A u t o a n t i b o d y Tes t i n g for Autoimmune Disease

Sally E. Self, MD
KEYWORDS
 Autoantibody  Autoimmune  Serology  Rheumatic

GENERAL REMARKS
The proper use and interpretation of serologic testing for diagnosing autoimmune diseases presents a challenge to clinicians for several reasons. Most laboratory tests for autoimmune disease are significantly less than 100% sensitive or specific. In addition, different techniques for the same antibody test may give different results, such as indirect immunofluorescence and multiplex bead assay for antinuclear antibody (ANA). This problem was recently highlighted in a recent New England Journal of Medicine case record in which a diagnosis of systemic lupus erythematosus was confounded by a false-negative ANA using one method.1 Another challenge is considerable interlaboratory variability in quantitative indirect fluorescence titers. Although attempts have been made at standardization, indirect immunofluorescence, used in testing for ANAs and antineutrophil cytoplasmic antibodies (ANCAs), remains subjective, and titers will vary enormously among laboratories. It is not uncommon for College of American Pathologists proficiency surveys show ANA titers varying from 1:32 to greater than 1:5120 on the same specimen. The biology of autoantibodies is an added problem. Low titers of autoantibodies are not uncommon in healthy individuals. The incidence and titer of autoantibodies in healthy individuals increase with age.2 Overlap also occurs in disease specificity among different autoantibodies. Autoantibody testing should be performed selectively and only when historical and clinical findings support the diagnosis of an autoimmune disease.

Therefore, this article discusses autoantibody testing in the context of the specific autoimmune disease.

SYSTEMIC LUPUS ERYTHEMATOSUS

Testing for ANAs is the screening test for patients in whom systemic lupus erythematosus is suspected. It has a sensitivity of greater than 95%.3 ANA testing can be performed using three major methods: indirect immunofluorescence, enzyme immunoassay (EIA), and multiplex bead flow cytometry. Indirect immunofluorescence on Hep-2 cells is the standard methodology and has the advantages of including all the nuclear antigens in the substrate and providing a pattern. The specificity will vary according to the screening titer used, with a reported false-positive rate of 32% when a 1:40 titer is used.2 Higher screening titers result in a higher specificity at the cost of sensitivity. Ideally, the screening titer should be determined by each laboratory based on the patient population and desired performance characteristics. However, this is not always practical. Indirect immunofluorescence is subjective and laborintensive. The titers reported vary considerably among laboratories because of the subjectivity. EIAs and multiplex bead assays are more objective, much less labor-intensive, and more suitable for large-volume testing. For these reasons, many of the large referral laboratories have embraced these technologies. The major disadvantage of these techniques is that they may not be able to detect all of the

Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 165 Ashley Avenue, Suite 309, Charleston, SC 29425, USA E-mail address: selfs@musc.edu Clin Chest Med 31 (2010) 415422 doi:10.1016/j.ccm.2010.04.001 0272-5231/10/$ see front matter 2010 Elsevier Inc. All rights reserved.

chestmed.theclinics.com

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antigens detectable with indirect immunofluorescence. Although the American College of Rheumatologists issued a position statement in February 2009 stating that ANA detected with indirect immunofluorescence is the gold standard, lively debate over this issue exists among laboratory directors.4 Clinicians must be aware of which test is being performed when they order an ANA; what the weaknesses are of that technique; and when it is worthwhile to order an alternate technology. The patterns seen when an ANA is performed using indirect immunofluorescence can provide information about the antigens involved.5 The homogeneous pattern is associated with antibodies against double-stranded DNA (dsDNA) and histones, and is the most specific pattern for lupus and drug-induced lupus. The speckled pattern is the most common pattern, but the least specific. Many different antigens will result in a speckled pattern, including SS-A/Ro, SS-B/La, U1 ribonucleoprotein (RNP), and Sm. The nucleolar pattern is most often seen in association with scleroderma, whereas the centromere pattern is seen in the limited cutaneous or CREST (calcinosis, Raynauds syndrome, esophageal immobility, sclerodactyly, and telangiectasia) variant of scleroderma (Fig. 1). The ANA patterns may be poorly reproducible, and more than one pattern may exist in a sample. Therefore, testing for antibody to specific lupus-associated antigens is generally indicated after a positive ANA, and is performed simultaneously with some of the more automated assays, such as the multiplex bead assays. More than 50 different autoantibody specificities have been reported in sera from patients with systemic lupus erythematosus, and on average a patient with lupus will have three different autoantibodies.6 Anti-native or anti-dsDNA and antiSm (Smith) are usually measured with enzyme immunoassays or multiplex bead assays. Both anti-dsDNA and anti-Sm are fairly specific for systemic lupus erythematosus, but only occur in 70% and 25% to 50% of cases, respectively. Both are associated with lupus renal disease. The titer of anti-dsDNA tends to correlate with activity of disease.6 Antibodies to histone are correlated with drug-induced lupus. Other autoantibodies seen in lupus include, among others, those directed against singlestranded DNA, nuclear RNP, SSA/Ro, SSA/La, Ku, Ki, proliferating cell nuclear antigen (PCNA), ribosomal RNP, and Hsp90 (heat shock protein). The Sm and nRNP antigens are closely associated. Although the Sm antigen can be isolated, the nRNP antigen will usually have contaminating Sm antigen. Some assays then report anti-Sm/RNP

Fig. 1. ANA patterns detected using indirect immunofluorescence. (A) Homogeneous pattern. Note that the metaphase plate (arrow) is positive. (B) Speckled pattern. The metaphase plate in the speckled pattern is negative (arrow). (C) Nucleolar pattern. (D) An example of the centromere pattern. The centromeres line up along the metaphase plate (arrow).

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for this reason. Anti-Ku antibodies are seen in 23% of patients with idiopathic pulmonary arterial hypertension (primary pulmonary hypertension).7 Anti-Ki is seen in approximately 10% of patients with lupus and is also associated with pulmonary hypertension.6 A summary of autoantibody specificities in lupus is presented in Table 1. The traditional way to test for these antibodies is to perform an ANA screen using indirect immunofluorescence, and then if positive, to follow-up with testing for antibodies to dsDNA and extractable nuclear antigens. The extractable nuclear antigen panel usually includes tests for antibodies to Sm, Sm/RNP, SSA(Ro), SSB(La), and Scl-70. Elevated titers of antibody to dsDNA have been associated with increased disease activity, as have decreased concentration of serum complement C3 and C4.6 Antiphospholipid antibodies (APAs) are seen in approximately one-third of patients with lupus and can be tested for in several ways.8 The antibodies to cardiolipin result in a false-positive test for syphilis when using cardiolipin-based assays (eg, rapid plasma reagin and venereal disease research laboratory tests). The lupus anticoagulant (which in vivo functions as a procoagulant) is measured through the prolongation of the partial thromboplastin time, which can be overcome by adding excess phospholipid. The dilute Russell viper venom test can also be used to test for a lupus anticoagulant. Enzyme immunoassays for APA are also available. Included in these are tests for antibodies to phospholipid-binding proteins, which may be the real target for APAs. The most notable of these is b2glycoprotein 1.

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Table 1 Selected autoantibodies in SLE Antigen Hep-2 cell nuclei dsDNA Nature ANA Native, double-stranded DNA Prevalence in SLE Associations >95% 40%60% Numerous autoimmune diseases High specificity for lupus, titers correlate with disease activity Drug-induced lupus High specificity for lupus MCTD Sjogren syndrome, subacute cutaneous lupus, neonatal lupus with heart block, SLE with interstitial pneumonia Sjogren syndrome MCTD, scleroderma, primary pulmonary hypertension Arthritis, pericarditis, and pulmonary hypertension in patients with SLE Polymyositis Neuropsychiatric SLE Lupus anticoagulant, arterial and venous thromboses, neurologic disease

Histones Sm Nuclear RNP (U1RNP) SS-A(Ro)

Small nuclear RNAs complexed with protein Small nuclear RNAs complexed with protein Protein associated with RNA

50%70% 20%30% 30%40% 30%50%

SS-B(La) Ku

Protein bound to small RNA 10%15% DNA binding proteins 10%39%

Ki

Nuclear protein

8%31%

PCNA/cyclin Cell cycle protein Hsp90 Heat shock protein P ribosomal protein, rRNP Ribosomal phosphoprotein ssDNA Single-stranded DNA b2-glycoprotein 1 Anionic proteins, cardiolipin

3% 50% 10% 70% 25%

Abbreviations: MCTD, mixed connective tissue disease; SLE, systemic lupus erythematosus; ssDNA, single-stranded DNA.

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Autoantibodies are seen in approximately 95% of patients with scleroderma and more than 30 specificities have been described. Most of these can be detected with a screening ANA using indirect immunofluorescence. The antigen specificity may be detected with enzyme immunoassay or multiplexed bead assay. The antigen specificities have been shown in some studies to correlate with pulmonary involvement, either interstitial fibrosis, pulmonary hypertension, or both.9,10 The nucleolar pattern according to immunofluorescence is relatively specific for systemic sclerosis, but is not seen in all cases. Target antigens giving the nucleolar pattern include anti-PM-Scl, antifibrillarin/anti-U3-ribonucleoprotein (anti-U3RNP), anti-Th/To, and the anti-RNA-polymerases (variously called anti-RNAP or Pol 1-3). Of the latter, the Pol 3 antibody is more often seen in scleroderma. The centromere pattern can also be seen through indirect immunofluorescence. The antigen is CENP-B, which is associated with the CREST syndrome. CREST designation may not be as useful clinically as once thought, and currently systemic sclerosis is divided between limited cutaneous systemic sclerosis and diffuse cutaneous systemic sclerosis. Anticentromere antibodies are seen in more than 50% of limited cutaneous systemic sclerosis, and seldom in diffuse cutaneous systemic sclerosis. Anti-Scl-70, an antibody against topoisomerase 1, gives a speckled pattern on ANA indirect immunofluorescence. Recently, antiplatelet derived growth factor receptor antibodies were described in systemic sclerosis.11 Although intriguing from a pathogenic viewpoint, the testing for these antibodies was based on a functional assay and not generally available. The clinical significance of the presence of these antibodies to organ involvement and prognosis has not been determined. A large series by Steen10 related the antigen specificity to the development of lung disease, either pulmonary hypertension, interstitial fibrosis, or both. Patients who had anticentromere antibodies had a low incidence of pulmonary fibrosis but were likely to develop pulmonary hypertension late in their illness, with more than half of those who die from scleroderma-related illness dying of pulmonary hypertension. Patients with anti-Th/To develop pulmonary hypertension and interstitial fibrosis. Patients with antibodies to U3-RNP tend to be black and have severe lung disease with both pulmonary hypertension and interstitial fibrosis. Patients with antibody to topoisomerase (Scl-70) tend to have interstitial fibrosis without pulmonary hypertension. Patients with anti-Pol 3 have a low incidence of pulmonary fibrosis. An association does not seem to be present between titer of any to the autoantibodies and disease severity, nor does there seem to be any use for monitoring the autoantibodies over time. Table 2 summarizes the autoantibodies seen in scleroderma. Several autoantibodies are associated with polymyositis/ scleroderma overlap syndrome, including antiPM-Scl, anti-U1-RNP, and anti-Ku.

POLYMYOSITIS/DERMATOMYOSITIS
Approximately one-third of patients with polymyositis and dermatomyositis will have interstitial lung disease.12 Of patients with polymyositis or dermatomyositis, between 60% and 90% will have a positive ANA. The patterns vary according to the antigen specificity. Anti-PM-Scl will give a nucleolar pattern, whereas antibodies to Jo-1, PL-12, and Ku will give a speckled pattern. The myositis-specific antibodies occur in 25% to 40% of patients with myositis. They can be divided into three groups: anti-tRNA synthetases (anti-Jo-1, anti-PL-7, anti-PL-12, and anti-OJ), anti-signal recognition particle (anti-SRP), and anti-Mi-2, a cytoplasmic antigen. Myositis-associated antibodies include anti-PM-Scl, anti-U1RNP, anti-U2RNP, and anti-Ku. Anti-SSA, anti-SSB, and anti-Sm antibodies may also be present. Antibodies to Jo-1 and SS-A(Ro) are significantly associated with interstitial lung disease.12 Table 3 summarizes the polymyositis/dermatomyositisassociated antibodies.

RHEUMATOID ARTHRITIS
The mainstays of the serologic testing for rheumatoid arthritis are rheumatoid factor and antibodies to cyclic citrullinated peptide (anti-CCP). As with other serologic tests for autoimmune disease, neither is entirely sensitive or specific. Rheumatoid factor is an antibody (usually an IgM isotype, although IgG and IgA rheumatoid factor do occur) to the Fc portion of the IgG antibody. It can be measured using several techniques: latex agglutination, enzyme immunoassay, or nephelometry. Rheumatoid factor may be positive in several other conditions, including chronic bacterial infections, viral infection (eg, hepatitis C), hematologic diseases, and chronic inflammatory diseases of uncertain origin. The incidence of rheumatoid factor in healthy individuals increases with age.13 Anti-CCP is measured using enzyme immunoassay. Antibody testing for CCP was developed

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Table 2 Selected autoantibodies in systemic sclerosis (scleroderma) Antigen Hep-2 cell nuclei Scl-70 Centromere Nature Classic ANA DNA topoisomerase 1 Centromere proteins Prevalence in Scleroderma 70%90% 70% Diffuse 13% Limited 8% Diffuse 57%82% Limited 1%11% Diffuse 8%19% Limited 2%5% 5% Diffuse 10% Limited Associations Numerous autoimmune diseases High incidence of pulmonary fibrosis Rare pulmonary fibrosis, but high incidence of pulmonary hypertension Pulmonary fibrosis and pulmonary hypertension Pulmonary hypertension Pulmonary fibrosis and pulmonary hypertension, may be missed on non-IIF ANAs Pol-3 associated with low incidence of pulmonary fibrosis

Th/To U1-RNP U3-RNP

Protein complexed with 7S and 8S RNA Spliceosome complex Fibrillarin

Pol-1, Pol-2, Pol-3

RNA polymerases

23%

Data from Kumar V, Abbas AK, Fausto N, et al. Diseases of the immune system. In: Robbins and Cotran pathologic basis of disease. 8th edition. Philadelphia: Saunders Elsevier; 2010. p. 215; von Muhlen CA, Nakamura RM. Clinical and laboratory evaluation of systemic rheumatic diseases. In: McPherson RA, Pincus MR, editors. Henrys clinical diagnosis and management by laboratory methods. 21st edition. Philadelphia: Saunders Elsevier; 2007. p. 91644; and Nakamura RM, Tan EM. Steen VD. Autoantibodies in systemic sclerosis. Semin Arth Rheum 2006;35(1):3542.

from the observation that patients with rheumatoid arthritis had antibodies against filaggrin derived from human skin.14 The target antigen in filaggrin was found to be citrulline, a modified arginine residue. The posttranslational conversion of arginine to citrulline is accomplished by the enzyme peptidylarginine deiminase (PAD).15,16 PAD normally occurs in an inactive intracellular form. PAD may leak out of apoptotic cells in the synovium of patients with rheumatoid arthritis and become activated, causing citrullination of extracellular arginine. Anti-CCP antibodies seem to be more specific for rheumatoid arthritis than rheumatoid factors (Table 4). The higher the titer of rheumatoid factor, the more specific it is. Monitoring titers of rheumatoid factor or anti-CCP antibodies generally has little value for indicating activity of disease. General markers of inflammation (acute-phase reactants)thrombocytosis, the erythrocyte sedimentation rate, and antibodies to C-reactive proteincan indicate disease activity.

60% to 95% of patients with Sjogren syndrome, and antibodies to SS-B/La are seen in 40% to 90%. The importance of these antibodies is attested by their inclusion as one of six criteria in the Revised International Criteria for Sjogren syndrome.17 These antibodies are generally measured with enzyme immunoassay, but they are included in some of the multiplex bead assays. Development of lymphoma occurs in up to 5% of patients with Sjogren syndrome. The development of lymphoma has been associated with low levels of complement C4 and the presence of type II (mixed) cryoglobulins.17

MIXED CONNECTIVE TISSUE DISEASE

Mixed connective tissue disease (alternatively called undifferentiated autoimmune rheumatic/ connective tissue disorder) is characterized by high titers of anti-RNP. Mixed connective tissue disease is characterized by combined features of systemic lupus erythematosus, scleroderma, and polymyositis. Anti-RNP antibodies are usually measured with enzyme immunoassay or multiplex bead assay. Anti-RNP antibodies may be seen in other autoimmune diseases, such as systemic lupus, rheumatoid arthritis, and systemic sclerosis.

SJOGREN SYNDROME
Antinuclear antibodies are seen in 50% to 80% of patients with Sjogren syndrome. Autoantibodies to SS-A/Ro are seen in greater than

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Table 3 Autoantibodies in PM/D Antigen Hep-2 nuclei Jo-1 Nature Anti-ANA tRNA synthetase Prevalence in PM/D 40%50% 20% Associations High incidence of interstitial lung disease

EJ, OJ, PL-7, PL-12 tRNA synthetases Mi-2 Nuclear protein SRP PM-Scl

U1nRNP SS-A(Ro) Ku

2%3% 15%35% polymyositis 5%9% dermatomyositis Signal recognition protein 5% polymyositis 0% dermatomyositis Nucleolar protein complex 8% polymyositis 25% polymyositis/ scleroderma overlap Spliceosome complex 4%17% Protein associated with 16% Interstitial lung disease RNA DNA binding protein 1% Dermatomyositis with overlap of Sjogren syndrome or SLE and pulmonary hypertension

Abbreviations: PM/D, polymyositis and dermatomyositis; SLE, systemic lupus erythematosus; SRP, signal recognition protein. Data from von Muhlen CA, Nakamura RM. Clinical and laboratory evaluation of systemic rheumatic diseases. In: McPherson RA, Pincus MR, editors. Henrys clinical diagnosis and management by laboratory methods. 21st edition. Philadelphia: Saunders Elsevier; 2007. p. 91644; Schnabel A, Reuter M, Biederer J. Interstitial lung disease in polymyositis and dermatomyositis: clinical course and response to treatment. Semin Arth Rheum 2003;32(5):27384; Hang L, Nakamura RM. Autoimmune diseases of muscle: myasthenia gravis and autoimmune myositis. In: Nakamura RM, Keren DF, Bylund DJ, editors. Clinical and laboratory evaluation of human autoimmune diseases. Chicago: ASCP Press; 2002. p. 2989.

ANCA-RELATED VASCULITIDES
The advent of ANCA testing in the 1980s contributed greatly to the diagnosis and understanding of Wegeners granulomatosis and microscopic polyangiitis.18,19 Indirect immunofluorescence

Table 4 Autoantibodies in rheumatoid arthritis Autoantibody Sensitivity Specificity

Rheumatoid factor (RF) 66%85% 72%82% Anti-CCP 56%74% 90%92% RF and anti-CCP 48% 96%
Data from Bas S, Genevay S, Meyer O, Gabay C. Anti-cyclic citrullinated peptide antibodies, IgM and IgA rheumatoid factors in the diagnosis and prognosis of rheumatoid arthritis. Rheumatology 2003;442:67780; and von Muhlen CA, Nakamura RM. Clinical and laboratory evaluation of systemic rheumatic diseases. In: McPherson RA, Pincus MR, editors. Henrys clinical diagnosis and management by laboratory methods. 21st edition. Philadelphia: Saunders Elsevier; 2007. p. 922.

testing for ANCA is performed with ethanol-fixed neutrophils as the substrate, which may result in two patterns: cytoplasmic (C-ANCA) and perinuclear (P-ANCA). The serum is then tested against formalin-fixed neutrophils, on which both CANCA and P-ANCA give a cytoplasmic pattern. Indirect immunofluorescence for ANCA is shown in Fig. 2. The antigen responsible for C-ANCA is usually Proteinase-3 (Pr-3), whereas the antigen usually detected by P-ANCA is myeloperoxidase. Myeloperoxidase is transported to a perinuclear location during ethanol fixation, but stays in the cytoplasm during formalin fixation. Anti-Pr3 and anti-myeloperoxidase antibodies may be tested with enzyme immunoassay or multiplex bead assay. Some other antibodies give a positive reaction on ethanol fixation, but are negative or give only a weak smudgy appearance when tested against formalin fixed neutrophils. These are called atypical ANCAs. ANCAs without myeloperoxidase or Pr-3 specificity may be caused by autoantibodies against elastase, lactoferrin, lactoperoxidase, cathepsin G, lysozyme, bactericidal/ permeability-increasing (BPI) protein, and

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Fig. 2. ANCA patterns detected using indirect immunofluorescence. (A) A C-ANCApositive serum placed on ethanol fixed neutrophils. (B) The same serum on formalin-fixed neutrophils. Both A and B show cytoplasmic labeling. (C) A P-ANCApositive serum on ethanol-fixed neutrophils. (D) The same serum on formalin-fixed neutrophil. The P-ANCA antigen (myeloperoxidase) acquires a perinuclear localization during ethanol fixation but stays in the cytoplasm during formalin fixation.

azurocidin, and uncharacterized antigens. The clinical significance of non-myeloperoxidase, non-Pr3 ANCAs has not been established. Two strategies for ANCA testing exist. The first is to perform indirect immunofluorescence screening, then, based on pattern, test for antiPr3 or myeloperoxidase (MPO). The indirect fluorescence is more sensitive and the antigenspecific testing is more specific. Like ANA testing, ANCA testing with indirect immunofluorescence is labor-intensive and subjective. The second strategy is to test for anti-MPO and anti-Pr3 using EIA, and then for ANCA using indirect fluorescence if indicated.

Table 5 shows the incidence of ANCA antibodies in Wegener granulomatosis, microscopic polyangiitis, and Churg-Strauss syndrome. There is an association of ANCA titers and activity of disease and relapse, but it is not strong enough to be very reliable.20

GOODPASTURE SYNDROME
Goodpasture syndrome is the presence of pulmonary hemorrhage and crescentic glomerulonephritis caused by the presence of antibodies to the glomerular basement membrane (anti-GBM). The anti-GBM antibodies can be shown in the

Table 5 Sensitivity of ANCAs in active vasculitis C-ANCA/anti-Pr3 Wegener granulomatosis Microscopic polyangiitis Churg-Strauss syndrome 56%95% 8%26% 33% P-ANCA/anti-MPO 5%23% 49%85% 33% C-ANCA/Pr3 or P-ANCA/MPO 85%95% 67%96% 56%

Data from Hagen EC, Daha MR, Hermans J, et al. Diagnostic value of standardized assays for anti-neutrophil cytoplasmic antibodies in idiopathic systemic vasculitis. Kidney Int 1998;53:74353; and Trevisin M, Pollock W, Dimech W, et al. Antigen specific ANCA ELISAs have different sensitivities for active and treated vasculitis and for nonvasculitic disease. Am J Clin Pathol 2008;129:4253.

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tissue using direct immunofluorescence, or in the serum using Western blot or enzyme immunoassay. Older tests using indirect immunofluorescence on normal renal tissue are unreliable and no longer clinically useful. The antigen in Goodpasture syndrome is the noncollagenous domain I type IV collagen. Western blot is more sensitive and specific than enzyme immunoassays,21 but the enzyme immune assay is more suitable for use in the routine clinical laboratory. The EIA also allows for quantitation of the autoantibody, which may be used in following therapy.
8. von Muhlen CA, Nakamura RM. Clinical and laboratory evaluation of systemic rheumatic diseases. In: McPherson RA, Pincus MR, editors. Henrys clinical diagnosis and management by laboratory methods. 21st edition. Philadelphia: Saunders Elsevier; 2007. p. 91644. 9. Ho KT, Reveille JD. The clinical relevance of autoantibodies in scleroderma. Arthritis Res Ther 2003;5: 8093. 10. Steen VD. Autoantibodies in systemic sclerosis. Semin Arthritis Rheum 2006;35(1):3542. 11. Svengliati BS, Santillo M, Bevilacqua F, et al. Stimulatory autoantibodies to the PDGF receptor in systemic sclerosis. N Engl J Med 2006;354:266776. 12. Schnabel A, Reuter M, Biederer J. Interstitial lung disease in polymyositis and dermatomyositis: clinical course and response to treatment. Semin Arthritis Rheum 2003;32(5):27384. 13. Wener MH. Rheumatoid factors. In: Rose NR, Hamilton RG, Detrick B, editors. Manual of clinical laboratory immunology. 6th edition. Washington, DC: American Society for Microbiology Press; 2002. p. 96172. 14. Vincent C, de Keyser F, Masson-Bessiere C, et al. Anti-perinuclear factor compared with the so-called antikeratin antibodies and antibodies to human epidermis filaggrin, in the diagnosis of arthritides. Ann Rheum Dis 1999;58:428. 15. Vossenaar ER, vanVenrooij WJ. Citrullinated proteins: sparks that may ignite the fire in rheumatoid arthritis. Arthritis Res Ther 2004;6:10711. 16. van Venrooij WJ, Prujin GJ. Citrullination, a small change for a protein with great consequences for rheumatoid arthritis. Arthritis Res 2000;2:24951. 17. Mitsias D, Moutsopoulos M. Sjogren syndrome. In: Shoenfelk Y, Cervera R, Gershwin ME, editors. Diagnostic criteria in autoimmune disease. Totowa (NJ): Humana Press; 2008. p. 3742. 18. Hagen EC, Daha MR, Hermans J, et al. Diagnostic value of standardized assays for anti-neutrophil cytoplasmic antibodies in idiopathic systemic vasculitis. Kidney Int 1998;53:74353. 19. Trevisin M, Pollock W, Dimech W, et al. Antigen specific ANCA ELISAs have different sensitivities for active and treated vasculitis and for nonvasculitic disease. Am J Clin Pathol 2008;129:4253. 20. Girard T, Mahr A, Noel L-H, et al. Are antineutrophil cytoplasmic antibodies a marker predictive of relapse in Wegeners granulomatosis? A prospective study. Rheumatology 2001;40:14751. 21. Collins AB, Colvin RB. Kidney and lung disease mediated by anti-glomerular basement membrane antibodies: detection by Western blot analysis. In: Rose NR, Hamilton RG, Detrick B, editors. Manual of clinical laboratory immunology. 6th edition. Washington, DC: American Society for Microbiology Press; 2002. p. 104953.

SUMMARY
Autoantibody testing should only be performed in the context of the clinical workup of patients who have a reasonable likelihood of having the disease for which the testing is relevant. Otherwise, the predictive value of a positive test is too low. Particularly with ANA and ANCA testing, clinicians must know the methodology through which the tests are being performed, and should develop a relationship with the laboratory pathologist so that inconsistent or surprising results can be investigated.

REFERENCES
1. Kroshinsky D, Stone J, Bloch D, et al. Case 5-2009: a 47-year-old woman with a rash and numbness and pain in the legs. N Engl J Med 2009;360(7):71120. 2. Tan EM, Feltkamp TEW, Smolen JS, et al. Range of antinuclear antibodies in healthy individuals. Arthritis Rheum 1997;40:160111. 3. Kumar V, Abbas AK, Fausto N, et al. Diseases of the immune system. In: Kumar V, Abbas AK, Fausto N, et al. Robbins and Cotran pathologic basis of disease. 8th edition. Philadelphia: Saunders Elsevier; 2010. p. 215. 4. Cheek W. Making sense of the ANA hodgepodge. CAP Today 2009;23(9):1110. 5. von Muhlen CA, Nakamura RM. Guidelines for selecting and using laboratory test for autoantibodies to nuclear, nucleolar, and other related cytoplasmic antigens. In: Nakamura RM, Keren DF, Byland DJ, editors. Clinical and laboratory evaluation of autoimmune diseases. Chicago: ASCP Press; 2002. p. 18398. 6. Nakamura RM, Tan EM. Clinical and laboratory evaluation of systemic lupus erythematosus and lupusrelated disorders. In: Nakamura RM, Keren DF, Byland DJ, editors. Clinical and laboratory evaluation of autoimmune diseases. Chicago: ASCP Press; 2002. p. 11139. 7. Isern RA, Yaneva M, Weiner E, et al. Autoantibodies in patients with primary pulmonary hypertension: association with anti-Ku. Am J Med 1992;93:30712.