Download Anti-Cyclic Citrullinated Peptide (anti-CCP) Antibodies

Anti CCP Antibodies
Anti-Cyclic Citrullinated Peptide (anti-CCP) Antibodies
Anti-CCP antibodies are potentially important surrogate markers for
diagnosis and prognosis in rheumatoid arthritis (RA), because they:
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are as sensitive as, and more specific than, IgM rheumatoid
factors (RF) in early and fully established disease
may predict the eventual development into RA when found in
undifferentiated arthritis
are a marker of erosive disease in RA
may be detected in healthy individuals years before onset of
clinical RA
First described as a marker for RA in 1964, anti-perinuclear factor
(APF) was directed to constituents of the keratohyaline granules near
senescent buccal mucosal cell nuclei, later found to contain the
“filament aggregating protein” filaggrin. Despite its specificity for
RA, because of exacting technical requirements, APF never became
widely used. Anti-keratin antibodies (AKA), first described in 1979,
bound filaggrin tightly bound to keratin in senescent esophageal cells.
As was APF, AKA had greater specificity for RA than RF.
Antibodies to the so-called Sa antigen, described first in 1994, bound
post-translationally modified vimentin, a cytoskeletal intermediate
filament protein found in mesenchymal cells. Since 1998 it has
become increasingly evident that all of the above antibodies likely
target citrullinated proteins.
Citrulline is a non-standard amino acid, created by de-imination of
arginine residues in several proteins by the action of peptidylarginine
deiminase (PAD). There are several isotypes of this enzyme; in the
inflammatory RA synovium, PAD 2 and PAD 4 are abundant. These
enzymes cause the local citrullination of synovial proteins, such as
fibrin. Interestingly, citrullinated peptides fit better in the HLA DR4
(DRB1*0401 or *0404) antigen binding grooves than the
corresponding arginine containing peptides, findings that link this
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immune response to the shared epitope hypothesis of RA
pathophysiology. Citrullinated extracellular fibrin in the RA synovium
may be one of the major autoantigens driving the local immune
response, suggested by the discovery of local production of anti-CCP
and anti-citrullinated filaggrin antibodies in the joint. Also, functional
haplotypes of PADI4 may be associated with RA.
The citrulline moiety is the true determinant on proteins recognized
by APF, AKA, and possibly anti-Sa. Detailed studies of citrullinated
filaggrin peptides showed that different patients with RA recognized
different linear citrullinated peptides, indicating a polyclonal
response. Flanking regions around the citrulline residue are important
for the reactivity, so not all sera are reactive with every peptide. The
first generation of ELISa for anti-CCP (CCP1), using several filaggrin
epitopes, had high specificity for RA and a sensitivity of 65-70% (1).
Various cyclic epitopes that mimic true conformational epitopes were
selected from libraries of citrullinated peptides for the widely
available 2nd generation anti-CCP assay (CCP2).
Diagnostic use of autoimmune serology in RA
RF has been used as a marker of RA for more than half a century.
IgM RF, the isotype most typically detected, is seen not only in RA
but also in various other conditions. IgA RF, more easily detected
than IgG RF, may be a better indicator of T-cell dependent affinity
matured antibodies directed to particular Fc-gamma epitopes relevant
to RA than IgM RF, but it has never gained wide interest. The
combined detection of IgM and IgA RFs in a serum is a strong
indicator of RA.
Most studies published to date comparing the sensitivity and
specificity of RFs and anti-CCP antibodies for the diagnosis of RA
have used the CCP1 assay. In general, the sensitivity of anti-CCP has
been comparable to RF (50-75%) with a higher specificity (90-95%).
More recent studies using the CCP2 assay show higher sensitivity for
RA than CCP1, with equally high specificity.
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Clinical use of anti-CCP
In many early cases of RA, clinical symptoms are milder and
nonspecific, and patients will not fulfill ACR classification criteria for
RA. Therefore, the detection of a disease-specific autoantibody like
anti-CCP could be of great diagnostic and therapeutic importance.
Anti-CCP antibodies may be detected in roughly 50-60% of patients
with early RA at ‘baseline’(e.g., at their initial encounter with a
specialist, usually after 3-6 months of symptoms). The specificity of
anti-CCP is around 95-98% as regards undifferentiated forms of
arthritis that do not develop into RA. IgM RF is often found in the
same patients, but with much lower specificity for RA. One study
using a CCP1 assay showed a sensitivity of 55% and a specificity of
97% specificity for RA, when both anti-CCP and IgM RF were
positive in the early stage of arthritis. More recent studies using the
CCP2 assay have shown even higher prevalence at the first visit to
clinics; in one study anti-CCP antibodies were found in 70% of such
patients. Interestingly, using stored samples, anti-CCP could be
detected 1.5 to 9 years before the onset of arthritis. A study using the
CCP2 assay found progression from undifferentiated polyarthritis to
RA in 93% of anti-CCP positive patients but only in 25% of antiCCP negative patients after 3 years of follow-up. In a study of
patients with RA or palindromic rheumatism, anti-CCP (CCP1) were
found in 55% of both conditions, indicating that palindromic
rheumatism is closely related to and often progresses to RA.
Several observations have indicated that anti-CCP positive early RA
patients may develop a more erosive disease than those without antiCCP. Other investigators have confirmed this, and suggested the
superiority of anti-CCP over IgM RF in predicting an erosive disease
course. The use of anti-CCP results in the decision whether a patient
should be treated aggressively at an early stage or not is an important
area for research. In addition, the relationship of levels of anti-CCP
antibodies and various therapeutic interventions is under
investigation.
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Conclusions
The use of anti-CCP antibodies may allow the clinical rheumatologist
to better predict the diagnosis and prognosis of individual patients
with RA. Whether this or other serologic tests will allow more
rational therapeutic decision-making and hence influence the longterm outcome of the disease will be determined by further study.
Testing
Rheumatoid arthritis is both common and chronic, with significant
consequences for multiple organ systems. Better understanding of its
pathophysiology has led to the development of targeted therapies that
have dramatically improved outcomes. The key to therapeutic success
lies in identifying individuals who will have severe destructive disease
as early as possible, so that effective treatment can be initiated before
irreversible damage occurs.
Anti-cyclic citrullinated peptide (anti-CCP) antibody testing is
particularly useful in the diagnosis of rheumatoid arthritis, with high
specificity, presence early in the disease process, and ability to identify
patients who are likely to have severe disease and irreversible damage.
However, its sensitivity is low, and a negative result does not exclude
disease. Anti-CCP antibodies have not been found at a significant
frequency in other diseases to date, and are more specific than
rheumatoid factor for detecting rheumatoid arthritis. We discuss antiCCP antibody testing in rheumatoid arthritis, with an emphasis on
diagnostic performance, prognostic capability, and relevance to
pathogenesis and new treatment paradigms in rheumatoid arthritis.
Rheumatoid arthritis (RA) is a common systemic autoimmune disease
with a prevalence of about 1% worldwide. The American College of
Rheumatology (ACR) criteria for the classification of RA are not very
well suited to diagnose RA at an early stage of the disease, because
these criteria rely heavily on the expression of clinical symptoms of
RA. In early RA these clinical parameters are often not (yet) manifest.
Therefore, a specific and sensitive (serological) marker, which is
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present very early in the disease, is needed. A good marker should
ideally not only indicate the development of the disease, but also be
able to predict its erosive or non-erosive progression. The serological
parameter that meets these requirements for a good and useful
marker for early RA is the anti-citrullinated protein antibody. The
sensitivity of this antibody is comparable to that of the rheumatoid
factor (RF) (approximately 80%), but its specificity is much higher,
about 98%. Several assays have been developed to detect this class of
auto-antibodies, which are termed anti-CCP because the most
sensitive test is based upon cyclic citrullinated peptides.
Most patients with rheumatoid arthritis (RA) have some marker of
the disease in the blood. This might include:
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positive rheumatoid factor (RF)
cyclic citrullinated peptide (CCP) antibody
elevated measures of inflammation
an increase erythrocyte sedimentation rate (ESR)
C-reactive protein (CRP).
On the other hand, occasionally a patient with RA may have active
disease and all these tests are normal. In general, those patients
without these abnormalities in the blood are less likely to develop
joint damage. Almost all patients with lupus have the presence of the
antinuclear antibodies (ANA) or if negative, an SSA antibody or antiphospholipid antibodies (associated with an increased risk of
thrombosis such as a blood clot or stroke or miscarriage). Once
again, there are patients, although rare, who have lupus despite these
negative tests. Most Sjögren’s syndrome patients will typically exhibit
an elevated ESR or have a positive ANA, RF, SSA or SSB antibody
in their blood.
It is important to remember that while blood tests are helpful in
confirming a diagnosis and assessment of disease activity, it is more
important to diagnose and treat based on the patient’s clinical
presentation.
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According to the Arthritis Foundation, if you have signs or
symptoms of arthritis for more than two weeks, it is time to see a
doctor. These warning signs include:
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joint pain
stiffness
swelling
difficulty moving a joint
Self treating is not recommended for patients with new symptoms
until the cause can be confirmed. The reason for this caution is that
arthritis may be a symptom of a more serious condition such as
lupus, rheumatoid arthritis, infection or malignancy. An accurate
diagnosis leads the way to proper treatment.
Some patients may have more than one problem causing their
arthritis such as:
• rheumatoid arthritis and fibromyalgia
• psoriatic arthritis and gout
The presence of a positive RF or CCP in this patient would help
confirm a diagnosis of rheumatoid arthritis (RA). On the other hand,
up to 30 % of patients with RA may not have these antibodies,
especially early in their disease. In addition, the presence of a RF,
especially at a low level is not uncommon in patients who do not
have, and never will develop RA. The anti-CCP antibody is more
likely to be associated with RA, so if elevated at a high level, the
patient without typical manifestations of RA may be more likely to
develop the disease.
The other two blood tests mentioned are the ESR and CRP. These
blood tests measure inflammation and are typically elevated in
patients with active RA. Normal levels do not rule out RA, but those
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patients may be less likely to develop damage in their joints than
patients with high levels of inflammation, especially an elevated CRP.
An anti-nuclear antibody (ANA) is an important test in our example
patient to evaluate for systemic lupus erythematosus or SLE. While
low levels of ANA are common in RA, high levels of ANA in this
example patient may indicate possible lupus, especially if the CCP
and RF are negative.
Finally, on subsequent visits, the RF and CCP are not typically reordered if positive. On the other hand, the ESR and CRP are
frequently ordered as they can help confirm (in addition to the
patients history and exam) whether the arthritis is active or in
remission.
Anti-Cyclic Citrullinated Peptide (Anti-CCP) antibodies are very
suggestive of a diagnosis of Rheumatoid Arthritis (RA) although their
absence does not exclude this diagnosis. Anti-CCP antibodies carry a
higher predictive value than Rheumatoid Factor for:
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Diagnosis of RA
Disease activity
Radiologic damage
Rheumatoid Factor (RF) is an antibody (classically IgM) to selfimmunoglobulin. Presence of RF in serum from a patient with
suspected RA increases the likelihood of the diagnosis of RA. Of
patients with RA, those with RF positivity tend to have more
aggressive arthritis than those without RF.
Rheumatoid Factor or anti-CCP antibodies may be present prior to
the onset of arthritis (particularly anti-CCP antibodies). With
established RA more patients will become positive over time. This
leads to a higher prevalence of anti-CCP and/or RF in patients who
have established RA, and in patients that have been referred to
specialist clinics. This leads to “sensitivity” of such assays being
higher in these patient groups when compared with most general
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practice settings. The sensitivity of anti-CCP antibodies for RA (3766%) is only marginally better than of RF (17-71%). Therefore CCP
should not be used as a screening test in patients unlikely to have RA.
Some RA patients who are RF negative are positive for anti-CCP (up
to one third in one series). Anti-CCP antibodies are associated with a
higher likelihood of radiologically evident joint damage, especially
when RF is also present. The additional value of anti-CCP testing to
other disease markers (radiology, RF) is relatively less in established
RA than early RA.
In some cases, a diagnosis may not be readily apparent on an initial
evaluation. Symptoms may resolve in time, or new symptoms may
develop that provide information and lead to a diagnosis.
Once a diagnosis is established and other associated conditions
excluded, self treatment may be an option. Nonetheless, it is always
best to discuss your therapy with a physician as patients may
experience adverse effects from self treatment. For example,
NSAIDS are available over-the-counter (OTC). We know that these
medications may increase the risk of bleeding ulcers, cardiovascular,
kidney and liver disease. Taking these medications without physician
supervision may put a patient at increased risk of developing a life
threatening adverse event.
In addition, while over the counter supplements and herbs may have
benefit in helping arthritic symptoms, many of these treatments have
not been extensively studied and pose a potential risk also.
Anti-CCP antibodies cannot be used to exclude a diagnosis of RA. In
patients in whom inflammatory arthritis is likely, the presence of antiCCP antibodies is highly specific (90-98%) for a diagnosis of RA, and
consideration for DMARD therapy is important. There is increasing
evidence that patients with RA benefit from early introduction of
effective disease modifying anti-rheumatic drug (DMARD) therapy.
Patient selection is important to provide treatment to patients who
have the most to gain from therapy, especially if commenced early in
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the course of the disease before bony erosions develop. However,
when a patient presents with arthritis of recent onset, a definite
diagnosis of the underlying pathology is very difficult.
Other Conditions
Some Juvenile Inflammatory Arthritis (JIA) patients are anti-CCP
positive, but at a lower prevalence than in adult RA, and the role of
anti-CCP for JIA is not as well defined. One series of Sjögren’s
syndrome (SS) patients were positive for anti-CCP in 8% (>60% of
SS cases have RF), but 90% of these fulfilled ACR criteria for RA
(and so were likely to have RA with secondary SS).
Anti-CCP passivity has been noted in other conditions (gout,
osteoarthritis, sarcoid, psoriatic arthritis) in some series, but at low
prevalence (<10% of cases). Some of these patients may develop
clinically evident RA in the future.
Patients with Polymyalgia Rheumatica (PMR) are usually anti-CCP
negative.
Combination Approach
A combination of markers has been shown to provide more clinical
information than single tests. Markers that have been shown to have
some predictive power include: Erythrocyte Sedimentation Rate
(ESR), anti-CCP, C Reactive Protein (CRP), and RF.
Testing for anti-CCP antibodies. The assay will provide a quantitative
value in arbitrary ELISA units (EU).
Reference range: < 20 EU
Weak positive:
20-39 EU
Moderate positive: 40-59 EU
Strong positive:
>60 EU
Specimen required: Serum
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The Role of Anti-CCP in the Laboratory Diagnosis of
Rheumatoid Arthritis
Rheumatoid Arthritis (RA) is one of the most common systemic
autoimmune diseases, affecting approximately 0.5–1.0% of the world
population.1 The American Rheumatism Association criteria for the
classification of RA includes:
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morning stiffness,
arthritis of three or more joint areas,
arthritis of hand joints,
symmetric arthritis,
rheumatoid nodules,
serum rheumatoid factor (RF), and
Radiographic changes.
A patient should have four of the seven criteria to be diagnosed with
RA and the first four criteria should be present for at least six weeks.
Until recently, the only serological test routinely performed for the
detection of RA was for the presence of IgM RF. RF is found in
approximately 50%–90% of these patients, but it is also found in
patients with infections, other autoimmune diseases, and some
healthy individuals with increasing frequency in older age groups,
thus limiting its specificity for RA.
Several studies have shown that anti-perinuclear auto-antibodies,
otherwise known as anti-keratin auto-antibodies, are found in
patients with RA. It has been discovered that these antibodies
recognize an epitope that contains the deamidated form of arginine
called citrulline. Enzyme-Linked Immunosorbent Assay (ELISA)
testing for these auto-antibodies directed against anti-cyclic
citrullinated peptide (anti-CCP) is reasonably sensitive (68%) and
highly specific (98%) in patients with RA. The pathogenesis of antiCCP antibodies in rheumatoid arthritis has been shown to be
attributable to the body’s humoral response to citrulline.
Citrullination is the post-translational conversion of arginine to
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citrulline by an enzyme called peptidylarginine deiminase (PAD). See
figure 1. PAD activation is assisted by calcium ions. PAD is normally
present as inactive intracellular enzymes. During programmed cell
death (apoptosis) in the synovial joints of patients with rheumatoid
arthritis, PAD may leak out of the dying cells. Once activated, PAD
will cause citrullination of extracellular arginine. In the synovium, the
citrulline acts as an antigenic stimulant to induce anticitrullinated
protein antibodies (ACPA) locally produced by plasma cells.7 The
ELISA that detects these autoantibodies uses synthetic cyclical
citrulline peptides.
Figure 1
The original ELISA for the anti-CCP sequence was not broadly
marketed due to low sensitivity and technical complexity. However,
the second generation anti-CCP test (often referred to in the
literature as CCP-2/CCP2) shows superior performance compared to
the original peptide.8 The vast majority of the laboratories that offer
this test utilize the second-generation CCP assay.
In 2005, a third generation of anti-cyclic citrullinated peptide (CCP3)
was made available for the laboratory diagnosis of RA. These assays
have been reported to recognize additional citrulline epitopes that are
not identifiable with the second-generation CCP assays. The CCP3
assays have had reported results of up to 5% increased sensitivity
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compared to the CCP2 assays.9,10 To the contrary, however, several
publications have shown similar diagnostic performance between the
CCP3 and CCP2 assays.11,12
Recently, Nishimura et al. performed a meta-analysis of published
studies regarding the diagnostic accuracy of anti-CCP and RF for
rheumatoid arthritis.13 Their results showed a positive likelihood ratio
of 12.46 and a negative likelihood ratio of 0.36 for anti-CCP antibody
in patients with RA. The same study showed a positive likelihood
ratio of 4.86 and a negative likelihood ratio of 0.38 for RF. These
results indicate that anti-CCP positivity alone is more specific than
IgM RF for the diagnosis of RA.
In addition to diagnostic value, several studies have shown that antiCCP may also add prognostic significance in the determination of
development of erosive disease in RA.14,15sp> Kroot, EJ et al showed
that anti-CCP positive patients developed significantly more severe
radiologic damage than those patients who were anti-CCP negative.14
Although the presence of anti-CCP is not currently required for the
diagnosis of RA, future classification criteria will most likely
incorporate its use as an adjunct to IgM RF as a laboratory diagnostic
tool. Additionally, RA patients with positive CCP status may benefit
from its prognostic value by receiving earlier customized treatment
regimens that could potentially delay the development of erosive
disease.
High positivity of anti-CCP antibodies in patients with Down
syndrome.
The aim of the present study was to evaluate the prevalence of anticyclic citrullinated peptide (CCP) antibodies in patients with Down’s
syndrome (DS) previously tested for IgM rheumatoid factor (RF) and
to correlate the results with clinical findings. Eighty-eight patients
with DS previously tested for IgM-RF were divided into two groups
matched for sex and age. Group A consists of 42 RF positive patients
and group B of 44 RF negative patients. The presence of anti-CCP
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antibody was determined using a second-generation enzyme-linked
immunosorbent assay. A total of 52.3% (45/86) of DS patients were
positive for anti-CCP antibodies. Twenty-four patients (57.1%) of the
RF positive group and 21 (47.7%) of the RF negative group
presented anti-CCP circulating antibodies. The concordance between
both tests was 54.6%. None of the patients had clinical evidence of
rheumatoid arthritis or juvenile idiopathic arthritis.
Although a high prevalence of anti-CCP antibodies was observed in
DS patients, no association has been found presently with clinical
disease. Careful follow-up of these patients will be necessary to clarify
the real significance of these findings.
Usefulness of anti-CCP antibodies in patients with hepatitis C
virus infection with or without arthritis, rheumatoid factor, or
cryoglobulinemia.
The presence of antibodies to cyclic citrullinated peptide (CCP) has
high specificity in the diagnosis of rheumatoid arthritis (RA).
Hepatitis C virus (HCV) infection may induce extra-hepatic
manifestations, such as polyarthritis that mimic RA. The aim of this
study was to determine the prevalence of anti-CCP antibodies in
HCV-infected patients with or without arthritis, rheumatoid factor
(RF), or cryoglobulinemia and to investigate whether anti-CCP
antibodies may be helpful in discriminating patients with RA from
patients with HCV-associated arthropathy. A total of 44 patients with
RA, 34 patients with HCV infections, and 42 control patients with
non-RA rheumatic diseases were recruited for the study. Anti-CCP
antibody levels were determined by enzyme-linked immunosorbent
assay. We found that, consistent with other reports, patients with RA
were more likely to have high titers of anti-CCP antibody than HCVinfected or control patients. A significant number of HCV-infected
patients with neither RF nor cryoglobulinemia were also positive for
anti-CCP antibodies (the three positive values were 36.10, 8.65, and
5.83 U/ml, P < 0.01 compared with the control patients). The
presence of cryoglobulinemia and/or RF in HCV-infected patients
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did not affect the anti-CCP outcomes. Although anti-CCP antibodies
remain to be a very useful tool in discriminating RA from non-RA,
HCV-infected patients with neither RF nor cryoglobulinemia may
have anti-CCP antibodies. Because of limited patient numbers, this
tentative conclusion may need further confirmation with inclusion of
more patient population.
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