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Dr.

Kaushik Mukhopadhyay
PGT, Dept. of Pharmacology
IPGME&R
TODAY’S TOPICS
• AN OVERVIEW OF RHEUMATOID ARTHRITIS
• OXIDATIVE STRESS AND RA
• OBJECTIVES
• MATERIALS & METHODS –

 STUDY POPULATON
 SAMPLE SIZE
 STUDY DESIGN
 STUDY PARAMETERS
 STUDY TECHNIQUE
 ANALYSIS PLAN

• CONCLUSION
• Rheumatoid arthritis (RA) is a chronic disabling inflammatory arthritis
affecting about 0.8% of the world population
• Key features –
 Symmetric, inflammatory, Autoimmune polyarthritis
 Presence of RA Factor, Anti-ccp antibody
 Females > Males
 Symptoms > 6 weeks
 Morning stiffness usually > 1 hour
 Elevated acute phase reactants – ESR, CRP
• Diagnosis by ‘The 2010 ACR-EULAR classification criteria for
rheumatoid arthritis’.



RHEUMATOID ARTHRITIS
Stage I:
Acute: synovial thickening;
confined to joint capsule
Stage II:
Persistent
inflammation  c/c
pain, joint damage
Stage III:
Inflammation and
damage limiting joint
function
Stage IV:
Permanent joint damage
and deformity  disability

Score
Target population (Who should be tested?): Patients who
1.have at least 1 joint with definite clinical synovitis (swelling)
*

2.with the synovitis not better explained by another disease



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
§


1 large joint


0
2-10 large joints

1
1-3 small joints (with or without involvement of large joints)
#

2
4-10 small joints (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
sw28 - Swollen Joint Count (0-28)
t28 - Tender Joint Count (0-28)
VAS disease activity (0-100mm)
DAS28 (ESR based) = 0.56*(t28)
+ 0.28*(sw28) + 0.70*Ln(ESR)
+ 0.014*VAS

ASSESSMENT OF DISEASE SEVERITY
RHEUMATOID ARTHRITIS &
OXIDATIVE STRESS
• Oxidative stress biomarkers of peripheral blood is increased in
active RA patients implying reactive oxygen species are possible
mediators of tissue damage
• ROS trigger a cascade of events through NFkB activation, which
up-regulate gene expression of pro-inflammatory cytokines that
mediates the immune responses causing inflammation
• Neutrophil granulocytes, macrophages and lymphocytes are
activated, and reactive oxygen (ROS) are produced locally
1. Estimation of ROS levels in peripheral blood
(neutrophils) in patients with high disease activity score
(DAS28) and also establish whether there is a
correlation between DAS28 and ROS levels before and
after therapy.

2. Correlation of ROS levels in peripheral blood
(neutrophils) with HAQ-DI before and after therapy.

OBJECTIVES
Materials
&
Methods
INCLUSION CRITERIA EXCLUSION CRITERIA



 Adult (age >18 years) Patient of either
gender who fulfils ‘The 2010 ACR-
EULAR classification criteria for
rheumatoid arthritis.’

 Patient with DAS28 (ESR based) score
>3.2 (moderate to severe disease
activity)
 Patients who have received oral or
intra-articular steroid in the past
eight weeks.
 Patients who have received any
antioxidant medication in the past
eight weeks.
 Patient with any other
autoimmune disorder
 Patient with any neurological
disorder that interferes with
normal physical functioning
 Patients with any other
arthropathies except RA

Subjects who attend the Outpatient unit of Rheumatology clinic of IPGME&R will be
screened for the following subject selection criteria –

• SAMPLE SIZE:
Our Primary objective is to evaluate correlation between ROS levels in
peripheral blood neutrophils and DAS28 score. The sample size has
been calculated based on the following assumptions-
 The population correlation coefficient of 0.8
 Sample correlation coefficient of 0.5
 Alpha = 0.05
 Power = 80%

we would need 30 subjects.
If there is a drop-out rate of 20% we would need to recruit –
38 patients.

• STUDY PERIOD:
The study is intended to be completed within a period of 18 months from
inception. Subject recruitment is expected to be completed within the first 12
months.

Study Design
After completion, Correlation of ROS from neutrophil with DAS28 & HAQDI
will be evaluated
• Longitudinal Observational study
Parameters To Be Measured
MEASUREMENT OF ROS
Peripheral blood (5 ml) will be collected from each subject with
heparin as anticoagulant.
Neutrophils will be isolated from blood using Hisep 1077 &
GranuloSep GSM 1119 according to manufacturer’s instructions.
Cell viability will be confirmed using trypan blue.
Neutrophils will be loaded with dichloro-dihydrofluorescein
diacetate (H
2
DCFDA) and incubated for 30 minutes at 37
o
C.
Cells will be then washed with phosphate buffered saline (PBS),
resuspended in PBS and acquired in a flow-cytometer.
Fluorescence will be measured in the log mode using CellQuest
Pro software and expressed as geometrical mean fluorescence
channel (GMFC).
• The study specific data will be collected in a case record form
(CRF).
• The data from the CRF and GMFC values will be transcribed onto
an excel database and analysed using SPSS statistical software.
• Pearson’s correlation coefficient (r) test or Spearman’s (р) test
would be used to analyse correlation between variables.
• Paired T-test or Wilcoxon’s signed rank test will be used for
paired situation.
• The level of significance would be set at 5% for all comparisons.
ANALYSIS PLAN
• We propose to undertake a longitudinal study that intends to find
whether the disease activity in RA patients under DMARD therapy
reflects oxidative stress in peripheral blood.



• If successful, this study may enrich already existing knowledge
about role of oxidative stress in pathophysiology of RA and
chance of future antioxidant trial in treatment of active RA.


CONCLUSION
REFERENCES
• www.das-score.nl

• http://www.rheumatology.org/practice/clinical/classification/ra/ra_2010
.asp. The 2010 ACR-EULAR classification criteria for rheumatoid arthritis.

• Harrison's Rheumatology. 2nd ed.


• Kundu S, Ghosh P, Datta S, Ghosh A, Chattopadhyay S, Chatterjee M.
Oxidative stress as a potential biomarker for determining disease activity in
patients with Rheumatoid Arthritis. Free Radic Res. 2012 ;46:1482-9.

• Desai PB, Manjunath S, Kadi S, Chetana K, Vanishree J. Oxidative stress and
enzymatic antioxidant status in rheumatoid arthritis: a case control study.
Eur Rev Med Pharmacol Sci. 2010;14:959-67.



THANK YOU