Right and wrong in antinuclear antibody tests. Timo Walle HUSLAB, Department of Immunology

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1 Right and wrong in antinuclear antibody tests Timo Walle HUSLAB, Department of Immunology

2 Antinuclear antibodies (ANA) Autoantibodies reacting with nuclear antigens Nucleoplasm Nuclear membrane Nucleoli (Mitotic spindle) Today, all antibodies reacting with Hep2 cells are often called ANA including antibodies to cytoplasmic structures Ribosomes Mitochondria Lysosomes Golgi Cytoskeleton Centrioles

3 Antinuclear antibodies in diagnostics Tentative diagnosis ANA screening IFA / EIA Staining pattern Titer/Units + - ANA to specific antigens (EIA screen?) SSA Jo-1 Ribosomal P prot dsdna, histone, nucleosome Sm, U1-RNP, SS-A, -B, Scl-70/Topo I, Jo-1 (Histidyl trna synthetase), Ribosomal P proteins etc.

4 Conditions associated with a positive ANA ANA very useful for diagnosis Systemic lupus erythematosus Systemic sclerosis ANA somewhat useful for diagnosis Sjögren's syndrome Polymyositis-dermatomyositis ANA very useful for monitoring or prognosis Juvenile chronic arthritis Raynaud's phenomenon ANA is a critical part of the diagnostic criteria Drug-associated lupus Mixed connective tissue disease Autoimmune hepatitis Solomon DH et al. Evidence-based guidelines for the use of immunologic tests: Antinuclear antibody testing. Arthr Rheum 2002;47,4,434

5 ANA not useful or has no proven value for diagnosis, monitoring or prognosis Rheumatoid arthritis Multiple sclerosis Thyroid disease Infectious disease Idiopathic thrombocytopenic purpura Fibromyalgia

6 ANA screening methods Indirect immunofluorescence assay (IFA) Substrate Secondary antibody Hep-2 Hep-2 cells + SSA/Ro60 Other cell lines (rarely used) Tissue sections Anti-human-IgG Anti-human-Ig

7 ANA screening methods EIA and other solid phase assays Antigens Nuclear extracts Purified nuclear antigens Recombinant proteins

8 IFA with Hep-2 substrate as screening test Today considered as the standard method for ANA Human laryngeal carcinoma (epithelial) cell line A vast array of different autoantigens in relatively native conformation Fixation/handling of the cells may affect by denaturing antigen determinants or extracting antigens (SSA) Sometimes allows presumptive estimate of the antibody specificity (centromere, nucleolus antigens, mitochondria etc.) In most cases further testing is needed to find the specificity of the ANA reaction Labor intensive, subjective (microscopy) Specificity should be set to an acceptable level, 5 % positive results in healthy population

9 ANA in healthy individuals Tan et al, Arthr Rheum 40: 1601, 1997 International units (WHO, IU/ml) can be used

10 Solid phase screening methods Easy to automate, objective results Able to detect only a limited number of ANA specificities Abs to rare antigens are often missed (nucleolus, nuclear membrane, mitotic spindle etc.) Mixtures of purified antigens are best suited to screening before specific ANA tests Nuclear extracts combined with defined antigens correlate best with IFA No information about the ANA specificity Is it right to call these tests ANA?

11 Antibodies to specific nuclear antigens ENA (extractable nuclear antigens) Original antigens saline extracts of nuclear proteins or RNAprotein complexes Sm (B/B, D1/2/3) RNP (70k, A, C) SS-A/Ro (Ro52, Ro60) SS-B/La Scl70/Topo I Jo-1 (and other aminoacyl trna synthetases) Ribosomal P proteins Original methods: Double immunodiffusion Counter-immunoelectrophoresis (Passive hemagglutination, Western blotting)

12 Solid phase techniques for specific ANA Methods EIA Bead arrays Line/dot blotting Microchip technology Antigens Purified proteins Recombinant proteins E. coli Baculovirus Synthetic peptides (immunodominant peptide)

13 Anti-dsDNA antibodies ANA and anti-dsdna (or anti-sm) antibodies are included in the ACR classification criteria for SLE More often present in active disease and in cases with renal involvment In some patients rising levels indicate impending disease flares may be useful in longitudinal assessment

14 RIA Anti-dsDNA techniques Farr assay (precipitation with ammonium sulphate) Detects high avidity Abs, all Ig isotypes Quantitative PEG RIA (precipitation with polyethylene glycol) Detects also antibodies with lower avidity IFA Crithidia luciliae kinetoplast Detects medium to high avidity Abs High disease specificity for SLE, low sensitivity Usually IgG (isotype can be determined) Semiquantitative

15 Anti-dsDNA techniques EIA (and other solid phase techniques) Detect also low avidity Abs Sensitive Lower disease specificity for SLE Quantitative Easy to automate Modification for analysis of high avidity Abs possible

16 dsdna antigens Bacterial Mammalian (calf thymus) Plasmid Bacteriophage Defined human oligonucleotides Denaturation of dsdna to ssdna may lead to unspecific reactions circular dsdna (plasmid or bacteriophage) is preferred Protein contamination

17 Anti-dsDNA Discrepant results with different techniques Avidity, Ig isotype, antigen At population level relatively good correlation, for individual patient sera discrepancies (up to 30 %) Different tests for different applications: Screening High sensitivity (EIA) Diagnosis High specificity (IFA, Farr, EIA with higher cut off?) Monitoring disease activity (Farr, EIA?) Cooperation between clinicians and laboratories

18 External quality assessment in ANA tests EQA schemes are usually able to provide consensus reports only Ideal samples - Single donor serum/plasma (not diluted) - Clinically diagnosed donors - Reactivity tested in a few specialized laboratories using several methods including conceivable golden standards Ideal samples are not readily available

19 Labquality ANA, ENA, dsdna Methods used for selecting samples Test ANA (titer, staining pattern) Anti-dsDNA Anti-Sm (B, D) Anti-U1-RNP (70k, A, C) Anti-SSA (Ro52, Ro60) Anti-SSB Anti-Scl-70/Topo I Anti-Jo1 (histidyl trna synthetase) Anti-Cenp-B Method IFA (Hep2) IFA (Crithidia luciliae), FEIA FEIA, Line blotting FEIA, Line blotting FEIA, Line blotting FEIA, Line blotting FEIA, Line blotting FEIA, Line blotting FEIA, Line blotting

20 Results: Right or wrong? Policy of Labquality Informative reports, no scoring Expected/intended results = results in the reference laboratory - should not be taken as the only right response Educational challenge Comparison between other laboratories (own method, all methods) Comparison between methods Before international standards for the specific ANAs are available the results can be considered right or wrong according to participant consensus The qualitative results for each ANA test can be evaluated when 80 % participant consensus is reached?

21 Use of international serum standards Standards/International units (IU) accepted by WHO WHO 66/233 (anti-nuclear factor) for homogeneous pattern in IFA WHO Wo/80 for anti-dsdna antibodies Other reference sera available for quality assurance Arthritis Foundation/Centers of Disease Control and Prevention (CDC) antinuclear antibody reference sera for IFA patterns and for defined ANA specificities Association of Medical Laboratory Immunologists (AMLI) consensus reference panel for defined ANA specificities