ISO 24421:2023
(Main)Biotechnology — Minimum requirements for optical signal measurements in photometric methods for biological samples
Biotechnology — Minimum requirements for optical signal measurements in photometric methods for biological samples
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.
Biotechnologie — Exigences minimales relatives aux mesures de signaux optiques dans les méthodes photométriques pour les échantillons biologiques
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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 24421:2023(E)
© ISO 2023
---------------------- Page: 1 ----------------------
ISO 24421:2023(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2023
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: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
© ISO 2023 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 24421:2023(E)
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
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ISO 24421:2023(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.
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.
iv
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ISO 24421:2023(E)
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.
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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, 2.13, modified — “measurement accuracy” and “accuracy of
measurement” deleted as terms. Note 4 to entry added.]
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ISO 24421:2023(E)
3.2
biological sample
material or object of biological origin
3.3
dynamic range
range of optical signal (3.6) values that can be measured quantitatively
[SOURCE: ISO 2041:2018, 3.4.17, modified — “optical signal” and “quantitatively” added to the
definition.]
3.4
light source
optical device emitting appropriate wavelength(s) in a specified spectral region
Note 1 to entry: A light source can be a part of an optical signal (3.6) measurement instrument.
[SOURCE: ISO 25178-604:2013, 2.3.1 modified — “wavelength(s)” replaced “range of wavelengths”.
Note 1 to entry added.]
3.5
optical reference
material, light source (3.4) or photodetector, sufficiently reproducible and stable with respect to optical
properties, that has been established to be fit for its intended use
EXAMPLE Light emitting diode (LED)-based reference light source (3.11), laser, slide of fluorescent glass,
fluorescent dye in solution or other matrix (e.g. fluorescent bead), slide embedded fluorescent material, reference
filter, reference cuvette, reference film, reference solution, power meter (3.9) (see Annex B).
Note 1 to entry: The term “optical reference” includes both uncalibrated references and calibrated standards.
Optical references can be distributed by an internal organization or prepared by a laboratory (e.g. in-house
standard, in-house reference material).
Note 2 to entry: Optical references can be used for verification (3.14) of optical signal (3.6) measurement
instruments (see Annexes D, E, G, H, I and J).
3.6
optical signal
light emitted or changes of light due to absorption caused by transmitting light through samples or
chromogenic substances
Note 1 to entry: The optical signal measurement involves, for example, bioluminescence, chemiluminescence,
fluorescence and absorption measurements. Annex A gives information about optical signals.
Note 2 to entry: In this document, the term “optical signal” focuses on light before detection.
3.7
optical signal intensity
strength of an optical signal (3.6)
Note 1 to entry: Intensity can be used to express the absolute strength or relative strength of an optical signal.
An appropriate unit can be used in order to express the intensity of a particular optical signal.
3.8
photometric method
analytical technique using optical signal (3.6) measurement(s) to determine components or biological
parameters of biological samples (3.2)
Note 1 to entry: The photometric method includes preanalytical, optical signal measurement and data analysis
procedures.
Note 2 to entry: Biological parameters of biological samples include, for example, cellular and metabolic activities,
and gene expressions.
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ISO 24421:2023(E)
Note 3 to entry: Examples for representative photometric methods are shown in Annex B.
Note 4 to entry: Analysis and assay results of photometric methods can be expressed qualitatively or
quantitatively.
Note 5 to entry: The term “radiometric” is widely used instead of “photometric” in the field of optical engineering
(e.g. IEC 60050-845).
3.9
power meter
optical power meter
measurement device to determine the radiant power of light used as an optical reference (3.5)
Note 1 to entry: The watt (W) is used as a unit to express radiant power.
3.10
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 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 5725-3:1994).
Note 3 to entry: Measurement precision is used to define measurement repeatability, intermediate measurement
precision, and measurement reproducibility.
Note 4 to entry: Sometimes “measurement precision” is erroneously used to mean measurement accuracy.
Note 5 to entry: ISO 5725-1:1994 uses a different definition for “precision”.
[SOURCE: ISO/IEC Guide 99:2007, 2.15, modified — “measurement precision” deleted as a term. Note 5
to entry added.]
3.11
reference light source
light source (3.4) used as an optical reference (3.5)
EXAMPLE Characterized or calibrated LED and laser.
3.12
reference material for calibration curve
material with known value of concentration or amount of a specific substance, for intended purpose
Note 1 to entry: It is identical to or commutable with the measurement object of a biological sample (3.2).
Note 2 to entry: Examples for expressing concentration and amount are mol/l and mol, respectively.
3.13
validation
confirmation, through the provision of objective evidence, that the requirements for a specific intended
use or application have been fulfilled
Note 1 to entry: The objective evidence needed for a validation is the result of a test or other form of determination
such as performing alternative calculations or reviewing documents.
Note 2 to entry: The word “validated” is used to designate the corresponding status.
Note 3 to entry: The use conditions for validation can be real or simulated.
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ISO 24421:2023(E)
Note 4 to entry: ISO/TS 16393:2019 uses the term “validation” in a different meaning in defining “validation
experiment”. ISO/IEC Guide 99:2007 uses a different definition for “validation”.
[SOURCE: ISO 9000:2015, 3.8.13, modified — Note 4 to entry added.]
3.14
verification
confirmation, through the provision of objective evidence, that specified requirements have been
fulfilled
Note 1 to entry: The objective evidence needed for a verification can be the result of an inspection or of other
forms of determination such as performing alternative calculations or reviewing documents.
Note 2 to entry: The activities carried out for verification are sometimes called a “qualification process”.
Note 3 to entry: The word “verified” is used to designate the corresponding status.
Note 4 to entry: ISO/IEC Guide 99:2007 uses a different definition for “verification”.
[SOURCE: ISO 9000:2015, 3.8.12, modified — Note 4 to entry added.]
4 Principles
4.1 General
Optical signal measurements, including bioluminescence, chemiluminescence, fluorescence and
absorption measurements, are used in photometric methods. Optical signal measurements are often
used for biological samples to determine a diverse set of biological parameters qualitatively and
quantitatively, including cellular and metabolic activities, and gene expressions (see Annex A for more
information). In the photometric methods, the optical signal intensity and spectrum from biological
samples are measured using instruments.
NOTE 1 Examples of instruments are luminometers, imaging analysers, fluorescence plate readers, flow
cytometers, microarray readers, spectrofluorometers, plate readers, spectrophotometers and DNA sequencers
(see Annex B).
Accuracy, precision, repeatability and reproducibility represent some of the important metrological
factors used for evaluating the effectiveness of photometric method applied.
Photometric methods can be qualitatively validated using positive and negative control materials.
NOTE 2 The performance characteristics of qualitative photometric methods and their validation can be
determined with appropriate statistical models depending on the method, structure of data and statistical
experience (e.g. ISO/TS 16393).
Accurate analysis and assay results are obtained by measuring the optical signal with an appropriate
selection of experimental materials, including the reagents generating the optical signal from the
sample, and the use of suitable instruments for the intended purpose.
Sample preparation is also an important factor governing the performance of a photometric method.
Optical signal measurements produce relative and absolute optical signal values that are functionally
related to the quantity of specific characteristics of biological samples or biological parameters. In
spectral-resolved measurements, spectral characteristics are indicative for the interaction of particular
molecules, structural elements of molecules, or molecular interaction with electromagnetic radiation of
different energy.
In some cases, calibration curves constructed using a reference material for calibration curve are
required for quantification of the absolute amount of biological sample. A calibration curve can be also
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ISO 24421:2023(E)
used to determine an effective amount of a test article (e.g. an amount that elicits 50 % response across
the calibration curve or ED ).
50
NOTE 3 Annex H gives an example for the construction of a calibration curve.
For measurement of biological samples, it is sometimes necessary to label or stain biological samples,
introduce a reporter gene into cells, tissues and whole organisms, or trigger chemical reactions.
NOTE 4 Reagent quality and its photophysical and chemical properties affects optical signals from the sample.
Activity of cells can sometimes affect optical signals.
NOTE 5 Ambient light radiation can cause deterioration of bioluminescent reagents, chemiluminescent
reagents, fluorescent materials and fading absorption.
When cells are used in photometric methods, the robustness of analysis and assay results is less reliable
if the cellular activity is unstable. In particular, optical signal measurement results are directly affected
by the stability of the cellular activity during long-term storage/subculturing and by the stability of
responsiveness to the target bioactive substance. The incident measuring light can also affect cellular
functions and properties, in particular if the cells are exposed to the light for a long period. Accordingly,
the reliability of optical signal measurement results can be increased by maintaining cell stability.
NOTE 6 Examples are assays to evaluate cellular activity, including viability, toxicity and metabolic activity by
means of cell-based assays.
NOTE 7 Relevant standards that describe representative methods by means of optical signal measurements
are listed in Annex C.
Preanalytical procedures applied before performing optical signal measurements, including cell lysis,
antigen-antibody reaction, dye labelling or staining, can affect analysis and assay results.
4.2 Instruments and measurements
Photodetectors, including photomultipliers, photodiodes and image sensors, have specific spectral
responsivities. Optical signals, including bioluminescence, chemiluminescence, fluorescence and
absorption, can be measured accurately by using spectrally suited photodetectors and colour filters.
NOTE 1 Annex K gives examples for spectral responsivity data of photodetectors.
The optical signal measurement instruments are affected by environmental conditions, including
laboratory temperature, and exposure to direct sunlight. Adjustment of the spatial resolution of an
instrument can be required depending on the application.
Optical signals can be measured quantitatively when the signal intensity is within the dynamic range of
the photodetector. Photodetectors have specific linear or nonlinear responsivities within this dynamic
range, which can be determined with test measurements.
NOTE 2 The limits of linearity can be determined statistically.
Most instruments perform relative measurements of optical signals. The output values, therefore,
depend on the instrument unless a reference material is available to establish a calibration curve. Only
when the instruments are absolutely calibrated in radiometric values including the number of photons,
can the measured optical signal values be expressed as absolute radiometric quantities.
Background signals can affect optical signal measurement results. Typical sources of background
signals are electrical noises (e.g. dark count and read-out noise) and optical noises (e.g. stray light and
external light).
Background signals can exist even in the absence of optical signals. Background signals are
automatically or manually subtracted after optical signal measurement.
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ISO 24421:2023(E)
4.3 Optical references
Optical references can be used to confirm the performance of optical signal measurement instruments,
including repeatability, intermediate precision, reproducibility, dynamic range and other related
instrument performance.
Consistency of optical characteristics between the optical reference and the biological sample increases
confidence in instrument performance and the analysis and assay results by measuring the optical
signals of biological samples.
NOTE Examples for optical references are an LED-based reference light source (see Annex D for an exemplary
luminometer qualification with LED), laser, slide of fluorescent glass, fluorescent dye in solution or other matrix
(e.g. fluorescent bead), slide embedded fluorescent material, reference filter, reference cuvette, reference film,
reference solution and power meter (see Annex B).
Optical references can be used for installation qualification, operational qualification and performance
qualification. Optical references can also be used to compare photodetector responsivity between
instruments.
Optical references can be used to calibrate optical signals to amounts or relative amounts, or potencies
of target biological samples.
5 Minimum requirements to support optical signal measurement
5.1 Elements of photometric methods
Standardized approaches should be followed to provide accurate analysis and assay results by
measuring the optical signal in photometric methods for analysis of biological samples.
Instruments, reagents, biological samples including cells, and other experimental materials used for
optical signal measurement in the photometric methods shall be selected for the intended purpose and
procedures. Reagents and biological samples shall be properly stored and maintained.
NOTE 1 Stability of reagents and biological samples can change during long-term storage and maintenance.
NOTE 2 In cells expressing reporter gene(s), including bioluminescence, chemiluminescence, fluorescence or
colorimetric reporter gene, expression level of the reporter gene(s) can change during storage and subculturing.
The copy number of the reporter gene(s) can also change du
...
DRAFT INTERNATIONAL STANDARD
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Voting begins on: Voting terminates on:
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Biotechnology — Minimum requirements for optical signal
measurements in photometric methods for biological
samples
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ISO/DIS 24421:2022(E)
DRAFT INTERNATIONAL STANDARD
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ISO/TC 276 Secretariat: DIN
Voting begins on: Voting terminates on:
Biotechnology — Minimum requirements for optical signal
measurements in photometric methods for biological
samples
ICS: 07.080
COPYRIGHT PROTECTED DOCUMENT
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© ISO 2022
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PROVIDE SUPPORTING DOCUMENTATION. © ISO 2022
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ISO/DIS 24421:2022(E)
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 V erification 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
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ISO/DIS 24421:2022(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 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
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 276, Biotechnology.
iv
© ISO 2022 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/DIS 24421:2022(E)
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 field 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. This document 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 common measurement scale
within a laboratory, between manufacturer and manufacturer, manufacturer and user, or user and user.
v
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DRAFT INTERNATIONAL STANDARD ISO/DIS 24421:2022(E)
Biotechnology — Minimum requirements for optical signal
measurements in photometric methods for biological
samples
1 Scope
This document provides minimum requirements to support accurate measurement of optical signals in
photometric methods used for qualitative or quantitative characterization of biological samples.
This document includes optical signal that is generated, for example, by bioluminescence,
chemiluminescence, fluorescence, and optical signal that is 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 terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at https:// 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 ‘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: JCGM 200:2012, 2.13, modified — Note 4 has been added.]
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3.2
biological sample
material or object of biological origin
3.3
dynamic range
range of optical signal (3.7) values that can be measured quantitatively
[SOURCE: ISO 2041:2018, 3.4.17, modified — Optical signal and quantitatively have been added for
clarification.]
3.4
intensity
strength of an optical signal (3.7)
Note 1 to entry: This term is used as a general term for the strength of an optical signal. It can be used to express
absolute strength or relative strength of an optical signal. An appropriate unit needs to be used in order to
express the intensity of a particular optical signal.
3.5
light source
optical device emitting an appropriate wavelength(s) in a specified spectral region
Note 1 to entry: A light source can be a part of an optical signal measurement instrument.
[SOURCE: ISO 25178-604:2013, 2.3.1 modified — Range of wavelengths has been changed to
wavelength(s) and Note 1 has been added.]
3.6
optical reference
material, light source (3.5), or photodetector, sufficiently reproducible and stable with respect to optical
properties, that has been established to be fit for its intended use
EXAMPLE LED-based reference light source, laser, slide of fluorescent glass, fluorescent dye in solution or
other matrix (e.g., fluorescent bead), slide embedded fluorescent material, reference filter, reference cuvette,
reference film, reference solution, and power meter (3.10) (see Annex B).
Note 1 to entry: Optical reference includes both uncalibrated reference and calibrated standard. Optical reference
can be distributed by an internal organization or prepared by a laboratory (e.g., in-house standard, in-house
reference material).
Note 2 to entry: Optical reference can be used for verification of optical signal measurement instruments (see
Annex D, E, G, H, I, and J).
3.7
optical signal
light with various wavelengths emitted or changes of light due to absorption caused by transmitting
light through samples or chromogenic substances
Note 1 to entry: The optical signal measurement involves, for example, bioluminescence, chemiluminescence,
fluorescence, and absorption measurements. Annex A gives information about optical signals.
Note 2 to entry: Optical signal, in this document, focuses on light before detection.
3.8
photometric method
analytical technique using optical signal (3.7) measurement(s) to determine components or biological
parameters of biological samples (3.2)
Note 1 to entry: Photometric method includes preanalytical, optical signal measurement, and data analysis
procedures.
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Note 2 to entry: Biological parameters of biological samples include, for example, cellular and metabolic activities,
and gene expressions.
Note 3 to entry: Examples for representative photometric methods are shown in Annex B.
Note 4 to entry: Analysis and assay results of photometric methods can be expressed qualitatively or
quantitatively.
Note 5 to entry: The term “radiometric” is widely used instead of “photometric” in the field of optical engineering
(e.g., IEC 60050-845).
3.9
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 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 5725-3:1994).
Note 3 to entry: ‘Measurement precision is used to define measurement repeatability, intermediate measurement
precision, and measurement reproducibility.
Note 4 to entry: Sometimes “measurement precision” is erroneously used to mean measurement accuracy.
Note 5 to entry: ISO 5725-1:1994 uses a different definition for precision.
[SOURCE: JCGM 200:2012, 2.15, modified —Note 5 has been added.]
3.10
power meter
optical power meter
measurement device to determine radiant power of light used as an optical reference (3.6)
Note 1 to entry: Power meter is a standard that is calibrated with responsivity value, including excitation laser
power.
Note 2 to entry: The watt (symbol: W) is used as a unit to express radiant power.
Note 3 to entry: Power meter is sometimes referred to as optical power meter.
3.11
reference light source
optical reference (3.6) used as light source (3.5)
Note 1 to entry: Optical reference includes LED reference light source and laser.
Note 2 to entry: In order to be used as an optical reference, reference light source is confirmed to emit stable and
reproducible power of light.
3.12
reference material for calibration curve
reference material with known value of concentration or amount for constructing a calibration curve
Note 1 to entry: Reference material for calibration curve is identical to or commutable with measurement object
of biological sample.
Note 2 to entry: Examples for expressing concentration and amount are mol/l and mol, respectively.
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3.13
validation
confirmation, through the provision of objective evidence, that the requirements for a specific intended
use or application have been fulfilled
Note 1 to entry: The objective evidence needed for a validation is the result of a test or other form of determination
such as performing alternative calculations or reviewing documents.
Note 2 to entry: The word “validated” is used to designate the corresponding status.
Note 3 to entry: The use conditions for validation can be real or simulated.
Note 4 to entry: ISO 16393:2019 uses the term validation in a different meaning in defining 'validation
experiment'. JCGM 200:2012 uses a different definition for validation.
[SOURCE: ISO 9000:2015, 3.8.13, modified — Note 4 has been added.]
3.14
verification
confirmation, through the provision of objective evidence, that specified requirements have been
fulfilled
Note 1 to entry: The objective evidence needed for a verification can be the result of an inspection or of other
forms of determination such as performing alternative calculations or reviewing documents.
Note 2 to entry: The activities carried out for verification are sometimes called a qualification process.
Note 3 to entry: The word “verified” is used to designate the corresponding status.
Note 4 to entry: JCGM 200:2012 uses a different definition for verification.
[SOURCE: ISO 9000:2015, 3.8.12, modified — Note 4 has been added.]
4 Principles
4.1 General
Optical signal measurements, including bioluminescence, chemiluminescence, fluorescence, and
absorption measurements, are used in photometric methods. Optical signal measurements are often
used for biological samples to determine a diverse set of biological parameters qualitatively and
quantitatively, including cellular and metabolic activities, and gene expressions (see Annex A for more
information). In the photometric methods, the optical signal intensity and spectrum from biological
samples are measured using instruments.
NOTE 1 Examples of instruments are luminometers, imaging analyzers, fluorescence plate readers, flow
cytometers, microarray readers, spectrofluorometers, plate readers, spectrophotometers, and DNA sequencers
(Annex B).
Accuracy, precision, repeatability, and reproducibility represent some of the important metrological
factors used for evaluating the effectiveness of photometric method applied.
Photometric methods can be qualitatively validated using positive and negative control materials.
NOTE 2 The performance characteristics of qualitative photometric methods and their validation can be
determined with appropriate statistical models depending on the method, structure of data and statistical
experience (e.g., ISO/TS 16393).
Accurate analysis and assay results are obtained by measuring optical signal with appropriate selection
of experimental materials, including the reagents generating the optical signal from the sample, and the
use of suitable instruments for the intended purpose.
Sample preparation is also an important factor governing the performance of a photometric method.
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Optical signal measurements produce relative and absolute optical signal values that are functionally
related to the quantity of specific characteristics of biological samples or biological parameters. In
spectral-resolved measurements, spectral characteristics are indicative for the interaction of particular
molecules, structural elements of molecules, or molecular interaction with electromagnetic radiation of
different energy.
In some cases, calibration curves constructed using a “reference material for calibration curve” are
required for quantification of the absolute amount of biological sample. A calibration curve can be used
also to determine an effective amount of a test article (e.g., an amount that elicits 50 % response across
the calibration curve or ED ).
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NOTE 3 Annex H gives an example for the construction of a calibration curve.
For measurement of biological samples, it is sometimes necessary to label or stain biological samples,
introduce a reporter gene into cells, tissues, and whole organisms, or trigger chemical reactions.
NOTE 4 Reagent quality and its photophysical and chemical properties affects optical signals from the sample.
Activity of cells can sometimes affect optical signal.
NOTE 5 Ambient light radiation can cause deterioration of bioluminescent reagents, chemiluminescent
reagents, fluorescent materials, and fading absorption.
When cells are used in photometric methods, the robustness of analysis and assay results is less
reliable if cellular activity is unstable. In particular, optical signal measurement results are directly
affected by the stability of cellular activity during long-term storage/subculturing and by the stability
of responsiveness to the target bioactive substance. The incident measuring light can also affect cellular
functions and properties, in particular if the cells are exposed to the light for a long period. Accordingly,
the reliability of optical signal measurement results can be increased by maintaining cell stability.
NOTE 5 Examples are assays to evaluate cellular activity, including viability, toxicity, and metabolic activity
by means of cell-based assays.
NOTE 6 Relevant standards that describe representative methods by means of optical signal measurements
are listed in Annex C.
Preanalytical procedures applied before performing optical signal measurements, including cell lysis,
antigen-antibody reaction, dye labelling or staining, can affect analysis and assay results.
4.2 Instruments and measurements
Photodetectors, including photomultipliers, photodiodes, and image sensors, have specific spectral
responsivities. Optical signals, including bioluminescence, chemiluminescence, fluorescence, and
absorption, can be measured accurately by using spectrally suited photodetectors and color filters.
NOTE 1 Annex K gives examples for spectral responsivity data of photodetectors.
The optical signal measurement instruments are affected by environmental conditions, including
laboratory temperature, and exposure to direct sunlight. Adjustment of the spatial resolution of an
instrument can be required depending on the application.
Optical signals can be measured quantitatively when the signal intensity is within the dynamic range of
the photodetector. Photodetectors have specific linear or non-linear responsivities within this dynamic
range, which can be determined with test measurements.
NOTE 2 The limits of linearity can be determined statistically.
Most instruments perform relative measurements of optical signals. The output values, therefore,
depend on the instrument unless a reference material is available to establish a calibration curve. Only
when the instruments are absolutely calibrated in radiometric values including the number of photons,
the measured optical signal values can be expressed as absolute radiometric quantities.
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Background signal can affect optical signal measurement results. Typical sources of background signal
are electrical noises (e.g., dark count and read-out noise) and optical noises (e.g., stray light and external
light).
Background signal can exist even in the absence of optical signal. Background signal is automatically or
manually subtracted after optical signal measurement.
4.3 Optical references
Optical references can be used to confirm performance of optical signal measurement instrument,
including repeatability, intermediate precision, reproducibility, dynamic range, and other related
instrument performance.
Consistency of optical characteristics between optical reference and biological sample incre
...
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