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ANA - PITFALLS
Carolien Bonroy
UZ Gent
6 juni 2015
National Symposium on Non-infectious serology
ANA - historically
For screening: indirect immunofluorescence (IIF) was the
only method
For identification: conventional and laborious methods
(e.g. western blotting, immunodiffusion,
immunoprecipitation) based on native antigens
Performed in specialized laboratories only
ANA - historically
Limited number of requests:
mostly requested by the reumatologists
always in a relevant context
Less equipment + QA/QC-requirements were limited
ANA - today
Different methods for screening are used
For identification: ≠ routine-applicable methods avaliable
solid phase assays (SPA) often use recombinant antigens
ANA - today
Performed in most clinical routine laboratories
An increasing number of requests not only by the reumatologist more frequently in a context of low pre-testprobability for
connective tissue disease
More equipment + more stringent QA/QC requirements for the routine laboratory
Due to the different setting, pitfalls in ANA testing today are not the same as before
Steps to establish and to perform a routine test
Before routine analysis: Test/method selection Method validation/implementation
Routine analysis:
Analysis based on a request Checking results with QC Reporting results
Pitfalls may arise in all the different steps! Only a selection is discussed today…
ANA Anti-ENA dsDNA antibodies
Relevant aspects: The different ANA screening tests have different (performance)
characteristics
The differences between antigens included in the SPA should be taken into account
Do we need to detect all these newer antibodies in a routine
context?
Aspects will be discussed in more details on next slides
Method selection - pitfalls
ANA screening: comparison characteristics IIF versus SPA
Method selection: differences between methods for ANA screening
Op De Beeck et al. (Autoimmunity reviews 2011): detection of ANA by IIF and SPA (FEIA) on diagnostic samples in comparison with healthy donors (HD), patients with chronic fatique syndrome (CFS) and diseased controls (DC)
Method selection: differences between methods for ANA screening
IIF versus SPA: IIF more sensitive than SPA?
Sensitivity Frequency in controls
SLE SSc SjS PM/DM HD CFS DC IIF (1:80) IIF (1:320)
93% 69%
97% 81%
83% 44%
47% 29%
8,7% 2,7%
7,9% 2,9%
25% 11,2%
SPA 74% 73% 89% 39% 2,7% 2,9% 3,7%
At low cutoff, IIF high sensitivity but low specificity At higher cutoffs, specifity of IIF ↑ but sensitivity ↓ Comparable specificity IIF and SPA at cutoff 1:320 SPA had
higher sensitivity in some CTD
When comparing SPA and IIF, the screening dilution used for IIF should be taken into account
Method selection: differences between methods for ANA screening
IIF versus SPA: added value of SPA? Bossuyt and Fieuws (ARD 2014)
The added value of solid phase assays depends on the context the test is used in
For SLE and SjS, the best strategy was performing both tests on all samples (red line) For SLE, AUC this strategy was not significantly higher than AUC of second best strategy
(the 2-step approach – dashed green line) ↔ In SjS, the difference between the AUC was significant.
Differences in antigens included in the SPA may be: The number of antigens
Origin of the antigen:
native vs. recombinant (≠ expression systems) vs. peptide Information often only available on request
The exact nature of the antigen:
Examples: SSA vs. Ro52/Ro60 Sm vs. Sm/RNP ….
The differences between antigens used in the solid phase assays should be taken into account
Method selection: differences between antigens used in the solid phase
Method selection: differences between antigens used in the solid phase
Anti-
Sm
Anti-RN
P
Migliori et al (Autoimmunity 2005)
Sm/RNP complex consists of:
Uridine rich U1-RNA
≠ proteins some common with most U-
snRNPs (=core proteins) specifically associated with U1-
snRNA (A, C and 68)
Anti-Sm antibodies directed against 7 core proteins (B/B’, D1, D2, D3, E, F and G), with B and D
most frequently recognized Ab against the D antigen = highest specificity for SLE Sm antibodies are frequently associated with RNP antibodies in SLE Anti-RNP antibodies • React with proteins A, C and/or 68 kDa • Isolated high titer anti-RNP are diagnostic for MCTD
Sm Sm/RNP Interpretation RNP
- + Antibody to RNP Antibodies to C-terminal motif
shared by protein A, C, B/B’
+ -
+ + Antibody to Sm and U1-RNP Antibody to Sm
+ -
+ - Antibody to conformational epitope of Sm not displayed on the Sm/RNP combination
Antibody to conformational epitope of Sm and/or RNP not displayed on the Sm/RNP combination
- +
Some assay detect antibodies against Sm and the SmRNP combination, but
not against RNP alone combination of both test results is needed to derive
which antibody is present exactly.
An increasing number of ‘newer’ antigens becomes available in
SPA
These newer assays ofter target low prevalent antibodies When applied widely (low pretest probability), high risk of out
of context results
Important: relevance of test results for some of these ‘newer’ antibodies strongly depend on the test method used see example
Do we need to detect all these newer antibodies in a routine context?
Method selection: need for detection of newer antibodies in routine
Example: anti-Th/To antibodies Historically: detected by RNA-IP + highly specific
for SSc, associated with the limited cutaneous form (low prevalence 1-6% in SSc!)
Method selection: need for detection of newer antibodies in routine
Target antigens different proteins of the mitochondrial RNA processing (MRP) and RNase P complex This complex consists of at least 10 proteinen: e.g. Rpp25, hPop1, Rpp20, Rpp38(=Rpp40),… Major autoantigens are Rpp25, hPop1, Rpp38
SPA with Rpp25 (CLIA) and hPop1 (LB) are now available: Bonroy et al. JIM 2012: sens 2,1%, spec 97,8%, kappa with RNA-IP=0,146 Mahler et al. Arth &Ther 2013: sens 2,9%, spec 99,5% Mahler et al: comparison on RNA-IP+ samples: 34% missed by LB, 20%
missed by CLIA
Do we need to detect all these newer antibodies in a routine context?
Method selection: need for detection of newer antibodies in routine
Good strategy: only encourage the use of evidence-based antibody assays
Before implementing these tests: critical review of literature!
Are there independent data on assay performance on these newer antibody assays available in literature?
Relevant question: can you give the correct, evidence-based advice to the clinician for each antibody detected?
This is of particular importance today, as these antibodies are often requested/detected in a context of low pre-test probability
Method validation - pitfalls
Method validation is not evident: Autoantibodies are also present in ‘healthy’ and may appear in a
preclinical phase (e.g. systemic sclerosis) difficult to define false negative, false positive,…
No consensus on the reference method: conventional techniques (used at time characterization antibodies) vs. recent assays based on recombinant antigens?
For IIF: semi-quantitative and subjective ( easier since automated IIF)
Method validation - pitfalls
Ideally, validation is performed in cooperation with the
reumatologists on clinically defined (consecutive) cohorts to avoid selection bias
BUT not possible for every laboratory
Important: proposed cutoff values may be suboptimal Examples: RNA polymerase III antibodies and PM/Scl
antibodies
Clinical evaluation of a set of routine-applicable assays for the detection of RNA polymerase III and PM/Scl antibodies
The proposed cutoff values of some commercial tests may be suboptimal
Lineblot (LB) Fluorescent enzyme immunoassay (FEIA) Bonroy et al. Clin Exp Rheum 2012 Bonroy et al. JIM 2012
Methods in comparison with a combination of conventional techniques (P-IP , WB
and DID)
Samples: 145 well characterized consecutieve SSc patients and 277 diseased controls (90 RA, 58 SLE, 50 SpA, 49 OA, 18 PMR, 12 AAV)
Results:
Technique Antibody Performance based on proposed cutoff criteria
After optimalisation
LB PM/Scl (PMScl-75 and PM/Scl-100)
Spec=88% (LR=1,4) Κ=0,167
Double reactivity Spec=>99% Κ=0,854
RNA polymerase III (RP11 en RP155)
Spec=96% (LR=2,5) Κ=0,831
Double reactivity Spec=100% Κ=1,000
FEIA PM/Scl-100 Spec=96% LR= 0,5
CO ↑>45 Spec=100% LR=∞
RNA polymerase III/RP155 Spec=98% LR=2,5
CO ↑>31 Spec=99,6% LR=14,8
Adapted cutoff values are used in routine
Steps to establish and to perform a routine test
Before routine analysis: Test/method selection Method validation/implementation
Routine analysis:
Analysis based on a request Checking results with QC Reporting results
Request - pitfalls
• Increased number of requests
• Origin not limited to the rheumatology department
• A decrease of pre-test probability significant consequences on the post testprobability
Mahler et al. 2014
Important not to encourage the request of ‘out of context’ autoantibodies
Analysis- pitfalls
High variability in ANA IIF results (within one laboratory)
Procedures needed to limit these variations
Subjectivity of reading
Lot variability between ANA kits
Influence of pipetting devices
Stability of the microscope,
automated IIF analyzer
Analysis- pitfalls
- Double reading
- Interpersonnel comparison exercices 3x/y
- Automated IIF
Subjectivity of reading
UZ Gent strategy:
Analysis- pitfalls
UZ Gent strategy:
- Lot-to-lot comparison before
acceptance
- Lot reservations for at least 6
months (ideally 1 year)
- Monitoring of the effect of lot
change by follow-up of: - % ANA positives
- median ANA immunofluorescence
signal
Lot variability between ANA kits
Maenhout and Bonroy et al. (CCLM 2014)
Report - pitfalls
As method details strongly influence performance and relevance of the results correct reporting is important
Always specify method and antigen details Use of IIF or SPA? IIF:
Screening dilution used in IIF? Staining nucleus and/or cytoplasma
reported? Titer: endpoint titer or scoring on 1 dilution
(e.g. based on ‘fluorescence intensity’ reading of an automated system)
SPA: Type of antigens included: Ro52 and/or Ro60, SmD and/or SmB,…
Suggestion: the registration of EQC samples in the LIS may allow for the detecting of errors in reporting (especially on the printed report)
In conclusion
Pitfalls in ANA testing today are not the same as before
IIF vs. SPA: both methods have their charactheristics and the details of the method should be specified on the report
Differences between antigens used in the SPA should be taken into account when selecting a test and interpreting the results
Relevant question: do we need to detect all these newer antibodies? Only encourage the use of evidence-based antibody assays Due to low prevalence of these antibodies importance of high pre-test
probability The proposed cutoff values of some commercial tests may be suboptimal
High variability within laboratory persist procedures needed to limit these variations