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The Role of the Laboratory in the Diagnosis of Rheumatoid Arthritis

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Published: August 2011

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Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic joint inflammation that ultimately leads to joint destruction. Diagnosis of early disease is important. While the diagnosis of RA is established primarily on clinical criteria (physical examination and radiographic evaluation), laboratory testing for cyclic citrullinated peptide antibodies contributes to early RA diagnosis.

Presenter: Dr. Melissa Snyder

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Introduction

Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.

Our presenter for this program is Dr. Melissa Snyder, Director of the Antibody Immunology Laboratory, in the Division of Clinical Biochemistry and Immunology at Mayo Clinic in Rochester, Minnesota. Dr. Snyder discusses clinical symptoms and classification of rheumatoid arthritis and the contribution of laboratory testing in diagnosis.

After viewing the Hot Topic, we invite you to participate in Beyond Hot Topic. This question and answer session will be posted online approximately 1 month after the Hot Topic presentation is posted. You can submit a question for Dr. Snyder using the information at the end of the presentation. Thank you Dr. Snyder.

Introduction to Rheumatoid Arthritis

Rheumatoid arthritis is a chronic inflammatory disease, with patients requiring life-long monitoring and treatment following diagnosis. It is heterogeneous in its clinical presentation, both in terms of its initial presentation and the overall disease course. Although most people are aware of the joint destruction that accompanies rheumatoid arthritis, we also need to appreciate that this disease is systemic, with the potential to involve a variety of organ systems. It is believed to have an autoimmune etiology, which is based primarily on the characteristics of the disease. However, the presence of a specific autoantigen has yet to be demonstrated.

There are multiple factors that are related to the pathogenesis of rheumatoid arthritis, and these are both environmental and genetic. Genetics is thought to account for approximately half of the risk for developing RA, with environmental exposures also playing a significant role.

Both the genetics and the cumulative environmental exposures together are likely responsible for the varied presentation of the disease.

Risk Factors Associated with Development of Rheumatoid Arthritis

As I just stated, there are multiple risk factors, both environmental and genetic, that may predispose an individual to developing rheumatoid arthritis.

There is a gender bias in rheumatoid arthritis, as is seen in many autoimmune diseases. Rheumatoid arthritis is 2 to 3 times more common in females than in males. Although this is thought to be related to hormonal differences, the specific cause of this bias is not known.

Cigarette smoking is another important risk factor. Although the reason for this is not well understood, recent studies have shown that cigarette smoke is associated with increased protein citrullination and may lead to the development of anti-CCP antibodies, which we will discuss shortly.

Lastly, as far as environmental factors go, infection may also play a role in the pathogenesis of rheumatoid arthritis. Although no specific bacteria or virus has been identified as a causative agent, it is hypothesized that infection, in certain individuals, may alter the immune response, resulting in a chronic autoimmune process.

With regard to the genetic component, individuals with a history of autoimmune disease in their family, or individuals with a first-degree relative with rheumatoid arthritis, are more at risk for developing the disease. Rheumatoid arthritis is a multigenic disease, meaning that the effect of many genes combine to predispose an individual to developing the disease.

Much of the genetic susceptibility has been linked to the MHC class II genes. The research that has gone on in this field has led to the shared epitope hypothesis. The MHC genes that are associated with rheumatoid arthritis, specifically certain HLA-DR4, HLA-DR1, and HLA-DR6 alleles, all share a similar amino acid sequence, referred to as the “shared epitope”, within the HLA-DR beta chain. Again, how this is related to the pathogenesis of rheumatoid arthritis is not known. However, it is thought that perhaps this sequence may be responsible for presenting a specific autoantigen that initiates or prolongs the immune response, or that perhaps these alleles result in preferential selection of autoreactive lymphocytes during development.

Another genetic risk factor that has been discovered is PTPN22. This gene encodes for a protein tyrosine phosphatase that is partially responsible for regulating T-cell activation. A specific polymorphism, which is designated as R620W, has been shown to be found more frequently in individuals with autoimmune disease, including rheumatoid arthritis, than in healthy individuals. Data also suggest that heterozygosity or homozygosity for this polymorphism predisposes patients with rheumatoid arthritis to having a more aggressive disease course.

Clinical Symptoms of Rheumatoid Arthritis

As far as the clinical features of rheumatoid arthritis are concerned, in most individuals, the disease begins with articular manifestations, generally as a symmetric arthritis. This process begins with inflammation in the synovial joint. Ultimately, without adequate treatment, the disease may progress to erosion and destruction of the cartilage and bone within the joint. In some patients, the disease progresses beyond the joint.

Probably the most common extra-articular manifestations are generally systemic in nature, with patients complaining of fever, fatigue, and unintentional weight loss.

For some patients, the disease will go on to affect other organ systems. I won’t go through each manifestation individually, but as you can see, almost any organ system can be affected, with the clinical consequences being potentially severe for the patient.

1987 Classification Criteria for Rheumatoid Arthritis

In 1987, the American College of Rheumatology established a set of classification criteria for rheumatoid arthritis. These criteria focused heavily on clinical symptoms.

The criteria included morning stiffness that lasted longer than 1 hour and arthritis of 3 or more joints, particularly the hand joints , that was accompanied by inflammation and swelling. In particular, symmetric arthritis was included as an important indicator of rheumatoid arthritis.

The presence of nodules was also an indication of the disease, as were radiographic changes that indicated the presence of erosions or boney decalcification.

The only laboratory test that was part of these criteria was rheumatoid factor.

Using these criteria, it was suggested that, if 4 out of the 7 features were present, then the patient could be classified as having rheumatoid arthritis. Although very useful, these criteria were limited in their ability to classify patients with early disease.

2010 Classification Criteria for Rheumatoid Arthritis

Just recently, new criteria for rheumatoid arthritis were published. One goal of these criteria was to improve the classification of patients with early, less severe disease, while still allowing for their application to patients with more established or advanced disease.

The first question that was asked was “Which patients should be evaluated using these criteria?” It was determined that these criteria should only be used in patients who have at least 1 joint with definitive clinical synovitis and for whom the synovitis is not caused by another disease process or trauma.

Once the appropriate patient is identified, the classification criteria uses a scoring system based on clinical and laboratory evaluation.

The clinical criteria focus on joint involvement, with the involvement of numerous small joints generating a score of up to 5 points, in comparison to involvement of between 2 and 10 large joints, which is only given a score of 1 point.

Duration of symptoms is also important, with symptoms lasting at least 6 weeks adding another point to the total score. The laboratory also plays a role in this classification system through the evaluation of autoantibody serology and acute phase reactants.

A low-positive result for either rheumatoid factor or anti-CCP antibodies is given a score of 2 points while a high-positive result for either antibody is given a score of 3 points.

Similarly, an elevated C-reactive protein or erythrocyte sedimentation rate adds another 1 point.

All the points for a given patient are then added together, and a score of 6 or higher leads to the classification of that patient as having rheumatoid arthritis.

Laboratory Evaluation of Patient with RA

To expand a little on the laboratory evaluation of a patient with rheumatoid arthritis , testing usually begins with a general assessment of the patient. This almost always includes a CBC with differential to determine if the patient has any cytopenias or elevated cell counts. Inflammatory markers are also often assessed to determine if the patient has evidence of an inflammatory process. Liver and renal function may also be evaluated. This is not usually for diagnostic purposes, but to help guide potential therapeutic interventions. Total immunoglobulins may also be evaluated. Patients with rheumatoid arthritis may have elevated total immunoglobulins, and a protein electrophoresis may be needed to rule out a possible monoclonal gammopathy.

The serology testing most useful for rheumatoid arthritis are rheumatoid factor and anti-CCP antibodies. These are not only useful diagnostically, but also prognostically, as high titers of antibodies may identify a patient at risk for more severe disease.

In addition, a clinician may want to perform a synovial fluid analysis. Although the results of this analysis may not be diagnostic for rheumatoid arthritis, it can be useful to rule out an infection or a crystalline arthritis.

At this point, I would like to expand on the serology testing for rheumatoid factor and anti-CCP antibodies.

Utility of Rheumatoid Factor

Rheumatoid factor is the classic marker used for the diagnosis of rheumatoid arthritis. Rheumatoid factor is an antibody, either an IgG, IgA, or IgM isotype, that recognizes the Fc portion of an IgG molecule. Most rheumatoid factor assays have reasonable sensitivity for rheumatoid arthritis, generally between 70 and 90%. However, the biggest problem with rheumatoid factor is a lack of specificity. It is found at relatively high frequencies in other connective tissue diseases. It can also be found in healthy individuals. The frequency of positivity for rheumatoid factor is age-related, and may approach 25% in individuals over 60 to 70 years of age. This lack of specificity led to investigations designed to find other diagnostic markers for rheumatoid arthritis.

Discovery of Anti-Citrulline Antibodies

As part of this investigation, several groups independently identified a novel antibody distinct from rheumatoid factor.

This antibody was originally characterized by indirect immunofluorescence using a variety of tissue substrates. One group named their finding “anti-perinuclear factor” because the antibody bound to perinuclear granules in human mucosal cells.

Another group named their finding “anti-keratin antibodies” due to the demonstration that the antibody bound to keratinized epithelial cells.

Ultimately, it was demonstrated that both antibodies recognized the same target antigen, which was a citrullinated form of filaggrin.

A third discovery was that of anti-Sa, which bound to placental or RA pannus tissue.

The target antigen of this antibody was discovered to be citrullinated vimentin.

Enzymatic Process of Citrullination

Citrullination is an enzymatic process that results in a post-translational modification of arginine residues that are found in protein molecules. This process is mediated by peptidyl arginine deiminase. In the presence of calcium, a positively charged arginine residue is deiminated by the enzyme, ultimately releasing ammonia and forming a neutral amino acid referred to as citrulline. It has been demonstrated by numerous groups that the antibodies originally characterized as anti-perinuclear factor, anti-keratin antibodies, and anti-Sa antibodies are all specific for the citrullinated form of a variety of proteins.

Diagnostic Performance of Anti-Citrulline Antibodies

After the discovery of these antibodies, various studies were performed to determine if they would be an improvement over rheumatoid factor as a diagnostic marker for rheumatoid arthritis.

The initial studies done using indirect immunofluorescence indicated that the antibodies lacked sensitivity in comparison to rheumatoid factor, generally being around 40 to 50% sensitive.

However, the specificity of these antibodies was a clear improvement, being above 95% in most studies.

Development of Clinical Assays for Anti-Citrullinated Protein Antibodies

As a result of the initial studies, development of clinical assays for anti-citrullinated protein antibodies focused on improving assay sensitivity while maintaining the high specificity.

This was accomplished through optimization of the protein or proteins used as the target antigen.

During the course of this work, it was discovered that formation of a cyclic peptide improved the sensitivity of the assays.

To form these circularized structures, cysteine residues were placed on either end of the peptide. When a disulfide bond is formed between the 2 cysteine residues, a cyclic peptide results. This is where the term “anti-cyclic citrullinated peptide antibody” or “anti-CCP antibody” come from. It is thought that the cyclic structure allows for maximum exposure of the citrullinated residue.

There have been several generations of anti-CCP antibody assays.

The first generation assay used a cyclic peptide that had been derived from the human fillagrin protein. Although this assay had improved sensitivity over the initial indirect immunofluorescence assays, the sensitivity was still poor in comparison to rheumatoid factor. This type of assay was never widely used in most clinical laboratories.

The second generation anti-CCP antibody assay had significantly improved sensitivity, primarily because these assays used a panel of citrullinated peptides that had been identified through screening of peptide libraries using sera from patients with rheumatoid arthritis. The second generation assays are still widely used as a clinical test.

The third generation assays are relatively new, and use a synthetic form of a cyclic citrullinated peptide. The third generation assays are generally quite comparable to the second generation assays, although some studies have demonstrated a small improvement in sensitivity, primarily in patients with early disease.

Sensitivity and Specificity Comparison Anti-CCP and RF

In a study performed in our laboratory at the Mayo Clinic, we compared the sensitivity and specificity of the second and third generation anti-CCP antibody assays with our nephelometric assay for rheumatoid factor. We looked at a population of healthy individuals, as well as patients with rheumatoid arthritis and patients with other systemic rheumatic diseases. The first item to point out is that the overall sensitivity of both anti-CCP antibody assays is comparable to rheumatoid factor, all of which are approximately 80%. However, a small number of patients were positive for anti-CCP and negative for rheumatoid factor, and vice versa, so using both laboratory tests may offer a small increase in sensitivity. The second item to mention is that the specificity of the anti-CCP assays is improved over rheumatoid factor in both the healthy individuals and in patients with other connective tissue diseases. Using healthy individuals, the specificity of both anti-CCP antibody assays approaches close to 100%. However, this specificity appears to decrease slightly when evaluating patients with other connective tissue diseases. This may be due to the presence of overlap syndromes in some of these patients, although further studies are needed to clarify this.

Summary

In summary, the new classification criteria for rheumatoid arthritis has been modified in an attempt to improve the sensitivity for early disease. Certain criteria more associated with advanced disease, such as the presence of nodules and evidence of radiographic changes, have been removed while additional laboratory criteria, including anti-CCP antibodies and inflammatory markers, have been included.

Our data suggest that the anti-CCP antibody test has similar sensitivity to rheumatoid factor, but significantly improved specificity. However, rheumatoid factor is still included in the classification criteria, and may be useful in the small sub-set of patients who are negative for anti-CCP antibodies.

As with all autoantibody markers, the results of the anti-CCP antibody and rheumatoid factor testing must be interpreted in conjunction with other information, including the clinical presentation of the patient and results from imaging and other diagnostic testing.

Questions?

I hope this presentation has provided you with useful information regarding rheumatoid arthritis and the available laboratory testing.

 


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