ISO 20391-1:2018

INTERNATIONAL ISO
STANDARD 20391-1
First edition
2018-01
Biotechnology — Cell counting —
Part 1:
General guidance on cell counting
methods
Biotechnologie — Dénombrement des cellules —
Partie 1: Lignes directrices générales relatives aux méthodes de
dénombrement des cellules
Reference number
ISO 20391-1:2018(E)
©
ISO 2018

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ISO 20391-1:2018(E)

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© ISO 2018
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ISO 20391-1:2018(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General concepts of cell counting . 4
4.1 General . 4
4.2 Total cell counting . 5
4.3 Differential cell counting . 5
4.4 Direct cell counting . 5
4.5 Indirect cell counting . 5
5 Considerations for cell counting measurements . 5
5.1 Selection of a cell counting method . 5
5.2 Considerations for selecting a cell counting method . 6
5.3 Sampling of cells for counting . 6
5.4 Preparation of cell samples for counting. 7
5.4.1 Environmental factors . . . 7
5.4.2 Procedures . 7
5.4.3 Quality and stability of reagents . 7
5.5 Performing a measurement . 8
6 Qualification, validation, and verification . 8
6.1 Instrument qualification . 8
6.2 Method validation and verification . 8
6.3 Reference materials . 9
6.3.1 Certified reference materials . 9
6.3.2 In-house reference materials . 9
6.3.3 Uses of reference materials . 9
7 Data processing, analysis, and reporting .10
7.1 Data processing and analysis .10
7.1.1 General.10
7.1.2 Image processing and analysis .10
7.1.3 Gating .10
7.1.4 Coincidence correction .10
7.2 Reporting .10
Annex A (informative) Description of common cell counting methods .11
Annex B (informative) Common cell counting methods for various measurement purposes .14
Bibliography .15
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ISO 20391-1:2018(E)

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 ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
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 documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2. www.iso.org/directives
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received. www.iso.org/patents
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on 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 WTO
principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary
information
This document was prepared by ISO/TC 276, Biotechnology.
A list of all the parts of ISO 20391 can be found on the ISO website.
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ISO 20391-1:2018(E)

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 (which is
Part 1 of a multi-part standard on cell counting) defines terms and provides general guidance for the
cell counting measurement process, including method selection, sample preparation, measurement,
qualification and validation, and data analysis and reporting.
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INTERNATIONAL STANDARD ISO 20391-1:2018(E)
Biotechnology — Cell counting —
Part 1:
General guidance on cell counting methods
1 Scope
This document defines terms related to cell counting for biotechnology. It describes counting of cells
in suspension (generally cell concentration) and cells adhered to a substrate (generally area density
of cells). It provides key considerations for general counting methods (including total and differential
counting, and direct and indirect counting) as well as for method selection, 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.
This document is not intended for counting of cells while in a tissue section or a biomaterial matrix.
Several sector/application-specific international and national standards for cell counting currently
exist. When applicable, the user can consult existing standards when operating within their scope
(specific measurement techniques and/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 terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
3.1
accuracy
closeness of agreement between a measured quantity value and a true quantity value of a measurand
Note 1 to entry: The concept of “measurement accuracy” is not a quantity and is not given a numerical quantity
value. 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 that are being attributed to the measurand.
[SOURCE: ISO/IEC Guide 99:2007, 2.13, modified]
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.
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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
area density
cell count of adherent cells on a surface, typically expressed as number of cells per unit area
3.5
attribute
physical, chemical, biological or microbiological property or characteristic
3.6
cell concentration
cell count per volume
Note 1 to entry: Typically used for cells in suspension.
3.7
cell count
discrete number of cells
Note 1 to entry: Cell count is typically expressed as cell concentration (3.6) or area density (3.4).
3.8
cell counting
measurement process to determine the cell count
3.9
cell suspension
cells dispersed in a liquid matrix
3.10
debris
fragments of cells and/or particles of biological or non-biological origin
3.11
differential cell count
number of a subset of cells, which have been distinguished from other cell subpopulations by at least
one distinct cell attribute 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.
3.12
direct cell counting
counting 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.
3.13
indirect cell counting
counting 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)
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3.14
limit of quantitation
LoQ
lowest amount of analyte in a sample that can be quantitatively determined with a suitable precision
and accuracy using a specific analytical method
Note 1 to entry: The limit of quantitation describes quantitative assay for low levels of cells in sample matrices.
3.15
linearity
ability to elicit test results that are directly, or indirectly by means of well-defined mathematical
transformations, proportional to cell count within a given range
3.16
measurand
quantity intended to be measured
[SOURCE: ISO/IEC Guide 99:2007, 2.3, modified]
3.17
precision
measurement precision
closeness of agreement between indications or measured quantity values obtained by replicate
measurements on the same or similar objects under specified conditions
[SOURCE: ISO/IEC Guide 99:2007, 2.15, modified]
3.18
proportionality
characteristic exhibited by a collection of measurements in which 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 1 to entry: When a set of measurements exhibits proportionality over a range of a given input, the
expected value of the measurements can be expressed as the input parameter multiplied by a fixed constant,
with no bias term.
3.19
reagent
substance used in chemical/biochemical analysis or other reactions
3.20
reference material
material sufficiently homogeneous and stable with reference to specified properties, which has been
established to be fit for its intended use in measurement or in examination of nominal properties
Note 1 to entry: Reference materials with or without assigned quantity values can be used for measurement
precision control whereas only reference materials with assigned quantity values can be used for calibration or
measurement trueness control.
[SOURCE: ISO/IEC Guide 99:2007, 5.13, modified]
3.21
reference method
thoroughly investigated measurement procedure shown to yield values having an uncertainty in
measurement commensurate with its intended use, especially in assessing the trueness of other
measurement procedures for the same quantity and in characterizing reference material
[SOURCE: ISO 17511:2003, 3.29, modified]
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3.22
repeatability
measurement precision under defined conditions of measurement
[SOURCE: ISO/IEC Guide 99:2007, 2.21, modified]
3.23
ruggedness
measure of a method’s capacity to remain unaffected by small, but deliberate, variations in method
parameters and provides an indication of its reliability during normal usage
[SOURCE: ICH Harmonised Tripartite Guideline, 1994]
3.24
selectivity
property of a measuring system, used with a specified measurement procedure, whereby it provides
measured quantity values for one or more measurands such that the values of each measurand are
independent of other measurands or other quantities in the phenomenon, body, or substance being
investigated
[SOURCE: ISO/IEC Guide 99:2007, 4.13, modified]
3.25
total cell count
count of all cells, independent of the attribute(s) of the cell
3.26
uncertainty
non-negative parameter characterizing the dispersion of values attributed to a
measurand, based on the information used
[SOURCE: ISO/IEC Guide 99:2007, 2.26, modified]
3.27
validation
confirmation, through the provision of objective evidence, that the requirements for a specific intended
use or application have been fulfilled
[SOURCE: ISO 9000:2015, 3.8.13, modified]
3.28
verification
confirmation, through the provision of objective evidence, that specified requirements have been
fulfilled
[SOURCE: ISO 9000:2015, 3.8.12, modified]
3.29
viable cells
cells within a sample that have an attribute 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
4 General concepts of cell counting
4.1 General
Various cell counting methods (as described in Annex A) can be broadly categorized as total or
differential cell counting, and direct or indirect cell counting.
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4.2 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).
4.3 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.
EXAMPLE Differential cell counting includes viable cell counting, counting of cells that express a specific
surface marker, or counting of cells that exhibit specific cell morphology.
4.4 Direct cell counting
Direct cell counting involves the recording of a signal or a set of signals from each cell (3.12). In this
context, the signal(s) can be electrical (as in impedance), optical (as in fluorescent or colorimetric), or
mechanical. The signal can be recorded manually by a user or automatically by an instrument. Due to
the large number of cells in a typical sample, certain direct cell counting methods require dilution of
samples. The cell count is then extrapolated based on a dilution factor.
4.5 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) (3.13).
EXAMPLE Indirect cell counting includes measurement of total cell mass, total DNA, and metabolic activity.
NOTE 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.
5 Considerations for cell counting measurements
5.1 Selection of a cell counting method
Many cell counting methods exist (see Annex A); these methods can be used to measure total or
differential cell count via direct or indirect cell counting (Figure 1 and Annex B).
Figure 1 — Cell counting categories
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Some methods can be employed for multiple categories based on the intended measurand for the stated
purpose.
EXAMPLE 1 Automated microscopy can be used for direct/total cell counting if the measurand is the total
number of objects/cells; it can be used for direct/differential cell counting if the measurand is the number of
labelled objects/cells; it can also be used for indirect/total cell counting if the measurand is percent confluence.
Some instruments and/or methods can provide a cell count for more than one counting category
simultaneously by detecting different measurands.
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. The user shall consult
available knowledge to select a method or methods suitable for the intended cell type, application,
and/or sample preparation procedure (fit-for-purpose).
NOTE Requirements for cell counting can vary by intended use. Intended use can be, for example, product
release or in-process cell counting.
Direct cell counting (both total and differential) requires well-dispersed cells for optimal performance.
The presence of debris and aggregated or agglomerated cells can lead to over- or underestimated cell
count. Whenever possible, a process should be established to prepare well-dispersed samples with
minimized debris, aggregate, and agglomerate content.
Indirect cell counting methods use a surrogate measure to evaluate the cell count. The accuracy of
these 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).
5.2 Considerations for selecting a cell counting method
Selection of the cell counting method depends on the intended purpose as well as sample and processing
factors. 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, and/or agglomerates;
— 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).
5.3 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).
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Measurements from a small sample size/fraction can have a larger sampling error. In some instances,
sampling errors can be reduced by taking a larger random sample size/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.
5.4 Preparation of cell samples for counting
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 and/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.
5.4.1 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 can alter its attribute and needs to be selected
accordingly.
5.4.2 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. 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 Excessive shear can rupture some cells.
5.4.3 Quality and stability of reagents
When possible, reagents used in sample preparation should be verified to ensure quality and consistency.
The quality of the reagent should be verified using available methods or reference materials.
Some reagents (e.g. fluorescent dye, buffer) might not be stable over time or under certain environmental
conditions. Cell counting measurements should be conducted within the accepted stability range of the
reagents.
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Formulation errors of some reagents can cause either overestimation or underestimation of the cell
count. Acceptable reagent concentration ranges should be determined.
Some reagents (e.g. antibodies) might not be consistent from lot-to-lot or from different suppliers. The
user should define acceptable specifications prior to using these reagents.
The binding efficiency of reagents (such as absorption of dyes) used in cell counting should be
considered, and when appropriate, specifications should be established.
5.5 Performing a measurement
Cell counting shall be performed on properly maintained instruments.
The instrument should be calibrated or verified at appropriate intervals.
Cell counting shall be performed using validated procedures. Appropriate instrument settings should
be established for the intended cell counting.
NOTE 1 Settings for one instrument might not be directly transferable to another instrument.
NOTE 2 Optimi
...