A microfabricated electrical differential counter for the selective enumeration of CD4+ T lymphocytes

Abstract

We have developed a microfabricated biochip that enumerates CD4+ T lymphocytes from leukocyte populations obtained from human blood samples using electrical impedance sensing and immunoaffinity chromatography. T cell counts are found by obtaining the difference between the number of leukocytes before and after depleting CD4+ T cells with immobilized antibodies in a capture chamber. This differential counting technique has been validated to analyze physiological concentrations of leukocytes with an accuracy of ∼9 cells per µL by passivating the capture chamber with bovine serum albumin. In addition, the counter provided T cell counts which correlated closely with an optical control (R(2) = 0.997) for CD4 cell concentrations ranging from approximately 100 to 700 cells per µL. We believe that this approach can be a promising method for bringing quantitative HIV/AIDS diagnostics to resource-poor regions in the world.

Fig. 1

Principle of electrical differential counting of CD4+ T cells. (a) Graphically-rendered image depicting the chip’s geometry, specifically its two electrical counters and capture chamber. Cross-sectional views of the chip during an experiment: (b) forward flow direction to obtain total leukocyte count at the entrance counter, (c) reverse flow direction and enumeration of uncaptured cells after leukocytes reach the exit sensor, and (d) finished experiment after all unbound leukocytes are washed from the capture chamber. The concentration of CD4+ cells can be obtained simply by normalizing the differential count by the known sample volume ((d), right panel). The right panels of (b) and (c) illustrate how cell flow direction at the entrance counter can be determined by the change in pulse signature polarity, with the pulse signature changing from up-down (b) to down-up (c) in time.

Fig. 2

(a–g) Microfabrication process flow for the electrical differential counter chip. (h) Fabricated chip compared to the size of a US dime (scale bar length is 10 mm). (i) Micrograph of an entrance counter region, depicting the three-electrodes setup, narrow counter channel, and 50 µm-high capture chamber (scale bar length is 100 µm).

Fig. 3

Experimental setup and process for electrical differential counting of CD4+ T cells. A dual lock-in amplifier is used to inject an AC signal and monitor its change through the chip’s counter regions, which are balanced by a Wheatstone configuration. Process: (1) the cells pump injects leukocytes into the chip (forward direction) to be enumerated at the entrance counter; (2) after cells are sensed at the exit counter, both valves are switched to reverse flow direction and the PBS wash pump purges the capture chamber of uncaptured cells, which are enumerated again by the entrance counter.

Fig. 4

Impedance data of the entrance counter for the entire duration of a reverse-flow differential counting experiment. The data can be separated into four regimes: (i) no cells present, (ii) cells beginning to enter the chip’s entrance, (iii) just after reversing flow and counting cells exiting back out the entrance, and (iv) near the end of purging the capture chamber of uncaptured cells. Magnifications of the impedance signal during forward (v) and reverse flow (vi) show how pulse signature polarity can be used to ensure when reverse counting begins.

Fig. 5

Maximum pulse density characterization. Concentration calculations were based on the number of pulses per s for a known flow rate. Error bars represent the standard deviations obtained from measurements of 9 (1 : 1000), 14 (1 : 500), 25 (1 : 200), and 23 (1 : 100) duration windows.

Fig. 6

Analysis of reverse differential counting for a passivated capture chamber. (a) Percent error decreases exponentially (dashed line, R² = 0.57) as the number of leukocytes entering the chip (forward count) increases. (b) Cumulative counts of forward and reverse-flow regimes for the particular experiment highlighted in (a). Error is calculated by normalizing the differential count (absolute difference between the forward and reverse counts) to the forward count.

Fig. 7

Reverse-flow differential counting of CD4+ T lymphocytes. (a) Comparison of electrical differential counting method and the optical controls. A linear fit (R² = 0.997) is not shown, as it is would be indistinguishable from the y = x correlation standard. (b) Cumulative counts of forward and reverse-flow regimes for the particular experiment highlighted in (a). The absolute number of captured CD4+ T cells was found simply by subtracting the reverse count from the forward count. The dotted line denotes a completed analysis (where reverse counting levels off) duration of approximately 7 minutes. (c) Bland–Altman analysis of the data in (a). The dash-dot lines denote upper and lower levels of agreement (ULA and LLA, respectively). The dashed line shows a bias of 9 cells toward the optical method. (d) Counting error decreases as the total number of captured CD4+ T cells increases. Listed statistics are for three regimes demarcated by dashed lines: 0 to 1000 captured helper T cells (0 to 200 cells per µL, assuming 5 µL of sample flowed into chip), n = 3; 1000 to 1750 (200 to 350 cells per µL), n = 5; and greater than 1750 (>350 cells per µL), n = 6.