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Chapter 2 Rheumatologic Laboratory Tests

Manual of Rheumatology and Outpatient Orthopedic Disorders

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Chapter 2 Rheumatologic Laboratory Tests

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Keith B. Elkon

Acute-phase reactants
Rheumatoid factor (RF)
Antinuclear antibodies (ANAs)
Complement
Other serologic tests

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. This chapter discusses erythrocyte sedimentation rate, C-reactive protein, auto-antibodies, complement, and other tests helpful in the serologic evaluation of rheumatic diseases. Synovial fluid analysis is discussed in Chapter 5, and uric acid metabolism in Chapter 37.

I. Acute-phase reactants

1. Erythrocyte sedimentation rate (ESR). The rate of fall in millimeters per hour of red blood cells (RBCs) in a standard tube (Westergren method) is a time-honored measurement of inflammation . Methods other than Westergren have been found to be less reliable. RBCs in inflammatory disorders tend to form stacks ( rouleaux ) that partly result from increased levels of fibrinogen and thus form sediment more rapidly . Falsely low ESRs are found in sickle cell disease, anisocytosis, spherocytosis, polycythemia, and heart failure. Prolonged storage of blood to be tested or tilting of the calibrated tube will 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. A normal ESR value tends to exclude active inflammatory disorders such as acute rheumatic fever , systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and temporal arteritis-polymyalgia rheumatica (TA-PMR). The ESR is of some use in following the course (including therapeutic responses) of chronic inflammatory disorders.

2. C-reactive protein (CRP) is an acute-phase reactant serum protein that is present in low concentration in normal serum and was originally identified by its precipitin reaction with pneumococcal C polysaccharide. It is now commonly measured by a latex agglutination test or rocket electrophoresis. CRP levels rise rapidly under an inflammatory stimulus and then fall when inflammation subsides. In SLE and scleroderma, CRP levels are inappropriately low, unless infection is present. CRP testing may be performed on freeze-stored serum, which is its major advantage in comparison with ESR testing.

II. Rheumatoid factor (RF) is primarily associated with RA but is also found in other disorders (Table 2-1). RFs are immunoglobulins with specificity for the Fc portion of immunoglobulin G (IgG). Multiple immunoglobulin classes have RF activity, but conventional serologic systems (agglutination) detect primarily polymeric (IgM and IgA) RF.

Table 2-1. Frequency of rheumatoid factor as measured by latex agglutination in rheumatic and nonrheumatic diseases

1. Method. Agglutination of IgG-coated latex particles is the method used in most laboratories for measuring polymeric RF. RF activity is measured by using serial dilutions of the test sera. There are many other serologic systems for detection of RF, including bentonite flocculation, hemagglutination, nephelometry, and radioimmunoassay.
2. Interpretation. About 75% of RA patients have IgM RF, and patients with extraarticular disease are invariably RF-positive. Because IgM RFs are not specific for RA and only 75% of patients with RA have IgM RFs, the test is helpful only when combined with clinical information (see Table 2-1). High-titer RF activity is also found in the sicca (primary Sjgren') syndrome and mixed cryoglobulinemia, whereas in the other diseases listed, RF titers are usually low.

III. Antinuclear antibodies (ANAs). A wide array of antibodies to nuclear and cytoplasmic cellular antigens is found in lupus and other rheumatic diseases by immunofluorescence, immunoassay, and immunodiffusion. Different profiles of ANAs in rheumatic diseases have been described and have been correlated with clinical features. The LE cell test is obsolete as a result of its poor sensitivity compared with ANA assay. (Some aspects of the clinical application of ANA data are discussed in Chapter 30.)

1. Indirect immunofluorescence for ANA testing
   1. Immunofluorescence technique employs a cellular substrate, traditionally a thin section of frozen rat liver or kidney, placed on a glass slide. A cytocentrifuge preparation of white blood cells or cells from tissue culture can also be used. Sensitivity and pattern discrimination are optimal on tissue culture cell substrates (e.g., Hep-2). Test sera diluted to titers of 1:20 or greater are added to the cells, incubated, and then washed off. Fluorescein-labeled antibodies reactive with all immunoglobulins or specific for human IgG, IgM, or IgA are then layered over the sections, incubated, and washed off. Immunofluorescence microscopy detects the presence of antibodies in the test sera bound to cell membrane and intracellular components . Through indirect immunofluorescence, a variety of patterns representing antibodies to different cellular antigens can be detected . Percentages of ANA positivity and patterns of immunofluorescence observed are shown in Table 2-2.
      
      

      
      

      Table 2-2. Patterns of immunofluorescence (ANA) ^a

      
      
      
   2. ANA studies are usually reported by pattern, intensity of fluorescence (1 to 4+), or titer. Values of 2 to 4+ or titers greater than 1:40 are usually considered significant. Once positive ANA activity has been documented in a patient's serum, there is seldom need to repeat the test unless major changes in the patient's therapy or condition have taken place. Steroid or immunosuppressive therapy may change the ANA titer, but most physicians do not rely on serial changes in ANA to monitor disease activity. The usefulness of an ANA study is not its specificity but rather its sensitivity and technical simplicity. The ANA study should therefore be performed as an initial screening test for auto-antibodies. Greater diagnostic specificity is obtained by determining the reactivity of ANA-positive sera with nuclear constituents such as deoxyribonucleic acid (DNA), Smith antigen (Sm), or nuclear ribonuclear protein (nRNP) (see section III. A. 3 ; B ; C ).
   3. A special case of ANA are auto-antibodies that bind relatively selectively to cytoplasmic proteins within neutrophils (antineutrophil cytoplasmic antibody, or ANCA). The screening test for these auto-antibodies is similar to that described above except that human neutrophils are used as the cell substrate. Two patterns of IIF are obtained: speckled cytoplasmic, called c-ANCA, which is highly specific for Wegener's granulomatosis (present in approximately 80% of cases), and perinuclear (p-ANCA), which is present in polyarteritis nodosa and crescentic-glomerulonephritis.
2. Anti-DNA antibodies
   1. Antibodies to DNA are measured by the Farr method (percent DNA binding). Equal volumes of complement-inactivated test serum and Escherichia coli double-stranded DNA (ds-DNA) radiolabeled with carbon 14 are incubated at 37C for 1 hour and refrigerated overnight. Saturated ammonium sulfate is then added to the mixture; this step precipitates out all antibodies and any bound DNA. The mixture is then centrifuged, and the top half is counted as supernatant (S). The remaining precipitate is redissolved in saline solution and counted as precipitate (P). Percent DNA binding is calculated by the following formula:

      Percent DNA Binding = [(P S)/(P + S)] — 100

   2. Other methods for measuring ds-DNA antibodies are outlined and reviewed by Chubick ( Ann Intern Med 1978;89:186). The most frequently used are millipore binding, Crithidia kinetoplast immunofluorescence, and solid-phase immunoassay. Because even highly purified ds-DNA may have single-stranded (ss) regions or nicks exposing single-stranded antigenic determinants , the conventional Farr assay probably measures a small amount of binding to ss-DNA. A positive Farr assay (>30%) is generally found only in SLE or mixed connective tissue disease (MCTD). The level of antids-DNA antibodies is also of some value in assessing activity of SLE. Antibodies to ss-DNA are found in SLE and a variety of other autoimmune conditions, including drug-induced SLE and liver diseases.
3. Antibodies to other intracellular constituents. The heterogeneity of anti-nuclear antibodies, evident from varied patterns of ANAs, has been verified by immunodiffusion and counterimmunoelectrophoresis studies in which soluble components derived from cells as extractable antigens (ENAs) are used. In addition to nucleic acid antigens, several protein antigens from both the nuclei and cytoplasm have been identified. Table 2-3 summarizes the current knowledge regarding the cellular location of the antigens, immunofluorescence patterns, methods of detection, and primary disease associations. In many cases, the protein antigens have been molecularly cloned and have been expressed as fusion proteins. Solid-phase immunoassays utilizing these recombinant antigens are replacing many of the older assays for detection of specific protein antigens. Several of the antinuclear specificities are associated with speckled ANA pattern [Sm, nRNP, Ro (SS-A), La (SS-B), RANA]. Most of them are observed with multiple nuclear substrates, but at least one antigen, rheumatoid arthritis nuclear antigen (RANA), is present only in human B-lymphocyte lines. The two antinuclear specificities most characteristic of SLE are antids-DNA and anti-Sm. High titers of anti-nRNP are usually associated with a mixed or undifferentiated pattern of MCTD; however, none of these reactivities can be considered diagnostic and must be interpreted in the context of the clinical presentation.
   
   

   
   

   Table 2-3. Antibodies to nuclear and cytoplasmic antigens

   
   
   

IV. Complement. The complement system, composed of at least 18 different plasma proteins, is a major effector of the humoral immune system. Activation of the system by immune complexes or polysaccharides can occur through either of two pathways, the classic or the alternative. Both pathways eventually cleave C3 with subsequent activation of the terminal components (C5b through C9), leading to lysis of target cells and the generation of multiple mediators of inflammation and anaphylaxis (Fig. 2-1). Serial measurements of complement levels may be helpful in assessing disease activity in SLE patients (see Chapter 30).

FIG. 2-1. Complement pathways

1. Measurement of complement or its components can be performed by functional (e.g., hemolytic) assays or by concentration with the use of antisera specific for individual components.
   1. Total hemolytic complement (CH50), measured in hemolytic units, assays the ability of the test serum to lyse 50% of a standardized suspension of sheep RBCs coated with rabbit antibody.
   2. By using stable intermediate complexes of complement components or sera known to be deficient in various components, functional assays of individual components have been developed.
   3. C3, C4, properdin, and factor B are usually measured by radial immunodiffusion.
2. Complement deficiency states. Deficiencies of early classic pathway components C1 through C4 (of which C2 and C4 are the most common) are associated with SLE-like syndromes and an increased incidence of infection. Deficiencies of terminal components C5 through C9 have also been associated with rheumatic syndromes and in 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.
3. Associations and interpretation. CH50 measurements are often low in SLE and cryoglobulinemia as a result of decreased production or consumption by circulating or fixed immune complexes. Because numerous complement components are heat-labile, all test sera must be kept frozen at 20C and assayed within 2 weeks or, preferably, frozen at 70C and assayed within 2 years . Table 2-4 summarizes the serum and synovial fluid CH50 determinations in various rheumatic diseases.
   
   

   
   

   Table 2-4. Complement activity (CH50) in serum and synovial fluid

   
   
   

V. Other serologic tests

1. Lupus anticoagulant. Lupus antibodies that inhibit the activation of thrombin can be identified by a prolonged partial thromboplastin time and a normal or slightly prolonged thromboplastin time in the absence of anticoagulant therapy. Antibodies in SLE sera that inactivate factors VII, XI, and XII have also been identified. Clinically significant bleeding is rare, whereas an association with vascular thrombosis is well documented.
2. False-positive serologic tests for syphilis and cardiolipin antibodies. False-positive serologic tests for syphilis are present in as many as 25% of SLE patients. The test is frequently positive in patients with the lupus anticoagulant; antibodies against phospholipid antigens are most likely responsible for both phenomena. Antibodies against cardiolipin are determined by immunoassays. High titers of IgG anticardiolipin antibodies in SLE are associated with thrombosis.
3. Hepatitis B (HB) and hepatitis C serologic tests (see Chapter 32). Syndromes associated with chronic HB surface (HBs) antigen include polyarteritis (approximately 30% of cases), essential mixed cryoglobulinemia (variable reported associations, up to 30%), and membranoproliferative glomerulonephritis. Patients with HBs-positive polyarteritis usually express HB early (HBe) antigen, have antibodies to HB core (HBc) antigen, and have immune complexes containing HBs reactants. Recently, an association between essential mixed cryoglobulinemia and hepatitis C virus infection has been reported.
4. Immune complex assays. Several procedures have been developed for the detection and quantification of immune complexes in sera and other biologic fluids. Principles employed include size and solubility of complexes (vs. monomeric immunoglobulins), reactivity of complexes with complement (or its components), and affinity of complexes for receptors on cell membranes. Among these procedures, the two that have been most extensively applied are the following:
   1. The Raji cell assay, which is based on the attachment of complexes to human lymphoblastoid cells through surface Fc and complement receptors.
   2. The C1q-binding assay (as well as related techniques that depend on the affinity of purified C1q for IgG aggregates).
5. Cryoglobulins. The simplest test for immune complexes, based on their diminished solubility in the cold, is cryoprecipitation of sera at 0 to 5C. Although it lacks sensitivity and is not readily quantified , cryoprotein screening is the basis for identification of the immune complex syndrome called mixed, or essential, cryoglobulinemia. Cryoglobulinemia is also a common feature of SLE. Blood to be studied for cryoproteins should not be processed by routine laboratory procedures; after clotting takes place in a 37C bath, serum should be separated by a brief centrifugation at room temperature and an aliquot examined for precipitation after 2 days at 0 to 5C. Turbidity caused by lipid can be distinguished by centrifugation in the cold (the lipid does not precipitate). Small amounts of cryoprotein (<30 g/mL) may be seen in a wide variety of pathologic sera and are of questionable significance. Sera with very large quantities of cryoprotein may contain monoclonal immunoglobulins; the sera can be subjected to cryocrit measurements in calibrated hematocrit tubes.

Bibliography

Carson DA, et al. Physiology and pathology of rheumatoid factors. Springer Seminar. Immunopathology 1981;4(2):161.

Christian CL, Elkon KB. Autoantibodies to intracellular proteins: clinical and biological implications. Am J Med 1986;80(1):53

Chubick A. An appraisal of tests for native DNA antibodies in connective tissue diseases. Ann Intern Med 1978;89(2):186

Cooper NR. The complement system. In: Fudenberg HH, ed. Basic and clinical immunology, 5th ed. Los Angeles: Lange, 1984

Harris EN, et al. Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet 1983;2(8361):1211

Pepys MB, Baltz ML. Acute phase proteins with special reference to C-reactive protein and related proteins (pentaxins) and serum amyloid A protein. Adv Immunol 1983;34:141

Plotz PH. Studies on immune complexes. Arthritis Rheum 1982;25(10):1151

Tan EM. Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv Immunol 1989;44:93.

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Copyright 2000 by Lippincott Williams & Wilkins
Stephen A. Paget, M.D., Allan Gibofsky, M.D., J.D. and John F. Beary, III, M.D.
Manual of Rheumatology and Outpatient Orthopedic Disorders

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