Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Whether it’s for monitoring rates of proliferation or cytotoxicity, or simply knowing how many cells to seed into a dish or a plate, counting is an often overlooked but essential activity of any lab dealing with cells. And for those who demand precision and accuracy, a hemocytometer just won’t cut it.
The most accurate and precise way to count cells uses the Coulter principle, also called the “electrical current exclusion” principle, “resistance measurement” principle, “impedance-based” measurement and the “electrical sensing zone” method. The method has been widely adopted—it remains the dominant technology used by clinical hematology analyzers to count cells, according to Fernando Chaves, director of global scientific affairs at Beckman Coulter—and has been incorporated into several research-oriented instruments, as well.
Coulter-based instruments today return information about particle number and size, and some have capabilities to yield additional data. Here is what you need to know about handheld and benchtop instruments for the research lab.
The Coulter principle
More than 50 years ago, Wallace Coulter discovered that as particles (such as cells) traveling through conductive fluid pass through a narrow opening separating two electrodes, they create an increase in resistance in the electrical circuit proportional to particle volume. This is the Coulter principle. If the resistance is plotted against time, then counting the spikes in the graph will indicate the number of particles that have traversed that gap; measuring the amplitude of a spike indicates the size of the particle that disrupted the circuit.
As the absolute particle count increases, the tighter the data tend to be, statistically speaking. Because Coulter principle-based counters typically count tens of thousands of cells in just a few seconds, they can boast very low coefficients of variance (CV)—less than 2% CV in the case of Beckman Coulter’s COULTER COUNTER® instruments, for example, compared with less than 6% for the company’s popular image-based Vi-CELL® XR instrument, notes senior global business manager Matthew Rhyner.
Home (in) on the range
The dynamic range of the instrument—that is, the size of the particles that can be analyzed—is generally considered to be roughly 2% to 80% of the opening separating the electrodes (with some vendors citing more conservative numbers). For example, the benchtop COULTER COUNTER Z™ series has several optional size openings to choose from, from a 50-µm aperture that lists a range of 1 µm to 30 µm, to a 200-µm aperture with a listed range of 4.0 µm to 120 µm. “The Z does a lot of mammalian cells,” says Rhyner. But using the standard 100-µm aperture, “you’re going to have trouble with bacterial or plant cells.”
The dynamic range of most impedance-based research instruments is suitable for typical mammalian cells, including blood cells—from erythrocytes to lymphocytes and granulocytes. Among the narrowest of these is Orflo’s smartphone-like Moxi Z™ Mini Automated Cell Counter, at 3 µm to 34 µm. But many instruments also offer the ability to change out the component containing that pore (what Beckman Coulter terms the “aperture tube”) to query larger or smaller particles. Roche’s benchtop CASY Model TT Cell Counter and Analyzer (PDF), for example, can be fitted with a “measuring capillary” ranging from 45 µm to 150 µm, and EMD Millipore’s pipettor-like Scepter™ 2.0 Handheld Automated Cell Counter uses disposable sensors with either a 40 µm or 60 µm opening. Beckman Coulter’s benchtop Multisizer™ series, with aperture tubes from 10 µm to 2000 µm and a dynamic range of 0.2 µm to 1,600 µm, “is really designed to handle anything, including industrial particles,” notes Rhyner.
Izon, a relatively new company from New Zealand, takes a different tack. Instead of a fixed aperture, “we put our hole into a plastic membrane, or tunable elastomer, almost like a rubber band,” says Darby Kozak, Izon’s chief scientist in North America. “You can apply a mechanical stretch and open and close the hole, giving you a larger dynamic range.”
Izon offers three portable benchtop analyzers: The qMicro can handle 4 µm to 100 µm particles, while the qNano has a dynamic range of 50 nm to 20 µm—useful for cells as well as submicron particles like viruses, exosomes and microvesicles. Offering the same dynamic range as the qNano, the qViro-X is designed for use with infectious agents. “You can take the entire unit and put it into an autoclave, or wash it down with something like picric acid or bleach,” Kozak explains. “You can completely decontaminate it.”
But is it alive?
The disadvantage of the Coulter method, “if there is one,” says Rhyner, “is that it doesn’t tell you much about the viability of the cells.” Hemocytometers and image-based counters like the Vi-CELL and Thermo Fisher Scientific's Countess®, for example, typically use a dye such as Trypan blue to distinguish live from dead cells—something not available in label-free Coulter counting.
Some vendors have supplemented the Coulter principle with other technologies. Clinical hematology analyzers, for example, typically use multiple optical and other measures to establish a variety of parameters such as morphology and the presence of a nucleus.
The handheld Orflo Moxi Flow™ is essentially the Moxi Z with an integrated single-color flow cytometer, enabling dead or apoptotic cells stained with propidium iodide or an antibody to annexin-V, for example, to be distinguished, says Don O’Neil, Orflo’s senior vice president for sales and strategic partnerships.
Other instruments use sophisticated electronics and/or software algorithms to help make such determinations. CASY devices, for example, use the fact that the membranes of dead cells no longer act as an electrical barrier, so these cells are recorded as being the size of their nucleus.
Cost
A hemocytometer can be had for under $100. Handheld Coulter-principle instruments start at less than $3,500 (with the Moxi Flow retailing for slightly less than $10,000). Benchtop counters start at about $12,000 for the qMicro and work their up to about $50,000 for a well-equipped Multisizer 4e.
But don’t forget consumables. The Moxi Z costs less than $1.50 per test to run, and the Moxi Flow costs about $2.50 per test. Disposable sensors for the Scepter are $3.50 each from the Fisher catalog. The cost of benchtop units’ consumables, on the other hand, ranges from about 20 to 25 cents per run (although Izon’s elastomer nanopores may need to be replaced every month or so, at a cost of about $50, notes Kozak).
Image: A cell-counting cassette for Orflo's Mozi Z mini automated cell counter.
Correction (9/10/14): The aperture rate for Beckman Coulter's Multisizer series was incorrectly given at 20–2000 μm; it should be 10–2000.