1. Rational Use of Laboratory
Tests
In Rheumatic Diseases
Jeffrey Carlin, MD
Division of Rheumatology,
Virginia Mason Medical Center
Director, Rheumatology Bioregistry
and Rheumatology Clinical Trials
Benaroya Research Institute
2.
3. Ideal Rheumatologic Test
• All patients with disease have positive test, all
without have negative test
• All patients with positive test result have disease,
all with negative do not
• Test correlates with organ system involvement
• Test correlates with disease activity
4. Test Statistics Definitions
• Sensitivity
• Proportion of patients with a disease with a positive lab
test
• Specificity
• Proportion of patients without the disease with a
negative lab test
• Positive predictive value
• Probability that a patient with a positive test has the
disease
• Negative predictive value
• Probability that a patient with a negative test does not
have the disease
5. Definitions
• Sensitivity describes the ability of a diagnostic test
to identify people who truly have a disease. Thus, a
high sensitivity test has few false negatives and is
effective at ruling conditions “out”
• Specificity describes the ability of a diagnostic test
to be correctly identify people who are well. Thus,
a high specificity test has few false positives and is
effective in ruling conditions “in”
6. Statistical Definitions
• Negative Probability Value: probability that an individual is not
affected with the condition when a negative test result is observed
• (True negative)/(True and false negatives)
• Positive Probability Value: probability that an individual is
affected with the condition when a positive test result is observed
• (True positive)/(True and false positives)
• Likelihood ratios: relative odds that a positive test represents a
correct clinical diagnosis or true positive (LR1) and that a negative test
represents a true negative (LR). Likelihood ratios greater than 5 and less
than 0.2 are typically the most clinically relevant.
• LR1 5 (Test sensitivity)/(1 Test specificity)LR 5 (1 Test sensitivity)/
(Test specificity)
7. Interpretation of Laboratory
Tests
• Sensitivity and Specificity are independent of disease
prevalence
• Predictive value is markedly dependent on disease
prevalence
• As the pretest probability increases so does the clinical
utility of a laboratory test
8. Question #1
82 YO female is referred to your office for a chronic frontal headache
for the past 6 months. Her generalists ordered an ESR that was
elevated at 40 (normal <20). She is otherwise healthy, does not
complain of jaw claudication, visual symptoms, pain/stiffness in
shoulders, neck, or hip girdle, or fatigue. Results of other lab testing
are shown below:
WBC 6.2 Creatinine 0.85 (eGFR 88)
Hg 13.1 RF Neg
Plts 275 CRP 1.3 mg/L
Which of the following most likely explains this person’s
abnormal ESR?
A. Her headache
B. Her hemoglobin
C. Her age
D. Her renal function
9. Tests of Acute Phase
Reactants• Erythrocyte Sedimentation Test
• C-Reactive Protein
10. Patterns of Response of Acute
Phase Reactants
Gabay C, Kushner I, NEJM , 1999;340:450
12. What Does the ESR Measure?
• Measures Acute Phase Proteins
– Fibrinogen most common
• 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
13. ESR’s
• Non-specific marker- elevated in rheumatic diseases,
infection, malignancy
• Can be artificially elevated by:
• Pregnancy
• Anemia
• Nephrotic Syndrome
• Benign/Malignant Monoclonal Gammopathies
• Age
• Obesity
• Can be normal in some inflammatory conditions
14. Formula for Age- Related
Normals
• Men:
ESR(mm/hr)= (age in years)/2
• Females
ESR (mm/hr)= (age in years + 10)/2
15. C- Reactive Protein
• Rapid rise in response to inflammatory stimuli
• Can be affected by:
• Obesity/Metabolic Syndrome
• Proinflammatory molecules produced in abdominal fat cells
stimulate immune respone
• Age
19. Rheumatoid Factors
• Autoantibodies to the Fc portion of IgG.
• Support a diagnosis of Rheumatoid Arthritis but
are not by themselves diagnostic.
• Are seen in about 75% to 80% of patients with RA
• Sensitivity 80%
• Specificity 95%
• PPV (unselected populations)- 20-30%
(RA population)- 80%
• NPV- 95%
• Are seen in conditions other than RA
• Acute phase reactant
22. RF: Clinical Significance
• Highly predictive of RA in patients with identified
rheumatic disease
• May be absent at the onset of disease in up to half of
patients with typical clinical picture of RA
• approx 20% remain seronegative
• many convert within 2 years
• Best used to confirm RA for typical presentation
• inflammatory polyarthritis, “gel phenomenon,” etc.
• Not useful to follow course of illness
• generally not helpful to repeat after diagnosis
• May be positive in “pre-clinical RA” and precede clinical
RA by years
24. Anti-CCP Antibody Assay
• Accuracy (Anti-CCP-2 Assay)
• Specificity 79% Sensitivity 96-98%
• Diagnosis more accurate when combined with RF+
• Present in 50-60% early RA patients
• Can be seen 1.5 -9 yrs pre-diagnosis of RA
• Predictive for progressive joint damage
• Present in up to 40% percentage of RF- patients with
erosions
• RF+, anti-CCP+ pts have very aggressive disease
25. 2010 ACR/EULAR Criteria for
Classification of RA
Domain/Criteria Points
Joint involvement (0-5)
1 medium/large joints 0
2-10 medium-large joints 1
1-3 small joints 2
4-10 small joints 3
>10 joints (at least 1 small) 5
Serology (0-3)
Neither RF nor anti-CCP + 0
At least one test low titer + 2
At least one test + high titer 3
Duration of Synovitis
< 6 weeks 0
> 6 weeks 1
Acute Phase Reactants
Neither CRP or ESR elevated 0
Abnormal ESR/CRP 1
Patients with
> 6 points
considered to
have definite dx
of RAA
Aletia, D et al Arth & Rheum 2010; 62:2586-2581
Low titer < 3X ULN
High titer >3X ULN
26. Question 2
You are consulted on a 32 YO African American female who presents
to the emergency room with new onset seizures. Her past history had
been notable for photosensitive rashes, oral ulcers, and worsening
proteinuria or uncertain etiology. She had consulted another
rheumatologist who had ordered the following tests:
ANA Neg (<1.4 IU) immunoassay
RF Neg (<20) nephelometry
ANCA Pr3 Neg (<20) immunoassay
ANCA MPO Neg (<20) immunoassay
C3 47 (>80)
C4 12 (>20)
Urinalysis 1+ blood
3+ protein
3-5 RBC
3-5 WBC
Spot UProtein 117 mg/DL
Spot UCreatinine 57 mg/DL
27. Question 2
In addition to working up proximal causes for her
seizures, your best step to clarify her underlying
diagnosis is to do which of the following:
A. Order a complement Ch50 test
B. Order an anti-smith antibody
C. Order an anti-dsDNA antibody
D. Repeat the ANA test by immunofluorescence
35. Automated Techniques
• Autoantibodies against nuclear
ribonucleoproteins/nuclear components
• SSA, SSB, Sm, RNP, SM-RNP, Jo-1, SCL-70, Centromere,
Chromatin, Ribosomal P (+ DNA)
• Useful for helping to confirm diagnosis
• used as adjunct to ANA
• Not useful for disease monitoring
• need not be repeated once identified
36. Identifying Anti-Nuclear Antibodies
Antigen Disease Association
Homogenous and Diffuse
DNA-histone complex (nucleosome) SLE (60%)
Drug-induced lupus (95%)
Peripheral Rim
dsDNA SLE
Speckled
RNA polymerase types II and III Systemic sclerosis
RNP MCTD (100%)
Scl-70 Systemic sclerosis (15%-70%)
Sm SLE (25%-30%)
SS-A Sjögren's syndrome (8%-70%)
SLE (35%-40%)
SS-B Sjögren's syndrome (14%-60%)
SLE (15%)
Nucleolar
Nucleolar RNA, RNA polymerase 1 Systemic sclerosis
Pm-scl Polymyositis
Centromere
CENP Limited scleroderma
38. ACR: Position Statement 8/2011
• The ANA by immunofluorescence is the gold standard
• Newer automated ANA’s that test for handfulls of antigens may
miss 100’s of potential autoantigens screened by IIF
• Laboratories should indicate
which method they use
40. Estimated Prevalence of ANA +
in the US Population
Satoh M et al Arth & Rheum 2012;64:2319-2127
41.
42. Anti-DFS70 Antibodies
• Isolated anti-DFS70 antibodies
are not associated with systemic
autoimmune disease (SARD)
• Anti-DFS70 antibodies are
responsible for as much as 12%
of positive Hep-2 results in
routine testing
• Testing for anti-DFS70 antibodies
can eliminate unnecessary reflex
and follow up testing
• Isolated anti-DFS70 antibodies
may be an exclusionary marker
for SARD
Fig. 1: Dense fine speckled pattern of resting cells (blue
arrow) Chromatin staining of mitotic cells (red arrow)
43. Incidence & Estimated New
Cases of CTD’s in B.C.
1999-2000
Guidelines and Protocols Advisory Committee
BCMA, 71/2000 www.health.gov.bc.ca/msp
Incidence per million Est. # new
pts./annum
SLE 56 226
Scleroderma 19 77
PM/DM <10 <40
45. Remember!
• A positive ANA does not mean the patient has a
connective tissue disease, but a negative ANA will
R/O CTD
• Only order an ANA if there is a high likelihood that
the patient has a CTD
46. Anti-phospholipid Antibodies
• ACL measured by ELISA for IgG, IgM, and IgA
Isotypes
• IgG isotype most associated with thrombosis
• LAC measured by phospholipid-dependent
screening test. If prolonged, add 1:1 mix with
normal plasma. If no correction = LAC
• Best association with thrombotic events
• Beta2 glycoprotein I measured by ELISA
50. European Criteria for
Spondyloarthropathy
• Inflammatory spine pain or synovitis
• And one or more of the following:
• Alternate buttocks pain
• Sacroiliitis
• Enthesopathy
• Positive family history
• Psoriasis
• IBD
• Recent episode of urethritis/cervicitis or gastroenteritis
51. HLA- B27 and Disease
(Caucasians)
Disease Association
Ankylosing spondylitis 90%
Reactive arthritis 60-80%
Inflammatory bowel disease 35-75%
Psoriatic arthritis
With spondylitis 50%
With peripheral arthritis 15%
Undifferentiated Spondyloarthropathy 70%
Anterior Uveitis 50%
52. HLA-B27 in the General
Population
• Caucasian 6-8%
• African-Americans 4%
• African Blacks 0%
• Japanese <1%
• Koreans 3-4%
• Native Americans 40-50%
(Haida, Navajo, Eskimos)
54. ANCA: General Information
• Immunofluorescence pattern caused by antibodies
against antigens in cytoplasm of neutrophils
• c-ANCA: anti-proteinase 3
• p-ANCA: anti-myeloperoxidase
• Specific antibodies detectable via ELISA
57. c-ANCA/PR-3 Antibodies
• Highly specific, fairly sensitive
• Polyangiitis with Granulomatosis (Wegener’s)
• Generalized >90%
• Limited - 60%
• Can be seen in Microscopic Polyangiitis
• Best to confirm diagnosis by biopsy
• lung, upper airways/sinuses, kidney, or other involved
organ system
• best used for patients with pulmonary-renal syndromes
or undiagnosed nephritis
58. p-ANCA/MPO Antibodies
• Fairly poor sensitivity and specificity
• A positive test for MPO antibodies and a
positive pANCA are consistent with
• Microscopic Polyangiitis
• Glomerulonephritis
• Polyangiitis with Granulomatosis
• Eosinophilic Granulomatosis with Polyangiitis (Churg
Strauss syndrome)
• Goodpasture syndrome.
• MPO and p-ANCA may also be present in other
autoimmune disorders such as
• Lupus
• RA
• Sjogren’s Syndrome
• Crohn’s disease
59. Take-home points
• Accept clinical diagnosis of non-CTD conditions
• OA, fibromyalgia, soft tissue rheumatism, entrapment
syndromes
• Tests most useful to confirm absence of CTD, but
beware impact of positive tests
• referrals, costs of additional testing, anxiety,
interference with treatment strategy
• Recognize low positive predictive values
• use in highly selected patient populations to confirm
suspected diagnosis
60. Finally:
While laboratory testing can help with
the diagnosis of rheumatic disease,
over-reliance on a lab test may lead the
clinician to an incorrect diagnosis.
