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A point of care diagnostic device for monitoring CD4 levels in HIV patients in a resource poor setting. Group 14: Lina Aboulmouna Peter DelNero Parker Gould Rosie Korman Chris Madison Stephen Schumacher Advisor: Dr. Kevin Seale, BME Vanderbilt University VIIBRE/ SyBBURE Nashville, TN.

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Group 14: Lina Aboulmouna Peter DelNero Parker Gould Rosie Korman Chris Madison

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A point of care diagnostic device for monitoring CD4 levels in HIV patients in a resource poor setting

Group 14:

LinaAboulmouna

Peter DelNero

Parker Gould

Rosie Korman

Chris Madison

Stephen Schumacher

Advisor: Dr. Kevin Seale, BME

Vanderbilt University

VIIBRE/SyBBURE

Nashville, TN


Problem Statement

  • The Gates Foundation has identified low-cost HIV testing as a primary global health goal (“Grand Challenges in Global Health”).

  • Current HIV testing methods are slow and expensive

  • Flow cytometers are not suitable for the point-of-care needs for developing countries

    • ~$30,000-$150,000 per flow cytometer.

  • Limitations include energy scarcity, untrained technicians, high capital-cost equipment, patient proximity, low throughput and long feedback period


What is a CD4+ Lymphocyte?

CD4 Antigen

CD4

White blood

cell

CD4

Other surface markers

Note: Image not to scale


CD4+ counts and Stage of HIV Infection

  • Patients with HIV who have CD4+ counts above 500cells/uL are in stage 1, CD4+ counts between 500cells/uL and 200cells/uL are in stage 2, and CD4+ counts of 200cells/uL and below are in stage 3 and are classified as having AIDS.


Performance Criteria

  • Prototype accurately determines CD4 count

  • Meets $2/test Gates Foundation challenge

  • Small sample volume (single finger-stick)

  • Generate results in minutes

  • Disposable and portable

  • Minimal energy requirements

  • Low technical expertise


Primary objective

  • Create a working prototype that accomplishes the specified goals and meets the performance criteria


Diagnostic Device Design

Input

PDMS

Outputs

Filter Device

Glass

Pump

Antibodies

Buffer

Blood

Mixer


Antibody conjugation protocol

  • Suspend antibodies and CMEUs (carboxylate-modified Europium nanoparticles) at 30 ugIgG/mg CMEU in the coating buffer: 10mM NaPO4 pH 8.0

  • Allow the antibodies to coat the CMEUs for 1-2 hours, with gentle shaking.

  • After the coating, spin down the CMEUs, remove supernatant, and resuspend in blocking buffer: either 10 mg/ml BSA in buffer, or 5% PEG in buffer

  • Wash 2 or 3 times:  Spin down the CMEUs at 10,000-12000g, remove supernatant, resuspend in blocking buffer

  • Spin down CMEUs, remove supernatant, resuspend in Conjugate Dilution Buffer


CD4 detection

Eu NP

ɑ-CD4 Antibody

conjugated to Eu nanoparticle

CD4

White blood

cell

CD4

Other surface markers

Well

Note: Image not to scale


Peristaltic Pump (hand-crankable)

1.

Stepper Motor

Alignment Screws

Set Screw

Shaft Coupler

Fluid Tubing

PDMS Washer

Thrust Bearing

PDMS Device

Polycarbonate Base

2.

3.

4.

5.

6.

7.

8.

9.


Peristaltic Pump (hand-crankable)

1.

4.

2.

3.

5.

6.

7.

8.

9.

Stepper Motor

Alignment Screws

Set Screw

  • Shaft Coupler

  • Fluid Tubing

  • PDMS Washer

Thrust Bearing

PDMS Device

Polycarbonate Base


FilterArray

White blood cell

Red blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells


FilterArray

White blood cell

Red blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells


FilterArray

White blood cell

Red blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells


FilterArray

White blood cell

Red blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells


FilterArray

White blood cell

Red blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells


FilterArray

White blood cell

Red blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells


FilterArray

White blood cell

Red blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells


Input

PDMS

Filter

Outputs

Glass

Filter with PDMS/Glass coverings


TRF (Time-Resolved Fluorescence) Imaging

In a nutshell:

  • Precisely timing a xenon excitation flash and image capture with long lifetime fluorophores(europium nanoparticles).

  • Reduces the impact of background fluorescence.


TRF imaging with a cell trap device

EuNP aggregate and water droplet

Possibly CD4+ cells, possibly EuNP aggregates


Goals

  • Convert DC pump to hand-cranked mechanical power generator mechanism

  • Integrate microfluidic platform with camera and pumps

  • Separation of white and red cells in whole blood sample

  • Fluorescent conjugation, labeling, and excitation of anti-CD4 antibodies

  • Digital image acquisition

  • Digital image analysis

  • Accomplish the primary objective


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