Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

The overall goal of this procedure is to directly quantify red blood cell aggregates in microcirculation, dynamically under controlled and constant shear rates. This is accomplished by first in training, red blood cell suspensions, and a double y microchannel with a phosphate buffer saline solution, creating a constant cheer in the blood layer. The second step is to visualize and record the flow with a high speed camera.

Next, use image processing techniques to process the recording and obtain an average aggregate size and the aggregate size distribution in the microchannel. The final step is to determine the blood velocity by following the displacement of fluorescent particles injected in the microchannel between two consecutive frames. Ultimately, the shear rate in the microchannel is calculated based on the blood velocity and the blood layer thickness to relate the aggregate sizes to the corresponding local shear rate.

The main advantage of this technique over existing methods like the light transmission method, is that the red blood cell aggregates are analyzed dynamically in the microscopic share flow as opposed to statistically with other techniques. This method can help answer key questions in the hemo radiological field, such as understanding the conditions of red blood cell aggregates formation and finding blood radiological behavior in micro circulation. To begin fabricate micro channels, 120 microns wide, 60 microns high, and seven millimeters in length according to standard photolithographic methods.

Next, collect blood cells from healthy volunteer donors and prepare red blood cell suspensions with five, 10, and 15%hematocrit levels as described in the accompanying text protocol, combine one milliliter of each red blood cell suspension with 60 microliters of a 1%solution containing fluorescent tracer particles. Gently shake the tube to mix the solution. Also combine the same volume of tracer particles to one milliliter of PBS at a pH of 7.4.

Load a 25 microliter glass syringe with the tracer solution containing red blood cells and a 100 microliter glass syringe. With the tracer solution in PBS, ensure that there are no bubbles present in the system. Next place the microchannel on an inverted microscope stage connected to a high speed camera capable of recording 18 frames per second or greater, and a microparticle image velocimetry system.

Connect each syringe via tubing to one of the inlet ports of the microfluidic channel and then load them into the holder of the same syringe pump. Program the syringe pump to dispense the 25 microliter syringe at a rate of two microliters per hour. This will also dispense the 100 microliter syringe at four times that rate due to their size difference.

Change the flow rate between experiments in order to test different shear rates. Use a calibration ridicule with the 20 x lens in place to calibrate the high speed camera. Then start the pump to begin dispensing the two solutions into the microchannel.

Determine an optimal camera exposure. Time for clear imaging of the aggregates. It is important to wait for the flow to reach steady state before acquiring any measurements.

If steady state is now reach within three to five minutes. Refill the syringe to a educate the mixture and keep red blood cells in suspension. Once steady state flow is reached, begin recording the movement of the red blood cells.

Record the flow for seven seconds. Using a program capable of image processing, enhance the contrast for each gray scale image taken using the histogram equalizer method. Next, convert the gray scale images into binary images.

Carefully set the threshold value by testing several values and choosing the value for which all the aggregates are properly detected. If necessary, fill in the holes corresponding to the gaps within one aggregate. Then detect the cells by determining neighboring white pixels in the binary image so that adjacent cells are accounted as aggregates.

Label the different red blood cell aggregates detected, and then convert the binary image into a red, green blue image for better visualization. Finally, overlay the red, green blue image onto the original image. In order to verify the efficiency of the image processing, calculate the area of each aggregate detected in the frame based on the number of pixels detected.

Process all the frames in the same manner. Next, average the aggregate sizes detected in each frame, and then the results of all frames. To obtain the average aggregate size for each recording of the red blood cell suspensions engines, convert the results to square micrometers using the ridicule image.

Then calculate the area of one red blood cell to determine a representative. Estimated number of red blood cells within each detected aggregate once the data is acquired. Using the high speed camera, switch to the double pulsed camera used for the microparticle image velocimetry system.

Use an imaging software such as the one shown here for image acquisition of the flow and the image processing for velocity field determination. Be sure to put on protective eyewear and then turn on the laser system and the camera. Place a micrometer under the microscope and use the resulting image to calibrate the system.

Then start the fluid flow and visualize the particles in both fluids. Find the middle plane of the microchannel by focusing on the fastest and brightest particles in the flow. Next, set the time interval between frames to about two milliseconds so that the fastest particles move five to 10 pixels between both images and begin recording the flow O.Set the software to acquire 100 pairs of images for all the different red blood cell suspensions and for different shear rates.

Following image pre-processing. Choose the correlation window size and shape as well as the percentage of images overlapping. Express the results as an average velocity field calculated from the 100 image pairs and the root mean square error in velocity.

Turn off the laser camera and computer. Once all the measurements and image processing has been performed, then calculate the sheer rate by first detecting and estimating the blood thickness in the micro channel based on the high speed camera recording. For this purpose, average all the frames in the specific recording to obtain a background image of the video.

Finally, delit the blood layer by visually inspecting the average image of all the frames and manually detect the edge of the blood layer. Then obtain the velocity value for the corresponding blood layer thickness manually from the extracted velocity profile, and calculate the shear rate by dividing the velocity value by the blood layer. Thickness shown here are the net motions of four red blood cell aggregates that were detected in three consecutive frames.

The large aggregates of more than 30 cells are shown in red. The medium aggregates between nine and 30 cells are shown in green, and small aggregates of fewer than nine cells are shown in blue. The velocity of an aggregate varies based on its position in the microchannel shown.

Here are the velocity profiles of the red blood cells suspended at 5%hematocrit, 10%hematocrit, and 15%hematocrit at a flow rate of 10 microliters per hour. You can see the velocity head towards zero at both edges as expected, and the inflection point between the two flow streams vary slightly between the hematocrit levels as the overall shear rate increases the size of the aggregates in each hematocrit level drops. This is due to the sheer force in the microchannel exceeding the aggregating force causing the red blood cells to disaggregate.

While attending this procedure, it's important to remember to ensure a good image quality of the flow in the micro channel and ensure a contrast between the red blood cells flowing and the image background. After watching this video, you should have a good understanding of how to analyze qualitatively and quantitatively red blood cell aggregates under controlled flow conditions.