ISO 20391-2:2019

Overview

ISO 20391-2:2019 - Biotechnology - Cell counting - Part 2 describes an experimental-design and statistical-analysis framework to quantify the performance of a cell counting measurement process. The standard defines a dilution-series approach that yields quality indicators based on measurement repeatability and the degree to which results follow an ideal proportional response to dilution. It is intended primarily for eukaryotic cells and applies to total, differential, direct and indirect cell counting methods provided the test material meets the experimental-design criteria (for example, cells suspended in solution). Note: this method does not directly provide absolute accuracy and is especially useful when reference materials or traceable methods are unavailable.

Key topics and technical requirements

• Experimental design: use of a dilution-series (stock solution and multiple dilution fractions) with guidance on sample preparation, labelling and measurement replication.
• Statistical methods: calculation and interpretation of mean cell count, measurement precision (e.g., %CV), model fit to a proportional response, coefficient of determination (R²), and the Proportionality Index (PI). The standard also describes smoothed residual calculations and additional quality metrics.
• Quality indicators: metrics derived from repeatability and proportionality that quantify method performance without a reference material.
• Data interpretation and reporting: recommended reporting elements, documentation of experimental parameters, and guidance on comparing PI values across methods or conditions.
• Supporting information: annexes provide practical tools - e.g., methods to assess pipetting error contributions, formulas for PI, and use-case examples for single-method evaluation and inter-method comparison.

Practical applications

• Method development & optimization: evaluate and refine counting protocols or instrument settings for a specific cell preparation.
• Validation, verification & performance qualification: generate evidence on precision and proportional response when reference standards are not available.
• Routine monitoring: track counting-process stability and detect drift or degradation in measurement quality.
• Instrument comparison & selection: compare multiple counting processes using common quality indicators (PI, %CV, R²) to inform purchasing or deployment decisions.

Who should use this standard

• Laboratory scientists and technicians performing cell counts
• Method developers and assay validation teams in biotech and biopharma
• Quality assurance/quality control professionals and regulatory affairs staff
• Clinical laboratories and advanced therapy manufacturers
• Instrument manufacturers and developers of cell counting technologies

Related standards

• ISO 20391-1 - Biotechnology - Cell counting - Part 1: General guidance on cell counting methods (normative reference)
• Sector- or application-specific national and international cell counting standards (consult when operating within their scope)

Keywords: ISO 20391-2:2019, cell counting, experimental design, dilution series, statistical analysis, proportionality index, quality indicators, method validation, measurement precision.