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.
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.
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 samples cannot be frozen for shipment or later analysis.
Epigenetic markers have recently emerged as powerful analytical tools for the study of phenotypic variations. Subsequently, human leukocyte populations have been shown to have cell-lineage DNA methylation patterns that allow for the precise characterization of WBC subsets. Subtyping based on epigenetic markers provides a promising alternative that does not depend on fresh blood sample collection or expensive antibodies and can be exploited as a biomarker for disease onset and susceptibility1,2,3,4,5.
Genome-wide studies have been performed for extensive mapping of methylated specific CG-rich regions in the genome (CpG islands) in leukocyte subtypes to identify candidate epigenetic markers specific to leukocyte subtypes. PCR protocols have been developed because of this reason for methylated gene regions, e.g., CD3Z and FOXP3, corresponding to CD3+ T-Cells and CD4+ CD25+ Regulatory T-Cells (T-Regs), respectively. Wiencke et al. have demonstrated the utility of duplex droplet PCR for epigenetic subtyping of T-Cells, yielding results that highly correlate with flow activated cell sorting (FACS) analysis6. This quantitative genetic analysis method relies on partitioning the template nucleic acid molecules and PCR reagents into thousands of discrete, volumetrically defined, sub-nanoliter sized droplets containing zero, one or more target nucleic acid copies, using water-in-oil emulsions enabled by microfluidics7,8. The PCR amplification is performed within each individual droplet and the endpoint fluorescence intensity of each droplet is measured, allowing absolute quantification of targets present in the sample. Droplet PCR has been established to be more precise, accurate, and technically simpler than standard qPCR, making it a more favorable DNA methylation-based method for clinical evaluation of T-Cells. Although a rapidly emerging subtyping methodology, multiplexed epigenetic analysis to probe for various methylated CpG regions simultaneously is lacking. This is necessary for routine leukocyte differential counts.
Herein, a thermoplastic elastomer (TPE) droplet microfluidic device is presented and employed for methylation-specific multiplex droplet PCR (mdPCR). The technology has been used to delineate specific leukocyte subtypes, CD3+ T-Cells and CD4+ CD25+ T-Regs, based on cell-lineage DNA methylation patterns, i.e., epigenetic variation of CD3Z and FOXP3 CpG regions, respectively. A detailed protocol for DNA extraction, bisulfite conversion and mdPCR is described in concert with a fabrication method for a TPE droplet generation device. Representative results of the method are compared to those of immunofluorescence staining highlighting the utility of the proposed approach.
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
2. Immunofluorescence staining and imaging protocol
3. DNA extraction and bisulfite conversion
4. Droplet generation device fabrication
NOTE: A microfluidic device used for droplet generation (CAD file provided in the Supplementary Information) was fabricated in a clean room (class 1,000) environment in thermoplastic elastomer (see Table of Materials) using hot embossing generated by the following protocol.
5. Droplet generation and PCR
NOTE: Table 1 outlines information on the forward and reverse primers along with the double-quenched hydrolysis probes for C-LESS, CD3Z and Foxp3 genes, which are required for the multiplex amplification of demethylated gene targets.
6. Fluorescence imaging and image analysis
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 subsequent thermoforming of thermoplastics using hot embossing. Once the epoxy mold was obtained, thermoplastic elastomer was embossed. Following the embossing, the TPE was demolded, and devices were cut. A flat TPE substrate was used to seal the device. Holes were punched in the top cover, and the two substrates were bonded. Finally, the necessary tubing for world-to-chip connections were inserted and the device was ready for use. Sample images of fabricated master mold, TPE material and embossed devices are shown in Figure 2. Assembled device with tubing interconnects was operated with a pair of independent programmable syringe pumps to generate emulsions for mdPCR. Methylation-specific mdPCR was performed using bisulfite treated DNA extracted from frozen PBMCs. Appropriate primers and hydrolysis probes for CD3Z, FOXP3, and C-Less genes were added to the PCR mix for emulsification. Following droplet generation of appropriate size (approximately 72 μm diameter), the emulsion was subjected to a thermal cycling protocol. Finally, the droplets were introduced into a glass capillary having 1 mm width and 50 µm height for imaging. This results in a monolayer distribution of droplets ideal for fluorescence image acquisition (Figure 3). Images were recorded for each of the four wavelengths. An image analysis was then performed to identify all droplets (Figure 4) meeting the threshold criteria established through ‘definetherain’ algorithm. The fluorescence intensity of all droplets in their respective fluorophore is then plotted and the threshold of positive and negative droplets was established (Figure 5). Subsequently, identification and counting of these droplets were performed which were above the selected threshold. The CPD values were then calculated and the percentage of CD3+ T-Cell and CD4+ 25+ T-Regs was established based on methylated CD3Z and FOXP3 copies, respectively, with respect to the CPD of total cells, or C-LESS gene (Figure 6). The percent values were then compared to those obtained through immunofluorescence imaging of T-Cell and T-Reg populations using appropriate antibodies.
FOXP3 Forward | GGG TTT TGT TGT TAT AGT TTT TG | |
FOXP3 Reverse | TTC TCT TCC TCC ATA ATA TCA | |
CD3Z Forward | GGA TGG TTG TGG TGA AAA GTG | |
CD3Z Reverse | CAA AAA CTC CTT TTC TCC TAA CCA | |
C-LESS Forward | TTG TAT GTA TGT GAG TGT GGG AGA GA | |
C-LESS Reverse | TTT CTT CCA CCC CTT CTC TTC C | |
FOXP3 Probe | /56-FAM/CA ACA CAT C/ZEN/C AAC CAC CAT /3IABkFQ/ | |
CDZ3 Probe | /56-HEX/CC AAC CAC C/ZEN/A CTA CCT CAA /3IABkFQ/ | |
C-Less Probe | /56-CY5/CT CCC CCT C/ZEN/T AAC TCT AT/3IABkFQ/ |
Table 1: Primer and hydrolysis probe design.
Reagent | Stock Solution | Master Mix Volume | Working Concentration |
Tris-HCl | 1 M | 2 µL | 20 mM |
KCl | 1 M | 10 µL | 100 mM |
MgCl2 | 25 mM | 16 µL | 4 mM |
C-LESS Primers | 10 µM | 10 µL | 1 µM each |
CD3Z Primers | 10 µM | 10 µL | 1 µM each |
FOXP3 Primers | 10 µM | 10 µL | 1 µM each |
C-LESS probe | 10 µM | 5 µL | 500 nM |
CD3Z probe | 10 µM | 5 µL | 500 nM |
FOXP3 probe | 10 µM | 5 µL | 500 nM |
HotStarTaq DNA Polymerase | 5 Units/µL | 8 µL | 0.4 Units/µL |
Bisulfite-treated DNA Target | Variable | Variable | Variable |
Nuclease-Free Water | Variable | ||
Total Volume | 100 µL |
Table 2: Master mix recipe for mdPCR.
Optical configuration | Exposure | DIA lamp | Florescent light |
Bright field | 50 ms | ON | OFF |
FAM | 300 ms | OFF | 20% |
HEX | 300 ms | OFF | 20% |
Cy5 | 400 ms | OFF | 20% |
Table 3: Settings for optical configuration and image acquisition.
Figure 1: Rapid prototyping of the droplet generator. Schematic illustration of the process used for the fabrication of (A) master mold and (B) TPE-based microfluidic emulsification device. Please click here to view a larger version of this figure.
Figure 2: Components involved in the fabrication process. (A) Silicon master, (B) PDMS replica, (C) epoxy mold, (D) TPE material extruded into sheets and packaged in rolls, (E) embossed TPE and (D) assembled device. Please click here to view a larger version of this figure.
Figure 3: Fluorescence image acquisition of droplets following thermal cycling. (A) Brightfield image of the droplets in a capillary that allowed monolayer image acquisition. (B) Cy5 filter image of C-LESS gene targets representing total cell count. (C) HEX filter image of methylated CD3Z gene target correlated to CD3+ T-Cell count. (D) FAM filter image of methylated FOXP3 gene target correlated to CD4+ CD25+ T-Reg count. Scale bar is 100 μm. Please click here to view a larger version of this figure.
Figure 4: Pipeline used for identifying droplets that meet threshold intensity. Images were first organized into appropriate fluorescence filters using file naming. The images were then converted to grayscale and relative intensities rescaled based on the minimum and maximum droplet fluorescence intensities. The threshold was then selected according to values obtained from ‘definetherain’ algorithm and the objects – or droplets – meeting the defined criteria were identified and quantified. Finally, the objects were filtered according to eccentricity and size to obtain the refined droplet count of spherical objects meeting 75 µm diameter size. The quantified objects and their intensities were then exported to a spreadsheet software for downstream analysis. Please click here to view a larger version of this figure.
Figure 5: Intensity scatter plots for fluorescence intensity of droplets following mdPCR. (A) C-LESS gene amplification with Cy5 hydrolysis probe representing the total cell count as the target region was devoid of cytosine residues and, therefore, resistant to bisulfite treatment. (B) Methylated CD3Z gene amplification with HEX hydrolysis probe, indicative of CD3+ T-Cell population. (C) Methylated FOXP3 gene amplification with FAM hydrolysis probe, representing the CD4+ CD25+ T-Reg population. All the scatter plots were subjected to ‘definetherain’ algorithm to set the appropriate threshold whereby the positives were within 3 standard deviations of the positive cluster mean. The amount of ‘rain’ was also established so that it does exceed 1% of the positive defined droplets. Each dataset for analysis consisted of ~5,000 droplets and was run in triplicate (see Supplementary Information for raw data and analysis). Please click here to view a larger version of this figure.
Figure 6: CPD values of all gene targets and determination of white blood cell subset percentage. (A) The calculated CPD values based on Poisson distribution of the positive droplet count as a ratio of total droplets. The input represents the theoretical CPD expected based on 740 ng of bisulfite treated DNA, 75 µm diameter droplets, assuming 6.6 pg for 1 gene copy. The input theoretical CPD was 0.25 and correlated with the C-LESS CPD representing the total cell count. (B) The ratio of CPD for CD3Z and FOXP3 with respect to C-LESS was then used to obtain the percent of CD3+ T-Cells and CD4+ CD25+ T-Regs, respectively. These ratios as percent of total leukocytes were then compared with values obtained by immunofluorescence imaging. As demonstrated, the values from mdPCR and immunofluorescence analysis correlate with no significant difference, with standard deviation errors from mdPCR being much less pronounced. Please click here to view a larger version of this figure.
Supplemental Information. Please click here to download these files.
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 manufacturing9,10,11,12,13. In addition, it is also optically clear and exhibits low fluorescence background in the visible spectrum which is an attractive feature for future integration of a complete sample-to-answer workflow using mdPCR. The PCR reagent recipes are also provided in a homebrew format to allow for facile multiplex gene quantification, while also maintaining stability throughout the thermal cycling protocol. As such one of the advantages of the presented devices and protocols is the flexibility in customizing the device design and reagents used for a particular application, which is difficult to achieve with commercial products and proprietary formulations. Furthermore, it removes the necessity of expensive instrumentation to perform the experiment.
Appropriate thermoplastic material selection of the microfluidic droplet generator is a critical parameter that can circumvent cumbersome surface treatment to render the device hydrophobic for efficient droplet formation. In addition, the selection of an optimized mdPCR buffer is also critical for maintaining droplet stability through the thermal cycling process – whilst not compromising the PCR efficiency and hydrolysis probe functionality. Further modification and optimization of the mdPCR buffer is, therefore, encouraged to arrive at highly specific and sensitive PCR amplification of selected gene targets, coupled with fluorescence based probe detection. This serves to increase signal-to-noise ratios, reducing the instances of ‘rain’ and simplifying the experimental analysis and identification of positive droplet populations. Troubleshooting of the experiment depends on assessing the critical steps from thermoplastic material selection to polymerase concentration to adapting the temperature ramp in the thermal cycling program in order to ensure droplet stability.
The current limitation of the described protocol is that it still necessitates manual sample transfer from the droplet generator to the thermal cycler and finally to a droplet imaging device and instrument such as a fluorescence microscope. Future efforts however are directed at the miniaturization of mdPCR and integration of these steps whereby droplet generation, PCR and imaging can be performed on a single automated platform.
The obtained results in Figure 6 demonstrate the versatility of this mdPCR approach for performing differential white blood cell quantification. The proposed method is more precise and reproducible than immunofluorescence imaging, which is often affected by user manipulation, giving rise to unwanted measurement errors. This is also the case for flow cytometry techniques that also rely on an immune-based method of detection which is highly dependent on cell viability, as well as multiple protocol steps prone to user error. Alternative methods such as real-time quantitative PCR (qPCR) for the quantification are often adversely affected by low copy number gene targets, a drawback that is remedied through mdPCR without the need for a dedicated calibration curve11. The presented mdPCR approach, therefore, presents a unique molecular approach to white blood cell differential counts that is based on methylation profiles of specific gene targets. Calibration curves for individual gene targets are also not necessary since mdPCR is based on an absolute quantification of binary positive and negative signals using Poisson distribution for CPD calculation.
The precise quantification of non-methylated gene islands, especially of low copy number genes such as FOXP3, presents a multitude of opportunities for clinical diagnosis. This is highlighted by the capacity of such an approach to identify known methylation patterns correlated to disease onset and progression. mdPCR, as a molecular approach with absolute quantification can be employed beyond methylation studies and can be applied to appropriately preserved samples, therefore removing the dependence on fresh clinical samples, and further expanding its applications. Future automation and miniaturization of this technique will be of great value for rapid and accurate clinical diagnosis and subsequent prognosis.
The authors have nothing to disclose.
The authors acknowledge financial support from the National Research Council of Canada.
Bio-Rad, Mississauga, ON | TFI0201 | PCR tube | |
RAN Biotechnologies, Beverly, MA | 008-FluoroSurfactant | Fluoro-surfactant | |
Silicon Quest International, Santa Clara, CA | |||
Oxford Instruments, Abingdon, UK | EMCCD camera | ||
Thermo Fisher Scientific, Waltham, MA | MA5-16728 | ||
Thermo Fisher Scientific, Waltham, MA | 22-8425-71 | ||
CellProfiler | Used for fluorescence image analysis | ||
Nikon, Japan | 10x objective | ||
American Type Culture Collection (ATCC), Manassas, VA | PCS-800-011 | ||
Ramé-Hart Instrument Co. (Netcong, NJ) | p/n 200-U1 | ||
Fisher, Canada | |||
Vitrocom, NJ, USA | 5015 and 5010 | Borosilicate capilary tube | |
(http://definetherain.org.uk/) | |||
Hamamatsu, Japan | LC-L1V5 | DEL UV light source | |
Dolomite | 3200063 | Disposable fluidic tubing | |
Dolomite | 3200302 | Disposable fluidic tubing | |
IDT, Coralville, IA | |||
Nikon, Melville, NY | Upright light microscope | ||
Cytec Industries, Woodland Park, NJ | |||
EV Group, Schärding, Austria | |||
Zymo Research, Irvine, CA | D5030 | ||
Photron, San Diego, CA | |||
IDT, Coralville, IA | |||
Gersteltec, Pully, Switzerland | SU-8 photoresist | ||
Fineline Imaging, Colorado Springs, CO | |||
Qiagen, Hilden, Germany | 203603 | ||
Image J | Used to assess droplet diameter | ||
Anachemia, Montreal, QC | |||
Excelitas, MA, USA | Broad-spectrum LED fluorescent lamp | ||
Galenvs Sciences Inc., Montreal, QC | DE1010 | ||
Hexpol TPE, Åmål, Sweden | Thermoplastic elastomer (TPE) | ||
Thermo Fisher Scientific, Waltham, MA | 13-400-518 | ||
Nikon, Japan | Used for image acquisition | ||
3M, St Paul, MN | Carrier Oil | ||
Thermo Fisher Scientific, Waltham, MA | R37605 | Blue fluorescent live cell stain (DAPI) | |
IDEX Health & Science, Oak Harbor, WA | P-881 | PEEK fittings | |
Sigma-Aldrich, Oakville, ON | 806552 | ||
Dow Corning, Midland, MI | |||
ThinkyUSA, CA, USA | ARV 310 | ||
Ihc world, Maryland, USA | IW-125-0 | ||
Zinsser NA, Northridge, CA | 2607808 | ||
Cetoni GmbH, Korbussen, Germany | |||
Sigma-Aldrich, Oakville, ON | 484431 | ||
Bio-Rad, Mississauga, ON | 1861096 | ||
Hitachi High-Technologies, Mississauga, ON | |||
Nikon, Melville, NY | Inverted microscope | ||
Nikon, Japan | |||
Loctite | AA 352 |