Course Authors

Peter Barland, M.D.

Dr. Barland reports no commercial conflict of interest.

Estimated course time: 1 hour(s).

Albert Einstein College of Medicine – Montefiore Medical Center designates this enduring material activity for a maximum of 1.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only the credit commensurate with the extent of their participation in the activity.

In support of improving patient care, this activity has been planned and implemented by Albert Einstein College of Medicine-Montefiore Medical Center and InterMDnet. Albert Einstein College of Medicine – Montefiore Medical Center is jointly accredited by the Accreditation Council for Continuing Medical Education (ACCME), the Accreditation Council for Pharmacy Education (ACPE), and the American Nurses Credentialing Center (ANCC), to provide continuing education for the healthcare team.

 

Learning Objectives

Upon completion of this Cyberounds^®, you should be able to:

 

Introduction

The antinuclear antibody test (ANA) is the single most sensitive test currently available for the laboratory diagnosis of a systemic connective tissue disease. However, the ANA test is also nonspecific, found in varying frequencies not only in all of the autoimmune diseases, but also in many chronic inflammatory conditions, and to a much lesser extent in normal patients as well.

While the titer of the ANA test (Table 1) and the pattern of nuclear staining (Figure 1) provides additional information which improves the specificity of the ANA test significantly, it is still not possible to make a clinical diagnosis based on an ANA test alone.

Figure 1.

Typical ANA patterns seen with indirect immunofluorescent staining of HEP 2 cells: homogeneous (top left) frequently seen with anti-histone antibodies found in patients with drug-induced ANA's and with anti-histone DNA complexes found in patients with rheumatoid arthritis and SLE; speckled (top right) seen with anti- RNP antibodies found in patients with mixed connective disease, with anti-SM antibodies found in patients with SLE and with other less well-characterized anti-nuclear antibodies found in scleroderma and polymyositis; centromere (bottom left) seen with anti- centromere antibodies found specifically in patients with CREST syndrome; and nucleolar (bottom right) seen with antibodies to ribonuclear proteins and RNA polymerases found primarily in patients with scleroderma.

The presence of a positive ANA in a particular clinical setting should trigger the search for more specific and frequentlly diagnostic autoantibodies all of which are part of the family of antinuclear antibodies. Conversely, low titers of antinuclear antibodies may be found in some normal individuals. Thus a positive ANA is not necessarily indicative of pathology.

The following four patients are representative of clinical situations where the interpretation of the ANA test is helpful in patient management.

• A 40-year-old woman presents with swollen painful wrists, metacarpal phalangeal joints and knees of four months' duration. Physical exam confirms the presence of synovitis and subcutaneous nodules. A recent laboratory report shows a high titer rheumatoid factor and high titer (1:640) ANA with a diffuse staining pattern.
• A 21-year-old medical student on oral contraceptives develops an eczematoid rash on her upper extremities and face. Her ANA test comes back positive at 1:40.
• A 36-year-old nurse developed Raynaud's phenomenon six months ago. The physical exam is normal. An ANA test comes back positive at 1:160 with a speckled staining pattern.
• A 39-year-old man with a history of Raynaud's phenomenon presents to the emergency room with new onset precordial chest pain. The physical exam reveals a pericardial friction rub and his CPK is elevated at 400 units/dL, but the MB fraction is negative. A call to his primary care physician reveals a recent ANA test was positive at 1:1280 with a speckled staining pattern.

At the end of these Cyberounds^® you will have the opportunity to make some decisions as to the next step in the workup of these patients based on what you have learned.

Methodology

The ANA test is performed using indirect immunofluorescent microscopy in which diluted patient serum is incubated with nuclei that have been made permeable to immunoglobulin by either fixation, sectioning or both fixation and sectioning. In addition, the nuclear antigens need to be immobilized by gentle fixation which does not denature the nuclear contents enough to alter their antigenicity. At the time when the test was first developed, it was widely believed that all nuclei contained the same antigens. As a result, a number of different substrates were employed as the source of nuclear antigens--usually tissue sections from laboratory animals such as rat liver or mouse kidney. However, it is now clear that there are important nuclear antigens that are species specific, while other antigens are found only in certain cell types. Still other antigens are constitutive (i.e. present all the time) in the nuclei of certain cells, while other antigens are inducible in the nuclei of certain cells after exposure to the recently described intercellular peptide messengers (cytokines) produced primarily by cells of the immunoregulatory system.

Almost all commercial laboratories and large hospital-based laboratories now employ a cultured human cell line, Hep 2, as the substrate for the ANA test. Because there is nonspecific binding of immunoglobulin to nuclear contents in undiluted serum, most laboratories screen for ANA at a dilution of 1:40.

The antinuclear antibodies are found in all of the major immunoglobulin classes (e.g. IgG, IgM, and IgA.). This observation has led to the use of polyclonal antisera that react with these three major immunoglobulins, conjugated to fluorescein as the staining agent. However, some laboratories use antisera conjugated with peroxidase as the conjugate. The peroxidase is used to develop an insoluble black precipitate which can be viewed under the light microscope. This method yields a more permanent record and obviates the need for a fluorescent microscope, although it adds to the complexity of the test and makes interpretations of patterns of nuclear staining more difficult.

With the Hep 2 cell substrate and polyclonal fluorescein conjugates, there is excellent reproducibility of the ANA test between laboratories, as determined by proficiency studies. Additionally, you're likely to achieve better than 90% reproducibility of the ANA titer +/- one doubling dilution.

Interpretation

A great many diseases are associated with a significantly increased frequency of a positive ANA test, although the prevalence of the ANA test in each of these conditions varies greatly. The major conditions associated with a positive ANA test and the prevalence of ANA positivity in these conditions are shown in Table 1.

Even among normal blood donors the incidence of a positive ANA is approximately 5% when the screening is done with a serum dilution of 1:40. However, these false positive ANA's are usually of low titer and less that 1% of the normal population will have a titer of > or = 1:160.

Usually the titer of the ANA test is >1:160 in the systemic connective tissue diseases. But neither the disease activity nor the patient's prognosis can be related to the titer of the ANA.

Let's take a look at some of the major connective tissue diseases and the expected ANA results for each.

Systemic Lupus Erythematosis

In systemic lupus erythematosis (SLE) the ANA is almost always positive in high titer. Before the use of Hep 2 cells approximately 5% of patients with clinically proven SLE had persistently negative ANA's. Now, however, using Hep 2 cells which constitutively contain the SS-A/Ro, SS- B/La and p16 antigens not found in resting liver and kidney nuclei, the incidence of ANA negative SLE is extremely small and certainly less than 1%. While a rim staining pattern of the nucleus frequently correlates with the presence of anti-DNA antibodies which are virtually pathognomonic for SLE, this correlation is too rough to be used as a substitute for the anti-DNA antibody assay.

Drug-Induced Lupus

As shown in Table 2, a number of drugs can induce ANA's. However, only a few of these induce a clinical syndrome of arthralgias, fever and serositis resembling SLE, although they differ from many patients with the spontaneous form of SLE by the fact that they never develop renal or CNS involvement.

Drug-induced ANA's almost always produce a diffuse staining pattern. The titer of the ANA does not distinguish between those patients who are symptomatic from those that are asymptomatic. Drug-induced ANA's are usually directed at histone antigens in the nuclear nucleosomes and produce a diffuse nuclear staining pattern. The ANA test may remain positive for many months after the drug is discontinued and therefore a careful drug history is important in evaluating a patient with an unexplained ANA.

Scleroderma

In scleroderma (systemic sclerosis) the ANA is positive in approximately 90% of patients and there is usually a speckled or nucleolar staining pattern (Figure 1 b & d). The presence of a nucleolar pattern is unusual enough to raise the suspicion of scleroderma in a patient with Raynaud's syndrome. The CREST syndrome, a subset of scleroderma characterized by sclerodactyly, Raynaud's phenomenon, telangiectasias, esophageal dysmotility and cutaneous calcinosis, is often accompanied by an anticentromere staining pattern on the ANA test characterized by each nucleus containing 23 fluorescently stained granules. In mitotic cells, these granules double in number and align along the chromosomes. While an occasional patient with the CREST syndrome will develop the more systemic form of the disease, the presence of an anti-centromere antibody is usually associated with persistently localized disease. Conversely, in patients whose only clinical symptom is Raynaud's phenomenon, the presence of a nonspecific ANA does not foretell the development of a more systemic autoimmune disease.

Rheumatoid Arthritis

The ANA test usually correlates with the presence of rheumatoid factor in rheumatoid arthritis When this is the case these patients seem to have the same amount of active joint disease and extra articular features of their rheumatoid arthritis as those patients who are rheumatoid factor positive and ANA negative. Approximately 5% of patients with rheumatoid arthritis are ANA positive and rheumatoid factor negative. These patients seem to behave clinically like rheumatoid factor positive patients.

Juvenile Rheumatoid Arthritis

In juvenile rheumatoid arthritis (JRA) ANA positivity is associated with pauciarticular disease and with extra- articular features of the disease including iridocyclitis (inflammation of the anterior uveal tract) and band keratopathy (opacification of the cornea leading to significant visual loss).

Discoid Lupus and Other Condtions

Approximately 25% of patients with discoid lupus without systemic symptoms have a positive ANA test. The majority of these patients do not develop clinical manifestations of SLE after a 10-year follow-up period. Similarly, most of the autoimmune inflammatory conditions directed at specific organs such as autoimmune hepatitis and autoimmune thyroiditis are accompanied by an increased prevalence of a positive ANA. However, the incidence of SLE and other systemic connective tissue diseases does not appear to be significantly increased in these patients. Similarly, patients with autoimmune hemolytic anemia and autoimmune thrombocytopenia or both (Evan's syndrome) frequently manifest a positive ANA without other systemic features even after prolonged follow-up.

Now that you've read a bit about ANA, let's revisit our four patients which will be referenced in the questions below.

• A 40-year-old woman presents with swollen painful wrists, metacarpal phalangeal joints and knees of four months' duration. Physical exam confirms the presence of synovitis and subcutaneous nodules. A recent laboratory report shows a high titer rheumatoid factor and high titer (1:640) ANA with a diffuse staining pattern. (answer)