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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