INTRODUCTION — Biologic markers, commonly termed "biomarkers," are biologic characteristics (eg, of blood or joint fluid) that can be objectively measured and serve as indicators of normal or pathologic processes or as measures of the response to therapy. In patients with rheumatoid arthritis (RA) the term is commonly applied to diagnostic or prognostic indicators, such as rheumatoid factor (RF), and to measures used to assess disease activity, such as acute phase reactants. The US National Institutes of Health has defined a biologic marker (biomarker) as "a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention" .
RA follows a variable disease course with regard to joint injury and functional outcomes, and early RA may be challenging to diagnose with accuracy. Thus, early identification of patients with RA and, in particular, those likely to assume a more rapidly destructive form of disease can help to target those patients most likely to benefit from early, aggressive intervention with disease-modifying agents. The response to therapy in patients with RA is typically assessed using a combination of subjective reporting and physical and laboratory findings; no single biologic measure has proven sufficient for the measurement of disease activity. These observations highlight the need for biologic markers in blood and joint fluids that may serve as reliable objective indicators of prognosis, the response to therapy, and the degree of ongoing disease activity.
This topic will review markers that are widely used in clinical practice and others proposed for such use that may serve as aids in the diagnosis of RA, for predicting prognosis, and for the assessment of disease activity. Genetic features of RA and clinical findings or associations that may have prognostic or diagnostic implications and the diagnosis and differential diagnosis of RA are presented separately, as are more detailed discussions of RF and acute phase reactants.
●(See "Acute phase reactants".)
BIOMARKERS: USE IN PROGNOSIS — The presence of RF or ACPA also predicts poorer functional and radiographic outcomes. However, prognosis varies widely within seropositive and seronegative patient populations.
Other commercially available biomarkers may provide additional useful information but require further study. (See '14-3-3eta' below.)
Rheumatoid factors — High-titer RF appears to be a predictor of a severe disease course, showing a stronger correlation with extraarticular manifestations, such as interstitial lung disease and vasculitis, than appears to be the case with ACPA. The presence of RF also increases the likelihood of a clinically significant response to rituximab after failure of tumor necrosis factor (TNF) inhibitor therapy . (See "Rheumatoid factor: Biology and utility of measurement" and "Epidemiology of, risk factors for, and possible causes of rheumatoid arthritis".)
RF may have some prognostic value with regard to disease manifestations and activity, as well as the severity of joint erosions. Seropositive RA (ie, RA associated with a positive RF test) is often associated with more aggressive joint disease and is more commonly complicated by extraarticular manifestations than seronegative RA [3-9]. As examples:
●Radiographic progression may be more rapid among patients with a positive RF at initial evaluation [12-14].
●A retrospective analysis of a case-control study of 135 women with early RA found that patients with persistently positive RF had more erosions, nodules, extraarticular disease, functional disability, and disease activity than seronegative or intermittently seronegative individuals over a mean period of six years of follow-up .
The presence of RF, particularly at high levels, may antedate the clinical development of RA . These issues are discussed in detail elsewhere. (See "Rheumatoid factor: Biology and utility of measurement", section on 'Healthy individuals' and "Epidemiology of, risk factors for, and possible causes of rheumatoid arthritis".)
Anti-citrullinated peptide antibodies — Enzyme-linked immunosorbent assays (ELISA) for antibodies against cyclic citrullinated peptides (CCP) are the most commonly used assays for ACPA (see 'Anti-CCP antibodies' below). Another ACPA assay that is commercially available detects antibodies against mutated citrullinated vimentin (MCV). (See 'Anti-MCV antibodies' below.)
A decrease in ACPA titers can be seen in patients treated effectively, particularly if treated early with nonbiologic or biologic disease-modifying antirheumatic drugs (DMARDs) but is less frequent and of a lesser magnitude than the decrease in IgM RF that may be seen in such patients .
As with RF, ACPA may be present prior to the appearance of symptoms of RA. This phenomenon is discussed separately (see "Epidemiology of, risk factors for, and possible causes of rheumatoid arthritis").
The relationships of ACPA to cigarette smoking and to the shared epitope are also discussed elsewhere. (See "Epidemiology of, risk factors for, and possible causes of rheumatoid arthritis", section on 'Cigarette smoking' and "Pathogenesis of rheumatoid arthritis", section on 'Citrullinated proteins and peptides'.)
Anti-CCP antibodies — In an ELISA assay for ACPA, arginine residues are replaced by citrulline in a mixture of CCP, increasing the sensitivity of the assay for ACPA. These anti-CCP assays have become the principal commercially available assay kits for the detection of ACPA.
The newer-generation assays, including the second-generation anti-CCP antibody assays (anti-CCP2), have improved sensitivity and specificity compared with the original anti-CCP assays.
ACPA have been reported in other disease including several autoimmune rheumatic diseases (eg, systemic lupus erythematosus [SLE], psoriatic arthritis) [18-27], tuberculosis [28-30], and sometimes chronic lung disease [28-33].
Anti-CCP and RA prognosis — ACPA-positive patients with early RA are at increased risk of progressive joint damage [9,14,34-36], and ACPA testing may predict erosive disease more effectively than RF [37,38]. This was illustrated in a series of 145 such patients in which there was more radiographically apparent damage after five years of observation in those with detectable ACPA compared with those who were RF-positive . The presence of ACPA was also predictive of more rapid radiographic progression in two studies of 183 and 279 Swedish patients with early RA [14,36] and in the BeSt (Behandelstrategieën voor Reumatoide Artritis [Treatment Strategies for Rheumatoid Arthritis]) trial of 508 Dutch patients .
Positive ACPA testing also appears to predict an increased risk for radiographic progression in patients with early oligo- or polyarthritis who are IgM-RF-negative. This was demonstrated in a prospective study that included 178 such patients . Radiographic progression (more than 5 units by Sharp score) was more frequent in the ACPA-positive patients than in those with a negative test result (40 versus 5 percent). The anti-CCP test for ACPA correctly predicted whether or not there would be worsening radiographic damage in 83 percent. Similar findings have been noted in other studies [40-42].
Anti-MCV antibodies — Testing for anti-MCV antibodies has very similar results to anti-CCP antibody testing . Anti-MCV antibodies recognize a naturally occurring isoform of citrullinated vimentin, which can be found in patients with RA and in which arginine residues are replaced by glycine [44,45], and since vimentin is expressed and appears to be implicated in pathogenesis, it is a candidate biomarker . Vimentin is a widely expressed intermediate filament. It becomes citrullinated through deimination, which occurs in macrophages undergoing apoptosis. (See "Investigational biologic markers in the diagnosis and assessment of rheumatoid arthritis", section on 'Autoantibodies'.)
The prognostic value of anti-MCV antibodies in RA was analyzed in a 2010 systematic review of 14 studies, most of which used a commercially available assay, finding similar performance to anti-CCP antibody testing . A report subsequent to the systematic review, which evaluated a longitudinal cohort of 238 patients with RA over 10 years, found that anti-MCV predicted joint damage and did so comparably with anti-CCP . The odds ratios (OR) for radiographic progression were increased in the presence of either anti-MCV or anti-CCP (OR 7.3, 95% CI 3.2-16.5 and OR 5.7, 95% CI 2.6-12.5, respectively). Additionally, the odds of progression increased not only based on the presence or absence of anti-MCV antibodies but also with increasing levels of anti-MCV antibodies. Similar observations were previously made in this cohort with anti-CCP antibodies .
Other anti-citrullinated peptide antibodies — Prior to the widespread use of anti-CCP antibody measurements and the measurement of anti-MCV antibodies, a number of antibodies were identified in the sera of patients with RA that led to the recognition of citrullinated peptides as important antigenic targets in patients with RA. Testing for these antibodies is not widely available, and they are mainly of historical interest, including antikeratin and antiperinuclear antibodies, which recognize epidermal filaggrin, an intermediate filament-associated protein for which a major determinant of antibody binding is the presence of citrulline, formed by a post-translational modification of arginine [49-63].
Antibodies to other citrullinated proteins include antibodies to citrullinated fibrinogen, citrullinated vimentin (anti-Sa), citrullinated synthetic type I or type II collagen telopeptides, and to alpha-enolase [44,45,64-73]. A commercial assay for anti-mutated citrullinated vimentin is also available. (See 'Anti-MCV antibodies' above.)
DISEASE ACTIVITY AND PROGNOSIS: ACUTE PHASE REACTANTS — The main clinically useful biologic markers in patients with rheumatoid arthritis (RA) that aid in assessing disease activity and predicting functional and radiographic outcomes include acute phase reactants, particularly the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) (see 'Erythrocyte sedimentation rate' below and 'C-reactive protein' below). Measurements of combinations of multiple acute phase proteins and other markers of the immune response in RA have also been used for this purpose. (See 'Multi-protein biomarker algorithms' below.)
In patients with RA, the ESR and CRP generally tend to both be elevated or not in the same patients, but one study found that results for the two tests were discordant (ESR >28 mm/hr with CRP ≤0.8 mg/dL or ESR ≤28 mm/hr with CRP >0.8 mg/dL) in about one-quarter of patients in a large practice-based registry, regardless of the level of disease activity .
The acute phase response, including the ESR and CRP, is discussed in more detail separately. (See "Acute phase reactants".)
Erythrocyte sedimentation rate — The ESR level tends to correlate with disease activity in RA as well as disease severity and may be useful for monitoring the therapeutic response [75-77]. Moreover, despite some shortcomings, an elevated ESR in patients with early RA is predictive of greater radiographic joint damage in subsequent years despite treatment with conventional disease-modifying antirheumatic drugs (DMARDs) [13,14,78].
The rate at which erythrocytes fall through plasma, the ESR, depends largely upon the plasma concentration of fibrinogen . However, the ESR can be greatly influenced by the size, shape, and number of red cells, as well as by other plasma constituents such as immunoglobulins. Thus, results may be imprecise and sometimes misleading. (See "Acute phase reactants", section on 'Erythrocyte sedimentation rate'.)
C-reactive protein — Assessment of the CRP has been advocated as an objective measure of disease activity in RA. Radiologic damage, as assessed by erosion counts in RA, is significantly more likely to progress when CRP and ESR are elevated, irrespective of the presence or absence of rheumatoid factor (RF) and irrespective of therapeutic intervention [13,14,80]. Unlike the ESR, CRP can be measured using stored serum samples, is independent of the hemoglobin concentration, and can be performed in automated serum analyzers.
Elevations of both ESR and CRP are stronger indications of radiologic progression than CRP alone . In one study of 147 patients, for example, absence or progression of radiographic joint damage after two years was correctly predicted in 83 percent of the patients using a combination of disease activity at presentation (assessed by ESR, CRP, or disease activity score) and DR4 and RF positivity .
Another study assessed progression of radiologic damage in 110 patients with early RA who had symptoms for less than one year . A highly significant correlation between radiologic progression and cumulative CRP production was noted . However, a wide variation in the relationship between the degree of radiographic change and cumulative CRP was noted between patients, particularly those with low CRP levels. This interindividual variability could not be accounted for by human leukocyte antigen (HLA)-DR4, positive RF, sex, or age and limits the value of serial measurements of acute phase proteins in predicting radiologic progression.
Multi-protein biomarker algorithms — A multi-biomarker disease activity (MBDA) test for RA has been developed that may provide a useful adjunct to clinical assessment and the use of single laboratory tests [84,85]. The combined use of multiple markers may provide advantages over the use of single markers for predicting disease activity and progression and for identifying patients with subclinical disease, although the cost-effectiveness and optimal role for this test in routine clinical practice remains to be established.
The commercially available MBDA test for RA (Vectra DA) was developed by initial testing of 130 candidate biomarkers in feasibility studies, then selecting 25 for algorithm training . Multi-biomarker statistical models outperformed individual biomarkers at estimating disease activity assessed by the disease activity score (DAS) using a 28 joint count and C-reactive protein level (DAS28-CRP). The final MBDA algorithm used 12 biomarkers to generate an MBDA score between 1 and 100.
Post hoc analyses of data from several clinical trials in patients with early RA showed that the MBDA score at baseline was a strong independent predictor of radiographic progression at one year [86-88]. High MBDA scores were better predictors of such progression than the DAS. As an example, in a study of 163 patients with RA in the Leiden Early Arthritis Cohort, at a total of 271 visits, patients with a high MBDA score were 2.3 times more likely (95% CI 1.1-3.7) to have joint damage progression during the subsequent year .
Whether MBDA has a useful role in informing a clinical management decision has been more controversial. In an analysis of patients with RA from the Swedish Pharmacotherapy (Swefot) trial with an inadequate response to methotrexate (MTX), those patients with greater MBDA score reduction were more likely to respond to triple therapy (MTX plus sulfasalazine plus hydroxychloroquine) than to MTX plus infliximab . However, post hoc analysis of data from the AMPLE ("abatacept versus adalimumab comparison in biologic-naïve RA subjects with background methotrexate") trial has been interpreted as indicating that the MBDA score cannot reliably be used to guide decision making in RA management, particularly for patients who receive abatacept or adalimumab as a first biologic agent . Furthermore, as interleukin (IL)-6 is a contributor to the MBDA score, caution must be exercised when interpreting MBDA response following use of IL-6R inhibitors, given that blood IL-6 rises after treatment and therefore lowers the magnitude of response observed for the MBDA score compared with the DAS28-CRP .
INVESTIGATIONAL AND OTHER BIOMARKERS — A number of other potential markers have been the subject of investigation in rheumatoid arthritis (RA). Broadly defined, these include:
●Immunologic or serologic abnormalities, in addition to rheumatoid factor (RF) and anti-citrullinated peptide antibodies (ACPA) (see 'Immune abnormalities and autoantibodies' below)
●Genetic factors (see 'Genetic factors' below)
●Acute phase reactants, induced as part of the inflammatory response, in addition to the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP)
●Macromolecules specific for joint associated tissues that are released or excreted as part of degenerative and reparative processes
Immune abnormalities and autoantibodies — A number of antibodies have been the subject of study in RA in addition to RF and ACPA but most are not available for routine clinical use and none has proven useful in routine clinical practice. Those of particular interest include:
●Anti-p68 (BiP) antibodies 
●Anti-RA33 antibodies [13,93-95]
●Gal 0 glycoforms [96-101]
●Antibodies to alpha-enolase 
●Antibodies to glucose phosphate isomerase (GPI) 
●Antibodies to ferritin 
●Antibodies to the enzyme peptidyl-arginine deiminase 4 (PADI4) 
Other immune changes investigated as potential biomarkers in RA include enhanced expression of CD40 ligand (CD154) on activated T cells  and markers of complement activation, including the concentration of covalently linked C1q-C4 complement components .
Genetic factors — Important associations have been noted between certain genetic polymorphisms and the susceptibility to and severity of RA. Of particular interest has been the association with certain human leukocyte antigen (HLA)-DR alleles (particularly HLA-DR4) encoding a conserved amino acid sequence in the third hypervariable region of the DRB1 chain, termed the shared epitope. These associations and their role in RA pathogenesis are discussed separately; genetic testing has yet to play a role in diagnosis or treatment of RA. (See "HLA and other susceptibility genes in rheumatoid arthritis".)
In addition to the shared epitope, other reported genetic associations include:
●Matrix metalloproteinase (MMP) genotype polymorphisms and RA disease severity 
●Interleukin (IL)-10 promoter genotype polymorphisms and RA severity but not susceptibility 
●Sulphoxidation status and risk of disease persistence among patients with early RA 
In established RA, a longitudinal genomic analysis using serial ribonucleic acid (RNA) sequencing of RA blood samples revealed that preinflammatory mesenchymal cells and B cells are present one to two weeks before clinical flare of the arthritis, suggesting that activation of the flare in synovitis takes place outside the joint .
Other acute phase markers — IL-6 increases during the acute phase response, and IL-6-targeted biologics are effective in treating RA. However, IL-6 levels have not been found to correlate with radiographic progression in RA, despite their close relationship to the acute phase response in RA patients . Measurement of IL-6 levels in blood or synovial fluid is not useful in routine clinical practice, although interest in further research remains.
Tissue-specific markers — A number of biochemical markers of joint damage have been described in RA. These molecules may be synthetic or degradative, their presence in body fluids arising as a consequence of metabolism of the tissue of origin. They are predominantly derived from a single tissue such as cartilage, bone, or synovium and can be detected principally by immunoassay of joint fluid, serum, or urine. Appropriate assays are not routinely available, and joint fluids are not readily available (apart from knee joints), in comparison with serum or urine. There is, as yet, limited information regarding the usefulness of tissue-specific markers as measures of disease activity or response to therapy in RA. However, some markers may be of prognostic value.
●Synovium-specific markers – In addition to 14-3-3eta, which is markedly enriched in synovial fluid compared with the serum (see '14-3-3eta' below), other products of the synovium may be markers of disease activity and have prognostic implications. These include serum hyaluronan [113-117] and MMP, including MMP-1 and MMP-3 [118,119]. Elevated serum levels of immunoglobulin soluble receptor (Fc gamma RIIIa) in RA are thought to be due to synovial macrophages and/or natural killer cells .
●Cartilage-specific markers – Markers of cartilage metabolism may have some prognostic value in patients with RA. Studies have focused upon elevated serum levels of cartilage oligomeric matrix protein (COMP), a member of the thrombospondin protein family, in early RA [121,122]; the aggrecan content of synovial fluid [123,124]; a putative marker of cartilage aggrecan synthesis, epitope 846, located on the chondroitin sulphate-rich area of the aggrecan molecule ; measurement of crosslinked c-terminal peptides from type II collagen (CTX-II) in urine [125,126]; and a peptide derived from the helical portion of type II collagen (HELIX-II) that may be detected in the urine .
The combination of cartilage and synovial biomarkers including both MMP-3 and CTX-II levels may have a better ability to predict radiographic progression than either alone .
●Bone-specific markers – As with cartilage, several bone-specific markers may be useful in patients with RA as markers of disease activity, prognosis, or both. These include bone sialoprotein, an osteoblast-derived protein preferentially expressed in juxtaarticular bone that may be measured in synovial fluid ; measurement of pyridinoline crosslinks, a marker of bone degradation, that may be detected in urine ; measurement of crosslinked carboxyterminal telopeptides of type I collagen (ICTP), a larger serum marker for bone collagen degradation [125,131-133]; and the initial ratio of serum levels of an inhibitor of osteoclast differentiation (osteoprotegerin) and a stimulatory protein (Receptor Activator of Nuclear factor Kappa B ligand [RANKL]) .
●Vascular markers – Serum vascular endothelial factor (VEGF) concentrations are elevated in RA patients compared with healthy controls and with patients with osteoarthritis [135-137], and elevated levels in patients with early RA correlate with the degree of radiographically assessed joint damage over the subsequent year .
●Plasma proteins – In a proteomic approach, 163 plasma proteins were evaluated in 44 patients with RA to determine which were associated with disease activity . Plasma proteins whose concentrations were correlated with disease activity included IL-6, oncostatin M, IL-2, macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF receptor superfamily member 9), C-C motif chemokine 23 (CCL23), transforming growth factor (TGF)-alpha, and chemokine C-X-C motif ligand 13 (CXCL13).
14-3-3eta — Serum 14-3-3eta, an isoform of the 14-3-3 family of intracellular chaperonin proteins, may be helpful diagnostically in RA as some data suggest that it may have similar sensitivity and specificity to RF and ACPA in distinguishing patients with RA from osteoarthritis, other autoimmune disorders, and healthy controls . However, in a cohort of patients with arthralgia, preselected for ACPA and/or RF positivity, the added predictive value of 14-3-3eta could not be established .
An assay for serum 14-3-3eta is commercially available but is not widely used; further study is needed to justify a role for the assay in routine clinical practice.
SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Rheumatoid arthritis".)
●Definitions – Biologic markers, commonly termed "biomarkers," are biologic characteristics (eg, of blood or joint fluid) that can be objectively measured and serve as indicators of normal or pathologic processes or as measures of the response to therapy. In patients with rheumatoid arthritis (RA), the term is commonly applied to diagnostic or prognostic indicators, such as rheumatoid factor (RF), and to measures used to assess disease activity, such as acute phase reactants. (See 'Introduction' above.)
●Use in diagnosis – The main clinically useful biologic markers for the diagnosis of RA are RF and antibodies to citrullinated peptides (ACPA).
●Assessing prognosis – The presence of RF or ACPA also predicts poorer functional and radiographic outcomes but neither is of sufficient specificity alone to establish the diagnosis of RA, and prognosis varies widely within seropositive and seronegative patient populations, respectively. Other commercially available biomarkers may provide additional useful information but require further study. (See 'Rheumatoid factors' above and 'Anti-CCP antibodies' above and '14-3-3eta' above.)
●Assessing disease activity – The main clinically useful biologic markers in patients with RA that aid in assessing disease activity and predicting functional and radiographic outcomes include acute phase reactants, particularly the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). Measurements of combinations of multiple acute phase proteins and other markers of the immune response in RA have also been used for this purpose. (See 'Erythrocyte sedimentation rate' above and 'C-reactive protein' above and 'Multi-protein biomarker algorithms' above.)
●Investigational biomarkers – Biologic markers that remain investigational include (see 'Investigational and other biomarkers' above):
•Immunologic (or serologic) abnormalities, including various ACPA that were under study prior to the development and widespread availability of anti-cyclic citrullinated peptides (CCP) and anti-mutated citrullinated vimentin (MCV) antibody assays, such as antiperinuclear factor, antikeratin antibodies, and anti-Sa antibodies; other autoantibodies; and other immunologic markers (see 'Immune abnormalities and autoantibodies' above)
•Genetic factors that may influence the development and severity of RA, including the human leukocyte antigen (HLA) class II shared epitope, matrix metalloproteinase (MMP) genotype, interleukin (IL)-10 promoter genotype, and sulphoxidation status (see 'Genetic factors' above)
•Other measures of the acute-phase response (eg, IL-6), elicited as part of the inflammatory process, in addition to measurement of the ESR and CRP (see 'Other acute phase markers' above)
•Macromolecules specific for joint-associated tissues, such as cartilage, bone, and synovium, that are released into the circulation or excreted in the urine as part of degenerative and reparative processes (see 'Tissue-specific markers' above)
•Serum 14-3-3eta, an isoform of the 14-3-3 family of intracellular chaperonin proteins, which has been proposed as an additional useful diagnostic marker (see '14-3-3eta' above)
ACKNOWLEDGMENT — The editorial staff at UpToDate acknowledge Ravinder N Maini, BA, MB BChir, FRCP, FMedSci, FRS, who contributed to an earlier version of this topic review.
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