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INTRODUCTION
Total Concept Diagnosis of Rheumatoid Arthritis
PT SALI POLAPA BERSAMA
By : Herlangi Sukma
RHEUMATOID ARTHRITIS
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Manifestation• Joint swelling
(symmetrical)• Pain• Loss of mobility• Fatigue
Consequences• Destruction of cartilage• Erosion of bones• Deformation• Disability• Death
RHEUMATOID ARTHRITIS
DESTRUCTIVE DISEASE
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Rheumatic Diseases
Rheumatism• „Rheuma“ is derived from the Greek
and describes a flowing pain
• A non-specific term for medical problems affecting the heart, bones, joints, kidney, skin and lung no injury or tumor
• Rheumatism, about 400 diseases, that often differ significantly in cause, symptoms, disease progression, therapy and health consequences
Rheumatoid Arthritis
PT. SALI POLAPA BERSAMA
• Morning stiffness lasting for at least 1 hr.
• Arthritis of 3 or more joint areas
• Arthritis of hand joints
• Symmetric arthritis
• Rheumatoid nodules
• Serum Rheumatoid Factor (RF)
• Typical radiographic changes
Arnett FC et al, Arthritis Rheum 31:315-324,1988
Classification criteria of RA (ACR-1987, rev.)
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What happens ?• Swelling of the synovial lining, causing
pain, stiffness, redness and swelling around the joint
• Rapid division and growth of cells (pannus), which causes the synovium to thicken
• Inflamed cells release enzymes that damage, bone & cartilage. The involved joint looses its shape and alignment
• Pain and loss of movement
Rheumatoid Arthritis
Rheumatoid Arthritis
RA/RIS/09-07PT. SALI POLAPA BERSAMA
Stages of Rheumatoid Arthritis
RA/RIS/09-07
Early Intermediate Late
RHEUMATOID ARTHRITISRHEUMATOID ARTHRITIS
Clinician Treatment :1. Symptoms
2. Causative
RA/RIS/09-07
RHEUMATOID ARTHRITISRHEUMATOID ARTHRITIS
Anamnesis and Clinical picture
Morning stiffness of jointsSymmetrical joint inflammationRheumatoid nodulesFeverMyalgia
Laboratory Tests :ESRCRPRheumatoid FactorsAnti-CCP
Imaging Technologies
X-RayUltrasoundMRT
- Medication- Fisiotherapy
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Rheumatoid Arthritis
Key facts• Rheumatoid arthritis (RA) is a chronic,
progressive and disabling autoimmune disease• RA causes stiffness, pain, loss of mobility,
inflammation, and erosion in the joints• RA affects 1 % of the population• over 40 % of RA patients are disabled within 3 years * RA is an autoimmune disease
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What is an Autoimmune Disease ?
• When your body is attacked ...
• .... by bacteria, viruses or parasites ....
• .... your immune system defends you
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The Loss of Immunological Tolerance
• Normal• Immune system attacks foreign antigens
but tolerizes the own cells
• Pathological• Immune system attacks foreign and self
• antigens and destroys the tissues and organs -
+
Autoimmune Disease ?
PT SALI POLAPA BERSAMA
This is Autoimmunity
Prevalence of Autoimmune Diseases?
According toRose et al., 1998, The Autoimmune diseases, 3rd Ed.US Census Bureau, Int. Data Base, 2004
Total Prevalence of Autoimmune diseases
• Canada - 1.0 Mio.• Brazil - 5.8 Mio.• Mexico - 3.3 Mio.• Germany - 2.6 Mio.• France - 1.9 Mio.• Russia - 4.5 Mio.
• Iran - 2.1 Mio.• Egypt - 2.4 Mio.• Ethiopia - 2.3 Mio.• India - 33.3 Mio• China - 41.0 Mio.• Thailand - 2 Mio.
PT SALI POLAPA BERSAMA
Prevalence of Autoimmune Diseases?
Autoimmune Disease (AID) Prevalence• Autoimmune thyroid disease 2.0 % • Rheumatoid arthritis 1.0 %• Systemic Lupus erythematodes 0.5 %• Celiac disease 0.4 %• Pernicious anemia 0.1 %
PT SALI POLAPA BERSAMA
J. DARMAWAN*,, K. D. MUIRDEN , H. A. VALKENBURG* and R. D. WIGLEY , 1993
0,2 % in 4683 rural and 0,3 % in 1071 urban in Semarang - Indonesia
What is an Autoimmune Disease ?
Organ specific• Hashimoto‘s
Thyroiditis• Celiac disease• Pernicious anemia• Goodpasture‘s
syndrome• AI Liver Diseases
Systemic• Rheumatoid arthritis• Systemic Lupus
Erythematodes (SLE)• Antiphospholipid
syndrome (APS)• Wegeners‘s
granulomatosis
PT SALI POLAPA BERSAMA
Laboratory investigations• ESR / CRP level
• Rheumatoid Factor (RF)
• Complete Blood Count (CBC)
• Electrolytes
• Creatinin level
• Hepatic panel (AST, ALT and albumin)
• Urinalysis
• Synovial fluid analysis
• Auto-antibodies
(ACR Subcommitee on Rheumatoid Arthritis Guidelines. Arthritis Rheum. 2002)
RHEUMATOID ARTHRITISRHEUMATOID ARTHRITIS
PT. SALI POLAPA BERSAMA
Laboratory investigations
• ESR / CRP level
• Rheumatoid Factor (RF)
• Complete Blood Count (CBC)
• Electrolytes
• Creatinin level
• Hepatic panel (AST, ALT and albumin)
• Urinalysis
• Synovial fluid analysis
• Auto-antibodies
(ACR Subcommitee on Rheumatoid Arthritis Guidelines. Arthritis Rheum. 2002)
RHEUMATOID ARTHRITISRHEUMATOID ARTHRITIS
HUMASED 40
IMTEC Hs CRP ELISAHs CRP (Fotometri)CRP (turbidimetri)Humatex CRP
Laboratory investigations
• ESR / CRP level
• Rheumatoid Factor (RF)
• Complete Blood Count (CBC)
• Electrolytes
• Creatinin level
• Hepatic panel (AST, ALT and albumin)
• Urinalysis
• Synovial fluid analysis
• Auto-antibodies
(ACR Subcommitee on Rheumatoid Arthritis Guidelines. Arthritis Rheum. 2002)
RHEUMATOID ARTHRITISRHEUMATOID ARTHRITIS
Humatex RF Latex (Rapid Test)RF latex reagent (turbidimetry)IMTEC IgM-RF ELISAIMTEC IgA-RF ELISAIMTEC IgG-RF ELISAIMTEC RF-Sreen IgGAM ELISA
Humatex RF Cat.No. 40052, 40050, 40053 • Latex agglutination qualitative, cut-off 20 IU/ml• Semi quantitative, diluted with Glycine-NaCl Buffer
sensitivity 12 IU/ml standard “International Reference Preparation of Rhematoid Arthritis Serum” (WHO).
1 : 2 24 IU/ml
1 : 4 48 IU/ml1 : 8 96 IU/ml1 : 16 192 IU/ml1 : 32 384 IU/ml
• Sensitivity : ≥ 12 IU/ml
Rheumatoid Arthritis
PT SALI POLAPA BERSAMA
RF HUMAN• Cat.No. 11261P • Photometric-Turbidimetry, with standard concentration
on the label. • Sensitivity : 20 – 100 IU/ml, no prozone up to 1,000 IU/
ml• Diluent, Latex, • Std. (conc. on label), calib. Curve 1 - 5
PT SALI POLAPA BERSAMA
Rheumatoid Arthritis
IMTEC RF IgG, IgA, IgM• ELISA Quantitative determination of Rheumatoid
Factors
• RF Ig G, Cat.no. ITC60007
• RF Ig A, Cat.no. ITC60010
• RF Ig M, Cat.no. ITC60003
Rheumatology
PT SALI POLAPA BERSAMA
Consisting of :
- CAL 1 – 5 = 12,5 – 25 – 50 – 100 - 200 U/ml
- CTRL +, CTRL - , BUFF WASH, BUFF DIL, CONJ., SUBS, STOP.Sol.
IMTEC RF Screen IgGAM• ELISA Quantitative determination of Rheumatoid
Factors
• Cat.no. ITC60000
Rheumatology
PT SALI POLAPA BERSAMA
Consisting of :
- CAL 1 – 5 = 12,5 – 25 – 50 – 100 - 200 U/ml IgGAM
- CTRL +, CTRL - , BUFF WASH, BUFF DIL, CONJ., SUBS, STOP.Sol.
Laboratory investigations
• ESR / CRP level
• Rheumatoid Factor (RF)
• Complete Blood Count (CBC)
• Electrolytes
• Creatinin level
• Hepatic panel (AST, ALT and albumin)
• Urinalysis
• Synovial fluid analysis
• Auto-antibodies
(ACR Subcommitee on Rheumatoid Arthritis Guidelines. Arthritis Rheum. 2002)
RHEUMATOID ARTHRITISRHEUMATOID ARTHRITIS. HUMACOUNT INSTRUMENT
Laboratory investigations
• ESR / CRP level
• Rheumatoid Factor (RF)
• Complete Blood Count (CBC)
• Electrolytes
• Creatinin level
• Hepatic panel (AST, ALT and albumin)
• Urinalysis
• Synovial fluid analysis
• Auto-antibodies
(ACR Subcommitee on Rheumatoid Arthritis Guidelines. Arthritis Rheum. 2002)
RHEUMATOID ARTHRITISRHEUMATOID ARTHRITIS
Laboratory investigations
• ESR / CRP level
• Rheumatoid Factor (RF)
• Complete Blood Count (CBC)
• Electrolytes
• Creatinin level
• Hepatic panel (AST, ALT and albumin)
• Urinalysis
• Synovial fluid analysis
• Auto-antibodies
(ACR Subcommitee on Rheumatoid Arthritis Guidelines. Arthritis Rheum. 2002)
RHEUMATOID ARTHRITISRHEUMATOID ARTHRITIS
Laboratory investigations
• ESR / CRP level
• Rheumatoid Factor (RF)
• Complete Blood Count (CBC)
• Electrolytes
• Creatinin level
• Hepatic panel (AST, ALT and albumin)
• Urinalysis
• Synovial fluid analysis
• Auto-antibodies
(ACR Subcommitee on Rheumatoid Arthritis Guidelines. Arthritis Rheum. 2002)
RHEUMATOID ARTHRITISRHEUMATOID ARTHRITIS
Laboratory investigations
• ESR / CRP level
• Rheumatoid Factor (RF)
• Complete Blood Count (CBC)
• Electrolytes
• Creatinin level
• Hepatic panel (AST, ALT and albumin)
• Urinalysis
• Synovial fluid analysis
• Auto-antibodies : Anti CCP, Anti RA33
(ACR Subcommitee on Rheumatoid Arthritis Guidelines. Arthritis Rheum. 2002)
RHEUMATOID ARTHRITISRHEUMATOID ARTHRITIS
Bezugslinie / Reference Line /Linea di rifertimento
Nukleosom / Nucleosomes
Histon / HistonesSmD1
U1-snRNPSS-A / Ro 60 kD
SS-B / LaScl 70Zentromer / CentromereJo-1
SS-A / Ro 52 kD
cut-off-Kontrolle / ControlFunktionskontrolle / Functional Control
ELISA
HUMAN-IMTEC - Key Notes
HUMAN-IMTEC - Quality
• Certified under DIN EN ISO 13485:2001 and DIN EN ISO 9001
• First manufacturer of IVD in Germany to meet the requirements of the European IVD directive
• Proven quality for over 15 years
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Human - IMTEC - Key Notes
Inovation – IMTEC
IMTEC products has protected and patented in 20 countries Germany and International.
It shows inovation and profesional competency
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Human - IMTEC-Product Line
The IMTEC product
range includes ...
Bezugslinie / Reference Line /Linea di rifertimento
Nukleosom / Nucleosomes
Histon / HistonesSmD1
U1-snRNPSS-A / Ro 60 kD
SS-B / LaScl 70Zentromer / CentromereJo-1
SS-A / Ro 52 kD
cut-off-Kontrolle / ControlFunktionskontrolle / Functional Control
ELISA LIALine Immuno Assay
IFAImmunofluorescence
PT SALI POLAPA BERSAMA
12.07.09
Human - IMTEC-Productline
ELISA - Principle•Antigens immobilized on a polystyrene microplate
•Sample incubation / Wash
•Secondary Ab / Wash
•Conjugate catalyzed color change of Substrate
•Stop solution will stop the reaction
•Optical density read-out with Microplate Reader (450/620 nm)
Human - IMTEC-Product Line
LIA - Principle• Antigens immobilized in a line on
a synthetic membrane
• Sample incubation / Wash
• Secondary Ab / Wash
• Precipitating Substrate - Visualization
• Visual comparison to Cut-Off Control line
Bezugslinie / Reference Line /Linea di rifertimento
Nukleosom / Nucleosomes
Histon / HistonesSmD1
U1-snRNPSS-A / Ro 60 kD
SS-B / LaScl 70Zentromer / CentromereJo-1
SS-A / Ro 52 kD
cut-off-Kontrolle / ControlFunktionskontrolle / Functional Control
PT SALI POLAPA BERSAMA
Human - IMTEC-Product Line
Immunofluorescence - Principle
• Cells or tissues immobilized on a glass slide
• Incubations of serial sample dilutions
• Secondary Ab - Marked with fluorescent-dye
• Microscopy
PT SALI POLAPA BERSAMA
Rheumatoid ArthritisELISA
• IMTEC-ANA SCREEN
- IMTEC ds DNA Ab
- IMTEC NUCLEOSOME
- -IMTEC ENA SCREEN
- -IMTEC ENA PROFILE
- -IMTEC ANTI CCP
- -IMTEC ANTI RA33
- -IMTEC RF Screen
- -IMTEC RF IG G
- -IMTEC RF IG M
- -IMTEC RF IG A- -IMTEC PR3 AB
- -IMTEC MPO-ANCA
LINE IMMUNOASSAY (LIA)
• -IMTEC ANA (LIA)
• -IMTEC ANA / dsDNA-LIA
IMTEC TESTSFor RA
Bezugslinie / Reference Line /Linea di rifertimento
Nukleosom / NucleosomesHiston / HistonesSmD1
U1-snRNPSS-A / Ro 60 kD
SS-B / LaScl 70Zentromer / CentromereJo-1
SS-A / Ro 52 kD
cut-off-Kontrolle / ControlFunktionskontrolle / Functional Control
RHEUMATOID ARTHRITISRHEUMATOID ARTHRITIS
UpdatedDiagnosis of
RA
Sensitivity and specificity of auto antibodies in RA
Marker Sensitivity (%) Specificity (%)
RF 50-90 Low*
APF 33 73-99
AKA 26-33 88-99
Anti Sa 22-40 98
Anti-CCP 50-76 95-98
* CCP = Cyclic Citrunillated Peptides
Anti-RA 33 36 98
RA-33 = hnRNP-A2
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• Antibodies directed against human γ-globulin Fc (mainly IgM, less often IgG or IgA)
• IgM RF calibrated against WHO reference preparation
• Sensitivity 80% in patients with RA• Specificity ranges from 80-90%• High titer predicts adverse outcome
erosive arthritis, vasculitis
• ELISA detects all isotypes
RHEUMATOID ARTHRITIS
RHEUMATOID FACTORS
- 39 -- 39 -
Why testing RF on ELISA ?
• Fully automatable • Detection of all relevant Ig-classes
The simultaneous detection of IgA and IgM class RF is nearly 100 % specific for RA *
• High sensitivity• Standardized method• Information on therapy outcome
Domer et al., Curr Opin Rheumatol 2004;16:246–253.
Bobbio-Pallavicini, F. et al. Ann Rheum Dis 2007;66:302
*
RHEUMATOID ARTHRITIS
RHEUMATOID FACTORS
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High IgA rheumatoid factor levels are associated with poor clinical response to TNFa inhibitors in RABobbio-Pallavicini, F. et al. Ann Rheum Dis 2007;66:302-307
DETECTION OF RF ISOTYPES
INCREASED RELEVANCE
High IgA rheumatoid factor levels are associated with greater risk of developing RAJonsson & Valdimarsson. Ann Rheum Dis 1993;52:161-164
Strong association between IgA RF and bone erosions – More radical treatment is neededBobbio-Pallavicini, F. et al. Ann Rheum Dis 2007;66:302-307
- 41 -
Rheumatic DiseasesRheumatoid Arthritis (~70%)Sjögren’s Syndrome (~90%)Lupus (~20%)Kryoglobuline Syndrome (90%)
Lung DiseaseInterstitial Fibrosis
InfektionsHepatitis C VirusAcute viral InfectionEndokarditisTuberkulosis
OtherSarcoidosisInflammationAging
RHEUMATOID FACTORS
NEED FOR SPECIFICITY
- 42 -- 42 -
• Autoantibodies directed against cyclic citrullinated peptide (derived from filaggrin)
• Sensitivity 60 - 80 %; Specificity > 95 %
• Prognostic marker positive up to 9 years before clinical manifestations
• approx. 30 % of CCP+ are RF IgM-
A Specific Marker for Rheumatoid Arthritis
DIAGNOSTIC ALGORITHM
ANTI-CCP
- 43 -- 43 -
Citrullination
• Deimination (Citrullination) of proteins by the enzyme peptidyl-arginin-deiminase (PAD).
• Posttranslational protein modification, leading to modified biochemical characteristics
• Citrullination mainly occurs for proteins of the cytoskeleton (i.e.. cytokeratin, vimentin, filaggrin)
• Citrullination is involved the regulatory process of apoptosis
DIAGNOSTIC ALGORITHM
ANTI-CCP
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Anti-CCP Prognosis of Disease Outcome
• Presence of CCP antibodies is associated with development of erosive arthritis.
• The presence of anti-CCP in high titer has important prognostic implications
• Anti-CCP helps in predicting which RA patients will have persistent disease, and erosive disease
DIAGNOSTIC IMPORTANCE
ANTI-CCP
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• Increasing sensitivity• Detecting RF / CCP seronegative patients• Specific for anti-nuclear antigen• Anti-RA33 associated with mild disease
heterogeneous nuclear ribonucleoprotein A2
• Single Early RA-Marker in 13 % of RA Patients• Specificity of 90 %
increasing to 96 % if negative for Anti-U1-snRNP
MOST SENSITIVE DIAGNOSIS OF RA
ANTI-RA33 ANTIBODIES
RHEUMATOID ARTHRITISRHEUMATOID ARTHRITIS
RF < 50 U/ml
Anti CCP -
Anti RA33 * ( + )
One of third RA patients is detectable of Anti RA33 and it shows similar specifity like RF
UPDATED DIAGNOSIS OF RA
* Anti RA33 is patented product HUMAN - IMTEC
Anti CCP +
Human – IMTEC ELISA SYSTEM
InfrastructureValidated Automation
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An attractive alternativeHumatemp
Automated Washer
Pipette
Humareader Plus
ELISYS UNOOr
Humareader Single
Humareader HS
Humawash Manual
IMTEC ELISA SYSTEM
12.07.09
Human - IMTEC Productline
The high diversity in diseases and tests requires flexible solutions for laboratories....
…HUMAN IS ONE OF THE CHOICESFOR FLEXIBLE SOLUTION….
PT SALI POLAPA BERSAMA
The complexities of Rheumatoid Arthritis
J Clin Invest, 116, 4, 2006
RA/RIS/09-07
- 51 -
Autoimmune market worldwide
Help out of the Jungle
Autoimmune Diagnostics is a complex and continuously changing field
Human is the guide to help and provide
up to date autoimmune diagnostics
- 52 -
Autoimmune market worldwide
Customized DiagnosticsEach laboratory requires customized solutions that fit best to their daily routine based on ...
• Turn-over• Test frequency• Test diversity• Official requirements
HUMAN is the right partner…..
Human - IMTEC Product Line
Our ToolsFlyers, Workshops,Seminars,Congress participation
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Laboratory investigations
• ESR / CRP level• Rheumatoid Factor (RF)• Complete Blood Count (CBC)• Electrolytes• Creatinin level• Hepatic panel (AST, ALT and albumin)• Urinalysis• Synovial fluid analysis
• Auto-antibodies
(ACR Subcommitee on Rheumatoid Arthritis Guidelines. Arthritis Rheum. 2002)
SUMMARY : Total Concept Diagnosis Of RASUMMARY : Total Concept Diagnosis Of RA
PT. SALI POLAPA BERSAMA
UPDATED DIAGNOSIS Of RA
12.07.09
End