Innovative, Multi-platform, Affordable CD4+ Enumeration Reagents that Need No Cold Chain: Impacting HIV/AIDS Management in Resource Poor Settings Dr.Anand Sivaraman, ReaMetrix India Pvt. Ltd. Bangalore 560092

HIV in India – Current Status HIV Prevalence rate in India 0.36% (source, NACO 2007 Bulletin) 2.5 Million HIV Infections Management of HIV Infection Providing Anti Retroviral Therapy (ART) Immunophenotyping CD4 T-Lymphocyte Count Viral Load Assay PCR – quantitate virus levels in blood Role of CD4+ T-Lymphocyte Count in HIV pathogenesis Assessing immune status of patients Indicating initiation of prophylactic ART therapy Assessing patient response to therapy

HIV Prevalence rate in India 0.36% (source, NACO 2007 Bulletin)

2.5 Million HIV Infections

Management of HIV Infection

Providing Anti Retroviral Therapy (ART)

Immunophenotyping

CD4 T-Lymphocyte Count

Viral Load Assay

PCR – quantitate virus levels in blood

Role of CD4+ T-Lymphocyte Count in HIV pathogenesis

Assessing immune status of patients

Indicating initiation of prophylactic ART therapy

Assessing patient response to therapy

Current Issues with CD4 testing in Resource Poor Settings High cost of testing Poorly developed cold chain in the transportation, storage & use of reagents Aging of blood samples that are transported to centralized testing facilities Complicated assay workflow with multiple critical pipetting steps leading to errors in CD4 count enumeration ISSUES Undertook a study of the anatomy of cost of CD4 testing Worked on a locally relevant innovative product delivery model – dried reagents Re-designed reagents to enable staining in collection centres Simplified assay workflow with a single critical pipetting step – reduced chance of human error ReaMetrix APPROACH

High cost of testing

Poorly developed cold chain in the transportation, storage & use of reagents

Aging of blood samples that are transported to centralized testing facilities

Complicated assay workflow with multiple critical pipetting steps leading to errors in CD4 count enumeration

Undertook a study of the anatomy of cost of CD4 testing

Worked on a locally relevant innovative product delivery model – dried reagents

Re-designed reagents to enable staining in collection centres

Simplified assay workflow with a single critical pipetting step – reduced chance of human error

Clinical Information Generation – Current Scenario Sample journey and cost accumulation Acquire Process Run Provide Results Transport Collection centre Path Lab Doctor Hospital Centralized Path Lab Syringe/Vacutainer/Overheads Cold chain/Courier Tech Labor + Reagent Capital Equipment Amortization Lab Infrastructure Overheads CD4 test example: ~2 to 5 times cost addition from reagent manufacture to cost to patient Average cycle time for results ~ 48 to 72 hours Layers of margin at every step

CD4 test example:

~2 to 5 times cost addition from reagent manufacture to cost to patient

Average cycle time for results ~ 48 to 72 hours

Innovations that helped reduce the cost of CD4 testing Optimized the manufacturing processes to enable a two-fold increase in antibody-conjugate yield Validated and standardized the use of open source bright green and red dyes Atto 488 (FITC equivalent) Phycoerythrin-Dyomics 649 (PE-Cy5 equivalent) Development of Dried Reagents Uses proprietary stabilizers in formulation Removes costs associated with maintaining cold chain in storage and transport

Optimized the manufacturing processes to enable a two-fold increase in antibody-conjugate yield

Validated and standardized the use of open source bright green and red dyes

Atto 488 (FITC equivalent)

Phycoerythrin-Dyomics 649 (PE-Cy5 equivalent)

Development of Dried Reagents

Uses proprietary stabilizers in formulation

Removes costs associated with maintaining cold chain in storage and transport

Innovation in Product Delivery : Issue of Aging of Blood samples In India, the real challenge lies in transporting blood samples from remote areas to centralized testing centers. Often times, this leads to “aged” blood samples (> 48 hrs), making it unusable Blood collection centres need access to cold-chain to enable storage of liquid reagents, if they were to stain samples and then ship them CD3 capping

In India, the real challenge lies in transporting blood samples from remote areas to centralized testing centers.

Often times, this leads to “aged” blood samples (> 48 hrs), making it unusable

Blood collection centres need access to cold-chain to enable storage of liquid reagents, if they were to stain samples and then ship them

Unitized (one test per tube) dried-down format : Eliminating the Issue of Aged Samples Dried reagents can be stored and transported at room temperature Blood can be collected, stained and fixed at the point of collection before being shipped to a central testing facility at room temperature The stained samples can be stored for up to 7 days at room temperate without any statistical difference in CD4/CD8 counts on 1 st day vs. the 7 th day.

Dried reagents can be stored and transported at room temperature

Blood can be collected, stained and fixed at the point of collection before being shipped to a central testing facility at room temperature

The stained samples can be stored for up to 7 days at room temperate without any statistical difference in CD4/CD8 counts on 1 st day vs. the 7 th day.

Simplification of Assay Workflow Methodology Employ simple Lyse/no wash protocol - single platform technology Unitized Format Pre-dispensed reagents in unitized form provided as ready to use tubes Convenience & Simplified Work Flow Just 2 pipetting steps – no separate pipetting of beads, reagents etc.

Methodology

Employ simple Lyse/no wash protocol - single platform technology

Unitized Format

Pre-dispensed reagents in unitized form provided as ready to use tubes

Convenience & Simplified Work Flow

Just 2 pipetting steps – no separate pipetting of beads, reagents etc.

ReaMetrix HIV Reagents Performance Assessment * * * * - values in cells per uL

Building in Robustness in Reagents: Shelf Life exceeds 1 year Dried Reagents are stable at room temperature for more than 12 months Enables use of dried reagents in resource poor settings without cold chain/ refrigeration – enables increased penetration into remote areas . Stability of ReaMetrix Dried Reagents at Room Temperature : do not require Cold storage

Dried Reagents are stable at room temperature for more than 12 months

Enables use of dried reagents in resource poor settings without cold chain/ refrigeration – enables increased penetration into remote areas .

ReaMetrix Reagents : Making the difference : Changing the Paradigm All reagents already formulated, unitized and dried Steps as simple as mix & read No cold chain in transportation or storage Long shelf life and reduced waste Less opportunity for pipetting errors – with fixed volume pipettes, sample processing is made very easy All this without compromising quality or affordability.

All reagents already formulated, unitized and dried

Steps as simple as mix & read

No cold chain in transportation or storage

Long shelf life and reduced waste

Less opportunity for pipetting errors – with fixed volume pipettes, sample processing is made very easy

All this without compromising quality or affordability.

In the words of the National Microbiology Reference Lab, Zimbabwe

Key Centres who have already Validated Reagents and Published Data from Validation National AIDS Research Institute (NARI), Pune Public Health Agency of Canada, Canada National Microbiology Reference Lab, Zimbabwe and CDC, Zimbabwe

National AIDS Research Institute (NARI), Pune

Public Health Agency of Canada, Canada

National Microbiology Reference Lab, Zimbabwe and CDC, Zimbabwe

Publications & Presentations 1. Manian, BS, et al, Affordable Diagnostics — Changing the Paradigm in India , Cytometry Part B (Clinical Cytometry) 74B (Suppl. 1):S117–S122 (2008) 2. Bhavani Venkatkrishnan, Pratibha Malini, K.L.Ravikumar, Anand Sivaraman, Comparative Evaluation of an Indigenous CD4 Reagent Kit in a Dry Format Requiring no Cold Chain, Proceedings of the International Conference on Fundamental and Translational Research on HIV/AIDS, Mumbai, October 2008. 3. Rajesh Anand, Jammi Narasimham, Bhavani Venkatakrishnan, Wilson D'Souza, Sowmya Narayanan, Donny Thomas, Anand Sivaraman, Multi-platform, Affordable CD4 Enumeration Reagents that need no Cold Chain: Enabling evidence-based Management of HIV/AIDS in Resource-poor Settings , Proceedings of the Association of Pathologists and Microbiologists (APCON), Chennai, December 2008.

1. Manian, BS, et al, Affordable Diagnostics — Changing the Paradigm in India , Cytometry Part B (Clinical Cytometry) 74B (Suppl. 1):S117–S122 (2008)

2. Bhavani Venkatkrishnan, Pratibha Malini, K.L.Ravikumar, Anand Sivaraman, Comparative Evaluation of an Indigenous CD4 Reagent Kit in a Dry Format Requiring no Cold Chain, Proceedings of the International Conference on Fundamental and Translational Research on HIV/AIDS, Mumbai, October 2008.

3. Rajesh Anand, Jammi Narasimham, Bhavani Venkatakrishnan, Wilson D'Souza, Sowmya

Narayanan, Donny Thomas, Anand Sivaraman, Multi-platform, Affordable CD4 Enumeration Reagents that need no Cold Chain: Enabling evidence-based Management of HIV/AIDS in Resource-poor Settings , Proceedings of the Association of Pathologists and Microbiologists (APCON), Chennai, December 2008.