Anti–citrullinated peptide antibody assays and their role in the diagnosis of rheumatoid arthritis

Authors


Introduction

The diagnosis of early rheumatoid arthritis (RA) has relied on clinical criteria, including history and physical examination findings and laboratory and radiographic results. Irreversible damage frequently occurs early in RA (1–5). With mounting evidence supporting early diagnosis and aggressive treatment to prevent damage and disability, there is a need to improve identification and diagnosis of early RA (6). Until recently, assays detecting rheumatoid factor (RF), antibodies directed against the Fc portion of the IgG molecule, have been the primary serologic tests for RA diagnosis. Anti–citrullinated peptide antibody (ACPA) assays, developed and commercialized in the past decade, are now being employed clinically. Since ACPAs are present before the onset of RA symptoms and are predictive of RA development, they are a valuable diagnostic test early in the course of the disease (4).

This review synthesizes currently available data regarding the diagnostic properties of RF and ACPAs for the diagnosis of early RA. We focus on ACPAs, given their recent development and their potential role in the improved identification of early, undifferentiated RA. Data included in this review were obtained from medical literature searches, Web sites of and contact with companies marketing the assays, and information and opinions obtained from experts in the field. We have included information on the biologic basis and development of ACPA assays, the available assays, and data concerning assay performance characteristics, in particular those published in peer-reviewed journals, but also those publicized by manufacturers. Diagnostic properties of these tests are reviewed, including but not limited to sensitivity, specificity, and positive and negative predictive values.

Rheumatoid factor

In 1940, Waaler observed that mixing serum from an RA patient with IgG-sensitized sheep erythrocytes inhibited hemolysis, but caused cell agglutination (7). Rose later reported that RA sera agglutinated sheep erythrocytes coated with rabbit anti-sheep erythrocyte antibodies more than sera from healthy individuals (8). These findings formed the basis of the earliest RA assay, the Rose-Waaler test. RF assays most commonly detect IgM antibodies directed against the Fc portion of the IgG molecule. The agglutination test measures IgM-RF only and remains the most widely used assay. Agglutination assays are reported as either titers or units. Cutoffs for positivity are determined by manufacturers and based on results from RA patients compared with healthy controls (4, 9). Agglutination assays have sensitivities for RA ranging from 70% to 85% and specificities ranging from 40% to 90%, as agglutination in individuals without RA may occur (10–12).

Other assays for RF have been developed, including enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays, and laser or rate nephelometry techniques (13). Assays for the detection of IgA-RF and IgG-RF are also available (14–19). The sensitivity of RF for RA diagnosis by these techniques is 50–90% and the specificity is 50–95%. These wide ranges reflect the differences in the populations tested (20–26). Studies directly comparing RF detection techniques in cohorts of established RA patients, healthy controls, and patients with noninflammatory joint disease have reported latex agglutination test performance to be similar to that of nephelometry and radioimmunoassays (12, 27). In a meta-analysis of 50 studies of RF assays from 1998 to 2005, the pooled likelihood ratios (dependent on both sensitivities and specificities) were quantitatively similar for IgM-RF, IgA-RF, and IgG-RF assays, and for using a higher versus lower RF titer for positivity (26) (Table 1).

Table 1. Positive and negative LRs for IgM-RF in the diagnosis of rheumatoid arthritis*
 Positive LR (95% CI)Negative LR (95% CI)References
  • *

    Adapted, with permission, from ref.26. LR = likelihood ratio; IgM-RF = IgM rheumatoid factor; 95% CI = 95% confidence interval; ELISA = enzyme-linked immunosorbent assay.

Pooled LR4.86 (3.96–5.97)0.38 (0.33–0.44)9, 14–17, 19, 25, 27, 39, 40, 44, 56, 93, 100, 101, 114–144
RF assay type   
 Nephelometry4.15 (2.95–5.84)0.32 (0.25–0.41)17, 25, 46, 93, 115, 117, 118, 120, 122, 124, 126, 127, 129–132
 Latex agglutination5.05 (3.01–8.50)0.39 (0.27–0.56)9, 27, 44, 101, 114, 116, 119, 121, 125, 133, 136–138, 144, 145
 ELISA6.13 (4.6–8.17)0.42 (0.34–0.51)14–16, 19, 38–40, 123, 128, 134, 135, 139, 140, 142, 143, 146
RF value, units/ml   
 ≥204.42 (3.02–6.47)0.39 (0.31–0.50)26
 ≥405.49 (2.25–13.38)0.50 (0.37–0.69)26
 ≥804.57 (4.60–8.17)0.42 (0.34–0.51)26

False-positive RF results commonly occur in the setting of chronic infections, malignancy, and other rheumatic diseases (21). RF is detected in the sera of 1–4% of healthy young persons and in a higher percentage of elderly persons without RA (28, 29). The RF assay, however, is widely available, relatively inexpensive, and understood by both primary care physicians and arthritis specialists (21).

Antibodies to citrullinated peptides

In 1964, Nienhuis and Mandema described an autoantibody they called antiperinuclear factor. Detected by indirect immunofluorescence test on human buccal mucosa cells, antiperinuclear factor recognized antigens present in keratohyalin granules surrounding the nucleus (30). Antiperinuclear factor was present in up to 90% of established RA patients, with 73–99% specificity (31). Young and colleagues later detected antikeratin antibodies using indirect immunofluorescence on cryosections of rat esophagus (32). The reported sensitivity of the antikeratin assay in RA patients was 36–59% and the specificity was 88–99% (31). Despite the high specificity for RA, these tests were not widely used because of difficulty in standardization of natural substrates and arbitrary interpretation of the indirect immunofluorescence pattern.

In 1995, Sebbag and colleagues demonstrated that both of these antibodies belonged to a family of autoantibodies directed against citrullinated filaggrin, an epithelial cell protein (33). Citrullination is a posttranslational modification of arginine to citrulline by the enzyme peptidyl arginine deiminase. This process occurs naturally during inflammation, apoptosis, and keratinization (9). Although filaggrin is not present in the synovium (34), several citrullinated proteins, including fibrinogen and fibronectin, are present in RA synovium, and other citrullinated epitopes have been identified as targets of highly RA-specific autoantibodies (35–37). In 1998, Schellekens and colleagues produced synthetic linear citrullinated peptides derived from human filaggrin, easily detected by ELISA with enhanced sensitivity and no loss of specificity (35). To improve antigen composition and antibody recognition, a cyclic citrullinated peptide (CCP) was developed (38).

The first commercially available ACPA assay (first-generation CCP [CCP-1]) was developed by Euro-Diagnostica (Arnhem, The Netherlands) and was used in early studies (2000 to 2001). This ELISA-based test employed a single CCP derived from filaggrin (38). The assay detected autoantibodies in 53% of established RA patients, with 96% specificity (38).

Peptide libraries were then screened for better epitopes. Since 2000, second-generation CCP (CCP-2) and third-generation CCP (CCP-3) assays have been developed. Several companies market these assays for RA diagnosis. CCP-3 assays rely on additional epitopes not present in the CCP-2 antigen sequence (39, 40). Apart from the main difference in substrate, both CCP-2 and CCP-3 use ELISA methods and similar dilutions (1:101), diluents, controls, conjugates, and reinterpretation. AxSYM Anti-CCP (Abbott, Dundee, UK) uses microparticle enzyme immunoassay for the semiquantitative determination of the IgG class of autoantibodies specific to CCP-2. Most studies, however, show no evident improvement of CCP-3 compared with CCP-2 assays (41–43). The compositions of many new CCP-3 peptides are not yet publicly available because patents are pending. The anti-CCP3.1 assay marketed by Inova Diagnostics (San Diego, CA) detects both IgG and IgA CCP-3 antibodies in an effort to increase sensitivity (41). Euro-Diagnostica has developed a point-of-care assay, employing a finger lancet to obtain a drop of blood for rapid office-based results.

Newer assays detect non-CCPs (41); the term ACPA has therefore replaced anti-CCP antibody. Citrullinated vimentin is present in synovial fluid and anti-Sa antibodies directed against it are detectable in RA synovium (44, 45). Anti-Sa antibodies have reported a sensitivity of 20–25% and a specificity of 95% in early RA (46). An ELISA for the detection of autoantibodies against mutated citrullinated vimentin (anti-MCV) has better sensitivity than anti-Sa antibodies. The sensitivity of anti-MCV is comparable (or even higher in some studies) to that of ACPA (82% versus 72%) (47), while specificity of anti-MCV is slightly lower than ACPA in several studies (90–92% versus 96–98%) (41, 48). Unlike ACPA assays, the anti-MCV levels may correlate with disease activity (47, 49).

Pathogenetic role of ACPA in RA

The roles of citrullinated peptides and autoantibodies to them in RA pathogenesis remain unclear. ACPAs are strongly associated with an increased risk of developing RA in healthy individuals and are detectable in the blood of healthy persons prior to clinical RA (14, 50, 51). Among those with RA, their presence is associated with more severe structural damage, radiographic progression, and poorer response to therapy (26, 38, 52–61). Geneticists and epidemiologists hold ACPA-positive RA to be a homogeneous phenotype of severe RA. ACPA is strongly associated with the HLA–DRB1 shared epitope (62) and PTPN22 (63, 64), strong genetic risk factors for RA, and smoking (65, 66), the strongest known environmental risk factor for RA. Smoking by individuals with inherited HLA–DRB1 shared epitope genes may trigger RA-specific immune reactions to citrullinated peptides, the generation of ACPAs and, ultimately, disease (65).

ACPA reproducibility and stability over time

In stored blood bank samples, Nielen and colleagues detected the presence of ACPA antibodies up to 14 years prior to RA onset, with gradually increasing prevalence and increased sensitivity and specificity for RA compared with RF (51). The duration of the preclinical, autoantibody-positive, symptom-free period prior to RA may increase with increasing age (60). In a 3-year study of 97 individuals with RA, ACPA status was relatively stable: 3 ACPA-positive subjects became negative, while 2 ACPA-negative subjects became positive (67). Decreases in ACPAs may be observed with some RA therapies, but generally patients do not lose their positive results (68–72). Although in some small studies ACPA levels paralleled RA disease activity (68, 69, 73–75), this has not been corroborated in subsequent studies and ACPA assay results are not clinically employed to monitor disease activity (70–72).

Currently available ACPA assay performance characteristics

Several ACPA assays are currently approved by the US Food and Drug Administration (Table 2). The ACPA assays employed by European and Canadian early arthritis cohorts are mainly CCP-2 assays (Diastat, Axis-Shield Diagnostics, Dundee, UK; Immunoscan-CCP Plus, Euro-Diagnostica; ELiA CCP, Phadia, Friedberg, Germany; Quanta Lite, Inova; etc.). Most of the currently available assays are kits employing a substrate derived from the synthetic cyclic peptide described by Schellekens and colleagues (38, 41), but differ in incubation time, volume and dilution of serum, type of conjugate and enzymatic substrate, and range of units reported and thresholds for positive results (41, 42, 76–78). To determine the diagnostic performance, manufacturers have tested established RA patients and healthy individuals meeting the 1987 American College of Rheumatology (ACR; formerly the American Rheumatism Association) criteria (79). Sensitivities range from 60% to 80% and specificities range from 85% to 99%. CCP-2 assays have slightly higher sensitivity than CCP-1 assays; the newest non-cyclic ACPA assays report similar performance compared with CCP-2 (42, 76–78, 80, 81).

Table 2. Examples of available ACPA assays on the market*
NameManufacturer, countryType of assay (generation)Sensitivity for RA, % (ref.)Specificity for RA, % (ref.)Within-assay CV, % (ref.)
  • *

    The 1987 American College of Rheumatology (formerly the American Rheumatism Association) criteria for RA (79) was used for calculating the sensitivity and specificity in most studies. ACPA = anti–citrullinated peptide antibody; RA = rheumatoid arthritis; CV = coefficient of variation; ELISA = enzyme-linked immunosorbent assay; N/A = not available; anti-CCP = anti–cyclic citrullinated peptide; anti-MCV = anti–mutated citrullinated vimentin.

DiastatAxis-Shield Diagnostics, UKELISA (2nd)76.5 (77)86.1 (77)13.6 (147)
CCPointEuro-Diagnostica, The NetherlandsColloidal gold  immunoassay (2nd)56.0 (148)98.8 (148)N/A
CCPlusEuro-Diagnostica, The NetherlandsELISA (2nd)70 (149)97.5 (149)0.4–5.1 (150)
EDIAEuro-Diagnostica, The NetherlandsELISA (2nd)66.7 (78)97 (78)1.9–7.9 (150)
RA anti-CCP ELISAEuro-Diagnostica, The NetherlandsELISA (2nd)76.5 (41)95.4 (41)12.6–34.3 (41)
EuroimmunEuroimmun,  GermanyELISA (2nd)72.5 (41)96.4 (41)6.4–12.1 (41)
Quanta LiteInova, USELISA (2nd)70 (77)91.3 (77)6 (147)
ELiA CCPPhadia, Sweden/ GermanyImmunocap  method (2nd)77.5 (41)95.9 (41)7.2–9.8 (41)
Quanta Lite CCP3Inova, USELISA (3rd)77.5 (41)87.8 (41)3.7–5.1 (41)
Quanta Lite CCP3.1Inova, USELISA (3rd)74 (132)89.6 (132)0.5–4.8 (132)
Org 548 anti-MCVOrgentec, GermanyELISA MCV74.5 (41)90.3 (41)8.4–12.3 (41)

Because ACPA assays are based on the detection of autoantibodies by ELISA or microparticle enzyme immunoassay or immunoenzymofluorometry, reactivity is related to the quantity of antibodies present in a nonlinear fashion. Although changes in antibody concentration are reflected in a corresponding rise or fall in results, the change is not proportional in most assays (i.e., a doubling of the antibody concentration will not double the reactivity) (41). In a head-to-head comparison of the technical performance of 6 different commercially available ACPA assays, Inova, Euro-Diagnostica, and Genesis (Cambridgeshire, UK) (41) demonstrated significant deviation from linearity; the best linearity was achieved by Euroimmun (Luebeck, Germany).

ACPA assay precision

Studies comparing different ACPA assays have concluded that the majority of assays are precise, with within-assay (intraassay) coefficients of variation (CVs) for most available assays ranging from 4% to 19% (41, 78). In a study by Coenen and colleagues comparing 6 ACPA assays, the greatest precision was found with the Genesis (4.8–5.9% intraassay CV) and Inova assays (3.7–5.1% intraassay CV), and the lowest with the Euro-Diagnostica assay (12.6–34.3% intraassay CV) (41, 78).

ACPA assay correlation

Although different antigens and methods are employed to quantitate and report ACPAs, the results, expressed as positive or negative values, are highly correlated among commercially available ACPA assays, with correlation coefficients ranging from 0.48 to 0.96 (41, 78) (Table 3). Vander Cruyssen and colleagues studied 4 ACPA assays, including the Inova CCP-3 assay. They found that the discrepancy between the ACPA assays was due to borderline results, interassay variability, and intertest variability. The lowest intertest discrepancy is observed between tests using the same substrate (82). If one false-positive ACPA was found in an individual without RA, there was a high probability that ACPAs would be negative in a different ACPA assay (82).

Table 3. Spearman's rho correlations comparing quantitative anti–citrullinated peptide antibody assays*
 Inova CCPInova 3 CCPEuroimmun CCPEuro-Diagnostica CCPAxis-Shield CCPEDIA CCPPharmacia CCPTriturus CCP
  • *

    Reproduced, with permission, from ref.78. CCP = cyclic citrullinated peptide; IgM-RF = IgM rheumatoid factor.

Inova 3 CCP0.86       
Euroimmun CCP0.840.76      
Euro-Diagnostica CCP0.960.830.8     
Axis-Shield CCP0.830.710.810.8    
EDIA CCP0.860.820.930.620.93   
Pharmacia CCP0.820.660.710.790.80.87  
Triturus CCP0.750.740.770.710.670.70.59 
IgM-RF nephelometry0.670.640.630.650.590.730.580.48

Development of an international reference range for standardized ACPA reporting

Given the variety of ACPA assays, quantitative results are not currently comparable between studies. Work is underway to develop standardized ACPA units. (83). Results were promising, but require additional confirmation in large numbers of samples and acceptance by assay manufacturers (Bizzaro N: personal communication).

ACPA assays in other diseases

While the specificity of ACPA assays for RA compared with healthy individuals is good, the potential for lower specificity in the setting of other inflammatory disorders such as psoriatic arthritis, scleroderma, systemic lupus erythematosus (SLE), and seronegative spondylarthritides is of concern (84). The presence of immune complexes or other immunoglobulin aggregates can cause increased nonspecific binding and false-positive results.

We identified and reviewed 63 studies that examined the cross-reactivity rate of ACPA in non-RA rheumatic diseases and common infections. The highest frequency of ACPA positivity in non-RA autoimmune conditions is found in psoriatic arthritis (9%), SLE (8%), and juvenile idiopathic arthritis (8%), as well as scleroderma and CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias; 7%), followed by Sjögren's syndrome (6%) and vasculitis (5%) (Table 4). Because many patients in these studies do not have long-term followup, they may have ultimately been diagnosed with ACPA-positive RA or an overlap syndrome. For example, 7 of 126 psoriatic arthritis patients with detectable ACPA had more severe, erosive disease and a high prevalence of the RA-associated HLA–DRB1 shared epitope (85). A high frequency of ACPA positivity has been observed in patients with erosive arthritis and overlap syndromes with features of scleroderma and SLE (41, 43, 78, 82, 86–90). ACPA in juvenile idiopathic arthritis has been associated with RF-positive disease similar to RA in adults (91).

Table 4. Detection of ACPAs in other diseases*
 NACPA positive, no. (%)References
  • *

    ACPA = anti–citrullinated peptide antibody; CREST = calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias syndrome.

Psoriatic arthritis1,343115 (8.6)41, 78, 82, 85, 86, 151–155
Systemic lupus erythematosus1,07884 (7.8)41, 78, 82, 88–90
Sjögren's syndrome60935 (5.7)41, 78, 86, 88, 90
Spondylarthropathy43110 (2.3)78, 88, 103, 148, 154
Scleroderma/CREST syndrome38026 (6.8)41, 43, 78, 86, 87, 90
Hepatitis C/cryoglobulinemia28510 (3.5)86, 156, 157
Osteoarthritis1824 (2.2)78, 86, 88, 90, 148
Hepatitis B1761 (0.6)158
Juvenile idiopathic arthritis16913 (7.7)86, 90, 151, 159–161
Polymyalgia rheumatica1460 (0)88, 90, 148, 162
Vasculitis/Wegener's granulomatosis1075 (4.7)78, 86, 88, 90, 103
Tuberculosis9633 (34.3)92, 163
Polymyositis/dermatomyositis750 (0)41, 78, 82, 86
Fibromyalgia742 (2.7)78, 148
Gout and pseudogout580 (0)86, 88, 148

The surprisingly high prevalence of ACPA in active tuberculosis has been studied by Kakamanu and colleagues (92). They reported that reactivity to non-citrullinated arginine-containing residues was common in tuberculosis, but not in RA. The mechanism of induction of ACPA in active pulmonary tuberculosis is known. ACPA levels decreased somewhat, but not rapidly, after treatment for tuberculosis (92).

Comparison of first-, second-, and third-generation and newer ACPA assays

Given the rapid evolution of ACPA assays, establishing the comparative sensitivity and specificity of the 3 generations of assays is crucial if they are to be used interchangeably. CCP-2 and CCP-3 assays offer slightly improved sensitivity over that of CCP-1 assays (84, 93), although they have similar specificity for RA (86–96%). CCP-2 and CCP-3 assays in most (41–43) but not all studies (90) have had similar performance characteristics, with sensitivities of 68–79% and specificities of 86–96% (26, 43, 75, 78, 81, 94).

New anti-MCV assays also have similar performance, with sensitivities of 70–82% and specificities of 90–98% (47, 48, 95, 96). Higher false-positive rates have been reported with Orgentec Diagnostika (anti-MCV; Mainz, Germany) and Inova Quanta Lite (CCP-3) assays (41). There is some lack of agreement between the results obtained from different ACPA assays on the same subjects, which could be partially attributed to borderline results and interassay variability. One study has shown 18% discrepancy between two different ACPA assays tested on RA patients (82).

Cost and availability of RF and ACPA assays

RF assays have been widely used for years and are familiar to general practitioners. They are relatively inexpensive and easy to obtain. Since 2000, when ACPA assays were first introduced, the availability of these tests has drastically increased and costs have decreased. They are now marketed almost worldwide by a variety of companies. The Diastat CCP-2 assay from Axis-Shield, for example, is sold globally. It has the approval of the US Food and Drug Administration, the European Medicines Agency, and the Japanese Ministry of Health and Welfare. The price per kit varies from market to market, but it is approximately $250–300 US dollars per 96 well kits. Immunoscan second-generation ACPA assay 96 well kits from Euro-Diagnostica are currently marketed for €350–400, $500–600 US dollars, or £250–300 in the UK. Fully automated and point-of-care assays are beginning to be marketed by several companies.

Konnopka and colleagues performed a cost-effectiveness analysis to address the incremental benefit of testing for ACPAs in addition to the current ACR (formerly the American Rheumatism Association) criteria for RA classification (79, 97). They developed a Markov model of the 10-year progression of RA in patients presenting with undifferentiated arthritis, and estimated the effects of ACPA testing on incremental costs and quality-adjusted life years, including the impact of late diagnosis and treatment. Their analysis revealed that the upfront use of ACPA testing, rather than waiting and testing after a few years of symptoms, was cost effective, and when indirect costs were incorporated, saved in the range of €1,000 per quality-adjusted life year. Although based on multiple assumptions, this study does provide evidence for changing the current approach to early inflammatory arthritis.

Diagnostic accuracy of ACPA assays

More than 300 studies have been published concerning the diagnostic accuracy of ACPA assays in RA diagnosis (26). These studies vary substantially in focus: some have addressed technical aspects, while others have compared the diagnostic accuracy in different populations of individuals (early or established RA; patients with other diseases or healthy controls). The studies are heterogeneous in their comparison of ACPA assay utility with other tests, including IgM-RF, IgA-RF, and IgG-RF (26), and their use of a gold standard for RA diagnosis (most often the existing 1987 ACR [formerly the American Rheumatism Association] criteria for the classification of RA &lsqbr;79&rsqbr;).

In studies of early or undifferentiated RA, ACPA testing is generally more specific than and equally sensitive to RF (Table 5). In cohorts containing both established and early RA, the performance characteristics of the two tests are comparable (Table 4). The definition of early arthritis or early RA has varied in these studies. In the majority of studies, early arthritis has been defined as a symptom duration of less than 2 years (median of approximately 2 months) and initial serologies of patients who developed RA have been compared with those who did not (14, 26, 38–40, 46, 51, 56, 59, 76, 98–110). Most of these data are from the prospective followup of early arthritis cohorts in Japan, The Netherlands, and Austria.

Table 5. Comparison of performance characteristics of IgM-RF and ACPA (CCP-2) assays in early RA cohorts and cohorts containing both early and established RA*
 Refs.ACPA (CCP-2)IgM-RFACPA (CCP-2) or IgM-RFACPA (CCP-2) and IgM-RF
  • *

    IgM-RF = IgM rheumatoid factor; ACPA = anti–citrullinated peptide antibody; CCP-2 = cyclic citrullinated peptide 2; RA = rheumatoid arthritis.

Early RA cohorts38, 46, 58, 76, 100, 101, 103, 105, 110, 118    
 Sensitivity range, % 41–6341–6652–6733–58
 Specificity range, % 91–10087–9772–8298–100
Early and established RA  cohorts9, 41, 82, 95, 101, 105, 119, 123, 132, 154, 164    
 Sensitivity range, % 41–7762–8770–8133–57
 Specificity range, % 88–9843–9680–9191–99

RF and ACPA: one, either, or both in early, inflammatory arthritis?

Given the substantial overlap between the diagnostic performance and utility of RF and ACPA for the diagnosis of RA, the marginal diagnostic value of adding one test to the other and the added value of performing both must be addressed. In particular, the challenge is to decide on the assay or combination of assays that offers superior performance for the identification of RA among patients presenting with early, undifferentiated inflammatory arthritis. Although correlated, RF and ACPA assays detect different underlying biologic phenomena in RA, and thus agreement between assay results is not static, but likely fluctuates during the disease course (102).

In our review of data from early RA cohorts, ACPA was slightly more specific than RF, but the two assays have equivalent sensitivity (Table 5). The positive predictive value for ACPA in the setting of early undifferentiated arthritis is 78–96% in early RA cohorts, with most values ranging from 90% to 95%, and the negative predictive value is 62–96% (38, 46, 58, 76, 100, 101, 103, 110). The positive predictive value for RF is broader, from 36% to 97%, with most values ranging from 70% to 80%, and the negative predictive value is 69–95%. Positive ACPA results may be particularly helpful in the setting of a negative RF. The positive predictive value of a positive ACPA test was 91.7% among 260 IgM-RF–negative early arthritis patients followed for one year (76). Employing both ACPA and RF positivity further increases specificity and positive predictive value to more than 95%, but substantially decreases sensitivity. When either ACPA or RF positivity is required, the sensitivity is somewhat increased (52–67%), but the specificity is similar to that of RF alone (72–82%) (101, 103).

In cohorts containing both established and early RA, the performance characteristics of RF and ACPA are comparable and the sensitivity of both RF and ACPA is improved (although the ranges of performance characteristics are large and the data are mixed). A strategy requiring either ACPA or RF may improve sensitivity for both early and established RA. In one study, the presence of either ACPA or RF increased testing sensitivity for RA from 66% (ACPA) and 72% (RF) to 81%, with a good specificity of 91% (9). The specificity of requiring both to be present is comparable to that of ACPA alone.

The addition of ACPA testing improved the sensitivity of the 1987 ACR (formerly the American Rheumatism Association) criteria (which rely on the presence of RF as one of the 11 possible criteria, 4 of which must be present) for the correct classification of subjects with early RA (79, 111). Adding ACPA results to the 1987 ACR (formerly the American Rheumatism Association) criteria increased the sensitivity for early RA (disease duration of ≤6 months) from 25% to 44% and did not change the specificity of 86%. ACPA also played an important role in a rule developed by van der Helm-van Mil and colleagues to predict which patients with undifferentiated arthritis would progress to RA (112). Five hundred seventy patients with undifferentiated arthritis in the Leiden Early Arthritis Center were selected and reassessed at one year for RA development. The prediction rule consisted of 9 variables: sex, age, location of symptoms, morning stiffness, tender joint count, swollen joint count, C-reactive protein level, and RF and ACPA positivity. ACPA was one of the strongest predictors, and if positive, a subject received 2 points (112). A modified form of this prediction rule was validated in 3 cohorts of patients with recent-onset undifferentiated arthritis and was found to have excellent discriminative ability to assess progression to RA (113).

ACPA assays are increasingly available and affordable. The assays have good predictive validity because ACPAs are associated with known genetic and epidemiologic risk factors for RA, and therefore identify a population of RA patients with more severe, erosive joint disease that is at high risk for rapid joint destruction. Positive and negative results are highly correlated between current assays. International standardization of reporting units is underway and will facilitate interassay comparisons. Both CCP-2 and CCP-3 assays have improved on CCP-1 assays and have comparable diagnostic utility, with sensitivities of 68–79% and specificities of 86–96% for RA (26, 75, 78, 81, 94).

We did not obtain all data, published and unpublished, from past comparisons of RF and ACPA assay performance or perform a formal meta-analysis. We did review published studies and presented sensitivity and specificity ranges of assays, alone and in combination, in both early and established RA cohorts. Our results suggest that ACPA assays offer a slight advantage over RF (including high-titer RF and combined IgM-RF, IgA-RF, and IgG-RF levels) due to higher specificity. RF and ACPA are two different autoantibody systems that do not measure or reflect the same underlying biology (102). Although there is substantial correlation between ACPA and RF seropositivity within patients, the ACPA assay may be especially valuable in predicting RA in patients who are RF negative but nevertheless have a high probability of RA (76). If the role of the assay is to aid in the identification of patients developing RA among those presenting with early undifferentiated symptoms, in a high-risk population with a high prevalence of disease (rather than screening the general population), the positive predictive value of the ACPA assay is ∼95% (76).

ACPA assays have high specificity, high predictive validity, high sensitivity, apparent cost-effectiveness, and good reproducibility for the diagnosis of early RA. In prior studies, accepting either ACPA- or RF-positive assay results for the diagnosis of RA did not improve on testing for RF alone, and requiring both assays to be positive for diagnosis is a very specific, but not extremely sensitive, approach. Ultimately, the decision to use one or both tests depends on the population tested, the indications for the testing, and the inherent tradeoff between sensitivity and specificity.

Discussion

ACPA assays have good predictive validity in that they are associated with the known genetic and epidemiologic risk factors for RA and identify a population of RA patients with more severe, erosive joint disease at high risk for more rapid joint destruction. RF and ACPA are two different autoantibody systems and do not measure or reflect the same underlying biology. ACPA assays are becoming increasingly available and less expensive. Cost-effectiveness analyses suggest that upfront testing of ACPA in patients presenting with undifferentiated arthritis is cost effective, in particular in terms of the saved indirect costs of delayed diagnosis. ACPA offers similar sensitivity but higher specificity for RA than RF in early RA. When used in the identification of patients potentially developing RA among those presenting with early undifferentiated symptoms, in a high-risk population (rather than screening the entire population), the prevalence of disease will be high and the positive predictive value of the ACPA assay is on the order of 95% (76). In the setting of relatively high clinical suspicion (pretest probability) and a positive ACPA result, the patient has a high likelihood of having or developing RA. If ACPA is negative, further testing may be indicated, depending on the level of clinical suspicion.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Aggarwal had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Aggarwal, Ringold, Costenbader.

Acquisition of data. Aggarwal, Liao, Nair, Ringold, Costenbader.

Analysis and interpretation of data. Aggarwal, Liao, Nair, Ringold, Costenbader.

Acknowledgements

This publication was made possible in part by the ACR-European League Against Rheumatism RA Classification Criteria Committee. We thank Gillian Hawker, MD, MSc, David Felson, MD, MPH, and Josef Smolen, MD, for their expert opinions and input.

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