New Perspectives on New (and Old) Rheumatology Serologies
Transcript of New Perspectives on New (and Old) Rheumatology Serologies
New Perspectives on New
(and Old)
Rheumatology Serologies
Robert W. Lightfoot, Jr., MD
Topics for Today’s Talk
1. The Old ANA
- What are the upper limits of normal for the ANA?
2. The New ANA (The Bead Assays)
- The problem of the false negative ANA
- The problem of the false positive ANA
3. The “ANA Profile” and Its Problems
4. The anti- CCP in Arthritis Diagnosis
Case #2
A 33 yr. old woman comes by the booth of the local lupus society at a health fair in your local shopping mall to be screened for SLE.
A 33 yr. old woman is referred
to you with 4 mos. of sustained
pain, stiffness and swelling in
the knuckles of the hand, the
wrists and the knees.
PE corroborates same and is
otherwise negative.
She is Normal Rheumatoid Arthritis
Case #1
Her ANA is + at 1:160Her ANA is + at 1:160
The most likely diagnosis is: The most likely diagnosis is:
What are the upper limits of
normal for the ANA?
The Bell Curve
2 S.D. 2 S.D.1 S.D. 1 S.D.
68% 2.5% 2.5%
The ANA
Hospital personnel, medical students 1%
Blood donors 3%
Elderly 10-15%
Miscellaneous diseases 6%
Arthritis, excluding RA, SLE 14%
Hospitalized, non-rheumatic 17%
Conn. Tissue diseases, not SLE 24%
Rheumatoid arthritis 30-40%
Relatives of SLE patients 33%
SLE 95%
uu uuuu1:20 1:40 1:80 1:160 1:320 1:640 1:1280
u
INDIRECT IF(IIF) ASSAY
HEP-2 CELL
PATIENT
SERUM
Fluorescein-tagged
anti-IgG, -IgA or -
IGM
ANA’S
There are between 100- 150
different antigens in the nucleus
that can be detected in the IIF ANA.
We only know what about 8 of those
antigens are.
What does an ANA of 1:160 tell
you?
Disease Present Disease Absent
Test Sensitivity & Specificity
SLE Present SLE Absent
ANA
95%+
97%Neg.
The ANA in Normals
ANA 1:40 positive
ANA 1:80 positive
ANA 1:160 positive
ANA 1:320 positive
Tan, EM, et al. Arthritis Rheum, 1997. 40:1601-1611.
Specificity
68.3%
86.7%
95%
96.7%
31.7%
13.3%
5%
3.3%
The Problem Is...
Lupus occurs in only 0.05% of the
general population
Ergo, 99.95% do not have lupus
SLE Present General Population
ANA Sensitivity & Specificity
97% Neg.
So...
Of the 0.05% of people who have SLE, 95% have a + ANA, or
0.0475% of people
Of the 99.95% of non-SLE, 3% have a + ANA, or
2.999% of people
2.999 / 0.0475 = 63/1
A ratio of 63:1:: Normal:SLE, i.e.,
98% of ANA positives do NOT have SLE
NOW…
Rheumatoid arthritis is
present in 1.5% of the
population
And, So...
Of the 1.5% of people who have RA, 30% are ANA + , or 0.45% of people,
Therefore,
The ratio of RA to SLE with positive ANA’s is:
0.45/0.0475, or >9 ANA + RA patients for every 1 ANA + SLE patient
And…
We could do similar calculations
for any pre-test percentage
likelihood a given patient has
lupus
SLE Present General Population
ANA Sensitivity & Specificity
97% Neg.
SLE Present General Population
ANA Sensitivity & Specificity
97% Neg.
SLE Present General Population
ANA Sensitivity & Specificity
97% Neg.
SLE Present General Population
ANA Sensitivity & Specificity
97% Neg.
Test Sensitivity & Specificity
SLE Present SLE Absent
ANA
95%+
97%Neg.
ANA PREDICTIVE VALUE FOR SLE
0%
20%
40%
60%
80%
100%
0% 20% 40% 60% 80% 100%
Pre-Test Probability
Po
st-T
est
Pro
ba
bil
ity
ANA +
ANA -
Sensitivity 95%, Specificity 97%
How can the ANA be
made more useful?
ANA PREDICTIVE VALUE
0%
20%
40%
60%
80%
100%
0% 20% 40% 60% 80% 100%
Pre-Test Probability
Po
st-T
est
Pro
ba
bil
ity
ANA +
ANA -
Sensitivity 95%, Specificity 97%
Every historical feature, every
physical finding (or lack
thereof) has its own sensitivity
and specificity for a given
illness.
At the end of the history and
physical exam, 98% of the
diagnostic testing has been
done.
Of the remainder, 98% of the
talent and wisdom required
is for differential diagnosis.
THE BEAD ASSAYS*
*ANA Choice
ANA Direct
Polystyrene microparticles of uniform size are used
as the solid phase.
SOLID-PHASE IMMUNOASSAYS
HISTONE SSA“n-DNA”
SSB SCL-70 RNP
SMITH
Unique bead sets can be conjugated with various, unique target
molecules of interest.
RNPSSBSSA Scl70
Thousands of each bead set are combined to form a
multiplex bead suspension.
The bead suspension is added to the wells of the microplate.
If present, antibody from the test sera will bind to the
antigen-coated bead.
Anti-human Ig reporter “tags” bound antibody.
AtheNA Multi-Lyte System
RNPSSBSSA Scl70
If the patient possesses antibody to more than one bead set, all the
relevant beads will be labeled with antibody and then conjugate.
AtheNA Multi-Lyte System
RNPSSBSSA Scl70
Beads flow through the flow cell, one bead at a time.
• Beads flow through the flow cell and light scatter will determine
color of each bead and if it fluoresces.
• The other determines the amount of classification dyes…the color
of the bead set (i.e., the antigen)
• One laser identifies the amount of fluorescence on the surface.
This flow analysis of the beads occurs at a rate of up to 20,000
beads/per second.
THE BEAD ASSAY AND
FALSE POSITIVES
Risks of “Panel” Testing*
No. of tests Percent of Times One is
Abnormal
1 5%
2 10%
4 19%
6 26%
10 40%
20 64%
*Galen & Gambino- “Beyond Normality”, Wiley & Sons 1975
“The more tests performed
on a healthy subject, the
more likely is the discovery
of an abnormal result.”
Beyond Normality- Galen, RS,& Gambino R, Wiley, 1975
In some labs, if a single “bead
antigen” assay is positive, the ANA is
reported as “positive”
Clues are-
There is no “ANA titer”
There are no “ANA units”
THE BEAD ASSAY AND
FALSE NEGATIVE ANA’s
OTHER SOLID-PHASE IMMUNOASSAYS
HISTONE SSA“n-DNA”
SSB SCL-70 RNP
SMITH? ?
Lab Report
ANA PositivenDNA Negative
SSA Positive
SSB Negative
Scl-70 Negative
Smith Negative
RNP Negative
Lab Report
ANA NegativenDNA Negative
SSA Negative
SSB Negative
Scl-70 Negative
Smith Negative
RNP Negative
Lab Report
ANA (IIF) PositivenDNA Negative
SSA Positive
SSB Negative
Scl-70 Negative
Smith Negative
RNP Negative
THE BEAD ASSAY AND FALSE
POSITIVE ANTI-nDNA
THE BEAD ANTI-“nDNA” ASSAY
Anti- nDNA
• The biggest problem with all anti-nDNA
assays is contamination of the antigen
with single-stranded portions.
• Antibodies to single-stranded DNA are
less specific than the ESR.
F-Anti-IgGSLE Serum
+ +
Crithidia luciliae Tube Dilution anti-nDNA Assay
Kinetochore
Nucleus
1. Any ANA screening test should include an
indirect immunofluorescent ANA screen.
2. For any bead assay positive for anti- “n-
DNA”, a better assay (? Crithidiae) should
be performed.
3. Any patient with an antibody to a single
“bead” antigen (e.g., anti-SSA), should
probably see a specialist.
(IIF)
THE ANTI-CCP ASSAY
peptidyl
arginine
deiminase
(PAD)
Ca++
+ H20+ NH3
+ H+
Arginine
NH2
C=NH2+
NH
CH2
CH2
CH2
O
CN
H
C
Citrulline
NH2
NH
C=O
CH2
CH2
CH2
O
CN
H
C
Known Citrullinated Proteins
• Myelin basic protein
• Filaggrin
• Keratin
• Histones
• Vimentin
• Fibrinogens/fibrins
• Type I Collagen in synovium
CCP Peptides
SHQESTRGRSRGRSGRSGS (306-324)
SHQESTXGRSRGRSGRSGS ( “ - “ )
SHQESTRGXSRGRSGRSGS ( “ - “ )
SHQESTRGRSXGRSGRSGS ( “ - “ )
SHQESTXGRSXGRSGRSGS ( “ - “ )
Lee & SchurRF+
RF-
From, Lee, DM and Schur, PH, Ann Rheum Dis 2003 62:870.
False Positive anti-CCP TestsPsoriatic arthritis 8.6%
-Psoriasis sans arthritis 0.7-17%
SLE 7.8%
Sjogren’s 5.7%
Spondyloarthropathy 2.3%
Scleroderma 6.8%
Hep C Cryoglobulinemia 3.5%
Osteoarthritis 2.2%
Juvenile polyarthritis 7.7%
Fibromyalgia 2.7%
Tuberculosis 34.3%
Arthritis Rheum 61 (11): 1472, 2009
Anti-CCP Specificity and PPV
TEST
Pos.
Likelihood
Ratio
Neg.
Likelihood
Ratio
Sens. Spec.
RF 4.9 0.38 69% 85%
Anti-CCP 12.5 0.36 67% 95%
Prognostic Value of a-CCP’s
• Of 936 patients seen in an Early (<2yrs.)
Arthritis Clinic (EAC), 590 (63%) could
be readily diagnosed, and 205 (21.9%)
had RA.
• 346 (37%) had undifferentiated arthritis
(UA).
• They were followed for 3 years.
Van Gaalen, et al., A&R 50:709, 2004
Prognostic Value of a-CCP
Criterion ACR Criteria ACR & a-CCP
AM stiffness > 1hr. 2.9 ns
Arthritis of > 3 jts. 5.8 5.0
Symmetric arthritis 2.6 6.1
IgM RF positivity 9.8 ns
Erosions on x-ray 7.6 8.7
Anti-CCP positive N/A 38.6
(Odds Ratios in Multivariate Analysis)
Van Gaalen, et al.,A&R, 2004
RF > 50IU
RF < 50IU
Ero
sion
Sco
re
0 2 yrs1 yr 3 yrs
0
10
20
30
40
50
60
70
Nell, et al., Ann Rheum Dis 2005
A-CCP +
A-CCP -
Ero
sion
Sco
re
0 2 yrs1 yr 3 yrs
0
10
20
30
40
50
60
70
Nell, et al., Ann Rheum Dis 2005
ANA
RF
a-CCP
I
N
T
E
R
P
R
E
T
+
+
+
–
–
–
–
–
+
+
–
–
–
+
+
+
+
–
+
–
+
–
+
–
?SLE
M
O
R
E
T
E
S
T
S
RA
?SLE
M
O
R
E
T
E
S
T
S
?RAOther
?SLE
M
O
R
E
T
E
S
T
S
RA
?SLE
RA RA RARA
Summary
• If your ANA Panel shows a negative
ANA, make sure an IIF ANA is done.
• If your ANA is “positive” without a titer,
make sure an IIF ANA is done.
• If your ANA is positive at a titer of
<1:320, more history and/or a panel may
be indicated.
• A positive anti-nDNA usually isn’t.
• A positive anti-CCP is strong evidence
for RA