Choosing the Correct ANA Technology for
your Laboratory
• Michael L. Astion, MD, PhD, HTBE
• Professor and Director of Reference
Laboratory Services
• University of Washington
• Dept of Laboratory Medicine
University of Washington
Acknowledgements
• Bio-Rad including Ginger
Weedin, Steve Binder
• Immunology Laboratory at
the Univ. of Washington
Dept. of Laboratory Medicine
• Mark Wener, M.D.
• Kathy Hutchinson, M.S.
Astion: Biases, Disclosures
Univ Washington:
• Director, Reference Laboratories
• Associate Director: Immunology,
Chemistry, Medical Informatics
• Intellectual property holder:
• The “Tutor” series including ANATutor, ANCA-Tutor… (Medical
Training Solutions)
• Grants: Optimizing use of lab tests
(CareCore National Insurance Co)
1992: Two happy faculty
celebrate launch of ANATutor
Consultant:
• Bio-Rad, since 1992 when ANA-Tutor
was 1st launched.
• Various non-profit hospitals
2006: Bald guy discussing
lab errors on UWTV
Overview
• ANA: Am Coll Rheum vs clinical lab perspective
• Review of selected systemic rheumatic diseases
• ANA methods for screening and follow-up testing: IFA, EIA
and multiplexing
• ANA testing algorithms: dsDNA, ENA, and the others
• Testing in Rheumatoid Arthritis (IgM-RF, Anti-CC)
• Specialist vs. primary care perspectives on systemic
rheumatic disease testing
• Conclusions
Perspective of the Amer Coll Rheumatology
ACR position statement on ANA testing, February 2009
www.rheumatology.org/publications/position/ana_position_stmt.asp?aud=mem
•IFA-ANA test should remain the gold
standard for ANA testing.
•Labs using multiplex or EIA assays for
ANAs must provide data to ordering
physicians on request that their assay has
the same or improved sensitivity and
specificity compared to the IFA ANA.
•In-house assays for ANA, anti-DNA, antiSm…should be standardized according to
CDC or WHO standards.
•Report your ANA method when reporting
results.
ANA testing: Clinical Lab Perspective
1. No gold standard for ANA testing.
2. Labs should be able to explain their ANA testing strategy –
including problems-- to ordering physicians.
3. ANA testing is one of the least standardized areas of lab
testing, for example different IVD kits for the “same”
antigen(s) vary significantly. Modest standardization
remains a goal.
4. At the care provider’s request, labs should describe their
ANA methods.
5. Labs should have > 1 ANA methods available, and should
encourage care providers to test by >1 method when test
results do not match clinical findings.
Binder SR. Autoantibody detection using multiplex technologies.
Lupus. 2006;15: 412–421.
Systemic Rheumatic Diseases
• Chronic, multi-organ
inflammatory diseases; causes
largely unknown.
• Autoantibodies (antibodies
against “self” antigens)
present. Some damage is
immune - mediated.
• Diagnosis is based on:
– Many clinical features, and
– Antinuclear antibody (ANA)
testing
– RF, Anti-CCP testing
Systemic Rheumatic Diseases
• Systemic Lupus Erythematosus
(SLE)
• Sjögren’s Syndrome
• Progressive Systemic Sclerosis
• Dermatomyositis / Polymyositis
• Mixed Connective Tissue
Disease
• Undifferentiated CTD
• Rheumatoid Arthritis*
*Tests = anti-CCP and Rheumatoid
factor rather than the ANA test
SLE: As regards ANA testing, it is the prototypical
systemic rheumatic disease
• Multisystem disease with variable presentation and course.
Manifestations include arthritis, skin rashes, renal disease,
neuropsychiatric disease.
• Lab tests:
• + ANA in >90% of cases on initial presentation
• Brisk, broad autoantibody response; average of 3 of 7
commonly tested antibodies present at Dx
• dsDNA, Sm, RNP, SSA, SSB, Ribo P, antiphospholipid…
• Specific ANA markers include Sm and dsDNA/chromatin
• Decreased complement in active disease
American College of Rheumatology (ACR):
11 criteria for classification* of SLE
1. malar rash, 2. discoid rash,
3. photosensitive rash
4. oral ulcers
5. arthritis (non-erosive)
6. serositis
7. renal disorder (proteinuria, casts)
8. Neurologic: seizures, psychosis
9. Hematologic disorder ( ↓WBCs,
hemolytic anemia)
10. Lab test: +ANA or +ACL
11. Lab test: + dsDNA or + Sm
Alopecia in SLE: common
but not diagnostic
*www.rheumatology.org/publications/classification/index.asp?aud=mem
Sjögren’s syndrome (SS)
•
Characteristic triad:
1. dry eyes,
2. dry mouth
3. arthritis / arthralgia
•
Pathology: lymphocytic
infiltrates in exocrine glands
•
Lab tests:
• + ANA
• + SSA
• + SSB
• + Rheumatoid Factor in 50
- 90% of cases
Salivary gland enlargement
Progressive Systemic
Sclerosis (Scleroderma)
• Signs/ symptoms due to fibrosis
• Variants:
• Diffuse = bad prognosis
• CREST = more limited form)
• Lab tests:
+ ANA (nucleolar or centromere)
+ anti-Scl70 in the diffuse variant
+ anti-centromere in CREST
+ anti-nucleolar antibodies (e.g.
RNA polymerase, fibrillarin), but
not commonly used clinically.
Upper: Digital pitting scars
Lower: Raynaud’s
Main teaching points about systemic
rheumatic diseases
1. Most Dx are based on an ACR “laundry list” and lab
testing is only one component of the Dx.
2. ANA test:
• Screens for most, but not all, of the diseases.
• Confirmatory testing beyond the ANA screen is often
(but not always) a part of Dx, Rx, and prognosis.
3. Autoantibodies are + before clinical onset and Dx
4. The disease symptoms (e.g. fatigue, hair loss) are
common but the diseases are not. This leads to over
testing.
Sensitivity of ANA-IFA for various conditions:
• SLE: 88-99%
• Sjogren’s Syndrome: 60-70%
• PSS (Scleroderma): 60-70%
• MCTD (>95%)
• Normal adults < 60 years old: 5% (No way,
more like 7% - 35%)
Autoantibodies occur before clinical onset of
disease
This is true in:
• SLE
• Rheumatoid Arthritis (e.g.
anti-CCP)
• SjÖgren’s syndrome
• Antiphospholipid
Syndrome
• Type 1 diabetes
• Probably many others
# Autoantibodies
Diagnosis
Time before Dx (years)
Arbuckle et al. Development of
Autoantibodies before the clinical onset of
Systemic Lupus Erythematosus. NEJM:
2003; 349:1526-1533
Methods of ANA testing
• ANA-IFA: ~ 65% of labs, the “? gold
standard”
• EIA: ~ 25% of labs, but > 40% of
assays (volume labs often choose
EIA)
• Multiplexing (beads): ~ 10% of labs,
but > 20% of assays as higher volume
labs are switching to this.
• Microarrays: currently for research
only, not yet available for clinical
autoantibody testing
Methods currently used in the UW Immunology Lab
ANA screen (IFA)
ANA follow-up testing:
Anti-cytoplasmic antibody
screen (IFA)
•
ENA screen: Sm/RNP, Sm,
SSA, SSB, (Multiplex)
•
RiboP, Chromatin (Multiplex)
•
dsDNA (multiplex and EIA)
•
Scl-70, Jo-1 : (EIA, switching
to multiplex)
•
Anti-centromere (IFA and
CEN B by multiplex)
GBM: multiplex
•
PCNA (IFA, CIE)
Sendouts
•
Anti-phospholipids
– Cardiolipins: IgG, IgA, IgM
all (EIA)
– ß2GP1(IgG, IgM): (EIA)
Thyroid: TPO, thyroglobulin
(EIA)
ANCA:
– IFA
– MPO, PR-3 (multiplex)
•
Histones
•
Myositis autoantibodies (Mi-2,
PL-7, PL-12 and 9 others)
•
RNA Polymerase I, II, III
Microscope-based ANA-IFA
(Cultured HEp-2 cells are the substrate for antibody binding)
ANA-IFA: two criteria for a positive test:
1) intensity > endpoint control, 2)identifiable staining pattern
Negative
+, Homogeneous pattern
End-point Control
ANA-IFA patterns loosely correlate with specific
antibodies and diagnoses
Homogeneous
Speckled
Centromere
Nucleolar
ANA-IFA titers
• The higher the
titer the more
likely the + ANA
test is clinically
significant.
• In patients with
disease, higher
titers suggest
more active
disease.
1:40
1:80
1:160
1:320
1:640
control
ANA-IFA: a problematic test
• Subjective: poor precision,
poor accuracy
• High false + rate
• Methods not standardized:
– Substrates
– Microscopes
– Photobleaching
• Time consuming
• Fatiguing
• Intense training and
competency assessment
Photobleaching (right part of
microscope field)
ANA-IFA results from “experienced” labs:
• Tan et al., Arthritis Rheum, 1997, 40:1601- 1611
• Methods:
– 15 different labs testing 125 normal sera
– each lab used their own HEp-2 IFA method.
• Results: Normal sera were positive in 32% at
1:40; 13% at 1:80, and 5% at 1:160
• Authors’ recommendations:
– Report all results at 1:40 and 1:80
– Report lab’s false + rate with the test result
ANA-EIA: Types of Tests
• Hep-2 nuclear extract (1st
generation)
• Hep-2 nuclear extract spiked
with extra antigens (2nd and
3rd generation kits)
• Recombinant antigens only
Multiplexing: Flow cytometric immunoassay based on multiplexed
fluorescent microspheres (beads)
Multiple antibodies can be detected in 1 reaction using multiple
antigen beads. Patient’s SSA antibody=Y; Detection antibody = Y
SSB
Sm
RiboP
Scl70
Sm
Y
Y
RNP
SSA
SSA
Control
SSB
RNP
dsDNA
CenB
Scl70
dsDNA
Anatomy of a bead used in the multiplex assay
Illustrations courtesy of Bio-Rad laboratories, used by permission.
Multiplex assay: The detection system counts many beads of
each autoantibody specificity, and determines which
autoantibodies are present in the patient’s serum. A particular
ANA screen may have up to 13 autoantibody specificities
depending on the manufacturer.
Illustrations courtesy of Bio-Rad laboratories, used by permission.
Multiplex testing: future
•
It is significantly impacting auto-antibody testing.
•
Multiplex instruments will become part of lab instrumentation
used by many labs.
• Automated
• Multiple results from one sample
• Allows consolidation of many assays onto 1 platform
• Many assays now available, or soon will be available
• vasculitis panel with anti-MPO, anti-PR3, anti-GBM
• celiac panel with IgA tissue transglutaminase, IgA
• various infectious disease panels
IFA vs. EIA vs. Multiplex
Key points about ANA testing
•
No gold standard method
•
There are differences between
methods (EIA, IFA, multiplex).
and within a method (e.g.,
“multiplex” assays not the
same.)
– Assay components differ
•
It is easy to find patients negative by
one method and positive by another.
•
You can’t make lab policy and
procedure based on 1 or 2 patients.
•
Labs need more than 1 method
available to them.
•
Clinicians should retest by the same
and a different method when results
do not match clinical findings.
•
When switching methods,
communicate to care providers the
differences between the new and old
methods. Individualize the
differences when possible!!
•
The ACR position on IFA is flawed
and unrealistic.
– Assay cutoffs differ
– Assay quality is variable
•
Literature is confusing:
– Methods are difficult to
compare/contrast
– Patient populations are
different and have biases
ANA-IFA vs. ANA-EIA vs. ANA-Multiplex:
A sampling of the large and expanding literature
EIA vs. IFA
–
–
–
–
–
–
Carey JL. Clin Lab Med, 1997, 17: 355-365
Emlen W et al. Arthritis Rheum, 1997, 40: 1612-1618
Homburger HA et al. Arch Pathol Lab Med, 1998, 122: 993-999
Russell AS et al. Clinical Exp Rheum, 2003, 21: 477-480
Tonuttia E et al. Autoimmunity, 2004, 37: 171-176
…
Multiplexing
–
–
–
–
–
Shovman O. et al. Ann N Y Acad Sci. 2005;1050:380-8.
Zandman-Goddard et al. Clin Dev Immunol. 2005;12:107-111.
Nifli A. et al. J Immunol Methods. 2006;311:189-197.
Desplat-Jego S. et al. Ann N Y Acad Sci. 2007;245-255.
Bardin N. et al. Autoimmunity. 2009;42:63-68.
Fact: A lab can successfully switch to a solid-phase
assay for ANA screening and confirmatory testing.
The keys to success are:
• Understanding the performance characteristics of the
new vs. old method as performed by your lab.
Communicating this understanding to care providers.
• Encourage rheumatologists to test by more than 1
method in difficult cases.
• dsDNA…
dsDNA assays: a difficult area
•
A pathogenic autoantibody
•
dsDNA is a specific test for SLE, part of ACR classification criteria
•
disease severity marker in SLE patients with nephritis
•
dsDNA Assays
–
Farr assay
–
EIA - regular (primary method used at UW)
–
EIA – high salt (primary method used at UW)
–
Crithidia
–
Multiplex (secondary method at UW)
•
Different dsDNA assays do not measure exactly the same thing.
•
It is useful to have >1 assay available for problem cases.
•
Best to follow a dsDNA+ patient by comparing results within the
same method.
•
If switching methods, establish patient-specific baselines using
the new method.
Should a lab implement a solid-phase assay
(ANA-EIA or multiplex)?
• A complex question; the answer depends on the lab.
• Clear Advantages of solid-phase:
– objective measurement, fast, easy to automate
• Clear Disadvantages of solid-phase assays:
– can require MD re-education due to loss of pattern/titer
• Complex issues:
– volume fatigue, cost, historical momentum, training,
cutoffs for positive results, change management
(especially with rheumatologists)
Comparing / Contrasting: EIA vs. Multiplexing
• Multiplexing is the most automated method
• Multiplexing requires the least amount of sample
• EIAs based on HEp-2 cell extracts have a broader
representation of antigens. Is this an advantage or
disadvantage?
• Complex issues:
– cost, volume fatigue, historical momentum,
training, cutoffs for positive results, change
management with rheumatologists
When the ANA-IFA, ANA-EIA, or ANAmultiplex screen is positive, what are some
approaches to confirmatory testing?
• Doc chooses the specific autoantibodies to test
• Lab offers “disease” panels, e.g. SLE panel
– E.g, Scleroderma panel would include Scl-70 and
Centromere B
– SLE panel would include multiple autoantibodies (ENA,
dsDNA, antiphospholipid, RiboP, etc)
• Create specific panels for individual physicians.
ANA test sequence
• Screen with IFA, EIA or
multiplex assay
• If (+), test (or release) panel
of individual autoantibodies
• Panels customized for
particular clients
• UW standard panel:
dsDNA, SSA, SSB, Sm, RNP
• UW custom panels also
include Scl70, Jo-1,
complement, others
•
•
•
•
•
•
•
•
•
•
•
•
dsDNA
Sm
RNP
SSA
SSB
Ribosomal P
Scl-70
Centromere B
Chromatin /Histones
Jo-1
Anti-phospholipids
More…
Conservative ANA testing sequence using
solid phase (EIA or multiplex) screen
• Start with solid phase assay
• If negative, report result and you are finished.
• If positive
– Do ANA-IFA to get pattern and titer
– Do follow-up testing for individual
autoantibodies
Aggressive ANA testing sequence using
solid phase assay (EIA or multiplex)
• Start with solid phase ANA assay
• If negative, report result and you are finished.
• If positive, do individual autoantibody
identification.
– Do not do a HEp-2 pattern and titer.
– This means physicians do not get “their
pattern”.
Multiplex testing: ANA testing
algorithm
• For confirmatory testing:
– “Release” the results of
the individual beads
– Perform additional
autoantibody tests if
not covered by the
multiplex ANA screen
Am Coll Rheum Criteria for Rheumatoid Arthritis
Arnett F, et al. Arth Rheum. 1988;31:315-24
www.rheumatology.org/publications/classification/ra/ra.asp?aud=mem
1. Morning stiffness > 1 hr
2. Joint swelling ≥ 3 joint areas at once
3. Swelling in hand (wrist, metacarpal phalangeal (knuckle), or proximal
interphalangeal joint)
4. Symmetric arthritis
5. Rheumatoid nodules
6. Positive test for rheumatoid factor (RF; most assays measure IgMRF, which is autoantibody IgM directed against IgG) *
7. Erosions or bony decalcification on x-ray of the hand and wrist
At least 4 of 7 criteria, with criteria 1 through 4 present for > 6 weeks.
*Anti-CCP, though commonly used and newer, is not currently in the criteria
Disease progression in RA can be severe with erosive
(deforming) arthritis and organ involvement.
Therefore, the goal in RA is early detection and
prognostication to choose Rx that will prevent disease
progression.
Lab Tests in Rheumatoid Arthritis
• Diagnostic / Prognostic
• IgM- RF (Rheumatoid
factor)
• Anti-CCP (cyclic
citrillunated peptide)
• Also useful
IgM-RF
Anti-CCP
Sensitivity
~69%
~67%
Specificity
~85%
~95%
• CRP
• ESR
Nishimura K et al. Meta-analysis:… Accuracy of anti-CCP and RF for
Rheumatoid Arthritis. Ann Intern Med 2007; 146: 797-808.
Rheumatoid Factor: Introduction
• Antibody directed against the Fc portion of IgG
• Most assays measure IgM-RF
• May be involved in disease pathogenesis
• Higher levels tend to be associated with poorer
prognosis
• Found in other rheumatologic (e.g. Sjogren’s
syndrome) and non-rheumatologic conditions,
especially Hepatitis C.
Anti-CCP Diagnostic Test
• Similar role as RF, similar sensitivity but more
specific
• !! Negative in patients with Hepatitis C
• Predicts severe and erosive disease
• Present in early synovitis
• Not perfectly correlated with RF, i.e. somewhat
different diagnostic information so
rheumatologists usually order both tests.
Clinical lab perspective on ANA testing
• ANA-IFA is subjective, requiring intensive training
• Some labs have little ANA-IFA expertise.
• Biggest ANA-IFA problem is hi false positive rates
• Survey specimens show large variations -and no official
“correct” answer- in reported pattern and titer.
• Methods: No standardization of substrate, reagents,
microscope, training, competency assessment
• Looking for better, automated methods
Perspective on ANA testing: Internists and
Family Practitioners (1)
• Not knowledgeable about:
– ANA testing
– Systemic rheumatic
diseases
• Usually order the ANA test
to rule out SLE
• They want the result to be
negative.
Perspective on ANA testing: Internists and
Family Practitioners (2)
• They would love for your
lab to change your cutoff
from 1:40 to 1:160.
• If ANA test is +, they will
refer to a Rheumatologist
Utilization issues regarding ANA testing
• Why are too many ANA screening tests ordered in
ambulatory settings?
– Patient pressure via googlification.
– Systemic rheumatic diseases are rare diseases with
common symptoms.
• The main impact on patients is the medical adventure
caused by false positive results.
Patient pressure
• Google search for “joint
pain” on January 19, 2010.
• This is the # 1 listing (after
the ads)
• What tests might an
insured “worried well”
patient want?
• “I’m waiting for my lab
results.”
ANA testing is common (about 0.5% of the population
each year) because the symptoms of SLE and other
rheumatic diseases are common.
• SLE: Most frequent symptoms at onset
– Fatigue
– Fever
– Weight loss
– Arthritis (joint inflammation)
– Arthralgia (joint pain)
3 Cases:
1. A 40 y.o. male presents with a burning feeling on urination.
He says that his new girlfriend was recently diagnosed
with “Lupus”. He read about lab tests for “Lupus” online
and wants an ANA test.
2. A 40 y.o. woman presents with 1 month of fatigue, hair
loss and a describes a “rash near her nose” although none
is now present.
3. A 40 y.o. woman presents with progressive fatigue, hair
loss, a malar rash, oral ulceration on the inside of her lip,
and joint pain bilaterally in her hands, wrists, ankles and
knees.
The ANA test is a not a good test for ruling in SLE since SLE
has a low prevalence in the population and the ANA test is not
specific. PPV = positive predictive value.
(50%,79%)
100%
80%
60%
40%
(10%,30%)
20%
0.1%
1.0%
10.0%
0%
100.0%
Prevalence or pretest probability
PPV
The ANA-IFA test is a good test for ruling out SLE since SLE
has a low prevalence in the population and the ANA test is 9599% sensitive for SLE. NPV = Negative Predictive Value.
100%
80%
NPV
60%
(10%,99%)
40%
(75%,83%)
20%
0%
0%
25%
50%
75%
100%
Prevalence or pretest probability
Perspective on ANA testing: Rheumatologists
• Knowledgeable about the use and interpretation
of the ANA test
– Comfortable with interpreting borderline ANA
screening tests (e.g. ANA-IFA positive at 1:40)
• Often not knowledgeable about lab methods
• Order the ANA test and the reflexive panel
• Manage patients with SLE and the other systemic
rheumatic diseases.
Conclusions
1.
There is no gold standard for ANA testing.
2.
Labs should be able to explain their ANA testing strategy –
including potential problems-- to ordering physicians.
3.
ANA testing is one of the least standardized areas of lab testing, for
example different IVD kits for the “same” antigen(s) vary
significantly. Modest standardization remains a goal.
4.
At the care provider’s request, labs should describe their ANA
methods.
5.
Labs should have > 1 ANA methods available, and should
encourage care providers to test by >1 method when test results
do not match clinical findings.
6.
Anti-CCP is a newer, more specific test for Rheumatoid Arthritis. It
is usually used in conjunction with IgM-RF testing.
Overall trends in autoantibody testing
• More automation:
– movement from IFA to EIA to multiplex flow
cytometry and other automated methods
• More assays:
–  of EIA, ,multiplex tests will be available
• Gradual progression:
– Steady, slow  in ANA-IFA
– EIA holds steady
– Steady, slow in multiplexing
– Intro of autoantibdoy microarrays (?3 – 5 years)
Thank you!