positive - CECentral

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 #1
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.
Case #2
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.
Her ANA is + at 1:160
Her ANA is + at 1:160
The most likely diagnosis is:
The most likely diagnosis is:
She is Normal
Rheumatoid Arthritis
What are the upper limits of
normal for the ANA?
The Bell Curve
2.5%
2.5%
68%
2 S.D.
1 S.D.
1 S.D.
2 S.D.
The ANA
Hospital personnel, medical students
Blood donors
Elderly
Miscellaneous diseases
Arthritis, excluding RA, SLE
Hospitalized, non-rheumatic
Conn. Tissue diseases, not SLE
Rheumatoid arthritis
Relatives of SLE patients
SLE
1%
3%
10-15%
6%
14%
17%
24%
30-40%
33%
95%
uuuuuuu
1:20
1:40
1:80
1:160
1:320
1:640
1:1280
INDIRECT IF(IIF) ASSAY
Fluorescein-tagged
anti-IgG, -IgA or IGM
PATIENT
SERUM
HEP-2 CELL
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?
Test Sensitivity & Specificity
Disease
SLE Present
ANA
95%+
Disease
SLE Absent
97%Neg.
The ANA in Normals
Specificity
ANA 1:40 positive
31.7%
68.3%
ANA 1:80 positive
13.3%
86.7%
ANA 1:160 positive
5%
ANA 1:320 positive
3.3%
Tan, EM, et al. Arthritis Rheum, 1997. 40:1601-1611.
95%
96.7%
The Problem Is...
Lupus occurs in only 0.05% of the
general population
Ergo, 99.95% do not have lupus
ANA Sensitivity & Specificity
SLE Present
General Population
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
ANA Sensitivity & Specificity
SLE Present
General Population
97% Neg.
ANA Sensitivity & Specificity
SLE Present
General Population
97% Neg.
ANA Sensitivity & Specificity
SLE Present
General Population
97% Neg.
ANA Sensitivity & Specificity
SLE Present
General Population
97% Neg.
Test Sensitivity & Specificity
SLE Present
SLE Absent
ANA
97%Neg.
95%+
ANA PREDICTIVE VALUE FOR SLE
Post-Test Probability
Sensitivity 95%, Specificity 97%
100%
80%
60%
ANA +
ANA -
40%
20%
0%
0%
20%
40%
60%
Pre-Test Probability
80%
100%
How can the ANA be
made more useful?
ANA PREDICTIVE VALUE
Sensitivity 95%, Specificity 97%
Post-Test Probability
100%
80%
60%
ANA +
ANA -
40%
20%
0%
0%
20%
40%
60%
Pre-Test Probability
80%
100%
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
“n-DNA”
SSB
HISTONE
SSA
SCL-70
RNP
SMITH
SSA
SSB
RNP
Scl70
Unique bead sets can be conjugated with various, unique target
molecules of interest.
Thousands of each bead set are combined to form a
multiplex bead suspension.
The bead suspension is added to the wells of the microplate.
AtheNA Multi-Lyte System
SSA
SSB
RNP Scl70
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
SSA
SSB
RNP Scl70
If the patient possesses antibody to more than one bead set, all the
relevant beads will be labeled with antibody and then conjugate.
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.
• One laser identifies the amount of fluorescence on the surface.
• The other determines the amount of classification dyes…the color
of the bead set (i.e., the antigen)
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
1
2
4
6
10
20
Percent of Times One is
Abnormal
5%
10%
19%
26%
40%
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 areThere is no “ANA titer”
There are no “ANA units”
THE BEAD ASSAY AND
FALSE NEGATIVE ANA’s
OTHER SOLID-PHASE IMMUNOASSAYS
“n-DNA”
SSB
?
HISTONE
SSA
SCL-70
RNP
SMITH
?
Lab Report
ANA
nDNA
SSA
SSB
Scl-70
Smith
RNP
Positive
Negative
Positive
Negative
Negative
Negative
Negative
Lab Report
ANA
nDNA
SSA
SSB
Scl-70
Smith
RNP
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Lab Report
ANA (IIF)
nDNA
SSA
SSB
Scl-70
Smith
RNP
Positive
Negative
Positive
Negative
Negative
Negative
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.
Crithidia luciliae Tube Dilution anti-nDNA Assay
Kinetochore
+
+
SLE Serum
Nucleus
F-Anti-IgG
1. Any ANA screening test should include an
indirect immunofluorescent ANA screen. (IIF)
2. For any bead assay positive for anti- “nDNA”, 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.
THE ANTI-CCP ASSAY
Arginine
Citrulline
NH2
NH2
peptidyl
arginine
deiminase
(PAD)
C=NH2+
NH
CH2
N
H
+ H20
C=O
NH
+ NH3
CH2
Ca++
CH2
CH2
CH2
CH2
C
C
C
O
N
H
C
O
+ H+
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 & Schur
From, Lee, DM and Schur, PH, Ann Rheum Dis 2003 62:870.
RF+
RF-
False Positive anti-CCP Tests
Psoriatic arthritis
-Psoriasis sans arthritis
SLE
Sjogren’s
Spondyloarthropathy
Scleroderma
Hep C Cryoglobulinemia
Osteoarthritis
Juvenile polyarthritis
Fibromyalgia
Tuberculosis
Arthritis Rheum 61 (11): 1472, 2009
8.6%
0.7-17%
7.8%
5.7%
2.3%
6.8%
3.5%
2.2%
7.7%
2.7%
34.3%
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
(Odds Ratios in Multivariate Analysis)
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
Van Gaalen, et al.,A&R, 2004
70
RF < 50IU
RF > 50IU
60
Erosion Score
50
40
30
20
10
0
Nell, et al., Ann Rheum Dis 2005
0
1 yr
2 yrs
3 yrs
70
A-CCP -
A-CCP +
60
Erosion Score
50
40
30
20
10
0
Nell, et al., Ann Rheum Dis 2005
0
1 yr
2 yrs
3 yrs
ANA
+
+
+
–
+
–
–
–
RF
+
–
+
+
–
+
–
–
a-CCP
+
+
–
+
–
–
+
–
I
N
T
E
R
P
R
E
T
RA
?SLE
RA
RA
M
O
R
RA
E
?SLE
T
E
S
T
S
?SLE
M
O
R
E
RA
?SLE ?RA RA
T
E
S
T
S
Other
M
O
R
E
T
E
S
T
S
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