positive - CECentral
Transcription
positive - CECentral
New Perspectives on New (and Old) Rheumatology Serologies Robert W. Lightfoot, Jr., MD Topics for Today’s Talk 1. The Old ANA - What are the upper limits of normal for the ANA? 2. The New ANA (The Bead Assays) - The problem of the false negative ANA - The problem of the false positive ANA 3. The “ANA Profile” and Its Problems 4. The anti- CCP in Arthritis Diagnosis Case #1 A 33 yr. old woman comes by the booth of the local lupus society at a health fair in your local shopping mall to be screened for SLE. Case #2 A 33 yr. old woman is referred to you with 4 mos. of sustained pain, stiffness and swelling in the knuckles of the hand, the wrists and the knees. PE corroborates same and is otherwise negative. Her ANA is + at 1:160 Her ANA is + at 1:160 The most likely diagnosis is: The most likely diagnosis is: She is Normal Rheumatoid Arthritis What are the upper limits of normal for the ANA? The Bell Curve 2.5% 2.5% 68% 2 S.D. 1 S.D. 1 S.D. 2 S.D. The ANA Hospital personnel, medical students Blood donors Elderly Miscellaneous diseases Arthritis, excluding RA, SLE Hospitalized, non-rheumatic Conn. Tissue diseases, not SLE Rheumatoid arthritis Relatives of SLE patients SLE 1% 3% 10-15% 6% 14% 17% 24% 30-40% 33% 95% uuuuuuu 1:20 1:40 1:80 1:160 1:320 1:640 1:1280 INDIRECT IF(IIF) ASSAY Fluorescein-tagged anti-IgG, -IgA or IGM PATIENT SERUM HEP-2 CELL ANA’S There are between 100- 150 different antigens in the nucleus that can be detected in the IIF ANA. We only know what about 8 of those antigens are. What does an ANA of 1:160 tell you? Test Sensitivity & Specificity Disease SLE Present ANA 95%+ Disease SLE Absent 97%Neg. The ANA in Normals Specificity ANA 1:40 positive 31.7% 68.3% ANA 1:80 positive 13.3% 86.7% ANA 1:160 positive 5% ANA 1:320 positive 3.3% Tan, EM, et al. Arthritis Rheum, 1997. 40:1601-1611. 95% 96.7% The Problem Is... Lupus occurs in only 0.05% of the general population Ergo, 99.95% do not have lupus ANA Sensitivity & Specificity SLE Present General Population 97% Neg. So... Of the 0.05% of people who have SLE, 95% have a + ANA, or 0.0475% of people Of the 99.95% of non-SLE, 3% have a + ANA, or 2.999% of people 2.999 / 0.0475 = 63/1 A ratio of 63:1:: Normal:SLE, i.e., 98% of ANA positives do NOT have SLE NOW… Rheumatoid arthritis is present in 1.5% of the population And, So... Of the 1.5% of people who have RA, 30% are ANA + , or 0.45% of people, Therefore, The ratio of RA to SLE with positive ANA’s is: 0.45/0.0475, or >9 ANA + RA patients for every 1 ANA + SLE patient And… We could do similar calculations for any pre-test percentage likelihood a given patient has lupus ANA Sensitivity & Specificity SLE Present General Population 97% Neg. ANA Sensitivity & Specificity SLE Present General Population 97% Neg. ANA Sensitivity & Specificity SLE Present General Population 97% Neg. ANA Sensitivity & Specificity SLE Present General Population 97% Neg. Test Sensitivity & Specificity SLE Present SLE Absent ANA 97%Neg. 95%+ ANA PREDICTIVE VALUE FOR SLE Post-Test Probability Sensitivity 95%, Specificity 97% 100% 80% 60% ANA + ANA - 40% 20% 0% 0% 20% 40% 60% Pre-Test Probability 80% 100% How can the ANA be made more useful? ANA PREDICTIVE VALUE Sensitivity 95%, Specificity 97% Post-Test Probability 100% 80% 60% ANA + ANA - 40% 20% 0% 0% 20% 40% 60% Pre-Test Probability 80% 100% Every historical feature, every physical finding (or lack thereof) has its own sensitivity and specificity for a given illness. At the end of the history and physical exam, 98% of the diagnostic testing has been done. Of the remainder, 98% of the talent and wisdom required is for differential diagnosis. THE BEAD ASSAYS* *ANA Choice ANA Direct Polystyrene microparticles of uniform size are used as the solid phase. SOLID-PHASE IMMUNOASSAYS “n-DNA” SSB HISTONE SSA SCL-70 RNP SMITH SSA SSB RNP Scl70 Unique bead sets can be conjugated with various, unique target molecules of interest. Thousands of each bead set are combined to form a multiplex bead suspension. The bead suspension is added to the wells of the microplate. AtheNA Multi-Lyte System SSA SSB RNP Scl70 If present, antibody from the test sera will bind to the antigen-coated bead. Anti-human Ig reporter “tags” bound antibody. AtheNA Multi-Lyte System SSA SSB RNP Scl70 If the patient possesses antibody to more than one bead set, all the relevant beads will be labeled with antibody and then conjugate. Beads flow through the flow cell, one bead at a time. • Beads flow through the flow cell and light scatter will determine color of each bead and if it fluoresces. • One laser identifies the amount of fluorescence on the surface. • The other determines the amount of classification dyes…the color of the bead set (i.e., the antigen) This flow analysis of the beads occurs at a rate of up to 20,000 beads/per second. THE BEAD ASSAY AND FALSE POSITIVES Risks of “Panel” Testing* No. of tests 1 2 4 6 10 20 Percent of Times One is Abnormal 5% 10% 19% 26% 40% 64% *Galen & Gambino- “Beyond Normality”, Wiley & Sons 1975 “The more tests performed on a healthy subject, the more likely is the discovery of an abnormal result.” Beyond Normality- Galen, RS,& Gambino R, Wiley, 1975 In some labs, if a single “bead antigen” assay is positive, the ANA is reported as “positive” Clues areThere is no “ANA titer” There are no “ANA units” THE BEAD ASSAY AND FALSE NEGATIVE ANA’s OTHER SOLID-PHASE IMMUNOASSAYS “n-DNA” SSB ? HISTONE SSA SCL-70 RNP SMITH ? Lab Report ANA nDNA SSA SSB Scl-70 Smith RNP Positive Negative Positive Negative Negative Negative Negative Lab Report ANA nDNA SSA SSB Scl-70 Smith RNP Negative Negative Negative Negative Negative Negative Negative Lab Report ANA (IIF) nDNA SSA SSB Scl-70 Smith RNP Positive Negative Positive Negative Negative Negative Negative THE BEAD ASSAY AND FALSE POSITIVE ANTI-nDNA THE BEAD ANTI-“nDNA” ASSAY Anti- nDNA • The biggest problem with all anti-nDNA assays is contamination of the antigen with single-stranded portions. • Antibodies to single-stranded DNA are less specific than the ESR. Crithidia luciliae Tube Dilution anti-nDNA Assay Kinetochore + + SLE Serum Nucleus F-Anti-IgG 1. Any ANA screening test should include an indirect immunofluorescent ANA screen. (IIF) 2. For any bead assay positive for anti- “nDNA”, a better assay (? Crithidiae) should be performed. 3. Any patient with an antibody to a single “bead” antigen (e.g., anti-SSA), should probably see a specialist. THE ANTI-CCP ASSAY Arginine Citrulline NH2 NH2 peptidyl arginine deiminase (PAD) C=NH2+ NH CH2 N H + H20 C=O NH + NH3 CH2 Ca++ CH2 CH2 CH2 CH2 C C C O N H C O + H+ Known Citrullinated Proteins • • • • • • • Myelin basic protein Filaggrin Keratin Histones Vimentin Fibrinogens/fibrins Type I Collagen in synovium CCP Peptides SHQESTRGRSRGRSGRSGS (306-324) SHQESTXGRSRGRSGRSGS ( “ - “ ) SHQESTRGXSRGRSGRSGS ( “ - “ ) SHQESTRGRSXGRSGRSGS ( “ - “ ) SHQESTXGRSXGRSGRSGS ( “ - “ ) Lee & Schur From, Lee, DM and Schur, PH, Ann Rheum Dis 2003 62:870. RF+ RF- False Positive anti-CCP Tests Psoriatic arthritis -Psoriasis sans arthritis SLE Sjogren’s Spondyloarthropathy Scleroderma Hep C Cryoglobulinemia Osteoarthritis Juvenile polyarthritis Fibromyalgia Tuberculosis Arthritis Rheum 61 (11): 1472, 2009 8.6% 0.7-17% 7.8% 5.7% 2.3% 6.8% 3.5% 2.2% 7.7% 2.7% 34.3% Anti-CCP Specificity and PPV TEST Pos. Likelihood Ratio Neg. Likelihood Ratio Sens. Spec. RF 4.9 0.38 69% 85% Anti-CCP 12.5 0.36 67% 95% Prognostic Value of a-CCP’s • Of 936 patients seen in an Early (<2yrs.) Arthritis Clinic (EAC), 590 (63%) could be readily diagnosed, and 205 (21.9%) had RA. • 346 (37%) had undifferentiated arthritis (UA). • They were followed for 3 years. Van Gaalen, et al., A&R 50:709, 2004 Prognostic Value of a-CCP (Odds Ratios in Multivariate Analysis) Criterion ACR Criteria ACR & a-CCP AM stiffness > 1hr. 2.9 ns Arthritis of > 3 jts. 5.8 5.0 Symmetric arthritis 2.6 6.1 IgM RF positivity 9.8 ns Erosions on x-ray 7.6 8.7 Anti-CCP positive N/A 38.6 Van Gaalen, et al.,A&R, 2004 70 RF < 50IU RF > 50IU 60 Erosion Score 50 40 30 20 10 0 Nell, et al., Ann Rheum Dis 2005 0 1 yr 2 yrs 3 yrs 70 A-CCP - A-CCP + 60 Erosion Score 50 40 30 20 10 0 Nell, et al., Ann Rheum Dis 2005 0 1 yr 2 yrs 3 yrs ANA + + + – + – – – RF + – + + – + – – a-CCP + + – + – – + – I N T E R P R E T RA ?SLE RA RA M O R RA E ?SLE T E S T S ?SLE M O R E RA ?SLE ?RA RA T E S T S Other M O R E T E S T S Summary • If your ANA Panel shows a negative ANA, make sure an IIF ANA is done. • If your ANA is “positive” without a titer, make sure an IIF ANA is done. • If your ANA is positive at a titer of <1:320, more history and/or a panel may be indicated. • A positive anti-nDNA usually isn’t. • A positive anti-CCP is strong evidence for RA