LAB TESTING IN RHEUMATOLOGY DR. PHILIP A. BAER SEACOURSES ASIA CME DECEMBER 2017

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2 LAB TESTING IN RHEUMATOLOGY DR. PHILIP A. BAER SEACOURSES ASIA CME DECEMBER 2017

3 COPYRIGHT 2017 BY SEA COURSES INC. All rights reserved. No part of this document may be reproduced, copied, stored, or transmitted in any form or by any means graphic, electronic, or mechanical, including photocopying, recording, or information storage and retrieval systems without prior written permission of Sea Courses Inc. except where permitted by law. Sea Courses is not responsible for any speaker or participant s statements, materials, acts or omissions.

4 LEARNING OBJECTIVES 1. Correctly order common rheumatology lab tests such as RF, CCP, ANA, ENA, HLA-B27, ESR, CRP, C4 2. Correctly interpret the results of these common lab tests 3. Integrate learnings from the Choosing Wisely Canada initiative into clinical practice in the field of rheumatic diseases

5 5 CASE STUDY 60 year old woman Reasons for referral Left knee OA Chronic low back pain with DDD Enclosures with referral Cumulative patient profile ECG X-rays left hip and knee Labs

6 6 CASE STUDY Labs CBC Chemistry panel TSH, B12, ferritin Urinalysis RF ANA Anti-dsDNA All negative

7 RHEUMATOID FACTOR (RF)

8 RHEUMATOID FACTOR Rheumatoid factor (RF) is an autoantibody that is directed against the organism s own tissues, most relevant in rheumatoid arthritis RF is an antibody against the Fc portion of IgG, which is itself an antibody RF and IgG join to form immune complexes which contribute to disease process

9 BACKGROUND: RHEUMATOID FACTOR TEST Early RA Established RA 50% RF +ve 50% RF -ve 25% RF -ve 75% RF +ve Some convert to RF +ve Wong KO, Bond K, Homik J, et al. Comparative Effectiveness Review No

10 RHEUMATOID FACTOR: POSITIVE TESTS RA 50-90% SLE 15-35% Sjogren s syndrome 75-95% Systemic sclerosis 20-30% Polymyositis/dermatomyositis 5-10% Cryoglobulinemia % MCTD 50-60% Age > % Bacterial endocarditis 25-50% Hepatitis 15-40% Sarcoidosis 3-33% Pulmonary silicosis 30-50% Primary biliary cirrhosis 45-70% Malignancy 5-25%

11 RHEUMATOID FACTOR Pre-test Prob Post-test Prob with + RF Post-test Prob with - RF 1% 16% 0.2% 25% 84% 7% 50% 94% 17% 75% 98% 39% 90% 99% 65% Assuming a sensitivity of 80% and specificity of 95% for RA (more recent data are sensitivity 65% and specificity 89%) From Shmerling R., Am J Med 1991.

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13 RF TESTS ARE + IN 5% OF PEOPLE OVER 40

14 WHY WAS RF TESTING DONE? Pre RF Diagnosis 45% 40% 35% 30% 25% 20% 15% Pre RF Diagnosis 10% 5% 0% RA suspected IA suspected Not RA/IA/CTD RF = Rheumatoid Factor IA = Inflammatory Arthritis CTD = Connective Tissue Disease

15 ACR CRITERIA FOR RA Morning stiffness 2. Arthritis of 3 or more joint areas 3. Arthritis of hand joints 4. Symmetric arthritis 5. Rheumatoid nodules 6. RF + 7. Radiographic changes

16 ACR RA CRITERIA PRESENT IN 235 PATIENTS TESTED FOR RF ACR Criteria Present

17 INFLAMMATORY ARTHRITIS IN PATIENTS SENT FOR RF TESTING Final diagnosis in 235 patients sent for RF testing NID RA Other IA Crystal ReA PMR NID=Non-inflammatory disease IA=Inflammatory arthritis ReA=Reactive arthritis, PMR=Polymyalgia rheumatica

18 REPEAT RF TESTING ONCE RF HAS BEEN + No value BCMA RA Guideline

19 ANTI-CYCLIC CITRULLINATED PEPTIDE ANTIBODY (ANTI-CCP)

20 BACKGROUND: CYCLIC-CITRULLINATED PEPTIDE ANTIBODY TEST Test detects the presence of autoantibodies to citrullinated peptides in serum Formation of antibodies to cyclic-citrullinated peptide (CCP) is very specific for adult patients with RA False positives are seen in active TB, Sjogren s, SLE, scleroderma, and inflammatory myositis

21 CITRULLINATED PROTEINS

22 ANTI-CCP PREVALENCE

23 ANTI-CCP TEST PARAMETERS

24 DIAGNOSTIC ACCURACY OF ANTI-CCP ANTIBODY AND RF FOR RA Anti-CCP RF Sensitivity 67% 69% Specificity 95% 85% + Likelihood Ratio Likelihood Ratio Anti-CCP antibodies are more specific than RF for diagnosing RA and may better predict erosive disease Nishimura et al. Ann Int Medicine 2007; 146:

25 ETA

26 INTRODUCTION TO FAMILY Ubiquitously expressed intracellular chaperonins Discovered as brain proteins Named based on chromatographic elution properties Seven different isoforms with ~ 50% AA homology exist b beta; g gamma; e epsilon; z zeta; h eta; q theta; and s sigma (stratifin) Relevant isoform in inflammatory arthritis is h (eta) 26

27 ETA PROTEIN IS SPECIFIC FOR RA Maksymowych. J Rheumatol. 2014;41(11):

28 14-3-3η ng/ml % Positive ARTHRALGIA: ETA PREDICTS RA DEVELOPMENT η Levels in Arthralgia Patients by Arthritis Development Frequency of η Positive Status by Arthritis Development Developed Arthritis (n=43) p<0.01 No Arthritis (n=101) 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% p<0.01 Developed Arthritis Did Not P=0.04 p< ng/ml 0.40 ng/ml 0.80 ng/ml Positive η Cut-off The Arthralgia group that developed arthritis had 3.4 times higher levels Arthralgia patients are 22% more frequently positive if they developed arthritis 28 Van Beers-Tas. Arthritis Research and Therapy. 2016;18:76

29 ETA INDICATIONS & TEST RESULTS Indication Diagnosis Prognosis Monitoring Definition A JOINTstat test result 0.19 ng/ml denotes a positive test result A JOINTstat test result 0.19 ng/ml denotes a higher risk for radiographic damage progression 0.50 ng/ml denotes an even higher risk for radiographic damage progression Serial decreases and a negative JOINTstat level may assist with assessing response outcomes

30 HLA-B27 ANTIGEN (HLA-B27)

31 RECOMMENDATION 2 2. Don t order an HLA-B27 unless spondyloarthritis is suspected based on specific signs or symptoms. Guidelines: Assessment of SpondyloArthritis International Society (ASAS) Guidelines 3e Initiative in Rheumatology

32 HLA-B27 TESTING HLA-B27 is positive in 5-14% of Caucasians Low back pain is a ubiquitous condition

33 HLA-B27 TESTING HLA-B27 is not useful as a single diagnostic test in a patient with low back pain in the absence of other spondyloarthropathy (SpA) signs or symptoms The post-test probability of a +ve HLA-B27 in a patient with chronic low-back pain alone would not exceed 30% If HLA-B27 is used, at least 2 SpA signs or symptoms, or the presence of positive imaging findings, need to be present to classify a patient as having axial SpA

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35 ANTI-NUCLEAR ANTIBODIES (ANA)

36 ANA TESTING Antinuclear antibodies were discovered in 1957 in the sera of patients with systemic lupus erythematosus Most commonly used method for testing is the indirect fluorescent assay (IFA) Positive ANA test result can reflect an interaction between serum antibodies and as many as 180 different nuclear antigens Only a minority of these antigens are well described and have established associations with disease, whereas many of the remaining antigen-antibody reactions may not be clinically relevant

37 ANA TESTING Should not be used to screen subjects without specific symptoms ANA are present in many nonrheumatic conditions such as infections, medications, other medical conditions, and even in healthy people (up to 20%) In patients with low-test probability, positive ANA results can be misleading and may precipitate further unnecessary testing, erroneous diagnosis or even inappropriate therapy Mahler, Journal of Immunology Research 2014

38 MISDIAGNOSIS USING ANA: SERIOUS CONSEQUENCES 137 patients with + ANA but no systemic illness 39 had received prednisone Some received doses of 60 mg/day Narain S, Richards HB, Satoh M, et al. Diagnostic accuracy for lupus and other systemic autoimmune diseases in the community setting. Arch Intern Med. 2004;164:

39 ANA-IFA TITRES The higher the titre, the more likely the + ANA test is clinically significant 1:40 1:80 1:160 1:320 In patients with disease, higher titres suggest more active disease 1:640 control

40 ANA POSITIVITY AT 1:40 IS COMMON 304 healthy individuals % Normal blood donors % 10,550 patients referred for ANA testing % 1 Marin GG, Cardiel MH, Cornejo H, Viveros ME. Prevalence of antinuclear antibodies in 3 groups of healthy individuals: blood donors, hospital personnel, and relatives of patients with autoimmune diseases.j Clin Rheumatol. 2009;15: Fernandez SA, Lobo AZ, Oliveira ZN, et al. Prevalence of antinuclear autoantibodies in the serum of normal blood donors. Rev Hosp Clin Fac Med Sao Paulo. 2003;58: Peene I, Meheus L, Veys EM, De Keyser F. Detection and identification of antinuclear antibodies (ANA) in a large and consecutive cohort of serum samples referred for ANA testing. Ann Rheum Dis. 2001;60:

41

42 ANA + 1/80: GENDER AND AGE 0verall 13.8%

43 ANA POSITIVITY AT 1:160 IS STILL FREQUENT Annual physical examinations 9.5% Hayashi N, Koshiba M, Nishimura K, et al. Prevalence of disease specific antinuclear antibodies in general population: estimates from annual physical examinations of residents of a small town over a 5-year period. Mod Rheumatol. 2008;18:

44 CONDITIONS ASSOCIATED WITH POSITIVE ANA

45

46 ANA FOR DIAGNOSIS OF SLE ANA is a very sensitive (95-100%) but non-specific (49-90%) test for SLE A positive ANA is insufficient to diagnose SLE

47 (50%,79%) 100% (10%,30%) 80% 60% 40% 20% PPV 0% 0.1% 1.0% 10.0% 100.0% Prevalence or pretest probability The ANA test is a NOT a good test for ruling in SLE since SLE has a low prevalence in the population and PPV = Positive Predictive Value the ANA test is not specific

48 NPV 100% 80% 60% 40% 20% (10%,99%) (75%,83%) 0% 0% 25% 50% 75% 100% The ANA-IFA Prevalence test is a or good pretest probability for ruling out SLE since SLE has a low prevalence in the population and the ANA NPV = Negative Predictive Value test is 95-99% sensitive for SLE

49 PRE-TEST AND POST-TEST PROBABILITY Mahler, Journal of Immunology Research 2014

50 The American Journal of Medicine Volume 126, Issue 4, Pages , April 2013

51 STUDY OUTLINE The goal was to evaluate the clinical utility of the positive ANA test result in a real-world setting Determine the final diagnoses of patients who were referred to a tertiary rheumatology clinic for evaluation of a positive ANA test result Investigate the positive predictive value of ANA testing in a cohort of adult patients referred to a rheumatology center specifically for a positive ANA test result Excluded those with a prior diagnosis of an ANAassociated rheumatic disease (AARD), second opinions, transfers of care

52 LIMITED VALUE OF + ANA 232 Patients with +ANA AARD 9% No AARD 91% AARD = ANA-associated rheumatic disease

53 PPV OF HIGHER ANA TITRES REMAINS LOW 30.00% 25.00% 20.00% 15.00% 10.00% AARD SLE 5.00% 0.00% 1:40 1:160 1:640

54 ANA+ PATIENT CHARACTERISTICS DO NOT PREDICT DISEASE

55 RESULTS SUMMARY 232 patients 5 SLE 21 AARD 23 non-aard rheumatic disease 28 anti-thyroid antibody + (of only 91 tested)

56 COMMON REASONS FOR ANA TESTING AND FINAL DIAGNOSES Other CTS OA FM 10 0 Widespread pain Hand pain Knee pain

57 ANA TESTING: BOTTOM LINE

58 RECOMMENDATION 1 1. Don t order ANA as a screening test in patients without specific signs or symptoms of systemic lupus erythematosus (SLE) or another connective tissue disease (CTD) Guidelines: American College of Pathologists British Columbia Ministry of Health American College of Rheumatology Italian Society of Laboratory Medicine Guidelines

59 In one Vancouver hospital, % of ANA done were positive, costing over $800,000 1, 551 repeat testing and simultaneous ordering Man et al. Clin Rheumatol 2013

60 RHEUMATOLOGY TESTS AND COSTS IN ALBERTA Test ANA RF Anti-CCP HLA-B27 Total costs Number 63,000 83,000 18, ordered Cost ($) 3,780,000 1,660, , ,340,000 1/50 Albertans had an ANA test in this period

61 ANA TESTING IN CANADA In Calgary, rheumatology referrals are all through a central triage service 643 (4.1% of 15,357) referrals over 3 years were for a positive ANA >1: (40.9%) seen by rheumatologist 63/263 (24%) had a diagnosis of ANA associated rheumatic disease 69 (26.2%) had no evidence of any disease Fitch-Rogalsky, PLOS online 2014

62 ANA-IFA PATTERNS Homogeneous Speckled ANA patterns loosely correlate with specific Centromereantibodies and diagnoses Nucleolar

63 EXTRACTABLE NUCLEAR ANTIBODIES (ENA) ENA Sm RNP Ro, La Topoisomerase Jo1 SLE MCTD/Overlaps SLE Sjogrens SLE Scleroderma Polymyositis Neonatal lupus Subacute cutaneous LE

64 FLOW CYTOMETRIC IMMUNOASSAY USING FLUORESCENT BEADS SSB Sm SSA dsdna RiboP Scl70 Control CenB RNP Y SSB Scl70 Sm SSA RNP dsdna PATIENT S SSA ANTIBODY=Y; DETECTION ANTIBODY = Y

65 Don t test ANA sub-serologies without a positive ANA and clinical suspicion of immune-mediated disease Tests for anti-nuclear antibody (ANA) sub-serologies (including antibodies to double-stranded DNA, Smith, RNP, SSA, SSB, Scl-70, centromere) are usually negative if the ANA is negative Exceptions include: anti-jo1, which can be positive in some forms of myositis occasionally, anti-ssa, in the setting of lupus or Sjögren s syndrome Broad testing of autoantibodies should be avoided; instead the choice of autoantibodies should be guided by the specific disease under consideration

66 COMPLEMENT STUDIES (C3, C4, CH50)

67 COMPLEMENT Reduced C3, C4, CH50 levels: Immune Complex disease Active SLE Cold agglutinin syndrome Recurrent infections If ordering in SLE, just order C4

68 ANA: CLINICAL SUSPICION OF SLE ANA + AntidsDNA Anti-ENA C4 Disease activity Diagnosis Diagnosis Disease Activity

69 ERYTHROCYTE SEDIMENTATION RATE (ESR)

70 THE ESR How is the test performed? Take a tube Fill with blood Sedimentation occurs

71 ESR: REFERENCE RANGE Adult females: 0-20 mm/h Adult males: 0-15 mm/hr Children(<10): 0-10 mm/hr Add 10 after age 60

72 ESR: NORMAL ADJUSTED FOR AGE

73 SOME CONDITIONS WITH VERY HIGH ESR: >100 MM/HR Multiple myeloma PMR, Giant cell arteritis Connective tissue disorders SLE, RA and other autoimmune diseases Tuberculosis Malignancies Severe anemia Infections

74 ESR: INFLUENCE OF DRUGS INCREASE ESR Dextran, methyldopa, oral contraceptives, penicillamine, procainamide, theophylline, and vitamin A DECREASE ESR Aspirin, cortisone, and quinine

75 ESR: NON-SPECIFIC TEST Erythrocyte sedimentation rate is not diagnostic of any particular disease ESR has a high sensitivity but low specificity Never base a diagnosis solely on an ESR value, either normal or high Interpretation of the result should always be guided by the patient's clinical history, examination findings and results of other tests done If high ESR is encountered without any obvious reasons, the patient should be reassured and the test repeated after a reasonable amount of time (a couple of months) There is no need to extensively search for an occult disease without repeating it again

76 C-REACTIVE PROTEIN (CRP)

77 CRP C-reactive protein (CRP) belongs to the pentraxin family of proteins, which has five identical subunits. It was named because it reacts with the somatic C polysaccharide of Streptococcus pneumoniae, and was first discovered in 1930 by Tillet and Francis. Plasma levels begin increasing within 4-6 hours following acute inflammatory stimulus

78 PCC-reactive protein pentamer

79 Rader, NEJM 2000; 343: 1181.

80 CRP vs. ESR Assessing and Measuring the Inflammatory Response

81 CRP AND ESR REFERENCE RANGES CRP (mg/l) < 8 ESR (mm/hour) Female Male Child Adult < 50 years Adult > 50 years

82 HOW DO ESR AND CRP DIFFER? Comparison of ESR and CRP Results affected by: ESR CRP Gender Yes No Age Yes No Pregnancy Yes No Temperature Yes No Drugs (e.g. steroids, salicylates) Yes No Smoking Yes No

83 ESR AND CRP These tests are most useful when you are RULING OUT an inflammatory disease (they are sensitive), but they are non-specific if positive In a meta-analysis involving 941 patients tested with an ESR for temporal arteritis, the LR+ was 1.1 (i.e., essentially useless), but the LR- was 0.2 (useful!) In other words, if your pre-test probability for temporal arteritis was 50%: your post-test probability with an abnormal ESR would be 50% your post-test probability with a normal ESR would be 18%

84 PEARLS

85 TAKE HOME POINT Serologic tests for rheumatologic diseases are SUPPORTIVE rather than DIAGNOSTIC

86 Patients in Edmonton referred to a single rheumatologist from primary care with 6-24 weeks of joint pain/stiffness/swelling and no prior diagnosis of a rheumatic disease and no prior serology

87 TESTING: RF, CCP, ANA, ESR, CRP Status Number + tests Diagnoses at 1 year Serology done SLE, 4 RA, 3 PMR Serology not done 798 n/a No rheumatic diseases Patients in Edmonton referred to a single rheumatologist from primary care with 6-24 weeks of joint pain/stiffness/swelling and no prior diagnosis of a rheumatic disease and no prior serology

88

89

90 RESOURCES

91

92

93 BARRIERS TO CHANGE: LAB TESTING Overconfidence in value of lab tests in ruling in/ruling out disease Ordering a panel of tests where only one test is indicated Lack of prompts on proper test ordering at the point of care (EMR) Patient demand for tests

94 YOU CAN ONLY FALL IN LOVE SIX TIMES IN YOUR LIFE. CHOOSE WISELY. QUESTIONS? Douglas Coupland

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