Biotechnology -- Cell counting

This document provides a method for evaluating aspects of the quality of a cell counting measurement process for a specific cell preparation through a set of quality indicators derived from a dilution series experimental design and statistical analysis. The quality indicators are based on repeatability of the measurement and the degree to which the results conform to an ideal proportional response to dilution. This method is applicable to total, differential, direct and indirect cell counting measurement processes, provided that the measurement process meets the criteria of the experimental design (e.g. cells are suspended in a solution). This method is most suitable during cell counting method development, optimization, validation, evaluation and/or verification of cell counting measurement processes. This method is especially applicable in cases where an appropriate reference material to assess accuracy is not readily available. This method does not directly provide the accuracy of the cell count. This method is primarily applicable to eukaryotic cells. NOTE Several sector/application specific international and national standards for cell counting exist. Where applicable, consulting existing standards when operating within their scope can be helpful.

Biotechnologie -- Dénombrement des cellules

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Status
Published
Publication Date
27-Aug-2019
Current Stage
6060 - International Standard published
Start Date
30-Jul-2019
Completion Date
28-Aug-2019
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ISO 20391-2:2019 - Biotechnology -- Cell counting
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INTERNATIONAL ISO
STANDARD 20391-2
First edition
2019-08
Biotechnology — Cell counting —
Part 2:
Experimental design and statistical
analysis to quantify counting method
performance
Biotechnologie — Dénombrement des cellules —
Partie 2: Conception expérimentale et analyse statistique pour
quantifier les performances de la méthode de dénombrement
Reference number
ISO 20391-2:2019(E)
ISO 2019
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ISO 20391-2:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

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Published in Switzerland
ii © ISO 2019 – All rights reserved
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ISO 20391-2:2019(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms, definitions, symbols and abbreviated terms ....................................................................................................... 1

3.1 Terms and definitions ....................................................................................................................................................................... 1

3.2 List of abbreviated terms and symbols .............................................................................................................................. 7

4 Principle ........................................................................................................................................................................................................................ 8

4.1 General ........................................................................................................................................................................................................... 8

4.2 Proportionality ....................................................................................................................................................................................... 9

4.3 Deviation from proportionality ................................................................................................................................................ 9

5 Experimental design ......................................................................................................................................................................................10

5.1 General ........................................................................................................................................................................................................10

5.2 Considerations for the cell counting measurement process ........................................................................10

5.3 Preparation of samples for the experimental design ..........................................................................................11

5.3.1 General...................................................................................................................................................................................11

5.3.2 Stock cell solution ........................................................................................................................................................11

5.3.3 Dilution fraction experimental design .......................................................................................................12

5.3.4 Considerations for generating dilution fractions .................. ............................................................13

5.4 Test sample labelling ......................................................................................................................................................................14

5.5 Measurement of the test sample...........................................................................................................................................14

6 Statistical methods ..........................................................................................................................................................................................15

6.1 General ........................................................................................................................................................................................................15

6.2 Mean cell count ....................................................................................................................................................................................16

6.3 Measurement precision ................................................................................................................................................................16

6.4 Proportional model fit ...................................................................................................................................................................16

6.5 Coefficient of determination ....................................................................................................................................................17

6.6 Proportionality index (PI) ..........................................................................................................................................................17

6.6.1 General...................................................................................................................................................................................17

6.6.2 Calculation of the smoothed residual (e ) ..............................................................................18

smoothed

6.6.3 Calculation of proportionality index (PI).................................................................................................18

6.7 Additional statistical analysis and quality metrics ...............................................................................................19

6.8 Data interpretation ...........................................................................................................................................................................19

6.8.1 General...................................................................................................................................................................................19

6.8.2 Interpretation of %CV .............................................................................................................................................19

6.8.3 Interpretation of R ...................................................................................................................................................19

6.8.4 Interpretation of PI values ....................................................................................................................................20

6.8.5 Comparison of PI values .........................................................................................................................................20

7 Reporting ...................................................................................................................................................................................................................20

7.1 Reporting of quality indicators ..............................................................................................................................................20

7.2 Documentation of experimental design parameters and statistical analysis method ...........21

7.3 Additional reporting elements on the cell counting measurement process ...................................22

Annex A (informative) Method to assess pipetting error contributions to dilution integrity ...............23

Annex B (normative) Method to calculate smoothed residual (e ) when a set of
smoothed

measured dilution fractions (DF ) is obtained ..................................................................................................27

measured

Annex C (informative) Example formulae for calculating PI .....................................................................................................29

Annex D (informative) Use case 1 — Evaluating the quality of a single cell counting

measurement process ..................................................................................................................................................................................31

© ISO 2019 – All rights reserved iii
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ISO 20391-2:2019(E)

Annex E (informative) Use case 2 — Comparing the quality of several cell counting

measurement processes ............................................................................................................................................................................38

Bibliography .............................................................................................................................................................................................................................52

iv © ISO 2019 – All rights reserved
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ISO 20391-2:2019(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 (see 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 (see 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 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.
A list of all parts in the ISO 20391 series 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.
© ISO 2019 – All rights reserved v
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ISO 20391-2:2019(E)
Introduction

Cell counting impacts many aspects of biotechnology, from biomanufacturing to medical diagnosis

and advanced therapy. The cell count can serve as an in-process quality control or be used in decision-

making. Cell count is also an important parameter in many cell-based assays, including activity and

potency, which are often normalized to the cell count to allow data comparison.

Cell count is generally expressed as a concentration and can reflect the total cell count of a cell

population (total cell count) or subpopulation (differential cell count). Advances in instrumentation

have resulted in a wide range of cell counting techniques/instruments for total and/or differential

cell counts. In the absence of a readily available reference material or ground truth, the accuracy of

a measurement method has been difficult to ascertain. This has been confounded by the complexity

of the biological preparation (e.g. cell type, sources, preparation, etc.). Several standards that address

sector/application-specific cell counting or the use of a specific measurement system exist (See

ISO 20391-1 and Reference [16] for further information). Some of these methods use a comparability

approach whereby the result from a newer cell counting test method is traced to the results obtained

from a more established cell counting method. While the comparability approach allows the data from

the second instrument to be benchmarked against those obtained from a primary (more established)

[17]

instrument, it does not address the quality of either measurement process . There remains a need to

develop strategies to provide assurance for the quality of a cell counting measurement process in the

[17]
absence of a reference material or reference method .

This document provides a method for evaluating aspects of the quality of a cell counting measurement

process through the use of a dilution series experimental design. From this experimental design, a set

of quality indicators are derived to assess the performance of a cell counting measurement process.

Specifically, the quality indicators assess precision and proportionality of cell counting measurement

processes. This approach is particularly useful when accuracy cannot be determined (i.e. in the absence

of a traceable reference method or traceable reference material) and is also relevant in aspects of

[17]

validating and monitoring the quality of cell counting measurement processes in general .

Information in this document is intended to provide confidence in the data produced by a chosen cell

counting measurement process. This approach can be useful for selecting or optimizing a measurement

process for a given cell preparation. This approach can also provide supporting performance parameters

that can be utilized during performance qualification of a particular cell counting measurement process.

vi © ISO 2019 – All rights reserved
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INTERNATIONAL STANDARD ISO 20391-2:2019(E)
Biotechnology — Cell counting —
Part 2:
Experimental design and statistical analysis to quantify
counting method performance
1 Scope

This document provides a method for evaluating aspects of the quality of a cell counting measurement

process for a specific cell preparation through a set of quality indicators derived from a dilution series

experimental design and statistical analysis. The quality indicators are based on repeatability of

the measurement and the degree to which the results conform to an ideal proportional response to

dilution. This method is applicable to total, differential, direct and indirect cell counting measurement

processes, provided that the measurement process meets the criteria of the experimental design (e.g.

cells are suspended in a solution).

This method is most suitable during cell counting method development, optimization, validation,

evaluation and/or verification of cell counting measurement processes.

This method is especially applicable in cases where an appropriate reference material to assess

accuracy is not readily available. This method does not directly provide the accuracy of the cell count.

This method is primarily applicable to eukaryotic cells.

NOTE Several sector/application specific international and national standards for cell counting exist. Where

applicable, consulting existing standards when operating within their scope can be helpful.

2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 20391-1, Biotechnology — Cell counting — Part 1: General guidance on cell counting methods

3 Terms, definitions, symbols and abbreviated terms
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:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1 Terms and definitions
3.1.1
accuracy

closeness of agreement between a measured quantity value and a true quantity value

of a measurand
[SOURCE: ISO/IEC Guide 99:2007, 2.13, modified — Notes deleted]
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ISO 20391-2:2019(E)
3.1.2
bias
estimate of a systematic measurement error
[SOURCE: ISO/IEC Guide 99:2007, 2.18]

Note 1 to entry: Systematic measurement error is a component of measurement error that in replicate

measurements remains constant or varies in a predictable manner. A reference quantity value for a systematic

measurement error is a true quantity value, or a measured quantity value of a measurement standard of

negligible measurement uncertainty, or a conventional quantity value.

Note 2 to entry: Also defined as the difference between the expectation of the test results and an accepted

reference value (ISO 3534-1).
3.1.3
cell concentration
cell count 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 or the count of a specific subset of cells within

the volume (e.g. viable cell number per ml).
3.1.4
cell count
discrete number of measured cells

Note 1 to entry: Cell count for cells in suspension is typically expressed as cell concentration.

3.1.5
cell counting
measurement process to determine the cell count
3.1.6
cell suspension
single cells or aggregates of cells dispersed in a liquid matrix
3.1.7
debris

fragments of cells and/or particles of biological or non-biological origin

3.1.8
differential cell count

cell count 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.
3.1.9
dilution fraction

ratio by which the concentration of solute in a solution has been reduced from an original concentration

Note 1 to entry: Dilution fraction can range from 0 to 1.

Note 2 to entry: Dilution fraction is also sometimes referred to as “dilution ratio” or “dilution factor”.

EXAMPLE The ratio by which the concentration of cells (solute) in a cell suspension (solution) has been

reduced from a starting concentration of cells in suspension.
2 © ISO 2019 – All rights reserved
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ISO 20391-2:2019(E)
3.1.10
dilution series

group of solutions that have increasing or decreasing concentrations of the same substance

Note 1 to entry: A dilution series can be generated by serial dilution or by independent dilution.

Note 2 to entry: For a cell suspension, a dilution series is a group of suspensions that have increasing or decreasing

concentrations of cells.
3.1.11
experimental design
process of planning a study to meet specified objectives

Note 1 to entry: Plan for assigning experimental conditions to participants and the statistical analysis associated

with the plan. Typically, this includes a specification of the independent variables, dependent variables, number

of participants and sampling strategy, procedure for assigning participants to experimental conditions, and

order in which test tasks are given.
3.1.12
independent dilution

dilution series where each dilution is conducted independently of other dilutions

Note 1 to entry: Generally independent dilution series are generated directly from a common stock solution at a

pre-specified (or target) dilution fraction.
3.1.13
intermediate precision

condition of measurement, out of a set of conditions that includes the same measurement procedure,

same location, and replicate measurements on the same or similar objects over an extended period of

time, but may include other conditions involving changes

Note 1 to entry: The changes can include new calibrations, calibrators, operators, and measuring.

Note 2 to entry: Operator bias refers specifically to error introduced by human operator experience.

[SOURCE: ISO/IEC Guide 99:2007, 2.22, modified — Note 3 deleted]
3.1.14
limit of quantitation
LOQ

lowest cell count 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 quantification describes quantitative assay for low levels of cells in sample matrices.

3.1.15
linearity

within a given range, ability of an analytical procedure to obtain test results which are directly

proportional to the concentration (amount) of analyte in the sample
[SOURCE: Reference [14], modified.]

Note 1 to entry: In cell counting the concentration of analyte refers to the concentration of cells (total or

differential) in the sample.

Note 2 to entry: When a set of measurements exhibits linearity over a range of a given input (while all other

inputs and measurement conditions are held constant), the expected value of the measurand can be expressed as

the sum of a constant bias term and the input parameter multiplied by a fixed constant.

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ISO 20391-2:2019(E)
3.1.16
measurand
quantity intended to be measured
[SOURCE: ISO/IEC Guide 99:2007, 2.3, modified — Notes and examples deleted.]
3.17
measured dilution fraction
dilution fraction verified by a traceable measurement

Note 1 to entry: For example, the volume of liquid can be verified by measuring the mass of the liquid (taking into

density) using a calibrated and traceable scale with appropriate sensitivity.
3.1.18
measurement process
entire process for obtaining a cell count

Note 1 to entry: A measurement process can include sample preparation procedures, the measuring system, its

settings (e.g. aperture choice, cell size gating, magnification, light exposure time etc.), and data analysis.

3.1.19
measurement precision

closeness of agreement between indications or measured quantity values 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 conditions of measurement,

intermediate precision conditions of measurement, or reproducibility conditions of measurement (see

ISO 5725-1).
[SOURCE: ISO/IEC Guide 99:2007, 2.15, modified — Notes 3 and 4 deleted.]
3.1.20
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

Note 1 to entry: In cell counting the concentration of analyte refers to the concentration of cells (total or

differential) in the sample.

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.
3.1.21
proportionality constant
constant multiplier that directly relates the measurand to an input parameter
3.1.22
proportionality index

measure of deviation from proportionality for a dilution series experimental design

Note 1 to entry: The proportionality index (PI) is specific to the cell preparation and cell counting measurement

process being evaluated.
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ISO 20391-2:2019(E)
3.1.23
p-value
output of a statistical hypothesis test

Note 1 to entry: The p-value is obtained in the following manner: The distribution of the test statistic under the

assumption that the null hypothesis is true, called the null distribution, is determined. The p-value is computed

from the null distribution as the probability of observing a test statistic that is as or more extreme than the test

statistic obtained from the actual data.
3.1.24
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 and example deleted.]
3.1.25
range
quantity interval bounded by rounded or approximate extreme indications
3.1.26
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 in measurement or in examination of nominal properties

[SOURCE: ISO/IEC Guide 99:2007, 5.13, modified — Notes and examples deleted.]
3.1.27
reference method
reference measurement procedure

measurement procedure accepted as providing measurement results fit for their intended use in

assessing measurement trueness of measured quantity values obtained from other measurement

procedures for quantities of the same kind, in calibration, or in characterizing reference materials

[SOURCE: ISO/IEC Guide 99:2007, 2.7]
3.1.28
repeatability
precision 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

[17]

successive measurements of the same measurand carried out under the same conditions of the measurement .

3.1.29
residual

numerical difference between the observed value of a dependent variable and the

predicted value
3.1.30
sample

one or more parts taken from a system and intended to provide information on the system

Note 1 to entry: Often the sample serves as a basis for decision on the system or its production.

Note 2 to entry: For example, a smaller volume or aliquot of cell suspension taken from a larger volume of cell

[17]
suspension .

[SOURCE: ISO 15198:2004, 3.22, modified — “population” replaced by “system”, Notes added.]

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ISO 20391-2:2019(E)
3.1.31
serial dilution

stepwise dilution of a substance in solution where the reduction of concentration is cumulative,

lessening with each subsequent dilution

Note 1 to entry: In a serial dilution series, all dilutions except for the first are dependent on the preceding dilution.

3.1.32
stock cell solution

sufficiently stable (over time) cell suspension at sufficiently high concentration to allow dilution into

working concentrations during experimentation
3.1.33
systematic error

component of measurement error that in replicate measurements remains constant or varies in a

predictable manner

Note 1 to entry: A reference quantity value for a systematic measurement error is a true quantity value, or a

measured quantity value of a measurement standard of negligible measurement uncertainty, or a conventional

quantity value.

Note 2 to entry: Systematic measurement error, and its causes, can be known or unknown. A correction can be

applied to compensate for a known systematic measurement error.

Note 3 to entry: Systematic measurement error equals measurement error minus rando

...

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