1Department of Mechanical Engineering, University of Louisville, 2Department of Bioengineering, University of Louisville
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White, W. N., Sethu, P. Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells. J. Vis. Exp. (34), e1656, doi:10.3791/1656 (2009).
Part 1. Microfluidic device fabrication
A silicon master mold of the microfluidic lysis device was created using standard photolithographic techniques with the equipment in the University of Louisville cleanroom . AutoCAD (Autodesk, Inc., San Rafael, CA) was used to generate a transparency mask (Fineline Imaging, Colorado Springs, CO) to create negative replicas of the channels. The microfluidic lysis device was fabricated from the silicon master mold using soft lithographic techniques with the elastomer poly(dimethylsiloxane) (PDMS; Dow Corning, Midland, MI). The elastomer with the replicated channels was released, and channel access holes were punched with a 20-gauge needle (Small Parts, Miramar, FL.). The PDMS wafer was irreversibly bonded to a glass slide via oxygen plasma and tested for experimentation.
Part 2. Protocol for running lysis device
After the microfluidic device has been fabricated and tested, experiments with erythrocyte lysis are ready to begin. The following section details the protocol for the microfluidic device.
2.1 Sample Collection
2.2 Prime microfluidic device with PBS
Figure 1. Schematic of device showing inlets for respective solutions and outlet.
2.3 Preparation of syringe pumps and solution
2.4 Erythrocyte lysis
2.5 Preparation for flow cytometric analysis
Whole blood has been run through the microfluidic lysis device and post-processing protocol, ridding of erythrocytes and enriching circulating nucleated cells (CNCs). Ideal results will yield a clean flow cytometry scatter plot with separate CNC populations. Displayed below in Figures 2 and 3 are scatter plot comparisons of a whole blood sample without erythrocyte lysis and with erythrocyte lysis by the microfluidic protocol with axes as forward scatter (FSC) versus side scatter (SSC) light. It can be seen that the microfluidic enrichment protocol is necessary to obtain a clean flow cytometry scatter plot.
Figure 2. Flow cytometry scatter plot of whole blood sample without erythrocyte lysis with FSC and SSC axes.
Figure 3. Flow cytometry scatter plot of circulating nucleated cells with microfluidic erythrocyte lysis labeled by populations with FSC and SSC axes. Region 1 (red) represents lymphocytes, Region 2 (green) represents monocytes, Region 3 (blue) represents granulocytes, and Region 4 (purple) has yet to be characterized.
Removal of red cell debris by pipette in the protocol is also important when necessary. Below in Figure 4 is a scatter plot showing a surplus of red cell debris. Although not clouding the scatter plot as in Figure 2, one must account for the added events due to erythrocyte debris during data analysis.
Figure 4. Flow cytometry scatter plot depicting red cell debris, seen as a cluster of events in Region 5 (cyan).
Staining cells for certain antibody phenotype markers will also generate characteristic results. In the following Figures are antibody phenotype stains for 3 distinct leukocyte populations: lymphocytes, monocytes, and granulocytes. Figure 5 depicts lymphocyte populations plotted with axes CD3 and CD4. Monocytes and granulocytes are shown in Figures 6-7 with axes as FSC versus CD14 and CD66b, respectively.
Figure 5. Flow cytometry scatter plot depicting lymphocytes with CD3 and CD4 axes.
Figure 6. Flow cytometry scatter plot depicting monocytes with FSC and CD14 axes.
Figure 7. Flow cytometry scatter plot depicting granulocytes with FSC and CD66b axes.
An automated microfluidic blood lysis protocol has been reported. Within the protocol are critical steps worth noting. In section P.2.1, it is important to discard the first blood vial collected as the sample contains dislodged mature endothelial cells acting as false positives. Also make sure to run the blood sample within an hour of collection. Priming the microfluidic device in section P.2.2, it is important to initially rid of the bubbles. Additionally, one should avoid introducing bubbles when attaching new syringes to the needles throughout the protocol. When filling the 1 ml syringe with blood in section P.2.4, one must remember to first add 1X PBS and the blood, all while keeping the syringe vertical to avoid mixture of the two solutions. Before starting the syringe pump pushing the blood sample, ensure the large syringe pump has been running so the system is at full speed. After the sample has finished running through the device and been centrifuged, carefully remove as much supernatant as possible, including red cell debris, without disturbing the white pellet. Lastly, in section P.2.5 follow the manufacturer s protocol to assure the correct concentration of antibody is added to the 100 μL sample.
Applications of the microfluidic protocol are immense in clinical research. Interrogating the immune status of an individual in terms of CNCs generates important information on condition of health and may help with diagnosis and understanding of disease pathogenesis. Accessing these CNC populations through blood sample collection is easier and more convenient than human expression analyses involving tumor tissues. To examine these nucleated cells in circulation one must be able to enrich the populations from other blood components, including erythrocytes, the primary function of the reported device. Hence, such studies would benefit greatly from the microfluidic blood lysis protocol.
Significance of the microfluidic protocol emerges in terms of applications. Because nucleated cell enrichment is required in clinical studies of blood, a protocol requiring little expertise and user-mediated steps that produces clear and consistent results is important. The microfluidic protocol ensures consistency through automation and controlled exposure of cells to harsh environments. With uniformity among clinical laboratories data will be directly comparable .
Research has been supported by the Wallace H. Coulter Foundation Early Career Award in Translational Research. Thanks to Tim Andrews, Pediatric Hematology/Oncology, for help with blood sample collection. Also thanks to Dr. Sam Wellhausen, Flow Cytometry Core, James Graham Brown Cancer Center, for help with running flow cytometry samples.
|Sylgard 184 Silicone Elastomer Base||Dow Corning|
|Sylgard 184 Silicone Elastomer Curing Agent||Dow Corning|
|30-gauge syringe needle||Small Parts, Inc.||NE-301PL-25|
|Tygon tubing (.010" ID)||Small Parts, Inc.||TGY-010|
|20-gauge syringe needle||Small Parts, Inc.||NE-201PL-25|
|Syringe Pump (Harvard PHD 22/2000)||Harvard Apparatus||702000|
|Syringe Pump (Harvard Pico Plus)||Harvard Apparatus||702213|
|Flow cytometry tubes||DB Biosciences||352052|
|Antibody stains||DB Biosciences|
|1.5 ml Eppendorf tubes||Fisher Scientific||05-402-25|
|50 ml tube||Fisher Scientific|
|1 ml syringe||Fisher Scientific|
|30 ml tube||Fisher Scientific|
|Paraformaldehyde (PFA)||Fisher Scientific|
|Bovine serum albumin (BSA)||Fisher Scientific|
|10X PBS||Fisher Scientific|