Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Epigenetic markers are used for white blood cell (WBC) subtyping through the quantification of DNA methylation patterns. This protocol presents a multiplex droplet polymerase chain reaction (mdPCR) method using a thermoplastic elastomer (TPE)-based microfluidic device for droplet generation allowing for precise and multiplex methylation-specific target quantification of WBC differential counts.

Abstract

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, along with a thermoplastic elastomer (TPE) microfluidic droplet generation device. Epigenetic markers are used for WBC subtyping which is of important prognostic value in different diseases. This is achieved through the quantification of DNA methylation patterns of specific CG-rich regions in the genome (CpG loci). In this paper, bisulfite-treated DNA from peripheral blood mononuclear cells (PBMCs) is encapsulated in droplets with mdPCR reagents including primers and hydrolysis fluorescent probes specific for CpG loci that correlate with WBC sub-populations. The multiplex approach allows for the interrogation of many CpG loci without the need for separate mdPCR reactions, enabling more accurate parametric determination of WBC sub-populations using epigenetic analysis of methylation sites. This precise quantification can be extended to different applications and highlights the benefits for clinical diagnosis and subsequent prognosis.

Introduction

Analysis of white blood cells (WBCs) composition is among the most frequently requested laboratory tests in hematological diagnostics. Differential leukocyte count serves as an indicator for a spectrum of diseases including infection, inflammation, anemia, and leukemia, and is under investigation as an early prognostic biomarker for several other conditions as well. Gold standard in WBC subtyping involves immunostaining and/or flow cytometry both of which require costly, instability-prone fluorescent antibodies and are often highly dependent on operator proficiency in sample preparation. Moreover, this method is applicable to fresh blood samples only, such that the sa....

Protocol

All the experiments performed in this study involving human samples were approved by the NRC’s Ethics Board and were done according to NRC’s policies governing human subjects that follow applicable research guidelines and are compliant with the laws in Québec, Canada.

1. Cell preparation

  1. Thaw the frozen human peripheral blood mononuclear cells (PBMCs) immediately by placing the cryovial in a water bath at 37 °C for 5 min.
  2. Invert the cryovial twice to.......

Representative Results

The TPE-based microfluidic droplet generator device was fabricated using the described protocol as shown in Figure 1. A transparency mask was used in photolithography to obtain silicon (Si) master. Soft lithography was performed to obtain an inverse PDMS replica of the Si master which was then used to fabricate the epoxy mold. Epoxy precursor was poured onto the PDMS and cured to crosslink and harden. This mold, representing the exact replica of the Si master was more resilient for subsequen.......

Discussion

The presented experimental protocol and methods allow for in-house mdPCR using a fabricated TPE droplet generator, a thermal cycler, and fluorescence microscope. The fabricated device using soft TPE to TPE bonding affords hydrophobic surface properties that are uniform across all channel walls, such that the final device does not require any surface treatment for subsequent use as a droplet generator. This material has been routinely employed in point-of-care platforms that necessitate compatibility with high throughput .......

Acknowledgements

The authors acknowledge financial support from the National Research Council of Canada.

....

Materials

NameCompanyCatalog NumberComments
Bio-Rad, Mississauga, ONTFI0201PCR tube
RAN Biotechnologies, Beverly, MA008-FluoroSurfactantFluoro-surfactant
Silicon Quest International, Santa Clara, CA
Oxford Instruments, Abingdon, UKEMCCD camera
Thermo Fisher Scientific, Waltham, MAMA5-16728
Thermo Fisher Scientific, Waltham, MA22-8425-71
CellProfilerUsed for fluorescence image analysis
Nikon, Japan10x objective
American Type Culture Collection (ATCC), Manassas, VAPCS-800-011
Ramé-Hart Instrument Co. (Netcong, NJ)p/n 200-U1
Fisher, Canada
Vitrocom, NJ, USA5015 and 5010Borosilicate capilary tube
(http://definetherain.org.uk/)
Hamamatsu, JapanLC-L1V5DEL UV light source
Dolomite3200063Disposable fluidic tubing
Dolomite3200302Disposable fluidic tubing
IDT, Coralville, IA
Nikon, Melville, NYUpright light microscope
Cytec Industries, Woodland Park, NJ
EV Group, Schärding, Austria
Zymo Research, Irvine, CAD5030
Photron, San Diego, CA
IDT, Coralville, IA
Gersteltec, Pully, SwitzerlandSU-8 photoresist
Fineline Imaging, Colorado Springs, CO
Qiagen, Hilden, Germany203603
Image JUsed to assess droplet diameter
Anachemia, Montreal, QC
Excelitas, MA, USABroad-spectrum LED fluorescent lamp
Galenvs Sciences Inc., Montreal, QCDE1010
Hexpol TPE, Ă…mĂĄl, SwedenThermoplastic elastomer (TPE)
Thermo Fisher Scientific, Waltham, MA13-400-518
Nikon, JapanUsed for image acquisition
3M, St Paul, MNCarrier Oil
Thermo Fisher Scientific, Waltham, MAR37605Blue fluorescent live cell stain (DAPI)
IDEX Health & Science, Oak Harbor, WAP-881PEEK fittings
Sigma-Aldrich, Oakville, ON806552
Dow Corning, Midland, MI
ThinkyUSA, CA, USAARV 310
Ihc world, Maryland, USAIW-125-0
Zinsser NA, Northridge, CA2607808
Cetoni GmbH, Korbussen, Germany
Sigma-Aldrich, Oakville, ON484431
Bio-Rad, Mississauga, ON1861096
Hitachi High-Technologies, Mississauga, ON
Nikon, Melville, NYInverted microscope
Nikon, Japan
LoctiteAA 352

References

  1. Teitell, M., Richardson, B. DNA methylation in the immune system. Clinical Immunology. 109 (1), 2-5 (2003).
  2. Suarez-Alvarez, B., Rodriguez, R. M., Fraga, M. F., LĂłpez-Larrea, C. DNA methylation: A promising landscape for immune system-rel....

Explore More Articles

Methylation specific Multiplex Droplet PCRPolymer Droplet GeneratorHematological DiagnosticsDifferential Leukocyte CountingEpigenetic Methylation MarkersImmunofluorescent StainingPeripheral Blood Mononuclear CellsBisulfite ConversionIon Chromatography Column

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved