Biotechnology -- Cell counting

ISO 20391-1:2018 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. ISO 20391-1:2018 is applicable to the counting of all cell types ? mammalian and non-mammalian (e.g. bacteria, yeast) cells. ISO 20391-1:2018 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).

Biotechnologie -- Dénombrement des cellules

General Information

Status
Published
Publication Date
18-Jan-2018
Current Stage
6060 - International Standard published
Start Date
29-Dec-2017
Completion Date
19-Jan-2018
Ref Project

Buy Standard

Standard
ISO 20391-1:2018 - Biotechnology -- Cell counting
English language
16 pages
sale 15% off
Preview
sale 15% off
Preview

Standards Content (sample)

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
---------------------- Page: 1 ----------------------
ISO 20391-1:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018

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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved
---------------------- Page: 2 ----------------------
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

© ISO 2018 – All rights reserved iii
---------------------- Page: 3 ----------------------
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.
iv © ISO 2018 – All rights reserved
---------------------- Page: 4 ----------------------
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.
© ISO 2018 – All rights reserved v
---------------------- Page: 5 ----------------------
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.
© ISO 2018 – All rights reserved 1
---------------------- Page: 6 ----------------------
ISO 20391-1:2018(E)
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)
2 © ISO 2018 – All rights reserved
---------------------- Page: 7 ----------------------
ISO 20391-1:2018(E)
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]
© ISO 2018 – All rights reserved 3
---------------------- Page: 8 ----------------------
ISO 20391-1:2018(E)
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.
4 © ISO 2018 – All rights reserved
---------------------- Page: 9 ----------------------
ISO 20391-1:2018(E)
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
© ISO 2018 – All rights reserved 5
---------------------- Page: 10 ----------------------
ISO 20391-1:2018(E)

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).

6 © ISO 2018 – All rights reserved
---------------------- Page: 11 ----------------------
ISO 20391-1:2018(E)

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.
© ISO 2018 – All rights reserved 7
---------------------- Page: 12 ----------------------
ISO 20391-1:2018(E)

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
...

Questions, Comments and Discussion

Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.