The overall goal of this experiment is to mimic platelet transfusion in vitro and test it using hemostasis on collagen, with hydrodynamic flow and real time microscopy. Patients with thrombocytopenia are treated with stored platelet concentrates. Our method is intended to help answer key questions in the field of thrombosis and hemostasis, with specific emphasis on the quality and function of such stored platelet concentrates.
The added value of this technique is that it mimics blood flow, and thus takes into account rheology influences on participating cells and biomolecules. The current method assesses platelet binding to collagen. It can, however, also be applied to other adhesive tissue, including atherosclerotic plaque, endothelial cells, or specific matrix proteins like Von Willebrand factor and fibrinogen.
Begin with preparing the biochip. Vortex and dilute a collagen stock one to 20 in the manufacturer's isotonic glucose solution to a final concentration of 50 micrograms per milliliter. Then, pipette 0.8 microliters into the lanes of a new disposable biochip.
Fill all the lanes on one end of the chip, and mark this as the outlet end. Then inspect the chip to make sure that each lane is filled 5/6 of the way with the solution, and no air bubbles. Then, incubate the chip at four degrees celsius for four hours or overnight in a humidified and closed container.
Later, block the coded channels by pipetting blocking buffer into the other end of the lanes. Then for each lane, cut 12-centimeter lengths of tubing, and attach a pin to each length of tubing to make them attachable to the biochip. Rinse the tube lengths with distilled water from a syringe and a connector.
Then, saturate them with blocking buffer and seal them into a humidified container for at least one hour. Next, prepare the pump and the manifold. First, rinse the pump and manifold with distilled water to remove any air bubbles.
Next, attach the biochip to the automated microscope stage. Put one end of the lengths into a 1.5 milliliter tube filled with HBS, then attach them with the other end to the biochip inlets. Put the manifold pins in the outlets of the biochip.
Then, rinse the system with HBS using the pump. Any remaining blocking buffer or poorly adhered collagen will be thus removed. This completes the flow chamber preparations.
Begin with pooling recent blood collections from healthy volunteers into a conical centrifuge tube. Spin the sample for about 15 minutes at 250 Gs.Do not use the brake, because it will disturb the loosely-packed lower phase. Remove and discard the platelet-rich plasma and the buffy coat.
Save the packed red blood cells containing as few platelets as possible, to make the reconstituted blood. Now, start thawing four milliliters of type AB plasma at 37 degrees celsius for five minutes and 20 seconds. Meanwhile, determine the hematocrit of the prepared packed red blood cells using an automated hematology analyzer.
Then, determine the platelet concentration of a blood bank prepared platelet concentrate to use for reconstitution of the whole blood. Calculate the required volumes for one milliliter of reconstituted blood with 40%hematocrit, and 250, 000 platelets per microliter. Now, using a clipped pipette tip, transfer the required volume of red blood cells, plasma, and platelet concentrate to the desired concentration, and a final volume of one milliliter.
Mix the reconstituted blood with gentle inversions, and perform a complete blood count. Then, get a prepared microcentrifuge tube containing Calcein AM, and add one milliliter of reconstituted blood to it. Mix the blood with dye using gentle inversions.
Then, incubate the reconstituted blood for five minutes at 37 degrees celsius. In the software, select the region of interest in the perfusion lane. Focus on the collagen fibers adhered at the bottom of the lanes at 100x magnification.
Ideally, use phase contrast or differential interference contrast. Choose the set current Z for selected tile regions option to digitally fix the selected Z positions. The optical focus prior to perfusion should be on ameloblast collagen, but this can be tricky.
An alternative is to set the microscope focus on the initial adhering platelets. Now, gently mix the samples and position them next to the biochip. Then, put the tubing connected to the observed lane into the reconstituted blood.
Next, using the software controlling the pump, apply a 50 dyne per square centimeter pressure to move the blood sample into the lane and start the experiment. During the experiment, record images every 15 seconds for five minutes. Determine the thrombus growth kinetics using image analysis software.
In this case, Zen 2012 is utilized. Begin with opening the plugin image analysis to determine the surface coverage of the platelets. Set the fluorescence threshold in the analyze interactive tab to define the pixel intensity that correlates with a positive signal, which would come from adhering platelets.
Then, use create tables to automatically generate a spreadsheet with a list of the surface areas of the positive signals for each time point. Save these spreadsheets in the XML format, and open them in a spreadsheet program for further calculations. At every time point, calculate the total surface areas of the selected objects, and express this value as the percentage of surface that is covered.
Then, plot the data as a function of the perfusion time, and calculate the slope by linear regression. This models the thrombus growth kinetics of that particular experimental condition. Three identical reconstituted whole blood samples were perfused simultaneously over collagen-coated surfaces.
This resulted in a coefficient of variation of 8.7%suggesting acceptable intra-assay and intra-laboratory variation for test comparison. Transfusion was simulated by reconstituting the blood with the deficient component. Several reconstitutions were performed with decreasing platelet concentrations.
As expected, lower platelet counts resulted in less adhesion as a function of perfusion time. Next the effect of decreased temperatures after blood collection and during perfusion were investigated. Thrombi were found to build more slowly when the blood was cooled to room temperature.
As compared to a sample kept at 37 degrees Celsius throughout the study. While circulating, platelets bind to vessel injury sites in conditions of elevated wall sheer stress. As expected, varying the sheer rate in the microfluidic flow chambers changed the total platelet adhesion.
These conditions also change the thrombus growth kinetics. After watching this video, one should have a good understanding of how to perform microfluidic flow chamber experiments using reconstituted blood to test the quality and function of stored platelet concentrates. This technique can be done in roughly seven hours.
Microfluidic flow chamber experiments paved the way for researchers in the field of platelet transfusion to explore the impact of donation, preparation and storage variables. One relevant example is pathogen inactivation. We have given example of just one experimental subject.
However, variations in calcium concentration, sheer stress and surface coatings can be used to address different research questions. Similar to this procedure other measurements in microfluidic flow chambers can be performed to answer additional questions. For instance the performance of stored platelets to coagulation on the flow.
