Common Rheumatologic

Tests: Evaluation and

Interpretation

Beth Valashinas, D.O.

Chief Rheumatology Fellow
University of North Texas Health
Science Center/ Plaza Medical
Center
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 SLE 57% 97% 16.3 0.49

Ab
Anti-Sm Ab SLE 25-30% high * *
Anti-Ro/SSA Sjogren’s, 8-70% 87% * *
Ab SCLE
Anti-La/SSB Sjogren’s, 16-40% 94% * *
Ab SCLE
Scl-70 Scleroderma 20% 100% >25 0.8
Anticentrom CREST 65% 99.9% 650 0.4
e
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_16
A.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 • % ANA-positive
– SLE – 99%
– Drug induced lupus – 95-100%
– MCTD – 95-100%
– Autoimmune liver – 60-100%
dz – 75-90%
– Sjogren’s syndrome – 30-80%
– Polymyositis – 30-50%
– RA
Adapted from Hobbs, K in West, S Rheumatology Secrets, 2002.
ANA
• Condition • % ANA-positive
– Multiple sclerosis – 25%
– Pts with silicone
breast implants
– Healthy relatives of – 15-25%
pts with SLE
– Neoplasms
– Normal elderly (>70 – 20%
yrs)
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

Scleroderm (-) Low (-) Rare Rare 10-15%

a titer
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 Frequency
Antigen
Polymyositis tRNA synthetase 20-30%
(anti-Jo-1, etc)
SLE Ribosomal P 5-10%
Wegener’s Serine proteinase- 90%
granulomatosis 3
(in neutrophils)
Microscopic Myeloperoxidase 70%
polyarteritis (in neutrophils)
Primary biliary Mitochondria 80%
cirrhosis
Hobbs, K. in West, S. Rheumatology Secrets. 2002.
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 • MPO negative
– Microscopic – Ulcerative colitis
polyarteritis – Autoimmune
– Pauci-immune GN disease
– Churg-Strauss – HIV
vasculitis – Chronic infections
– Drug-induced or neoplasms (rare)
syndromes
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 • Non-rheumatologic
diseases conditions
– RA (80-85%) – Chronic hepatitis
– Sjogren’s (75-95%) – Pulmonary disease
– MCTD (50-60%) – Neoplasms
– Aging
– Scleroderma (20-30%)
– Cryoglobulinemia
– Sarcoidosis (15%)
(40-100%)
– Polymyositis (5-10%)
– 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 • Frequency of +RF
– 20-60 years – 2-4%
– 60-70 years – 5%
– >70 years – 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 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
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 Appearanc Total WBC %PMNs
e Count/mm
3

Normal Clear, 0-200 <10%

viscous
Non- Clear to sl. 200-2000 <20%
inflammator turbid
y
Inflammator Slightly 2000- 20-70%
y turbid 50,000
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 Calcium
urate (MSU) pyrophosphate
dihydrate
(CPPD)
Shape Needle Rhomboid or
rectangular
Birefringence Negative Positive
Crystal color Yellow Blue
parallel to axis
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
References
1. ACR AD HOC Committee on Immunologic testing in the rheumatic diseases: an introduction. Arthritis
Care and Research. August 15, 2002, Vol. 47, No. 4 pp.429-433.
2. Gitlin JD, Colten HR: Molecular biology of the acute phase plasma proteins. In Pick E, Landy M [eds]:
Lymphokines. Vol. 14. San Diego, Academic Press, 1987, pp 123-153.)
3. Morley JJ, Kushner I: Serum C-reactive protein levels in disease. Ann N Y Acad Sci 389:406-418, 1982.
4. Macy EM, Hayes TE, Tracy RP: Variability in the measurement of C-reactive protein in healthy subjects:
implications for reference intervals and epidemiological applications. Clin Chem 43:52-58, 1997.
5. Morely JJ, et al. Serum C-reactive protein levels in disease. Ann N Y Acad Sci 1982;389:406-418.
6. M. R. Arbuckle, J. A. James, K. F. Kohlhase, M. V. Rubertone, G. J. Dennis, J. B. Harley (2001)
Development of Anti-dsDNA Autoantibodies Prior to Clinical Diagnosis of Systemic Lupus
Erythematosus. Scandinavian Journal of Immunology 54 (1-2) , 211–219.
7. M. J. Nielen, et al. Specific Autoantibodies Precede the Symptoms of Rheumatoid Arthritis. A Study of
Serial Measurements in Blood Arthritis Rheum 2004,50:380-386.
8. Shmerling RH. Rheumatic disease: choosing the most useful diagnostic tests. Geriatrics 1996;51:22-
6,29-30,32.
9. Braun J, Bollow M, Remlinger G et al. Prevalence of spondylarthropathiesin HLA-B27 positive and
negative blood donors. .Arthritis Rheum 1998;41:58–67.
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