ANA-tomy of Autoimmunity:Revisiting ANA

ANA-tomy of Autoimmunity:
Revisiting Antinuclear Antibodies
Allan D. Corpuz, MD, FPCP, FPRA
PRA Annual Convention 2024
29 Feb 2024
RHEUM
2024

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Disclosures
None
#PROrheum24

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Objectives
1. Compare different methodologies used in
determining the presence of antinuclear
antibodies (ANA)
2. Discuss the use of the International
Consensus on ANA Patterns (ICAP) in
interpreting ANA results
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ANA
#PROrheum24
ANA has a normal physiologic function

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Defining ANA
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Autoimmune antibodies that bind nuclear components
• Double-stranded DNA
• Small nuclear ribonucleoproteins
(eg, SS-A/Ro, SS-B/La, RNP,Smith antigen)
• Enzymes (eg, topoisomerase/Sc|70)
• Histone proteins
• Centromeric proteins
>150 epitopes identified to-date
https://www.rheumatology.org/Portals/0/Files/Methodology%2001%20Testing%20Antinuclear% 20Antibodies%20Position%20Statement.pdf
Kavanaugh A, Guidelines for clinical use of the antinuclear antibody test and tests for specific autoantigens to nuclear antigens. Arch Pathol Lab Med
2000;124:71-81.

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Applying ANA to Clinical Diagnosis
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Kavanaugh et al. Arch Pathol Lab Med 2000; 124:71-81

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#PROrheum24
Evolution of ANA request from the 1950s to 2022 according to the
involvement of autoimmune phenomena in different diseases and
technological development.
Mahler, M. J. Immunol. Res. 2014, 2014, 315179.
Claessens, J. Autoimmun. Rev. 2018, 17, 533-540.
Guidelines for Immunologic Laboratory Testing in the Rheumatic Diseases: An Introduction. Arthritis Rheum. 2002, 47, 429 433.

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THE PAST OF ANA TESTING:
LE Cell and Latex Agglutination

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LE CELL PREPARATION

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ANA: The LE Cell
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LE Preparation/LE Cell
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COLLECTION OF SAMPLE:
• Heparinized bone marrow • Heparinized venous blood •
Oxalated venous blood • Defibrinated venous blood •
Clotted venous blood • LE factor and donor cells
LE Factor:
An Antibody found in the serum found in SLE Patients.
• An LE cell test is considered positive when ~ 2%-30%
of the cells seen on the slide in the neutrophil count are
LE cells.
• A smear is considered positive when 10 or more
characteristic LE cells are seen during a 15-minute
search, associated with the presence of extracellular,
amorphous, nuclear masses.

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LATEX AGGLUTINATION

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Latex particles are bound with native DNA by means of an
intermediary albumin matrix. These coated latex particles
combine with any antibodies to native DNA in serum to give
a visible agglutination.
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http://www.vitroscience.cl/pdf/stanbio/RAPET _sLE.pdf
Latex Agglutination

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Benefits and Limitations of
Latex Agglutination
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Benefits Limitations
Simple and rapid Low sensitivity and specificity
Inexpensive Limited information
Ease of Use Not suitable for screening or
specific antibody detection
Can be used as secondary test in
resource limited settings

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THE PRESENT
OF ANA TESTING
Indirect Immunofluorescence
(Manual and Automated)
and
Solid Phase Assays

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#PROrheum24
INDIRECT
IMMUNOFLUORESCENCE (IIFA)
ASSAYS USING HEP-2 CELLS

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MANUAL MICROSCOPY
ANA IFA

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Indirect Immunofluorescence Assay (IIFA)
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ANA bind epitopes in the substrate → ANA bound with a secondary, fluorophore-
labeled antibody nuclear → fluorescence if ANA present
• Preferred substrate - Human Epithelial-2 cells
• Serial titers in1:2 increments, starting at 1:40 or 1:80
• [Check laboratory guidelines - setting a maximal dilution may be
required.]
Bio-Rad Diagnostics

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Understanding the Hep-2 Cell
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Bio-Rad Diagnostics

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Understanding the Cell Cycle
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Bio-Rad Diagnostics

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Steps in Reading an ANA IFA
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Bio-Rad Diagnostics

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ANA Screening by IFA
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Single serum may produce multiple patterns depending on
dilutions
Rim pattern might be seen at low dilution and homogeneous
pattern at higher dilution
May be the other way around: Frequently homogeneous and/or
peripheral pattern seen at low dilution and speckled pattern at
higher dilution
Despite its sensitivity, HEp-2 IIF has a high inter-observer
variability and low specificity
Mahler, M. J. Immunol. Res. 2014, 2014, 315179.]

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Antinuclear Antibodies
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Negative
Normal titer less than 1:40 dilution
(cut-off may be increased up to 1:160)
Positive
Normal patient
without
underlying
abnormality: 3-
30%
Systemic
Rheumatic
Diseases (SRDs)
(SRDs)
Infections Miscellaneous
conditions
Medications
More common
in older women
SLE
RA
MCTD
Sjogren’s
Syndrome
Necrotizing
vasculitis
TB
Chronic active
hepatitis
Subacute
bacterial
endocarditis
HIV Infection
T1 DM
Multiple
Sclerosis
Pulmonary
fibrosis
Silicone gel
implants
Pregnancy
Elderly patients
Phenytoin
Ethosuximide
Pirimidone
Methyldopa
Hydralazine
Penicillamine
Carbamazepine
Procainamide
Thiazides
Griseofulvin
Chlorpromazine
INH
Quinidine
Gold salts
Minocycline
Pashnina, I.A.Antinuclear Autoantibodies in Health: Autoimmunity Is Not a Synonym of Autoimmune Disease. Antibodies 2021, 10, 9.

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Prevalence of ANA (HEp-2 IFA) in Healthy Individuals
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• A multicenter study of normal individuals (Tan EM, et
al 1997)
- 31.7% positive at screening titer of 1:40
• ANA prevalence in the US population of individuals
ages 12 years and older (Satoh M, et al 2012)
• 95% confidence interval (12.2-15.5%)

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The Problem with ANA IFA:
The Lack of Standardization and Discrepancies between Laboratories
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Abeles et al: Clin Rheumatol (2016) 35:1713-1718

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Lack of Harmonization in HEp-2 IFA Screening,
Estimating, and Reporting of Titers
Naides SJ, et al. J Rheumatol. 2020;47:1768-73.
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• No consensus on screening titer
• College of American Pathologists'
(CAP) proficiency testing program
• ~50% screen at 1:40
• Different dilution schemes
• 2-fold, 4-fold etc
• Different reporting
• Qualitative
• Semi-quantitative or quantitative
• Different end-point titers
1:640, 1:1280, 1:2560, 1:5120

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AUTOMATED ANA IFA
Computer Assisted Diagnosis
Systems

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Automated ANA IFA Evaluation System
#PROrheum24
• Provides automated pattern recognition, end-point titer
suggestions, and reduction of interpretation bias.
• Offers walkaway capability and high throughput for ANA
evaluation by automated processing.
• Reduces turnaround time as compared to manual IFA.
• Provides classification of anti-nuclear antibody (ANA) and
anti-cytoplasmic antibody patterns, including the
interpretation of mixed patterns and titers.
doi: 10.3389/fimmu.2021.638863

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Study I
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• In a peer-reviewed study, the automated pattern recognition of HEp-2 cell-
based IIF was compared to conventional visual interpretation in a total of
351 sera.
• In the discrimination of positive from negative samples, concordant results
between visual and automated evaluation were obtained for 349 sera
(99.4%, kappa = 0.984).
• The system missed out none of the 272 antibody-positive samples and
identified 77 out of 79 visually negative samples (analytical
sensitivity/specificity: 100% / 97.5%).
• 94.0% of all main antibody patterns were recognized correctly by the
software.
Voigt, J. et al. Clin Dev Immunol. 2012;2012:651058.

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Study II
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• The authors compared 3745 serum samples using NOVA
View archived images with manual analysis by microscopy.
• Agreement of the interpretation of an automated ANA IIF
testing platform and manual microscopy was 96.9%
• The authors concluded that automation and standardization
made ANA IFA more consistent.
• However, confirmation of the digital immunofluorescence
images by expert technicians was essential.
Zheng B. et al. Clin Chem Lab Med 2017

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The International Consensus on
ANA Patterns (ICAP)

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ICAP: Efforts to Standardize IIFA Reporting
#PROrheum24
Chan EKL, Damoiseaux J, de Melo Cruvinel W, et al. Report on the second International Consensus on ANA Pattern (ICAP)workshop in
Dresden 2015. Lupus 2016;25:797-804.
Herold M, Klotz W, Andrade LEC, Conrad K, et al. International consensus on antinuclear antibody patterns: defining negative results and
reporting unidentified patterns. Clin Chem Lab Med 2018;56(10): 1799-1802.
Andrade LEC, Klotz W, Herold M, Conrad K, et al. International consensus on antinuclear antibody patterns: definition of the AC-29
pattern associated with antibodies to DNA topoisomerase I. Clin Chem Lab Med 2018;56(10): 1783-8.
International Consensus on ANA Pattern (ICAP)
• Goal: standardize HEp-2 ANA reporting practice
• Consensus document, 2016
• 28 initial Anti-Cell/AC patterns
• Nuclear, cytoplasmic, and mitotic
categories
• Organized by category, pattern, and level of
training/expertise
• Regarding negative & unidentified patterns, 2018;
AC-0 (Negative) added
• AC-29 (DNA Topoisomerase I) added, 2018

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Nomenclature and Classification Tree
https://anapatterns.org/trees-2021.php
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Efforts to Standardize IIFA Reporting
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www.anapatterns.org
AC-1 - Nuclear homogeneous
AC-2 - Nuclear dense fine speckled
AC-3 - Centromere
AC-9 - Clumpy nucleolar

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ICAP Classifications: Linking Pattern, Antigen, and
Disease
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www.anapatterns.org

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Points for Consideration
https://anapatterns.org/

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#PROrheum24
Interested users should
note that they must first
register for a free login
account
at www.ANApatterns.org

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For Healthcare Professionals:
 Improved Consistency: ICAP reduces inter-observer variability in pattern
interpretation, leading to more consistent and reliable diagnoses.
 Enhanced Diagnostic Accuracy: By associating specific patterns with
particular autoimmune diseases, ICAP helps refine diagnoses and reduce false
positives or negatives.
 Streamlined Communication: Using standardized terminology
facilitates communication between clinicians, pathologists, and researchers, improving
patient care and collaboration.
 Guidance for Further Testing: ICAP recommendations indicate which
patterns require further specific antibody testing for better disease characterization.

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For Healthcare Patients
 Accurate Diagnosis: Standardized interpretation improves diagnostic
accuracy, potentially leading to faster and more appropriate treatment.
 Improved Prognosis: Early and accurate diagnosis can improve long-term
outcomes for patients with autoimmune diseases.
 Reduced Uncertainty: Clear interpretations and potential disease
associations aid in understanding the diagnosis and its implications.

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 ICAP is not a definitive diagnostic tool. Other clinical findings and specific
antibody testing are necessary for accurate diagnosis.
 ICAP is an ongoing initiative, and updates may occur as new
knowledge emerges.
 Expertise in pattern recognition and understanding of individual clinical
context remain crucial for accurate interpretation.

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Benefits and Limitations of IIFA Testing
#PROrheum24
Patterns correspond to disease
states
Specificity can be low, particularly
at low titers
Sensitive Batched, manual preparation
common
Standardization in process Automation now available, but
expensive
Dark room/space & technical
expertise needed

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THE SOLID PHASE ASSAYS

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Enzyme Linked Immunosorbent Assays (ELISA)
#PROrheum24
Direct: epitopes on solid phase capture ANA in specimen
→ enzyme-conjugated detection antibody binds ANA →
signal generated → colorimetric detection
• Semi-quantitative result vs index-based cutoff
• Qualitative interpretation - Positive, Equivocal,
Negative
ANA screening by EIA
• HEp-2 cellular or nuclear homogenate coats
the wells
ANA sub-serology testing by EIA
• Purified antigen in wells(eg, SS-A/Ro, SS-B/La,
dsDNA...)
• Commonly used as second-level tests after initial
ANA result is positive

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Solid Phase Assays Compared
#PROrheum24
Solid-phase assays (ELISA, FEIA, and CIA) and line or dot blots as screening and
confirmation methods for autoantibody detection.
Diagnostics 2022, 12(3), 647; https://doi.org/10.3390/diagnostics12030647
Jeong, S. J. Immunol. Res. 2018, 2018, 9094217.

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ANA Screening:
SPAs vs Hep-2 IFA Method
Can initial screening for ANA
by non-Hep-2 IFA methods
replace Hep-2 IFA without loss
of critical information?
Reference
#PROrheum24

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SPAs vs. HEp-2 IFA Methods
Optimal Approach Dependent in Type of ANA-CTD
#PROrheum24
ANA-CTD
SLE
↑Hep-2 IFA+
SPA1,2
MCTD
↑Hep-2 IFA+
SPA1,2
SSc
•↑Hep-2 IFA+
SPA1,2
SJs
•↓Hep-2 IFA+
SPA1,3
↑ = high sensitivity
↓ = sub-optimal sensitivity
1. Low levels of confirmatory antibodies in SPAs questionable
2. HEp-2 IFA: high sensitivity, morphological relevance of pattern in confirming SPA
3. HEp-2 IFA may miss SS-A/Ro (Ro52 and Ro60). Direct testing for antibodies to Ro52,
Ro60 and SS-B/La appropriate

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Performance Characteristics ANA
Methods for Three Main ANA-CTDs
Jeong S, et al. Semin Arthritis Rheum. 2018;48:334-342.
#PROrheum24
ANA-CTD Methods # of Patients Sensitivity (%) Specificity (%)
SLE Hep-2 IFA 2839 95.2 83.3
SPA 89.4 89.1
SSc Hep-2 IFA 1002 93.6 84.2
SPA 85.4 92.8
SJs Hep-2 IFA 610 88.7 86.8
SPA 88.4 89.9
All 3 CTDs Hep-2 IFA 3976 88.4 78.9
SPA 87.4 79.7
• Sensitivity and specificity between SPA and HEp-2 were not significant in most subgroups
• Summary sensitivity of SLE presented statistically significant changes

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ELISA
(in ANA associated SARDs)
#PROrheum24
• Know:
1. Type of ELISA
• 2. Antigenic source
• 3. Definition of cut-off points
de Almeida Brito, F.; Santos, S.M.E.; Ferreira, G.A.; Pedrosa, W.; Gradisse, J.; Costa, L.C.;
Neves, S.P.F Diagnostic Evaluation of ELISA and Chemiluminescent Assays as Alternative
Screening Tests to Indirect Immunofluorescence for the
Detection of Antibodies to Cellular Antigens. Am. J. Clin. Pathol. 2016, 145, 323-331.
HEP 2 Assay ELISA with
mixture of
purified Antigens
ELISA with
HEp2 Extract as
Ag source
Sensitivity 87.4% 76% 90%
Specificity 72.3% 90.4% 50%

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Combining Test Information
#PROrheum24
• HEp2 IFA may detect Abs in patients with ANA-associated
SARDs that are undetectable using SPAs
• Therefore, use information in combination rather than in
isolation
• 8% of ANA-associated SARDs will have a negative FEIA but a
highly positive HEp2 IFA
• LR for ANA-associated SARDs: HEp2 IFA (+) near cutoff value
or (-) HEp2 IFA but (+) FEIA BETTER than weakly (+) or (-)
HEp2 IFA & (-) FEIA
Bossuyt, X. Ann. Rheum. Dis. 2020, 79, E65.

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Combining Test Information
#PROrheum24
Combining the results obtained using
SPAs and HEp-2 IIF is more powerful than
performing either assay alone, and could
have a diagnostic value, since double-positive
results or double-negative results by both
techniques can better confirm or rule out
disease, respectively.

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Benefits and Limitations of ELISA (SPAs) Testing
#PROrheum24
Automatable Widely variable clinical
performance
Relatively inexpensive Limited pre-defined ANA
specificities
High-capacity No pattern given (if screening)
Very sensitive and/or specific Best when automated (expensive)
Good for SARDs with known
autoAbs (eg Sjogren’s Syndrome)

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THE FUTURE
OF ANA TESTING
Multiplex Flow Immunoassays

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#PROrheum24
MULTIPLEX FLOW IMMUNOASSAY
Addressable laser bead immunoassay
(ALBIA)
and
Particle-based multianalyte technology
(PMAT)

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Multiplexed Immunoassay (MIA)
#PROrheum24
Direct, but in a liquid, bead-based phase
• Detection usually fluorescence-based
• Simultaneous testing for 10-12 most common ANA
antigens (eg, dsDNA, SS-A, SS-B, Sm, RNP....)
• Semi-quantitative signal vs index-based cutoff
• Qualitative interpretation: Positive, Equivocal, Negative

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Multiplexed Assays (ALBIA and PMAT)
#PROrheum24
Olsen, N.J.; Choi et al. Emerging Technologies in Autoantibody Testing for Rheumatic
Diseases. Arthritis Res. Ther. 2017, 19, 172.
Cavazzana, I.; et al. Evaluation of a Novel Particle-Based Assay for Detection
of Autoantibodies in Idiopathic Inflammatory Myopathies. J. Immunol. Methods
2019, 474, 112661.

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Antigens used in Commercial Multiplex
ANA Screening Kits
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Binder SR (2006) Lupus 15:412-421

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Multiplexed Immunoassay (MIA)
#PROrheum24
Rapid Limited epitopes represented 
limits clinical performance as first-
level test
Automated Testing systems can be very
expensive
Random-access
Ability to report specific
antigens/epitopes targeted
Well-suited for basic sub-serology
testing

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#PROrheum24
The Future: Biomarkers for the
“Seronegative Gap”

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#PROrheum24
The Future: Machine Learning
and Applied Artificial
Intelligence

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#PROrheum24
Schematic representation of a machine-
learning process
Stafford, I.S.Systematic Review of the Applications of Artificial Intelligence and Machine
Learning in Autoimmune Diseases. NPJ Digit. Med. 2020, 3, 30.
Kingsmore, K.M.An Introduction to Machine Learning and Analysis of Its Use in Rheumatic
Diseases. Nat. Rev. Rheumatol. 2021, 17, 710-730.
As autoimmune diseases are
chronic, multifactorial conditions,
through machine learning, a
branch of the wider field of
artificial intelligence, it is possible
to extract patterns within
patient data, and exploit these
patterns to predict patient
outcomes for improved clinical
management
Limitations
- Technical
- Availability
- Governance
- Reproducibility
- Interpretation
Toh, T.S. Looking beyond the Hype: Applied Al and
Machine Learning in Translational Medicine. EBioMedicine
2019, 47, 607615.

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Many Testing Options –
(Still) One Clinical Gold Standard
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American College of Rheumatology's (ACR's) Position
Statement (2015)
• IIFA remains the gold standard for ANA testing
• Clinical performance data needed for locally-
used methods
• Data available on-request
• Non-lIFA methods used for ANA detection
must be demonstrably equivalent or superior to
lIFA in terms of sensitivity
ALSO:
• Call for standardized methodology and/or reporting
• Method used stated in the result report
https://www.rheumatology.org/Practice-Quality/Clinical-Support/Clinical-Practice-Guidelines
https://www.rheumatology.org/Practice/Clinical/Position/Position Statements/

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Conclusion/Summary
#PROrheum24
ANA testing supports Rheumatologists and other medical specialists in their
efforts to diagnose, monitor, and predict outcomes for an array of disease states,
most of which are connective tissue disorders.
Though there are many approaches to ANA testing, the indirect
immunofluorescence assay (lIFA) remains the gold standard. However, given all
available technologies, use different techniques as an advantage, not a limitation.
Continuing efforts to standardize reporting for ANA testing by IIFA are driven by
ICAP.
Laboratories need to generate and provide locally-sourced clinical performance
data for ANA testing methods used.

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Conclusion/Summary

ANA-tomy of Autoimmunity:Revisiting ANA

ANA-tomy of Autoimmunity:Revisiting ANA

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