Parallel microchannel-based measurements of individual erythrocyte areas and volumes

Abstract

We describe a microchannel device which utilizes a novel approach to obtain area and volume measurements on many individual red blood cells. Red cells are aspirated into the microchannels much as a single red blood cell is aspirated into a micropipette. Inasmuch as there are thousands of identical microchannels with defined geometry, data for many individual red cells can be rapidly acquired, and the fundamental heterogeneity of cell membrane biophysics can be analyzed. Fluorescent labels can be used to quantify red cell surface and cytosolic features of interest simultaneously with the measurement of area and volume for a given cell. Experiments that demonstrate and evaluate the microchannel measuring capabilities are presented and potential improvements and extensions are discussed.

FIGURE 1

A schematic diagram of the HEMA array. The array consists of 300 rows of ∼300 parallel microchannels (only the middle part of the array is shown). The overall size of the array is 10 mm × 5 mm. Most of the microchannels in the array are 6-μm wide, so the red cells can easily pass through (a). The channels in the middle section have a wedge shape designed to arrest the cells (b). Note that there are wide channels in every row so that flow through the array does not cease even when all wedge-shaped channels fill with cells.

FIGURE 2

Scanning electron micrograph of the negative of the wedge-shaped channels etched in the silicon wafer. Since the resolution of the pattern transfer on the wafer was limited to ∼100 nm, the slope of the wedges is not perfectly smooth but has small steps. The etching process produced vertical but slightly rippled walls. The scale bar indicates 20 μm.

FIGURE 3

Scanning electron micrograph of the wedge-shaped channels cast in silicone elastomer. The silicone cast is a good replica of the silicon master (Fig. 2). The dark vertical lines on the channel walls are most likely the steps in the slope of the wedge shape. The scale bar indicates 20 μm.

FIGURE 4

HEMA experimental setup. The microchannel array embedded in the silicone (a) is sealed facedown to a standard glass microscope slide (b) covered with a thin film of the same silicone elastomer (c). The entrance to the array is cut open (d). The exit section of the array is connected through a hole in the glass slide (e) to a water reservoir (f). The flow through the array is provided by aspiration pressure from a vacuum source coupled to the reservoir. The array module is transparent so that the microchannels can easily be visualized with a standard light microscope (g).

FIGURE 5

Red cells arrested in wedge-shaped microchannels as observed during a typical experiment. The arrows indicate the channel entrance (A) and exit (B), and the top (C) and the bottom (D) of a trapped cell. For each cell, the positions C and D are measured relative to A and B. These measurements are used to calculate cell area and volume. The microchannels on the figure are 80 ± 1 μm long and 3.4 ± 0.1 μm deep. The channel width at the entrance is 3.44 ± 0.2 μm and the width at the exit is 0.95 ± 0.2 μm.

FIGURE 6

The fluorescently labeled RBC membrane of an arrested cell as viewed with a confocal microscope. A superposition of 24 Z-planes obtained with X-Y-Z scan is shown. The nominal distance between the Z-planes was 0.2 μm. No creases in the membrane are observed.

FIGURE 7

Schematic representation of a three-dimensional model used for computing the area and volume of the arrested cells (A). The cells are considered to be rectangular wedges with rounded edges and caps. A cross-section of the main cell body (B). The round edges have a radius R_E. The caps of the cell were taken as quasihemispheres, with an effective radius R_C, made to fit smoothly with the membrane of the main body.

FIGURE 8

The mean change in cell position with changes in osmolarity of the buffer flowing through the array. The experiment was carried out as follows. Once the cells were arrested in the microchannels, the osmolarity of the perfusing buffer was periodically changed. Buffers of three different osmolarities were used (292, 259, and 365 mmol/kg). When a buffer of lower osmolarity was introduced the cells swelled and moved upward, and the opposite was true of shifts to higher osmolarity. The positions were measured after equilibrium was attained. The mean difference in position was calculated for 40 cells. The mean cell position correctly follows the changes in buffer osmolarity.

FIGURE 9

The mean relative change of cell volume and area from the same experiment as presented in Fig. 8. The relative changes were calculated with respect to the values measured at the beginning of the experiment. As expected, the calculated volumes follow the change of osmolarity whereas the changes in the calculated areas are much smaller. The small nonrandom changes in the area may reflect the systematic errors of the HEMA.

FIGURE 10

The relationship between MCV measured with the HEMA and with the standard method. The two sets of values are plotted against each other with the line of unity (A), and as their percent difference vs. their average value (B). In the second panel, the solid line represents the mean error (ē ≈ + 1.1%, with 95% confidence limits at −0.4% and +2.6%) and the dashed lines define the 95% confidence interval for the distribution of data. The error bars represent the combined measurement error of the difference of the two methods (≈(0.65² + 1.28²)^1/2 ≈ 1.44%), where the random error in the standard method (0.65%) was determined from multiple measurements made on the 10 blood samples and the random HEMA error (1.28%) was determined from the data associated with Table 1.

FIGURE 11

The relation between cell volume and surface area in a typical measurement. The solid line represents the regression line fitted to the relation A = k × V + A₀. The measured values in this case are: k = 1.1 μm²/fL (the mean area-to-volume ratio was 1.37 μm²/fL), A₀ = 26.8 μm², and the correlation coefficient between area and volume was 0.96.

FIGURE 12

The histograms of the distributions of area (a) and volume (b) and the correlation between the two (c) for a sample with two different subpopulations of red cells mixed at a 1:1 ratio. The two subpopulations are the small cell size fraction and the large cell size fraction obtained via counterflow centrifugation. The small fraction was additionally dehydrated with ionophore A23187 and calcium buffer. The solid lines represent curves fitted to the data obtained from the measurements performed on each of the subpopulations alone (see Table 2).