Cell counting is a tedious process that needs to be performed properly, be it automated or manual, in academia or industry, as reliable results are required to continue the research. In order to help with this process, CytoSMART Technologies provides five important tips to get more out of cell counting.
For users, these tips may surprise them, even if they are adept at automated or manual cell counting. This article shows how cells can be counted in a faster and better way, which perhaps could make this process more enjoyable.
Tip #1: Sample preparation, improving the first step that counts
Users who want to determine cell concentration either manually or automatically will need an appropriate sample to obtain accurate values, because an incorrect sample will lead to invalid counts.
So, what are the measures that can be used to prevent the analysis of incorrect samples? The following steps will help users to obtain more accurate results and have less variation between operators.
Standardize your protocols
Cell counting process varies from one operator to another, right from trypsinizing cells to the way pipettes are managed. Cell counting accuracy from one sample to the other can be increased by standardizing protocols across the entire facility and ensuring that these protocols are properly followed through routine assessments.
While this may take some time and effort to get everybody to follow the protocol, more accurate counts will be obtained over time which will ultimately lead to better experimental results and save costs.
Maintain a homogenous suspension
When a sample is being analyzed, it is assumed that the whole suspension is represented by the cell distribution within the sample. However, when a cell suspension is allowed to rest for a short time, cells will gravitate toward to the bottom of the tube.
This creates a concentration gradient inside the suspension and any sample taken will not be representative. However, a homogenous distribution can be regained by using the traditional "finger-flick" or by using a vortex prior to sampling.
Some operators re-suspend the cell suspension when collecting a sample with the pipette. Regardless of the type of procedure performed, this should be made the standard protocol.
Eliminate causes of debris inclusion
In many counting protocols, some amount of debris is likely to be present. If this is not properly adjusted, it can become a major cause for misclassification – both for when a cell is excluded as debris (false-negative) or when debris is included as a cell (false-positive).
Minimizing the level of debris present in the sample will improve the accuracy of results. Accurate results can also be obtained either through improved detection parameters for automated systems or reducing misclassification by training for manual counting.
In order to improve the sample and thus the results, protocols should be standardized and followed as best as possible. This will not only reduce the errors caused by sample preparation, but also the variation between operators.
In addition, maintaining the sample’s homogeneity as best as possible and excluding the debris can significantly increase the accuracy of the count.
Tip #2: A better understanding of the total cost of cell counting
The manual counting procedure is quite simple — users can harvest the cells, dilute them, place them on a hemocytometer slide, view through the microscope, and start clicking.
This procedure can be performed by anybody with basic cell culture experience. It is also a cost-effective way to obtain accurate counts and thus considerable results. However, is it the cheapest way?
Manual yet accurate cell count calls for a certain level of expertise. The ability to differentiate between dead cells, live cells, debris, and cell clumps takes time and training.
Automated cell counters were launched to improve productivity in the lab. However, better automated results involve a great deal of financial cost, raising the question whether manual counting is less expensive than automated counting.
Which counting setup is financially best?
Regardless of the type of counting setup, three cost factors should always be considered which include the initial purchase cost, operator cost, and operating cost such as consumables. So far, manual counting is considered to be the cheapest option regarding purchasing.
Users have their setup that includes a clicker and a glass counting chamber costing about €150. This setup does not feature the required bright-field microscope, which is seen as a standard lab apparatus.
The operating cost is a single coverslip for each measurement, which reduces to approximately €0.02. To put it simply, this setup is inexpensive. So, why are automated cell counters are still financially interesting? When considering operator cost for both automated and manual setup, something interesting occurs.
“The times, they are a-changing"
Even an experienced operator will spend about four minutes to examine the cells, but an automated setup will take mere seconds. This considerably reduces the amount of operator cost, particularly with increasing amounts of counts.
Despite an initial purchasing cost of about €3000, an automated setup can still have a high return on investment with an increasing amount of cell counts. This even exceeds the increased operating costs, because automated setups need more expensive and custom disposable counting consumables that start from €0.35 per count - 15 times more than the cover slip needed by manual counting.
If the total costs are calculated for both automated and manual setups, including the initial purchase cost, operator cost, and operating cost, a break-even point was seen where automated counting is more expensive due to its high operating costs (Figure 1).
Figure 1. Representation of total cost of both manual and automated counting both included their respective purchase cost, operating cost and operator cost. Around 2600 performed counts a break-even point is reached.
When comparing cell counters, the initial cost of a counting setup is a major factor. However, simply looking at the initial purchase price may cost more in the long run. It was shown that a better financial comparison between different systems can be made by incorporating the entire cost of a setup.
Users are advised to make their own comparison, because purchase cost, operator cost, and operating cost can vary based on their location and situation. The break-even points can vary considerably when a comparison is made between automated and manual systems, or in some situations may not occur at all.
Tip #3: A little something to assist you
Manual counting is a simple procedure which involves harvesting of cells. During the first cell count, users are likely to make mistakes – either misusing the pipette or adding too much trypsin, or an incorrect calculation has left a reduced number of cells in the flask.
Users are not ingrained with all the knowledge, but with time, they become more proficient and more experienced in cell counting. They still have to improvise at times; maybe the T-175 flasks ran out and users had to rapidly switch to T-25 to maintain their cells. However, the question is how many mL enters into a T-25 again?
In order to help users to deal with this situation, a cell concentration cheat sheet is provided. This template includes a useful calculator as well as standard information about flasks.
Tip #4: Accuracy of cell counters explained
Accuracy is considered to be an important aspect when researching lab equipment for purchase. A critical question is how to determine the system’s accuracy? And what is the difference between accuracy, precision, and trueness? While these three terms are used interchangeably, they have very different meanings.
To start with, let us examine the definition to be used. According to the ISO definition, accuracy is defined as the "closeness of the agreement between the result of a measurement and a true value of the measurand". Accuracy is the sum of two subcomponents known as precision and trueness.
Previously called bias, trueness is described as the "closeness of agreement between the expectation of a test result or a measurement result and a true value", meaning that trueness defines the systemic error of quantitative measurements.
A representation of trueness is shown In Figure 1. The average value is close to the bullseye, but the individual points are not because of lack of precision.
Precision is described as the "closeness of agreement between independent test/measurement results obtained under stipulated conditions". Therefore, it is a measure of repeatability and relates to the robustness of a quantitative device.
Precision is shown as repeating values being near each other but not close to the bullseye (Figure 2). The standard deviation or variance is generally used to describe a device’s precision.
To conclude, only a device that measures with a high degree of precision and a high degree of trueness will be able to provide accurate results.
Figure 2. Various kinds of accuracy presented with increasing trueness and precision. A. With only high precision, a system will underscore or overscore the actual sample. B. Only with high trueness and precision, a system is accurate. C. With low trueness and low precision, very low accuracy is achieved. D. With only high trueness, the average value is correct but many samples are needed.
How to measure the trueness of a cell counter
The trueness of a cell counter is defined by the degree of closeness between the measured average value of the measurements of the device and the actual concentration. In order to determine this, a sample with a known concentration has to be analyzed with the device.
Users must be aware that cell counters will function differently at the extremes of their operating concentration range. Based on this fact, a basic test for trueness assessment is carried out by diluting a sample of known concentration several times.
This will generate a calibration curve, which will show the trueness of the cell counter within the entire operating range, and not just at a specific concentration.
If such a calibration curve is produced, a cell counter with a high trueness will reveal two things – A linear relationship between theoretical concentration and measured concentration, and a slope close to one.
How to measure the precision of a cell counter
Since users know how to determine the trueness, the precision of a cell counter will be described. As previously mentioned, the robustness of the system is defined by the precision of a cell counter. Therefore, if the same sample is counted multiple times, similar results should be obtained.
This is statistically represented by the coefficient of variation (CV value) and can be measured from Equation 1, where x̄ is the mean of the dataset and σ is the standard deviation.
The comparison should be made between this measured CV value and its theoretical CV value, which can be calculated from Equation 2.
The theoretical CV value represents a reference value for the calculated CV value to determine the precision. This calculated CV value has to be close to the theoretical value, but it should never be lower than the theoretical CV value.
The CV value measured will always be higher due to an increased standard deviation because of the error caused by the operator and the system, in addition to the theoretical CV value.
This article has shown how the accuracy of a cell counter can be determined. It also demonstrated that accuracy is composed of two components – precision and trueness.
It is shown how these two components are related to the performance of a cell counting system. With this information on hand, users can review the information provided by manufacturers and test a system.
To examine the trueness of a device, a calibration curve has to be performed. A cell counter with a high trueness will reveal a linear relationship between the theoretical concentrations and measured concentrations.
To examine the precision of a device, users should look for the CV value provided by system manufacturers and compare it to the theoretical value. The measured CV value should always be lower than the theoretical value.
Tip #5: Switch between manual and automated
"Sure you can come over to our lab with your automated cell counter, but I warn you, I am a manual counting cheerleader!" This is part of a conversation held with one of CytoSMART’s clients. It appears that some individuals have prejudice regarding manual counting or automated cell counter. To investigate this properly, users must answer one important question.
What do they want to count? The answer to this question will provide the type of counting most suitable for them. Users probably want to measure a single cell type many times each day or examine a range of bacteria and cells.
While they are making a decision between automated and manual automated setups, whatever they want to count, the following four aspects should be taken into account.
The lowest initial purchase and operating costs are associated with manual counting, thanks to its re-usability. Particularly when it comes to a small number of counts, manual counting is the best choice regarding budget (see Tip 2).
However, this does not include the time required by the operator to do the count and the time needed to gain experience in manual counting. Accordingly, when a number of counts increases, the cost can also increase in total.
In the case of automated counting, the initial purchase cost cannot be easily resolved as they are more expensive by a factor of 1,000x. One advantage of automated counting is that it considerably reduces the processing time for each sample.
The necessity for consumables is an additional cost that can significantly exceed the initial purchase cost when the device is employed for a large amount of cell counts. With increasing numbers of counts, automated systems may be more cost-effective than manual counting due to its reduced operator costs.
The results obtained must be accurate for both automated and manual setups. Benefits of manual counting are versatility, early problem detection, and accurate classification.
Since the operator observes the cells directly, any errors can be assessed quickly; however, the operator is also its disadvantage, since inter-operator variation can be high and results can be subjective. Even if the same grid is counted from the same hemocytometer, the counting protocol may differ considerably from one person to another.
Advantages of automated counting are the lower error rate per sample and lack of subjectivity associated with manual cell counting.
Being automated, automated counting has a high reproducibility when compared to manual counting, but the prospect of misclassification is higher since there is no human element classifying cells. This can make the automated systems precise but with a low level of trueness, thus underestimating the true concentration.
Manual counting is highly versatile, as it can be easily adapted to many situations. At the same time, it is also limited by human operators. These limitations are the time required to carry out a count and proper counting through experience.
Automated cell counting facilitates a higher throughput of samples, making it possible to perform more counts. This leads to less misclassification and higher productivity between samples, but also limits its versatility available with a certain system.
Since the use of the first hemocytometer in the 19th century, reporting has remained the same for manual counting. The operator simply has to write the results in the lab journal. However, a lot has changed on the automated side, enabling for more detailed and faster reporting of the cell counts.
With present setups, more detailed analysis reports are available, such as graphical display of the cells counted. In addition, data can be reanalyzed at a later time, thanks to cloud data storage. This can provide innovative results that are otherwise overlooked.
Before deciding between manual and automated counting systems, users must first define the type of cells that need to be counted. Once they are clear about what they should count and in what quantity, they can properly assess the setups available to them.
Users must focus on details of both automated and manual systems with regard to their versatility, accuracy, costs, and reporting, and how these are related to their needs.
Once users are able to relate this information to their specific situation, they will be able to select a suitable system that meets their requirements. This will enable them to make the best decision, whether to continue with manual counting or switch to automated counting.
A final tip
Before attempting a new setup, users should arrange for a trial to see if this setup fits their requirements.
Produced from materials originally authored by Bart Fischer, Applications Specialist at CytoSMART Technologies.
CytoSMART Technologies BV, based in The Netherlands, develops smart solutions for live-cell imaging in cell culture laboratories. The company was founded in 2012 by a group of researchers at the Eindhoven University of Technology, who were frustrated about the lack of inexpensive small imaging systems to support biological cell culture.
Live-cell imaging solutions have remained prohibitively large, complex and very expensive, especially when comparing them to smaller and more cost-effective house-hold imaging systems such as photo cameras and smartphones.
Inspired by recent advances in optics, microelectronics and information technology, the founders of CytoSMART believed that it was time to create new compact imaging systems for biological cell culture.
- Systems that are small, easy to use and available to everyone.
- Systems that make advanced live-cell imaging and image analysis possible for all types of cell culture and enable researchers to get a better grip on the complexity of biological cell culture.
- Systems that can measure and analyze data in almost real-time. Allowing researchers to act immediately, for better and more reproducible results with cell culture.
CytoSMART offers the following products for time-lapse live-cell imaging and cell counting:
- The CytoSMART Lux2, a small, easy-to-use and incubator proof microscope for live-cell imaging.
- The CytoSMART Exact, an automated cell counter for mammalian cells.
- The CytoSMART Omni, a compact automated system for full 96 well live-cell imaging inside an incubator
If you have any question regarding a CytoSMART product, please visit our website at www.cytosmart.com or contact us via email at: firstname.lastname@example.org.
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