It is something that many of us perform multiple times a day and is the foundation of most cell-based assays, but cell counting, or more precisely ‘accurate cell counting’ can be very difficult. In this blog we explore the different methods, their use, and their limitations to help determine the fit for application system(s) that best suit your lab’s needs.
Cell counting and viability determination is critical for almost all clinical and translational research applications, however, there are many pitfalls in the process of cell counting and this can have a profound effect assay performance. In part to address these issues, there have been advances in cell counting over the last decade, enabling the process to become highly automated.

The Gold Standard

Despite technical advances, the hemacytometer remains the Gold Standard for many laboratories performing cell-based research. The hemacytometer was invented by Louis-Charles Malassez to enumerate blood cells, however, this method can be applied to enumerate cells within a diverse array of sample types including blood, cell cultures, urine, and water samples. Typically, a viability dye such as Trypan Blue is used to distinguish live from dead cells prior to loading of the sample onto the hemacytometer.
At its core, a hemacytometer is a specimen slide characterized by an etched grid that generates a 9-square indentation outlining a 1mm x 1mm area, with each square then intersected into cross sections to create the smallest subdivision of 0.00025mm2. When covered with a coverslip this forms a chamber of height of 0.1mm-and therefore creates a space of a known, set volume.

Typically, cells are counted using a 4x or 10x objective lens over each of the four corner 1mm x 1mm squares and the average calculated. From this, the cell counts are determine based on:

Average Cell Number x 104 x dilution factor

For statistical robustness, the goal is to have roughly 100-200 cells per square, – in some instances, the cell density may not be high enough to achieve this, whereas in others a dilution step may be required, in which case the selected dilution factor may be critical.
The accuracy of cell counting using this method is influenced by a number of factors principally around human variability in mixing, pipetting, and handling, as well as instrumentational and material variations between labs and users. Variations between dilution factors may be >55%, and variations between operators have been reported as high as 52% (Freund and Carol, 1964). In order to overcome these high Coefficients of Variation, multiple counts of the same sample are recommended. But really, how feasible is this in a high throughput working lab?
So, in order to address some of these limitations, I’ve put together some simple and practical steps that can help improve the performance of cell counting using a hemacytometer:

  1. Thorough mixing of samples prior to sample collection and counting of multiple aliquots to help provide statistical significance and robust determination of cell counts.
  2. Minimizing of clumps within the samples prior to cell counting for example using thorough pipetting or filtration of cell sample.
  3. When using viability dyes it is important not to wait too long before cell counting; trypan blue is a common dye for dead cells- however, long exposures to this toxic dye may eventually stain all cells, dead or alive, if left longer than around 10 minutes. So, make sure you organize your workflow appropriately. Alternatively, consider using a membrane-impermeable DNA-binding dye such as 4’-6’-diamidino-2-phenylindole (DAPI) which will only be taken up by dead cells.
  4. Calibration of pipettes- key to many laboratory practices, pipettes maintenance and calibration should never be overlooked.
  5. Careful serial dilutions of samples for analysis for example wiping of excess drops from the outer surface of the pipette tips.
  6. Appropriate placement of coverslip and loading of the sample onto the hemacytometer to ensure no air bubbles or fibers are captured within the counting field. Another consideration is that larger cell types may concentrate along the edges of the collection field, which may affect the determined cell count.
  7. Cleaning of the hemacytometer and coverslip with alcohol to remove debris and ensure optimal sample distribution across the counting field. Since the sample loads into the counting field by capillary action, any debris or air-pockets can easily disrupt this process.
  8. Standard Operating Procedures- since this is a very manual process, individual approaches can impact the reproducibility of the cell count; this can be minimized by adopting standard processes.
  9. Make a plan for those inevitable line riders- within any given run it is inescapable that some cells will lie on the borders of the larger squares. Formulate a plan on which of the two boarders to include in your Standard Operating Procedure; this will help ensure that you have a systematic plan to include 50% of the line riders, and therefore help ensure more accurate and reproducible results.

Considerations when Selecting an Automated Cell Counting System

With so many factors and subjectivity involved in manual cell counting, it is not surprising that many automated cell counting systems are now commercially available that overcome many of these issues associated with inter-observer variability and hemacytometer cell distribution. Their capabilities have improved vastly over recent years, providing a time-efficient and cost-effective alternative to manual counting. However, each system has pros and cons, so it is important to understand and consider your specific lab needs and identify platforms that support your requirements.
Automated Cell Counters typically fall in to four main types: impedance (Coulter Principal), Optical (light scattering); flow cytometry and direct imaging using specialty stains. Each platform has distinctive applications and strengths, so it is important to look into each system’s features and ensure that the instrumentation that you adopt fits the needs of your laboratory.

How do Automated Cell Counters Work?

Impedance cell counters work by counting and sizing cells via changes in electrical impedance as a cell passes through an aperture. They represent one of the earliest types of automated cell counters and are still common in many hospital laboratories. Optical methods of cell counting use laser light scatter by a cell that is detected by photodetectors. These systems are very similar in nature to flow cytometers and can differentiate between various blood cell types for example based on simple scatter plots.
More sophisticated flow cytometry cell counters utilize forward scatter for identify cells, and their size and volume, coupled with side scatter to provide information on the inner complexity of the cell. Side fluorescence can also be employed to interrogate RNA and DNA content within the cells. Hand-held flow cytometers have also been built to support HIV-testing and monitoring in Africa for example. Many state- of-the-art stain-free cell enumeration platforms utilize proprietary algorithms to use transmitted light signatures to describe a number of commonly used cell types from CHO and HeLa to THP-1 and embryonic stem cells. Both Coulter and Flow Cytometry -based cell counters can be used over a wide range of sample and cell types. They best perform on cell samples that are not clumpy which can cause coincidence to misread the cell number. One major advantage of Coulter and Flow Cytometry cell counting methods is the wide range of cell types that they are compatible with, these including cell culture and primary cells, PBMCs, yeast and bacteria, but require instrument or gating adjustments to identify the population of interest.
In recent years, there has been a resurgence in Imaging Cell Counters. These rely on autofocusing and exposure technology, coupled with machine-learning algorithms to provide precise and accurate cell counts over a broad range of cell types, cell sizes and cell densities within a sample. This level of sophistication may have highest value when it comes to cell samples that are highly heterologous or that have a higher propensity to clump, since the systems are readily able to decipher the boundaries between clumps of cells and identify debris, dead/dying cells and cells of various sizes.
Many of these imaging technologies employ fluorescence-based cell counting that provides superior ability to distinguish debris over Trypan Blue. One of the optimum combinations of Acridine Orange (AO) and Propidium Iodide (PI) is used by a number of cell counting platforms. AO is membrane permeable and stains the nuclear DNA of viable cells, as opposed to PI that only stains dead cells. The fluorescent signal from each of these dyes is readily resolved, and the positive staining of nucleated particles, helps to support accurate cell viability determinations. This is particularly useful for PBMC enumeration since RBCs would be readily distinguishable based on their lack of PI staining.
There is a lot to consider, so before selecting a specific cell counting platform, consider these key performance criteria:

  1. Throughput-whereas some systems have limited sample throughput, other have up to 24-channel slides for much higher throughput. How many samples does your lab need to enumerate per day?
  2. Types of samples to be analyzed- consider the heterogeneity of the sample cell sizes and complexity.
  3. Optimal Precision verses Throughput- is a challenge to achieve both in the same instrument. For your application, are precise cell counts critical, or will a +/- of 20% suffice?
  4. Regulatory Compliance – for example any instrumentation used in the production of cell therapies require complete 21 CFR Part 11 compliance for GMP regulatory compliance. FlowMetric is a GLP-compliant organization, so audit trials are an important consideration for us- does your lab require this level of tracking?
  5. Staining Considerations-for example are you enumerating nucleated versus non-nucleated cell staining?
  6. Logistical considerations- does the lab require a benchtop or is a handheld cell counter more convenient?
  7. The required dynamic range of cell enumeration- some of the most advanced systems can now accurately count samples with cell densities as low as 1 x104 cell per mL up to 1 x 107 cells per mL without the need to specialize preparation.
  8. User-friendly interface, for target cell identification, data reporting and data management.

Final Thoughts

Although many automated cell counters support similar basic functions, they are not alike, and many do not adhere to GLP/GMP or 21 CRF part 11 compliance requirements. There are currently two sets of ISO guidelines relating to cell counters: Part 1 defines the terms and provides general guidelines for the cell counting process, method selection, sample preparation, measurement, qualification, validation, data analysis and reporting (ISO 20391-1:2018 (en) Biotechnology-Cell Counting -Part 1. General Guidance on Cell Counting Methods) and Part 2 that provides guidelines for evaluating the quality indicators of a cell counting measurement process (ISO 20391-2:2019 Biotechnology -Cell Counting- Part 2: Experimental Design and Statistical Analysis to Quantify Counting Method Performance).
Whenever evaluating a cell counting platform it is important to review the entire workflow from sample collection, diluting and staining to data acquisition, reporting and data management. As with any laboratory process, training and standard procedures are an essential component of performance. With so many different platforms and instrumentation commercially available, cell counting has never been easier and more reliable- enabling researchers to feel confident in the enumeration and viability determination that can have such a significant impact on their downstream work.
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