ISO 20391-2:2019

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
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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
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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
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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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