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

© 2008 Reametrix, Inc. All rights

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
CD4 T-Lymphocyte Count

Providing Anti Retroviral Therapy (ART) Immunophenotyping 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

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High cost of testing

Current Issues with CD4 testing in Resource Poor Settings ReaMetrix APPROACH
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

Poorly developed cold chain in the transportation, storage & use of reagents Aging of blood samples that are facilities transported to centralized testing Complicated assay workflow with multiple critical pipetting steps leading to errors in CD4 count enumeration

Simplified assay workflow with a chance of human error

single critical pipetting step – reduced

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Clinical Information Generation – Current Scenario
Sample journey and cost accumulation
Acquire Transport Process Run Provide Results

Collection centre Path Lab Hospital
Syringe/Vacutaine r/Overheads Cold chain/Courier

Centralized Path Lab
Tech Labor + Reagent Capital Equipment Amortization

Doctor

Lab Infrastructure Overheads

Layers of margin at every step

• CD4 test example:

• Average cycle time for results ~ 48 to 72 hours

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

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Innovations that helped reduce the cost of CD4 testing
Optimized the manufacturing processes to enable a two-fold increase in antibodyconjugate 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

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Innovation in Product Delivery : Issue of Aging of Blood samples

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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
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Dried reagents can be stored and transported at room temperature

Unitized (one test per tube) dried-down format : Eliminating the Issue of Aged Samples

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 1st day vs. the 7th day.

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

Convenience & Simplified Work Flow

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ReaMetrix HIV Reagents Performance Assessment
* * *

* ­ values in cells per uL

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Building in Robustness in Reagents: Shelf Life exceeds 1 year

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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

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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.

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In the words of the National Microbiology Reference Lab, Zimbabwe

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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

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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.

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