C-reactive protein (CRP) is composed of five identical subunits that are linked together. This protein is present in animals that trace their evolutionary origins for hundreds of millions of years (such as the horseshoe crab). CRP is normally present in plasma in only trace amounts: approximately 0.2 mg per dL. Levels increase dramatically and quickly after a stimulus. Moderate elevations occur in most connective tissue diseases (1 to 10 mg per dL). Very high levels are seen in bacterial infections and systemic vasculitis (15 to 20 mg per dL). Diabetes, obesity, and cigarette smoking can increase CRP levels in variable amounts. CRP is not thought to be altered by age or gender. The CRP levels can increase within 4 to 6 hours and normalize within 1 week in response to a stimulus. These changes occur much more quickly compared to the erythrocyte sedimentation rate (ESR). CRP levels fall when inflammation subsides. Because a substantial stimulus is required for CRP elevation, a normal value does not exclude an inflammatory process. Many clinicians prefer sending ESR concomitantly with CRP. Of interest, in systemic lupus erythematosus (SLE) and other connective tissue diseases, CRP levels are lower than one would expect for the amount of inflammation present. CRP was initially identified by its ability to form a precipitin reaction with pneumococcal polysaccharide. It is now measured by either
latex agglutination or rocket electrophoresis. In contrast to the ESR, CRP can be assayed on specimens that have been stored by freezing. This is an advantage compared with ESR, which must be performed on fresh blood. Generally for CRP, the upper limit of the reference range is age/50 in men and age/50 +0.6 in women. Recently, high-sensitivity CRP has become available. This test is much more sensitive, and can detect slight elevations of CRP that are technically within the normal range, but may have clinical relevance with respect to coronary artery disease. However, it has not been proven cost-effective or having additional benefit in routine monitoring of rheumatologic diseases.

Although an elevated CRP is highly associated with inflammation, ESR has been the most widely used indicator of inflammation and the acute phase response. ESR is performed by placing anticoagulated blood in a vertical glass tube and measuring the rate of red blood cell (RBC) settling. Normally, RBCs repel each other because the electrical charges on the surface of all RBCs are the same. When inflammation is present, there is an increase in the concentration of asymmetrically charged proteins that bind to the RBCs and thus prevent this repulsion. The RBCs, therefore, tend to aggregate. Aggregated clumps of cells settle more rapidly than individual cells, thus providing a higher ESR. Fibrinogen is the protein that is most responsible for elevations in ESR in acute states of inflammation. However, in chronic inflammation, decreased serum albumin and hematocrit levels also contribute to an increased ESR. An increase in immunoglobulin (Ig) such as that seen with myeloma or monoclonal gammopathies can also lead to an ESR elevation, although such changes may not necessarily indicate an inflammatory state. The ESR is influenced by anemia, polycythemia, and alterations in the size and shape of erythrocytes. Falsely low levels are seen in sickle cell disease, anisocytosis, spherocytosis, polycythemia, and heart failure. Prolonged storage of blood before testing or tilting of the calibrated tube will tend to an increase in the ESR. The Westergren method is thought to be the most reliable. This method measures the fall of RBCs in millimeters per hour in a standardized tube. Normal is considered 0 to 15 for males and 0 to 20 for females. However, the ESR also increases with age; thus, “normal” levels are variable. Levels up to 40 mm per hour are common in healthy elderly people. A rule of thumb is that the age-adjusted upper limit of normal of ESR is age divided by 2 for men and age plus 10 divided by 2 for women. The ESR is probably most helpful if it is normal. Active inflammatory disorders such as acute rheumatic fever, SLE, rheumatoid arthritis (RA), temporal arteritis, and infections tend to have elevated ESRs. Although elevations of the ESR in septic arthritis and crystal-induced arthritis are the rule, a normal ESR does not completely rule out these entities. Joint aspiration is required. From this, it is clear that the ESR and CRP are neither diagnostic nor specific. Nonetheless, they can be helpful in evaluating patients when RA or other systemic and local inflammatory conditions are being considered. ESR and CRP are a part of the metrics used to measure and follow disease activity in RA patients along with signs and symptoms.

Other acute phase reactants include ferritin, fibrinogen, serum amyloid A, etc. Although markers of inflammation, they are not routinely measured because they are nonspecific.

Rheumatoid factors (RFs) are autoantibodies that are directed against the Fc fragment of immunoglobulin G. It is believed that they are synthesized in response to immunoglobulins (Igs) that have been conformationally altered after reaction with antigen. They are most commonly associated with RA but are also found in other disorders.

Historically, the test was performed by coating sheep red blood cells or latex particles with human IgG and measuring the dilution of patient serum that will still aggregate the particle. Newer methods include radioimmunoassay, enzyme-linked immunoassay (ELISA), and nephelometry. These methods increase sensitivity and specificity.

RF is one of the laboratory tests most frequently ordered in the evaluation of patients with joint complaints. The test is positive in 75% to 90% of patients with RA. In early RA, only 50% of patients may have a positive RF. However,
these data are taken from highly selected populations and might be subject to referral bias. The high specificity that is also reported in studies from these populations may not be observed in patients who have weak indications for the test, who may be elderly, or who have other diseases that may cause a false-positive RF. In fact, the prevalence of false-positive RFs in patients older than 75 years is between 2% and 25%. Although there may be an increased likelihood of developing RA in a RF-positive asymptomatic individual, the use of RF as a screening test performs poorly because of the high frequency of false-positive test results. RFs are most common in patients with RA, but they are also present in normal sera as well as in sera of patients with acquired immunodeficiency syndrome, hepatitis, various parasitic diseases, chronic bacterial infections such as tuberculosis and subacute bacterial endocarditis (SBE), tumors, chronic lymphocytic leukemia, and other hyperglobulinemic states such as cryoglobulinemia, primary Sjögren syndrome, chronic liver disease including chronic hepatitis C, sarcoidosis, and some chronic pulmonary diseases. For example, when RF is found in a patient with fever, arthralgia, and a heart murmur, SBE may be a more likely cause of a positive RF than RA. When found in patients with RA, RFs are usually specific for human IgG, are of high affinity, and include not only IgM RFs but also IgG, IgA, and IgE variants.

High levels of RF have been associated with a worse prognosis; there tend to be more involved joints when first seen by a physician, more erosions, and greater ligamentous instability. However, RF titer does not correlate with disease activity and thus cannot be used to assess disease activity. A high level of RF is considered a risk factor for RA vasculitis. Other laboratory findings that are common in RA include leukocytosis (usually with a normal differential), thrombocytosis, anemia (normochromic, normocytic), normal uric acid (if the patient is not taking salicylates, which can raise uric acid levels), a negative ANA and anti-DNA antibody, and normal or elevated serum complement.

Anti-cyclic citrullinated peptide (CCP) antibodies are autoantibodies directed against the acids formed by posttranslational modification of arginine. During inflammation, the amino acid arginine can be enzymatically converted into citrulline, in proteins such as vimentin, in a process called citrullination. If their shapes are significantly altered, the proteins may be seen as antigens by the immune system, thereby generating an immune response. IgG anti-CCP antibodies are measured by ELISA using synthetic citrullinated peptides. The 2010 American College of Rheumatology (ACR)/EULAR Rheumatoid Arthritis Classification Criteria now include anti-CCP testing. Anti-CCPs have the same sensitivity as RF, but the advantage is that they are more specific. Also anti-CCPs can be detected in early RA and are better predictors of erosive disease than RF. There are patients who are both RF and CCP negative but have clinical signs of RA. They are labeled seronegative RA. As with RF, CCP levels cannot be used to monitor disease activity. However, RF and CCP combined can help in the diagnosis of RA. A false-positive CCP can sometimes be seen in diseases like tuberculosis, chronic lung diseases, and other connective tissue diseases. The newer generation assays, anti-CCP antibody assays (anti-CCP2), have improved sensitivity and specificity compared with the original anti-CCP assays.

There have been several other antibodies described for diagnosing RA: 14-3-3ε autoantibodies, alone and in combination with the 14-3-3ε protein, RF, and/or anti-CCP identified most patients with early and established RA.¹ A number of other autoantibodies have been the subject of investigation in RA—anti – mutated citrullinated vimentin, Ig-binding protein, glucose-6-phosphate isomerase, type 2 collagen, mannose-binding lectin, ferritin, anti-RA33, anti-p68, anti-alpha enolase, antibodies to the enzyme peptidylarginine deiminase 4, etc. All of these require further studies for them to be widely used.

Lupus is another autoimmune disease which may have major joint manifestations. It usually causes a nonerosive, nondeforming symmetric arthropathy. Multiple joints are typically involved. The ankle and foot are less commonly involved than with RA. In addition, systemic features are more common. These include rash, fever, central nervous system involvement, renal disease, and serositis. Unlike RA, many types of autoantibodies are typically found in SLE and related syndromes. ANAs are a hallmark of SLE. These are antibodies directed against the cell nucleus. The ANA is positive in 95% to 99% of patients with lupus. The indirect immunofluorescence ANA is the method most commonly used. It is performed by diluting test sera and incubating with substrate cells. Traditionally, thin sections of frozen rat kidney or liver are used. However, cytocentrifuged preparations of cells from tissue culture such as Hep-2 can give optimal sensitivity and pattern discrimination. Any bound ANAs are then detected by fluorescein-tagged antihuman Ig. The substrate cell is then viewed under a fluorescence microscope. ANAs are reported by either intensity of fluorescence (i.e., 1+ to 4+) or by maximal dilution of serum giving a positive result. Values of 2+ or greater or titers of greater than 1:40 are considered abnormal. The pattern of the ANA is also reported (e.g., speckled, diffuse, rim, centromere). ANAs have different targets, and the pattern of immunofluorescence can provide differential diagnostic information.

Homogeneous patterns are least specific, and can be seen in up to 5% of normal patients, especially women and the elderly. In this setting, they are usually present in low titers. The rim pattern is characteristic of SLE. Nucleolar and speckled patterns are also seen and are associated
with scleroderma, CREST (Calcinosis cutis, Raynaud phenomenon, Esophageal dysmotility, Sclerodactyly, and Telangiectasia), mixed connective tissue disease (MCTD), and other diseases. Once a patient has been documented to have a positive ANA, it is usually not necessary to repeat the test unless a major change in therapy or status is detected. The ACR recommends testing for ANA only when there is a suspicion of a connective tissue disease like lupus. Positive tests must always be interpreted in context with the history, physical exam, and other laboratory tests. It should be noted that steroids and other immunosuppressant agents may lower ANA titers. Extractable nuclear antigens (ENAs) sometimes help sort the diagnosis.

Many diseases can produce anti-single-stranded DNA antibodies. These include liver diseases and drug-induced lupus. However, for the most part, only SLE (and MCTD) is associated with high-titer anti-dsDNA antibodies. These occur in nearly all SLE patients. In contradistinction to the ANA, levels of dsDNA antibodies are frequently used in monitoring disease activity, so it is often repeated in frequent intervals. Many other laboratory tests may be abnormal in SLE, and can be used in conjunction with the more specific tests for assessing disease activity. These include a low WBC (usually with neutropenia and lymphopenia), anemia (sometimes an autoimmune hemolytic anemia with positive Coombs), and thrombocytopenia. As suggested by the variety of ANA immunofluorescent patterns, ANAs may be directed against a number of different cellular constituents. Immunodiffusion and counterimmunoelectrophoresis can be performed using soluble components of cells (i.e., ENAs). The ENAs include anti-Ro, anti-La, anti-Smith and anti-ribonuclear protein (RNP). Anti-RNP is often accompanied by a speckled ANA and is seen in patients with SLE, or an overlap disease that is known as MCTD. Anti-Ro (SSA) and anti-La (SSB) are associated with Sjögren syndrome. In addition to nucleic acids, other protein antigens from both nuclei and cytoplasm have been shown to be targets of ANAs. The antinuclear specificities that are most common with SLE are anti-double-stranded DNA and anti-Smith. Anti-histone antibody and single-stranded DNA antibodies may be found in drug-induced lupus.

Many patients with lupus may have concomitant antiphospholipid syndrome (APLS). However, APLS may exist in patients without lupus too. Patients with APLS often present with ischemic toes, arterial and venous clots, or even emboli, resulting in strokes. It is characterized by elevated partial thromboplastin time (PTT), false-positive Venereal Disease Research Laboratories test, and antiphospholipid antibodies.

A factor has been known to exist in the serum of some SLE patients that prolongs the PTT. This inhibitor was frequently associated with false-positive serologic tests for syphilis. The factor could be absorbed from plasma by phospholipids. This factor became known as the lupus anticoagulant, even though approximately half of the patients with this serologic abnormality do not have SLE. In addition, although the inhibitor acts as an anticoagulant in vitro, patients with the lupus anticoagulant were not prone to excess bleeding. In fact, the opposite was found: they were prone to both arterial and venous thromboses as well as thrombocytopenia and fetal loss or miscarriage.