Title: CD4%20for%20monitoring%20HIV%20Progression
1CD4 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
2BD Activities
Brazil -COOPEX -MOH -BDB
3HIV/AIDSWhat is CD4/CD8
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6Flow 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)
7What Is Flow Cytometry?
- A technology that simultaneously measures
multiple characteristics of single cells at a
rapid rate
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9Lysed Whole Blood
Neutrophils
Side Scatter
Monocytes
Lymphocytes
Forward Light Scatter
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12Two-Color Cell Analysis
10 4
10 3
CD8 PE
10 2
10 1
10 0
10 0
10 1
10 2
10 3
10 4
CD4 FITC
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14Four color analysis
CD8 PE
SSC Height
CD45 PerCP
CD3 FITC
CD8 PE
CD4 Cy5
CD3 FITC
CD4 Cy5
1542 Million People Living with AIDS
16India 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
17India 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
18HIV/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 -
19HIV Testing paradigm
Screen
Confirm
CD4
Viral load
Est. annual tests
15-20M
1-2M
4-6M
5-8M
(includes genotyping)
20PCR Viral load
- It is a technique that imitates a cells
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. Its
used for - The development of new drugs
- To establish a patients 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
21CD4 and viral load
22HIV/AIDS Pandemic
- Situational Analysis
- Unmet Needs
- Affordable drugs/ART
- Affordable CD4
23Flow Cytometry
- GOLD STANDARD FOR CD4/CD8
- Monitoring in HIV/AIDS Patients
- Conceptually established by BD Worldwide
24FACSCount - Complete SystemIVD approved ( US FDA
)
25FACSCount - Complete System
Premeasured Reagents
26FACSCount - Complete System
FACSCount Reagents and Controls
FACSCount Workstation
27FACSCount
Measuring Absolute CD4, CD8 and CD3 Counts In
Three Easy Steps
28FACSCount - Clinical Need
- Safety
- Minimal Sample Preparation
- Fixed Samples
- Absolute Counts
- Limited Handling of
- Biohazardous Samples
29FACSCount - Clinical Need
- Easy to use
- Absolute Counts
- Unit Dose Reagents
- Minimal Hands-On Time
- No Formal Training Required
30FACSCounts in India
- 35 Placements
- 23 Instruments through NACO
- 12 Instruments in Private Path
Laboratories
31Reference Range of Lymphocytes Subsets In Healthy
Individuals (India)
- MALES
- CD3 617 2485
- CD19 125 701
- CD4 337 1090
- CD8 174 1240
- Ratio 0.61 2.68
- HIV Patients-Low CD4
- FEMALES
- CD3 898 2786
- CD19 119 721
- CD4 424 1050
- CD8 255 1353
- Ratio 0.95 2.40
32FACSCount - Analysis
- A hard copy results
- The control
- Patient sample
33HIV 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
34Performance of Competitive Tests
System Correlation w/FCM
FACSCount . 98
Coulter manual .74-.91
Zymmune .92-.94
Capcellia NA
Dynabeads .94
Viral Load N/A
35Pharmas taking action
- Abbott Laboratories
- Bristol-Meyers Squibb
- GlaxoSmithKline
- Hoffmann La-Roche Ltd
- Boehringer Ingelheim
- Merck Co., Inc
- Pfizer
- Cipla
- Ranbaxy
- Matrix
36- 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
37Application 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 T-cells
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
38Affordable Testing for HIV/AIDS in the Developing
WorldFebruary 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.
39BD BiosciencesAccomplishing 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
40HIV/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
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