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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

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choosing the correct ana technology for your laboratory
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
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
Astion: Biases, Disclosures

Univ Washington:

  • Director, Reference Laboratories
  • Associate Director: Immunology, Chemistry, Medical Informatics
  • Intellectual property holder:
    • The “Tutor” series including ANA-Tutor, ANCA-Tutor… (Medical Training Solutions)
    • Grants: Optimizing use of lab tests (CareCore National Insurance Co)

Consultant:

  • Bio-Rad, since 1992 when ANA-Tutor was 1st launched.
  • Various non-profit hospitals

1992: Two happy faculty celebrate launch of ANA-Tutor

2006: Bald guy discussing lab errors on UWTV

overview
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
slide5

Perspective of the Amer Coll RheumatologyACR position statement on ANA testing, February 2009www.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, anti-Sm…should be standardized according to CDC or WHO standards.
  • Report your ANA method when reporting results.
ana testing clinical lab perspective
ANA testing: Clinical Lab Perspective
  • No gold standard for ANA testing.
  • Labs should be able to explain their ANA testing strategy –including problems-- to ordering physicians.
  • 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.
  • At the care provider’s request, labs should describe their ANA methods.
  • 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.
slide7

Binder SR. Autoantibody detection using multiplex technologies. Lupus. 2006;15: 412–421.

systemic rheumatic diseases
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 diseases1
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
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
American College of Rheumatology (ACR):11 criteria for classification* of SLE
  • malar rash, 2. discoid rash, 3. photosensitive rash
  • oral ulcers
  • arthritis (non-erosive)
  • serositis
  • renal disorder (proteinuria, casts)
  • Neurologic: seizures, psychosis
  • Hematologic disorder ( ↓WBCs, hemolytic anemia)
  • Lab test: +ANA or +ACL
  • Lab test: + dsDNA or + Sm

Alopecia in SLE: common but not diagnostic

*www.rheumatology.org/publications/classification/index.asp?aud=mem

sj gren s syndrome ss
Sjögren’s syndrome (SS)
  • Characteristic triad:
    • dry eyes,
    • dry mouth
    • 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
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
Main teaching points about systemic rheumatic diseases
  • Most Dx are based on an ACR “laundry list” and lab testing is only one component of the Dx.
  • 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.
  • Autoantibodies are + before clinical onset and Dx
  • 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
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
Autoantibodies occur before clinical onset of disease

Diagnosis

This is true in:

  • SLE
  • Rheumatoid Arthritis (e.g. anti-CCP)
  • SjÖgren’s syndrome
  • Antiphospholipid Syndrome
  • Type 1 diabetes
  • Probably many others

# Autoantibodies

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
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:

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)

PCNA (IFA, CIE)

Anti-phospholipids

Cardiolipins: IgG, IgA, IgM all (EIA)

ß2GP1(IgG, IgM): (EIA)

Anti-cytoplasmic antibody screen (IFA)

Thyroid: TPO, thyroglobulin (EIA)

ANCA:

IFA

MPO, PR-3 (multiplex)

GBM: multiplex

Sendouts

Histones

Myositis autoantibodies (Mi-2, PL-7, PL-12 and 9 others)

RNA Polymerase I, II, III

Methods currently used in the UW Immunology Lab
slide20
ANA-IFA: two criteria for a positive test:1) intensity > endpoint control, 2)identifiable staining pattern

+, Homogeneous pattern

Negative

End-point Control

ana ifa patterns loosely correlate with specific antibodies and diagnoses
ANA-IFA patterns loosely correlate with specific antibodies and diagnoses

Homogeneous

Speckled

Centromere

Nucleolar

ana ifa titers
ANA-IFA titers

1:80

1:40

  • The higher the titer the more likely the + ANA test is clinically significant.
  • In patients with disease, higher titers suggest more active disease.

1:160

1:320

1:640

control

ana ifa a problematic test
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
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
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
slide26

Scl70

Sm

Y

Y

dsDNA

SSB

SSA

CenB

RiboP

Control

RNP

SSB

Scl70

Y

Y

RNP

Sm

dsDNA

SSA

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

anatomy of a bead used in the multiplex assay
Anatomy of a bead used in the multiplex assay

Illustrations courtesy of Bio-Rad laboratories, used by permission.

slide28

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
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
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

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 flawed and unrealistic.

Key points about ANA testing
ana ifa vs ana eia vs ana multiplex a sampling of the large and expanding literature
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.
slide33
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
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
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
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
slide37
When the ANA-IFA, ANA-EIA, or ANA-multiplex 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…

ANA test sequence
conservative ana testing sequence using solid phase eia or multiplex screen
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
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
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
slide42

Am Coll Rheum Criteria for Rheumatoid ArthritisArnett F, et al. ArthRheum. 1988;31:315-24www.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 IgM-RF, 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

slide43

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
Lab Tests in Rheumatoid Arthritis
  • Diagnostic / Prognostic
    • IgM- RF (Rheumatoid factor)
    • Anti-CCP (cyclic citrillunated peptide)
  • Also useful
    • 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.

Rheumatoid Factor: Introduction
anti ccp diagnostic test
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
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
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
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
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
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.”
slide52
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
3 Cases:
  • 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.
  • 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.
  • 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.
slide54

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%)

(10%,30%)

slide55

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 95-99% sensitive for SLE. NPV = Negative Predictive Value.

(10%,99%)

(75%,83%)

perspective on ana testing rheumatologists
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
Conclusions
  • There is no gold standard for ANA testing.
  • Labs should be able to explain their ANA testing strategy –including potential problems-- to ordering physicians.
  • 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.
  • At the care provider’s request, labs should describe their ANA methods.
  • 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.
  • 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
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)
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