Antibodies Against Citrullinated Peptides
A Role in the Diagnosis of Rheumatoid Arthritis
Published: September 2008Print Record of Viewing
Dr. Snyder discusses the use of anti-citrulline antibody in the Mayo connective tissue disease cascade algorithm to improve the specificity for diagnosis of rheumatoid arthritis.
Presenter: Dr. Melissa Snyder
- Director of the Antibody Immunology Laboratory, in the Division of Clinical Biochemistry and Immunology at Mayo Clinic
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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. Dr. Snyder discusses the use of anti-citrulline antibody in the Mayo connective tissue disease cascade algorithm to improve the specificity for diagnosis of rheumatoid arthritis.
Introduction to Connective Tissue Diseases
Rheumatoid Arthritis is an inflammatory disease characterized by an autoimmune etiology. It is classified as a connective tissue disease, or also known as systemic rheumatic disease. It is a chronic disease with patients requiring lifelong monitoring and treatment following diagnosis. Although most people are aware of the joint destruction that accompanies Rheumatoid Arthritis, we also need to be aware that this disease is systemic with the potential to involve a variety of organ systems. Rheumatoid Arthritis is only one of several connective tissues diseases which include lupus, Sjögren syndrome, and scleroderma among others. It can be a challenge to distinguish between these diseases as pts, especially those early in the disease, may present with relatively similar clinical symptoms.
Clinical Symptoms of Rheumatoid Arthritis
As I just stated, Rheumatoid Arthritis is a systemic disease. In most individuals, the disease begins as articular manifestations generally as a polyarthritis. This process begins with inflammation within 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 pts, the disease progresses beyond the joint. Some of the more common extra-articular manifestations include the development of nodules, osteoporosis, and vasculitis. Some individuals may develop rheumatoid lung disease and cardiac disease as well.
Epidemiologic Features of Rheumatoid Arthritis
Connective tissue diseases in general are relatively common disorders. Within this group of diseases, Rheumatoid Arthritis is one of the most common. In Caucasian populations in the US and Western Europe, the prevalence of Rheumatoid Arthritis is approx 1%. The prevalence is lower in other populations, such as what we see in some Asian countries. However, Rheumatoid Arthritis has been found at increased prevalences in some North American Indian tribes.
There is a gender bias in Rheumatoid Arthritis as is seen in many auto-immune diseases. Rheumatoid Arthritis is two to three times more common in females than males. Although this is thought to be related to hormonal differences, the specific cause to this bias is not known.
There is a genetic component to Rheumatoid Arthritis also. 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 affect of many genes combined to cause the disease. The largest known genetic contributor to Rheumatoid Arthritis is the HLA complex. There are specific HLA-DR alleles that are known to be associated with an increased risk of the development of the disease. However, it is important to remember that possession of these alleles is not sufficient to develop the disease as other genes certainly play a role.
An important piece of information to remember is that Rheumatoid Arthritis is associated with significant morbidity and mortality. It has been clearly demonstrated that patients with Rheumatoid Arthritis have a shortened life expectancy as compared to the general population, much of which is probably related to the extra-articular aspects of the disease. To minimize this, early diagnosis and prompt treatment is critical.
Diagnostic Criteria for Rheumatoid Arthritis
The American College of Rheumatology has established a set of Diagnostic Criteria for Rheumatoid Arthritis. These criteria focus heavily on clinical symptoms. The criteria include morning stiffness that lasts longer than 1 hour, and arthritis of 3 or more joints, particularly the hand joints, that is accompanied by inflammation and swelling.
In particular, Symmetric arthritis is an important indicator of Rheumatoid Arthritis. The presence of nodules is also an indication of the disease as are radiographic changes that indicate the presence of erosions and boney decalcification.
The only laboratory test that is currently part of the criteria is Rheumatoid factor (RF). I have included anticyclic citrullinated peptide (anti-CCP) antibodies on the list on this slide, although it is not part of the official diagnostic criteria. It is possible that it may be included at some point in the future, although many clinicians are using it now as a replacement for Rheumatoid factor.
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 70-90%; however the biggest problem with Rheumatoid Arthritis is its 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 60-70 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 nodal 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 (APF) because the antibody bound to perinuclear granules in human mucosal cells.
Another group named their finding anti-keratin antibodies (AKAs) due to the demonstration that the antibody bound to 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 Rheumatoid Arthritis 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 found in protein molecules. This process is mediated by peptidyl arginine deiminase (PAD). In the presence of calcium, a positively charged argentine 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 are all specific to 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 40-50%. However, the specificity of these antibodies was a clear improvement, generally 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 protein or proteins used as a 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, cystine residues were placed on either end of the peptide. When a disulfide bond was formed between the two cystine residues, a cyclic peptide resulted. This is where the term anticyclic citrullinated peptide antibody, or anti-CCP antibody, comes from. It is thought that the cyclic structure allows for maxim exposure of the citrullinated residue, thus improving analytic sensitivity.
There have been several generations of anti-CCP assays. The first generation assay uses a cyclic peptide that happens to derive from the human filaggrin protein. Although this assay had improved sensitivity over the initial indirect immunofluorescent 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 use a panel of citrullinated peptides that had been identified through screening of peptide libraries using serum from patients with Rheumatoid Arthritis. The second generation assays are still widely used as a clinical assay. The third generation assays are relatively new and use a synthetic form of 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 the specificity of the second and third generation anti-CCP antibody assays with our net filametric 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 sensitivity of both anti-CCP antibody assays is comparable to Rheumatoid factor, all of which being approximately 80%.
The second item to mention is that the specificity of the anti-CCP antibodies is improved over Rheumatoid factor in both the healthy individuals and the 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 point.
Diagnostic Algorithm for CTDs
Because of the similar sensitivity to Rheumatoid factor but the improved specificity, anti-CCP antibodies are the analyte of choice in helping to establish a diagnosis of Rheumatoid Arthritis. At Mayo Clinic, we offer this test individually as well as part of our connective tissue disease cascade. This cascade is designed to assist clinicians who are faced with a pt who appears to have systemic Rheumatic disease but in whom the clinical symptoms are not sufficient to narrow it down to a differential diagnoses.
When a test is ordered for a pt, two tests are done at the first level of testing: the anti-CCP antibody test and an ANA. A positive result for the anti-CCP antibody test is consistent with Rheumatoid Arthritis, although the result must be taken in the context of other lab results and the clinical picture of the pt. The other side of the cascade is more useful for other rheumatic diseases such as lupus and scleroderma. In the cascade, a positive ANA reflexes to the second level of testing. This second level of testing focuses on detection of discreet antibodies, that recognize specific antigens known to be associated with various connective tissue diseases. The pattern of discreet antibodies that are detected in conjunction with the anti-CCP antibody test can help the clinician narrow down the specific disease process that may be occurring in that particular patient.
In summary, the anti-CCP antibody test is useful in establishing the diagnosis of Rheumatoid Arthritis. It has similar sensitivity to Rheumatoid factor, but significantly improved specificity. As with all antibody markers, the result of the anti-CCP antibody test must be interpreted in conjunction with other information, including the clinical presentation of the pt and results from other diagnostic testing such as the ANA, discreet antibodies and inflammatory markers.