A POINT OF CARE DIAGNOSTIC DEVICE FOR MONITORING CD4 LEVELS IN HIV PATIENTS IN A RESOURCE POOR SETTINGGroup 14:Lina AboulmounaPeter DelNeroParker GouldRosie KormanChris MadisonStephen Schumacher

Advisor: Dr. Kevin Seale, BME

Vanderbilt UniversityVIIBRE/SyBBURENashville, 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?

Note: Image not to scale

White blood cell

CD4

CD4

CD4 Antigen

Other surface markers

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.

CD4+ count Stage Patient’s status

>500 cells/uL Stage 1 HIV-infected

200-500cells/uL Stage 2 HIV-infected

<200cells/uL Stage 3 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

Outputs

PDMS

Glass

Buffer

Antibodies

BloodMixer

Pump

Filter Devic

e

ANTIBODY CONJUGATION PROTOCOL1. Suspend antibodies and CMEUs (carboxylate-

modified Europium nanoparticles) at 30 ug IgG/mg CMEU in the coating buffer: 10mM NaPO4 pH 8.0

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

3. 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

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

5. Spin down CMEUs, remove supernatant, resuspend in Conjugate Dilution Buffer

CD4 DETECTION

Note: Image not to scale

White blood cell

CD4

CD4

Eu NP

ɑ-CD4 Antibody conjugated to Eu nanoparticle

Other surface markers Well

1. Stepper Motor2. Alignment

Screws3. Set Screw4. Shaft Coupler5. Fluid Tubing6. PDMS Washer7. Thrust Bearing8. PDMS Device9. Polycarbonate

Base

1.

2.

4.

6.

9.8.7.

PERISTALTIC PUMP (HAND-CRANKABLE)

3.

5.

PERISTALTIC PUMP (HAND-CRANKABLE)

1. Stepper Motor

2. Alignment Screws

3. Set Screw

1.

2.4.

6.

9.

8.7.

3.5.

7. Thrust Bearing

8. PDMS Device

9. Polycarbonate Base

4. Shaft Coupler

5. Fluid Tubing

6. PDMS Washer

Tunable pore size to selectively trap CD4+ cells

FILTERARRAY White blood

cellRed blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells

FILTERARRAY White blood

cellRed blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells

FILTERARRAY White blood

cellRed blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells

FILTERARRAY White blood

cellRed blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells

FILTERARRAY White blood

cellRed blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells

FILTERARRAY White blood

cellRed blood cell

Silicon

Pore

Tunable pore size to selectively trap CD4+ cells

FILTERARRAY White blood

cellRed blood cell

Silicon

Pore

Input

Outputs

PDMS

Filter

Glass

Filter with PDMS/Glass coverings

TRF (TIME-RESOLVED FLUORESCENCE) IMAGINGIn 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

Possibly CD4+ cells, possibly EuNP aggregates

EuNP aggregate and water droplet

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