely seen in other conditions. Tests for these autoantibodies employ citrulline peptides that are in a cyclic peptide, i.e. antibodies to cyclic citrullinated peptide (CCP)

13 Early Diagnosis & Prediction of RA

14 Contribution of RA Diagnostic Criteria Visser, et al, Arthritis Rheum 2002

15 Summary of Diagnostic Use of AntiCCP and RF in RA Pooled Sensitivity Specificity Data Anti-CCP 77% 97% RF 74% 78% From Vossenaar & van Venrooij, Conclusions: AntiCCP about equally sensitive as RF, more specific than RF for diagnosis of RA

16 Evaluation of Monoarthritis If recent history of trauma and compatible exam, evaluate as traumatic injury Consider imaging, possibly joint fluid analysis for blood and fat. Presence of fat indicates fracture. If trauma and history do not explain, synovial fluid analysis (arthrocentesis and lab exam) recommended.

17 Why Joint Aspiration? Rule out infection Confirm crystals Confirm type of arthritis crystal Sodium urate (gout) Calcium pyrophosphate dihydrate deposition (CPPD pseudogout) Apatite not identified by usual microscope techniques in most labs Blood, trauma

18 Intracellular Urate Crystal

19 What If Crystal Exam Negative? Repeat synovial fluid examination improves the diagnostic sensitivity: 3 /8 CPDD (37.5%) and 2/17 (11.8%) MSU crystal SF specimens were picked up by repeat examination alone At least 3/5 of the positive cases detected on repeat exam alone were clinically significant Reason for delayed visibility unclear Just another look, more detailed exam? Possible maturation/change of crystal to enhance birefringence? (Peter Simkin)

20 Serum Uric Acid (Urate) Level Pre-puberty mean uric acid 3.5 mg/dl Males post-puberty mean 5.2 mg/dl Upper limit of normal (95%ile) ~ 7.0 mg/dl Females adult, pre-menopausal mean 4.0 Female adults, post-menopausal mean 4.7 Upper limit of normal (95%ile) ~ 6.0 mg/dl Hyperuricemia common in middle age, with 5% of men 7.0 Most with modest hyperuricemia do not develop gout (~1/3 men with urate >8 eventually develop gout) (Framingham Study data, Hall et al Am J Med 1967; 42: 27-37) Men with urate>9.0 mg/dl: gout in 22% after 5 years (Campion et al, Am J Med 1987; 82:421)

21 Spot Urine Urate Sometimes Used to Assess Urate Production & Distinguish Overproducer (ratio>0.6) from Underexcretor (ratio <0.6) of Uric Acid Peter Simkin, Annals of Internal Med, 1979

22 Synovial Fluid Classification Type of Fluid Appearance Leukocytes/mm 3 Normal Noninflammatory Clear, colorless, viscous Clear, yellow, viscous < Inflammatory Cloudy, yellow, ,000 decreased viscosity Septic Purulent Usually >50,000 (>95% PMNs) Note: viscosity assessment usually done by person performing arthrocentesis, not by laboratory.

23 Synovial Fluid WBCs in Non-gonococcal Septic Arthritis Synovial Fluid WBC Sensitivity Specificity LR+ LR- >100,000/mm 3 29% 99% >50,000/mm 3 62% 92% >25,000/mm 3 77% 73% >17,500/mm 3 83% 67% >10,000/mm 3 90%??? PMN 90% 73% 79% Total synovial fluid wbc and % PMN s lab predictors of septic joint, but only mild elevation in wbc in some patients. WBC 17,500 has been proposed as cut-off for active consideration of infection in most patients. Median value in septic arthritis is 70,000. LR+ = positive likelihood ratio, LR- = negative likelihood ratio Adapted from Margaretten, JAMA 2007; Li, Emerg Med J 2007; Söderquist, Scand J Infect Dis. 1998

24 Objective 2 Recognize the serologic associations of rheumatic diseases.

25 Major Autoimmune Rheumatic Diseases and Autoantibodies Autoantibody Type Disease Examples Other immunology laboratory Clinical Features Antinuclear Antibodies Systemic lupus erythematosus AntidsDNA,, low complement, anticardiolipin rashes, kidney, CNS, lung, blood cells, thrombosis, others Limited cutaneous systemic sclerosis (CREST syndrome) Anti-centromere Calcinosis cutis Raynaud's Esophageal disease Sclerodactyly (scleroderma of only the distal fingers) Telangiectasis Generalized systemic sclerosis AntiScl70 diffuse skin fibrosis, kidneys, lung, GI tract Sjogren syndrome AntiSSA (Ro) AntiSSB (La) dry eyes, dry mouth, Arthralgia/arthritis Polymyositis/ Dermatomyositis Jo-1 muscle weakness, frequently lung fibrosis, frequently rashes, arthritis, Raynaud's Antineutrophil Cytoplasmic Antibody (ANCA) Wegener's granulomatosus canca pattern, antiproteinase 3 lung, kidney, sinus, nose, eyes Microscopic polyangiitis panca pattern, antimyeloperoxidase kidneys, lung, sometimes other (e.g. nerves, skin) Anticardiolipin Antiphospholipid syndrome Lupus inhibitor, antiβ2- glycoprotein I arterial and venous thromboses, recurrent spontaneous abortion, thrombocytopenia Anticytoplasmic SLE Anti-ribosomal P CNS lupus (psychosis, depression) Autoimmune liver disease Anti-mitochondria, antismooth muscle Abs Elevated tests of liver damage (e.g. AST, ALT), other autoimmune features Rheumatoid Factor Rheumatoid arthritis RF, ESR, C-reactive protein Arthritis, sometimes extra-articular features. Anti-CCP

26 AutoAbs, ANA Subsets & Dx Frequency (%) of Positive Test Result in Clinical Diagnosis or Condition = Sensitivity for Listed Condition Antibody Nl SLE Drug LE MCTD Sjogren SSc CREST DM/ PM ANA dsdna RA Histones Nl = normal reference population. MCTD = mixed connective tissue disease. SSc = systemic sclerosis or scleroderma CREST = limited scleroderma with calcinosis, Raynaud s, esophageal dysmotility, sclerodactyly, telangiectasia MD/PM= dermatomyositis/ polymyositis RNP Sm 30 Ro (SSA) La (SSB) Scl Centromere Jo-1 30 Ribosomes 1 20 RF <

27 Autoantibodies in Systemic Lupus Central to the epidemiologic classification of SLE via American College of Rheumatology (ACR) criteria 2 of the criteria are autoantibodies Central to diagnosis and evaluation of SLE Some used as prognosticators and as activity markers. Clues to pathogenesis and etiology of SLE. Present years before first symptom & dx

28 Immunofluorescence ANA on HEp-2 Cells Negative Positive, Homogeneous pattern, strong intensity Weak intensity

29 Classic Autoantibodies in SLE ANA + 99% sensitive Specificity 80-95% Much lab variability Anti-dsDNA ~80% sensitive Specificity 95%+ Anti-Sm ~20% sensitive Specificity very high

30 Multiple Autoantibodies in SLE Multiple autoantibody response typical Average of about 3 of 7 commonly tested antibodies present at Dx ANA, dsdna, Sm, RNP, SSA, SSB, Ribosomal P, antiphospholipid Specific ANA markers include Sm and dsdna

31 Antibodies to dsdna Antibodies to double-stranded, native DNA (anti-dsdna) is the most important antibody in SLE High specificity Disease activity marker, with elevations often preceding a flare Associated with diffuse proliferative lupus nephritis and more severe flares Plays pathogenic role in immune complexes within kidney

32 Evaluation of a Positive Test for Antibodies to Nuclear Antigens Flow Diagram for Clinical (ANA) Use of ANA Test Positive ANA Result High Probability of Autoimmune Rheumatic Disease Low Probability of Autoimmune Rheumatic Disease Identify specific ANA antigen Search for other evidence of disease or organs involved Consider ancillary tests, e.g. complement, other autoantibodies eg anticardiolipin, antiribosomal, anti- C1q, Coomb s test (antirbc) Low titer or transient ANA Ignore, reassure patient Search for alternative diagnosis or explanation, e.g. HCV, drug, autoimmune thyroid dx Follow Patient High titer persistent ANA Search for other evidence of disease or organs involved Identify Antigen

33 Anti-Phospholipid Syndrome Arterial and/or venous thrombosis Recurrent miscarriage during second and third trimesters Thrombocytopenia Positive test for antibodies to phospholipids and/or lupus anticoagulant (= lupus inhibitor test) At increased risk for neurological or cardiac disease

34 Antiphospholipid Syndrome: Diagnostic Criteria Clinical Criteria Venous thrombosis Arterial thrombosis Recurrent fetal loss Thrombocytopenia Laboratory Criteria IgG acl (mod/high titer) IgM acl (mod/high titer) Lupus anticoagulant Anti-β2-glycoprotein I IgG or IgM (>99%ile) Dx by presence of one clinical and one laboratory criterion & apl present on 2 occasions measured 3 months apart Miyakis S, et al, 2006, J Thromb Haemost 4:

35 Interpretation of Standardized Anticardiolipin Assays International standards for IgG & IgM anticardiolipin Units: acl-igg (GPL) and acl-igm (MPL) units APL Syndrome associated with medium (>40 GPL or MPL) or high (greater than 80 GPL or MPL) levels of anticardiolipin antibodies.

36 Interpretation of Low Positive Anticardiolipin Assay Results The clinical significance of relatively low positive results (20-40 GPL or MPL units) is unclear, Lower levels not used to classify the disorder by epidemiological criteria. Interpretation and use of IgA anticardiolipin and other antiphospholipid antibodies unclear and not recommended Many more false positives, relatively few true positives

37 Conclusions - Diagnosis of APLS Lupus anticoagulant at high titer (strongly positive) best predictor for thrombosis. Higher titer greater significance Role of non-standard antibodies (e.g. anti-phosphatidyl serine, antiprothrombin, etc.) remains uncertain Prognostic value only fair

38 Complement Used mainly to assess disease activity in lupus and some forms of vasculitis (e.g. cryoglobulinemia) in which complement is low because of consuming (using up) the complement proteins Elevated levels of complement common in inflammatory conditions without complement consumption Low levels most commonly due to consumption, during flares of immune complex disease Low levels also may occur because of congenital deficiency Three commonly-available complement measures Total hemolytic complement (CH50) is a test of complement function C3 and C4 measurement quantifies mass concentration (not function) of the C3 and C4 complement proteins C4 tends to be most sensitive to changes C4 sometimes is low because of inherited partial deficiency, which is common among patients of northern European background

39 CLASSICAL PATHWAY Complement Cascade ALTERNATIVE PATHWAY Immune Complexes C1q,r,s CH50, total hemolytic complement measures function of entire pathway C4 C2 C3 and C4 measured as antigen or mass. C3 C5 D B C6, C7, C8, C9 Bacterial Surfaces Negative Charges C3b C5b-9 Membrane Attack Complex

40 Clinical Complement Use Test Total hemolytic complement (CH50) Classical Activation (SLE) Alternate Pathway (sepsis) Inherited Deficient C2 Poor Specimen Handling Function of proteins is heatsensitive C3 N N C4 N N N Factor B (not routine) N- N N

41 ANCA Testing ANCA = anti-neutrophil cytoplasmic antibody Immunfluorescence antibody (IFA) test: staining of cytoplasm of normal polymorphonuclear leukocytes (PMNs) Two patterns c-anca= granular staining throughout cytoplasm Antigen recognized is usually PMN granule constituent proteinase-3 (Pr-3), tested by ELISA p-anca = perinuclear staining of cytoplasm Many antigens Most common and important is PMN granule constituent myeloperoxidase (MPO), tested by ELISA

42 ANCA Patterns in IFA Test c-anca Pattern p-anca Pattern

43 ANCA Clinical Use Useful for certain types of vasculitis Wegener s granulomatosis Microscopic polyangiitis (renal and pulmonary vasculitis) Churg-Strauss syndrome c-anca immunoflourescence pattern and antibodies to proteinase-3 (Pr3) are very specific p-anca immunoflourescence pattern and antibodies to myeloperoxidase (MPO) less specific Not sufficiently sensitive to rule out vasculitis

44 ANCA in Wegener's Granulomatosis c-anca is a sensitive marker for Wegener's, found in about 95% of patients with multisystem, active disease prevalence falls to about 35% of patients with inactive Wegener's or Wegener's with only 1 or 2 organs effected (so-called "limited Wegener's"). c-anca is quite specific for Wegener's (>95% specific) occasionally found in some other forms of vasculitis and rarely in other conditions

45 Sensitivity of ANCA for Wegener s Granulomatosis Classical, multi-system WG Active Disease Inactive (treated) Disease 95% 65% Limited WG 65% 35% Adapted from Nölle, et al, Ann Int Med 1989

46 Categories of Myopathy Myositis/inflammatory muscle disease Autoimmune Primary Most commonly identified autoantibody: anti-jo-1 Other autoantibody tests less readily available Associated with other autoimmune disease Secondary to malignancy/paraneoplastic syndrome Drug/chemical related: statins, alcohol, many others Also non-inflammatory drug myopathies Glucocorticoids (e.g. prednisone), statins, etc. Metabolic Myopathies

47 Laboratory Tests of Muscle Damage or Inflammation (Myositis) Muscle-specific markers (elevated in both skeletal and cardiac damage) Creatine kinase (CK) Elevations rarely due to macro-ck, i.e. CK that appears to have high molecular weight due to anti- CK autoantibody Myoglobin Normal reference range higher in males than females, and in African-Americans than whites Some muscle disease markers are also in liver, therefore not specific for muscle disease Transaminases (AST>>ALT in muscle disease) Aldolase

48 Metabolic Causes of Myopathy Laboratory Tests Thyroid function tests TSH (supplemented by T4, T3, free T4 if necessary) Potassium Hypokalemic myopathy Phosphate Hypophosphatemic myopathy Severe vitamin D deficiency Phosphate binding laxatives or other agents Specialized tests Lactate, pyruvate, ammonia (as part of ischemic forearm muscle test) Some metabolic myopathies also associated with modest elevations in CK and/or myoglobin

49 Objective 3 Apply the concepts of sensitivity, specificity, likelihood ratio, and the receiver operator characteristics (ROC) curve of a diagnostic test to the clinical practice of laboratory testing.

50 Theoretical & Mathematical Aspects of Diagnostic Tests: Sensitivity & Specificity Sensitivity = Frequency of positive tests in patients with disease being diagnosed = True Positives/Total with Disease= TP/(TP+FN) Specificity = Frequency of negative tests in subjects or patients without the disease being diagnosed = True negatives/total without disease = TN/(TN+FP) Subject with Dx Subject without Dx Positive Test Result True positive (TP) False positive (FP) Negative Test Result False negative (FN) True negative (TN)

51 Does a Positive ANA = SLE? NO! Pretest probability in random population = prevalence=0.1% If ANA 95% sensitive & 95% specific SLE SLE No Yes ANA ~50,000 ANA - 50 ~949, ,000 1,000,000 + ANA post-test probability=950/50,000=~1/50=~2%

52 What If ANA Only 80% Specific Test? Pretest probability in random population = prevalence=0.1% ANA 95% sensitive & 80% specific SLE SLE No Totals Yes ANA ~199, ,750 ANA - 50 ~799, , ,000 1,000,000 + ANA post-test probability=950/200750=~1/211=~0.5%

53 Theoretical ANA Distribution in SLE, Normal, & Disease Control Populations Proportion with Given ANA Result (Relative) Distributions of ANA results overlap, i.e. no single value that is negative for all normal individuals and positive for all SLE patients. Normals Disease Control ANA Test Result, 1:x SLE Pts

54 Theoretical Distribution of ANA in SLE, Normal Subjects, & Disease Controls Proportion with Given ANA Result (Relative) Normals Disease Control 1:40 1:80 1:160 1:640 ANA Titers. Area under the curves to the left of titer line indicates proportion with negative results, area to the right have positive results at that titer. SLE Pts ANA Test Result, 1:x

55 Receiver Operating Characteristics (ROC) Curve With most diagnostic tests, there is a trade-off between sensitivity and specificity If the threshold (cut-off) for calling a test result positive is lower (moved to left, e.g. from 1:80 to 1:40), there will be more positive results, leading to higher sensitivity (more true positives) and lower specificity (more false positives). Conversely, if the threshold for calling a test result positive is higher (moved to right, e.g. from 1:80 to 1:160), there will be fewer positive results, leading to lower sensitivity (fewer true positives) and higher specificity (fewer false positives). This relationship over a range of thresholds (cut-offs) is called the receiver operating characteristics (ROC) curve. To compare utility of diagnostic tests, both the sensitivity and specificity need to be compared, and comparison patient groups should be equivalent. Often, specificity is higher in a normal population than in disease control population.

56 Receiver Operating Characteristics (ROC) Curve: ANA in SLE vs Normals & Disease Controls TP Rate (Sensitivity, %) 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 1:640 1:160 1:40 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% SLE vs Normals SLE vs Disease Control FP Rate (1-Specificity, %)

57 ROC Curve: ANA in SLE vs Normals & Disease Controls at 1:40 Cutoff in Representative Lab 1:40 1:160 Distribution (Relative) 1:80 1: ANA Test Result, 1:x TP (Sensitivity, %) 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% Distribution of ANA in Normals Distribution of ANA in SLE Patients Distribution of ANA in Disease Controls S i 2 1:640 1:160 1:40 0% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% FP (1-Specificity, %)

58 Theoretical & Mathematical Aspects of Diagnostic Tests: Likelihood Ratio Positive likelihood ratio (LR+) = true positive rate/false positive rate=tp/fp Negative likelihood ratio (LR ) = false negative rate/true negative rate=fn/tn Subject with Dx Subject without Dx Positive Test Result True positive (TP) False positive (FP) Negative Test Result False negative (FN) True negative (TN)

59 Likelihood Ratios Positive likelihood ratio (LR+) = true positive rate/false positive rate=tp/fp Higher is better LR+> 5 considered to be good test Negative likelihood ratio (LR ) = false negative rate/true negative rate=fn/tn Lower is better LR < 0.2 considered good test LR+ or LR- close to 1.0: test not predictive LR multiplied by pre-test odds = post-test odds

60 Example of Use of Likelihood Ratio with Low Pre-test Probability At ANA 1:40 threshold, 95% sensitive & 95% specific in representative lab LR+ = (TP rate/fp rate) = 95%/5% = 19 LR = (FN rate/tn rate) = 5%/95% = Patient with estimated pre-test probability of SLE of 1% (0.01), i.e. odds of 1:99. If ANA is negative at 1:40, then post-test odds 0.01 x 0.05 = =1:2000, i.e. odds very strongly against SLE If ANA is positive at 1:40, then post-test odds 0.01 x 19 = 0.19 = 1:5, i.e. odds still strongly against having SLE

61 Example of Use of Likelihood Ratio with SLE & High Pre-test Probability At ANA 1:40 threshold, 95% sensitive & 95% specific in representative lab LR+ = (TP rate/fp rate) = 95%/5% = 19 LR = (FN rate/tn rate) = 5%/95% = Patient with estimated pre-test probability of SLE of 50%, i.e. odds of 1:1=1.0 If ANA is negative at 1:40, then post-test odds 1.0 x 0.05 = :19 If ANA is positive at 1:40 in this patient, then post-test odds 1 x 19 = 19 = 19:1, i.e. odds strongly in favor of SLE diagnosis.

62 Clinical Algorithm for Lab Testing in SLE Very low pre-test probability: do not test, chance of false-positive is high Reasonable pre-test probability: screen with ANA in lab that documents that lab technique generates less than 10% positive rate in general population, or use ANA cutoff with similar rate If negative ANA, highly unlikely to be SLE If negative ANA and high clinical suspicion of SLE, use other approaches and tests to help confirm diagnosis If positive with high titer ANA and several lupus autoantibodies, much more likely to reflect SLE. Clinicians rarely perform quantitative likelihood calculations for individual patients, but estimate odds informally based on clinical impression supported by laboratory results.

63 Evaluation of a Positive Test for Antibodies to Nuclear Antigens Flow Diagram for Clinical (ANA) Use of ANA Test Positive ANA Result High Probability of Autoimmune Rheumatic Disease Low Probability of Autoimmune Rheumatic Disease Identify specific ANA antigen Search for other evidence of disease or organs involved Consider ancillary tests, e.g. complement, other autoantibodies eg anticardiolipin, antiribosomal, anti- C1q, Coomb s test (antirbc) Low titer or transient ANA Ignore, reassure patient Search for alternative diagnosis or explanation, e.g. HCV, drug, autoimmune thyroid dx Follow Patient High titer persistent ANA Search for other evidence of disease or organs involved Identify Antigen

64 Reasons for Repeatedly False- Positive Autoantibody Tests 1. Method too sensitive or cut-off too low in lab (depends on lab choice of cut-off) 2. Sticky serum, non-specific binding (technical reasons for assay positivity in patient with sticky immunoglobulins ) 3. Normal individual with high level of natural or normal autoantibodies (even normals have some autoantibodies) 4. Preclinical disease (antibodies precede diagnosis) 5. Alternative antibody-positive diagnosis (similar autoantibodies in different autoimmune diseases)

65 ANA-IFA Tests In Experienced Labs : 15 experienced ANA labs tested normal sera each lab used their own HEp-2 IFA method. Normal sera positive rates 1:40 titer 32% of 125 normal sera positive 1:80 titer 13% of 125 normal sera positive 1:160 titer 5% of 125 normal sera positive Authors recommendations: Report all results at 1:40 and 1:80 Report lab s false positive rate along with the test result Tan et al., Arthritis Rheum, 1997, 40:

66 ANA Results in Hypothetical Population Rheum: SLE, SSc, SS, etc. NonRheum: Thyroid, HCV, Unexplained ANA + Negative Adapted from NEJM Oct, 2003

67 Autoantibodies Occur Before Clinical Disease & Dx This is true in: SLE Rheumatoid Arthritis Sjögren s syndrome Antiphospholipid Syndrome Type 1 diabetes Autoimmune thyroid disease Pernicous anemia Probably many others # Autoantibodies Diagnosis Time before Dx (years) Arbuckle et al. Development of Autoantibodies before the clinical onset of systemic lupus erythematosus. NEJM: 2003; 349:

68 Cutting Edge Diagnostics, 2004

69 The Follow-up Area of Indeterminate Results For autoantibody interpretation, often helpful to consider indeterminate range of results, neither positive nor negative Wiik et al, 2004

70 Objective 4 Identify lab tests that are routinely used in monitoring activity of rheumatic diseases.

71 Lab Tests In Rheumatology Diagnostic use Primarily used to confirm a clinical impression Rarely diagnostic by themselves Changes probability of diseases Prognostic use Improves ability to prognosticate Monitoring Used to monitor disease activity, response to treatment, drug toxicity

72 ESR & CRP Useful for Monitoring RA Inflammation Support clinical evaluation of disease activity Correlates with other measures of disease activity, and objective As part of disease activity scores (such as DAS) Prognostic: persistent elevations predict increased erosive disease

73 Autoantibody Levels Generally Are Not Used to Monitor RA RF does decrease with treatment, but levels change slowly and are not useful as part of routine clinical management AntiCCP levels may decline if treatment initiated early in disease, but later in disease course do not fluctuate generally.

74 Monitors of Lupus Activity Serology AntidsDNA Elevations may precede flare of disease Elevations particularly associated with diffuse proliferative lupus nephritis, not as closely associated with other disease flares Levels may fall or rise at the time of a flare Complement Levels of complement serum proteins (C3 and C4) and function (total hemolytic complement, CH50) decline during disease flares, because of consumption No measure is perfectly correlated with disease

75 Monitors of Lupus Activity Target Urinalysis Organ Monitoring Urine protein quantitation 24 hour urine protein is traditional measure Spot protein/creatinine ratio is equally good measure and more convenient Serum creatinine CBC All cytopenias can be monitored

76 Monitoring of ANCA-Associated Vasculitis ESR, C-reactive protein as non-specific measures of inflammation Levels of ANCA (antimpo, antipr3, and/or immunofluorescence titer) all may be followed In some series, may predict disease course, with elevations heralding disease flare Imperfect predictor: should not, by themselves, dictate therapy

77 Monitoring of Myositis Levels of creatine kinase (CK) and/or myoglobin are good measures of inflammation and on-going muscle damage in most cases. Exceptions With severe disease and muscle atrophy, CK and myoglobin may be low despite ongoing muscle inflammation because of very low muscle mass. Occasionally, mild elevations may persist because of some permanently leaky muscle cells, despite absence of inflammation. Aldolase is both a muscle and liver marker, therefore less specific for following muscle disease, but often still useful to follow. Generally, only one test (usually, CK) is used to monitor disease, unless confusing or uncertain correlation with disease.

78 Objective 5 List laboratory tests that are part of monitoring for toxicity of antirheumatic therapies.

79 Medications & Laboratory Baseline Tests Lab tests should be performed before starting some drugs, primarily to check for risk factors for toxicity and safety. For drugs that are hepatotoxic, baseline liver testing (transaminases, hepatitis virus serology) recommended Methotrexate, leflunamide, others Many immunosuppressives have the potential for marrow toxicity, so baseline testing is important. Methotrexate, azathioprine, cyclophosphamide For azathioprine, thiopurine methyltransferase (TPMT,major azathioprine metabolizing enzyme) genotyping or phenotyping suggests drug dose range and geneticallydetermined risk of toxicity HIV status should be assessed before starting immunosuppressive drugs if not already known.

80 Medications & Laboratory Monitoring Tests Some lab tests are performed to monitor drug toxicity and/or efficacy. Cytopenias are typically dose-dependent for immunosuppressives, therefore increase monitoring frequency when dose increased Some side effects are not clearly dose-dependent e.g. sulfasalazine and gold cytopenias, gold proteinuria Some monitoring is recommended by drug manufacturers in the product insert ACR has produced practice guidelines as part of quality movement recommendations.

81 Baseline Lab for Drugs Drug CBC with Platelets Creatinine AST or ALT Alkaline Phosphatase Albu - min CXR Other Methotrexate X X X X X X Sulfasalazine X X X Urinalysis Leflunomide X X X X TNF antagonists (etanercept, infliximab, adalimumab) Anakinra X PPD Azathioprine X X X X TPMT Cyclophosphamide X X Urinalysis Gold (IM) X X X Urinalysis Cyclosporine X X X X BP Glucocorticoids Glucose, BP, DEXA NSAIDs (daily dosing) X (if renal risk) CBC = complete blood count = hemoglobin (Hb), hematocrit (Hct), white blood cell count ALT = amino transferase AST = amino transferase CXR = chest x-ray PPD = purified protein derivative skin test TPMT = thiopurine methyltransferase genotype or phenotype BP = blood pressure DEXA = bone densitometry X PPD Hb or Hct if GI risk Adapted from Cohen S, et al. Practice View, Fall, 2006, ACR

82 Drug Toxicity Monitoring Lab Tests Drug CBC with Platelets Creatinine AST or ALT, Albumin Urinalysis Other Methotrexate Sulfasalazine 12 Leflunomide Azathioprine 4-12 Cyclophosphamide 4-12 Annually (consider cytology) Gold (IM) Cyclosporine 4-8 BP Glucocorticoids BP, glucose annually NSAIDs (daily dosing) If renal risk, within 1 st 12 weeks, then at least yearly Hct or Hb if GI risk in first year (Frequency of testing, in weeks) Adapted and modified from Cohen S, et al. Practice View, Fall, 2006, ACR

83 Variations in Methotrexate Toxicity Monitoring ACR Quality Assurance Guidelines, 2006 CBC, creatinine, transaminases, & albumin every 8 weeks OR Practice Review 1(20) Fall, 2006 ACR RA Management Guidelines, 2002 CBC, creatinine, LFT s monthly x 6, then every 1-2 months. If elevated transaminases, every 2-4 weeks Arthritis Rheum 2002; 46: OR British Society of Rheumatology National Guidelines For the Monitoring of Second Line Drugs 2000 CBC & LFT s every 2 weeks until 6 weeks after last dose increase, then monthly. Metabolic panel (electrolytes, BUN) every 6-12 months; more frequent if suspect renal impairment US Rheumatologists Monitoring Survey Every 4-6 weeks: 35%. Every 6-8 weeks: 38%. More than 8 weeks: 23%. Less than 4 weeks: 4% Arthritis Rheum 2003; 48:

84 Objectives 1. Describe laboratory tests which would be most useful in the evaluation, diagnosis, and prognosis of frequently occurring rheumatic diseases. ESR, CRP, anticcp, RF, synovial fluid exam, uric acid 2. Recognize the serologic associations of rheumatic diseases ANA, ANA subsets, antidna, anticardiolipin, complement, ANCA 3. Apply the concepts of sensitivity, specificity, and the receiver operator characteristics (ROC) curve of a diagnostic test to clinical practice of laboratory testing. Be able to calculate approximate post-test likelihood, given a test result and a pre-test likelihood. 4. Identify lab tests that are routinely used in monitoring activity of rheumatic diseases. ESR, CRP, antidsdna, complement, urine, ANCA, CK 5. List laboratory tests that are part of monitoring for toxicity of anti-rheumatic therapies. CBC, liver tests, urine, other