From the HSS Manual of Rheumatology and Outpatient Orthopedic Disorders
The laboratory studies outlined in this chapter are helpful in the diagnosis and treatment of rheumatic diseases. They should be interpreted in the context of a careful history and physical examination.
A test should be performed only if it will likely change the diagnosis, prognosis or therapy, not out of clinical or academic interest. Performing a large battery of screening tests, with no guidance by the specific clinical picture, will clearly beget false positives, and possibly incorrect diagnoses and treatments. One does not “chase or treat” a test but, rather, the patient and her entire clinical picture. Tests are placed into the context of a person and their problems.
This chapter discusses erythrocyte sedimentation rate, C-reactive protein, auto-antibodies, complement, and other tests helpful in the serologic evaluation of rheumatic diseases.
- Erythrocyte sedimentation rate (ESR). The ESR (Westergren method) is a time-honored measurement of inflammation.
- Method of detection: The ESR measures the rate of fall, in millimeters per hour, of red blood cells (RBCs) in a standard tube. Prolonged storage of the blood to be tested, or tilting of the calibrated tube, will falsely increase the ESR.
- Normal Westergren ESR values are 0 to 15 mm/h for male subjects and 0 to 20 mm/h for female subjects. The ESR increases with age, and values up to 40 mm/hr are not uncommon in healthy elderly people. In inflammatory disorders, red blood cells tend to form stacks (rouleaux) and these stacks cause the red cells to sediment more rapidly. This stacking may result from increased levels of fibrinogen.
- Measurement of the ESR can be helpful in evaluating the extent or severity of inflammation, and in monitoring changes in disease activity over time. This test, cannot, however, be used to definitely confirm or exclude any particular disease. Falsely low ESRs are found in sickle cell disease, anisocytosis, spherocytosis, polycythemia, and heart failure. Very high levels are seen in patients with a monoclonal gammopathies.
- C-reactive protein (CRP): The CRP is an acute-phase reactant serum protein that is present in low concentration in normal serum. It was originally identified by its precipitin reaction with pneumococcal C polysaccharide.
- Method of detection: CRP is most commonly measured using an immunoassay or laser nephelometry.
- CRP levels rise rapidly under an inflammatory stimulus (especially that provided by interleukin-6), and then fall when the inflammation subsides. Normal levels for healthy adults are less than 0.2 mg/dl although levels of up to 1 mg/dl are not uncommon. Moderate elevations (1-10 mg/dl) can be seen in inflammatory conditions such as rheumatoid arthritis and temporal arteritis. Elevations of greater than 15-20 mg/dl are generally seen in bacterial infections.
- Although serum CRP levels are elevated in some patients with active SLE, most lupus patients show only modest or no CRP elevation, even in the face of active disease. Highly elevated CRP levels in SLE patients should prompt consideration of a superimposed infection, although an elevated level should not be considered proof of infection.
- Rheumatoid factor (RF): RFs are immunoglobulins with specificity for the Fc portion of immunoglobulin G (IgG). Multiple immunoglobulin classes have RF activity, but the RF detected by standard laboratory testing is an IgM antibody.
- Method of detection: There are many methods to measure RF. Those most commonly used include the enzyme-linked immunosorbent assay (ELISA), agglutination of IgG-coated latex particles, or nephelometry.
- Interpretation. Fifty to seventy-five percent of rheumatoid arthritis (RA) patients have IgM RF, as do 3-5% of normal subjects. RF positivity is associated with the HLA-DR4 haplotype, and with more aggressive disease. Patients with extra-articular disease are invariably RF-positive. IgM RFs are also commonly seen in patients with primary Sjögren syndrome, and mixed cryoglobulinemia, as well as in patients with chronic infections such as subacute bacterial endocarditis and chronic hepatitis (See Table 4-1).
Table 4-1. Frequency of rheumatoid factor as measured by latex agglutination in rheumatic and nonrheumatic diseases.
- Anti-cyclic citrullinated peptide antibodies (anti-CCP). Citrulline is formed by the deamination of the amino acid, arginine. Antibodies directed against citrullinated peptides have been found in serum of many patients with RA.
- Method of detection: ELISA detection of antibodies to citrullinated antigens is the technique most commonly utilized.
- Interpretation: The sensitivity of this test in patients with rheumatoid arthritis is 40-70%, but the specificity may be as high as 98%. In patients with undifferentiated arthritis, the presence of anti-CCP antibodies is an important predictor for RA. Ninety-three percent of such patients will develop RA within 3 years. Some, but not all patients with anti-CCP antibodies will also have a positive RF. Similarly, some, but not all patients with a positive RF will have anti-CCP antibodies. The presence of both RF and anti-CCP best predict a poorer radiologic and functional outcome for RA patients.
- Antinuclear antibodies (ANAs). The production of antibodies to a wide array of nuclear antigens is characteristic of lupus and the other connective tissue diseases. Because the ANA test detects any antibody binding to nuclear constituents, it is extremely useful as a screening test for these diseases.
- Method of detection: Indirect immunofluorescence (IFA) is the most common method of ANA testing. This technique employs a cellular substrate, now most commonly a human epithelial cell line (Hep-2), which is placed on a glass slide. The patient’s serum is then applied (allowing autoantibodies to bind to the Hep-2 cells) and then tagged with a fluorescent-labeled anti-Fc IgG which can then be visualized under a microscope.
- ANA studies are usually reported as a pattern (homogeneous, speckled, rim or nucleolar) and by titer, or intensity of fluorescence (1 to 4+). Titers of at least 1:40 are considered positive, but higher titers (>1:160) are usually present in patients with lupus or other connective tissue diseases. Up to 20% of healthy elderly subjects, particularly women, have a positive ANA (usually <1:160). ANA titers do not correlate well with disease activity, therefore, once a positive ANA has been documented in a patient’s serum, there is seldom need to repeat the test.
- The pattern of the ANA reflects the nuclear distribution of the antigen(s) being bound. A homogeneous pattern is common in lupus, but the least specific pattern for that disease. A rim pattern usually suggests the presence of antibodies to chromatin (DNA or histone proteins) and is seen in lupus, both systemic and drug-induced. Speckled ANAs may reflect antibodies to RNA-associated proteins and can be seen in lupus, mixed connective tissue disease (MCTD) and Sjogren syndrome. Nucleolar ANAs are characteristic of scleroderma.
- A positive ANA can also be a nonspecific finding in a variety of other autoimmune conditions such as thyroiditis, juvenile arthritis, and psoriasis. Thus, the ANA is a very useful diagnostic screening test, with high sensitivity for lupus and other connective tissue diseases, but with very low specificity for those diseases, particularly when positive in only low titer.
iv Extractable nuclear antigen (ENA) and DNA antibody testing (see below) can be used to further evaluate the patient with a positive ANA. The prevalence and patterns of ANAs in various disease states are summarized in Table 4-2.
Table 4-2. Frequency of Antinuclear Antibodies as Measured by Indirect Immunofluorescence Assay
- Anti-DNA antibodies: Antibodies against “native”, or double stranded DNA (ds-DNA) are characteristic of patients with lupus. These antibodies bind to the deoxyribose phosphate backbone of DNA. Antibodies against single stranded DNA (ss-DNA), in contrast, bind to exposed purine and pyrimidine bases. Whereas anti-ds-DNA antibodies are highly specific for lupus, antibodies to ss–DNA can be seen in a wide variety of inflammatory conditions, such as drug-induced lupus and chronic hepatitis.
- Method of detection
- Until the 1990s, the FARR assay was the most common method used to measure antibodies to ds-DNA. This test has high specificity for lupus. Because it requires the use of radiolabeled carbon, however, it has been supplanted by other assays that are safer to perform.
- The Crithidia assay uses the hemoflagellate, C. luciliae, as a substrate for indirect immunofluorescence. Its kinetoplast contains a concentrated focus of stable, circularized ds-DNA, without contaminating RNA or nuclear proteins. This test is, therefore, sensitive and specific for antibodies to ds-DNA.
- Many laboratories now use an ELISA assay to measure antibodies to ds-DNA because the test is inexpensive and easy to perform. Because purified DNA can denature in the plastic ELISA plate wells, however, (exposing purine and pyrimidine bases), antibodies to ss-DNA can at times cause a false positive result. (A “positive” test for antibodies to ds-DNA by ELISA in the presence of a negative ANA, for example, strongly suggests the presence of antibodies to ss-DNA only.) Thus the specificity of this test for antibodies to ds-DNA, and for lupus itself, is less than that of the FARR or Crithidia assay.
- Interpretation: Anti-ds-DNA antibodies occur in approximately 75% of SLE patients, but are rare in normal subjects, or patients with other inflammatory and autoimmune conditions. In SLE, anti-ds-DNA antibodies strongly correlate with the presence of nephritis, and their levels can rise and fall with disease activity. This makes the test a useful, but not infallible test for monitoring disease activity and response to therapy.
- Anti-Histone antibodies: These antibodies are seen in over 50% of lupus patients, but they are seen in 100% of patients with drug-induced lupus. A negative test is useful, therefore, in ruling out drug-induced lupus.
- Antiphospholipid antibodies (the lupus anticoagulant test, anticardiolipin antibodies, and anti-B2-gylcoprotein antibodies) which are seen in approximately 40% of lupus patients, are associated with thrombosis and pregnancy morbidities. See Chapter 31 for a detailed discussion of this topic.
- Extractable Nuclear Antigens (ENA). Anti-nuclear antibodies are heterogeneous, and a variety of techniques have been used to determine the antigens to which these antibodies are directed. Early studies used techniques to separate nuclear components into those that were soluble, the “extractable” antigens (ENAs), and those that were insoluble, namely chromatin (DNA and histones).
- Method of detection: Antibodies to ENAs were initially studied using a variety of techniques including immunodiffusion and counterimmunoelectrophoresis. Many of the specific antigens have now been identified and the proteins cloned. As a result, most laboratories now use ELISA assays to detect antibodies to particular ENAs.
- Interpretation: Some of the antigens to which antibodies are commonly detected and their interpretations are listed below.
- Smith (Sm): Antibodies to this antigen are not very sensitive for lupus (seen in only 20-30% of patients), but are highly specific for this disease.
- Ribonuclear protein (RNP): Antibodies to this antigen are detected in 30-40% of lupus patients, and very high titers are also characteristic of patients with mixed connective tissue disease (MCTD).
- SS-A/Ro: Antibodies to this antigen are detected in 40% of lupus patients, and are associated with photosensitivity, subacute cutaneous lupus, neonatal lupus, and Sjögren syndrome.
- SS-B/La: Antibodies to this antigen are detected in only 10-15% of patients with lupus. They are strongly associated with Sjögren syndrome, as well as neonatal lupus.
- Anticentromere antibodies: These antibodies can be detected by IFA or ELISA assay. When an IFA assay is used, the substrate must include proliferating cells so that centromere antigen is expressed. These antibodies are seen in 30% of scleroderma patients and are associated with the limited form of the disease (CREST).
- Antitopoisomerase antibodies: Also called anti-Scl 70 antibodies, these are seen in about 30% of scleroderma patients and are associated with diffuse disease.
- Anti-Jo-1 antibodies: This antibody, to histidine tRNA synthetase, is seen in about 25% of patients with myositis and is associated with interstitial lung disease.
- Antineutrophil cytoplasmic antibodies (ANCA): These antibodies, which are directed against cytoplasmic proteins, are helpful in the diagnosis of some forms of vasculitis.
- Method of detection. These antibodies were first detected via an immunofluorescence assay (IFA) that used alcohol-fixed neutrophils as the cell substrate. Two patterns of IFA were observed: a speckled cytoplasmic pattern, called c-ANCA, and a perinuclear pattern called p-ANCA. In patients with vasculitis, these patterns were shown to reflect antibodies to either proteinase-3 (PR-3: c-ANCA) or myeloperoxidase (MPO: p-ANCA). Many laboratories now perform ELISA assays using these proteins as antigens.
- Interpretation. C-ANCA is highly specific for Wegener’s granulomatosis (present in approximately 80% of cases), whereas p-ANCA is seen primarily in microscopic polyarteritis nodosa, Churg-Strauss vasculitis, and crescentic glomerulonephritis. A positive p-ANCA by IFA can also be seen in other diseases, such as inflammatory bowel disease and tuberculosis. In these cases the positive immunofluorescence is due to antibodies to cytoplasmic antigens other than MPO or PR-3, and ANCA testing by ELISA will be negative. Therapeutic decisions and definitions of disease activity should be based upon the clinical presentation and not solely the ANCA titers.
The complement system is a major effector of the humoral immune system. Activation of the system by immune complexes, polysaccharides or oligosaccharides can occur through three pathways; the classical pathway, the alternative pathway and the lectin pathway. All three pathways eventually cleave C3 with subsequent activation of the terminal components (C5b through C9). The complement system has three main physiologic functions: defending against bacterial infection, bridging innate and adaptive immunity, and disposal or clearance of cell debris and antigen/antibody complexes (Fig. 4-1).
Figure 4-1. Complement pathways. The three pathways of complement activation: the MB-lectin pathway, which is triggered by mannan-binding lectin (MBL), a normal serum constituent that binds some encapsulated bacteria; the classical pathway, which is triggered by antibody (Ab), by binding of CRP to C1q or by direct binding of complement component C1q to the pathogen surface; and the alternative pathway, which is triggered directly on pathogen surfaces. All of these pathways generate the effector molecules of complement. The three main consequences of complement activation are opsonization of pathogens mediated by C3bi, the recruitment of inflammatory cells mediated by C3a, and direct killing of pathogens through the membrane attack complex (MAC).
- Method of detection: Measurement of complement or its components can be performed by functional (i.e. hemolytic) assays or by antigenic assays which measure individual components.
- Total hemolytic complement (CH50), measured in hemolytic units, assays the ability of a test serum to lyse 50% of a standardized suspension of sheep RBCs coated with rabbit antibody. All nine components of the classical pathway (i.e. C1 through C9) are required for a normal result.
- C3, C4, properdin, and factor B are measured by immunoassays including nephelometry, and ELISA techniques.
- Interpretation: Evaluation of C3, C4 and CH50 is useful in lupus patients, some of whom will have low values when their disease process is active, and normal values when the disease is quiescent. CH50 measurements are also low in mixed cryoglobulinemia, and in urticarial (“hypocomplementemic”) vasculitis. In contrast, most patients with other systemic autoimmune disorders do not have low complement levels.
- Complement deficiency states. Deficiencies of early classic pathway components, C1, C4 and C2 are associated with SLE and SLE-like syndromes. Lupus patients with genetic complement deficiencies will never “normalize” that complement component level, despite quiescent disease. Deficiencies of terminal components C5 through C9 are associated with an increased incidence of infection, particularly with Neisseria. Deficiency of the inhibitor of C1 esterase is associated with hereditary angioedema, and deficiency of C3b inactivator is associated with increased incidence of infection.
Cryoglobulins are immunoglobulins present in the serum that precipitate in the cold and dissolve again on rewarming.
- Types: Three different types of cryoblobulins have been defined.
- Type I: Monoclonal Immunoglobulin: This type of cryoglobulin is typically seen in myeloproliferative disorders such as multiple myeloma and Waldenstrom’s macroglobulinemia.
- Type II: Mixed cryoglobulin (immune complex of a polyclonal immunoglobulin and a monoclonal rheumatoid factor). Type II cryoglobulins are associated with Hepatitis B or C in most cases.
- Type III: Mixed polyclonal cryoglobulin (immune complex of a polyclonal immunoglobulin and a polyclonal rheumatoid factor). The cryoglobulin most often associated with autoimmune diseases such as lupus and Sjögren syndrome, and also with hematologic malignancies, Hepatitis C as well as other infections.
- Method of detection:
- Blood to be studied for cryoproteins should be processed under carefully controlled conditions because if the sample is incorrectly processed, the result may be falsely negative. Blood being tested for cryoglobulins must be allowed to clot at 37ºC, so that the cryoproteins remain soluble in the serum. (The cryoprotein will precipitate and become trapped in the clot if coagulation is allowed to occur at room temperature). Subsequently, the serum can be separated by a brief centrifugation at room temperature.
- After storage for 2 days at 0º to 5ºC, an aliquot is examined for the presence of a white precipitate in the bottom of the tube. The amount of cryoblobulins is quantified by performing a “cryocrit”. This is performed by centrifuging the serum in a graduated tube at 4oC and 2500 rpm, and then calculating the percentage of cryoglobulins. Note that there is not a good correlation between cryocrit and disease activity or severity
- Interpretation: Low levels of cryoglobulin (<30 mg/mL) can be found in a wide variety of inflammatory conditions (such as lupus) where they are of questionable significance. Cryoglobulins can also be pathogenic, such as in mixed essential cryoglobulinemia. In this vasculitic syndrome, cryocrits are usually in the 0.5-1.5% range. Sera with very large quantities of cryoprotein (cryocrits 3-5%) usually contain monoclonal immunoglobulins, and can be associated with Waldenstrom's macroglobulinemia or multiple myeloma.