Download Common Rheumatologic Tests: Evaluation and Interpretation

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Common Rheumatologic Tests:
Evaluation and Interpretation
Disclosures
• Nothing to disclose
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
Background
• Defining attributes of a test
– Sensitivity
– Specificity
– Positive predictive value
– Negative predictive value
– Likelihood ratios
– Pretest and posttest probabilities
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
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)
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 physician’s office
– As pretest probability increases, so does the
clinical utility of a specific test
Lane, SK and Gravel, JR. American Family Physician. 65;6,1073,2002.
Attributes of a test
• Likelihood ratio
– LR for a negative test result:
(1-sensitivity)/specificity
– LR for a positive test result:
sensitivity/(1-specificity)
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. 47:429, 2002.
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
Performance characteristics of
specific ANAs
Antigen
Condition
Sensitivity
Specificity
+ LR
- LR
Anti-dsDNA
Ab
SLE
57%
97%
16.3
0.49
Anti-Sm Ab
SLE
25-30%
high
*
*
Anti-Ro/SSA
Ab
Sjogren’s,
SCLE
8-70%
87%
*
*
Anti-La/SSB
Ab
Sjogren’s,
SCLE
16-40%
94%
*
*
Scl-70
Scleroderma
20%
100%
>25
0.8
Anticentrome CREST
65%
99.9%
650
0.4
Anti-U-3 RNP Scleroderma
12%
96%
3
0.92
Colglazier, CL et al. Southern Medical Journal.2005
Acute phase reactants
• Heterogeneous group of proteins
synthesized in liver in response to
inflammation
– Fibrinogen
– Haptoglobin
– C-reactive protein
– Alpha-1-antitrypsin
Acute phase protein response
Adapted from Gitlin JD, Colten HR in Pick E, Landy M [eds]: Lymphokines.14;123,1987.
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
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
Noninflammatory conditions with
elevated ESR
• Aging
• Female sex
• Obesity
• Pregnancy
Rule of thumb
• Age-adjusted upper limit normal for ESR
– Male: age/2
– Female: (age + 10)/2
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.
Causes of extremely low ESR
• ESR ~ 0mm/hr
– Agammaglobulinemia
– Afibrinogenemia/dysfibrinogemia
– Extreme polycythemia (Hematocrit >65%)
– Increased plasma viscosity
Approach to elevated ESR
• Complete H&P
• 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)
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)
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
Rule of thumb
• CRP <0.2 mg/dL: normal
• 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 Sci;389,1982.
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)
Normal SPEP
erl.pathology.iupui.edu/LABMED/IMAGES/SPE_16A.JPG
SPEP- acute inflammation
erl.pathology.iupui.edu/LABMED/IMAGES/SPE_16A.JPG
SPEP- Polyclonal gammopathy
erl.pathology.iupui.edu/LABMED/IMAGES/SPE_16A.JPG
Antinuclear antibodies (ANA)
• Initial LE test in 1940s
– Incubate bare nucleus with pt’s 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
LE Cell
Current ANA measurement
• Fluorescence microscopy
– HEp-2 cells (derived from human epithelial
tumor cell line) incubated with pt’s serum
– Fluoresceinated Ab added, binds to pt’s Abs
bound to nucleus
– Amount of ANA determined by dilution of the
pt’s serum - the greater the dilution (titer) at
which nuclear fluorescence detected, the
higher the ANA concentration
ANA
• Arbitrary definition of positive ANA is the
level that exceeds that seen in 95% of the
population
• Titers usually “positive” at 1:40 to 1:80
• Clinically significant titers (with HEp-2
cells) ~1:160
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.
ANA
• Condition
–
–
–
–
–
–
–
SLE
Drug induced lupus
MCTD
Autoimmune liver dz
Sjogren’s syndrome
Polymyositis
RA
• % ANA-positive
–
–
–
–
–
–
–
Adapted from Hobbs, K in West, S Rheumatology Secrets, 2002.
99%
95-100%
95-100%
60-100%
75-90%
30-80%
30-50%
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
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 ~50:1
Hobbs, K. in West, S Rheumatology Secrets. 2002..
ANA
• Clinical value of ordering an ANA test can
be dramatically enhanced when there is a
reasonable pre-test probability of an
autoimmune disease
ANA patterns
• Homogeneous (diffuse)
– SLE, drug-induced SLE, other diseases
ANA patterns
• Rim (peripheral)
– SLE, autoimmune hepatitis
ANA patterns
• Speckled
– SLE, MCTD, Sjogren’s, Scleroderma, other dz
ANA patterns
• Nucleolar
– Scleroderma, hepatocellular carcinoma
ANA patterns
• Centromere
– Limited scleroderma (CREST)
Drug-induced ANAs
• Common drugs that cause positive ANAs
– Procainamide
– Hydralazine
– Phenothiazines
– Diphenylhydantoin
– Isoniazid
– Quinidine
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
Lupus Profile
dsDNA RNP
SM
SS-A
SS-B
CENTROMERE
SLE
60%
30%
30%
30%
15%
Rare
RA
(-)
(-)
(-)
Rare
Rare
(-)
MCTD
(-)
>95%
(-)
Rare
Rare
Rare
Scleroderma (-)
Low
titer
(-)
Rare
Rare
10-15%
CREST
(-)
(-)
(-)
(-)
(-)
60-90%
Sjogren’s
(-)
Rare
(-)
70%
60%
(-)
Hobbs, K. in West, S Rheumatology Secrets. 2002.
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 Sjogren’s 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%
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
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
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 <1mo-9.3 years)
– 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) , 211–219.
Evaluation of pt with positive ANA
and generalized arthralgias
• H & P - any signs of CTD?
• If ANA titer > 1:160, consider lupus
profile
• Other possible tests: CBC, CMP, C3, C4,
SPEP, RF, ESR, UA, lupus anticoagulant,
anticardiolipin antibody
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
Antiphospholipid antibodies
• ACL measured by ELISA assay for IgG,
IgM, and IgA isotypes
• LAC measured by phospholipid-dependent
screening test, if prolonged, add 1:1 mix
with normal plasma - if no correction, LAC
present
• Beta 2-glycoprotein I measured by ELISA
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.
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.
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
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
Anticytoplasmic Antibodies
• Often more helpful in diagnosis than
antibodies against nuclear antigens
• Seen with multiple autoimmune diseases
and several forms of vasculitis
Anticytoplasmic antibodies
Disease
Cytoplasmic
Antigen
Frequency
Polymyositis
tRNA synthetase
(anti-Jo-1, etc)
20-30%
SLE
Ribosomal P
5-10%
Wegener’s
granulomatosis
Serine proteinase-3 90%
(in neutrophils)
Myeloperoxidase
70%
(in neutrophils)
Microscopic
polyarteritis
Primary biliary
cirrhosis
Mitochondria
Hobbs, K. in West, S. Rheumatology Secrets. 2002.
80%
Anti-neutrophil cytoplasmic
Antibodies (ANCA)
• C-ANCA
– Most commonly seen in Wegener’s
granulomatosis, microscopic polyarteritis,
rarely Churg-Strauss vasculitis
ANCA
• P-ANCA
– seen in multiple diseases as well as vasculitis
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)
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
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
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
Conditions associated with a
positive rheumatoid factor
• Rheumatologic diseases
–
–
–
–
–
–
RA (80-85%)
Sjogren’s (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.
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.
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
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
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 50:380, 2004.
Complement
• Cascade of proteins activated by many
agents, including immune or antigenantibody 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
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)
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
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
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
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 <800 mg/24 hrs suggests
underexcretion
Serum uric acid levels
• Important considerations
– Only 15% of pts. with hyperuricemia develop
gout
– If uric acid level>10mg/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
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%
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. Geriatrics;51:22, 1996.
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 Rheum;41:58, 1998.
Synovial fluid analysis
• Studies to perform
– Gram stain and culture
– Total leukocyte count with differential
– Polarized microscopy
Synovial fluid analysis
Fluid type
Appearance Total WBC %PMNs
Count/mm3
Normal
Clear,
viscous
0-200
<10%
NonClear to sl.
inflammatory turbid
200-2000
<20%
Inflammatory Slightly
turbid
200050,000
20-70%
Pyarthrosis
>50,000
>70%
Turbid
Adapted from Spencer, RT in West, S. Rheumatology Secrets, 2002
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
Polarized light microscopy
Gout
Pseudogout
Crystal
Monosodium
urate (MSU)
Shape
Needle
Birefringence
Negative
Calcium
pyrophosphate
dihydrate (CPPD)
Rhomboid or
rectangular
Positive
Crystal color
parallel to axis
Yellow
Blue
Adapted from Spencer, RT in West, S. Rheumatology Secrets, 2002
CPPD and MSU crystals
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
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