Transcript Document

CD4 for monitoring HIV Progression
31st Annual National Conference of
Indian Association of Preventive &
Social Medicine,
27th – 29th Feb 2004
PGIMER, Chandigarh
Dr. Arvinder Singh
BD Biosciences
BD Activities
China
Brazil
-CDC
-MOH
-BDB
-COOPEX
-MOH
-BDB
BD Bio-IS
India
-Value
Proposition
-Education and training
-QC/QA protocols
-Application protocols
-Reference range studies
Africa
-WHO -Countries
-DOD -NGOs
-BDB
Other
Countries
-WHO
-NGOs
-BDB
NGOs
-AAACP
-IAVI
-HVTN
-JHU
-MSF
-WJCF
-AMFAR
-GMHC
-ICMR
-NACO
-Hospitals / Path
labs
-BDB
GOVT
-NIH
-HRF
-DOD
-WHO
-UNAID
-USAID
Thailand
-MOH
-COE
-BDB
HIV/AIDS
What is CD4/CD8
BLOOD
T CELLS
T HELPER
CD4
RBC
WBC
PLATELETS
LYMPHOCYTES
MONOCYTES
GLANULOCYTES
B CELLS
T CYTOTOXIC
CD8
NK CELLS
Flow Cytometry Clinical
Applications
• HIV immunophenotyping (CD4 absolute counts)
• Cytokine analysis
• Leukemia and lymphoma diagnosis
- minimum residual disease detection
• Cell cycle and DNA ploidy analysis
• Stem Cell Analysis
• HLA – B 27 Typing
• Residual white blood cell detection
• Reticulocyte enumeration
• Flow crossmatching (organ transplantation)
What Is Flow Cytometry?
A technology that simultaneously
measures multiple characteristics
of single cells at a rapid rate
400
600
Neutrophils
200
Monocytes
Lymphocytes
0
Side Scatter
800
1000
Lysed Whole Blood
0
200
400
600
800
Forward Light Scatter
1000
10 2
10 1
10 0
CD8 PE
10 3
10 4
Two-Color Cell Analysis
10 0
10 1
10 2
CD4 FITC
10 3
10 4
CD8 PE
Four color analysis
CD3 FITC
CD8 PE
CD45 PerCP
CD4 Cy5
CD3 FITC
42 Million People Living with AIDS
India – Current Situation HIV
• HIV prevalence rate is 0.7%
• India has had a sharp increase in the
estimated number of HIV infections, from a
few thousand in the early 1990s to a working
estimate of about 3.8 million children and
adults living with HIV/AIDS in 2001
• Estimates range from 8 - 15 million HIV
infected adults and children living with HIV
in 2010
• Given India's large population, a mere 0.1
percent increase in the prevalence rate would
increase the number of adults living with
HIV/AIDS by over half a million people
India – Current Situation HIV
• About 90% of the total reported AIDS cases
occur in the sexually active and economically
productive 15 to 44 age group
• Men account for 79% of HIV infections in
India
• The predominant mode of HIV transmission is
through heterosexual contact
• In 2001, the HIV infection rate went above one
per cent in six states
– Maharashtra, Tamil Nadu and Manipur - account for 75% of
estimated HIV cases
– the burden of AIDS cases is beginning to be felt in states
affected early - Mumbai and Manipur have recorded 20 to 49
per cent bed occupancy by HIV positive people in certain
hospitals
HIV/AIDS Pandemic
Disease Management
Vaccines, Drugs, Education
• Antiretroviral Therapy (ART)
Controls disease progression
Increasing drug resistance patterns
Expensive (Big Pharma – western countries)
Trend to affordable (generic) drugs in developing world
• CD4 T-Lymphocyte Count
Flow cytometry is gold standard
Alternate technologies – currently inadequate
FacsCount is considered system of choice
CD4 count is the single most important objective criteria to guide
clinical decision-making on when to use ART
HIV Testing paradigm
Screen
Est.
annual
tests
Confirm* CD4
15-20M 4-6M
*(includes genotyping)
5-8M
Viral load
1-2M
PCR Viral load
It is a technique that imitates a cell’s ability to replicate
DNA by generating multiple copies of specific sequences of
DNA through amplification. In clinical diagnostics, a
specimen of genetic material can be repeatedly copied by
PCR to provide sufficient material to detect the presence or
absence of a virus as well as to quantitate its levels in the
blood. It’s used for:
– The development of new drugs
– To establish a patient’s disease status and the likelihood of disease
progression
– To determine when to start treatment
– To help the physician decide which drugs to use in a treatment regimen
– To assess whether a treatment regimen is working
CD4 and viral load
HIV/AIDS Pandemic
Situational Analysis
Unmet Needs
– Affordable drugs/ART
– Affordable CD4
Flow Cytometry
GOLD STANDARD FOR
CD4/CD8
Monitoring in HIV/AIDS Patients
Conceptually established by BD
Worldwide
FACSCount Complete System
IVD approved ( US FDA )
FACSCount - Complete
System
Premeasured Reagents
FACSCount - Complete
System
FACSCount Reagents and Controls
FACSCount Workstation
FACSCount
Measuring Absolute
CD4, CD8 and CD3 Counts
In Three Easy Steps
FACSCount - Clinical
Need
Safety
• Minimal Sample Preparation
• Fixed Samples
• Absolute Counts
• Limited Handling of
Biohazardous Samples
FACSCount - Clinical
Need
Easy to use
• Absolute Counts
• Unit Dose Reagents
• Minimal Hands-On Time
• No Formal Training Required
FACSCounts in India
35 Placements
• 23 Instruments through NACO
• 12 Instruments in Private Path
Laboratories
Reference Range of Lymphocytes
Subsets In Healthy Individuals (India)
• MALES
CD3+
617 – 2485
CD19+ 125 – 701
CD4+
337 – 1090
CD8+
174 – 1240
• FEMALES
CD3+
898 – 2786
CD19+ 119 – 721
CD4+
424 – 1050
CD8+
255 – 1353
Ratio
0.61 – 2.68
HIV Patients-Low CD4
Ratio
0.95 – 2.40
FACSCount - Analysis
A hard copy results
• The control
• Patient sample
HIV Monitoring
BD provides a gold standard system of
reliable products and support services
for CD4+ T-cell testing at affordable
pricing.
• Appropriate Technology
– Flow Cytometry
• Capacity Building
– Training, Support,
Service
• Reliable Laboratory
Systems
– GMP/GLP (IVDP)/GCP
Performance of Competitive Tests
System
FACSCount
Coulter manual
Zymmune
Capcellia
Dynabeads
Viral Load
Correlation
w/FCM
. 98
.74-.91
.92-.94
NA
.94
N/A
Pharmas taking action
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Abbott Laboratories
Bristol-Meyers Squibb
GlaxoSmithKline
Hoffmann La-Roche Ltd
Boehringer Ingelheim
Merck & Co., Inc
Pfizer
Cipla
Ranbaxy
Matrix
• General concensus is that flow cytometry based CD4 count is the most
accepted technique to HIV monitoring
– Quantitative – more cells counted, more reliable result
– Specific – mAb, identify CD4 T-lymphocytes most precisely
– QA/QC – internal and external - necessary for accreditation
– Throughput – manual operation is acceptable as technician costs are
not significant and does not limit test demand
• Existing flow cytometry systems (FACSCalibur or similar) are
adequate/accepted for Central Reference facilities – usually at the sites of
principal government services
– Perform broad clinical testing plus Research studies associated with
international partners
• Affordable lower cost platform required at Provincial and Local clinic sites
– dedicated clinical test platform
– Capability to perform CD4 count ( - ideal with broader test menu )
– Current FACSCount system should be viewed as maximal technology
complexity – sample preparation potential issue – reagent cost
limitation
– Challenge from Partec / Guava like technology or alternative assays
Application Comparison
BD TriTest Three Colour
Leucogating Two Colour
Monoclonal Ab panel
CD3/CD4/CD45
CD45/CD4
Type of Platform
Dual/Single*
Dual/Single*
Type of Assay
Lyse no Wash
Lyse Wash
Isotype controls
No Isotype Control
No Isotype Control
Gating strategy
CD45/SS (heterogeneous)
Expert lymphocyte auto-gating
software
CD45/SS (heterogeneous)
Advantage
CD3 is an additional lineage marker
for T-lymphocytes increasing the
confidence level on defining CD4 Tcells
Cell Type Enumeration
CD3 T-cells, CD4 T-cells, (absolute
and % of CD45)
CD4 T-cells (absolute and % of
CD45)
Analysis
Automated with MultiSet software
Manual
Affordable Testing for HIV/AIDS in the Developing World
February 5, 2004
On January 14, 2004, the William J. Clinton Presidential Foundation announced that BD, through
BD Biosciences, had entered into an agreement with the Foundation to provide affordable CD4
testing, both reagents and instruments, to the poorest and most severely HIV/AIDS afflicted areas
in the developing world.
The CD4 immunocytometry test has been used to establish decision points for antiretroviral (ARV)
therapy, to monitor efficacy of various treatments, as a prognostic indicator, and as one criterion for
initiating prophylaxis for opportunistic infections. The viral load test helps measure how effective
ARV therapies are in suppressing the virus and can alert clinicians about the need to adjust
dosages or change regimens.
BD Biosciences
Accomplishing our Mission –
HIV/AIDS
BD Biosciences is Helping With the Global AIDS
Crisis by:
• Supporting the Brazilian AIDS prevention
program
– Example program for reducing mortality
and new infections in the developing world
– Our flow instruments and reagents are used
as the gold standard to monitor disease
progression in HIV-infected individuals
• Collaborating with the International AIDS
Vaccine Initiative (IAVI)
– Speed the development and distribution of
preventive AIDS vaccines
– Our flow instruments and reagents are used
to detect immune response to the vaccine
HIV/AIDS
PANDEMIC
2010
80+ million PLWHA
India (15+ million) China (15+ million)
Negative growth rates in most of Africa due to AIDS mortality
WW economic impact
2002
Brazil
Achieves 50% reduction in AIDS deaths over last 10 years
Accessible drugs and health care
$500,000 million savings on AIDS impact