Systemic Lupus Erythematosus

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Systemic Lupus Erythematosus

Learning Objectives

At the conclusion of this chapter, the reader should be able to:

• Compare the different forms of lupus, citing manifestations, incidence, and other features.

• Name the two most common drugs that can cause drug-induced lupus.

• Explain the epidemiology and signs and symptoms of SLE.

• Describe the immunologic manifestations of SLE, including diagnostic evaluation.

• Discuss the laboratory evaluation of antinuclear antibodies.

• Analyze selected SLE case studies. Correctly answer case study related multiple choice questions

• Be prepared to participate in a discussion of critical thinking questions.

• Describe the principle, sources of error, limitation, and clinical application of the antinuclear antibody visible method

• Describe the principle and clinical applications of the rapid slide test for antinucleoprotein and autoimmune enzyme immunoassay ANA screening test

• Correctly answer end of chapter review questions.

Key Terms

Systemic lupus erythematosus (SLE) is the classic model of an autoimmune disease. SLE is a systemic rheumatic disorder and the term used most often for the group of disorders that includes SLE and other abnormalities involving multiple systems (e.g., joints, connective tissue, collagen vascular system) in the disease process. Table 29-1 lists the American College of Rheumatology criteria for the classification of SLE.

Table 29-1

1997 Update of the 1982 American College of Rheumatology Revised Criteria for Classification of Systemic Lupus Erythematosus

Criterion Definition
1. Malar rash Fixed erythema, flat or raised, over the malar eminences, tending to spare the nasolabial folds
2. Discoid rash Erythematous raised patches with adherent keratotic scaling and follicular plugging; atrophic scarring may occur in older lesions
3. Photosensitivity Skin rash as a result of unusual reaction to sunlight by patient history or physician observation
4. Oral ulcers Oral or nasopharyngeal ulceration, usually painless, observed by physician
5. Nonerosive arthritis Involving two or more peripheral joints, characterized by tenderness, swelling, or effusion
6. Pleuritis or pericarditis
7. Renal disorder
8. Neurologic disorder
9. Hematologic disorder
10. Immunologic disorder
11. Positive antinuclear antibody An abnormal titer of antinuclear antibody by immunofluorescence or an equivalent assay at any point in time and in the absence of drugs known to be associated with drug-induced lupus syndrome

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From Hochberg MC: Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus (letter), Arthritis Rheum 40:1725, 1997 and The American College of Rheumatology www.rheumatology.org 2012.

Different Forms of Lupus

There are several forms of lupus, including discoid, systemic, drug-induced, and neonatal lupus.

Discoid (cutaneous) lupus is always limited to the skin and is identified by biopsy of the rash that may appear on the face, neck, and scalp. Discoid lupus does not generally involve the body’s internal organs but can evolve into the systemic form of the disease, even if treated. Evolution to systemic lupus cannot be predicted or prevented. The antinuclear antibody (ANA) test may be negative or positive at a low titer. Discoid lupus accounts for approximately 10% of all cases of lupus.

Systemic lupus is usually more severe than discoid lupus and can affect the skin, joints, and almost any organ or body system, including the lungs, kidneys, heart, and brain. Systemic lupus may include periods in which few, if any, symptoms are evident (remission) and other times when the disease becomes more active (flare). Most often, when people mention “lupus,” they are referring to the systemic form of the disease. Approximately 70% of lupus cases are systemic. In about 50% of these cases, a major organ will be affected.

Drug-induced lupus occurs after the use of certain prescribed drugs (Box 29-1). The most frequently used drugs associated with drug-induced lupus are hydralazine hydrochloride and procainamide hydrochloride. Factors such as the rate of drug metabolism, the drug’s influence on immune regulation, and the host’s genetic composition are all believed to influence pathogenesis. Some drugs (e.g., oral contraceptives, isoniazid) induce serum antinuclear antibodies (ANAs) without symptoms. High antibody titers may exist for months without the development of clinical symptoms.

Box 29-1   Drugs That Can Produce Clinical and Serologic Features of Systemic Lupus Erythematosus

Procainamide-induced disease does not induce antibodies to double-stranded deoxyribonucleic acid (dsDNA). The ANAs in the drug-induced syndromes are histone-dependent and are never the only ANAs present in the blood. Even with discontinuation of the drug, antibody titers usually remain elevated for months or years.

Only about 4% of patients who take these drugs will develop the antibodies suggestive of lupus. Of those 4%, only an extremely small number will develop overt drug-induced lupus. The symptoms of drug-induced lupus are similar to those of systemic lupus, but milder. Patients with drug-related lupus have a predominance of pulmonary and polyserositic signs and symptoms. Patients with drug-induced lupus have no associated renal or central nervous system (CNS) disease. In addition, lupus-inducing drugs do not appear to exacerbate idiopathic SLE. The symptoms usually fade when the medications are discontinued.

Neonatal lupus is a rare condition acquired from the passage of maternal autoantibodies, specifically anti-Ro/SS-A or anti-La/SS-B, that can affect the skin, heart, and blood of the fetus and newborn. Neonatal lupus is associated with a rash that appears within the first several weeks of life and may persist for about 6 months before disappearing. Congenital heart block can occur but is much less common than a rash. Neonatal lupus is not systemic lupus.

Etiology

The cause of SLE is unknown (idiopathic). Although no single causative agent has been identified, a primary defect in the regulation of the immune system is considered important in the pathogenesis of the disorder. Genetic predisposition can be a factor. Hormones and environmental factors that may trigger the disease include infections, antibiotics (especially sulfonamides and penicillin derivatives), ultraviolet (UV) light, extreme stress, and certain drugs. A combination of these factors may be synergistic.

Antibodies directed against T lymphocytes, including the membrane molecules that mediate their responses, are regularly detected in patients with SLE. Their role in the pathogenesis of autoimmunity is still unclear.

Hormonal Influences

Hormonal factors may explain why lupus occurs more frequently in women than in men. Lupus is often called a woman’s disease because a disproportionate number of women between puberty and menopause suffer from SLE. The increase in disease symptoms may be caused by hormones, particularly estrogen. There is a risk that the disease will worsen during pregnancy and the immediate postpartum period. In addition, postmenopausal therapy is associated with an increased risk for developing SLE. The exact reason for the greater prevalence of lupus in women, and the cyclic increase in symptoms, is unknown.

A condition called the antiphospholipid syndrome can be secondary to lupus and may complicate pregnancy. Antibodies against specific autoantigens often present on coagulation factors can cause blood to clot faster than normal or, in some cases, not at all. Antiphospholipid antibodies can be found in many patients with lupus and pose a particular risk to pregnant lupus patients because their presence is often associated with miscarriages.

Both the developing fetus and the pregnant mother with lupus are at increased risk of various complications during and after pregnancy. Passive placental transfer of maternal antibodies can produce transient abnormalities such as hepatosplenomegaly, cytopenia, and a photosensitive rash in the newborn. These conditions do resolve themselves in the newborn after the antibody titer declines (see earlier discussion of neonatal lupus).

Epidemiology

Lupus can occur at any age and in either gender, although it occurs 10 to 15 times more frequently in women than in men after puberty. The Lupus Foundation of America estimates that approximately 1.4 million Americans have a form of lupus. The overall incidence of SLE is estimated to be 50 to 70 new cases/year/1 million population.

Racial groups such as blacks, Native Americans, Puerto Ricans, and Asians (particularly Chinese) demonstrate an increased frequency of SLE. Lupus is two to three times more prevalent among people of color. The incidence of SLE in black women between the 20 and 64 years old is 1 in 245. The reasons for ethnic differences are not clear.

The prevalence rate, based on a total population, is 1 in 2000, but it is 1 in 700 for women between 20 and 64 years old; 80% of those with systemic lupus develop it between ages 15 and 45 years.

Survival is estimated to be higher than 90% at 10 years after diagnosis. The highest mortality rate is in patients with progressive renal involvement or CNS disease. The two most frequent causes of death are renal failure and infectious complications.

Signs and Symptoms

SLE is a disease of acute and chronic inflammation. Symptoms of SLE often mimic other, less serious illnesses. Fever is one of the most common clinical manifestations of SLE. Disease activity accounts for more than 66% of febrile episodes in patients with SLE. Antibodies with elevated titers that are characteristic of lupus disease activity rather than infection include anti-dsDNA and anti–ribosomal P antibodies, as well as reduced levels of complement and leukopenia.

Many of the clinical manifestations of SLE are a consequence of tissue damage from vasculopathy mediated by immune complexes. Other conditions (e.g., thrombocytopenia, antiphospholipid syndrome) are the direct effects of antibodies to cell surface molecules or serum components.

Manifestations of the disease range from a typical mild illness limited to a photosensitive facial rash and transient diffuse arthritis to life-threatening involvement of the CNS or renal, cardiac, or respiratory system (Fig. 29-1). In the early phases, it is often difficult to distinguish SLE from other systemic rheumatic disorders, such as progressive systemic sclerosis (PSS), polymyositis, primary Sjögren’s syndrome, primary Raynaud’s phenomenon, and rheumatoid arthritis. Polyarthritis and dermatitis are the most common clinical manifestations.

image
Figure 29-1 Signs and symptoms of systemic lupus erythematosus (SLE).

The course of the disease is highly variable. It usually follows a chronic and irregular course, with periods of exacerbations and remissions. Clinical signs and symptoms can include fever, weight loss, malaise, arthralgia (joint pain) and arthritis (inflammation of the joints), and the characteristic erythematous, maculopapular (“butterfly”) rash over the bridge of the nose (Table 29-2). In addition, there is a tendency toward increased susceptibility to common and opportunistic infections. Multiple organ systems may be affected simultaneously.

Table 29-2

Systemic Lupus Erythematosus Symptoms

Symptom Percentage of Cases
Achy joints (arthralgia) 95
Frequent fevers >37.8° C (100° F) 90
Arthritis (swollen joints) 90
Prolonged or extreme fatigue 81
Skin rashes 74
Anemia 71
Kidney involvement 50
Pain in the chest on deep breathing (pleurisy) 45
Butterfly-shaped rash across the cheek and nose 42
Sun or light sensitivity (photosensitivity) 30
Hair loss 27
Abnormal blood clotting problems 20
Raynaud’s phenomenon (fingers turning white and/or blue in the cold) 17
Seizures 15
Mouth or nose ulcers 12

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Adapted from Lupus Foundation of America: General Lupus Fact Sheet, 2012 (http://www.lupus.org/webmodules/webarticlesnet/?z=8&a=351org).

The onset of lupus can be caused by sun exposure, resulting in sudden development of a rash and then possibly other symptoms. In some patients, an infection, even a cold, does not improve, and complications then arise. These complications may be the first signs of lupus. In some women, the first symptoms develop during pregnancy or soon after delivery.

Cutaneous Features

Approximately 20% to 25% of patients with SLE develop dermal disorders as the initial manifestation of the disease. As many as 65% of patients will develop a cutaneous abnormality during the course of the disease. The characteristic erythematous, maculopapular butterfly rash across the nose and upper cheeks is the cutaneous feature for which the disease is named—lupus erythematosus, the “red wolf” (Fig. 29-2). This rash may also be observed on the arms and trunk. Exposure to UV light will worsen erythematous, as well as other types of, cutaneous lesions.

image
Figure 29-2 Facial rash over bridge of nose in patient with active SLE. (From Behrman R, Kliegman R, Jenson HB: Nelson’s textbook of pediatrics, ed 17, Philadelphia, 2004, Saunders.)

The spectrum of cutaneous abnormalities includes urticaria, angioedema, nonthrombocytopenic purpura associated with the presence of cryoglobulins, scale formation, and ulcerations of oral and genital mucous membranes. Although neither the collection of immunoglobulins and complement at the dermal-epidermal junction nor the presence of specific antibody nuclear ribonucleoprotein (RNP), Sm, native DNA, and single-stranded DNA appears to play a direct role in the pathogenesis of cutaneous lupus lesion, Ro (SS-A) and perhaps La (SS-B) antibodies may be prominent factors.

Diffuse or patchy alopecia is also a common cutaneous manifestation. Hair loss is caused by pustular lesions of the scalp and is usually related to the stress of the disease process. Although the cause of pustular lesions is unknown, these inflammatory infiltrates are characterized by the presence of predominantly Ia-positive (activated) T lymphocytes with both CD4+ and CD8+ phenotypes.

Approximately 2% to 3% of SLE patients demonstrate lupus panniculitis. This condition is characterized by tender or nontender subcutaneous nodules that sometimes ulcerate and discharge a yellowish lipid material. In addition, various nonspecific skin changes are observable secondary to vascular insults. Raynaud’s phenomenon is demonstrated by approximately one third of patients with SLE and appears to be increased in those who have antibodies to nuclear RNP in their serum.

The presence of lesions does not distinguish between the limited cutaneous (discoid lupus erythematosus) and cutaneous manifestations of SLE. The term discoid lupus is used to differentiate the benign dermatitis of cutaneous lupus from the cutaneous involvement of SLE. In discoid lupus, the round lesion is an erythematous inflammatory dermatosis. These lesions are primarily located in light-exposed areas of the skin.

Renal Characteristics

Complement-mediated injury to the renal system is a usual consequence of the high levels of immune complexes in the blood that are deposited in tissues such as the kidneys. Renal disease progression is highly unpredictable. It may be acute, but more typically it progresses slowly. As the kidneys degenerate, the urinary sediment is typical of acute glomerulonephritis and later of chronic glomerulonephritis. Acute glomerulonephritis is characterized by the presence of erythrocytes, leukocytes, and granular and red blood cell (RBC) casts in urinary sediment. The presence of proteinuria may lead to nephrotic syndrome. If end-stage renal disease (renal failure) occurs, it can be managed by dialysis or allograft transplantation.

The systemic necrotizing vasculitis of SLE involves small blood vessels and leads to renal involvement. The most common method of classification of the renal involvement of SLE is the World Health Organization (WHO) system, which is based on histopathologic criteria. The stages of renal disease range from the earliest and least severe form, class II, characterized by mesangial deposits of immunoglobulin and C3, to class V, the most severe form of involvement.

Neuropsychiatric Features

In SLE, various neuropsychiatric manifestations develop secondary to involvement of the central and peripheral nervous systems. CNS involvement in SLE includes inflammation of the brain or intracranial blood vessels (vasculitis) and ischemic complications of vasculitis.

The most common abnormalities are disturbances of mental function, ranging from mild confusion, with memory deficiency and impairment of orientation and perception, to psychiatric disturbances such as hypomania, delirium, and schizophrenia. The most common manifestations are cognitive dysfunction, headache, seizures, and psychiatric conditions. Aseptic meningitis, stroke, encephalopathy, movement disorders, and myelopathy can be observed.

Seizures of the grand mal type may be the initial manifestation of SLE and may be present long before the multisystem disease develops. In addition, some patients may have epilepsy and severe headaches.

Antiribosomal P antibodies have been detected in patients with lupus suffer from psychosis or depression.

Late-Onset Lupus

Lupus can occur at any age, in either gender, and in any race. The average age of onset is 59 years; the average age at diagnosis is 62 years. Late-onset lupus affects women eight times more often than men. Late-onset lupus is found primarily in whites, but it occurs in all races.

Symptoms in most cases are relatively mild, but symptoms of lupus in older people can mimic those of other diseases (e.g., rheumatoid arthritis, Sjögren’s syndrome, polymyalgia rheumatica). Distinguishing among these disorders can be difficult and may result in a delayed or missed diagnosis. Drug-induced lupus occurs more often in older people because they are more likely to have conditions (e.g., high blood pressure, heart disease) that require treatment that may cause the symptoms of lupus. Symptoms generally fade when the medication is discontinued. Patients with late-onset lupus have a good survival rate and rarely die of the disease or complications of therapy when treated conservatively.

Immunologic Manifestations

B lymphocytes, T lymphocytes, and dendritic cells are involved in the pathogenesis of SLE. The pathogenesis of this systemic autoimmune disorder is characterized by the loss of tolerance to nuclear antigens, deposition of immune complexes in tissues, and multiorgan involvement.

Patients with SLE are known to produce multiple autoantibodies. There are two leading hypotheses, not mutually exclusive, as to why so many different antibodies develop. One hypothesis supports the belief that antibody-forming B lymphocytes are stimulated in a relatively nonspecific manner, so-called polyclonal B cell activation. The second hypothesis is that the immune response in SLE is specifically stimulated by antigens. The most compelling evidence in its favor is that the antibody molecules formed over time show evidence of the gene rearrangement and somatic mutation characteristic of an antigen-driven response. Recent studies have suggested that the neutrophilic leukocyte activity is implicated in linked biochemical and cellular events. Findings suggest that in SLE, anti–self antibodies activate neutrophils that consequently release neutrophil extracellular traps (NETs) containing complexes of DNA and antimicrobial peptide. These complexes activate plasmacytoid dendritic cells, which leads to interferon-α release and perpetuation of inflammation and disease. In the future, NETs may serve as a biomarker or predictor of tissue damage in SLE.

Laboratory features of SLE are the presence of ANAs, immune complexes, decreased complement level, tissue deposition of immunoglobulins and complement, circulating anticoagulants, and other autoantibodies. The human antineutrophil cytoplasmic antibodies (ANCAs), described for the first time in 1982, are directed against antigenic components mainly present in primary granules of neutrophils. ANCAs are serologic markers of primary necrotizing systemic vasculitis, particularly in Wegener’s granulomatosis. In addition, these antibodies have a prognostic interest because, in most cases, their titer is correlated with clinical activity during the disease.

Cellular Aspects

SLE is a disease that results from defects in the regulatory mechanism of the immune system. Studies of the immunopathogenesis of lupus nephritis have demonstrated a variety of aberrations in T cell and B cell function. It is uncertain whether the disease represents a primary dysfunction of T cells or B cells, but alterations in function do occur. Lymphocyte subset abnormalities are a major immunologic feature of SLE. Among the T cell subsets, a lack of or reduced generalized suppressor T cell function and hyperproduction of helper T cells occurs. The formation of lymphocytotoxic antibodies with a predominant specificity for T lymphocytes by patients with SLE at least partially explains the interference with certain functional activities of T lymphocytes associated with SLE. Lymphocytotoxic antibodies are capable of destroying T lymphocytes in the presence of complement and coating peripheral blood T cells.

The regulation of antibody production by B lymphocytes, ordinarily a function of the subpopulation of suppressor T cells, appears to be defective in patients with SLE. Although no single cause can be implicated in the pathogenesis of SLE, patients exhibit a state of spontaneous B lymphocyte hyperactivity, with ensuing uncontrolled production of a wide variety of antibodies to host and exogenous antigens. Host response to some antigens, such as vaccination with influenza, is normal in many cases and the patient manifests a specific, well-controlled humoral immune response.

Humoral Aspects

Circulating immune complexes are the hallmark of SLE. Patients with SLE exhibit multiple serum antibodies that react with native or altered self antigens. Demonstrable antibodies include antibodies to the following:

SLE is characterized by autoantibodies to almost any organ or tissue in the body. These antibodies may not be specifically diagnostic for SLE. In addition, some may have pathologic significance.

Antibodies to host antigens, particularly nuclear antigens such as DNA, are the principal type of antibody produced in SLE. ANAs are a heterogeneous group of antibodies produced against a variety of antigens within the cell nucleus. ANAs may be found in diseases other than SLE (e.g., other rheumatic or nonrheumatic diseases), as well as in some patients undergoing specific drug therapy and in healthy older individuals. The absence of ANAs almost excludes the diagnosis of SLE unless the patient is being chemically immunosuppressed. ANA titers and specific anti-DNA antibodies fluctuate during the course of the disease. In some cases, a rise in titer may forewarn of an impending disease flare-up.

Antigens to which antibodies are formed are present on nucleic acid molecules (DNA and RNA) or proteins (histones and nonhistones) and on determinants consisting of nucleic acid and protein molecules. Drug-induced cases of lupus have a high incidence of antibodies to histones. Some of these antibodies are directed against the double-stranded helical DNA (native DNA or dsDNA). The presence of anti–native DNA (anti-nDNA) antibodies was reported in 1957. High titers of dsDNA are seen primarily in SLE and closely parallel disease activity. Most SLE patients simultaneously demonstrate antibodies to nucleoprotein and native DNA.

Other nuclear antibodies are directed at the determinants of single-stranded DNA (ssDNA). Antibody titers of 1:32 or higher indicate a substantial concentration of antibody in an autoimmune response. Antibody to the Smith (Sm) antigen, a nuclear acidic protein extractable by aqueous solution, is considered a marker for SLE because anti-Sm has been found almost exclusively in patients with SLE. The presence of anti-Sm is seen in 25% to 30% of patients with SLE, but it rarely occurs in those with other systemic rheumatic (collagen) diseases.

The ANA antideoxyribonucleoprotein (anti-DNP) gives rise to the LE cell, which is found in more than 90% of untreated patients with active SLE. SLE patients with serositis may form LE cells in vivo. The LE cell testing procedure is now an obsolete test. In SLE patients with serositis, LE cells formed in vivo may be observed in aspirate fluid (e.g., pleural fluid). LE cells have been shown to be an expression of the interaction between IgG antibodies and DNP. Anti-DNP is referred to as the LE serum factor.

Antibodies to the Robert (Ro) soluble substance–A (SS-A) nuclear antigens are associated with SLE skin disease and the neonatal SLE syndrome. Antibodies to the Lane soluble substance–B (SS-B) antigens are associated with SLE and with primary and secondary forms of Sjögren’s syndrome. Their presence with SS-A antigen in SLE indicates mild disease. When present as the only antibody, SS-B is associated with primary Sjögren’s syndrome.

Autoantibodies to RBCs result in hemolytic anemia and can be detected by the anti–human globulin (AHG) test. Membrane-specific autoantibodies to neutrophils and platelets and autoantibodies to lymphocytes (cold-reactive type) are specific for SLE. Antibody titers correlate with disease activity.

Immunologic Consequences

Antibodies combine with their corresponding antigens to form immune complexes. When the mononuclear phagocyte system is unable to eliminate these immune complexes completely, immune complexes accumulate in the blood circulation. These circulating immune complexes are deposited in the subendothelial layers of the vascular basement membranes of multiple target organs, where they mediate inflammation. The sites of deposition are determined in part by the following physiochemical properties of the particular antigens or antibodies involved:

After deposition, the immune complexes seem to initiate a localized inflammatory response that stimulates neutrophils to the site of inflammation, activates complement, and results in the release of kinins and prostaglandins. These activities become the basis of antibody-dependent, cell-mediated tissue injury.

Diagnostic Evaluation

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 prothrombin 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:

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 29-3). 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.

Table 29-3

Antibodies in Systemic Rheumatic Diseases

Systemic Lupus Erythematosus Progressive Systemic Sclerosis Polymyositis Rheumatoid Arthritis
Antinuclear antibodies Antinuclear antibodies Antinuclear antibodies Antinuclear antibodies
Anti–native DNA Anti–Scl-1 Anti–Jo-1 Rheumatoid factors
Anti-Sm — — —

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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 29-4). There are 11 criteria for the diagnosis of SLE and, for a definitive diagnosis, patients must meet at least four of these criteria (see Table 29-1). 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.

Table 29-4

Immunologic Assays for Detection and Monitoring of SLE

Name of Assay Reference Range
Antibodies
Anti–double-stranded (ds) DNA antibody Negative (1:10)
Anti-La (SS-B) antibody Negative
Anti–liver cytosol antibody <15 U/mL
Anti–liver-kidney microsomal (LKM) antibody <1:40
Antimitochondrial antibody Negative
Antinuclear antibody Negative (1:40)
Anti–ribosomal P protein antibody <20 U/mL
Antiribonucleoprotein (anti-RNP) antibody Negative
Anti-Ro (SS-A) antibody Negative
Anti-Smith IgG Negative
Anti–soluble liver antigen antibody <5 U/mL
Complement
Total complement 63-145 U/mL
C3 86-145 mg/dL
C4 20-58 mg/dL

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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 antibodies 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 29-5.

Table 29-5

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

Antibody Disease Incidence (%)
Centromere-kinetochore CREST variant of progressive systemic sclerosis (PSS) 70-90
Diffuse scleroderma 10-20
Jo-1 Polymyositis 31
Ki antigen Systemic lupus erythematosus (SLE) 20
Ku Polymyositis/scleroderma overlap 55
Ma antigen SLE 20
Mi-I Dermatomyositis 11
NuMa (nuclear mitotic apparatus) antigen Rheumatoid arthritis (RA)  
Sjögren’s syndrome  
Carpal tunnel syndrome  
SLE 3
Proliferating cell nuclear antigen (PCNA) RA 90
RANA (RA-associated nuclear antigen) PSS 20
Scl-70 SLE 30
Sm (Smith) Sjögren’s syndrome 70
SS-A/Ro SLE 50
Other connective tissue diseases  
Sjögren’s syndrome 40-50
SS-B/La SLE 15
Mixed connective tissue disease >95
UI-RNP SLE 35

image

CREST, Calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia.

Adapted from Reimer G, Tan E: Antinuclear antibodies. In Stein J, editor: Internal medicine, Boston, 1987, Little, Brown.

Laboratory Evaluation

Demonstrable antibodies include antibodies to nuclear components, 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. The 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 (see Color Plates #13-#16 and Figure 29-3), 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.

image
Figure 29-3 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.

Interpretation of Staining Patterns of Major Rheumatic Autoantibodies

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 29-6).

Table 29-6

Antinuclear Antibody Patterns and Disorders

ANA Staining Pattern Antibody Specificities Related Disorders
Homogeneous nDNA
dsDNA
ssDNA
DNP
Histones		 Systemic lupus erythematosus (SLE)
Rheumatoid arthritis (RA)
Sjögren’s syndrome
Mixed connective tissue disease (MCTD)
Peripheral or rim nDNA
dsDNA
DNP		 Active SLE
Sjögren’s syndrome
Speckled Smith (Sm)
RNP		 SLE
RA
Sjögren’s syndrome
Progressive systemic sclerosis (PSS)
MCTD
Nucleolar 4-6S RNP Scleroderma
Sjögren’s syndrome
Undiagnosed illnesses manifesting Raynaud’s phenomenon
Discrete, speckled Centromere
DNA, RNA, ENA		 CREST variant of PSS

CREST, Calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia.

Treatment

For most patients with lupus, effective treatment and prevention methods can minimize symptoms, reduce inflammation, and maintain normal body functions. For photosensitive patients, avoidance of (excessive) sun exposure and the regular application of sunscreens will usually prevent rashes. Regular exercise helps prevent muscle weakness and fatigue. Immunization protects against specific infections. Support groups and counseling can help alleviate the effects of stress. Lupus patients should avoid smoking, excessive consumption of alcohol, overuse or underuse of prescribed medication, and postponing regular medical checkups.

Medications are often prescribed for patients with lupus, depending on the organ(s) involved and the severity of involvement. Common medications include the following:

• Nonsteroidal antiinflammatory drugs (NSAIDs). NSAIDs are prescribed for a variety of rheumatic diseases, including lupus. Examples include acetylsalicylic acid (aspirin), ibuprofen (Motrin), and naproxen (Naprosyn). These drugs are usually recommended for muscle and joint pain and arthritis. Newer NSAIDs contain a prostaglandin in the same capsule (Arthrotec). The other NSAIDs work in the same way as aspirin, but may be more potent.

• Acetaminophen. Acetaminophen (Tylenol) is a mild analgesic that can often be used for pain. It has the advantage of causing less stomach irritation than aspirin but is not nearly as effective at suppressing inflammation as aspirin.

• Steroids (e.g., prednisone) are used to reduce inflammation and suppress activity of the immune system. Side effects occur more frequently when steroids are taken over long periods at high doses. These side effects include weight gain, a round face, acne, easy bruising, thinning of the bones (osteoporosis), high blood pressure, cataracts, onset of diabetes, increased risk of infection, stomach ulcers, hyperactivity, and increased appetite.

• Antimalarials. Chloroquine (Aralen) or hydroxychloroquine (Plaquenil), typically used to treat malaria, may also be useful for some individuals with lupus. Antimalarials are most often prescribed for the skin and joint symptoms of lupus.

• Immunomodulating drugs. Azathioprine (Imuran) and cyclophosphamide (Cytoxan), cytotoxic drugs, act in a manner similar to that of corticosteroids in that they suppress inflammation and tend to suppress the immune system.

Other agents (e.g., methotrexate, cyclosporin) can be used to control the symptoms of lupus. Some of these are used in conjunction with apheresis, a blood-filtering treatment. Apheresis has been tried by itself in an effort to remove specific antibodies from the blood, but the results have not been promising.

Studies have suggested that immunosuppressive therapy targeted against the calcineurin pathway of T helper (Th) cells, such as tacrolimus, may be effective in the treatment of primary membranous nephropathy.

Newer agents are directed toward specific cells of the immune system. These include agents that block the production of anti-DNA or that suppress the manufacture of antibodies through other mechanisms. Examples are IV immunoglobulin injections, which are given on a regular basis to increase platelet numbers.

Clinical Trials

Drugs are being investigated as therapy for SLE. Currently, 58 clinical trials are being conducted and new research trials are always being initiated. A projection calls for quadruple the number of drugs to treat SLE by 2015.

Belimumab (Benlysta; formerly called LymphoStat-B) is a human monoclonal antibody that specifically recognizes and inhibits the biological activity of B lymphocyte stimulator (BLyS). BLyS is a naturally occurring protein discovered by human genome scientists that is required for B lymphocytes to develop into mature plasma B cells, which produce antibodies, the body’s first line of defense against infection. Retrospective and prospective studies have shown elevated levels of BLyS in the blood of many SLE patients and in the blood and joint fluid of RA patients. In lupus, RA, and certain other autoimmune diseases, elevated BLyS levels are believed to contribute to the production of autoantibodies. Preclinical and clinical studies have demonstrated that B cell antagonists can reduce autoantibody levels and help control autoimmune disease activity.

CASE STUDY 1

A 39-year-old black woman with SLE was diagnosed with the illness 20 years ago. Her initial manifestations of illness developed during the postpartum period of her second pregnancy. The pregnancy had been complicated by proteinuria, believed to be caused by toxemia of pregnancy.

The patient had polyarthralgia, alopecia, and erythematous rashes of the face, arms, and legs. A renal biopsy was performed because her urinalysis revealed proteinuria and RBC casts. The renal biopsy revealed diffuse, proliferative glomerulonephritis. In addition to abnormal laboratory results related to renal function, she manifested ANA (titer 1:1280) and antibodies to DNA and the C3 component of complement.

Questions

See Appendix A for the answers to multiple choice questions.

CASE STUDY 2

image Antinuclear Antibody Visible Method

Principle

This test is an indirect immunoenzyme method that uses tissue culture cells (human epithelial cells) as a substrate for the detection and titration of circulating ANAs in human serum. Patient serum samples are diluted in buffer and added to microscope slide wells with Hep-2 (human epithelial) cells cultured in them. Hep-2 cells are characterized by extremely large nuclei and the presence of mitotic figures to aid in detection. If specific antibodies are present, stable antigen-antibody complexes are formed that bind AHG labeled with horseradish peroxidase (HRP). The presence of HRP is indicated by a reaction with 3,3′-diaminobenzidine stain. The resulting dark-brown to black staining patterns of the nuclei can be seen with a light microscope. The presence of one or more types of circulating autoantibodies is the hallmark of SRDs.

Reporting Results

A grading scale similar to the one shown in Box 29-2 may be helpful in establishing the criteria for each laboratory. Positive specimens should be confirmed by repeating the test with twofold dilutions of serum. All positive ANA patterns should be titered to end point dilution to detect possible mixed antinuclear reactions that may not be apparent when interpreting a single screening dilution. The end point titer is the last serial dilution in which 1+ coloration with a clearly discernible pattern is detected.

Box 29-2   Grading Reactions

Negative

No cytoplasmic or nuclear specific stain observed. The cells may be slightly colored because of some nonspecific reaction of the peroxidase staining reagent.

Sources of Error

False-negative results can occur if the ANA happens to be specific for an antigen other than the one used in the procedure. False-negative results may also occur if the substrate is fixed in acetone and is inadequately washed. Without fixation, however, some soluble nuclear antigen may be lost. False-negative results may also be related to the binding of antinuclear factor to circulating immune complexes and to a low antibody titer.

False-positive interpretations may occur because of nonspecific staining, which may resemble a speckled pattern of reactivity. These staining reactions occur whenever the conjugate or serum contains antibodies to other tissue antigens. Careful rinsing and removal of excess fluoresceinated conjugate minimize the risk of some nonspecific staining reactions.

Although IIF is considered to be the gold standard, it suffers from being a nonstandardized manual test, has subjective interpretation of results and has low reproducibility. Recently, manufacturers have automated the preevaluation and evaluation phases of IIF ANA testing, including using automatic fluorescent image analysis to provide a virtual titer, which eliminates the process of staining a series of diluted samples manually. EIAs and solid-phase methods (e.g., microarrays and bead-based assays) are popular. However, IIF currently remains the gold standard of testing.

Clinical Applications

In the evaluation of patients with connective tissue disease, the ANA must be interpreted with caution. Under proper testing conditions, a negative ANA generally rules out SLE. A negative ANA result can result from autoimmune disease in remission or nuclear autoantibodies not detectable with indirect immunofluorescent or peroxidase immunoenzyme procedures.

The significance of a positive ANA depends on the titer and to a lesser extent on the observed pattern (see Table 29-6). There is no general agreement on the significance of the various patterns and it should be noted that some patterns may mask other patterns in high concentration. Interpretation of ANA patterns can provide additional information about the type of nuclear component reacting.

Because of the sensitivity of the Hep-2 cell substrate, some apparently normal individuals may show a low degree of staining at the 1:40 screening dilution. ANA titers of 1:10 to 1:80 usually have little significance but may be seen in patients with RA or scleroderma. ANAs are known to be gender- and age-dependent; therefore, a positive low-titer result may be normal for certain individuals in the absence of other clinical signs and symptoms. If a specimen is positive at a 1:10 dilution, it should be retested at dilutions from 1:20 to 1:320. The higher the antibody titer, the more likely is the diagnosis of connective tissue disorder. Changes in the antibody titer can also be used to observe disease activity.

If the ANA test is positive, additional immunologic evaluation is necessary to determine the specificity of the reaction. These evaluations include double immunodiffusion, counterimmunoelectrophoresis, passive hemagglutination, radioimmunoassays, and identification of nuclear antigens by immunoprecipitation or immunoblotting. These evaluations may demonstrate the presence of more than one ANA specificity reaction in the serum. An LE cell preparation is not useful because it is positive in only 75% of patients with confirmed SLE.

image Rapid Slide Test for Antinucleoprotein

Principle

The SLE latex test provides a suspension of polystyrene latex particles coated with deoxyribonucleoprotein (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, PSS, Sjögren’s syndrome, MCTD, drug-induced lupus).

Chapter Highlights

• Systemic lupus erythematosus (SLE) is the classic model of autoimmune disease.

• No single cause of SLE has been identified, but a primary defect in immune system regulation is considered important in its pathogenesis. Other influences include the effect of estrogens, genetic predisposition, and extraneous factors.

• SLE is a disease of acute and chronic inflammation. Lymphocyte subset abnormalities are a major immunologic feature of SLE. The regulation of antibody production of B lymphocytes, ordinarily a function of the subpopulation of T suppressor cells, appears defective in SLE.

• Circulating immune complexes are the hallmark of SLE. Patients exhibit multiple serum antibodies that react with native or altered self antigens. Demonstrable antibodies include antibodies to nuclear components, cell surface and cytoplasmic antigens of polymorphonuclear and lymphocytic leukocytes, erythrocytes, platelets, and neuronal cells, and IgG.

• Antibodies also combine with their corresponding antigens to form immune complexes. When the mononuclear phagocyte system is unable to eliminate them entirely, these immune complexes accumulate in the blood circulation. These circulating immune complexes are deposited in the subendothelial layers of the vascular basement membranes of multiple target organs, where they mediate inflammation.

• The antinuclear antibody (ANA) procedure is a valuable screening tool for SLE. Demonstration of ANAs can indicate various systemic autoimmune connective tissue disorders characterized by antibodies that react with different nuclear components, such as double-stranded DNA, single-stranded DNA, and Sm antigen. ANAs can be found in SLE, MCTD, PSS (or scleroderma), Sjögren’s syndrome, polymyositis-dermatomyositis, and RA. A small percentage of patients with neoplastic diseases may also demonstrate the presence of ANAs.

• ANAs are classified into antibodies to DNA, antibodies to histones, antibodies to nonhistone proteins, and antibodies to nuclear antigens. Antibodies to DNA can be divided into two major groups:

• Detection of autoantibodies by immunofluorescence is extremely sensitive and may show positive results when ANA procedures (e.g., complement fixation or precipitation) yield negative results. At present, immunofluorescence is the most widely used technique for ANA screening.

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