Common Rheumatologic Tests: Evaluation and Interpretation

1
Common Rheumatologic Tests Evaluation and
Interpretation

• Beth Valashinas, D.O.
• Chief Rheumatology Fellow
• University of North Texas Health Science Center/
  Plaza Medical Center

2
Disclosures

• Nothing to disclose

3
Introduction

• Immunologic laboratory testing in rheumatology is
  useful for supporting or refuting a clinically
  suspected diagnosis
• Shotgun approaches or screening tests often
  lead to false positives, and further unnecessary
  workups/referrals

4
Background

• Defining attributes of a test
• Sensitivity
• Specificity
• Positive predictive value
• Negative predictive value
• Likelihood ratios
• Pretest and posttest probabilities

5
Attributes of a test

• Sensitivity
• Proportion of patients with a disease who have a
  positive test result
• Specificity
• Proportion of patients without a disease who have
  a negative test result
• Both sensitivity and specificity are independent
  of disease prevalence

6
Attributes of a test

• Predictive value
• likelihood of disease or lack thereof based on a
  positive or negative test result
• Negative predictive value (NPV)
• True negative/(true negative false negative)
• Positive predictive value (PPV)
• True positive/(true positive false positive)

7
Predictive value

• Predictive value is significantly affected by
  disease prevalence
• Predictive value of a positive rheumatologic test
  in patient with polyarthralgias is likely to be
  higher in a rheumatology practice than in a
  family physicians office
• As pretest probability increases, so does the
  clinical utility of a specific test

Lane, SK and Gravel, JR. American Family
Physician. 656,1073,2002.
8
Attributes of a test

• Likelihood ratio
• LR for a negative test result
• (1-sensitivity)/specificity
• LR for a positive test result
• sensitivity/(1-specificity)

9
Likelihood ratio

• Provides additional measure by allowing
  calculation of posttest probability based on
  pretest probability and test result
• Decision to use a test should be based upon
  whether posttest probability will be
  significantly different from the pretest
  probability given a positive or negative test
  result

ACR AD HOC Committee. Arthritis Care and
Research. 47429, 2002.
10
Attributes of a test

• If a test has a high positive likelihood ratio
  (e.g., 10), and the test result is positive, then
  the posttest probability of the test will be
  greatly increased
• If the likelihood ratio is only 1, then no
  difference would be expected between pretest and
  posttest probabilities

11
Performance characteristics of specific ANAs
Colglazier, CL et al. Southern Medical
Journal.2005
12
Acute phase reactants

• Heterogeneous group of proteins synthesized in
  liver in response to inflammation
• Fibrinogen
• Haptoglobin
• C-reactive protein
• Alpha-1-antitrypsin

13
Acute phase protein response
Adapted from Gitlin JD, Colten HR in Pick E,
Landy M eds Lymphokines.14123,1987.
14
Common markers of inflammation

• ESR
• Measures distance (in mm) that RBCs fall within
  specified tube (Westergren or Wintrobe) over 1
  hour
• Indirect measure of changes in acute-phase
  reactants and quantitative Igs
• Decreases by 50 in 1 week after inflammation
  resolves

15
Mechanism of elevated ESR

• If higher concentration of asymmetrically charged
  acute-phase protein or hypergammaglobulinemia
  occurs, dielectric constant of plasma increases
  and dissipates inter-RBC repulsive forces, leads
  to closer aggregation of RBCs, so they fall
  faster, and cause ESR elevation

Hobbs, K in West, S. Rheumatology Secrets,2002
16
Noninflammatory conditions with elevated ESR

• Aging
• Female sex
• Obesity
• Pregnancy

17
Rule of thumb

• Age-adjusted upper limit normal for ESR
• Male age/2
• Female (age 10)/2

18
Causes of markedly elevated ESR

• ESR 100
• Infection, bacterial (35)
• CTD (GCA, PMR, SLE, vasculitides (25)
• Malignancy lymphomas, myeloma, etc (15)
• Other causes (25)

Hobbs, K in West, S. Rheumatology Secrets,2002.
19
Causes of extremely low ESR

• ESR 0mm/hr
• Agammaglobulinemia
• Afibrinogenemia/dysfibrinogemia
• Extreme polycythemia (Hematocrit 65)
• Increased plasma viscosity

20
Approach to elevated ESR

• Complete HP
• Routine labs (CBC, CMP, UA)
• Up-to-date cancer screening/health maintenance
• Repeat ESR
• If still elevated without other association
• Consider SPEP, CRP
• Recheck in 1-3 months (up to 80 normalize)

21
C-reactive protein (CRP)

• Pentameric protein
• Trace concentrations in human plasma
• Highly conserved over hundreds of millions of
  years of evolution
• Properties of recognition and activation
• Activates classic complement pathway
• Modulates behavior of phagocytic cells (both
  inflammatory and non-inflammatory influence)

22
CRP

• Acute phase reactant produced by liver
• Response to IL-6, other cytokines
• Rises and falls quickly
• Elevation within 4 hr of tissue injury
• Peak at 24-72 hr
• Half-life 18 hr

23
Rule of thumb

• CRP
• CRP 0.2-1.0 mg/dL indeterminate (may be seen in
  smoking, DM)
• CRP 1.0 mg/dL inflammatory
• Levels 10mg/dL suggest bacterial infection (up
  to 85), or possibly systemic vasculitis,
  metastatic cancer

Morely JJ, et al. Ann N Y Acad Sci389,1982.
24
Serum protein electrophoresis (SPEP)

• Quantifies the acute-phase response
• Increase in alpha-1 and -2 zones (alpha-1
  antitrypsin and haptoglobin)
• Increase in beta-gamma area (fibrinogen and CRP)
• Decrease in pre-albumin, albumin, and the beta
  zone (transferrin)

25
Normal SPEP
erl.pathology.iupui.edu/LABMED/IMAGES/SPE_16A.JPG
26
SPEP- acute inflammation
erl.pathology.iupui.edu/LABMED/IMAGES/SPE_16A.JPG
27
SPEP- Polyclonal gammopathy
erl.pathology.iupui.edu/LABMED/IMAGES/SPE_16A.JPG
28
Antinuclear antibodies (ANA)

• Initial LE test in 1940s
• Incubate bare nucleus with pts serum, allowing
  ANAs to bind to nucleus
• Then add normal PMNs and if sufficient Ab have
  bound to nucleus, nucleus is opsonized and PMNs
  engulf the material
• LE cell is PMN containing phagocytosed nucleus

29
LE Cell
30
Current ANA measurement

• Fluorescence microscopy
• HEp-2 cells (derived from human epithelial tumor
  cell line) incubated with pts serum
• Fluoresceinated Ab added, binds to pts Abs bound
  to nucleus
• Amount of ANA determined by dilution of the pts
  serum - the greater the dilution (titer) at which
  nuclear fluorescence detected, the higher the ANA
  concentration

31
ANA

• Arbitrary definition of positive ANA is the level
  that exceeds that seen in 95 of the population
• Titers usually positive at 140 to 180
• Clinically significant titers (with HEp-2 cells)
  1160

32
ANA

• High sensitivity in SLE, but poor specificity
• Positive ANA has predictive value of only 11
  (positive LR 2.2)
• ANA found in 5-10 of pts without CTD
• Healthy pts, chronic infections (e.g., Hep C),
  multiple meds, etc.

33
ANA

• Condition
• SLE
• Drug induced lupus
• MCTD
• Autoimmune liver dz
• Sjogrens syndrome
• Polymyositis
• RA

• ANA-positive
• 99
• 95-100
• 95-100
• 60-100
• 75-90
• 30-80
• 30-50

Adapted from Hobbs, K in West, S Rheumatology
Secrets, 2002.
34
ANA

• Condition
• Multiple sclerosis
• Pts with silicone breast implants
• Healthy relatives of pts with SLE
• Neoplasms
• Normal elderly (70 yrs)

• ANA-positive
• 25
• 15-25
• 20

Adapted from Hobbs, K in West, S. Rheumatology
Secrets, 2002
35
ANA

• Is the ANA a good screening test for SLE?
• If 5 of normal U.S. population has positive
  ANA, then over 12.5 million normal people in
  U.S. are ANA positive
• Prevalence of SLE is only 1/1000, so only
  250,000 individuals with SLE and positive ANA
• If entire population was screened, more normal
  individuals would be detected with positive ANA
  than SLE pts. by 501

Hobbs, K. in West, S Rheumatology Secrets. 2002..
36
ANA

• Clinical value of ordering an ANA test can be
  dramatically enhanced when there is a reasonable
  pre-test probability of an autoimmune disease

37
ANA patterns

• Homogeneous (diffuse)
• SLE, drug-induced SLE, other diseases

38
ANA patterns

• Rim (peripheral)
• SLE, autoimmune hepatitis

39
ANA patterns

• Speckled
• SLE, MCTD, Sjogrens, Scleroderma, other dz

40
ANA patterns

• Nucleolar
• Scleroderma, hepatocellular carcinoma

41
ANA patterns

• Centromere
• Limited scleroderma (CREST)

42
Drug-induced ANAs

• Common drugs that cause positive ANAs
• Procainamide
• Hydralazine
• Phenothiazines
• Diphenylhydantoin
• Isoniazid
• Quinidine

43
Lupus or ANA profile

• If screening ANA is positive and additional info
  needed to further delineate type of autoimmune
  disease
• In extremely rare instances, ANA may be negative
  but SS-A antibodies may be detected in pts. with
  an SS-A associated disease

44
Lupus Profile
Hobbs, K. in West, S Rheumatology Secrets. 2002.
45
Lupus Profile

• Antibodies to dsDNA are associated with lupus
  nephritis, and often parallel disease activity
• Antibodies to SS-A/Ro and SS-B/La are commonly
  associated with Sjogrens syndrome
• Anti-Ro/SSA antibodies increase risk for neonatal
  lupus/congenital heart block (CHB), especially
  when in conjunction with anti-La/SSB Ab
• Overall risk is 5

46
Antibodies to ribonuclear protein (RNP)

• Target is spliceosomal snRNPs in nucleoplasm
• Seen in SLE, scleroderma, mixed connective tissue
  disease (MCTD)
• High levels very suggestive of MCTD
• MCTD is overlap disease with features of SLE,
  scleroderma, and polymyositis

47
Anticentromere and SCL-70 Ab

• Anticentromere Ab
• up to 98 pts with limited scleroderma (CREST)
• 22-36 pts with diffuse scleroderma
• Anti-SLC70 (anti-topoisomerase I)
• 22-40 pts with diffuse scleroderma
• longer disease duration, association with cancer,
  pulmonary fibrosis, digital pitting scars,
  cardiac manifestations

48
Anti-dsDNA Ab prior to Dx of SLE

• Serum from 130 SLE patients
• 55 had anti-dsDNA Ab prior to SLE Dx
• Mean onset of Ab 2.7 years prior to Dx
• (range
• 58 of cases with at least 2 positive samples had
  significant rise in anti-dsDNA within 6 months of
  Dx

M. R. Arbuckle, et al. Scandinavian Journal of
Immunology 54 (1-2) , 211219.
49
Evaluation of pt with positive ANA and
generalized arthralgias

• H P - any signs of CTD?
• If ANA titer 1160, consider lupus profile
• Other possible tests CBC, CMP, C3, C4, SPEP,
  RF, ESR, UA, lupus anticoagulant, anticardiolipin
  antibody

50
Antiphospholipid antibodies

• Heterogeneous group of Ab that bind to plasma
  proteins, have affinity for phospholipid surfaces
• Anticardiolipin Ab (ACL)
• Lupus anticoagulant (LAC)
• Beta 2-glycoprotein I

51
Antiphospholipid antibodies

• ACL measured by ELISA assay for IgG, IgM, and IgA
  isotypes
• LAC measured by phospholipid-dependent screening
  test, if prolonged, add 11 mix with normal
  plasma - if no correction, LAC present
• Beta 2-glycoprotein I measured by ELISA

52
Antiphospholipid antibodies

• Conditions with positive aPL
• 8 normal population
• chronic infections e.g., HIV, Hep C
• Medications e.g., phenothiazines, hydralazine,
  phenytoin, procainamide, quinidine
• 20 pts. with systemic vasculitis
• 15 pts. with recurrent miscarriage
• 50 pts. with SLE

Hansen, KE. in West, S Rheumatology Secrets,
2002.
53
Antiphospholipid antibodies

• 50 pts. with SLE and aPL will develop a
  thrombotic event
• 3-7 pts. per year who have aPL will experience
  a new thrombotic event
• Overall positive predictive value of an aPL for
  future CVA, venous thrombosis, or recurrent MC is
  between 10-25

Hansen, KE. in West, S. Rheumatology Secrets,
2002.
54
Cryoglobulins

• Immunoglobulins that precipitate in cold
  temperatures
• May cause hyperviscosity or vasculitis
• Symptoms include fatigue, arthralgias/arthritis,
  cutaneous vasculitis or purpura, neuropathies,
  visceral organ involvement, and digital ischemia

55
Cryoglobulins

• Type I- Monoclonal Ig (IgG or IgM)
• Lymphoproliferative disorders
• Type II- Monoclonal IgM directed against
  polyclonal IgG
• Majority associated with Hepatitis C
• Type III- Mixed polyclonal IgG and IgM
• Connective tissue diseases, chronic infections

56
Anticytoplasmic Antibodies

• Often more helpful in diagnosis than antibodies
  against nuclear antigens
• Seen with multiple autoimmune diseases and
  several forms of vasculitis

57
Anticytoplasmic antibodies
Hobbs, K. in West, S. Rheumatology Secrets.
2002.
58
Anti-neutrophil cytoplasmic Antibodies (ANCA)

• C-ANCA
• Most commonly seen in Wegeners granulomatosis,
  microscopic polyarteritis, rarely Churg-Strauss
  vasculitis

59
ANCA

• P-ANCA
• seen in multiple diseases as well as vasculitis

60
P-ANCA

• MPO positive
• Microscopic polyarteritis
• Pauci-immune GN
• Churg-Strauss vasculitis
• Drug-induced syndromes

• MPO negative
• Ulcerative colitis
• Autoimmune disease
• HIV
• Chronic infections or neoplasms (rare)

61
ANCA

• If pt. tests positive to ANCA, evaluation of
  specific antigen testing for MPO and PR3 should
  be undertaken
• If C-ANCA is not against PR3 or P-ANCA is not
  against MPO, must consider causes other than
  vasculitis

62
Rheumatoid factor

• Autoantibody directed against the Fc (constant)
  region of an IgG molecule
• Multiple isotypes, including IgM, IgG, IgA, and
  IgE
• IgM RF is routinely measured using latex
  agglutination titers, nephelometry, and ELISA

63
Rheumatoid factor

• Very low levels normal, but higher production
  secondary to chronic immune stimulation
• RF positive in 80 of patients with RA
• Multiple other causes of positive RF

64
Conditions associated with a positive rheumatoid
factor

• Rheumatologic diseases
• RA (80-85)
• Sjogrens (75-95)
• MCTD (50-60)
• Scleroderma (20-30)
• Sarcoidosis (15)
• Polymyositis (5-10)

• Non-rheumatologic conditions
• Chronic hepatitis
• Pulmonary disease
• Neoplasms
• Aging
• Cryoglobulinemia
• (40-100)
• Infections
• AIDS, Mono, TB, syphilis, parasites, endocarditis

Adapted from Kathryn Hobbs, from Rheumatology
Secrets, 2002, p.60.
65
Frequency of RF positivity in normal population

• AGE
• 20-60 years
• 60-70 years
• 70 years

• Frequency of RF
• 2-4
• 5
• 10-25

Adapted from Kathryn Hobbs in West, S.
Rheumatology Secrets, 2002.
66
Anti-CCP antibodies

• ELISA assay based on filaggrin from human skin or
  synthetic citrullinated peptides
• Target amino acid in filaggrin is citrulline, a
  post-translationally modified arginine residue
• High specificity and moderate sensitivity for RA

67
Anti-CCP antibodies

• Sensitivity 68 for RA
• Specificity 98 for RA
• Can be seen in active TB, other CTD
• Clinical implications
• Predictive of more aggressive disease with more
  progressive joint damage

68
Early antibody production as indicator of future
disease?

• Longitudinal study of 79 RA patients
• 50 produced anti-CCP Ab and/or IgM-RF prior to
  onset of disease
• Positive results occurred median of 4.5 years
  (range 0.1-13.8) before symptom onset
• Elevated levels of either IgM-RF or anti-CCP may
  imply high risk for development of RA

M. J. Nielen, et al. Arthritis Rheum 50380, 2004.
69
Complement

• Cascade of proteins activated by many agents,
  including immune or antigen-antibody complexes
• May be decreased due to
• Increased consumption (proteolysis)
• Increased levels of circulating immune complexes
  activate classical pathway
• Decreased production
• Hereditary deficiency or liver disease

70
Hereditary complement deficiencies

• May see SLE-like disease with deficiencies in
  C1-C4
• Terminal complement (C5-9) deficiencies lead to
  recurrent infections
• Deficiency in C1 INH leads to angioedema
  (hereditary or acquired)

71
Diseases associated with low complement levels

• Rheumatic diseases
• SLE, systemic vasculitis, cryoglobulinemia,
• RA (rare)
• Glomerulonephritis
• Post streptococcal and membranoproliferative
• Infectious diseases
• Bacterial sepsis, SBE, Hepatitis B, other
  viremias, parasitemias

72
Complement level assessment

• C3 and C4 generally decreased with increased
  disease activity in SLE
• Decreased levels may predict impending disease
  flares
• C4 lowers before C3 and remains lower longer
• CH50 not useful as disease activity marker

73
Serum uric acid levels

• Age- and sex-dependent
• Concentration levels rise with puberty in males
  and menopause in females
• Age of onset
• Peak for males 40-50 years
• Peak for females 60 years

74
Serum uric acid levels

• Hyperuricemia
• 7.0 mg/dL in males
• 6.0 mg/dL in females
• 24 hour urine collection
• Urate 800 mg/24 hrs suggests overproduction
• Urate

75
Serum uric acid levels

• Important considerations
• Only 15 of pts. with hyperuricemia develop gout
• If uric acid level10mg/dL, risk increases to
  30-50
• In 10 of patients with acute gout, serum uric
  acid levels are normal
• Need joint aspiration and polarized light
  microscopy to diagnose with certainty

76
Asymptomatic hyperuricemia

• Treatment indications
• Acute overproduction e.g., tumor lysis syndrome
• Severe hyperuricemia e.g., uric acid levels
  12mg/dL
• Risk of uric acid nephrolithiasis is 50

77
HLA-B27

• Sensitivity
• 95 for AS
• 80 for Reactive Arthritis
• 70 for SpA associated with psoriasis
• 50 for SpA associated with IBD
• 70-84 for uSpA

Shmerling RH. Geriatrics5122, 1996.
78
HLA-B27

• Specificity
• Low given prevalence is 8 in Caucasian
  population
• In patients with inflammatory back pain, HLA-B27
  positivity yields
• 20-fold increased risk of SpA
• 15-fold higher risk of radiological sacroiliitis

Braun J, et al. Arthritis Rheum4158, 1998.
79
Synovial fluid analysis

• Studies to perform
• Gram stain and culture
• Total leukocyte count with differential
• Polarized microscopy

80
Synovial fluid analysis
Adapted from Spencer, RT in West, S. Rheumatology
Secrets, 2002
81
Synovial fluid analysis

• Noninflammatory joint effusions
• OA, joint trauma, mechanical derangement, AVN
• Inflammatory synovial fluid
• Multiple rheumatic disorders
• Infectious arthritis
• Pyarthrosis
• Joint sepsis
• Pseudosepsis in gout, reactive arthritis or RA

82
Polarized light microscopy
Adapted from Spencer, RT in West, S. Rheumatology
Secrets, 2002
83
CPPD and MSU crystals
84
Conclusions

• Immunologic laboratory tests facilitate diagnosis
  and provide information regarding specific
  disease manifestations, disease activity and
  prognosis
• Clinical utility of laboratory evaluation can be
  enhanced by the employment of evidence-based
  guidelines
• A thorough history and physical examination
  remain the best screening and diagnostic tools

85
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