Staring down a black hole: A primary care perspective of rheumatologic lab testing A Primary Care Primer to Interpreting Rheumatologic Lab Tests Annual Review Family Medicine 2014 Jonathan Graf, M.D. Associate Professor of Clinical Medicine University of California, San Francisco Division of Rheumatology San Francisco General Hospital Demystifying Rheumatology Lab Sample ABIM question Tests • Understand basic principles of how given test is performed Typical ABIM Board Examination Question – What type of test is it? On Rheumatology Lab Testing – What does the test measure? No Idea – What are the test’s limitations? Rh. Factor • Know the patients being tested ANA – Pretest likelihood that they have disease for which they are being tested? ANCA 1
Sedimentation Rate ESR: Technique Sample question: What is the highest • Aspirating the diluted EDTA- Erythrocyte sedimentation rate ever recorded? blood (in citrate) to the 200 mm mark of a Westergren tube A. 100 59% • Placing the tube in a vertical B. 200 position in a Westergren rack in a location that is free of C. 400 vibration and that is not D. I have no Idea!!!!! exposed to direct sunlight. 18% 13% 11% • After exactly one hour, reading Answer: the distance the erythrocytes - Technically speaking: 200 MM/hr have fallen. 100 200 400 I have no Idea!!!!! - Practically speaking: About 150 What does an ESR Measure? • Measures Acute Phase Proteins – Fibrinogen most common – Produced in liver as part of an inflammatory response under control of cytokines like Il-6, Il-1, TNF • RBC’s serve as proxy for fibrinogen levels – Fibrinogen interacts with RBC to make them sediment faster • Many other factors that affect serum fibrinogen levels or RBC morphology can affect the ESR 2
Causes of Elevated ESR’s ESR - Tidbits • Women generally have slightly higher ESRs then Men • Pregnancy (increased Fib levels) • Anemia (Plasma counter flow altered) • ESRs rise with age: ESR < Age/2 (+5 in women) • Macrocytosis (cells fall faster) • Diabetes • ESRs can be affected by room temperature and laboratory technique • End Stage Renal Failure • Malignancy • Although ESRs are non-specific….. • Infections – ESRs part of diagnostic criteria for Polymyalgia Rheumatica & • Autoimmune inflammatory diseases Giant Cell Arteritis – Especially Vasculitis, PMR, RA – ESRs can be useful in following disease activity or response to therapy for rheumatoid arthritis and osteomyelitis Measuring the Acute Phase C Reactive Protein Response Directly • What is it? – Acute phase protein produced by the liver • How is it measured? – Directly via an ELISA or nephelometrey (unlike ESR) • Advantages – Rises and falls more rapidly in association with acute phase response – Not affected by anemia, renal failure, or other conditions that affect ESR – Unclear if always more sensitive than ESR for various CVD’s 3
Timing of CRP vs. ESR Response Comparison Between ESR & CRP ESR CRP Results affected by Gender Yes No Age Yes No Pregnancy Yes No Temperature Yes No Drugs (eg. steroids, salicylates) Yes No Smoking Yes No - CRP and ESR measure somewhat different aspects of inflammatory response. - They usually but not always correlate with each other. Examples of Autoantibodies Autoantibodies: Target self-antigens Self Antigens: Components of cells PM Complex Organelles Plasma Membrane Antiphospholipid 1,000’s of proteins Cytoplasm Antimitochondrial Complex Ribonuclear Nucleolus Anti Topoisomerase I Proteins Neutrophilic Cytoplasm Anti Pr3 (ANCA) Nucleic Acids Nucleus Anti dsDNA Phospholipids 4
How is an ANA Performed?? What is an Anti-Nuclear Antibody? • Autoabs directed specifically against intra-nuclear antigens • Hep-2 cells fixed to slide & permeabolized • Most commonly (not always) detected • Incubated in patient by immunofluorecence on intact cells serum • Washed vigorously to remove serum • Fluorescently labeled • If an ANA is detected, the specific antigen may or may not Anti-hum Ig secondary Ab be known (most ANA’s aren’t known – only detected by fluorescence inside of an intact nucleus) • Wash again • Detect florescence of • When an ANA screen is positive, one then uses more bound secondary Ab specific tests against known antigens to determine if that ANA is relevant to medical disease (Subserology) ANA Patterns ANA Patterns: Homogeneous • Depends upon what molecule(s) are recognized by patient antibodies – DNA is homogeneously distributed – Centromeres seen in dividing cells – Extractable nuclear antigens are speckled throughout cell 5
ANA Patterns: Speckled ANA Patterns: Nucleolar Testing for Anti-Nuclear Abs More ANA Facts • General screening test for antibodies against most • ANA is not nearly as specific for SLE as it is nuclear antigens sensitive – Autoimmune thyroid disease • Most of the other specific antibody tests for SLE are test for ANA’s – Other Collagen-Vascular diseases (>90% of SSc) – Medications • If ANA negative, with few exceptions (SSA), No need to – Malignancies test for other antibodies – Infections (viral) • Newest generation of IIF ANA’s, use human cell lines, – Normal people (especially low titers) are 95-99% sensitive for SLE • ANA negative SLE is rare 6
ABIM Choosing Wisely Campaign 2013 Antinuclear Antibodies and SLE http://www.choosingwisely.org/ • “An initiative of the ABIM Foundation…specialty societies have • Only one of eleven ACR classification criteria for created lists of “Things Physicians and Patients Should Question” — SLE evidence-based recommendations that should be discussed to help make wise decisions about the most appropriate care based on a – 2/11 criteria………………..50% Specificity patients’ individual situation.” – 3/11 criteria………………..75% Specificity – 4/11 criteria………………..95% Specificity • When working up SLE, the ANA should only be ordered with good pretest, clinical suspicion for SLE – In a patient with arthritis, ANA is no better than coin flip • If ANA negative, no need to check ANA “panel.” Homogeneous Patterns: Anti- When the ANA is Positive dsDNA Abs • Further differentiating the specific target may be of use, in the right clinical context • 50-60% sensitive for SLE • 90-95% specific for SLE • Most tests/sub-serologies are done by • 1/11 SLE “criteria” specific ELISA or immunoblot • Presence and titer can – Patient serum is incubated with target antigen correlate with renal/systemic disease – Antibodies remaining bound to the target antigen are detected with labeled antisera flares • Possible direct implication Homogeneous Pattern in GN • If detected, the specific target of the ANA, with the right clinical picture, can help clarify a diagnosis and/or serve a predictive role 7
Speckled: Extractable Nuclear Anti-Histone Antibodies (Histones are bound to DNA) Antigens • Diected against one or more proteins or protein- • Acid extractable DNA complexes in nucleosome (histone + nuclear antigens dsDNA) – U1SNRnP • Can be seen in SLE and Drug-induced LE • Anti-Smith – Not specific for Drug-LE • Anti-RNP – Very Sensitive (practically required to even consider – SSA (RO) the diagnosis of drug-induced LE) – SSB (La) • Strong negative predictive value (not positive) Speckled Particles • Can be seen with or without disease, with other diseases (SLE) • 95% cases of procainamide LE • Hydralazine, INH, Aldomet, Dilantin, Tegretol U1snRNP Particle Anti-Smith Antibodies • Complex • Poor sensitivity for SLE (20-30%) macromolecule of RNA and proteins • Very high Specificity for SLE (95-99%) • Includes target sites for both anti-Smith • May identify a subset of patients with more and anti-RNP Abs severe disease and/or renal involvement • Helps explain why many SLE patients have antibodies to both Smith and RNP 8
Anti-SSA (Ro) and SSB (La) Anti-RNP Antibodies Key Associations You Have to Know • 100% sensitivity for patients with MCTD • Sjogren’s syndome (diagnostic criterion) – 88-96% of patients with primary SS have SSA – 70-80% with primary SS have SSB • 40-60% patients with SLE – Much lower percentage for secondary SS pts. – More raynaud’s phenomenon, less renal – Primary SS usually dual Ab positive involvement, “less severe disease” • Increased incidence of vasculitis, purpura, lymphoma, etc… – More interstitial lung disease • Associated with neonatal lupus – Features of myositis, scleroderma, and – Implicated in pathogenesis, although not only factor arthritis – Mothers with SLE, Sjogren’s, or asymptomatic – Rash and congenital heart block Anti-Ro (SSA) Skin Disease Anti-Centromere Antibodies Subacute cutaneous lupus erythematosus • Newer ANA assays use a cell line that rapidly divides • ANA’s may recognize components of mitotic spindle • Most IIF can detect Anti- Centromere Abs now • Any doubt, order specific ELISA Papulosquamous Annular Courtesy ACR Image Bank 9
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