A Novel, Portable Device to Count CD4+ T Cells using Flow Cytometry and a

Cellphone

Overview1) Purpose2) Theory3) Fabrication - Materials and Techniques4) Testing5) Limitations6) Biocompatibility

Background - Traditional Flow Cytometry

Clinical Uses

Brown, Michael, and Carl Wittwer. “Flow Cytometry: Principles and

Clinical Applications in Hematology .” Clinical Chemistry, vol. 46, no.

8, 2000, pp. 1221–1229.

Flow Cytometry and HIV in Resource Scarce Countries

● CD4+ T cells play important role in adaptive immune system● HIV attacks and kills CD4+ T cells● Crucial to monitor CD4 count over HIV/AIDS disease lifetime● CD4+ Count determines what medications a patient should be on● Over 22 million of the predicted 33 million living with HIV are in Sub-Saharan

Africa● Flow Cytometry can be used to count CD4+ T cell count

Barnett, David, et al. “CD4 Immunophenotyping in HIV Infection.” Nature Reviews Microbiology, vol. 6, no. 11, Nov. 2008, pp. S7–S15., doi:10.1038/nrmicro1998.Glencross, Deborah, et al. “CD45-Assisted PanLeucogating for Accurate, Cost-Effective Dual-Platform CD4+ T-Cell Enumeration.” Cytometry, vol. 50, no. 2, 2002, pp. 69–77., doi:10.1002/cyto.10068.

Microfluidic device design

A. Capillary uptake for blood drop input

B. Buffer/ stain solution pump in

C. Blood sample-reagent mixing

D. WBC being focused to centerline; RBC being focused to sidewall

E. RBC being pumped out of system through outlet 2, outlet 3

Outlet 2

Outlet 3

F. Fluorescent excitation of WBC

G. Side scatter light receive

H. DetectorsI. Mobile phone receive data

J. Digital signal processing (machine learning)

K. User interface (cell phone App) measurement outputs

365 nm LED array

Focusing lensesSlit plate

View area 1

Side scatter

Outlet 2

Outlet 3

Inlet

Outlet 1

Microfluidic channel modeling

Zhang, Jun, Li, Weihua, Li, Ming, Alici, Gursel, & Nguyen, Nam-Trung. (2014). Particle inertial focusing and its mechanism in a serpentine microchannel. Microfluidics and Nanofluidics, 17(2), 305-316.

Microfluidic channel modeling in COMSOLConcentrate WBC to centerline while stain them as flowing through serpentine channel.

Jun Zhang, et al(2017). IEEE Transactions on Biomedical Circuits and Systems

5th turn 10th turn 12th turn

Total length: 15.2 mmCell size in modeling: 10 micron

Microfluidic channel fabrication

A patterned SU-8 template on glass

PMMA exposed to acetonitrile for 2 min then had an SU-8 template pressed into the surface for 10 min, which provided appropriately imprinted channels and a suitable surface for bonding.

Sun, Peeni, Yang, Becerril, & Woolley. (2007). Rapid prototyping of poly(methyl methacrylate) microfluidic systems using solvent imprinting and bonding. Journal of Chromatography A, 1162(2), 162-166.

Fluorescent Image on a Cell Phone

Designed attachment

HONGYING ZHU, MAVANDADI S., COSKUN A.F., YAGLIDERE O., OZCAN A., Optofluidic fluorescent imaging cytometry on a cell phone, Analytical Chemistry 83(17), 2011, pp. 6641–6647.

Procedure of receiving fluorescent signal

● The microfluidic chip is inserted in the devices.

● The view area is illuminated from the side by inexpensive

LEDs.

● A fluorescent microscope movie of the flow cells can be

acquired using the cell-phone camera unit by CMOS.

● The digital frames of these fluorescent movies are rapidly

processed to determine the count of labeled WBC, which

can be used to estimate the density of the target fluorescent

objects within the sample solution.

HONGYING ZHU, MAVANDADI S., COSKUN A.F., YAGLIDERE O., OZCAN A., Optofluidic fluorescent imaging cytometry on a cell phone, Analytical Chemistry 83(17), 2011, pp. 6641–6647.

Fluorescent Image on a Cell PhoneAdvantage :

Compact, lightweight and cost-effective

Rapid and approachable

The system is compatible with fluorophores that have different excitation/emission wavelengths.

HONGYING ZHU, MAVANDADI S., COSKUN A.F., YAGLIDERE O., OZCAN A., Optofluidic fluorescent imaging cytometry on a cell phone, Analytical Chemistry 83(17), 2011, pp. 6641–6647.

Digital Analysis of Fluorescent FlowIn order to reduce errors in detection , we averaged the number of detected particles and their velocities over multiple frames.

HONGYING ZHU, MAVANDADI S., COSKUN A.F., YAGLIDERE O., OZCAN A., Optofluidic fluorescent imaging cytometry on a cell phone, Analytical Chemistry 83(17), 2011, pp. 6641–6647.

Data Analysis of flow cytometry image ● Linear Discriminant Analysis

○ Features: Cell Morphology (Size, Shape, etc.), Protein Content, DNA content, etc. ○ Use of hyperplane to distinguish classes (whether disease is present or not, etc.)

Limitations● Different diseases need different set ups.● Downsizing typically reduces complexity of testing● Error categorizing each cell to get accurate count

Conclusion- Miniaturized whole device within 100 mm³- Flow cytometer with NO sheath solution required, and high throughput- Inexpensive LED array illuminate cells- Cell phone receive and process data- Patients can easily have access to flow cytometry results on cell phone

application