© 2008 Reametrix, Inc. All rights reserved.

Innovative, Multi-platform, Affordable CD4+ Innovative, Multi-platform, Affordable CD4+ Enumeration Reagents that Need No Cold Chain: Enumeration Reagents that Need No Cold Chain:

Impacting HIV/AIDS Management in Resource Poor Impacting HIV/AIDS Management in Resource Poor SettingsSettings

Dr.Anand Sivaraman, Dr.Anand Sivaraman, ReaMetrix India Pvt. Ltd.ReaMetrix India Pvt. Ltd.

Bangalore 560092Bangalore 560092

2© 2008 Reametrix, Inc. All rights reserved.

HIV in India – Current StatusHIV in India – Current Status

HIV Prevalence rate in India 0.36% (source, NACO 2007 Bulletin)2.5 Million HIV Infections

Management of HIV InfectionProviding Anti Retroviral Therapy (ART)Immunophenotyping

CD4 T-Lymphocyte CountViral Load Assay

PCR – quantitate virus levels in blood

Role of CD4+ T-Lymphocyte Count in HIV pathogenesisAssessing immune status of patientsIndicating initiation of prophylactic ART therapyAssessing patient response to therapy

3© 2008 Reametrix, Inc. All rights reserved.

Current Issues with CD4 testing in Resource Poor Current Issues with CD4 testing in Resource Poor SettingsSettings

High cost of testing

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

ISSUESUndertook a study of the anatomy of cost of CD4 testingWorked on a locally relevant innovative product delivery model – dried reagentsRe-designed reagents to enable staining in collection centres

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

ReaMetrix APPROACH

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Clinical Information Generation – Current ScenarioSample journey and cost accumulation

Acquire Process Run Provide Results Transport

Collection centre

Path LabDoctor

Hospital

Centralized Path Lab

Syringe/Vacutainer/Overheads

Cold chain/Courier

Tech Labor + Reagent

Capital Equipment

Amortization

Lab InfrastructureOverheads

• 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

5© 2008 Reametrix, Inc. All rights reserved.

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 ReagentsUses proprietary stabilizers in formulationRemoves costs associated with maintaining cold chain in storage and transport

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Innovation in Product Delivery : Issue of Aging of Blood samplesInnovation 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

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

Dried reagents can be stored and transported at room temperatureBlood 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.

8© 2008 Reametrix, Inc. All rights reserved.

Simplification of Assay WorkflowSimplification of Assay Workflow

MethodologyMethodologyEmploy simple Lyse/no wash protocol - single platform technologyUnitized FormatUnitized FormatPre-dispensed reagents in unitized form provided as ready to use tubes

ConvenienceConvenience & & Simplified Work FlowSimplified Work FlowJust 2 pipetting steps – no separate pipetting of beads, reagents etc.

9© 2008 Reametrix, Inc. All rights reserved.

ReaMetrix HIV Reagents Performance AssessmentReaMetrix HIV Reagents Performance Assessment

** *

* ­ values in cells per uL

10© 2008 Reametrix, Inc. All rights reserved.

Building in Robustness in Reagents: Shelf Life exceeds 1 yearBuilding 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 Stability of ReaMetrix Dried Reagents at Room Temperature : do Reagents at Room Temperature : do

not require Cold storagenot require Cold storage

11© 2008 Reametrix, Inc. All rights reserved.

ReaMetrix Reagents : ReaMetrix Reagents : Making the difference : Making the difference : Changing the ParadigmChanging the Paradigm

All reagents already formulated, unitized and driedSteps as simple as mix & readNo cold chain in transportation or storageLong shelf life and reduced wasteLess opportunity for pipetting errors – with fixed volume pipettes, sample processing is made very easyAll this without compromising quality or affordability.

12© 2008 Reametrix, Inc. All rights reserved.

In the words of the National Microbiology Reference Lab, Zimbabwe

13© 2008 Reametrix, Inc. All rights reserved.

Key Centres who have already Validated Reagentsand Published Data from Validation

National AIDS Research Institute (NARI), PunePublic Health Agency of Canada, CanadaNational 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.