ana

Indications for antinuclear antibody (ANA) testing

Dr. MJ Amengual. Immunology section, Laboratory Department. Corporació Sanitària Parc Taulí (CSPT). Dr. B Marí. Systemic Disease Unit, Department of Internal Medicine.CSPT ([email protected]).

Evolution of requests for ANA testing. CSPT

Requests for AAB 1997-2009

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Evolution of requests for tests to detect antibodies to extractable nuclear antigen (ENA). CSPT

Requests for ENA testing 1997-2009

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Which is the clinical usefullness of ANA testing?

Diagnosis of systemic autoimmune diseases (SAD).

They are included in the diagnostic criteria for some diseases, e.g. SLE

ANA testing is not useful for the follow-

up of patients diagnosed with SAD(except anti-DNA in SLE and possibly anti-PR3 in Wegener’s granulomatosis)

Tests for determining ANA

Indirect immunofluorescence (IIF).

“Gold standard”.

Enzyme-linked immunosorbent assay (ELISA). Less sensitive and less specific.

3. Washing

6. Visualization using fluorescence microscopy

1. Slides with specific substrate

2. Incubation with test and control serums

4. Incubation with anti-Igs marked with FITC

5. Washing

Negative control

1. Substrate used.

2. Type of fixation used.

3. Conjugation (a-IgG, a-IgG + a-IgM) .

4. Microscopy.

5. Subjectivity of the observer.

Problems of standardization in IIF

Mouse tissue

(liver, kidney, stomach)

HEp-2 cell culture(human laryngeal carcinoma)

Types of substrates used in ANA determinations

1. Substrate used.



4. Microscopy.



Type of fixation used

Triple rat or mouse tissue: liver/kidney/stomach.

Fixation

HEp-2 cell lines.

Requires fixation and permeabilization

Fixatives:

1. Ethanol

2. Formalin

3. Acetone

4. Methanol

Ethanol Formalin

Anti-MPO

Variability in the IIF pattern in function of the type of fixation

Formalin

Anti-PR3 Anti-MPO

Fixation with formalin does not enable distinction between the two main patterns of ANCA antibodies

1. Substrate used.



4. Microscopy.



Isotopic specificityAnti-IgG-FITC

(Anti-IgG + Anti-IgM)- FITC

Ratio of FITC to Protein3:1

Concentration of the specific antibody30-60 µg/ml

Characteristics of conjugation in IIF

1. Substrate used.


3. Conjugation (a-IgG, a-IgG + a-IgM)

4. Microscopy.



IIF using HEp-2 substrate

NEGATIVENUCLEAR POSITIVITY

(>1/80)CYTOPLASMIC

POSITIVITY

SPECKLED NUCLEAR

MEMBRANE

NUCLEOLAR

THICK/ THINDIFFUSE

PLEOMORPHIC PUNCTIFORM

Induced by virus or drugs.

Follow-up at 1-2 months

dsDNAnucleosomes

Suspected SAD

Ro

ENAs

Ro LaSm U1-RNP

Other RNP

Mi-2

RA33U2-RNP

Possible SAD

or

nonspecific

Histones

SSSLE

SSSLE

MCTD

SLE

+-

Drug-ind

SLE

scSLE

Others

PCNA

RASAD

DM

SLE

With/withoutNUCLEOLAR

Scl-70

Ku

Sp100

Centromere

+ - gp210

others FibrillarinU3-RNP

Diffuse SS

HOMO SPECPUNT

PM-SclTo/ThC23 B23

Laminins RNApolNOR-90

Ribo-somal

Jo-1PL7PL12

SLE

ASSSSPM

MyositisDiffuse SS

SADHCAAFSSLE

CBP

limited SSCREST

Drug-ind.

SLE

- +

SRP

PM

HOMOGENEOUS /HOMOGENEOUS with

PERIPHERAL reinforcement

chromatin in

metaphase

HOMO GRANPUNT

PBCDiffuse SS

SAF

PDH FLCLAL

POSITIVE (≤1/80)

SLEMyositis

SLE

Homogeneous pattern, peripheral reinforcement



(>1/80)CYTOPLASMIC

POSITIVITY

SPECKLED NUCLEAR

MEMBRANE

NUCLEOLAR

THICK/ THINDIFFUSE




dsDNAnucleosomes

Suspected SAD

Ro

ENAs

Ro LaSm U1-RNP

Other RNP

Mi-2

RA33U2-RNP

Possible SAD

or

nonspecific

Histones

SSSLE

SSSLE

MCTD

SLE

+-

Drug-ind

SLE

scSLE

Others

PCNA

RASAD

DM

SLE


Scl-70

Ku

Sp100

Centromere

+ - gp210


Diffuse SS

HOMO SPECPUNT

PM-SclTo/ThC23 B23


Ribo-somal

Jo-1PL7PL12

SLE

ASSSSPM

MyositisDiffuse SS

SADHCAAFSSLE

CBP

limited SSCREST

Drug-ind.

SLE

- +

SRP

PM



chromatin in

metaphase

HOMO GRANPUNT

PBCDiffuse SS

SAF

PDH FLCLAL

POSITIVE (≤1/80)

SLEMyositis

SLE

Speckled pattern



(>1/80)CYTOPLASMIC

POSITIVITY

SPECKLED NUCLEAR

MEMBRANE

NUCLEOLAR

THICK/ THINDIFFUSE




dsDNAnucleosomes

Suspected SAD

Ro

ENAs

Ro LaSm U1-RNP

Other RNP

Mi-2

RA33U2-RNP

Possible SAD

or

nonspecific

Histones

SSSLE

SSSLE

MCTD

SLE

+-

Drug-ind

SLE

scSLE

Others

PCNA

RASAD

DM

SLE


Scl-70

Ku

Sp100

Centromere

+ - gp210


Diffuse SS

HOMO SPECPUNT

PM-SclTo/ThC23 B23


Ribo-somal

Jo-1PL7PL12

SLE

ASSSSPM

MyositisDiffuse SS

SADHCAAFSSLE

CBP

limited SSCREST

Drug-ind.

SLE

- +

SRP

PM



chromatin in

metaphase

HOMO GRANPUNT

PBCDiffuse SS

SAF

PDH FLCLAL

POSITIVE (≤1/80)

SLEMyositis

SLE

aa--PMSclPMScl aa--FibrillarinFibrillarin

aa--NORNOR--9090 aa--SclScl--7070

Nucleolar patterns



(>1/80)CYTOPLASMIC

POSITIVITY

SPECKLED NUCLEAR

MEMBRANE

NUCLEOLAR

THICK/ THINDIFFUSE




dsDNAnucleosomes

Suspected SAD

Ro

ENAs

Ro LaSm U1-RNP

Other RNP

Mi-2

RA33U2-RNP

Possible SAD

or

nonspecific

Histones

SSSLE

SSSLE

MCTD

SLE

+-

Drug-ind

SLE

scSLE

Others

PCNA

RASAD

DM

SLE


Scl-70

Ku

Sp100

Centromere

+ - gp210


Diffuse SS

HOMO SPECPUNT

PM-SclTo/ThC23 B23


Ribo-somal

Jo-1PL7PL12

SLE

ASSSSPM

MyositisDiffuse SS

SADHCAAFSSLE

CBP

limited SSCREST

Drug-ind.

SLE

- +

SRP

PM



chromatin in

metaphase

HOMO GRANPUNT

PBCDiffuse SS

SAF

PDH FLCLAL

POSITIVE (≤1/80)

SLEMyositis

SLE

anti-Jo-1

Antisynthetase syndrome

Microscopic examination

The pattern of ANA gives a general idea of the antigenic specificity of the antibodies

Low sensitivity and specificity due to:Subjectivity in the technique itself.

Different dilutions can result in different patterns.

One pattern can mask another.

Different SAD can have the same IIF patterns.

Titration of ANA

Dilutions of the serum sample are used for titration.

The ANA titer is directly proportional to the number of antibodies.

Reference values: <1/40

Positivity:

Low titers: 1/80 to 1/160

Moderate titers: 1/320 to 1/640

High titers: >1/640

Titers 1/40: 25%-30%

Titers 1/80: 10%-15%

Titers 1/160 or higher: <5%

Presence of ANA in the general population


• What are ANAs?

• Usefulness of ANAs in diagnosis

• Interpreting a positive ANA

• Clinical utility of ANAs and antigenic specificities

• ANA-negative patients

• Recommendations for requesting ANA testing

• Conclusions

• What are ANAs?

� ANAs are antibodies directed to specific structures in the nucleus of the cell:

• They indicate the presence of an immunologic alteration.

• They can be detected in the blood of patients with different systemic autoimmune diseases (SAD), but also in patients with indeterminate syndromes and in a small proportion of healthy subjects.

• Diagnostic tests for ANAs are time consuming, but they are very useful in the diagnosis of SAD if used appropriately.

Indications for antinuclear antibody testing (ANA)

Classification

Anti-ENA

Anti-Scl70, CenpB

Anti.Sm, Anti-U1RNPAnti-Ro/SS-A, Anti-La/SS-B

Anti-Jo1

Anti-DNAAnti-Histone

ANA

Anti-RNP I/IIIAnti-Th/ToAnti-Pm-SclAnti-U3RNP

Usefulness of ANAs in diagnosing SAD

• Clinical situations associated with ANA (+)

Hashimoto’s thyroiditis ........................... .46%Graves-Basedow disease...................50%Autoimmune primary cholangitis.................30%Autoimmune hepatitis............................63%-91%Primary biliary cirrhosis.............................10%-40%Primary pulmonary artery hypertension.......40%Idiopathic pulmonary fibrosis........................10%Multiple sclerosis.................................... 5%-10%Idiopathic thrombocytopenic purpura......20%-30%

Viruses: HIV, HCV, EBV, CMV, PV B19, Chronic bacterial infectionsMycobacterial infections

Hydralazine, Procainamide , Immunomodulators, Antithyroids, Isoniazid , Beta-blockers, Minocycline, Penicillamine

Hematologic diseasesSolid tumors (adenocarcinomas)

Organ-specific diseases Infections

Drugs Neoplasms


• ANA (+) in healthy subjects

�3% to 30% of healthy subjects are ANA+ (depending on age and ANA titer).

0

10

20

30

40

50

60

70

ANA(-) 1:40 ene-00 160 320

healthy subjects

ANA (-) 1:40 1:80 1:160 1:320

�40%-60% of women >65 years old with multiple diseases are ANA+

3%

31%

13%5%

(*)

*Tan el al Arthritis and Rheumatism, vol 40, 1997

• The identification of ANAs does not mean the patient has a SAD …


Positive pred

ictive value 11%

Interpretation of ANA (+)

• Usefulness of ANA titer

1/1280 Systemic erythematous lupus (SLE)Mixed connective tissue disease (MCTD)Rheumatoid arthritis (RA)Systemic sclerosis (SS)Autoimmune HepatitisSjogren Syndrome (SjS)Polymyositis (PM)NeoplasmsHIV infectionBacterial infections

1/40 35% healthy subjects

• Usefulness of ANA titer

% Sensitivity and specificity of an ANA titer of 1:160

0

20

40

60

80

100

SLE SS SjS Controls

sensitivity specificity

Identifies 95% of SLE, SS, SjS, and controls

Tan el al. Arthritis and Rheumatism, vol 40, 1997

Interpretation of a positive ANA test

• Usefulness of IIF patterns

IIF pattern Antigen SAD

Homogenous DNA, Histones SLEPerinuclear reinforcement dsDNA SLESpeckled Sm, RNP SLE, MCTD

Ro/SS-A, La/SS-B Sjogren, SLE Centromere, Scl-70 Scleroderma

Mi-2 DMCytoplasmic speckled Jo-1 PMNucleolar RNA polymerase I-III, fibrillarin Scleroderma

Th/To, Scl-70 Pm-scl Scleromyositis

Centromeric CENP-A, CENP-B, CENP-C Limited sclerodPerinuclear ring gp210 PBC, SLE, PMNuclear dots Sp100 PBC

Interpretation of a positive ANA test

• ANA as a predictor of SAD

A considerable proportion (78%) of ANA+ patients without SAD remain ANA+ 10 years later, and only a small percentage (5%) develop a systemic autoimmune disease

ANAs are not useful for ruling out a SAD



Determination of antibodies prior to the diagnosis of SLE in 130 patients



In patients with Raynaud’s phenomenon (primary or associated with SAD symptoms) ANA+ may predict the development of a SAD:

e.g., anticentromere+ is associated with development of CREST (sensitivity 60% and specificity 98%); anti-Scl 70+ isassociated with development of diffuse scleroderma(sensitivity 30% and specificity 100%).


Clinical usefulness of ANAs

• Sensitivity of ANAs in diagnosing SAD

Very useful in the diagnosisSLE 95% Diagnostic criterionSystemic sclerosis 85% Not a diagnostic criterion

Useful in the diagnosisSjogren syndrome 40%-70% Association w/ neonatal SLE/CSLEDermato/Polymyositis 60% Not a diagnostic criterion

Useful in monitoring/prognosisRaynaud’s phenomenon 64% Not a diagnostic criterion

20%-30% develop SAD

ANA necessary for the diagnosis (100%)Drug-induced SLEMixed connective tissue diseaseAutoimmune hepatitis

Solomon DH et al.

Antigenic specificities

• Anti-dsDNA

A positive anti-DNA test is one of the classification criteria for SLE.

In general, positivity for ANA on ELISA is confirmed by IF with Crithidia(more specific).

Prognostic utility: anti-DNA are associated with SLE nephropathy.

High titers are correlated with disease activity, with a degree of variability among individuals.

Recommendations for serial monitoring: * 1-3 months if the patient is active, with other parameters likecomplement levels, proteinuria,..

* 4-6 months in cases with low activity

• Anti-Sm

Anti-Sm are a criterion for the classification of SLE.

Sensitivity for the diagnosis is only 15%-30%.

Very specific for SLE: they are uncommon in healthy subjects or those with other diseases.

They are often found together with high ANA titers.

They are not useful for monitoring.

Due to molecular similarities, Sm+ patients are usually also anti-RNP+ (anti-RNPs are observed in 30%-40% of SLE, nonspecific)


• Anti-RNP

Anti-RNPs are a criterion for the classification of mixed connective tissue disease.

Low specificity.Sensitivity: 100% in MCTD and 30% in SLE.

Prognostic utility: They identify Overlap syndromes (sclero-myositis)Lower association with nephropathy in SLE

Usually associated with high ANA titers.

They are not useful for monitoring.


They are criteria for the classification of Sjogren syndrome (SjS)

Specificity 87% in SjS and SLE

They are useful for prognosis:

*Associated to extraglandular involvement in SjS *20% SLE

*100% neonatal lupus *50-80% subacute cutaneous LE

They are not useful for monitoring

• Anti-Ro/SS-A, Anti-La/SS-B

Skin involvement in SLE Subacute cutaneous lupus Neonatal lupus


• Anticentromere (Cenp-B)

Very useful in the diagnosis of scleroderma (limited type)

Specificity 98% for the CREST variant: Calcinosis, Raynaud, Esophagus, Sclerodactyly, Telangiectasis.

Sensitivity:

* CREST: 60% * Healthy controls: 30%

* Other SAD: 30% * Raynaud’s phenomenon: 25%

Not useful for monitoring

Prognostic utility:

* Less skin involvement in SS *15% in Primary Biliary Cirrhosis (40% sclerod.traits)

* Benign course *10% associated to pulmonary hypertension


• Anti-topoisomerase (Scl-70)

Useful in the diagnosis of diffuse scleroderma (dSS)

Specificity approaching 100% by immunodiffusion

Sensitivity:

* CREST: 60% * Healthy controls: 20%

* Other SAD: 25% * Raynaud’s phenomenon: 30%


Prognostic utility:

* Worse prognosis * More skin involvement

* Pulmonary fibrosis * Nephropathy


• Antinucleolar antibodies (ANoA)

Characteristic pattern on IIF. Very specific for sclerodermaClinical impact remains to be demonstrated

Anti-RNA polymerase I/III

I: rapidly progressing typesIII: diffuse types with little visceral involvement

Anti-U3-RNP

Diffuse cutaneous typesPulmonary artery hypertension

Anti-Pm-scl

Overlap syndromes Scleroderma/Polymyositis

Anti-Th/To

Limited cutaneous typesPulmonary artery hypertension


• Anti-Jo-1 (antisynthetase)

Useful in the diagnosis of the antisynthetase syndrome, dermatomyositis (DM), polymyositis (PM), and interstitial lung disease

High specificitySensitivity in PM 10% and in DM 5%

Antisynthetase syndrome: Raynaud, myositis, mechanic’s hands, arthritis, interstitial lung disease (15%)

High percentage with ANA (-)


• Anti-Mi-2

They are not a criterion for classification, but they are very specific for idiopathic inflammatory myopathy of the dermatomyositis type.

Prognostic utility: better response to immunosuppressor treatment



ANA-negative patients

The negative predictive value of ANAs is high, but it is important to note that up to 20% of anti-Ro (+) patients can be ANA (-).

0/163 ANA- patients with nonspecific systemic inflammatory symptoms/signs without any of the symptoms/signs included in thecriteria for the classification of SAD developed a SAD after 1 year follow-up (Tampoia et al. Arch Pathol Lab Med 2007; 131: 112-116).


Kumar, et al. Diagnostic Pathology 2009, 4: 1-10.

• Conclusions-1

1. ANA determination is useful in the diagnosis of SAD

2. The degree of clinical suspicion is very valuable in the interpretation of a positive result; isolated ANA+ has little clinical value.

3. ANA positivity is not conclusive evidence for SAD, because ANAs can be found in other diseases and in healthy subjects

4. ANA values fluctuate independently of disease activity and are not useful for monitoring; thus, serial determinations are not necessary once a positive result is obtained.


• Conclusions-2

5. No specific titers have been established for each disease, but, in general, high titers are associated with SAD.

6. A negative result does not rule out the diagnosis of a SAD. Up to 20% of anti-Ro+ patients can be ANA negative at IIF.

7. The study of antigenic specificities according to the IIF pattern is appropriate in the presence of characteristic symptoms and signs or strong suspicion of SAD.

8. ENAs have significant diagnostic and prognostic value in SAD.


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