Cell counting: A practical guide to evaluating cell counting methods

Cells have become an essential aspect of research, cancer therapies, and biomanufacturing. With the increasing use of cells as a product and recent approval of CAR-T-cell- therapies by the Food and Drug Administration (FDA), there is a need for accurate cell counts. These counts allow for proper dosage, which could otherwise result in unwanted effects in patients. For this reason, multiple federal agencies, including the FDA, came together at the “Synergizing Efforts in Standards Development for Cellular Therapies and Regenerative Medicine Products” meeting to develop standards for cell counting and viability measurement. Since then, the International Organization for Standardization (ISO) has published two cell counting standards (1) to serve as practical guides for evaluating and analyzing cell counting and viability.

Given the heterogeneous nature of cells, the cell counting needs for various applications (e.g. Cell and Gene therapy) are not unique. Because there is no certified reference material to determine cell concentration, ISO cell counting standards are a valuable tool for evaluating and selecting fit-for-purpose cell counting methods.

ISO Cell Counting Standard Part 1: General Guidance on Cell Counting Methods

Part 1 of the ISO Cell Counting Standard focuses on defining concepts that help users select the most appropriate cell counting method for their sample type.

six key factors

Six key factors can be derived from the ISO Cell Counting Standard Part 1:

1) Intended use of the cell counting procedure (cytotoxicity, PBMC isolation, or immunophenotyping).

2) Preparation for the specialized content of cell samples (Does the sample contain a heterogeneous cell population, suspension cells, aggregates?).

3) All intended measurements are taken for the specific sample.

4) An appropriate cell counting system with the ability to count the sample of interest.

5) The entirety of the cell counting method process from sample preparation, dilution, staining to counting.

6) Continuous operator training for consistent results.

Together these allow the user the proper foundation to select the best cell counting method appropriate for both the sample and intended use.

cell counting method

Any changes to the parameters in the entire process may impact the cell counting results.

***Quality of cell count and viability is determined by the entire cell counting process, not the instrument***

ISO Cell Counting Standard Part 2: Experimental Design and Statistical Analysis to Quantify Counting Method Performance

Part 2 of ISO Cell Counting Standard focuses on providing guidance and a protocol for evaluating the cell counting measurement process resulting from a dilution series and subsequent statistical analysis. Key parameters derived from this analysis are the Coefficient of Determination (R2). This is a value that determines the linearity of the values related to the dilution. Other parameters evaluated by the ISO Cell Counting Standard Part 2 are Precision, Coefficient of Variation [CV], and Proportionality Index (PI), a measure of how cell counts will scale proportionally with dilution series.

How to compare two cell counting methods

While ISO Cell Counting Standards provide a guide for selecting and evaluating the most appropriate cell counting method, the Bland-Altman analysis method allows the user to compare the performance of two different cell counting methods. This is particularly useful when new cell counting instrumentation is introduced into the laboratory or when systems are being upgraded after many years. The Bland-Altman method allows the user to determine how closely the results of one method agree with the results of the second method. This is represented as a bias between two methods indicating which method counts higher or lower. In short, when comparing two cell counting methods, the Bland-Altman analysis can determine the bias between two methodslimits of agreement (LoA), and confidence interval (CI). As described above, bias determines the average difference in measurement between the two cell counting methods. LoA provides a measurement of how extensively these differences may vary. At the same time, the CI determines whether these differences are statistically significant. To have a successful Bland-Altman analysis, the sample size (n=72) needs to be taken into consideration to ensure enough data is generated to perform adequate statistical analysis and prediction models.

Sample preparation and experiment execution

The article “Practical application of cell counting method performance evaluation and comparison derived from the ISO Cell Counting Standards Part 1 and 2” (1) summarizes all the practical considerations needed to design and execute an experiment to evaluate the performance of two cell counting methods.

recommended sample preparation

Schematic of recommended sample preparation for evaluating two cell counting methods.

In short, the cell type and the evaluation to be performed are determined and the cell counting methods are compared. An example would be to evaluate the viability of PBMCs on two fluorescent cell counters using the viability dye AO/PI. Once the cells and methods are selected, six independent dilutions are generated from the stock concentration. The concentration of the dilution series should span the typical concentration ranges of the target cell being evaluated to compare cell counting methods properly. Three replicate samples are generated from each dilution sample generated in the previous step. Each replicate is measured four times in each cell counting method. This will result in 72 measurements per method, which are used to calculate R2, CV, and PI. If the Bland-Altman comparative analysis is also incorporated to evaluate method performance, the bias, LoA, and CI can also be determined. The cell counting methods used in this article were the high-throughput cell counter Cellaca MX and the whole-well Celigo Image Cytometer.

cellaca and celigo

The Cellaca MX high-throughput cell counter for measuring cell concentration and viability. Celigo image cytometry for performing high-throughput cell-based assays. Table summarizing results from ISO Cell Counting Standards Part 2 and Bland-Altman Comparative analysis.

Limitations to consider

Not having a certified reference material for cells is a challenge and the main obstacle to validating a cell counting method. The same experimental design needs to be applied for cell counting methods #1 and #2 to derive comparable data. Any deviation in the protocol for one method could significantly affect the interpretation of results like R2.

The ISO Cell Counting Standards Part 1 and 2 provide a framework for any user experienced in cell counting to evaluate the performance of any cell counting method. This protocol can be applied to various fields like virology, immune-oncology, gene, and cell therapy, when looking to validate cell counting methods for pre-clinical trials, clinical trials, and process development, among others. At Nexcelom Bioscience, the Field Application Scientists (FAS) and the Research and Development team have helped many researchers apply the ISO Cell Counting Standards to their workflows. Based on ISO Cell Counting Standards Part 2, Nexcelom Bioscience has developed the StandardsGo program. In this program, the FAS can consult and guide the user through the whole process of evaluating their cell counting method. From experimental design to data analysis, the FAS will support the user by providing the data necessary to make decisions that will make their workflows more efficient and accurate.

Learn more about understanding ISO cell counting standards in the related Nexcelom Webinar on Demand: Applying ISO Cell Counting Standards for Cell and Gene Therapy Products. Contact us for more information or to request a live demonstration today.


  1. International Organization for Standardization (ISO). ISO Cell Counting Standard Part 1. ISO 20391-1:2018 Biotechnology — Cell counting — Part 1: General guidance on cell counting methods. ISO Cell Counting Standard Part 2. ISO 20391-2:2019 Biotechnology — Cell counting — Part 2: Experimental design and statistical analysis to quantify counting method performance
  2. Practical application of cell counting method performance evaluation and comparison derived from the ISO Cell Counting Standards Part 1 and 2.  Y Huang, J Bell, D Kuksin, S Sarkar, LT Pierce, D Newton, J Qiu & LLY Chan. Cell & Gene Therapy Insights 2021; 7(9), 937–960 (DOI: 10.18609/cgti.2021.126)
  3. Practical Cell Counting Method Selection to Increase the Quality of Cell Counting Results Based on ISO Cell Counting Standards Part I Nexcelom Website