Understanding trends in autoantibody testing through the lens of
systemic lupus, celiac disease, and the antiphospholipid syndrome
• Michael L. Astion, MD, PhD, HTBE
• Medical Director, Dept of Pathology and
Laboratories, Seattle Children’s Hospital
Acknowledgements
•
Bio-Rad
•
Immunology Laboratory at
the Univ. of Washington
Dept. of Laboratory Medicine
•
Colleagues at Seattle
Children’s Hospital
•
Mark Wener, M.D.
•
Kathy Hutchinson, M.S.
Overview
•
Introduction to autoimmune diseases
•
Illustration of autoantibody testing principles using:
• Systemic Lupus Erythematosus
• Celiac Disease
• Anti-phospholipid syndrome
•
Conclusions
Autoimmune Diseases
•
Definition: Immunologic destruction of tissues by the body’s
own immune system
•
Complex, interrelated, multi-factorial diseases with genetic and
environmental components
•
About 80 autoimmune diseases have been described
•
•
•
•
Common ones include autoimmune thyroid disease and celiac disease
Most of the 80 are uncommon
Some of the 80 are primary and some are secondary to other causes
Patients with 1 autoimmune Dx are at higher risk for others.
Autoimmune Diseases
• Hard to Dx, sometimes taking years
• Usually diagnosed by specialists
• Tend to have patterns of flares and remissions
• Often treated by suppressing the immune system
Autoimmune diseases are associated ---in complex and
varying ways--- with circulating autoantibodies.
•
Diagnosis
3 kinds of autoantibodies
causing)
• Those that are an effect
of disease
• Naturally occurring
• Thus, interpretation of
autoantibody testing is
difficult!
•
Autoantibodies present
before disease onset
# Autoantibodies
• Pathogenic (disease
Arbuckle et al. Development of Autoantibodies
before the clinical onset of Systemic Lupus
Erythematosus. NEJM: 2003; 349:1526-1533
Autoimmune Disease: More than 80 diseases/syndromes
that can be classified into 3 overlapping categories
• Systemic Rheumatic Diseases
• Organ Specific Diseases
• an oversimplification as usually effects
more than one organ
• E.g. celiac disease, Hashimoto’s thyroiditis
• Intermediate group associated with pulmonary
– renal syndromes
Systemic Rheumatic Diseases
•
Chronic, multi-organ
inflammatory diseases; causes
largely unknown.
•
Diagnosis based on:
• Many clinical features, and
• ANA testing
• RF / Anti-CCP testing
•
Rheumatologic Dx is difficult
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: the prototypical systemic rheumatic disease
•
Multisystem disease with variable presentation/ course.
Often includes arthritis, rashes, and renal, neuropsychiatric
disease.
•
Lab tests:
• + ANA in 99% of cases
• Brisk, broad autoantibody response; average of 3 of 7
commonly tested antibodies present at Dx
• dsDNA, Sm, RNP, SSA, SSB, Ribosomal P, APL
• Specific ANA markers include Sm and dsDNA
• ↓complement in active disease
American College of Rheumatology (ACR):
11 criteria for classification* of SLE
1.
2.
3.
4.
5.
6.
7.
malar rash,
discoid rash
photosensitive rash
oral ulcers
arthritis (non-erosive)
serositis
renal disorder (proteinuria,
casts)
8. Neurologic: seizures, psychosis
9. Hematologic disorder ( ↓WBCs,
hemolytic anemia)
10. Lab test: +ANA
11. Lab test: + dsDNA or + Sm
Alopecia in SLE:
common but not
diagnostic
*www.rheumatology.org/publications/classification/index.asp?aud=mem
•
Google search for “joint pain”
on January 19, 2013.
•
This is the # 1 listing (after
the ads)
•
What tests might an insured
“worried well” patient want?
•
“I’m waiting for my lab
results.”
Methods currently used in the UW Immunology Lab
ANA screen (IFA)
Anti-CCP: multiplex
ANA follow-up testing:
• ENA screen: Sm/RNP, Sm,
SSA, SSB, (Multiplex)
• dsDNA (EIA, multiplex)
• RiboP, histones/chromatin,
Scl-70, Jo-1 : multiplex)
• Anti-centromere (IFA,
multiplex.
• PCNA (IFA, CIE)
• Anti-phospholipids
• Cardiolipins: IgG, IgA, IgM
all (EIA)
• ß2GP1(IgG, IgM): (EIA)
Rheumatoid Factor: Nephelometry
Anti-cytoplasmic antibody screen
(IFA)
Thyroid: TPO, thyroglobulin (EIA)
ANCA:
• IFA
• MPO, PR-3 (IFA,multiplex)
GBM: (multiplex)
Celiac (Tissue Transglutaminase
by EIA)
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%)
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
Pyrite (fool’s gold)
ANA-IFA: a problematic test that is still very useful
•
•
Subjectivity affects precision
and accuracy
High false + rate is common
•
Methods not standardized:
• Substrates
• Microscopes
• Photobleaching
•
Time consuming
•
Fatiguing if done in high volume
•
Requires a commitment to
training and competency
assessment
Photobleaching (right part of
microscope field)
ANA-IFA Results from “Experienced” Labs:
Results from a Classic Study
• 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: Normal sera
were positive in 32% at
• 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 is becoming part of the mix of
methods for autoantibody testing
•
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 IgA panel, celiac IgG panel
• various infectious disease panels
IFA vs EIA vs Multiplex
Key points about ANA testing.
There is more than 1 path to the top of the mountain.
•
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
• Assay cutoffs differ
• Assay quality is variable
•
Literature is confusing:
• Methods are difficult to
compare/contrast
• Patient populations are
different and have biases
•
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 important but
flawed.
Should a lab implement a solid-phase assay (ANAEIA 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
• Am Coll of Rheumatology loves IFA. This matters.
•
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 ANA-multiplex
screen is positive, what are some approaches
to confirmatory testing?
•
Screen with IFA, EIA or
multiplex assay
•
If (+), test (or release) panel
of individual autoantibodies
•
•
•
•
•
•
•
•
•
•
•
•
dsDNA
Sm
RNP
SSA
SSB
Ribosomal P
Scl-70
Centromere B
Chromatin /Histones
Jo-1
Anti-phospholipids
More…
Conservative vs aggressive 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
• Conservative: Do ANA-IFA to get pattern and titer, then
do follow-up testing for individual autoantibodies
• Aggressive: NO pattern/titer. Just do follow-up testing.
Send out ANA-IFA when needed.
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
Celiac Disease: Mechanism
•
Gluten= wheat storage
protein. It is eaten.
•
Gliadin = alcohol soluble
fraction of wheat gluten
•
Gliadin + TTG in gut
Deamidated gliadin
Deamidated gliadin
Immune rxn in those with disease
Tissue injury in small bowel
Symptoms
Celiac Disease: Clinical features
Diarrhea, Steatorrhea (fat
malabsorption)
Abdominal cramping, pain, and
distention
Vitamin, mineral deficiencies if
symptoms are not treated
Occasionally see anemia, arthritis,
rash…
 risk: other autoimmune diseases,
intestinal lymphoma
Celiac Disease: Diagnosis
Gold standard:
• Symptoms, abnormal small bowel biopsy (villous atrophy,
lymphocytes) in response to gluten challenge
• Research method, not done in practice
• Celiac disease afflicts 0.5% - 1% of the population, but alternative
practitioners think it is much higher.
Actual method of Dx:
• Symptoms + antibody tests on gluten-containing diet
• If Ab tests are +, do small bowel biopsies (not just endoscopy).
• Biopsy + = celiac disease if Ab tests and biopsy improve on
gluten-free diet.
Rubio-Tapia A et al. ACG Clinical Guideline…celiac disease. Am J. Gastroenterol. 2013; 108:656-676
Celiac Disease: Lab Testing
Autoantibodies in Celiac Disease
• IgA, Anti-tissue transglutaminase (ATTG).
• IgG Anti-tissue transglutaminase
• IgA, Anti-endomysial antibodies (AEMY) are directed
against tissue transglutaminase, so equivalent to ATTG
• IgA, Anti-deamidated gliadin peptide (DGP)
• IgG, Anti-deamidated gliadin peptide (DGP)
*Recommended first test is IgA ATTG
• Sensitivity, Specificity of IgA, ATTG is ~95%
*NIH Consensus Statement on Celiac Disease. NIH Consens State Sci Statements. 2004 Jun 28-30;21(1) 122.
Rubio-Tapia A et al. ACG Clinical Guideline…celiac disease. Am J. Gastroenterol. 2013; 108:656-676
Evidence-based testing for celiac disease
Rubio-Tapia A et al. ACG Clinical Guideline…celiac disease. Am J.
Gastroenterol. 2013; 108:656-676
Current Laboratory Testing Methods for
Celiac Disease
•
EIA or Multiplex methods
•
IgA-based detection
• ATTG
• DGP
• IgA level
•
IgG based detection
• ATTG
• DGP
•
Advantage of multiplexing is that it
requires less specimen.
•
IFA (monkey esophagus) for AEMY
EIA
Multiplex approaches to celiac disease
• Start with a panel consisting
• IgA (to detect IgA deficiency)
• IgA, Anti-tissue transglutaminase
• IgA, Anti-DGP
• Only If IgA deficient, or child < 2 years,
run kit for:
• IgG, Anti-tissue transglutaminase
• IgG, Anti-DGP
Celiac disease: Treatment
•
Gluten-free diet for life (no
wheat, barley, or rye)
•
Difficult Rx! (no birthday
cake)
•
Thus, unwise to Dx this
disease unless symptomatic
•
Also, treat Vitamin D
deficiency and other
nutritional problems caused
by small bowel injury
2 kids enjoying birthday cake
Celiac Disease: Monitoring Treatment
Follow up titers: IgA, Anti-tissue transglutaminase
1 follow-up small bowel biopsy
Individual values of IgA, Anti-tissue transglutaminase
CS= controls
CD = patients with celiac disease (CD),
GFD = celiac disease patients on gluten-free diet.
Basso D et al. J Pediatr Gastroenterol Nutr. 2006;43:613-618
Overbundling in autoantibody testing:
Example, Celiac disease (CD)
Guidelines: in vast majority of cases only 1 - 3 tests needed.
• IgA anti-tissue transglutaminase or IgA anti-endomysial antibody
• IgA level
• IgA deamidated gliadin peptide
Occasionally needed but not frontline tests
• HLA DQ2, HLA DQ8 are rarely needed to rule out CD
• IgG versions of autoantibody tests useful in IgA deficiency states
*Rubio-Tapia A et al. ACG Clinical Guideline…celiac disease. Am J. Gastroenterol. 2013; 108:656-676
National Institute for Health and Clinical Excellence (NICE). Coeliac disease. Recognition and assessment of
coeliac disease. London (UK): National Institute for Health and Clinical Excellence (NICE); 2009 May. 86 p.
(Clinical guideline; no. 86). [87 references]
NIH Consensus Statement on Celiac Disease. NIH Consens State Sci Statements. 2004 Jun 28-30;21(1) 1-22.
Celiac overbundling from 1 specialty lab. In >95% of cases, 1 – 3
tests are needed, but 5-test bundles were used 98% of the time.
•
•
•
3 insurance databases (2008, 2009)
> 4 million members
N= 760 had celiac testing from this specialty lab
Average age 32 years (range 0 – 82)
Most common diagnosis codes: Diarrhea (n=135); Abdominal pain (n=104),
Celiac disease (n=82); flatulence (n =29), constipation (n=23).
Total Amount spent on:
• celiac autoantibody tests
• Total IgA level related to celiac testing
$195,215
Portion of the total spend accounted for by 5-test
celiac panels consisting of
•4 celiac autoantibody tests
•Total IgA level related to celiac testing
$192,025
% of celiac tests in 5 test bundles
98.4%
Causes of unusual or unexplained venous
thrombosis (venous thrombophilia)
1. Activated protein C resistance (30 - 40% of patients)
2. Increased Factor VIII activity (20 - 25%)
3. Prothrombin mutation (5 - 10%)
4. Protein C deficiency (3 - 5%)
5. Protein S deficiency (3 - 5%)
6. Antiphospholipid antibody syndrome (3 - 5%)
7. Antithrombin deficiency (1 - 3%)
Antiphospholipid Syndrome (APS): Diagnosis requires at
least 1 clinical and 1 laboratory criteria.
Clinical criteria
• Recurrent arterial or venous thrombosis such as stroke, DVT,
pulmonary embolism…
• Pregnancy loss or premature birth
Lab criteria (Persistent, medium to high level results are key!)
• Persistently positive lupus anticoagulant
• 2 positive EIA tests (12 weeks apart) for anticardiolipin, IgG or IgM
• 2 positive EIA tests (12 weeks apart) for anti-beta2GP1, IgG or IgM
Miyakis S et al. International consensus statement on an update of the classification criteria for
definite antiphospholipid syndrome (APS). J Thromb Haemost. 2006; 4(2):295-306.
APS continued…
Secondary APS is associated
with systemic lupus. Primary
APS is not.
The Rx of APS is anticoagulation
to prevent further thrombotic
events.
A patient with alopecia in SLE. This
person is also at high risk for APS.
The following lab tests are NOT currently included in
APS classification*. Although associated with APS.
they do not add (and might hinder) diagnostic power…
•
IgA, anti-cardiolipin
•
IgA, anti-beta2GP1
•
Antibodies to:
• Prothrombin
• Phosphatidylserine
• Phosphatidylserine-prothrombin complex
• Phosphatidylethanolamine
• Phosphatidylinositol
*Miyakis S et al. International consensus statement on an update of the classification criteria for
definite antiphospholipid syndrome (APS). J Thromb Haemost. 2006; 4(2):295-306.
Keeling D. et al. British committee for standards in hematology. Guidelines…antiphospholipid
syndrome. Br J Haematol 2012;157:47-58.
Methods for APS testing
EIA or Multiplexing
for…
•IgG, IgA, IgM
anticardiolipin
•IgG, IgA, IgM antibeta2GP1
•Advantage of
multiplexing is that it
requires less specimen
and reagents.
Univ. Washington test requisition
Conclusions
1.
No gold standard for ANA testing. Labs should be able to explain
their ANA testing strategy –including potential problems-- to
ordering physicians. 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.
2.
The foundation of celiac disease testing is the IgA, anti-Tissue
Glutaminase test. Celiac testing is the most interesting testing
from a social perspective because it is underused and overused at
the same time!
3.
The foundation of testing for anti-phospholipid syndrome are IgG,
and IgM assays for anti-cardiolipins and anti-beta2GP1, as well as
testing for the lupus anticoagulant.
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, but still a very useful test!
• EIA holds steady
• Steady, slow in multiplexing
Thanks!
•