Testing in the Rheumatic DiseasesTesting in the Rheumatic Diseases

Salahuddin Kazi, M.D.Salahuddin Kazi, M.D.

Questions to Answer When Applying a Valid Questions to Answer When Applying a Valid Diagnostic Test to a Specific Patient*Diagnostic Test to a Specific Patient*

• Is the test available, affordable, accurate and Is the test available, affordable, accurate and precise in our setting?precise in our setting?

• Can we generate a clinically sensible Can we generate a clinically sensible estimate of our patient’s pre-test probability?estimate of our patient’s pre-test probability?

• Will the resulting post-test probability affect Will the resulting post-test probability affect our management and help our patient?our management and help our patient?

*Evidence Based Medicine: 2*Evidence Based Medicine: 2ndnd Edition, Sacket et al, 2000 Edition, Sacket et al, 2000

Test Statistics - A ReviewTest Statistics - A Review

• SensitivitySensitivity - The proportion of affected - The proportion of affected individuals with a positive testindividuals with a positive test

• SpecificitySpecificity - The proportion of unaffected - The proportion of unaffected individuals with a negative testindividuals with a negative test

• Utility lies at the extremesUtility lies at the extremes - SpPin “High - SpPin “High specificity; positive test rules in” and SnNout specificity; positive test rules in” and SnNout “High sensitivity; negative test rules out”“High sensitivity; negative test rules out”

Gold StandardGold Standard

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True NegTrue NegFalse NegFalse Neg

SensitivitySensitivity

= a/ a+c= a/ a+c

SpecificitySpecificity

= d/ b+d= d/ b+d

Positive PredictivePositive Predictive

Value = a/ a+bValue = a/ a+b

Negative PredictiveNegative Predictive

Value = d/ c+dValue = d/ c+d

Prevalence = a+c/ a+b+c+dPrevalence = a+c/ a+b+c+d

Likelihood RatiosLikelihood Ratios

• The likelihood that a given test result would The likelihood that a given test result would be expected in a patient be expected in a patient withwith the target the target disorder compared with the likelihood that disorder compared with the likelihood that the same result would be expected in a the same result would be expected in a patient patient withoutwithout the target disorder the target disorder

• +LR +LR = sensitivity/(1-specificity)= sensitivity/(1-specificity)• -LR -LR = (1-sensitivity)/specificity= (1-sensitivity)/specificity

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Pre-testprobability

Likelihoodratio

Likelihood Ratio NormogramLikelihood Ratio Normogram

Case #1Case #1

• A 32 y/o woman describes a 6 week history of pain A 32 y/o woman describes a 6 week history of pain and stiffness in her handsand stiffness in her hands

• No history of fever, rash, dysuria, conjunctivitis, No history of fever, rash, dysuria, conjunctivitis, travel or exposure. No prior renal disease, seizures, travel or exposure. No prior renal disease, seizures, or serositis. Her mother has deforming arthritis.or serositis. Her mother has deforming arthritis.

• On exam there is warmth and soft-tissue swelling of On exam there is warmth and soft-tissue swelling of the 2the 2nd nd and 3and 3rdrd MCPs bilaterally, the 3 MCPs bilaterally, the 3rdrd right PIP, and right PIP, and the left wrist. There seems to be a small effusion in the left wrist. There seems to be a small effusion in the left knee.the left knee.

• Labs: SMA6 and U/A - normal; mild anemia; CRP Labs: SMA6 and U/A - normal; mild anemia; CRP 2.7; ANA - 1:40, diffuse; Rheumatoid Factor - 3202.7; ANA - 1:40, diffuse; Rheumatoid Factor - 320

Rheumatoid FactorRheumatoid Factor

• Anti-IgG - can be all Ig classesAnti-IgG - can be all Ig classes• Specific for Fc portion of IgGSpecific for Fc portion of IgG• Can be polyclonal (typical of autoimmunity) Can be polyclonal (typical of autoimmunity)

or monoclonal (typical of lymphoid or monoclonal (typical of lymphoid malignancy)malignancy)

• Causes immune complex damageCauses immune complex damage• Reported as “units” or titerReported as “units” or titer

RF Test CharacteristicsRF Test Characteristics

• Sensitivity for RA is ~80%Sensitivity for RA is ~80%• Specificity is 85-95%Specificity is 85-95%• +LR of 5-16 depending on population studied+LR of 5-16 depending on population studied• High titer is associated with more severe RA High titer is associated with more severe RA

with extra-articular manifestationswith extra-articular manifestations• Monitoring titer as an indicator of disease Monitoring titer as an indicator of disease

activity is not appropriateactivity is not appropriate

Conditions Associated with RFConditions Associated with RF

• Normal individuals (5%), especially with age (15%)Normal individuals (5%), especially with age (15%)• Rheumatoid arthritis (85%), Sjögren’s Syndrome, Rheumatoid arthritis (85%), Sjögren’s Syndrome,

SLE (25-50%)SLE (25-50%)• Viral Infections: Hepatitis C (25-50%), Viral Infections: Hepatitis C (25-50%),

mononucleosis, HIV, influenzamononucleosis, HIV, influenza• Bacterial Infections: IE (25-50%), TB (10-25%), Bacterial Infections: IE (25-50%), TB (10-25%),

leprosy, syphilis, brucellosisleprosy, syphilis, brucellosis• Parasites: Typanosomiasis, malaria, Parasites: Typanosomiasis, malaria,

schistosomiasis, etc.schistosomiasis, etc.• Other: Sarcoidosis, pulmonary fibrosis (10-25%), Other: Sarcoidosis, pulmonary fibrosis (10-25%),

chronic liver diseasechronic liver disease

Case #1 – Using the Test ResultsCase #1 – Using the Test Results

• Chronic, inflammatory, symmetrical Chronic, inflammatory, symmetrical polyarthritis of the hands in a young womanpolyarthritis of the hands in a young woman

• What’s your pre-test probability that this What’s your pre-test probability that this patient has RA?patient has RA?

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Likelihoodratio

In this case, a test In this case, a test with a moderate with a moderate +LR makes the +LR makes the diagnosis almost diagnosis almost certain in a patient certain in a patient with a high pre-test with a high pre-test probabilityprobability

Highly positive RF takes a 50% pre-test probability to Highly positive RF takes a 50% pre-test probability to a >95% post-test probabilitya >95% post-test probability

Case #2Case #2

• A 64-year-old female was evaluated for generalized A 64-year-old female was evaluated for generalized joint pain and muscle pain, fatigue, fever and chills joint pain and muscle pain, fatigue, fever and chills for the past 6-8 weeksfor the past 6-8 weeks

• No rash, Raynaud’s, weight lossNo rash, Raynaud’s, weight loss• Graves’ disease 18 years ago - radioactive iodineGraves’ disease 18 years ago - radioactive iodine• Family history: SLE, thyroid diseaseFamily history: SLE, thyroid disease• PE: Tender joints but no joint swellingPE: Tender joints but no joint swelling• Labs: Labs:

– CBC, Chem 7, LFT’s, UA, TSH - all normalCBC, Chem 7, LFT’s, UA, TSH - all normal– ESR 18 mm/h, CRP <0.8, RF negative, ANA ESR 18 mm/h, CRP <0.8, RF negative, ANA

positive 1:80, homogeneous patternpositive 1:80, homogeneous pattern

Anti-Nuclear AntibodiesAnti-Nuclear Antibodies

• Began with the demonstration of the “LE Began with the demonstration of the “LE cell” by Hargraves in 1948cell” by Hargraves in 1948

• Includes antibodies to a number of antigens, Includes antibodies to a number of antigens, including native DNAincluding native DNA

• Performed by indirect immunofluorescencePerformed by indirect immunofluorescence• Reported as “negative” - usually less than a Reported as “negative” - usually less than a

certain titer, or as a titer and patterncertain titer, or as a titer and pattern

ANA - CharacteristicsANA - Characteristics

• Sensitivity - 95-100%Sensitivity - 95-100%

• Specificity - Depends on titer used as cut-offSpecificity - Depends on titer used as cut-off– 15-30% of normals have ANA of 1:4015-30% of normals have ANA of 1:40

– 5% have ANA of 1:1605% have ANA of 1:160

• +LR is ~20; utility for SLE is based on prevalence:+LR is ~20; utility for SLE is based on prevalence:– General population 50/100,000General population 50/100,000

– Young, African-American women 400/100,000Young, African-American women 400/100,000

– Children/elderly men 1/100,000 Children/elderly men 1/100,000

Pattern Related Antigen Specificities Homogeneous Chromatin, Histone, DNA, Ku Peripheral or Rim DNA, Lamins Speckled RNP, Sm, Ro, La, Ku

Topoisomerase I (Scl-70) Nucleolar RNA Pol 1, Fibrillarin, PM-Scl Centromere CENPs Cytoplasmic Ribosomal P, Aminoacyl t-RNA syn-

thetases

Immunofluorescence Patterns of ANAsImmunofluorescence Patterns of ANAs

HomogeneousHomogeneous Rim or PeripheralRim or Peripheral

NucleolarNucleolar SpeckledSpeckled

Causes of a Positive ANACauses of a Positive ANA

Rheumatic Disease Percentage Positive

Disease Specific Ab’s

SLE >95% Anti-Sm, Anti-dsDNA

Systemic Sclerosis 60%-90% Anti-centromere Anti-Scl-70

Sjögren’s Syndrome 75% Anti-Ro, Anti-La

Mixed CTD 95%-99% Anti-RNP

Poly/Dermatomyositis 25% Anti-Jo-1

RA 15%-35% Rheumatoid Factor

Interpreting a Positive ANAInterpreting a Positive ANA

Pretest likelihood of lupus

ANA titer Action

80 Ignore Low 160 Observe; look for an

alternative explanation 80 Observe; look for an

alternative explanation Moderate

160 Check for disease-specific antibodies

Negative Observe; look for an alternative explanation

High Positive, any titer

Check for disease-specific antibodies

Other Causes of Positive ANAOther Causes of Positive ANA

Non Rheumatic DiseaseNon Rheumatic DiseaseInfectionsInfections

Inflammatory bowel Inflammatory bowel diseasedisease

Autoimmune hepatitisAutoimmune hepatitis

Pulmonary fibrosisPulmonary fibrosis

Endocrine diseasesEndocrine diseases

Hematologic diseasesHematologic diseases

Neoplastic diseasesNeoplastic diseases

End-stage renal diseaseEnd-stage renal disease

Post-transplantPost-transplant

Healthy PeopleHealthy PeoplePregnancyPregnancy

Older peopleOlder people

Family history of Family history of rheumatic diseaserheumatic disease

Drug inducedDrug induced

Case #2:Why is the ANA Positive?Case #2:Why is the ANA Positive?

• History and PE: Does not suggest a CTDHistory and PE: Does not suggest a CTD• Labs: normal except for positive ANALabs: normal except for positive ANA• Pretest probability of SLE is lowPretest probability of SLE is low• Posttest probability for SLE remains lowPosttest probability for SLE remains low• Look for an alternative explanationLook for an alternative explanation

– Elderly femaleElderly female– Positive family history of rheumatic diseasePositive family history of rheumatic disease

• Reassure: ANA result is a normal findingReassure: ANA result is a normal finding

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Although an ANA Although an ANA >1:160 has a high >1:160 has a high +LR, it should not +LR, it should not be used to screen be used to screen patients without patients without clinical evidence of clinical evidence of autoimmune autoimmune diseasedisease

Ordering an ANAOrdering an ANA

• To confirm the diagnosis of SLE when the To confirm the diagnosis of SLE when the clinical suspicion is highclinical suspicion is high

• To exclude SLE when the clinical suspicion To exclude SLE when the clinical suspicion is moderate (2 or 3 lupus criteria)is moderate (2 or 3 lupus criteria)

• Avoid ordering it when the clinic suspicion Avoid ordering it when the clinic suspicion for SLE is low - a positive result can cause for SLE is low - a positive result can cause diagnostic confusion and unnecessary diagnostic confusion and unnecessary anxietyanxiety

Anti-DNA AntibodiesAnti-DNA Antibodies

• Detect antibodies to native (double Detect antibodies to native (double stranded) DNAstranded) DNA

• Typical methods are ELISA and Typical methods are ELISA and immunofluorescence on immunofluorescence on CrithidiaCrithidia

• Can have both diagnostic and Can have both diagnostic and prognostic significanceprognostic significance

Anti-DNA - CharacteristicsAnti-DNA - Characteristics

Sensitivity - 60% for SLESensitivity - 60% for SLE

Specificity - 97%Specificity - 97%

Low titers seen in 2-5% of RA, Sjögren’s, Low titers seen in 2-5% of RA, Sjögren’s, scleroderma, relatives of SLE pts., etc.scleroderma, relatives of SLE pts., etc.

Average +LR of 16 and -LR of 0.49 means that a Average +LR of 16 and -LR of 0.49 means that a positive anti-DNA has a large impact, but lack positive anti-DNA has a large impact, but lack of one doesn’t exclude SLEof one doesn’t exclude SLE

Anti-DNA - PrognosisAnti-DNA - Prognosis

• SLE Disease activity: Useful, but with small SLE Disease activity: Useful, but with small +LR (~4)+LR (~4)

• Nephritis: Associated, but with very small Nephritis: Associated, but with very small +LR (~1.7)+LR (~1.7)

• Rising titers may predict a flare of disease Rising titers may predict a flare of disease activity in some, but not all, patientsactivity in some, but not all, patients

• Clinical correlation is advisedClinical correlation is advised

Anti-ENAAnti-ENA

• Small nuclear RNPSmall nuclear RNP– Sm: Seen in 15-30% of SLE; specificSm: Seen in 15-30% of SLE; specific– U1-RNP: 30-40% of SLE; also RA, Sjögren's, U1-RNP: 30-40% of SLE; also RA, Sjögren's,

scleroderma, and overlap syndromesscleroderma, and overlap syndromes

• Anti-Ro and anti-LaAnti-Ro and anti-La– Subacute cutaneous LESubacute cutaneous LE– Sjögren's syndromeSjögren's syndrome– Neonatal lupus with congenital heart blockNeonatal lupus with congenital heart block

5’ 3’

Sm Antigens

RNP Antigens

33kDa (A)33kDa (A)

EE FF

GG

28kDa (B)28kDa (B)

16kDa (D)16kDa (D)

70kDa70kDa

CC

U1RNA

28kDa (B’)28kDa (B’)

““ENA”-Extractable Nuclear AntigensENA”-Extractable Nuclear Antigens

Anti-Scl-70/Anti-CentromereAnti-Scl-70/Anti-Centromere

• Scl-70 = Topoisomerase I; seen in 40-70% of Scl-70 = Topoisomerase I; seen in 40-70% of patients with diffuse scleroderma; worse patients with diffuse scleroderma; worse prognosis with more organ involvementprognosis with more organ involvement

• Centromere - 70-85% of patients with limited Centromere - 70-85% of patients with limited scleroderma; associated with Raynaud’s scleroderma; associated with Raynaud’s syndromesyndrome

• Neither is diagnostic by themselves Neither is diagnostic by themselves

Case #3Case #3

• A 48-year-old male has chronic sinusitis with A 48-year-old male has chronic sinusitis with occasional bloody drainageoccasional bloody drainage

• You order a c-ANCAYou order a c-ANCA– Positive at 1:80Positive at 1:80

• The chest radiograph, creatinine and The chest radiograph, creatinine and urinalysis are normalurinalysis are normal

• What is the likelihood that he has Wegener’s What is the likelihood that he has Wegener’s granulomatosis?granulomatosis?

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1. Documented WG2. Pulmonary-Renal Syndrome3. Systemic Necrotizing Vasculitis4. Rapidly Progressive GN5. GN6. Hospitalized Patient

Jeanette: Amer J Kidney Dis 18:164, 1991

Positive Predictive Value of ANCAPositive Predictive Value of ANCA

This PatientThis Patient

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Wegener's is rare Wegener's is rare (~0.4/100,000). (~0.4/100,000). Without signs of Without signs of progressive, progressive, necrotizing vasculitis, necrotizing vasculitis, even a test with a high even a test with a high likelihood ratio is not likelihood ratio is not helpfulhelpful

ANCA CharacteristicsANCA Characteristics

• C-ANCA (Proteinase-3)C-ANCA (Proteinase-3)– 90% specificity and 50-90% sensitivity for 90% specificity and 50-90% sensitivity for

active Wegener's granulomatosisactive Wegener's granulomatosis

• P-ANCAP-ANCA– MPO - 60% of microscopic polyangiitis, Churg-MPO - 60% of microscopic polyangiitis, Churg-

StraussStrauss– Cathepsins, lactoferrin, elastaseCathepsins, lactoferrin, elastase

• Should not take the place of tissue biopsyShould not take the place of tissue biopsy

ANCAs and Rheumatic Autoimmune DiseasesANCAs and Rheumatic Autoimmune Diseases

• P-ANCA (not directed against MPO) reported in:P-ANCA (not directed against MPO) reported in:– RA, SLE, PM/DM, Sjögren's syndrome, Juvenile RA, SLE, PM/DM, Sjögren's syndrome, Juvenile

chronic arthritis , Reactive arthritis, Relapsing chronic arthritis , Reactive arthritis, Relapsing polychondritis*polychondritis*

• C-ANCAC-ANCA– very rare in these diseasesvery rare in these diseases

• ANCA is not associated with increased ANCA is not associated with increased frequency of vasculitis in the autoimmune frequency of vasculitis in the autoimmune rheumatic diseasesrheumatic diseases

*Ann Intern Med 126:866-873, 1997*Ann Intern Med 126:866-873, 1997

ANCA and Inflammatory Bowel DiseaseANCA and Inflammatory Bowel Disease

• P-ANCA and some atypical patterns (not P-ANCA and some atypical patterns (not directed at MPO)directed at MPO)– Ulcerative colitis - 40% to 80%Ulcerative colitis - 40% to 80%– Crohn’s Disease - 10% to 40%Crohn’s Disease - 10% to 40%

• Does not facilitate the differential diagnosis Does not facilitate the differential diagnosis of patients with inflammatory bowel diseaseof patients with inflammatory bowel disease

• Correlation of titers with disease activity is Correlation of titers with disease activity is not sufficiently reliablenot sufficiently reliable

How are ANCAs detected?How are ANCAs detected?

• Indirect immunofluorescence (IIF)Indirect immunofluorescence (IIF)– c-ANCA or p-ANCA patternc-ANCA or p-ANCA pattern

• Enzyme linked immunosorbent assay Enzyme linked immunosorbent assay (ELISA)(ELISA)– specific antigens detectedspecific antigens detected– PR3: (c-ANCA on IIF)PR3: (c-ANCA on IIF)– MPO: (p-ANCA on IIF)MPO: (p-ANCA on IIF)

• ANCA testing is problematic because of lack ANCA testing is problematic because of lack of standardization between laboratoriesof standardization between laboratories

SummarySummary

• Connective tissue diseases have a low Connective tissue diseases have a low prevalenceprevalence

• Unselected “screening” of patients with Unselected “screening” of patients with “arthritis panels” will result in large numbers “arthritis panels” will result in large numbers of false positivesof false positives

• Estimation of clinical pre-test probability and Estimation of clinical pre-test probability and the knowledge of test characteristics are the knowledge of test characteristics are useful tools for rationally ordering and useful tools for rationally ordering and interpreting the results of diagnostic testsinterpreting the results of diagnostic tests