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 2018
© ISO 2018
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ii © ISO 2018 – All rights reserved

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
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
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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
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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.
iv © ISO 2018 – All rights reserved

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.
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.
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
concent
...