Name: Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells (Video) | JoVE Journal
Uploaded: 2009-12-31T14:00:00.000+00:00
Duration: 9 min 53 s
Description: Learn an automated microfluidic blood lysis protocol for enriching circulating nucleated cells from peripheral blood. This method minimizes cell loss and preserves sample quality for downstream flow cytometry analysis.

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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.

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 [1]. 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
<ol>
 <li>Collect 4 milliliters of blood sample from median cubital vein, on the anterior forearm vein of patient with heparin as anticoagulant in two 2 ml green top vaccutainers.</li>
 <li>Discard first 2 ml tube as it contains dislodged mature endothelial cells (false positives).</li>
 <li>Resuspend the second tube to mix heparin with blood by turning up and down; save immediately on ice.</li>
 <li>Transfer 1 ml of blood to a 1.5 ml Eppendorf tube. Process the blood sample immediately (within one hour of collection).</li>
</ol>
2.2 Prime microfluidic device with PBS
<ol>
 <li>Obtain a microfluidic device bonded to glass. Press-fit access tubing (Small Parts, Miramar, FL) of slightly larger diameter in the inlets and outlet (Figure 1).</li>
 <li>Attach a 30-gauge syringe needle (Small Parts, Miramar, FL) to the tubing on each of the inlets, providing the macro to micro interface.</li>
 <li>Fill a 1 ml syringe with 1X PBS and make sure to get rid of bubbles by flicking the syringe needle. Connect the PBS-loaded 1 ml syringe to inlet 1 on the microfluidic device. Push the syringe containing the 1X PBS gently by hand until the solution flows out of inlet 2, and then immediately clamp inlet 2 with a standard office binder clip</li>
 <li>Continue pushing the 1X PBS until fluid reaches the outlet port of the microfluidics device. Once the solution flows out of the outlet, clamp the outlet tubing with another binder clip.</li>
 <li>Continue pushing the 1 ml syringe until the 1X PBS flows out of inlet 3, and clamp the inlet 3 port with another office binder clip. The microfluidics device is now fully primed.</li>
</ol>
<img src="/files/ftp_upload/1656/1656fig1.jpg" alt="Microfluidic device diagram; inlets for PBS, deionized water, sample; fluid flow analysis." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Schematic of device showing inlets for respective solutions and outlet.
2.3 Preparation of syringe pumps and solution
<ol>
 <li>To calibrate the pumps follow these steps:
 <ol class="lalpha">
 <li>For the large Harvard syringe pump (Harvard, PHD 22/2000 Part # 702000): Set the diameter configuration to 22.5 mm, flow rate 600 μl/min. </li>
 <li>For the small Harvard Syringe pump (Harvard, Pico Plus Part # 702213): Set the diameter configuration to 4.61 mm, flow rate 20 μl/min. </li>
 </ol>
 </li>
 <li>Fill one 30 ml syringe with sterile deionized water. This can be accomplished by withdrawing the solution directly from the 50 ml conical tube. (Label the syringe)</li>
 <li>Fill a second 30 ml syringe with 2X phosphate-buffered-saline (PBS), 2% paraformaldehyde (PFA) using the procedure outlined in step 9. (Label the syringe)</li>
 <li>Fill a 1 ml syringe with 1X PBS from the aliquots stored in the 15 ml conical tubes, using the procedure outlined in step 9.</li>
 <li>Connect the 30 ml deionized water syringe to inlet 2 and the 30 ml 2X PBS syringe to inlet 3 (make sure you don t trap bubbles in the tubing or microfluidics device). Remove the clamp from inlets 2 and 3 and the outlet tubing. Connect the 1X PBS 1 ml syringe into inlet 1. Avoid introducing bubbles by filling the syringe needle with liquid, connecting the syringe, and flicking the syringe needle to rid of bubbles.</li>
</ol>
2.4 Erythrocyte lysis
<ol>
 <li>Set all of the syringes on the proper Harvard pumps as outlined below:
 <ol class="lalpha">
 <li>Set the two 30 ml syringes (one containing sterile, de-ionized water and the other with 2X PBS, 2% PFA) together on the large Harvard syringe pump.</li>
 <li>Set the 1 ml syringe (containing 1X PBS) on the small Harvard syringe pump. </li>
 <li>Place the outlet tubing in a waste collector (50 ml conical tube that is labeled waste ). </li>
 </ol>
 </li>
 <li>Turn on the large Harvard syringe pump (with the 30 ml syringes) and let it run for one minute. (Make sure there are no bubbles and no leakage in the device or in the tubing).</li>
 <li>Turn on the small Harvard syringe pump and let it run for an additional minute. (Make sure there are no bubbles and no leakage in the device or in the tubing). Stop the pumps. The microfluidics device is now ready to be used.</li>
 <li>Remove the 1 ml syringe containing 1X PBS from the small Harvard syringe pump.</li>
 <li>After obtaining a blood sample, fill the 1 ml syringe with 0.05 ml of sterile 1X PBS without trapping any bubbles. Next, fill the syringe with 0.5 ml of blood obtained from the Eppendorf tube. (The 1 ml syringe will now contain a final volume of 0.55 ml of the blood and 1X PBS buffer.) Note: Keep the syringe vertical while filling to avoid mixing of the 0.05 ml PBS with the blood. </li>
 <li>Connect the syringe containing the blood to inlet 1 and mount the syringe carefully (avoid pushing the blood through the tubing into the device) on the small Harvard syringe pump (the syringe is mounted vertical into the pump). </li>
 <li>Remove the outlet tubing from the waste tube and put a clean 50 ml centrifuge tube in its place and set it on a bucket of ice. </li>
 <li>Switch on the large Harvard syringe pump first and let it run for 1 minute. Start collecting the sample from the outlet into the 50 ml centrifuge tube. </li>
 <li>Switch on the small Harvard syringe pump.</li>
 <li>Once the blood sample in the 1 ml syringe has completely traveled through the device, stop both pumps. This should take approximately 20 minutes.</li>
 <li>Centrifuge the collected sample for 5 minutes at 350 x g at room temperature with the brake off.</li>
 <li>Remove supernatant by placing pipette tip at the opposite side of the white pellet. Remove as much supernatant as possible, especially red cell debris, without disturbing the white pellet.</li>
 <li>Resuspend the sample in 1 ml of flow buffer (1X PBS, 1% BSA [bovine serum albumin], 2% PFA) by pipetting up and down. Transfer sample to a 1.5 ml Eppendorf tube.</li>
 <li>Microcentrifuge the collected sample for 5 minutes at 4 x g at room temperature. Use same precision as before to remove the supernatant.</li>
 <li>Resuspend the sample in 1 ml of flow buffer by vortexing.</li>
</ol>
2.5 Preparation for flow cytometric analysis
<ol>
 <li>For sample analysis using flow cytometry, add 100 μl of sample to a flow cytometry tube.</li>
 <li>To the 100 μl of sample add specified antibody (ensure correct concentration).</li>
 <li>Allow sample to incubate for 30 minutes at 40 C. </li>
 <li>Wash twice with flow buffer prior to flow cytometry. Wash step includes adding 250 μl of flow buffer, vortexing, centrifuging for 5 minutes at 350 x g, and removing supernatant with one fluid turn of flow cytometry tube. </li>
 <li>Resuspend the pellet in 250 μl of flow buffer. Sample is ready for analysis by flow cytometry.</li>
</ol>
Representative Results
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. 
<img src="/files/ftp_upload/1656/1656fig2.jpg" alt="Flow cytometry result, scatter plot, FSC vs SSC, cellular analysis, dot distribution diagram." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2. Flow cytometry scatter plot of whole blood sample without erythrocyte lysis with FSC and SSC axes.
<img src="/files/ftp_upload/1656/1656fig3.jpg" alt="Flow cytometry, SSC vs FSC dot plot, cell population analysis, scatter diagram." data-altupdatedat="1788235205000" data-alt="Figure 3"> 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.
<img src="/files/ftp_upload/1656/1656fig4.jpg" alt="Flow cytometry scatter plot; FSC vs SSC analysis; Cell populations R2, R3, R4 differentiated." data-altupdatedat="1788235205000" data-alt="Figure 4"> 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.
<img src="/files/ftp_upload/1656/1656fig5.jpg" alt="Flow cytometry graph, CD3 vs CD4 antigen expression analysis, data on immune cell populations." data-altupdatedat="1788235205000" data-alt="Figure 5"> Figure 5. Flow cytometry scatter plot depicting lymphocytes with CD3 and CD4 axes.
<img src="/files/ftp_upload/1656/1656fig6.jpg" alt="Flow cytometry dot plot; FSC vs. CD14; cell population analysis, scatter data visualization." data-altupdatedat="1788235205000" data-alt="Figure 6"> Figure 6. Flow cytometry scatter plot depicting monocytes with FSC and CD14 axes.
<img src="/files/ftp_upload/1656/1656fig7.jpg" alt="Flow cytometry dot plot; CD66b vs FSC, displaying cell population analysis, colored clusters." data-altupdatedat="1788235205000" data-alt="Figure 7"> Figure 7. Flow cytometry scatter plot depicting granulocytes with FSC and CD66b axes.

<ol><li>White, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+application+of+microfluidic+leukocyte+enrichment+protocol+in+mild+phenotype+sickle+cell+disease+%28SCD%5BTitle%5D)+AND+%22Biomedical+Microdevices%22%5BJournal%5D)">Clinical application of microfluidic leukocyte enrichment protocol in mild phenotype sickle cell disease (SCD.</a> Biomedical Microdevices. 11 (2), 477-483 (2009).</li>
<li>Sethu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Continuous+Flow+Microfluidic+Device+for+Rapid+Erythrocyte+Lysis%5BTitle%5D)+AND+%22Anal.+Chem%22%5BJournal%5D)">Continuous Flow Microfluidic Device for Rapid Erythrocyte Lysis.</a> Anal. Chem. 76 (21), 6247-6253 (2004).</li>
</ol>

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...

<table><tbody><tr><td>Sylgard 184 Silicone Elastomer Base</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>Sylgard 184 Silicone Elastomer Curing Agent</td><td>Dow Corning</td><td></td><td></td></tr><tr><td>30-gauge syringe needle</td><td>Small Parts, Inc.</td><td>NE-301PL-25</td><td></td></tr><tr><td>Tygon tubing (.010&quot; ID)</td><td>Small Parts, Inc.</td><td>TGY-010</td><td></td></tr><tr><td>20-gauge syringe needle</td><td>Small Parts, Inc.</td><td>NE-201PL-25</td><td></td></tr><tr><td>Syringe Pump (Harvard PHD 22/2000)</td><td>Harvard Apparatus</td><td>702000</td><td></td></tr><tr><td>Syringe Pump (Harvard Pico Plus)</td><td>Harvard Apparatus</td><td>702213</td><td></td></tr><tr><td>Flow cytometry tubes</td><td>DB Biosciences</td><td>352052</td><td></td></tr><tr><td>Antibody stains</td><td>DB Biosciences</td><td></td><td></td></tr><tr><td>1.5 ml Eppendorf tubes</td><td>Fisher Scientific</td><td>05-402-25</td><td></td></tr><tr><td>50 ml tube</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>1 ml syringe</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>30 ml tube</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>10X PBS</td><td>Fisher Scientific</td><td></td><td></td></tr></tbody></table>

automated microfluidic blood lysis protocol for enrichment of circulating nucleated cells

Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and endothelial cells, provide an ideal platform for an updated status on the immune condition of an individual. The microfluidic protocol allows for enrichment of CNCs without selective cell loss and sample preparation variability due to user-mediated steps. Briefly, the protocol includes device fabrication, sample collection, device setup, and running blood through the microfluidic chamber. Within the device whole blood is rapidly mixed with deionized water for approximately 10 seconds in a 50 micron x 150 micron microfluidic channel. In this time span erythrocytes are lysed due to hypotonic conditions. Herringbone structures on the bottom of the channel ensure thorough mixing and exposure of cells to a constant environment. Remaining cells are returned to isotonic conditions at the exit of the device, fixed using 2% paraformaldehyde, centrifuged to separate erythrocyte debris from CNCs, and suspended in flow buffer for staining and analysis by flow cytometry. Results show clean flow cytometry scatter plots with CNC populations saved. Significance of this device and protocol comes in the study and understanding of disease pathogenesis by analysis of CNC populations. Hence, automation, effectiveness, and simplicity of the microfluidic protocol are demonstrated.

An automated microfluidic device was developed for circulating nucleated cell enrichment from peripheral blood via erythrocyte lysis that ensures isolation of high quality sample without cell loss.

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and ...

To enrich for circulating nucleated cells from blood. The microfluidic device is first primed with one XPBS to rid of bubbles, deionized water, and 2%paraldehyde in two XPBS are then run through the device via syringe pumps prior to loading of the blood sample. Blood is then processed through the microfluidic device and collected post-processing of the sample includes centrifugation and removal of supinate.Finally, the sample is analyzed via flow cytometry to produce fax plots. Hi, I'm William White from the laboratory of Microscale Biotechnology in the Department of Bioengineering at the University of Wool. Today we'll show you a procedure for the enrichment of circulating nucleus cells by lysis of whole blood via microfluidics.We use this procedure in our laboratory to study inflammation in various disease processes as to ensure enrichment of leukocytes and other circulating nucleated cells without loss or activation. So let's get started. To begin this procedure, press fit access tubing of slightly larger diameter than the channel access holes in the inlet and outlet holes.Then to provide the macro to micro interface, attach a 30 gauge syringe needle to the tubing on each of the inlets. Next, fill a one milliliter syringe with one XPBS making sure to get rid of bubbles. Connect the syringe to inlet one and push the syringe gently by hand until one XPBS flows out of inlet two.Then immediately clamp inlet two with an office binder clip. Continue pushing the syringe until one XPBS solution reaches the outlet port of the microfluidic device. Once the solution flows outta the outlet, clamp the outlet tubing with another binder clip.Continue further pushing the one milliliter syringe until one XPBS flows out of inlet three and clamp the inlet three port with a third office binder clip. The microfluidic device is now fully primed. Start calibrating the syringe pumps with the large Harvard syringe pump, setting the diameter configuration to 22.5 millimeters and the flow rate to 600 microliters per minute.For the small Harvard syringe pump. Set the diameter configuration to 4.61 millimeters and the flow rate to 20 microliters per minute. Next, fill one 30 milliliter syringe with deionized water by withdrawing a solution directly from the 50 milliliter conical tube.Remember to label the syringe, fill another 30 milliliter syringe with two XPBS 2%paraform aldehyde solution. Again, withdraw directly from a conical tube and label the syringe. Now connect the water containing syringe to inlet two and the 2%para formaldehyde in two XPBS syringe to inlet three while making sure no bubbles are trapped in the tubing or in the microfluidic device.Then remove the clamps from inlet two, inlet three, and the outlet tubing with all three syringes connected. Proceed to put the two large syringes together on the large Harvard syringe pump and set the one milliliter syringe on the small Harvard syringe pump. Place the outlet tubing in a 50 milliliter conical waste collection tube.Now turn on the large Harvard syringe pump holding the 30 milliliter syringes and let it run for one minute. Check that there are no bubbles and no leakage in the device or in the tubing. Then turn on the small Harvard syringe pump and let it run for an additional minute here too.Checking for bubbles and leakage. Stop the pumps after two minutes. As the microfluidic device is now ready to be used with blood samples, the blood samples to be analyzed were collected as described in the accompanying written protocol and are used within one hour of collection.The first tube of collected blood is discarded to rid of false positive results. Remove the one milliliter syringe from the small Harvard syringe pump, then inject one XPBS from the one milliliter syringe filling the syringe needle. To avoid introduction of bubbles, fill a one milliliter syringe with 50 microliters of one XPBS without trapping any bubbles.Then fill the syringe with 0.5 milliliters of blood. Keep the syringe vertical while filling to avoid mixing of the PBS with the blood. Next, connect the syringe containing the blood back to inlet one.Wipe away any excess fluid and mount the syringe vertically on the small Harvard syringe pump. Be careful not to push the blood through the tubing into the device. Remove the outlet tubing from the waste tube and place it in a clean 50 milliliter centrifuge tube sitting in an ice bucket.Now start the run switch on the large Harvard syringe pump first and let it run for one minute. Then switch on the small Harvard syringe pump. The sample will start collecting from the outlet tubing into the 50 milliliter centrifuge tube.Let the blood sample in the one milliliter syringe travel completely through the device, which takes approximately 20 minutes. Then stop both pumps. Now that the run is complete, centrifuge the collected sample for five minutes at 350 times G at room temperature with the break off.Remove the supernatant by placing the pipette tip at the opposite side of the white pellet. Remove as much supernatant as possible, especially red cell debris without disturbing the white pellet. Resuspend the sample in one milliliter of flow buffer containing 1%BSA and 2%para formaldehyde in one XPBS by pipetting.Up and down, transfer the sample to a 1.5 milliliter eend orph tube micro centrifuge, the collected sample for five minutes at four times g. At room temperature, remove the supernatant as carefully as in the previous centrifugation step. Finally, add one milliliter of flow buffer vortex to resuspend the sample and proceed to analyze the sample by flow cytometry.Now we'll show some representative results in the form of fax plots from a blood sample enriched by the microfluidic device with analysis using flow cytometry. The importance of enrichment by lysis can be seen by a fax plot where red blood cells cloud. The scatterplot produced by the microfluidic enrichment protocol is a clean scatterplot with defined nucleated cell populations found in circulation.The red region represents lymphocytes, green represents monocytes, blue represents granulocytes, and the purple region has yet to be characterized. Failure to remove red cell debris in the protocol produces a cluster of events in the fax plot seen in the cyan region. Populations of cells can be visualized in separate fax plots.Lymphocytes sustained with CD three and CD four. Monocytes sustained with CD 14 and granulocytes sustained with CD 66 P.We've just shown you how to set up and run a blood sample with the microfluidic blood lysis device. When doing this procedure, it's important to remember to run the blood sample immediately to not allow the PBS to mix with the blood prior to entering the device and to avoid introduction of bubbles into the channels of the device.So that's it. Thank you for watching and good luck with your experiments.

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Learn an automated microfluidic blood lysis protocol for enriching circulating nucleated cells from peripheral blood. This method minimizes cell loss and preserves sample quality for downstream flow cytometry analysis.

Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

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