Microfluidic-Based Novel Optical Quantification of Red Blood Cell Concentration in Blood Flow

Abstract

The optical quantification of hematocrit (volumetric percentage of red blood cells) in blood flow in microfluidic systems provides enormous help in designing microfluidic biosensing platforms with enhanced sensitivity. Although several existing methods, such as centrifugation, complete blood cell count, etc., have been developed to measure the hematocrit of the blood at the sample preparation stage, these methods are impractical to measure the hematocrit in dynamic microfluidic blood flow cases. An easy-to-access optical method has emerged as a hematocrit quantification technique to address this limitation, especially for the microfluidic-based biosensing platform. A novel optical quantification method is demonstrated in this study, which can measure the hematocrit of the blood flow at a targeted location in a microchannel at any given instant. The images of the blood flow were shot using a high-speed camera through an inverted transmission microscope at various light source intensities, and the grayscale of the images was measured using an image processing code. By measuring the average grayscale of the images of blood flow at different luminous exposures, a relationship between hematocrit and grayscale has been developed. The quantification of the hematocrit in the microfluidic system can be instant and easy with this method. The innovative proposed technique has been evaluated with porcine blood samples with hematocrit ranging from 5% to 70%, flowing through 1000 µm wide and 100 µm deep microchannels. The experimental results obtained strongly supported the proposed optical technique of hematocrit measurement in microfluidic systems.

Keywords: biosensing platform; grayscale; hematocrit quantification; microfluidics; optical measurement.

Figure 1

The schematic representation of the experimental setup was used to demonstrate the optical technique for the quantification of hematocrit.

Figure 2

(a) PDMS microchannel 100 μm high and 1000 μm wide, with inlet and outlet ports. (b) Schematic drawing of the microchannel used in the experiments detailing the dimensions.

Figure 3

Comparison of images captured during the flow of blood with 15% hematocrit in the microchannel (a) from the eyepiece of the microscope and (b) from the camera (1) before white balance and (2) after white balance.

Figure 4

Schematic representation of the grayscale measurement to quantify the hematocrit during blood flow in the microchannel: (a) the frame captured from the video of the blood flow in the microchannel (with 1000 µm width and 100 µm depth); (b) cropped image from Figure 4a in RGB format; (c) converted grayscale image from Figure 4b; (d) the plot of the grayscale histogram with average grayscale of 51.84.

Figure 5

Plot of the luminous exposure vs. grayscale for blood samples with varied hematocrit of 20%, 35%, and 50%.

Figure 6

Comparison of the average grayscale of different hematocrit blood images from donor 1 (blue) and donor 2 (red) at 6000, 8000, and 12,000 lux·µs. Images of the blood from donor 1 and donor 2 were taken with 20 lux and 40 lux illuminance, respectively. Each data point takes the average of three technical replicates, and the error bar shows the standard deviation.

Figure 7

Plots of experimental results of grayscale vs. hematocrit of the blood at different luminous exposures (Hv) within (a) the low luminous exposure range (<6000 lux·µs), with an average R² = 0.992 (the square of the correlation coefficient indicates that 99.2% of the variation in the grayscale can be explained by hematocrit), (b) the medium luminous exposure range (6000 lux·µs~15,000 lux·µs), with an average R² = 0.990, and (c) the high luminous exposure range (>15,000 lux·µs), with an average R² = 0.983. Each data point takes the average of three technical replicates.