The manifestations of SLE expressed in laboratory findings are numerous. Histologic, hematologic, and serologic abnormalities reflect the multisystem nature of this disease.

Histologic Changes

 The earliest pathologic abnormalities are those of acute vasculitis. Supportive tissue becomes edematous, initially infiltrated with neutrophils and later with plasma cells and lymphocytes. Persistent inflammation results in local deposition of a cellular homogeneous material, histologically similar to fibrin. Nuclear debris from resulting cellular necrosis reacts with ANAs (see later in this section) to form hematoxylin bodies. The presence of immunoglobulins in vascular lesions, predominantly IgM and IgG, can be demonstrated by indirect immunofluorescence.

Renal pathology can also be observed in SLE. The two basic renal abnormalities that manifest are as follows: (1) proliferative glomerulonephritis, which resembles the renal changes in immune complex nephritis; and (2) membranous nephritis.

Hematologic and Hemostatic Findings

 In SLE, a moderate anemia (normocytic normochromic anemia) representing chronic disease is a consistent factor. Some patients display coating of erythrocytes, which can be demonstrated by a positive AHG test, but actual hemolysis is infrequent. Lymphocytopenia is common and often reflects disease activity. Thrombocytopenia (50 to 100 × 10⁹/L) may also be seen.

Hemostatic Testing

Lupus anticoagulants, antiphospholipid antibodies, are often seen in association with SLE. Antiphospholipid antibodies develop in up to 20% of patients with SLE. These form a group of antibodies detected by tests for lupus anticoagulant and anticardiolipin antibodies.

Circulating anticoagulants are believed to be associated with the presence of false-positive serologic test results for syphilis. Because of the presence of lupus anticoagulant, patients with SLE frequently demonstrate prolonged pro thrombin time (PT) and partial thromboplastin time (PTT) results, but lupus anticoagulant rarely causes hemostatic problems. Inhibitors are not necessarily associated with bleeding unless some other defect is present. Because lupus anticoagulant is an inhibitor or prothrombin activator, it is often associated with excessive thrombosis rather than with bleeding. Patients with SLE have a high incidence of thrombotic episodes. Although less common, specific coagulation factor antibodies directed against coagulation factors VIII, IX, XI, and XII have been described. Thrombocytopenia can also occur because of the removal of antiphospholipid antibody coated platelets.

Serologic Findings

 Serologic testing frequently reveals high levels of anti-DNA antibodies, reduced complement levels, and the presence of complement breakdown products of C3 (C3d and C3c). In addition, cryoglobulins, which in some cases represent immune complexes, are frequently present in the serum of patients with SLE. Because monoclonal gammopathies have occasionally been described, a marked increase in gamma globulins may result in a hyperviscosity syndrome or renal tubular acidosis. Serum cryoglobulins of a mixed IgG-IgM type are found in patients with hypocomplementemia. The level of cryoglobulin correlates well with the severity of SLE. The following procedural results are helpful in assessing renal disease:

 • Antibody to double-stranded DNA

• Levels of C3 and C4 (with C4 probably being the most sensitive result)

• Cryoglobulin levels

A general correlation exists between abnormal results in each of these procedures and disease activity in many patients, but considerable disagreement surrounds the usefulness of these measurements in predicting renal disease activity. The best laboratory procedures for monitoring the activity of renal disease are the serum creatinine level, urinary protein excretion, and careful examination of urine sediment.

Complement

Inherited deficiencies of several complement components are associated with lupus-like illnesses. Some but not all deficiencies are coded for by autosomal recessive genes of the sixth chromosome, which are in linkage disequilibrium with human leukocyte antigen (HLA)–DRw2. The association of complement deficiencies with SLE may represent the fortuitous association of linked HLA-D region genes, rather than some unusual susceptibility induced by the complement deficiency.

Serum levels of complement typically are reduced, particularly during states of active disease. Deficiencies involving classic and alternative pathway complement components in SLE patients have resulted from consumption of components at the tissue sites of immune complex deposition, impaired synthesis, or both. A depressed level of complement is not specific for the diagnosis of SLE but is a helpful guide in treating patients. Levels of complement (C3, C4) are generally reduced in relationship to disease activity and fluctuation in these levels is often used to monitor disease activity. Patients with decreased levels are at risk for renal and CNS involvement. Deficiencies of C1, C3, and C4 are associated with SLE and other rheumatic diseases.

Antibodies

 Nonspecific elevation in immunoglobulin levels, particularly IgM and IgG, frequently occurs in SLE. An actual deficiency of IgA appears to be more common in SLE than in normal individuals.

The ANA procedure (discussed in detail in the next section) is a valuable screening tool for SLE; it has almost replaced the LE cell test because of its wider range of reactivity with nuclear antigens, as well as its greater sensitivity and quality control characteristics.

Antinuclear Antibodies

 Characteristics and Implications. ANAs are a heterogeneous group of circulating immunoglobulins that include IgM, IgG, and IgA. These immunoglobulins react with the whole nucleus or nuclear components (e.g., proteins, DNA, histones) in host tissues; therefore, they are true autoantibodies. Generally, ANAs have no organ or species specificity and are capable of cross-reacting with nuclear material from human beings (e.g., human leukocytes) or various animal tissues (e.g., rat liver, mouse kidney). ANAs are found in other diseases (e.g., rheumatoid arthritis), are associated with certain drugs, and are found in older adults without disease (Table 1). Thus, assays for ANAs are not specific for SLE. ANAs are present in more than 95% of SLE patients. Because the detection of ANAs is not diagnostic of only SLE, their presence cannot confirm the disease, but the absence of ANAs can be used to help rule out SLE. The significance of the presence of ANAs in a patient’s serum must be considered in relation to the patient’s age, gender, clinical signs and symptoms, and other laboratory findings.

Table1. Antibodies in Systemic Rheumatic Diseases

Systematic Classification. ANAs can be divided into four groups to provide a systematic classification: antibodies to DNA, antibodies to histone, antibodies to nonhistone proteins, and antibodies to nucleolar antigens. Antibodies to DNA. Antibodies to DNA can be divided into two major groups: (1) antibodies that react with native (double stranded) DNA; and (2) antibodies that recognize denatured (single-stranded) DNA only.

Antibodies that react with native DNA appear to interact with antigenic determinants present on the deoxyribose phosphate backbone of the beta helix of DNA. These autoantibodies characteristically stain the kinetoplast of the hemoflagellate Crithidia luciliae, a substrate used to detect anti–native DNA antibodies by indirect immunofluorescence. This procedure continues to be the gold standard for testing. Antibodies reactive with denatured DNA probably react with the purine and pyrimidine bases of DNA. These bases are readily accessible on ssDNA; they are buried within the beta helix of dsDNA and are therefore inaccessible. Anti–denatured DNA antibodies are unable to cross-react with native DNA. Conformational changes in the deoxyribose phosphate backbone of denatured DNA appear to be important for antigenicity.

Antibodies to Histones. Antibodies to histones have been shown to react with all major classes of histones—H1, H2A, H2B, H3, and H4. Antihistone antibodies can be induced by drugs such as procainamide and hydralazine. Procainamide induced lupus erythematosus is characterized by IgG antibodies against the histone complex H2A-H2B in symptomatic patients with SLE. In asymptomatic patients, the antibody may be restricted to the IgM class. Antibodies specific to other nuclear antigens are usually absent in drug-induced lupus in contrast to patients with SLE, who have ANAs of multiple specificity.

Patients with SLE are characterized by the presence of antibodies to multiple antigens, including Sm, RNP, dsDNA, chromatin, and SS-A/Ro (Table 2). There are 11 criteria for the diagnosis of SLE and, for a definitive diagnosis, patients must meet at least four of these criteria. Two of the criteria are a positive ANA and the detection of antibodies to Sm, dsDNA, or cardiolipin. Antibodies to Sm are detected in 20% to 30% of SLE patients and antibodies to dsDNA may occur in up to 60% of patients. Antibodies to Sm and RNP typically occur together because they react with different proteins that are associated in an RNP particle called a spliceosome. A positive Sm indicates a high probability of SLE.

Table2. Immunologic Assays for Detection and Monitoring of SLE

The presence of antibodies to dsDNA is one of the criteria for the diagnosis of SLE, and these antibodies are associated with active disease. The presence of dsDNA is a major concern in patients with SLE. The formation and deposition of immune complexes can affect various organ systems. Antibodies to dsDNA have also been reported in rheumatoid arthritis patients being treated with the tumor necrosis factor-α (TNF α) inhibitors. Patients with SLE have antibodies to chromatin more often than antibodies to dsDNA. These chromatin anti bodies are also associated with glomerulonephritis and have been identified, along with dsDNA antibodies, in immune complexes eluted from patients’ kidneys. Patients with drug induced lupus develop antibodies to chromatin and, in some cases, to the histone component of chromatin, but not to dsDNA.

The demonstration of only antihistone antibodies may be useful in distinguishing drug-induced lupus from SLE.

Antibodies to Nonhistone Proteins. Another primary class of ANAs in systemic autoimmune disorders is characterized by reactivity with soluble nonhistone nuclear protein and RNA-protein complexes. Clinically important antibodies that react with nuclear nonhistone proteins are listed in Table 3.

Table3. Antibodies to Nonhistone Proteins (NhPs) and NhP-RNA Complexes in Systemic Rheumatic Diseases

Antibodies to Nucleolar Antigens. The antibodies to nucleolar antigens are as follows:

• U3-RNA-protein complex (enzyme-transcribing ribosomal genes in the nucleolus)

 • 7-2-RNP

• RNA polymerase I

• PM-Scl

These antinucleolar antibodies are primarily associated with polymyositis-scleroderma overlap, where they have the highest incidence and titers. However, they are rarely demonstrated in PSS, dermatomyositis, or scleroderma.

Demonstrable antibodies include antibodies to nuclear com ponents, cell surface and cytoplasmic antigens of polymorphonuclear and lymphocytic leukocytes, erythrocytes, platelets, and neuronal cells, and IgG. The detection of ANAs is a valuable screening tool for SLE.

Immunofluorescence is extremely sensitive and may show positive results in patients in whom procedures for ANAs (e.g., complement fixation or precipitation) give negative results. At present, immunofluorescence is the most widely used technique for ANA screening. Serologic testing frequently reveals high levels of anti-DNA antibodies, reduced complement levels, and the presence of complement breakdown products of C3 (C3d, C3c).

In addition, cryoglobulins, which may represent immune complexes, are frequently present in the serum of patients with SLE. T he level of cryoglobulins correlates well with the severity of SLE. Assays helpful in assessing renal disease associated with SLE are antibodies to dsDNA, levels of C3 andC4, and cryoglobulins.

Indirect Immunofluorescent Tests for Antinuclear Antibody

 Indirect immunofluorescent tests for ANA are based on the use of fluorescein-conjugated antiglobulin. These methods are extremely sensitive. In one assay, the serum specimen is delivered into a well on a microscope slide that contains a mouse liver substrate. Substrates of rat or mouse liver or kidney, or cell-cultured fibroblasts, can also be used as the antigen and are fixed to the slides. If antibody is present in the serum of the patient, the unlabeled antibody will attach to the nuclei of the cells in the substrate. After the substrate is washed in buffer, the slide is incubated with fluorescein-labeled goat AHG. If the patient’s antibodies have attached themselves to the nuclear antigens in the substrate, the fluorescein-tagged goat AHG will attach to these antibodies. Fluorescence will be seen microscopically using UV light. The slides should be examined as soon as possible. If immediate examination is not possible, the slides can be stored in the dark at 4° C (39° F) for up to 48 hours before being read.

Several different patterns of fluorescence reactivity are seen (Figure 1), depending on whether the ANAs have reacted with the whole nucleus or with nuclear components, such as the nuclear proteins, DNA, or histone (a simple protein). This difference in nuclear fluorescence pattern reflects specificity for various diseases. Patterns are described as being diffused or homogeneous, peripheral, speckled, or nucleolar fluorescence. Nuclear rim (peripheral) patterns correlate with antibody to native DNA and DNP and bear a correlation with SLE, SLE activity, and lupus nephritis. Homogeneous (diffused) patterns suggest SLE or another connective tissue disorder. Speckled patterns are found in many diseases, including SLE. Nucleolar patterns are seen in patients with PSS and Sjögren’s syndrome.

Fig1. Illustration of Antinucleoprotein antibody patterns.

After ensuring that the results for positive and negative control specimens are providing the expected reactions, the results for the patient are reported. Results from the screening tests are reported as positive or negative. The normal person is expected to have a negative reaction—no green or gold fluorescence is observed. The degree of positive fluorescence may be semiquantitated on a scale of 1+ to 4+. Positive samples give a green-gold fluorescence of a characteristic pattern (homogeneous, peripheral, speckled, or nucleolar).

Indirect Immunofluorescent Technique

 The detection of autoantibodies by immunofluorescence has become an extremely valuable tool. This method is extremely sensitive and may be positive in cases in which procedures for ANAs, such as complement fixation or precipitation, are negative. At present, the indirect immunofluorescent method on a Hep-2 cell substrate is the primary screening test for the diagnosis of systemic rheumatic diseases (SRDs). A negative indirect immunofluorescence result almost rules out a diagnosis of SLE, but the patterns observed on Hep-2 slides can provide a key to the diagnosis of other SRDs.

Principles. The antigen in the substrate tissue is fixed to a slide for testing. ANA is not specific for a particular organ; therefore, any tissue containing nuclei may be used as substrate. The tissues most often used are rat or mouse liver or kidney, or cell cultured fibroblasts grown on slides. If antibody is present in a patient’s serum, the unlabeled antibody will attach to the nuclei in the substrate. After the substrate is washed in buffer, it is incubated with fluorescein-tagged goat AHG. If the patient’s antibodies have affixed themselves to the nuclear antigens of the substrate, the fluorescein-tagged goat AHG will attach to these antibodies. When the slide is examined microscopically, fluorescence will be visible on UV light.

 Interpretation of Staining Patterns of Major Rheumatic Autoantibodies

 • Double-stranded DNA (dsDNA)

• Chromatin, Sm

• RNP, SS-A/Ro

 • SS-B/La

 • Scl-70

 • Centromere

 • Jo-1, cyclic citrullinated peptide (CCP)

 Because ANAs react with the whole nucleus or with nuclear components (e.g., proteins, DNA, histone), reaction patterns reflect the distribution of the various antigens in the nuclei. Major ANAs are detected on all Hep-2 slides, but detection of antibodies to SS-A/Ro varies according to the fixation method. Alcohol diminishes or destroys the SS-A/Ro speckled ANA pattern, leading to a negative ANA. It is always important to include a control for antibodies to SS-A/Ro. Several patterns of reactivity can be observed when a slide is examined in the ANA procedure (Table 1).

Table1. Antinuclear Antibody Patterns and Disorders

Diffused or Homogeneous Pattern. The diffused or homogeneous pattern characterizes anti–DNA nucleoprotein antibodies (i.e., antibodies to nDNA, dsDNA, ssDNA, DNP, or histones). Antibodies to DNP have been shown to have the same specificity as the LE factor. Although vacuoles may be seen, the whole nucleus fluoresces evenly. This pattern is typically seen in rheumatoid disorders. High titers of homogeneous ANA suggest SLE, whereas low titers may be found in SLE, rheumatoid arthritis (RA), Sjögren’s syndrome, and mixed connective tissue disease (MCTD).

Peripheral Pattern. The peripheral (marginal or rim) pattern results from antibodies to DNA—nDNA, dsDNA, or DNP. The central protein of the nucleus is only lightly stained or not stained at all, but the nuclear margins fluoresce strongly and appear to extend into the cytoplasm. This pattern is associated with SLE in the active stage of the disease and in Sjögren’s syndrome.

Speckled Pattern. The speckled pattern occurs in the presence of antibody to any extractable nuclear antigen devoid of DNA or histone. The antibody is detected against the saline extractable nuclear antigens, anti-RNP and anti-Sm. A grainy pattern with numerous round dots of nuclear fluorescence, without staining of the nucleoli, is seen in this pattern type.

Antibodies to Sm antigen have been shown to be highly specific for patients with SLE and appear to be marker antibodies. Anti-RNP has been found in patients with a wide variety of rheumatic diseases, including SLE, RA, Sjögren’s syndrome, PSS, MCTD, and dermatomyositis.

Nucleolar Pattern. The nucleolar pattern reflects an antibody to nucleolar RNA (4-6S RNP). A few round smooth nucleoli that vary in size will fluoresce when examined under UV light. The nucleolar pattern is present in about 50% of patients with scleroderma (PSS), Sjögren’s syndrome, and SLE. This pattern can also be observed in undiagnosed illnesses manifesting Raynaud’s phenomenon.

Centromere. The anticentromere antibody reacts with centromeric chromatin of metaphase and interphase cells. The particular pattern on tissue culture cells is discrete and speckled. T his antibody appears to be highly selective for the CREST variant of PSS. The CREST syndrome (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia) is a variant of systemic sclerosis characterized by the presence of calcinosis, Raynaud’s phenomenon, esophageal motility abnormalities, sclerodactyly, and telangiectasia. This antibody is found infrequently in the serum of patients with SLE, MCTD, and PSS.

Rapid Slide Test for Antinucleoprotein

The SLE latex test provides a suspension of polystyrene latex particles coated with DNP. When the latex reagent is mixed with serum containing the ANAs, binding to the DNP-coated latex particles produces macroscopic agglutination. The procedure is positive in SLE and SRDs (e.g., RA, scleroderma, Sjögren’s syndrome).

Autoimmune Enzyme Immunoassay

The autoimmune enzyme immunoassay (EIA) provides a qualitative screening test for the presence of ANAs. In one well, the assay collectively detects total ANAs against dsDNA (nDNA) histones, SS-A/Ro, SS-B/La, Sm, Sm/RNP, Scl-70, Jo-1, and centromeric antigens, along with sera positive for immunofluorescent assay (IFA) Hep-2 ANAs. This assay serves as an alternative to the IFA for screening a patient’s serum for ANAs.

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Immunologic Manifestations of Systemic Lupus Erythematosus

Signs and Symptoms of Systemic Lupus Erythematosus

Different Forms of Lupus

Renal Disorders

Neuromuscular Disorders

Autoimmune Hematologic Disorders