ISO 20391-2:2019

Biotechnology — Cell counting — Part 2: Experimental design and statistical analysis to quantify counting method performance

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 — Partie 2: Conception expérimentale et analyse statistique pour quantifier les performances de la méthode de dénombrement

General Information

• Status: Published
• Publication Date: 27-Aug-2019
• ICS: 07.080 - Biology. Botany. Zoology
• Technical Committee: ISO/TC 276 - Biotechnology
• Drafting Committee: ISO/TC 276/WG 3 - Analytical methods
• Current Stage: 9020 - International Standard under periodical review
• Start Date: 15-Jul-2024
• Completion Date: 15-Jul-2024

Standards Content (Sample)

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 2019
© ISO 2019
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
Phone: [phone]
Fax: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved

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
Annex E (informative) Use case 2 — Comparing the quality of several cell counting
measurement processes .38
Bibliography .52
iv © ISO 2019 – All rights reserved

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.
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.
S
...

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2019-08
ISO/TC 276/WG 3
Secretariat: DIN
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
© ISO 2019 – All rights reserved i

Deleted: /FDIS
© ISO 2019
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
Phone: [phone]
Fax: [phone]
Email: [e-mail]
Website: www.iso.org
Deleted: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved

Deleted: /FDIS
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reference not valid.¶
Contents
Introduction 6¶
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Foreword . 5
2 Normative references 1¶
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3.1 Terms and definitions 1¶
1  Scope . 1
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2  Normative references . 1
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4 Principle 9¶
3  Terms, definitions, symbols and abbreviated terms . 1
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3.2  List of abbreviated terms and symbols . 7  4.2 Proportionality 9¶
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4  Principle . 9
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4.1  General . 9  5 Experimental design 11¶
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4.2  Proportionality. 9
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4.3  Deviation from proportionality . 10
5.2 Considerations for the
cell counting measurement
5  Experimental design . 11
process 11¶
5.1  General . 11
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5.2  Considerations for the cell counting measurement process . 11
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5.3  Preparation of samples for the experimental design . 12
5.3.1 General 12¶
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5.3.2  Stock cell solution . 12
5.3.3 Dilution fraction
5.3.3  Dilution fraction experimental design . 13
experimental design 13¶
5.3.4  Considerations for generating dilution fractions . 14
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5.4  Test sample labelling . 15
valid.¶
5.4 Test sample
5.5  Measurement of the test sample . 15
labelling 15¶
6  Statistical methods . 16
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6.1  General . 16
6 Statistical methods 16¶
6.2  Mean cell count . 16
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6.3  Measurement precision . 17
valid.¶
6.4  Proportional model fit . 17
6.2 Mean cell count 16¶
6.5  Coefficient of determination . 18
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valid.¶
6.6  Proportionality Index (PI ) . 18
6.4 Proportional model
6.6.1  General . 18
fit 17¶
6.6.2  Calculation of the smoothed residual (esmoothed) . 19
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6.6.3  Calculation of proportionality index (PI) . 19
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6.7  Additional statistical analysis and quality metrics . 20
6.6 Proportionality Index
(PI) 18¶
6.8  Data Interpretation . 20
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6.8.1  General . 20
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6.8.2  Interpretation of %CV . 20
6.6.2 Calculation of the
6.8.3  Interpretation of R . 20
smoothed residual
6.8.4  Interpretation of PI values . 21
(esmoothed) 19¶
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6.8.5  Comparison of PI values . 21
valid.¶
7  Reporting . 21  6.7 Additional statistical
analysis and quality
7.1  Reporting of quality indicators . 21
metrics 20¶
7.2  Documentation of experimental design parameters and statistical analysis method . 22
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7.3  Additional reporting elements on the cell counting measurement process . 23
valid.¶
6.8.1 General 20¶
Annex A (informative)  Method to assess pipetting error contributions to dilution integrity . 24
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A.1  General . 24
6.8.3 Interpretation of
R 20¶
... [1]
© ISO 2019 – All rights reserved iii

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A.2 Example procedure for pre-evaluating pipetting error contributions to dilution
integrity using a calibrated scale to obtain accurate volume estimates upon
pipetting . 24
A.3 Example procedure for obtaining a measured DF during the evaluation of PI using a
calibrated scale to obtain accurate volume estimates . 26
Annex B (normative) Method to calculate smoothed residual (e ) when a set of
smoothed
measured dilution fractions (DFmeasured) is obtained . 28
Annex C (informative) Example formulae for calculating PI . 30
C.1 Detailed example of the calculation of PI based on sum of the absolute value of
scaled smoothed residuals . 30
C.2 Detailed example of the calculation of PI based on R of smoothed residuals . 30
C.3 Additional examples of the calculation of PI when measured dilution fraction is
utilized . 31
Annex D (informative) Use case 1 — Evaluating the quality of a single cell counting
measurement process . 32
D.1 General . 32
D.2 Description of experimental design and statistical analysis . 32
D.3 Raw Data and Data Analysis for Use Case 1 . 33
D.4 Example Report for Use Case 1 . 33
D.5 Interpretation . 38
Annex E (informative) Use case 2 — Comparing the quality of several cell counting
measurement processes . 39
E.1 General . 39
E.2 Description of experimental design and statistical analysis . 39
E.3 Raw Data and Data Analysis for Use Case 2 . 40
E.4 Example Report for Use Case 2 . 42
E.5 Comparison of cell counting methods evaluated in Use Case 2 . 50
E.5.1 Comparing quality indicators between cell counting methods . 50
E.6 Interpretation . 51
E.7 Consistency of results with underlying simulated model . 52
Bibliography . 54
iv © ISO 2019 – All rights reserved

Deleted: /FDIS
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).
Deleted: www.iso.org/directives
Attention is drawn to the possibility that some of the elements of thi
...