The overall goal of this microfluidic experiment is to simultaneously measure platelet deposition in fibrin formation on a reactive surface under flow conditions using real-time video microscopy. This method can help answer key questions in the field of transfusion medicine about the effects of platelet concentrate preparation on the procoagulant nature of transfused blood. The main advantage of this technique is that it facilitates simultaneous analysis of platelet function and coagulation in the flow, allowing the interplay between both processes to be examined.
Though this method can provide insight into contact pathway induced platelet procoagulation, it can also be applied to the study of the extrinsic pathway by coating with lipidated tissue factor. Demonstrating the procedure will be Rosalie Devloo, a technician from the laboratory. Begin the microfluidics flow chamber setup by pipetting 0.8 microliters of freshly reconstituted collagen into five sixths of the length of each channel on one end of a new disposable microfluidic biochip with eight straight parallel channels and labeling this end of the microchip as the outlet.
Incubate the biochip at four degrees Celsius for at least four hours in a humidified and sealed container. At the end of the incubation, fill the coded channels in an equal number of Y-shaped channels in a mixing biochip with blocking buffer. Return the microchip to the container for one hour at room temperature.
Then, place a pin in one end of two, eight centimeter long and one, two centimeter long pieces of tubing and in both ends of one, 46 centimeter long piece of tubing for each channel in use. Next, use the Nanopump software to rinse all the pump fluidics in the connected tubing with distilled water and a syringe in syringe connector pin to fill all the other tubing with blocking buffer. Then, fix the coded biochip onto the automated microscope stage using a laboratory scissor jack to fix the mixing biochip at the same height as the microscope stage.
Use the pins in the eight centimeter tubes to fix these pieces of tubing to the mixing biochip inlet and put the free end of each tube in a vial of HEPES buffered saline. Now, connect the coded biochip to the mixing biochip with the 46 centimeter piece of tubing in a flexible, but straight, line. Attach a syringe connector pin to the luer-compatible tubing of the rinsing pump.
Then, connect the pin to the free end of the two centimeter piece of tubing and fix the other end of the tubing to the outlet of the coded biochip. Then, rinse all the tubing and the channels with one milliliter of HBS. To prepare the perfusion pumps, open the Microfluidic Perfusion Pump Driver software and double click on the syringes icon to initialize the pumps.
Select the type of syringe that will be used and disconnect the rinsing pump and the two centimeter tubing connected to it. Connect the disconnected two centimeter tubing with its syringe connector pin to the luer lock of a piece of thick-walled waste tubing and use a syringe to fill the thick-walled tubing with standard pepsin solution. Then, secure the open end of the thick-walled tubing with a Kocher clamp.
Pin the tubing to the outlet of the coded biochip. Disconnect the two tubes from the mixing flow chamber together and discard the containers of HBS. After obtaining the blood sample in evacuated sodium citrate containers, centrifuge the tubes and transfer the platelet rich plasma into a new centrifugation tube.
Spin down the containers with platelet rich plasma again. Then, transfer the supernatant platelet-poor plasma to a different centrifuge tube. Next, use a 21 gauge needle to perforate the bottom of the sodium citrate containers to allow the packed red blood cells to drip into a new conical tube.
When all of the packed red blood cells have been collected, gently reconstitute blood by combining separated packed red blood cells with platelet-poor plasma and add blood bank platelets prepared for transfusion to obtain samples with a 40%hematocrit and 2.5 times 10 to the fifth platelets per microliter concentration to a 2.5 milliliter final volume and obtain a complete blood count. Next, add 13 microliters of fluorophore-labeled fibrinogen to a polystyrene conical tube. Followed by one milliliter of the reconstituted blood.
Mix another one milliliter of blood with two microliters of fluorescent platelet dye and add the labeled blood samples together. Then, gently invert the mixed blood samples and incubate the cells for 10 minutes at 37 degrees Celsius. Before beginning the perfusion assay, focus the microscope optics to the collagen fibers adhered to the bottom of the first channel.
Using the acquisition software, define a region of interest in the selected channel. Load one milliliter of pre-warmed coagulation buffer into a one milliliter syringe and mount the syringe onto the perfusion pump. After a second gentle inversion, load the blood into a two milliliter syringe and mount the syringe onto the perfusion pump.
Then, use a syringe connector pin to attach both syringes to one piece of eight centimeter tubing and prime the connectors and tubes at 400 microliters per minute. When all the tubing is filled with either coagulation buffer or blood, pin the other end of both tubes to the split legs of the Y-shaped channel in the mixing biochip. Then, remove the Kocher clamp at the outlet of the waste tube and initiate the perfusion at 4.4 microliters per minute for the coagulation buffer and 44 microliters per minute for the reconstituted blood.
Record images every 10 seconds for 30 minutes in real-time, terminating the pumps and image acquisition after 30 minutes or when the signal becomes saturated. Then, discard all of the tubing and syringes as biohazardous waste. This video provides an excellent example of the buildup of platelet thrombi and fibrin deposition.
At the beginning of the perfusion, the platelets adhered to the reactive surface, resulting in a steady increase in recorded green fluorescence. During this adhesion phase, there's little violet fluorescence indicating that the fibrin is only marginally formed. Upon the initiation of coagulation, the violent fluorescing fibrin deposits rapidly with the platelet green fluorescence accumulating at about the same rate.
A moment of coagulation onset can therefore be extrapolated as a determinant of the platelet procoagulant potential. The addition of corn trypsin inhibitor to the reconstituted blood does not affect the platelet adhesion but the coagulation does not start for the total duration of the perfusion experiment. By increasing the number of platelets in a reconstituted sample, the rate of adhesion, accumulation and coagulation increases linearly.
The moment of onset is significantly shortened by increasing the platelet concentration, suggesting that a threshold number of activated deposited platelets is required to trigger coagulation. Further, in a paired analysis of channels coated with collagen only, or in combination with recombinant lipidated tissue factor, the coagulation onset is significantly faster, although neither the overall rate of the coagulation, nor the platelet accumulation, is different between the conditions. After watching this video, you should have a good understanding of how to perform a microfluidic flow chamber experiment with double-labeled reconstituted blood for the simultaneous analysis of platelet function and coagulation.
After it's development, this technique paved the way for researchers in the field of transfusion medicine to explore the interactions between platelets and coagulation under flow. In this video, we focused on demonstrating a standardized experiment. But changes in the surface coating, shield stresses or calcium concentration can be introduced to answer specific research questions of interest.
