Rheumatoid arthritis is an autoimmune disease that causes inflammation and destruction of joints. Early diagnosis through laboratory tests like rheumatoid factor, anti-CCP antibodies, erythrocyte sedimentation rate, and C-reactive protein is important so aggressive treatment can be started to control symptoms and prevent joint damage. The 2010 ACR/EULAR classification criteria uses scores based on the number and type of joints involved, serology results, markers of inflammation, and duration of symptoms to classify a patient as having rheumatoid arthritis.

1. Role of Laboratory Test in the
Diagnosis of Rheumatoid Arthritis
Kusworini Handono
Medical Faculty, Brawijaya University
Malang

2. Rheumatoid Arthritis
• Systemic rheumatic autoimmune
• Progressive and irreversible joints destruction
• May have potentially devastating consequences
• Average lifetime earnings loss = 50%
• 40%-85% of RA patients will be unable to work within 8-10
years of disease onset
• Early diagnosis and aggressive management can lead to
successful control and remission

3. Epidemiology
• Prevalence:  0.8% of the population
• Affects all races, prevalence increases with age (incidence
of RA is > 6x in 60-64 yo ♀ compared to 18-29 yo ♀)
• Onset is most frequent during the 4th and 5th decades of
life (80% at 35 – 50 yo)
• ♀ : ♂ = 3 : 1 (sex differences diminish in the older age
group)

4. Etiology
The cause of RA remains unknown
• Genetic predisposition : HLA-DR1, HLA-DR4
• Environmental
• Smoking
• Infectious agents : mycoplasma, EBV, CMV, parvovirus,
rubella virus
• Hormonal
• Immunologic

5. Pathogenesis of Rheumatoid Arthritis
Rheumatoid
Factors,
B cell anti-CCP Immune complexes
Complement
T cell
IFN- &
Neutrophil
other
Antigen- cytokines Macrophage
presenting
cells
Mast cell
B cell or
macrophage Synoviocytes TNF Chondrocytes
IL-1
Pannus
Osteoclast
Production of collagenase and other
Articular neutral proteases
cartilage Bone
Adapted from Arend WP, Dayer JM. Arthritis Rheum. 1990;33:305–15

6. Pathophysiology
Healthy joint Rheumatoid joint
Femur
 T cells
Capsule
 B cells
Cartilage Plasma
cell  IL-6
Synovial
membrane  TNF-
Synovial villi
 IL-1
Tibia Angiogenesis Pannus
Synoviocytes Neutrophils
Eroded
bone
1. Choy E and Panayi G. N Eng J Med 2001;344:907-916

7. Radiographic features
Characteristic pattern:
• Small joints involvment : PIP, MCP, wrist
• Juxtaarticular osteopenia
• Loss of articular cartilage
and bone erosions

9. Factors Suggesting Poor Prognosis
• >20 swollen joints • Joint erosions
• High RF titer • Presence of rheumatoid
• Elevated anti-CCPs nodules
• Elevated ESR • Socioeconomic
• Elevated CRP characteristics
• Late implementation of • Smoking
treatment • Poor functional status

10. Laboratory Diagnostic
• No laboratory tests are specific for diagnosing RA
• Rheumatoid factors
• Sensitivity 60 - 90 %, Specificity 50 - 60 %
• The presence of rheumatoid factor is not specific for RA
• RF is found in 5% of healthy persons
• Number of conditions besides RA are associated
with the presence of RF

11. Laboratory Diagnostic
• Antibodies to CP
• Similar sensitivity and a better specificity for RA than RF
• Can be found in ± 1.5% of normal individuals
• A useful test to confirm a diagnosis of RA and to estimate
prognosis
• CBC
• ESR
• Acute-phase reactant : CRP

12. 1987 revised ACR criteria for
classification of rheumatoid arthritis (RA)
1. Morning stiffness in and around joints lasting at least 1 hour before maximal
improvement;
2. Soft tissue swelling (arthritis) of 3 or more joint areas observed by a physician;
3. Swelling (arthritis) of the proximal interphalangeal, metacarpophalangeal or
wrist joints;
4. Symmetric swelling (arthritis)
5. Rheumatoid nodules
6. The presence of rheumatoid factor (RF)
7. Radiographic erosions and/or periarticular osteopenia in hand and/or wrist joints
• Criteria 1 through 4 must have been present for at least 6 weeks.
• Rheumatoid arthritis is defined by the presence of 4 or more criteria, and no further qualifications (classic,
definite, or probable) or list of exclusions are required. In addition, a “classification tree” schema is
presented which performs equally as well as the traditional (4 of 7) format. The new criteria demonstrated
91–94% sensitivity and 89% specificity for RA when compared with non-RA rheumatic disease control
subjects.
Arnett FC et al, Arthritis Rheum 31:315-324,1988

15. Classification criteria for RA (score-based algorithm:
add score of categories A-D; a score of ≥ 6/10 is
needed for classification of a patient as having definite
RA)
A. Joint involvement
B. Serology
C. Acute phase reactants
D. Duration of symptoms
Aletaha et al. Arthritis & Rheumatism.2010;62:2569-81

16. A.Joint involvement Score
1 large joint 0
2-10 large joints 1
1-3 small joints (with or without involvement of large joints) 2
4-10 small joint (with or without involvement of large joints) 3
>10 joints (at least 1 small joint) 5
B.Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low positive RF or low positive ACPA 2
High positive RF or high positive ACPA 3
C.Acute Phase Reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
D.Duration of symptoms
< 6 weeks 0
≥ 6 weeks 1
Aletaha et al. Arthritis & Rheumatism.2010;62:2569-81

17. Marker of Inflammation : Acute Phase Reactan
• Erythrocyte Sedimentation Rate / ESR
OR
• C - Reactive Protein / CRP

18. Erythrocyte Sedimentation Rate (ESR)
• ESR is the rate at which red blood cells
precipitate in a period of 1 hr.
• Test based on : inflammatory and necrotic
processes cause an alteration in blood
proteins  aggregation of RBCs
• Recent method  automatic

19. Erythrocyte Sedimentation Rate (ESR)
• Reference values :
Men : 0-15 mm/h
Women : 0-20 mm/h
Child : 0-10 mm/h
• Indonesian Reference value : ???
• ACR/EULAR 2010 criteria :
Normal : score 0
Abnormal : score 1

20. C-reactive protein (CRP)
• CRP is a protein found in the blood in response
to inflammation (an acute-phase protein)
• Produced by the liver and adipocytes
• Increment is due to a rise of TNFα, IL-6, which
is produced predominantly by macrophages as
well as adipocytes.
CRP is an annular
pentameric disc in
shape.

21. C-reactive protein (CRP)
• Reference value : < 0.3 mg/dl
• Indonesian Reference value : ???
• ACR/EULAR 2010 criteria :
Normal : score 0
Abnormal : score 1

22. Serology Markers
• Rheumatoid Factor / RF
OR
• Anti Citrullinated Protein Antibody / ACPA

23. Rheumatoid Factor
• Autoantibodies reactive with epitopes in the Fc portion of
IgG
• IgM, IgG and IgA isotype
• The RF assay : Aglutination, nephelometry, ELISA, ICT
• In the general population, a test sensitivity is 60- 90% and
specificity of 50-60%

24. IgM Rheumatoid Factor

25. Other causes of RF positivity
l Other connective tissue l Tuberculosis
diseases
l Liver diseases
l Viral infections
l Sarcoidosis
l Leprosy
l Mixed essential
l Leishmaniasis cryoglobulinemia
l Subacute bacterial
endocarditis
Akill M, Amos RS, BMJ 310:587–590, 1995

26. Rheumatoid Factor
• Reference value : 1 : 20 or 23 u/mL
• Indonesian reference value : ????
• ACR/EULAR 2010 criteria
Negative : less than or equal to the ULN  score 0
Low positive : higher than ULN but < 3 x ULN  score 2
High positve : higher than 3 x ULN  score 3
• If the RF value only in -/+, a positive result should be scored
as low positive.

27. Promising biomarkers for RA :
ACPAs (Anti-Citrullinated Protein-Antibodies)
• Anti – PF (Perinuclear Factor)
• Anti - Keratin
• Anti - Fillagrin
• Anti - citrullinated Fibrin/Fibrinogen
• Anti - citrullinated alpha enolase
• Anti - CCP 1
• Anti - CCP 2
• Anti - CCP 3
• Anti - CCP 3.1
• Anti - CCP 3.2
• Anti - Vimentin (Anti-Sa)
• Anti - MCV (Mutated Citrullinated Vimentin)

28. Anti Cyclic Citrullinated Peptide Antibodies
(anti-CCP)
• Antibodies reactive with citrullinated fillagrin epitopes
• Useful in detecting patient with early RA (sensitivity 50-70%,
specificity 95-98%)
• Detection assay : ELISA, ICT
• 1 st generation CCP : Ag pure cit fillagrin (sen 60 – 70 %, spec
85 %)
• 2 nd generation CCP : Ag synthetic (spec 96 %)
• 3 rd generation CCP : Ag ?? (spec 98%)

30. Reference Value (Quanta Lite)
• Negative :  20 U
• Weak Positive : 20 – 39 U
• Moderate Positive : 40 – 59 U
• Strong Positive :  60 U
• Indonesian Reference Value ???
30

31. ACR/EULAR 2010 : ACPA
• Negative : less than or equal to the ULN  score 0
• Low positive : higher than ULN but < 3 x ULN  score 2
• High positve : higher than 3 x ULN  score 3

32. Detection of Anti Citrunillated Protein Antibody (ACPA) using
anti-CCP2 and anti-CCP3 in patients with Rheumatoid Arthritis
in Saiful Anwar Hospital
(Kusworini Handono et al, 2010)
• 59 RA patients (ACR 1987), 83.1% women, age 50.13 ± 11.0 yo,
dur of symptom 8.5 ± 6.6 mo, joints involvement 5.6 ± 2.2
• ACPA detection using anti-CCP2 and anti-CCP3 (Quanta Lite)
• Result :
- No difference level of anti-CCP2 and anti-CCP3 in RA
patients (148.06 vs 185.01 U/ml ; p<0.05)
- Sensitivity of anti-CCP2 55,9%
- Sensitivity of anti-CCP3 66,1%
• Conclusion : anti-CCP3 slightly sensitive than anti-CCP2 in
detection of ACPA

33. Rheumachec : World First Rapid Test for
Rheumatoid Arthritis (RA)
• Rapid test–immediate answer : 15 minutes to the result
• „Point of Care Testing“ (POCT) : Using whole blood, right at
the side of the patient
• According to the instruction for use : “for professional use
only” – not for use by the patient
• IVD (in vitro diagnostics) for rheumatoid arthritis
• Use 2 markers : anti-MCV and RF

34. Biomarkers used for rheumachec - 2-1. :
Anti-MCV
• Vimentin : part of cytoskeleton, is citrullinated by
macrophages during apoptosis, triggers autoantibodies
• One of the native citrullinated isoforms is MCV, a highly
specific marker for RA
• Native protein with 45 potential epitopes, compared to the
synthetic construct CCP with 1-2 epitopes only

36. Conclusion
• RA is an autoimmune rheumatic desease with progressive
and irreversible joints destruction
• Early diagnosis and agressive theraphy are important lead to
better outcomes
• Dx RA -> ACR/EULAR 2010 criteria based on 4 domain : joint
involvement, serology (RF and ACPA), acute phase reactans
(ESR and CRP) and duration of symptoms
• Measurement of ACPA : anti-CCP or anti-Vimentin have a
good sensitivity and specifisity