CD 4 for monitoring HIV Progression 31 st

CD 4 for monitoring HIV Progression 31 st Annual National Conference of Indian Association of Preventive & Social Medicine, 27 th – 29 th Feb 2004 PGIMER, Chandigarh Dr. Arvinder Singh BD Biosciences

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HIV/AIDS What is CD 4/CD 8

Flow Cytometry Clinical Applications • HIV immunophenotyping (CD 4 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 CD 8 PE 10 3 10 4 Two-Color Cell Analysis 10 0 10 1 10 2 CD 4 FITC 10 3 10 4

CD 8 PE SSC Height Four color analysis CD 3 FITC CD 8 PE CD 4 Cy 5 CD 45 Per. CP CD 4 Cy 5 CD 3 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 1990 s 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 • CD 4 T-Lymphocyte Count Flow cytometry is gold standard Alternate technologies – currently inadequate Facs. Count is considered system of choice CD 4 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* 15 -20 M 4 -6 M *(includes genotyping) CD 4 5 -8 M Viral load 1 -2 M

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

CD 4 and viral load

HIV/AIDS Pandemic Situational Analysis Unmet Needs – Affordable drugs/ART – Affordable CD 4

Flow Cytometry GOLD STANDARD FOR CD 4/CD 8 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 CD 4, CD 8 and CD 3 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 CD 3+ 617 – 2485 CD 19+ 125 – 701 CD 4+ 337 – 1090 CD 8+ 174 – 1240 • FEMALES CD 3+ 898 – 2786 CD 19+ 119 – 721 CD 4+ 424 – 1050 CD 8+ 255 – 1353 Ratio 0. 61 – 2. 68 HIV Patients-Low CD 4 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 CD 4+ 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 • • • Abbott Laboratories Bristol-Meyers Squibb Glaxo. Smith. Kline Hoffmann La-Roche Ltd Boehringer Ingelheim Merck & Co. , Inc Pfizer Cipla Ranbaxy Matrix

• General concensus is that flow cytometry based CD 4 count is the most accepted technique to HIV monitoring – Quantitative – more cells counted, more reliable result – Specific – m. Ab, identify CD 4 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 CD 4 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 Tri. Test Three Colour Leucogating Two Colour Monoclonal Ab panel CD 3/CD 45 CD 45/CD 4 Type of Platform Dual/Single* Type of Assay Lyse no Wash Lyse Wash Isotype controls No Isotype Control Gating strategy CD 45/SS (heterogeneous) Expert lymphocyte auto-gating software CD 45/SS (heterogeneous) Advantage CD 3 is an additional lineage marker for T-lymphocytes increasing the confidence level on defining CD 4 T-cells Cell Type Enumeration CD 3 T-cells, CD 4 T-cells, (absolute and % of CD 45) Analysis CD 4 T-cells (absolute and % of CD 45) Automated with Multi. Set 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 CD 4 testing, both reagents and instruments, to the poorest and most severely HIV/AIDS afflicted areas in the developing world. The CD 4 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