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August 01, 2012 (Vol. 32, No. 14)

Hemocytometer Counting Alternative

Novel System Offers Cell Sizing, Count, and Assessment of Health

  • Cell counts are routinely performed in life science, clinical, and industrial laboratories to monitor cell growth rates, to measure seeding densities, to establish counts for data normalization, and to determine initial counts for experimental protocols. Traditionally, these counts are performed manually using hemocytometers. However, in addition to being laborious, this approach suffers from large errors and variability.

    Alternatives to hemocytometer-counting include high-end flow cytometers, Coulter-counting systems, and, more recently, imaging-based systems. The former systems are prohibitively expensive in both initial costs and the significant maintenance requirements. In contrast, imaging-based systems present researchers with the trade-off of realizing lower cost and ease-of-use at the expense of count precision and accuracy.

    Orflo Technologies recently introduced the Moxi Z mini-automated cell counter as a new alternative that bridges the gap in performance vs. cost and usability. Specifically, the Moxi Z utilizes the gold-standard Coulter Principle to provide three parameters of information regarding cell cultures: 1) precise cell sizing, 2) overall culture health, and 3) highly accurate cell counts.

  • Single-Cell Resolution—3D Cell Sizing and Health

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    Figure 1. Moxi Z cell sizing and health: (A) Sizes of precision calibrated beads were measured using both Type M (red markers) and Type S (blue markers) cassettes and were compared to manufacturer-reported sizes. Inset: Moxi Z size histogram for a multibead sample. (B) Comparison of healthy (blue, MPI=0.96) and necrotic-spiked (red, MPI=0.74) populations of Jurkat cells illustrating changes in MPI values with health. Histogram curve-fitting (dashed lines) identified potential sub-populations.

    At the core of the Moxi Z technology is a precise, volumetric (3D) electrical measurement of each cell or particle as it passes through an aperture (Coulter Principle). The resulting output is a high-resolution histogram that provides exact sizing information for each particle as well as a valuable perspective of the culture composition. The accuracy (r2>0.999) and precision of the Moxi Z particle sizing was illustrated through comparison (Figure 1A) of Moxi Z measured diameters of precision calibrated beads with manufacturer-reported values (3.0 µm, 4.17 µm, 5.6 µm, 7.5 µm, 10.1 µm, 15.6 µm, and 25.0 µm diameters). Furthermore the high-resolution histogram (e.g., Figure 1A inset—five bead mixture) demonstrates the size discrimination capabilities of the instrument.

    The quality of the sizing information provided by the Moxi Z strongly contrasts with the imprecision inherent in 2D image-based approaches that attempt to estimate cell size using image interpolation. Consequently, the Moxi Z uniquely enables size-based discrimination and analysis of cell sub-populations and a correspondingly more robust means of separating debris counts from cell counts. This degree of information allows researchers to quantitatively monitor and study size-based changes to their cell populations.

    The histogram sizing and shape also provides a means of monitoring culture health. One example of this is shown in Figure 1B, highlighting differences in histogram shapes for a healthy Jurkat population (Figure 1B, blue) versus a mix of healthy and heat-killed (60°C for 30 min, followed by overnight incubation at 37°C) Jurkat cells (Figure 1B, red). Increases in counts in the 4–8 µm range of the histogram reflect increased dead cell and debris counts.

    The histogram change is quantified as an MPI value, that is a ratio of the core cell population to the total particle population. Changes in the MPI values and histogram shape can additionally uncover potential microbial contamination in cultures through their contributions to smaller particle counts as they colonize/aggregate.

    In this regard, the MPI and monitoring of changes in histogram shapes can provide a level of quality control for the cultures by potentially signaling size changes associated with cellular mutations, cell cycle, variations in media composition, or other environmentally induced health changes.

  • Count Precision and Accuracy

    Click Image To Enlarge +
    Figure 2. Moxi Z’s precise and accurate cell counts: Plots of the Moxi Z measured concentrations (n=3, Type S cassettes) vs. analogous system measurements (100 µm aperture).

    Cell count information serves as the foundation for experimental protocols such as in the determination of the quantities of (costly) reagents and the cell seeding densities necessary for downstream processing. Count information is also often applied to the normalization of results in data analysis, thereby imposing a strict requirement for both consistency and accuracy.

    To gauge the Moxi Z counting performance, Moxi Z counts were compared to equivalent counts from a high-cost system that has long been used as a reference-standard in cell counting. Initial concentrations of 2e6–2.5e6 cells/mL for varying cell types were created through dilution in HBSS. Subsequent concentration levels were established through ratiometric serial dilutions and measured on both systems.

    As shown in Figure 2, the Moxi Z achieves a comparable count accuracy (r2 > 0.994) to that of the higher-cost system across a broad dynamic range (3e3–2.5e6 cells/mL). Furthermore, compared to manual and imaging-based approaches, the Moxi Z demonstrates a much higher level of repeatability with coefficients of variations of <5% vs. 30–40% for image-based systems (data not shown). As a result, the Moxi Z assures researchers a level of consistency and accuracy in their count data that is irrespective of the technician performing the counts.

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