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.

1. 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). 2. Attach a 30-gauge syringe needle (Small Parts, Miramar, FL) to the tubing on each of the inlets, providing the macro to micro interface. 3. 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 4. 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. 5. 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.

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. (Label the syringe) 3. 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) 4. 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. 5. 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.  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. 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.

Discussion
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 [2].