A point of care diagnostic device for monitoring CD4 levels in HIV patients in a resource poor setti...
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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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Group 14 lina aboulmouna peter delnero parker gould rosie korman chris madison

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

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

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

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

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

Primary objective

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


Diagnostic device design

Diagnostic Device Design

Input

PDMS

Outputs

Filter Device

Glass

Pump

Antibodies

Buffer

Blood

Mixer


Antibody conjugation protocol

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

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

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 crankable1

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


Group 14 lina aboulmouna peter delnero parker gould rosie korman chris madison

FilterArray

White blood cell

Red blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells


Group 14 lina aboulmouna peter delnero parker gould rosie korman chris madison

FilterArray

White blood cell

Red blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells


Group 14 lina aboulmouna peter delnero parker gould rosie korman chris madison

FilterArray

White blood cell

Red blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells


Group 14 lina aboulmouna peter delnero parker gould rosie korman chris madison

FilterArray

White blood cell

Red blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells


Group 14 lina aboulmouna peter delnero parker gould rosie korman chris madison

FilterArray

White blood cell

Red blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells


Group 14 lina aboulmouna peter delnero parker gould rosie korman chris madison

FilterArray

White blood cell

Red blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells


Group 14 lina aboulmouna peter delnero parker gould rosie korman chris madison

FilterArray

White blood cell

Red blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells


Group 14 lina aboulmouna peter delnero parker gould rosie korman chris madison

Input

PDMS

Filter

Outputs

Glass

Filter with PDMS/Glass coverings


Trf time resolved fluorescence imaging

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

TRF imaging with a cell trap device

EuNP aggregate and water droplet

Possibly CD4+ cells, possibly EuNP aggregates


Goals

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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