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