ISO 24421:2023

INTERNATIONAL ISO
STANDARD 24421
First edition
2023-07
Biotechnology — Minimum
requirements for optical signal
measurements in photometric
methods for biological samples
Biotechnologie — Exigences minimales relatives aux mesures
de signaux optiques dans les méthodes photométriques pour les
échantillons biologiques
Reference number
© ISO 2023
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ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principles . 4
4.1 General . 4
4.2 Instruments and measurements . 5
4.3 Optical references . 6
5 Minimum requirements to support optical signal measurement . 6
5.1 Elements of photometric methods . 6
5.2 Verification of optical signal measurement instruments . 7
5.2.1 Optical references . 7
5.2.2 Dynamic range . 7
5.2.3 Background signals . 8
5.3 Optical signal measurement of biological samples . 8
5.3.1 Optical signal measurement . 8
5.3.2 Calibration curve. 8
5.3.3 Photometric methods . . 9
5.3.4 Personnel . 9
5.4 Data analysis and reporting . 9
Annex A (informative) Principles of bioluminescence, chemiluminescence, fluorescence
and absorption .11
Annex B (informative) List of optical references, optical signal measurement instruments
and representative photometric methods .14
Annex C (informative) List of relevant standards describing representative methods
by means of optical signal measurements .15
Annex D (informative) Example of qualification of luminometer using LED reference light
source . .16
Annex E (informative) Example of application of reference light source for comparison
measurements of bioluminescent sample using luminometers .18
Annex F (informative) Example of determination of well-to-well crosstalk in multi-well
plates .20
Annex G (informative) Examples of dynamic range determination of luminometer .22
Annex H (informative) Example of construction of calibration curve and dynamic range
determination of fluorescence plate reader .25
Annex I (informative) Example of dynamic range determination of a flow cytometer .27
Annex J (informative) Example of calibration of reference light sources and luminometers .29
Annex K (informative) Examples of spectral properties of photodetectors .32
Bibliography .34
iii
Foreword
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This document was prepared by Technical Committee ISO/TC 276, Biotechnology.
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iv
Introduction
This document defines terms and provides general guidance for accurate measurement of optical
signals used for analysis of biological samples in photometric methods. These photometric methods
can use optical signal measurements, including bioluminescence, chemiluminescence, fluorescence or
absorption measurement, that can be applied in the fields of biotechnology, life science and medicine.
A measured optical signal value is applied for evaluating biological parameters qualitatively or
quantitatively, including cellular and metabolic activities, and gene expressions. Photometric methods
are used in applications such as toxicity testing, environmental risk assessment, biomanufacturing,
drug development, regenerative medicine and biobanking.
There are significant needs for both manufacturers and users for high quality optical signal measurement
in photometric methods in industry to increase confidence in the repeatability, intermediate precision
and reproducibility for analysis of biological samples. While repeatability of the photometric method is
already sufficient for qualitative characterization of biological samples, quantitative characterization
requires more accurate intermediate precision and reproducibility of optical signal measurement. It
requires proper optical signal measurements, and it also requires assessment of deviations from the
ideal proportionality of the optical signal and the output of the photometric method. Requirements
for proper optical signal measurement are an important component of the description of specific
applications of photometric methods.
This document provides a general framework to support proper measurement of an optical signal in
a photometric method. It focuses on the utilization of optical references and relevant technical issues
for optical signal measurement in photometric methods, including procedures for verification of
instruments, continual performance monitoring of instruments and photometric method validation.
Optical references can be used to verify instruments to increase confidence in the repeatability,
intermediate precision, and reproducibility of optical signal measurement. For example, an optical
signal emitted from biological samples can be compared on a common measurement scale within a
laboratory, between manufacturer and manufacturer, manufacturer and user, or user and user.
v
INTERNATIONAL STANDARD ISO 24421:2023(E)
Biotechnology — Minimum requirements for optical signal
measurements in photometric methods for biological
samples
1 Scope
This document specifies minimum requirements to support accurate measurement of optical signals in
photometric methods used for qualitative or quantitative characterization of biological samples.
This document is applicable to optical signals that are generated, for example, by bioluminescence,
chemiluminescence and fluorescence, and optical signals that are detected as changes of light due to
absorption.
This document addresses the verification of optical signal measurement instruments used in
photometric methods for measurement of biological samples including considerations for the use of
optical references.
This document does not provide sector- or application-specific performance criteria for the workflow
of measuring biological samples. When applicable, users can also consult existing sector- or application-
specific standards, or both.
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 terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
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 “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: The term “measurement accuracy” should not be used for measurement trueness and the term
“measurement precision” should not be used for “measurement accuracy”, which, however, is related to both
these concepts.
Note 3 to entry: “Measurement accuracy” is sometimes understood as closeness of agreement between measured
quantity values that are being attributed to the measurand.
Note 4 to entry: ISO 5725-1:1994 uses a different definition for “accuracy”.
[SOURCE: ISO/IEC Guide 99:2007, 2.
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