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Published: January 3, 2011
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The Coulter Principle and the development of IVDs

The Coulter Principle’s cell-volume monitoring capabilities provide new and evolving benefits in clinical and research applications, from basic cell biology to physiological testing and diagnostics.

By: Matthew N. Rhyner

The Coulter Principle provides a method for counting and sizing cells, and its proper application has contributed to advances in IVDs. The principle can be applied to every stage of test development, from studies of basic cellular biology, through preclinical physiological testing, and ultimately in routine clinical diagnostics. With a better understanding of the Coulter Principle and its contributions, IVD manufacturers may find new ways of applying the technique to their own IVD tests. 

In this article, the Coulter Principle is first described mechanistically. Second, recent discoveries supported by the Coulter Principle and related to IVD test development are highlighted. Finally, contemporary instruments pertinent to this discussion are presented. This article should give a better understanding and appreciation of the ways in which the Coulter Principle can be used to develop new IVD tests.
 
The Coulter Principle
The Coulter Principle relies on the fact that objects placed in an electric field will modify the current flow in that field. In order to turn this observation into a useful tool, Wallace H. Coulter identified several conditions that must be met. First, the electric field should be established in a conductive liquid. Second, the current path should be physically constricted so that the presence of particles or cells in the field will cause the current to change in a detectable manner. Furthermore, the particles must be dispersible in the liquid and diluted enough so that only one particle at a time enters the detection area. 
Figure 1. Simplified schematic of a Coulter Counter.
Figure 1. Simplified schematic of a Coulter Counter. As the vacuum pulls the suspension through the aperture in A, electric current flows between the electrodes. As individual particles travel through the sensitive zone (points 1-8) in B, voltage pulses are generated as shown in C and D. The large cell in C creates a larger amplitude pulse than the small cell in D since it causes greater electrical resistance through the aperture. The stirrer helps to keep the particles suspended, and liquid from the reservoir can be used to flush out the aperture tube between runs. 
Typically, the particles have an electrical conductivity that is different than the liquid’s. In most implementations, suspended particles are forced through a constriction simultaneously with an electric current while the current is measured. As individual particles pass through the constriction, discrete resistance pulses are generated, the magnitude of which are proportional to the particles’ volume. This process is illustrated in Figure 1, which shows a hypothetical comparison between the voltage peaks generated by large and small cells. Thus, the approach can allow accurate count and volume measurements with relatively few restrictions.
The Coulter Principle is flexible enough to be applied in many different ways with a host of cells, particles, and equipment. Due to this flexibility, the principle is used to characterize everything from ink toners to subvisible protein aggregates. This versatility is also important to IVDs since it offers investigators important options when they design experiments. What is important to the rest of this discussion is the ability to greatly vary liquid properties and to accurately measure volume changes over time. These capabilities are particularly useful in the early stages of IVD development, in which basic biological studies apply the Coulter Principle to study cellular function.
(The summary above provides a general description of the Coulter Principle. A more detailed description may be found in Coulter’s 1953 patent or his article in the Proceedings of the National Electronics Conference.1,2 Another source of information is a historical account of the discovery that was published to commemorate the 50th anniversary of Coulter’s patent.3)
 
Understanding Cellular Homeostasis
An important step in the development of a good commercial IVD test is a thorough understanding of the mechanism by which a disease affects normal cellular processes. One of the most important processes is the maintenance of cell volume. While changes in cell volume occur as a normal part of the cell cycle, they can also be indicative of a disorder. Since the Coulter Principle allows investigators to study these volume changes on the same time scale as they occur, it is frequently used for such investigations. These studies often focus on characterizing both the extracellular and intracellular factors that cause changes in cell volume.4
In healthy individuals, extracellular solute concentrations typically fluctuate within well-defined boundaries. However, in several diseases, the fluctuations become extreme. For example, in sickle cell anemia, overactive ion channels that are stimulated by hypoxic external conditions lead to severe red blood cell shrinkage.5 This initial shrinkage causes polymerization of one type of abnormal hemoglobin (HbS) and leaves the cell with its characteristic sickle shape. Through cell-volume and -count measurements, the Coulter Principle can help to identify the presence of sickle-cell anemia. 
Similarly, it can be used to study the mechanisms of many diseases caused by extracellular factors. Using the Coulter Principle as implemented in a Coulter Counter, the effects of extracellular solutes can be studied by varying composition of the suspending liquid and analyzing data collected across a range of compositions. Several investigators have used this approach to monitor the resulting changes in cell volume as a function of time.6
Intracellular factors also cause changes in cell volume. One well-known example is the swelling of cells as they begin mitosis. As cells progress through metaphase, anaphase, and telophase, they must continuously adjust their internal solute concentrations to maintain equilibrium with their environment. Data generated with a Coulter Counter has been used to suggest that changes in intracellular ion concentration can stimulate a cell to advance from metaphase to anaphase.7 Similarly, the Coulter Principle has helped to demonstrate that volume changes during apoptosis are driven by intracellular factors.8 Several other studies have utilized a similar approach to study other cellular functions.9 
Figure 2. The concept of regulatory volume changes using potassium as a model solute. In A, the intracellular and extracellular potassium concentrations are normal, and the cell is in homeostasis. In B, the extracellular potassium level has increased, leading to a regulatory volume decrease. In C, the intracellular potassium level has increased, leading to a regulatory volume increase. In both B and C, the regulatory volume changes involve the cells’ cytoskeleton, ionic protein pumps, and other regulatory proteins, which must act in a coordinated manner.
Examples of such volume regulatory processes are shown in Figure 2. Regulatory volume changes involve the concerted action of protein pressure transducers, ion pumps, cytoskeletal rearrangement, and various other actions designed to maintain equilibrium. The Coulter Principle, in conjunction with other techniques, has been used to characterize such regulatory volume changes. Examples include studies of cytoskeletal rearrangement, the activity of intracellular signaling cascades, and the activity of ion channels.10-12
 
Diseases Affecting Cell Volume
While the Coulter Principle’s contributions to an understanding of the molecular mechanisms of homeostasis and disease can lead to the development of IVD tests for specific proteins or cell markers, another equally important contribution is the physiological characterization of diseases. While the previous section focused on understanding intracellular disease mechanisms, this section discusses characterizing diseases using cell volume measurements, including diabetes, brain injury, and infertility.
The Coulter Principle continues to play an important role in the battle against diabetes and has helped investigators to understand better its mechanisms and diagnose susceptible individuals. For example, one study of harvested adipose cells obtained cellular volume distributions for both insulin-resistant and insulin-sensitive obese individuals.13 The investigators observed a bimodal cellular population and, contrary to expectations, found that insulin-resistant individuals had a smaller mean adipose cell size, due to a larger proportion of smaller cells. When combined with gene expression profiling, this finding led the investigators to conclude that a primary defect in the insulin-resistant adipose cells caused a less complete differentiation of the adipose tissue. As a consequence, the diabetic patients were unable to store properly triacylglycerol, thereby leading to their disease. Other studies utilizing the Coulter Principle have implicated the roles of solutes such as sorbitol in complications of diabetes, which include neuropathy, retinopathy, and cataract formation.5 These patient-based studies have furthered the understanding of diabetes and could lead to improved testing methods.
Since brain injuries force changes in cell volume, and many injuries can be modeled well in vitro, the Coulter Principle is a useful tool to study their mechanisms and impact. For example, sodium and potassium levels have long been known to cause changes in glial cell volume. However, these are not the only factors involved as volume changes have been observed in normotonic conditions.14 Rather, glial cell volume is a function of temperature, metabolite concentration, cell membrane moiety stability, blood oxygen concentration, and several other parameters. These discoveries have led to advances in treatments of stroke and cardiac arrest, such as the induction of mild hypothermia to reduce swelling after a coma.15 
Infertility in farm animals can cause economic hardship, and in humans, it can be extremely frustrating. As part of the effort to alleviate these burdens, new tests are continually being developed to ascertain infertility’s cause. Since fertility is a function of sperm count and volume, the Coulter Principle is a common tool used to study the disorder. Using the Coulter Principle, investigators have linked infertility to extracellular solutes, seasonal changes, and common pharmaceuticals.16-18 Several authors have proposed new clinical fertility tests based on the response of sperm to external stress factors.19 
 
IVDs Utilizing the Coulter Principle
The Coulter Principle’s most significant impact is demonstrated in routine clinical applications. Coulter originally developed the principle with the express purpose of counting and sizing red blood cells. With these two parameters, clinicians can detect anemias, iron deficiency, preleukemia, folate deficiency, and liver disease.20 As new preparative reagents have been developed, more parameters were added to the analyses conducted by the Coulter Principle. 
For example, in the newest hematology analyzers by Beckman Coulter, proprietary reagents are combined with radio-frequency measurements and light scattering to analyze and identify most normal types of white blood cells. The sensitivity and specificity of this multiparameter analysis adds to the list of diseases that can be characterized with data collected using the Coulter Principle (e.g., several types of acute myeloid leukemia, hypereosinophilia syndrome, infectious mononucleosis, agranulocytosis, essential thrombocytemia, non-Hodgkins lymphoma, and others).21 
 
Contemporary Instrumentation
As discussed throughout this article, the Coulter Counter is a useful tool for studying the mechanisms and characteristics of disease. Figure 3 shows the Multisizer 4 by Beckman Coulter, which is the most advanced Coulter Counter available. This instrument incorporates several innovations, both in the physical implementation of the Coulter Principle and the subsequent data analysis. The instrument uses an accurate, wide-range metering pump, which allows it to draw as little as 50 µL of electrolyte suspension through the aperture. Several available aperture tubes (from 20 µm to 2000 µm in diameter) allow measurements of organisms as small as bacteria or as large as flocculated yeast. Furthermore, the instrument is compatible with a wide range of solvents, which can be important for applications involving beads or other raw materials sometimes used in IVD assays. In addition, the instrument implements several sample handling techniques, such as a constant physical distance between the aperture and sample container, which improve precision among experiments.
Figure 3. The Beckman Coulter Multisizer 4. The housing of this Coulter Counter is engineered to reduce the effects of external noise while preserving ease of use. The sample stand’s design ensures consistent aperture position relative to the sample beaker among runs. The apertures are interchangeable to allow a broad measurement range (about 0.400 to 1200 µm). In addition to several options within the control software, the control cluster can be used to modify experimental conditions during testing.
In addition to enhancements in the experimental set-up, the signal processing utilized by the Multisizer 4 provides more capabilities than traditional analog Coulter Counters. The resistance pulses generated in the aperture are processed by sensitive and fast pre-amplification and amplification circuits that have a wide dynamic range. This allows data collection over the entire dynamic range of the aperture (about 2-60% of its diameter) in a single run. 
Following this step, the pulses are digitized and stored as 24-bit voltage data on a computer. Using the intuitive Multisizer 4 control software, this stored data can be re-binned to provide high resolution analyses. These analyses can also include techniques such as averaging many runs together; merging results from multiple aperture tubes into a single size distribution, computing statistics, or exporting the processed data to a spreadsheet software. Another advantage of this digital pulse processing is the ability to monitor changes in pulse height over time, which can be utilized in the regulatory volume change studies described earlier. 
 
Clinical Instruments
While the Multisizer 4 implements the Coulter Principle with a single, direct current (dc) energy source, the UniCel DxH 800 Coulter Cellular Analysis System by Beckman Coulter simultaneously applies the principle with two different energy sources, dc and radio frequency (RF), and adds a third energy source (laser light) for supplemental analysis. A conceptual illustration of the flow cell that makes this feat possible is shown in Figure 4. The electrodes simultaneously pass both dc and RF signals through the flow cell. As in the Multisizer 4, the dc signal measures total cellular volume. In a unique implementation of the Coulter Principle, the RF signals allow analysis of intracellular organelles by conductivity. Finally, the various light scatter angles enable classification of specific organelles by type. 
Figure 4. The UniCel DxH 800 Coulter Cellular Analysis System. The heart of the instrument is its electro-optical flow cell, a conceptual illustration of which is shown in A. The flow cell utilizes three energy sources to make several simultaneous measurements of cellular properties. Dc and RF signals are sent between the lower and upper electrodes, and represent two distinct implementations of the Coulter Principle. Laser light provides the third energy source. The sheath flow centers individual cells in the flow cell, improving the accuracy and reproducibility of all acquired data. In B, the specimen transport module gives the UniCel DxH 800 a small footprint and the option to link multiple systems together.
All of the measurements are made simultaneously for individual cells as they pass through the center of the flow cell. The data is collected and processed digitally. Similar to the Multisizer 4, digital pulse processing provides a wealth of information that was difficult to obtain previously. For every pulse generated, the mean pulse height, the full width at 75% maximum height, the full width at 50% maximum height, and the area under the pulse are stored. Thus, four pieces of information are collected for each of the five light scatter angles, the RF signal, and the dc signal. The DxH 800 System ultimately has up to 406 unique two-dimensional plots of results on which to perform an analysis. This increase in the amount of data provided allows more accurate classification of cell type and improves confidence in analytical results.
In addition to its data collection and analysis, the UniCel DxH 800 is engineered for reliability, efficiency, and ease of use in a clinical hematology laboratory. The magnetically driven specimen-transport module allows connection of several DxH 800s in a work cell. The module contains bypass and
return capability, allowing global bi-directional transport of samples between instruments. This enables autoreflex and autorepeat of samples requiring further analysis. Another advantage of the system is that if one instrument should temporarily become unavailable, analyses can continue uninterrupted on the back-up instruments. As shown in Figure 4, the specimen transport module is entirely encased in a protective housing and reduces the footprint of the DxH 800. 
 
Conclusion
The Coulter Principle is useful for IVD test development from basic biological studies to clinical application. While the discussion in this article focused on selected examples, they were chosen to demonstrate ways in which the Coulter Principle could be used in the development of many IVD tests. With continual advances in instrumentation, the future promises many more developments resulting from the application of the Coulter Principle to the field of IVDs.
 
Matthew N. Rhyner, PhD, is the technical marketing manager in the Particle Characterization Business Center at Beckman Coulter Inc. (Brea, CA). He can be reached at mnrhyner@beckman.com.
 
 
References
1. WH Coulter, “Means for Counting Particles Suspended in a Fluid,” U.S. Patent #2,656,508, October 20, 1953.
2. WH Coulter, “High Speed Automatic Blood Cell Counter and Cell Size Analyzer,” Proceedings of the National Electronics Conference, 12 (1957): 1034-1042.
3. MD Graham, “The Coulter Principle: Foundation of Industry,” Journal of the Association for Laboratory Automation 8, no. 6 (2003): 72-81.
4. KM Strange, “Cellular Volume Homeostasis,” Advanced Physiological Education 28 (2004): 155-159.
5. ML McManus, KB Churchwell, and KM Strange, “Regulation of Cell Volume in Health and Disease,” The New England Journal of Medicine 333, no. 19 (1995): 1260-1266.
6. DB Light, et al., “Cell Swelling Increases Intracellular Calcium in Necturus Erythrocytes,” Cell Science 116 (2003): 101-109.
7. S Grinstein, A Dupre, and A Rothstein, “Volume Regulation by Human Lymphocytes: Role of Calcium,” Journal of General Physiology 79 (1982): 849-868.
8. E Maeno, et al., “Normotonic Cell Shrinkage Because of Disordered Volume Regulation is an Early Prerequisite to Apoptosis,” Proceedings of the National Academies of Science 97, no. 17 (2000): 9487-9492.
9. JG Izant, “The Role of Calcium Ions During Mitosis,” Chromosoma 88, no. 1 (1983): 1-10.
10. R Franco, MI Panayiotidis, and LD Ochoa de la Paz, “Autocrine Signaling Involved in Cell Volume Regulation: The Role of Released Transmitters and Plasma Membrane Receptors,” Journal of Cellular Physiology 216 (2008): 14-28.
11. AK Hansen and HK Galtun, “Aquaporin Expression and Cell Volume Regulation in the SV40 Immortalized Rat Submandibular Acinar Cell Line,” European Journal of Physiology 453 (2007): 787-796.
12. GP Downey, et al., “Volume Regulation in Leukocytes: Requirement for an Intact Cytoskeleton,” Journal of Cellular Physiology 163, no. 1 (2005): 96-104.
13. T McLaughlin, et al., “Enhanced Proportion of Small Adipose Cells in Insulin-Resistant vs Insulin-Sensitive Obese Individuals Implicates Impaired Adipogenesis,” Diabetologia 50 (2007): 1707-1715.
14. SI Mori, et al., “Impaired Activity of Volume-Sensitive Anion Channel During Lactacidosis-Induced Swelling in Neuronally Differentiated NG108-15 Cells,” Brain Research 957, no. 1 (2002): 1-11.
15. SA Bernard, et al., “Treatment of Comatose Survivors of Out-of-Hospital Cardiac Arrest with Induced Hypothermia,” The New England Journal of Medicine 346 (2002): 557-563.
16. CH Yeung, M Anapolski, and TG Cooper, “Measurement of Volume Changes in Mouse Spermatozoa Using an Electronic Sizing Analyzer and a Flow Cytometer: Validation and Application to an Infertile Mouse Model,” Journal of Andrology 23, no. 4 (2002): 522-528.
17. AM Hossain, et al., “Time Course of Hypo-Osmotic Swellings of Human Spermatoza: Evidence of Ordered Transition Between Swelling Subtypes,” Human Reproduction 13, no. 6 (1998): 1578-1583.
18. CH Yeung, et al., “Human Sperm Volume Regulation. Response to Physiological Changes in Osmolality, Channel Blockers, and Potential Sperm Osmolytes,” Human Reproduction 18, no. 5 (2003): 1029-1036.
19. JK Webb, et al. “Coulter Counter-Based Evaluation of Sperm Volume to Assess Sperm Viability of Bull Semen and Application to X/Y Sperm Sorting,” Theriogenology 69, no. 8 (2008): 990-1000.
20. “Red Cell Distribution Parameters; Technical Bulletin 9617, 2007” (Brea, CA: Beckman Coulter Inc. 2007); available from Internet: www.beckmancoulter.com.
21. HD Alexander, et al., “Cell Sizing in Chronic Lymphoproliferative Disorders: An Aid to Differential Diagnosis,” Journal of Clinical Pathology 45 (1992): 875-879. 

 


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