ISO 20391-1:2026

• Abstract:

  This document provides standard terminology related to cell counting for biotechnology. This document describes counting of cells in suspension (generally cell concentration) and cells adhered to a substrate (generally area density of cells). This document provides key considerations for general counting methods (including total and differential counting, and direct and indirect counting) as well as for fit for purpose method selection, sources of variability in the measurement process, and data analysis and reporting. This document is applicable to the counting of all cell types – mammalian and non-mammalian (e.g. bacteria, yeast) cells. NOTE Several sector or application-specific international and national standards for cell counting currently exist. When applicable, the user can consult existing standards when operating within their scope (e.g. specific measurement techniques or applications).

• Status: Published
• Publication Date: 27-Jul-2026
• ICS: 07.080 - Biology. Botany. Zoology
• Technical Committee: ISO/TC 276 - Biotechnology
• Drafting Committee: ISO/TC 276 - Biotechnology
• Current Stage: 6060 - International Standard published
• Start Date: 28-Jul-2026
• Due Date: 20-Jun-2026
• Completion Date: 28-Jul-2026

• Revises: ISO 20391-1:2018 - Biotechnology — Cell counting — Part 1: General guidance on cell counting methods
• Effective Date: 24-Jun-2023

Overview

ISO 20391-1:2026 – Biotechnology - Cell counting - Part 1: General requirements and recommendations for cell counting analytical methods defines standard terminology and provides comprehensive guidance on analytical methods for cell counting in biotechnology. Published by the International Organization for Standardization (ISO), this standard covers the measurement and evaluation of cell counts for both cells in suspension (cell concentration) and cells adhered to a substrate (area density). ISO 20391-1:2026 outlines requirements and recommendations for selecting appropriate analytical methods, minimizing variability, managing data, and ensuring accurate reporting. It is applicable to the counting of all cell types, including mammalian and non-mammalian cells such as bacteria and yeast.

Key Topics

ISO 20391-1:2026 addresses several critical aspects of cell counting in biotechnology:

• Standard Terminology: Establishes definitions for key terms such as cell count, cell concentration, area density, direct and indirect cell counting, differential and total cell counting, and others to support uniformity and clarity in communication.

• Cell Counting Methods: Describes primary categories of cell counting:

  • Total Cell Counting: Measurement of all cells, regardless of type or attributes.
  • Differential Cell Counting: Enumeration of specific cell subsets distinguished by unique attributes.
  • Direct Counting: Identifies cells by detecting signals directly from individual cells (e.g., optical, electrical, or mechanical signals).
  • Indirect Counting: Measures surrogate signals (such as total DNA content or metabolic activity) and relates them to cell count through mathematical models.

• Measurement Process: Outlines steps such as sample collection, preparation, measurement, and analysis, discussing the potential sources of variability, including manual operations, reagent quality, and sample heterogeneity.

• Fit-for-Purpose Method Selection: Recommends that method selection be driven by the intended application, cell type, sample characteristics, and available instrumentation, considering factors like accuracy, precision, sample preparation, and detection limits.

• Data Analysis and Reporting: Highlights the importance of rigorous data processing, including image analysis, gating, correction for coincidence, and standardized reporting of results to support reproducibility and comparability.

Applications

ISO 20391-1:2026 supports a wide array of applications in biotechnology where reliable and accurate cell counting is essential:

• Biomanufacturing: Monitoring cell concentration in bioreactors for quality assurance in production processes.
• Cell-Based Therapies: Assessing potency and efficacy by quantifying viable and total cells in therapy products.
• Bioassays and Research: Standardized cell enumeration enables normalization and comparison of experimental results.
• Clinical Diagnostics: Enables the differential counting of specific cell subpopulations for diagnostic or monitoring purposes.
• Quality Control: Provides a basis for qualification, validation, and ongoing verification of cell counting methods to ensure consistency and regulatory compliance.

The guidance provided by the standard is broadly applicable to laboratories, manufacturers, researchers, and quality assurance professionals involved in cell counting across all domains of biotechnology.

Related Standards

For specialized techniques or sector-specific requirements, users are encouraged to consult other relevant international or national standards in conjunction with ISO 20391-1:2026. These may include:

• ISO 8196-3: Methods for milk and milk products (flow cytometry cell counting).
• ISO 13366-1: Microscopic methods for somatic cell counting in milk.
• ASTM F2149-16, ASTM F2944-12: Standards related to cell evaluation and counting in medical devices and biomaterials.
• DIN 58932: German standards for blood cell enumeration.
• CLSI H44-A2, USP Biological Tests: Standards for laboratory methods in hematology and biological testing.

By defining general requirements and recommendations, ISO 20391-1:2026 supports harmonization, reproducibility, and comparability in cell counting results throughout the biotechnology industry.

International
Standard
ISO 20391-1
Second edition
Biotechnology — Cell counting —
2026-07
Part 1:
General requirements and
recommendations for cell counting
analytical methods
Biotechnologie — Dénombrement des cellules —
Partie 1: Exigences générales et recommandations relatives aux
méthodes analytiques de dénombrement des cellules
Reference number
© ISO 2026
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
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Phone: [phone]
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Website: www.iso.org
Published in Switzerland
ii
Contents Page
Foreword .iv
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General concepts of cell counting . 6
4.1 General .6
4.1.1 Categorization of cell counting analytical methods .6
4.1.2 Total cell counting .7
4.1.3 Differential cell counting .7
4.1.4 Direct cell counting .7
4.1.5 Indirect cell counting .8
4.2 Cell counting as a measurement process .8
4.3 Cell count as a measurand .8
5 Considerations for selecting a fit for purpose cell counting analytical method . 9
6 Source of variability in a cell counting analytical method .10
6.1 General .10
6.2 Sampling of cells for counting .10
6.3 Preparation of cell samples for counting .11
6.3.1 General .11
6.3.2 Environmental factors .11
6.3.3 Procedures .11
6.3.4 Quality and stability of reagents . 12
6.4 Performing a measurement . 12
7 Qualification, validation, and continued verification .12
7.1 Instrument qualification . 12
7.2 Analytical method qualification, validation and continued verification. 13
7.3 Reference materials .14
7.3.1 General .14
7.3.2 Certified reference materials .14
7.3.3 In-house reference materials .14
7.3.4 Uses of reference materials . .14
8 Data processing, analysis, and reporting .15
8.1 Data processing and analysis . 15
8.1.1 General . 15
8.1.2 Image processing and analysis . 15
8.1.3 Gating . 15
8.1.4 Coincidence correction . 15
8.2 Reporting . 15
Annex A (informative) Description of common cell counting analytical methods . 17
Annex B (informative) Common cell counting analytical methods for various measurement
purposes .20
Annex C (informative) Example of intermediate measurements and measurands in a cell
counting analytical method .21
Bibliography .22

iii
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
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The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types
of ISO document should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
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Any trade name used in this document is information given for the convenience of users and does not
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For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO's adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 276, Biotechnology, Subcommittee SC 1,
Analytical Methods.
This second edition cancels and replaces the first edition (ISO 20391-1:2018), which has been technically
revised.
The main changes are as follows:
— Title updated;
— Scope clarified to not specifically exclude cells in biomaterial matrix;
— additional examples added for differential cell counting and indirect cell counting;
— subclause (4.2) added describing cell counting as a measurement process with Figure 2 providing
examples of steps in the measurement process for cell counting;
— subclause (4.3) added describing cell count as a measurand, explaining intermediate examinations and
intermediate measurements that can occur as a part of a cell counting analytical method (new Annex C),
and describing quantity values for cell count;
— new requirement was added in 4.3 regarding documenting intermediate examination criteria (e.g. gating
strategy, threshold values);
— Clause 5 was updated to address the concept of fit for purpose considerations for method selection;
— organization of the document was updated to include Clause 6 on sources of variability in cell counting
analytical methods with a new figure, Figure 3; Clause 6 incorporates ISO 20391-1:2018, 5.3, 5.4, and 5.5;
— requirement in ISO 2039101:2018 that cell counting be performed using validated procedures was
removed and replaced with the requirement that the cell counting method be at minimum performed
using qualified procedures;
iv
— Clause 7 of this document (previously ISO 20391-1:2018, Clause 6) was updated for clarity and consistency
with other biotechnology analytical method standards;
— requirement in ISO 20391-1:2018 that the cell counting measurement method be validated, was removed
and replaced with requirements for documentation of method qualification strategies;
— requirements in ISO 2039101:2018 that a validation plan and results be documented and maintained and
include method performance parameters for the intended use were removed and replaced with similar
requirements for qualification instead;
— requirement regarding the establishment of reference values for in-house reference materials was added
in 7.3.3;
— reporting clause was updated with new requirements for reporting cell count values.
A list of all the parts of ISO 20391 can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

v
Introduction
Cell counting (or cell enumeration) is a fundamental measurement that broadly impacts many aspects of
biotechnology, from biomanufacturing to advanced therapy. The cell count (or discrete number of cells) is
often expressed as cell concentration (i.e. cell count per volume) when in suspension and area density of
cells (i.e. cell count per unit area) when adhered to a surface. Cell count is critical in evaluating potency and
efficacy for cell-based therapy. The cell concentration within a bioreactor can serve as a quality assurance
metric in cell-based manufacturing processes. Many cell-based bioassays need to be normalized to the
respective cell count to allow data inter-comparability. This document (Part 1 of the ISO 20391 series on
cell counting) defines terms and provides general requirements and recommendations for cell counting,
including fit for purpose method selection, sources of variability, measurement, qualification and validation,
and data analysis and reporting.

vi
International Standard ISO 20391-1:2026(en)
Biotechnology — Cell counting —
Part 1:
General requirements and recommendations for cell counting
analytical methods
1 Scope
This document provides standard terminology related to cell counting for biotechnology. This document
describes counting of cells in suspension (generally cell concentration) and cells adhered to a substrate
(generally area density of cells). This document provides key considerations for general counting methods
(including total and differential counting, and direct and indirect counting) as well as for fit for purpose
method selection, sources of variability in the measurement process, and data analysis and reporting.
This document is applicable to the counting of all cell types – mammalian and non-mammalian (e.g. bacteria,
yeast) cells.
NOTE Several sector or application-specific international and national standards for cell counting currently exist.
When applicable, the user can consult existing standards when operating within their scope (e.g. specific measurement
techniques or applications).
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
accuracy
closeness of agreement between a measured quantity value (3.28) and a true quantity value (3.28) of a
measurand (3.23)
Note 1 to entry: The concept of “measurement accuracy” is not a quantity (3.27) and is not given a numerical quantity
value (3.28). A measurement is said to be more accurate when it offers a smaller measurement error.
Note 2 to entry: “Measurement accuracy” is sometimes understood as closeness of agreement between measured
quantity values (3.28) that are being attributed to the measurand (3.23).
[SOURCE: ISO/IEC Guide 99:2007, 2.13, modified — Note 2 was removed.]

3.2
agglomerate
two or more cells clustered weakly together and detected as a larger object
Note 1 to entry: Agglomerates of cells can be separated into nominally single cells without causing significant damage
to the cell.
3.3
aggregate
two or more cells clustered together (tightly or loosely) and detected as a larger object
Note 1 to entry: Aggregates of cells are generally more difficult to be separated into single cells.
3.4
analytical method
investigative procedure for qualitatively or quantitatively measuring or assessing the presence, amount, or
functional activity of a target entity (the analyte)
[SOURCE: ISO 23033:2021, 3.3]
3.5
area density
cell count (3.10) of adherent cells on a surface, typically expressed as number of cells per unit area
3.6
attribute
physical, chemical, biological or microbiological property or characteristic
3.7
calibration
operation that, under specified conditions, in a first step, establishes a relation between the quantity values
(3.28) with measurement uncertainties provided by measurement standards and corresponding indications
with associated measurement uncertainties and, in a second step, uses this information to establish a
relation for obtaining a measurement result from an indication
Note 1 to entry: A calibration may be expressed by a statement, calibration function, calibration diagram, calibration
curve (3.8), or calibration table. In some cases, it may consist of an additive or multiplicative correction of the indication
with associated measurement uncertainty (3.37).
[SOURCE: ISO/IEC Guide 99:2007, 2.39, modified — Notes 2 and 3 to entry were removed.]
3.8
calibration curve
expression of the relation between indication and corresponding measured quantity value (3.28)
[SOURCE: ISO/IEC Guide 99:2007, 4.31, modified — Note to entry removed.]
3.9
cell concentration
cell count (3.10) per volume
Note 1 to entry: Typically used for cells in suspension (e.g. cell number per ml).
Note 2 to entry: Cell concentration can refer to the total cell count (3.35) or the count of a specific subset of cells within
the volume (e.g. viable cell (3.39) number per ml).
3.10
cell count
discrete number of cells
Note 1 to entry: Cell count for cells in suspension is typically expressed as cell concentration (3.9) or area density (3.5).

3.11
cell counting
measurement process to determine the cell count (3.10)
3.12
cell suspension
single cells or aggregates of cells dispersed in a liquid matrix
3.13
debris
fragments of cells or other particles of biological or non-biological origin
3.14
differential cell count
cell count (3.10) of a subset of cells, which have been distinguished from other cell subpopulations by at least
one distinct cell attribute (3.6) identified in the measurement
Note 1 to entry: The concentrations derived from a differential cell count can be expressed in absolute concentration
or as a relative measure (i.e. percentage) with respect to the total cell number or another predefined population.
3.15
differential cell counting
cell counting (3.11) method in which a differential cell count (3.14) is evaluated.
3.16
direct cell counting
cell counting (3.11) method in which one signal is (or several signals are) detected for each single event
Note 1 to entry: Each single event should represent a single cell in an idealized measurement.
3.17
examination
process of experimentally obtaining one or more values that can reasonably be
attributed to a nominal property (3.25) together with any other available relevant information
Note 1 to entry: Definition based on Reference [1], NORDIN G, et al. (2018) Vocabulary on nominal property (3.25),
examination, and related concepts for clinical laboratory sciences (IFCC-IUPAC Recommendations 2017). Pure Appl.
Chem. 2018, 90(5), 913-935.
3.18
fit for purpose
fitness for the intended purpose
in line with prearranged requirements for an intended use (3.20)
[SOURCE: ISO 20387:2018, 3.24, modified — Note to entry deleted and "fitness for the intended purpose"
given as an admitted term.]
3.19
indirect cell counting
cell counting (3.11) method during which a signal (or a set of signals) is measured from a population of cells
and that signal is then related to cell number based on a measurement-specific mathematical model (e.g.
calibration curve (3.8))
3.20
intended use
intended purpose
use for which a product, process, or service is intended according to the specifications, instructions,
information or multiple of them provided by the manufacturer or user
[SOURCE: ISO 23033:2021, 3.26]

3.21
limit of quantitation
LoQ
lowest cell count (3.10) in a sample (3.33) that can be quantitatively determined with a
suitable precision (3.24) and accuracy (3.1) using a specific analytical method (3.4)
Note 1 to entry: LoQ is used particularly for the determination of impurities or degradation products or both.
3.22
linearity
within a given range, ability of an analytical procedure to obtain test results that are directly, proportional
to the concentration (amount) of analyte in the sample (3.33)
3.23
measurand
quantity (3.27) intended to be measured
[SOURCE: ISO/IEC Guide 99:2007, 2.3, modified — Notes to entry and examples deleted.]
3.24
precision
closeness of agreement between indications or measured quantity values (3.28) obtained by replicate
measurements on the same or similar objects under specified conditions
Note 1 to entry: Measurement precision is usually expressed numerically by measures of imprecision, such as standard
deviation, variance, or coefficient of variation (CV) under the specified conditions of measurement.
Note 2 to entry: The ‘specified conditions’ can be, for example, repeatability (3.31) conditions of measurement,
intermediate precision conditions of measurement, or reproducibility conditions of measurement (see
ISO 5725-1:1994).
[SOURCE: ISO/IEC Guide 99:2007, 2.15, modified — Notes 3 and 4 to entry deleted.]
3.25
nominal property
property of a phenomenon, body, or substance, where the property has no magnitude
Note 1 to entry: The nominal property is one for which only comparability by equivalence applies.
[SOURCE: ISO/IEC Guide 99:2007, 1.30, modified — Note 1 to entry added; Notes to entry 1 and 2 and
examples deleted.]
3.26
proportionality
ability of an analytical procedure, irrespective of range, to obtain test results which are directly proportional
to the concentration (amount) of analyte in the sample (3.33)
Note 1 to entry: In cell counting (3.11), the concentration of analyte refers to the concentration of cells (total or
differential) in the sample (3.33).
Note 2 to entry: A collection of measurements exhibit proportionality with respect to a given input parameter when
the ratio of the expected value of the measurement to the value of the input parameter at which the measurements
were taken remains constant as the value of the input parameter changes (while all other inputs and measurement
conditions are held constant).
Note 3 to entry: When a set of measurements exhibits proportionality over a range of a given input, then, Y = cX where
Y, the expected value of the measurements is expressed as the input parameter (X) multiplied by a fixed constant (c),
with no bias term.
3.27
quantity
property of a phenomenon, body, or substance, where the property has a magnitude that can be expressed
as a number and a reference
[SOURCE: ISO/IEC Guide 99:2007, 1.1, modified — Notes to entry and example deleted.]
3.28
quantity value
value of a quantity
number and reference together expressing magnitude of a quantity (3.27)
[SOURCE: ISO/IEC Guide 99:2007, 1.19, modified — Notes to entry and examples deleted.]
3.29
reagent
substance used in chemical/biochemical analysis or other reactions
3.30
reference material
reference standard
material sufficiently homogeneous and stable with reference to specified properties, which has been
established to be fit for its intended use (3.20) in measurement or in examination (3.17) of nominal properties
[SOURCE: ISO/IEC Guide 99:2007, 5.13, modified — Notes to entry and examples deleted.]
3.31
repeatability
precision (3.24) of the results of measurement under defined conditions of measurement
Note 1 to entry: Repeatability can also be considered as the closeness of the agreement between results of successive
[17]
measurements of the same measurand (3.23) carried out under the same conditions of the measurement .
Note 2 to entry: Repeatability conditions of a measurement refers to condition of measurement, out of a set of
conditions that includes the same measurement procedure, same operators, same measuring system, same operating
conditions and same location, and replicate measurements on the same or similar objects over a short period of time.
3.32
ruggedness
degree of reproducibility of test results obtained by the analysis of the same samples (3.33) under a variety
of normal test conditions
Note 1 to entry: Normal test conditions can include for example: different laboratories, different analysts, different
instruments, different reagent (3.29) lots, different analysis days, different elapsed times, different temperatures etc.
[SOURCE: ISO 23033:2021, 3.45]
3.33
sample
one or more parts taken from a system
3.34
selectivity
property of a measuring system, used with a specified measurement procedure, whereby it provides
measured quantity values (3.28) for one or more measurands (3.23) such that the values of each measurand
(3.23) are independent of other measurands (3.23) or other quantities in the phenomenon, body, or substance
being investigated
[SOURCE: ISO/IEC Guide 99:2007, 4.13, modified — examples and notes to entry deleted.]
3.35
total cell count
cell count (3.10) of all cells, independent of the attribute(s) (3.6) of the cell

3.36
total cell counting
cell counting (3.11) method in which the total cell count (3.35) is evaluated
3.37
uncertainty
non-negative parameter characterizing the dispersion of the quantity values (3.28) being
attributed to a measurand (3.23), based on the information used
[SOURCE: ISO/IEC Guide 99:2007, 2.26, modified — notes to entry deleted.]
3.38
validation
confirmation, through the provision of objective evidence, that the requirements for a specific intended use
(3.20) or application have been fulfilled
[SOURCE: ISO 9000:2015, 3.8.13, modified — notes to entry deleted.]
3.39
viable cell(s)
cells within a sample (3.33) that have an attribute (3.6) of being alive (e.g. metabolically active, capable of
reproduction, possessed of intact cell membrane, or with the capacity to resume these functions) defined
based on the intended use (3.20)
Note 1 to entry: Non-viable cells can be considered those cells within a sample (3.33) that do not exhibit an attribute
(3.6) or attributes (3.6) of being alive based on the intended use (3.20)
4 General concepts of cell counting
4.1 General
4.1.1 Categorization of cell counting analytical methods
Various cell counting methods (as described in Annex A) can be broadly categorized as total cell counting or
differential cell counting, and direct cell counting or indirect cell counting, or combination thereof (Figure 1
and Table B.1). Some methods can be employed for multiple categories based on the intended quantity for
the stated purpose.
EXAMPLE 1 Automated microscopy can be used for direct and total cell counting when the quantity is the total
number of objects or cells; it can also be used for direct and differential cell counting when the quantity is the number
of selectively labelled objects or cell subpopulations; it can also be used for indirect and total cell counting when the
quantity is percent confluence.
Some instruments and methods can provide a cell count for more than one counting category simultaneously
by detecting different attributes.
EXAMPLE 2 Total and viable cell count can be determined at the same time based on differences in optical
properties, labels, morphology, etc.
Each method has inherent noise and bias that can affect accuracy and precision.

Figure 1 — Cell counting categories
4.1.2 Total cell counting
Total cell counting involves the measurement of all cells, independent of the attribute(s) of the cell.
Criteria should be applied to distinguish cells from debris (cellular and non-cellular in origin).
4.1.3 Differential cell counting
Differential cell counting involves the measurement of a subset of cells that have been distinguished from
other cells by at least one distinct cell attribute. Differential cell counting can be conducted through direct
or indirect methods or combination thereof.
NOTE Differential cell counting can include viable cell counting, counting of cells that express a specific surface
marker, or counting of cells that exhibit specific cell morphology.
EXAMPLE Differential cell counts for tissue stem cells, committed progenitor cells, and terminally arrested cells
in a sample have been estimated by first conducting total cell counting of the cell cultures over time (via direct or
indirect methods) followed by mathematical modelling and computer simulation of the count data (indirect methods)
[2]
to estimate counts in the differential cell populations .
4.1.4 Direct cell counting
Direct cell counting involves the recording of a signal or a set of signals from each cell. In this context, the
signal(s) can be for example, electrical (as in impedance), optical (as in fluorescent or colorimetric signal), or
mechanical. The signal can be recorded manually by a user or automatically by an instrument.
Criteria should be applied to distinguish cells from debris or to distinguish cells in different subsets of
interest.
Due to the large number of cells in a typical sample, certain direct cell counting methods utilize dilution of
samples or sub-sampling. The cell count is then extrapolated based on a dilution or other scaling factor.
Direct cell counting can occur directly in cell culture. In some instances, cell counting can also be facilitated
by cells being placed in media or under conditions that are optimized for counting but do not necessarily
support long term maintenance of cell viability or function.

The presence of debris and aggregated or agglomerated cells can lead to over- or underestimated cell count.
When applicable, a process should be established to prepare well-dispersed samples with minimized debris,
aggregate, and agglomerate content.
NOTE In some direct cell counting (both total and differential) well-dispersed cells are needed for optimal
performance. Some other direct cell counting methods aim to distinguish individual cells within aggregates,
agglomerates, or other 2-D and 3-D configurations where cells are closely packed.
4.1.5 Indirect cell counting
Indirect cell counting involves the recording of a signal or a set of signals from all cells or a subset of cells
in the sample and then relating that signal to a cell count based on measurement specific mathematical
model(s) (e.g. calibration curve) (see ISO 11843-1:1997 for additional information on calibration).
NOTE 1 Indirect cell counting can include measurement of total cell mass, total DNA absorbance, genomic DNA
copy number, and metabolic activity. Indirect cell counting can also include mathematical inference from cell division
or proliferation rate.
Indirect cell counting uses a surrogate measure to evaluate the cell count. The accuracy of these analytical
methods depends on the accuracy of the measurement as well as the accuracy of the calibration curve. For
example, when total DNA quantity is used to estimate the cell count, the ability to accurately measure the
total DNA within a sample and establish an accurate relationship between DNA and cell number is important.
When possible, the calibration should be established using appropriate reference material(s).
NOTE 2 Uncertainty in the cell counts derived from indirect cell counting can arise from the mathematical model(s)
(e.g. calibration curve), in addition to other sources of measurement errors.
4.2 Cell counting as a measurement process
Cell counting is a measurement process that can include steps such as cell sample collection, test sample
[3]
preparation, data collection and data analysis (Figure 2). Each step of the measurement process can
introduce sources of random and systematic variability and should be considered in method optimization
and control. Manual processes can be a significant source of bias and variability and should be automated
when reasonable to do so.
Figure 2 — A measurement process for cell counting with examples of steps that can be a part of the
measurement process.
4.3 Cell count as a measurand
In a cell counting analytical method, the measurand, or the quantity that is intended to be measured, is the
cell count, typically expressed as a cell concentration.

A cell count quantity value can be expressed as:
a) a discrete number of cells with unit cells;
b) a concentration of cells with unit cells/V (e.g. cells/mL);
c) an area density of cells with unit cells/A (e.g. cells/cm ).
NOTE 1 Representation of cell count quantity can also follow the convention of 1/V or 1/A, omitting the phrase
“cells” to better follow International System of Units (SI) convention.
The measurement process to arrive at the measurand can include several intermediate measurements
and examinations (See Annex C for a schematic example). Intermediate measurements and intermediate
examinations should be considered for their contribution to systematic and random measurement error,
and measurement bias.
Intermediate examination processes that identify cells for enumeration should be optimized. If a cell
counting method includes examination steps, intermediate examination criteria (e.g. gating strategy,
threshold values) shall be documented.
Control materials can be used to support intermediate examination and measurements processes to enable
comparability between measurements or measurement systems and properly establish gates, thresholds or
criteria used in establishing examination criteria.
NOTE 2 Examination results in values that can be attributed to a nominal property (a property with no magnitude,
for example a category) while measurement results in values that can be attributed to a quantity (a property expressed
as a number with units).
EXAMPLE In differential direct cell counting, the cell types can be considered as a nominal property based on
certain pre-selected attributes that define the different cell types. In these methods, intermediate measurements can
be taken to provide criteria for examination to identify the different cell types within the sample, to establish the
subpopulations of cells over which to conduct the intermediate measurement of enumeration. Once the appropriate
cells have been enumerated, concentration of cells within the sub-population can be calculated based on another
intermediate measurement of volume over which the count was conducted or based on an assumed volume. Each
of these intermediate measurements and examinations can contribute to measurement error and bias and impact
comparability between counting methods.
5 Considerations for selecting a fit for purpose cell counting analytical method
The user shall consult available knowledge to select a method or methods suitable for the intended cell type,
application, and sample preparation procedure (fit for purpose).
Several sector, method, or application-specific international and national standards for cell counting
currently exist and can serve to support the development of fit for purpose cell count analytical methods
when operating within their scope. See for example, ISO 8196-3:2009, ISO 13366-1:2008, and ISO 10718:2022,
[4] [5] [6] [7] [8]
ASTM F2149-16 (2024), ASTM F2944-12, ASTM D4455-85 (2014), ASTM F2739-08, H20-A2,
[9] [10] [11] [12] [13]
DIN 58932-1, DIN 28932-2, DIN 58932-3, DIN 58932-4, DIN 58932-5, Reference method for
[14] [15]
enumeration of erythrocytes and leucocytes, platelet counting by the RBC/platelet ratio method, USP
[16] [17]
Biological Tests/<127> and CLSI H44-A2 .
Selection of the cell counting analytical method depends on the intended purpose as well as sample and
processing factors (fit for purpose considerations). These can include:
— intended purpose for cell counting;
— counting category(ies);
— appropriate measurand(s);
— appropriateness of instrumentation to assess defined measurand(s), including the limit of quantitation
(LoQ);
— sample characteristics, including cell attributes and potential effects of sample heterogeneity;

— potential impact on the measurement due to the presence of debris, aggregates, or agglomerates or
combination thereof;
— potential impact on the measurement due to bioprocessing and pre-measurement processing: including
storage, transfer, cryopreservation (including the freeze and thaw process);
— potential impact on the measurement due to ancillary materials and other components in the cell sample
(e.g. media, beads).
NOTE Intended purpose can be, for example, product release or in-process cell counting.
6 Source of variability in a cell counting analytical method
6.1 General
Cell counting analytical methods have several sources of variability arising from the analytical procedure,
cell samples, analyst, environment, analytical reagents, and equipment (Figure 3).
Figure 3 — Cause-effect diagram highlighting considerations for sources of variability in a cell
counting analytical method.
6.2 Sampling of cells for counting
The cell count is often determined from one or several sample(s) taken from the larger whole.
Proper sampling procedures should be used to minimize sampling errors associated with measuring a cell
sample rather than measuring the entire batch or lot (e.g. master cell bank, whole cell population).
Measurements from a small sample size or fraction can have a larger sampling error. In some instances,
sampling errors can be reduced by taking a larger random sample size or fraction or multiple samples
especially for measuring cells per area.
When taking an aliquot from cells in suspension, the suspension should be sufficiently homogeneous that
the aliquot is representative of the suspension. Heterogeneity in the cell suspension can lead to aliquots that
are not representative of the larger whole. A potential cause of heterogeneity is sedimentation of larger cells
or other cell sub-population.
6.3 Preparation of cell samples for counting
6.3.1 General
Cell counting processes can require preparation (e.g. mixing, lysing, staining) of the cell sample prior to
counting.
Aspects of a sample preparation process, such as environmental factors, procedures, and reagents can
introduce variability in cell counting.
A sample preparation process can alter the cell sample in systematic or random ways, reducing its
representativeness of the larger whole or altering the cell attribute associated with the counting measurand,
leading to misinterpretation of measurement results.
The presence of debris can lead to an overestimation of the number of cells. The influence of debris on cell
count measurements should be considered, and when possible, debris should be removed or accounted for
before or during counting.
The presence of aggregates or agglomerates can lead to undercounting of cells. Sample preparation
procedures should be established to prepare well-dispersed samples prior to taking an aliquot.
6.3.2 Environmental factors
Environmental factors that could change the sample in ways that affect cell counting should be minimized.
Environmental factors can include temperature, humidity, light exposure, sterility conditions, and airflow.
EXAMPLE The temperature at which a cell sample is held during execution of the cell counting method can alter
the cells attributes and would need to be selected accordingly.
6.3.3 Procedures
The effect of equipment and consumables on cell counting should be considered. Appropriate containers
and transferring apparatus should be selected to minimize loss of cells associated with sample transfer.
Transferring procedures (e.g. pipetting) should be suitable to an acceptable level of sample loss.
The mixing methods (e.g. mode, speed, duration) as well as wait/hold time in between processes can alter
the cell attribute associated with the counting measurand.
EXAMPLE 1 Sonication of reagents and vigorous mixing can introduce microbubbles that can cause false counts in
impedance counting.
Cell mixing procedures should be designed to minimize the effect on the counting measurand.
Errors in measuring cell suspension or diluent volume should be minimized when diluting cells.
Procedures to stain, lyse, disaggregate, disperse, or otherwise manipulate the cells should be evaluated for
their effects on the cell counting measurand. Potential effects on cell counting should be minimized.
EXAMPLE 2 Excessive shear can rupture some cells.
For counting of cells embedded in biomaterial matrices, penetration of analytical reagents (e.g. dyes) should
be evaluated and considered when interpreting cell counting results. Additionally, biomaterial matrices can
interfere with cell counting methods, introducing debris, and other sources of noise in the measurement that
can reduce the quality of the analytical method. Effects of biomaterial matrix materials on the measurement
process should be considered.
NOTE Additional information on cell counting-based methods in biomaterials scaffolds can be found in ASTM
[7]
F2739-08 .
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