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INTRODUCTION

Total Concept Diagnosis


of Rheumatoid Arthritis
By :
Herlangi Sukma

PT SALI POLAPA BERSAMA

RHEUMATOID ARTHRITIS

RHEUMATOID ARTHRITIS

DESTRUCTIVE DISEASE
Manifestation
Joint swelling
(symmetrical)
Pain
Loss of mobility
Fatigue
Consequences

Destruction of cartilage
Erosion of bones
Deformation
Disability
Death

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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

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Rheumatoid Arthritis
Classification criteria of RA (ACR-1987, rev.)

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

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Rheumatoid Arthritis
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

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Rheumatoid Arthritis

PT. SALI POLAPA BERSAMA

RA/RIS/09-07

RHEUMATOID ARTHRITIS
Stages of Rheumatoid Arthritis

Early

Intermediate
RA/RIS/09-07

Late

RHEUMATOID ARTHRITIS
Clinician Treatment :
1. Symptoms
Anamnesis and
Clinical picture
Morning stiffness of joints
Symmetrical joint inflammation
Rheumatoid nodules
Fever
Myalgia

2. Causative
Laboratory Tests :
ESR
CRP
Rheumatoid Factors
Anti-CCP
Imaging Technologies

- Medication
- Fisiotherapy

X-Ray
Ultrasound
MRT

RA/RIS/09-07

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

- 12 -

+
-

Autoimmune Disease ?

This is Autoimmunity
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Prevalence of Autoimmune Diseases?


Total Prevalence of Autoimmune diseases
Canada - 1.0 Mio.

Iran - 2.1 Mio.

Brazil - 5.8 Mio.

Egypt - 2.4 Mio.

Mexico - 3.3 Mio.

Ethiopia - 2.3 Mio.

Germany - 2.6 Mio.

India - 33.3 Mio

France - 1.9 Mio.

China - 41.0 Mio.

Russia - 4.5 Mio.

Thailand - 2 Mio.
According to
Rose et al., 1998, The Autoimmune diseases, 3rd Ed.
US Census Bureau, Int. Data Base, 2004

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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 %

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

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What is an Autoimmune Disease ?


Organ specific
Hashimotos
Thyroiditis
Celiac disease
Pernicious anemia
Goodpastures
syndrome
AI Liver Diseases
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Systemic
Rheumatoid arthritis
Systemic Lupus
Erythematodes (SLE)
Antiphospholipid
syndrome (APS)
Wegenerss
granulomatosis

RHEUMATOID 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)

PT. SALI POLAPA BERSAMA

RHEUMATOID ARTHRITIS
Laboratory investigations
ESR / CRP level
Rheumatoid Factor (RF)
Complete Blood Count (CBC)

IMTEC Hs CRP ELISA


Hs CRP (Fotometri)
CRP (turbidimetri)
Humatex CRP

Electrolytes
Creatinin level
Hepatic panel (AST, ALT and albumin)
Urinalysis
Synovial fluid analysis
Auto-antibodies
(ACR Subcommitee on Rheumatoid Arthritis Guidelines. Arthritis Rheum. 2002)

HUMASED 40

RHEUMATOID ARTHRITIS
Laboratory investigations
ESR / CRP level
Rheumatoid Factor (RF)
Complete Blood Count (CBC)
Electrolytes

Humatex RF Latex (Rapid Test)


RF latex reagent (turbidimetry)
IMTEC IgM-RF ELISA
IMTEC IgA-RF ELISA
IMTEC IgG-RF ELISA
IMTEC RF-Sreen IgGAM ELISA

Creatinin level
Hepatic panel (AST, ALT and albumin)
Urinalysis
Synovial fluid analysis
Auto-antibodies
(ACR Subcommitee on Rheumatoid Arthritis Guidelines. Arthritis Rheum. 2002)

Rheumatoid Arthritis
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/ml

1: 8

96 IU/ml

1 : 16

192 IU/ml

1 : 32

384 IU/ml

Sensitivity : 12 IU/ml
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Rheumatoid Arthritis
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

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Rheumatology
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

Consisting of :
- CAL 1 5 = 12,5 25 50 100 - 200 U/ml
- CTRL +, CTRL - , BUFF WASH, BUFF DIL, CONJ., SUBS, STOP.Sol.

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Rheumatology
IMTEC RF Screen IgGAM

ELISA Quantitative determination of Rheumatoid


Factors

Cat.no. ITC60000

Consisting of :
- CAL 1 5 = 12,5 25 50 100 - 200 U/ml IgGAM
- CTRL +, CTRL - , BUFF WASH, BUFF DIL, CONJ., SUBS, STOP.Sol.

PT SALI POLAPA BERSAMA

RHEUMATOID ARTHRITIS
Laboratory investigations

HUMACOUNT INSTRUMENT

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 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 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 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 ARTHRITIS
Laboratory investigations
ESR / CRP level
Rheumatoid Factor (RF)
Complete Blood Count (CBC)

ELISA

Electrolytes
Creatinin level
Hepatic panel (AST, ALT and albumin)

Bezugslinie / Reference Line /


Linea di rifertimento
Funktionskontrolle / Functional Control
cut-off-Kontrolle / Control
Nukleosom / Nucleosomes

Urinalysis

Histon / Histones
SmD1

Synovial fluid analysis

SS-A / Ro 60 kD

Auto-antibodies : Anti CCP, Anti RA33


(ACR Subcommitee on Rheumatoid Arthritis Guidelines. Arthritis Rheum. 2002)

U1-snRNP
SS-A / Ro 52 kD
SS-B / La
Scl 70
Zentromer / Centromere
Jo-1

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


ELISA

LIA
Line Immuno Assay

Bezugslinie / Reference Line /


Linea di rifertimento
Funktionskontrolle / Functional Control
cut-off-Kontrolle / Control
Nukleosom / Nucleosomes
Histon / Histones
SmD1
U1-snRNP
SS-A / Ro 60 kD
SS-A / Ro 52 kD
SS-B / La
Scl 70
Zentromer / Centromere
Jo-1

The IMTEC product


IFA

range includes ...

Immunofluorescence

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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)

12.07.09

Human - IMTEC-Product Line


LIA - Principle

Bezugslinie / Reference Line /


Linea di rifertimento
Funktionskontrolle / Functional Control

Antigens immobilized in a line on


a synthetic membrane

cut-off-Kontrolle / Control

Sample incubation / Wash

U1-snRNP

Secondary Ab / Wash
Precipitating Substrate Visualization
Visual comparison to Cut-Off
Control line

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Nukleosom / Nucleosomes
Histon / Histones
SmD1
SS-A / Ro 60 kD
SS-A / Ro 52 kD
SS-B / La
Scl 70
Zentromer / Centromere
Jo-1

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

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Rheumatoid Arthritis
IMTEC TESTS
For RA

ELISA

IMTEC-ANA SCREEN

IMTEC ds DNA Ab

IMTEC NUCLEOSOME

-IMTEC ENA SCREEN

-IMTEC ENA PROFILE

Bezugslinie / Reference Line /


Linea di rifertimento
Funktionskontrolle / Functional Control

-IMTEC ANTI CCP

cut-off-Kontrolle / Control

-IMTEC ANTI RA33

-IMTEC RF Screen

-IMTEC RF IG G

SS-A / Ro 60 kD

-IMTEC RF IG M

-IMTEC RF IG A

SS-B / La
Scl 70

-IMTEC PR3 AB

-IMTEC MPO-ANCA

LINE IMMUNOASSAY (LIA)

-IMTEC ANA (LIA)

-IMTEC ANA / dsDNA-LIA

Nukleosom / Nucleosomes
Histon / Histones
SmD1
U1-snRNP
SS-A / Ro 52 kD

Zentromer / Centromere
Jo-1

RHEUMATOID ARTHRITIS

Updated
Diagnosis 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

36

98

Anti-RA 33

* CCP = Cyclic Citrunillated Peptides

RA-33 = hnRNP-A2

RHEUMATOID ARTHRITIS

RHEUMATOID FACTORS
Antibodies directed against human
-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


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RHEUMATOID ARTHRITIS

RHEUMATOID FACTORS
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;16:246253.
Bobbio-Pallavicini, F. et al. Ann Rheum Dis
2007;66:302

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DETECTION OF RF ISOTYPES

INCREASED RELEVANCE
High IgA rheumatoid factor levels are associated
with poor clinical response to TNFa inhibitors in RA
Bobbio-Pallavicini, F. et al. Ann Rheum Dis 2007;66:302-307

High IgA rheumatoid factor levels are associated


with greater risk of developing RA
Jonsson & Valdimarsson. Ann Rheum Dis 1993;52:161-164

Strong association between IgA RF and bone


erosions More radical treatment is needed
Bobbio-Pallavicini, F. et al. Ann Rheum Dis 2007;66:302-307

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RHEUMATOID FACTORS

NEED FOR SPECIFICITY


Rheumatic Diseases

Infektions
Hepatitis C Virus
Acute viral Infection
Endokarditis
Tuberkulosis

Rheumatoid Arthritis (~70%)


Sjgrens Syndrome (~90%)
Lupus (~20%)
Kryoglobuline Syndrome (90%)

Other

Lung Disease

Sarcoidosis
Inflammation
Aging

Interstitial Fibrosis

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DIAGNOSTIC ALGORITHM

ANTI-CCP

A Specific Marker for Rheumatoid Arthritis

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-

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DIAGNOSTIC ALGORITHM

ANTI-CCP
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
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DIAGNOSTIC IMPORTANCE

ANTI-CCP
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

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MOST SENSITIVE DIAGNOSIS OF RA

ANTI-RA33 ANTIBODIES
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
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RHEUMATOID ARTHRITIS

UPDATED
DIAGNOSIS OF RA
RF < 50 U/ml
Anti CCP +

Anti CCP -

Anti RA33 *
(+)
One of third RA patients is
detectable of Anti RA33 and it
shows similar specifity like RF

* Anti RA33 is patented product HUMAN - IMTEC

Human IMTEC ELISA SYSTEM


Infrastructure
Validated Automation

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IMTEC ELISA SYSTEM


An attractive alternative
Humareader Single

Humawash Manual

Humareader Plus

Or
Automated Washer

Humareader HS

Pipette

ELISYS UNO

Humatemp

Human - IMTEC Productline

The high diversity in diseases and tests requires flexible


solutions for laboratories....

HUMAN IS ONE OF THE CHOICES


FOR FLEXIBLE SOLUTION.

PT SALI POLAPA BERSAMA


12.07.09

The complexities of Rheumatoid Arthritis

J Clin Invest, 116, 4, 2006


RA/RIS/09-07

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

- 51 -

Autoimmune market worldwide


Customized Diagnostics
Each 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..


- 52 -

Human - IMTEC Product Line


Our Tools
Flyers, Workshops,
Seminars,
Congress participation

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SUMMARY : Total Concept Diagnosis Of RA


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

UPDATED DIAGNOSIS Of RA

(ACR Subcommitee on Rheumatoid Arthritis Guidelines. Arthritis Rheum. 2002)

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End

12.07.09