RHEUMATOID FACTOR AND ANTI-CCP ANTIBODY

EXAMINATION FOR DIAGNOSIS OF

RHEUMATOID ARTHRITIS

Diah Nurul Hidayati, S.Ked

I1A005037
Department of Internal Medicine of Ulin General Hospital Banjarmasin

Abstract
Rheumatoid Arthritis (RA) is the commonest inflammatory joint disease,
affecting nearly1% of the adult population worldwide. It is characterized by
multiple deformities and is associated with considerable morbidity and mortality.
The etiology of rheumatoid arthritis is not fully understood.The RF autoantibody
system, directed against the Fc part of immunoglobulin (Ig) G molecules, has had
a central role in the diagnosis and prognosis of RA.Positive RF was found in 80%
of those with rheumatoid arthritis.The first generation CCP test (CCP1) used in
early diagnostic studies (2000–2001) contained a single cyclic citrullinated
peptide derived from filaggrin as the substrate It could detect ACPA in 68% of
patients with established RA with a very high specificity (98%). To improve the
sensitivity of the CCP test, several dedicated libraries of citrulline-containing
peptides were screened with a pool of RA sera. This screening finally lead to the
development of the second generation CCP test (CCP2) that displays sensitivities
comparable to that of RF (approximately 80%) but with superior specificity
(98%). The comparison of two currently used CCP ELISAs in patients with early
arthritis, the CCP2 test had better diagnostic properties and prognostic relevance
than the CCP1 test.

Key Words: Rheumatoid arthritis, rheumatoid factor, anti-citrullinated protein

antibodies

INTRODUCTION

Rheumatoid Arthritis (RA) is the commonest inflammatory joint disease,

affecting nearly1% of the adult population worldwide. It is characterized by
multiple deformities and is associated with considerable morbidity and mortality .1
The morbidity and mortality it causes are a consequence of local and systemic
inflammatory processes that damage cartilage, bone and soft tissue, as well as
blood vessels and viscera .2
Onset usually occurs between 30 and 50 years of age. Incidence in the
United States is estimated as 25 per 100,000 persons for men and 54 per 100,000
persons for women. Rheumatoid arthritis is responsible for an estimated 250,000
hospitalizations and 9 million physician visits each year .3

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 Female sex, a positive family history, older age, silicate exposure, and
smoking are associated with an increased risk for developing rheumatoid arthritis.
Consumption of more than three cups of coffee daily—particularly decaffeinated
coffee—also may contribute. High vitamin D intake, tea consumption, and oral
contraceptive use are associated with decreased risk.3
The etiology of rheumatoid arthritis is not fully understood. Evidence points
to a complex interplay between environmental and genetic factors. In
monozygotic twins, there is a more than 30 percent concordance rate for
rheumatoid arthritis development .3 The precise etiology of RA remains unknown,
there is strong evidence for autoimmunity since several autoantibodies are
associated with the disease.1
Better molecular markers for diagnosis and prognosis are needed to identify
RA patients earlier and fine-tune therapeutic choices to the individual patient.
Serological testing for rheumatoid factor is complicated by moderate sensitivity
and specificity,and high rates of positivity in other chronic inflammatory.3
Rheumatoid factor (RF) has been widely used as a screening test for patients with
arthritis. Although RF is prognostically useful, as it correlates with functional and
radiographic outcomes in both RA and early inflammatory polyarthritis , as a
diagnostic test it performs poorly, with low sensitivity and moderate specificity.4
Besides the rheumatoid factor (RF), another group of autoantibodies has recently
been detected in serum of patients with RA patients: the anti-cyclic citrullinated
peptide antibodies (anti CCP).1

Diagnostic criteria
In clinical trials, rheumatoid arthritis is diagnosed formally using seven
American Rheumatism Association (ARA) criteria in figure 1. In typical
outpatient practice, a definitive diagnosis using these criteria may be difficult to
obtain early in the disease process. During the initial visit, patients should be
asked about degree of pain, duration of stiffness and fatigue, and functional
limitations. A careful joint examination looking for the characteristics described
above is vital.3

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 Figure 1. ARA criteria for rheumatoid arthritis 3

Pathophysiology of Rheumatoid Arthritis

The synovial membrane in patients with rheumatoid arthritis is characterized by
hyperplasia, increased vascularity, and an infiltrate of inflammatory cells,
primarily CD4+ T cells, which are the main orchestrator of cell-mediated immune
responses. Antigen-activated CD4+ T cells stimulate monocytes,macrophages,
and synovial fibroblasts to produce the cytokines interleukin-1, interleukin-6, and
TNFa and to secrete matrix metalloproteinases through cell-surface signaling by
means of CD69 and CD11 as well as through the release of soluble mediators
such as interferon-γ and interleukin-17. Interleukin-1, interleukin-6, and TNFα are
the key cytokine that drive inflammation in rheumatoid arthritis. 5,6 Most of these
cytokines, including TNFa and interleukin-1, can be detected in synovial fluid
from patients with rheumatoid arthritis. Both TNFa and interleukin-1 are likely to
have primary roles in the pathogenesis of rheumatoid arthritis. The serum and
synovial concentrations of both cytokines are high in patients with active
rheumatoid arthritis.5 Furthermore, TNFa and interleukin-1 are potent stimulators
of mesenchymal cells, such as synovial fibroblasts, osteoclasts, and chondrocytes,
that release tissue-destroying matrix metalloproteinases. Interleukin-1 and TNFa
also inhibit the production of tissue inhibitors of metalloproteinases by synovial
fibroblasts.These dual actions are thought to lead to joint damage. Perhaps by
inducing the production of interleukin-11, TNFa stimulates the development of
osteoclasts, which are responsible for bone degradation.5
These activated macrophages, lymphocytes, and fibroblasts,as well as their
products, can also stimulate angiogenesis, which may explain the increased

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vascularity found in the synovium of patients with rheumatoid arthritis.
Endothelial cells in the synovium are activated and express adhesion molecules
that promote the recruitment of inflammatory cells into the joint. This process is
enhanced by the release of chemokines, such as interleukin-8, by inflammatory
cells in the joint. Finally, joint damage, associated with the development of locally
invasive pannus tissue, occurs through the actions of proteases, growth factors,
and activated osteoclasts. 5,6

Figure 2. Cytokine pathways in inflammatory arthritis5

RACTION OF THE RHEUMATOID FACTOR WITH ANTIGENANTIBODY

Pathophysiology Joint Damage in RA
Rheumatoid arthritis is characterized by progressive joint damage that is
mediated by several mechanisms (Fig. 2 and 3). Early erosion of cartilage and
bone is associated with the formation of a proliferating pannus. The interface
between pannus and cartilage is occupied predominantly by activated
macrophages and synovial fibroblasts that express matrix metalloproteinases and
cathepsins.5
Interleukin-1 and TNF-a stimulate the expression of adhesion molecules
on endothelial cells and increase the recruitment of neutrophils into the joints.

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Neutrophils release elastase and proteases, which degrade proteoglycan in the
superficial layer of cartilage.The depletion of proteoglycan enables immune
complexes to precipitate in the superficial layer of collagens and exposes
chondrocytes.Chondrocytes and synovial fibroblasts release matrix
metalloproteinases when stimulated by interleukin-1, TNF-a, or activated CD4+ T
cells. Matrix metalloproteinases, in particular stromelysin and collagenases, are
enzymes that degrade connective-tissue matrix and are thought to be the main
mediators of joint damage in rheumatoid arthritis. In animals, activated CD4+ T
cells stimulate osteoclastogenesis, and they may cause joint damage
independently of interleukin-1 and TNF-a in patients with rheumatoid
arthritis.5CTERACTION OF THE RHEUMATOID FACTOR WITH
ANTIGENANTIBO
COMPLEXES AN

D
AGGREGATED GAMMA
G Figure 3. Pathophysiology of Rheumatoid arthritis 5

INTERACTION OF THE RHEUMATOID FACTOR WITH ANTIGENANTIBO

Examination for Rheumatoid arthritis
Rheumatoid Factor (RF)
The key to early recognition of autoimmunity lies within the humoral
immune system. Since blood samples are taken from clinic-visiting (pre-)patients,
screening for serological RA-indicators can be performed quite easily. In the
American College of Rheumatology (ACR) criteria for RA one serological marker

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is included: rheumatoid factor (RF). The RF autoantibody system, directed against
the Fc part of immunoglobulin (Ig) G molecules, has had a central role in the
diagnosis and prognosis of RA during recent decades because RF can be detected
in the majority of RA patients. However, it is becoming more and more clear that
the presenc of RF is not restricted to patients with RA, but that it can also be
detected in subsets of patients suffering from other diseases and even in a
percentage of healthy (especially elderly) individuals 7.
Rheumatoid factor (rheumatoid factor, RF) are immunoglobulins that react
with IgG molecules. Because the patient also contain IgG in serum, the RF
including autoantibodi. RF causing factors is unknown for sure, although
complement activation due to the interaction of RF with IgG an important role in
rheumatoid arthritis (rheumatoid arthritis, RA) and other diseases with a positive
RF. Most of the IgM RF, but can also be IgG or IgA. 8 The combined detection of
IgM and IgA RFs in a serum is a strong indicator of RA. However, IgA RFs are
not widely available.1
Positive RF was found in 80% of those with rheumatoid arthritis. RF
levels are very high indicating a poor prognosis with severe joint disorders and the
possibility of systemic complications. 8
RF often found in other autoimmune diseases, such as LE, scleroderma,
dermatomiositis, but levels are usually lower than levels of RF in rheumatoid
arthritis. Low levels of RF are also found in non-immunological diseases and the
elderly (above 65 years). 8
RF test is not used for monitoring treatment for common test results
remain positive, despite clinical recovery has occurred. Also, it took about 6
months for a significant increase in titer. For the diagnosis and evaluation of RA
often used CRP and ANA tests. RF test patients for serum examined using Latex
Agglutination method or nephelometry and ELISA.8,9 According to the FDA, latex
agglutination is sensitive but can result in a fairly high number of false positives.
The ELISA and nephelometry methods have the advantage of objective
instrument measurement on a single sample dilution.9
Reference Values in adult; chronic inflammatory disease; 1/20-1/80
positive for rheumatoid arthritis conditions and other diseases;> 1 / 80 positive for
rheumatoid arthritis; child: usually not done  and elderly: slightly increased .
Value of reference may be different for each laboratory, depending on the method
used. 8

Limitations of Rheumatoid Factor (RF)

RF are antibodies directed to the constant region of immunoglobulins of
the IgG class and are found in 70-80 % of patients with RA. IgM RF, the isotype
most typically detected, is seen not only in RA but also in various other conditions
like other autoimmune diseases, infections and in up to 5-10% of healthy
individuals. The combined detection of IgM and IgA RFs in a serum is a strong
indicator of RA . However, IgA RFs are not widely available.1

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 High levels of RF can happen in rheumatoid arthritis, LE, dermatomiositis,
scleroderma, infectious mononucleosis, leukemia, tuberculosis, sarkoidosis,
cirrhosis of the liver, hepatitis, syphilis, chronic infections, the elderly.8
RF test results are often still found to be positive, regardless of whether clinical
recovery has occurred. RF test results can be positive in a variety of clinical
problems, such as collagen disease, cancer, cirrhosis of the liver. Elderly may
have increased titer of RF, without suffering from any disease.8
Although RF antibodies can be detected in the majority of RA patients,
they are not very specific for RA . Because of their early presence and high
specificity, anti-CCP antibodies represent a superior marker for the diagnosis and
prognosis of RA.10
The Anti-Cyclic Citrullinated Peptide Antibodies (Anti CCP)
The history of anti-citrullinated protein antibodies started exactly four
decades ago when the APF (anti-perinuclearfactor) antibodies were described.
These antibodies, as well as the independently described anti-keratin antibodies
(AKA)2. In 1979, Young et al. reported that RA contained antibodies that reacted
to the keratinized layer of epithelium. These antibodies were called antikeratin
antibodies, and were only found in RA patients.2 Subsequent studies demonstrated
that anti-keratin antibodies and antiperinuclear factor recognized a similar epitope,
and were perhaps the same antibody. It was also discovered that conversion of
arginine to citrulline on peptides was essential for anti-keratin antibody and
perinuclear factor binding. Therefore, antiperinuclear factor and anti-keratin
antibodies can be broadly categorized as anti-citrullinated-peptide antibodies.2The
first citrulline-binding autoantibodies in RA were discovered by Nienhu is et al. In
1964, as an autoantibody able to bind to perinuclear granules in normal human
buccal mucosa cells, and were named antiperinuclear factor. Antiperinuclear
factor was found in 48% of patients with RA, and only 1% of healthy controls.2
The first generation CCP test (CCP1) used in early diagnostic studies
(2000–2001) contained a single cyclic citrullinated peptide derived from filaggrin
as the substrate It could detect ACPA (anti-citrullinated protein/peptide
antibodies) in 68% of patients with established RA with a very high specificity
(98%). Because filaggrin is not expressed in the synovium, it is most likely not the
natural citrullinated antigen for ACPA. Other peptides, not related to filaggrin,
could therefore potentially provide better epitopes for detection of ACPA. Via
screening of a number of peptide libraries, novel citrullinated peptides were
obtained and incorporated into a second generation CCP test (CCP2).11
A major breakthrough came from the development of an ELISA that used
a synthetic citrullinated peptide derived from filaggrin . This procedure greatly
simplified the detection of anti-citrullinated protein antibodies and allowed for
better standardization. The use of a cyclic filaggrin derived peptide, in which the
citrulline-containing epitope is optimally exposed, resulted in an enhanced
sensitivity . Using this first generation CCP test (CCP1) a sensitivity of 68% could
be reached with a satisfying high specificity for RA of 97-98% . Studies using the
CCP1 test have been reviewed by van Boekel et al12 . To improve the sensitivity
of the CCP test, several dedicated libraries of citrulline-containing peptides were
screened with a pool of RA sera. This screening finally lead to the development of

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the second generation CCP test (CCP2) that displays sensitivities comparable to
that of RF (approximately 80%) but with superior specificity (98%).10
The literature of the last 4 years confirms that the anti-CCP2 test is a very
useful marker for the early and specific diagnosis of rheumatoid arthritis (RA).
Walther J. et al 11 studies said that the anti-CCP2 test is very specific for RA (95–
99%) and has sensitivity comparable to that of the rheumatoid factor (70–75%).
The antibodies can be detected very early in the disease and can be used as an
indicator for the progression and prognosis of RA11.Van Gaalen FA et al 13 have
compared the two currently used CCP ELISAs in patients with early arthritis. The
CCP2 test had better diagnostic properties and prognostic relevance than the
CCP1 test. 13

Clinical use of anti-CCP antibodies

The current therapeutic strategy uses increasingly aggressive regimens
early in the course of the disease as most of bony damage occurs in first two years
in 90% of patients. Thus, early diagnosis is crucial. The 1987 American College
of Rheumatology (ACR) criteria are rarely met during the first few months of the
disease. 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 is important. Anti-CCP
antibodies may be detected in roughly 50-60% of patients withearly RA usually
after 3-6 months of symptoms.10
Anti-CCP antibodies were positively correlated with higher erythrocyte
sedimentation rate (ESR), C-reactive protein (CRP), swollen joint count, and
worse physician global assessment ratings. Presence of rheumatoid factor was
positively correlated with increased ESR and CRP, but there was no association
with other disease activity markers. Statistically significant correlations were seen
between anti-CCP positivity and higher CRP, ESR, and disease activity
measurements.2 Titres were more likely to decrease in patients showing a greater
degree of clinical improvement. In summary, a decrease in anti-CCP titre can be
seen with RA treatment, however, the decrease is usually modest and should not
drive treatment decisions. Anti-CCP positive patients usually remain positive
despite treatment.2
Several observations have indicated that anti-CCP positive early RA
patients may develop more erosive disease than those without anti-CCP. Other
investigators have confirmed this,and suggested the superiority of anti-CCP over
IgM RF in predicting an erosive disease course. The likelihood of a total Sharp
score increase after six years was significantly greater among patients with anti-
CCP but not rheumatoid factor. Anti-CCP has been shown to be an independent
predictor of radiological damage and progression. The combination of anti-CCP
and IgM RF increased the ability to predict erosive and progressive disease in
another study.10

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CONCLUSION

Rheumatoid arthritis (RA) is a severe, progressive, systemic inflammatory

disease of unknown etiology. Although the precise etiology of RA remains
unknown, there is strong evidence for autoimmunity since several autoantibodies
are associated with the disease. Examination that Commonly use for diagnose RA
is rheumatoid factor (RF) test. Positive RF was found in 80% of those with
rheumatoid arthritis. RF often found in other autoimmune diseases, such as Lupus
Eritematous, scleroderma, dermatomiositis . So it is not to spesific for diagnose
RA. Besides the rheumatoid factor (RF), another group of autoantibodies has
recently been detected in serum of patients with RA patients: the anti-cyclic
citrullinated peptide antibodies (anti CCP). First generation CCP test (CCP1) a
sensitivity of 68% . To improve the sensitivity of the CCP test, several studies
have found the development of the second generation CCP test (CCP2) that
displays sensitivities comparable to that of RF (approximately 80%) but with
superior specificity (98%). Comparison two generation of anti-cyclic citrullinated
peptide antibodies have shown that anti-CCP2 test had better diagnostic properties
and prognostic relevance than the CCP1 test

REFERENCE
1. Pooja Khosla, S Shankar, Lalit Duggal. Anti ccp antibodies in rheumatoid
arthritis. J Indian Rheumatol Assoc 2004 ; 12 ; 143 -46
2. Niewold T.b., m.j. harrison ,paget. Anti-CCP antibody testing as a
diagnostic and prognostic Tool in rheumatoid arthritis. Q J Med 2007;
100;193–201
3. Rindfleisch J. Adam, m.d., daniel muller,. Diagnosis and Management of
Rheumatoid Arthritis. 2005 ; 72 ; 1037-47
4. Quinn M. A.et al. Anti-CCP antibodies measured at disease onset help
identify seronegative rheumatoid arthritis and predict radiological and
functional outcome. Rheumatology 2005;45:478–80
5. Ernest H.S, Choy M.D, Gabriel S, Panayi M.D .Cytokine pathways and
joint inflammation in rheumatoid arthritis. N Engl J Med, 2001;344;12;
907-16
6. Scott , Kingsley . Tumor Necrosis Factor Inhibitors for Rheumatoid
Arthritis. n engl j med 2006;7;704-12
7. Zendman A.JW, van Venrooij and Pruin. Use and significance of anti-
CCP autoantibodies in rheumatoid arthritis. Rheumatology 2006;45;20–25
8. Anonymous, Faktor reumatoid .2010. http://labkesehatan.blogspot.com
9. Sullivan Ellen. Rheumatoid Arthritis:Test for Anti-CCP Antibodies
Joining RF Test as Key Diagnostic Tools. Labmedicine . 2006;37 ;1;17-9
10. Pruijn, Erik R. Vossenaar, Jan W. Drijfhout, Walther J. van
Venrooij,Albert J.W. Zendman. Anti-CCP Antibody Detection Facilitates
Early Diagnosis and Prognosis of Rheumatoid Arthritis. Current
Rheumatology Reviews, 2005;1; 1-7

9
11. Walther J., van Venrooij, Albert J. W. Zendman. Anti-CCP2 Antibodies:
An Overview and Perspective of the Diagnostic Abilities of this
Serological Marker for Early Rheumatoid Arthritis. Clinic Rev Allerg
Immunol ,2008; 34:36–39
12. Van Boekel MA, Vossenaar ER, van den Hoogen FH, van Venrooij WJ.
Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and
diagnostic value. Arthritis Res 2002; 4:87-93.
13. Van Gaalen FA, Visser H, Huizinga TW. A comparison of the diagnostic
accuracy and prognostic value of the first and second anti-cyclic
citrullinated peptides (CCP1 and CCP2) autoantibody tests for rheumatoid
arthritis. Ann Rheum Dis. 2005;64:1510-2.

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