2021 CombiStats Session1 05OCT2021

THE EUROPEAN
DIRECTORATE FOR THE

QUALITY OF MEDICINES
& HEALTHCARE
(EDQM)
A Beginner’s Guide to CombiStatsTM
Webinar 1
05 October 2021
2 © EDQM, Council of Europe, 2021. All rights reserved.

Table of Contents
Introduction
Data Entry & Experimental Design
Analysis & Interpretation of Results
Combination of Assay Results
Files Management

Introduction

The CombiStats Software
Statistical analysis of data of biological dilution assays in accordance with Ph. Eur.
Chapter 5.3
• Developed and validated by A. Daas (EDQM statistician)
• Validation: numerical examples (Ph. Eur., D.J. Finney)
• User Manual: detailed presentation of CombiStats
First release in 1999 to OMCLs, in 2005 to non-OMCLs

• About 750 licences per year in more than 60 countries
Agreed reference in its domain, contributing to mutual recognition of statistical

results by all interested parties

Bioassays Overview
Bioassay An experiment that measures the biological response to a given stimulus
The observed response leads to an immediate

Qualitative qualitative conclusion, usually involving no calculation Example: urine pregnancy test
The observed response leads to a numerical

Quantitative assessment of some property of the material tested
Measures the dose needed to produce

Direct assay a given response, usually involving Example: stop heartbeat is rats.
simple calculations
Measures the response obtained Example: in-vivo, in-vitro

Indirect assay for doses applied at given levels assays with a calibration line
(so called x-point dilution
assay)
CombiStats

Indirect Assays - Example
A guinea-pic antiserum is assayed against a standard serum Standard S

Preparation to be
(0.4 IU/mL) using an enzyme-linked immunosorbent assay examined T
technique (ELISA). Dil. Obs.1 Obs.2 Dil. Obs.1 Obs.2
1/10 2.912 2.917 1/2.5 2.914 2.921
1/20 2.579 2.654 1/5 2.586 2.662

10 two-fold dilutions of each serum were applied on a 96-
well ELISA plate. Each dilution was applied twice. 1/40 2.130 2.212 1/10 2.133 2.220
1/80 1.651 1.638 1/20 1.654 1.640
1/160 1.073 0.973 1/40 1.078 0.974
1/320 0.585 0.666 1/80 0.587 0.674
1/640 0.463 0.356 1/160 0.465 0.361

What is the potency of the guinea-pic antiserum (T)? 1/1280 0.266 0.234 1/320 0.268 0.238
1/2560 0.228 0.197 1/640 0.232 0.200
What are the confidence limits of estimated potency? 1/5120 0.176 0.215 1/1280 0.183 0.222

Indirect Dilution Assays
Several preparations
Ref. preparation Test preparation(s)

Known concentration Conc. to be determined Relative
Potency
(RP)
Ref. prep.: internal standard (IS), Test prep.: candidate IS, Test Preparation
certified reference material (CRM), CRM or BRP,
manufactured batches, etc. - Relative Potency (RP) ~ 1/4
biological reference preparation (BRP),
etc. - Potency ~ 0.1 IU/ml

Regression models
Common structure in CombiStats
• X = Several doses for one or more preparations

Y = f (X)
• Y = Single or repeated measurements
Quantal response Quantitative response

Y = Proportion of responder Y = continuous/discrete data
E.g. in-vivo & in-vitro assay E.g. ELISA (absorbance)
Raw data: pos./neg. Aggregated

Binary Proportions

Data Entry

Ribbon
File Menu Window Menu

Create new data sheet Sheets in cascade,
Open existing template Edit Menu horizontal, vertical
Open data sheet Save, Print
Undo/Redo Tools Menu Examples
Copy selection Wizard (options) Manual
Copy data sheet Calculate Web Page
Paste selection Graph
Protect sheet Combine assays

Size of Tables

Orientation of Tables
Doses vertical
Doses horizontal

Table Layout
Required for calculation
Header
[Optional]
[Sample information]
Assigned or assumed potency
[Pre-dilution steps]
Doses

Results
Assigned and Estimated Potency Values
Purpose of the assay: estimate the potency of unknown sample(s), knowing the
potency of a standard
• Potency of Sample X calculated vs. BRP1
Unknown potency value
Estimated as 34 IU/ml
against BRP1
Known Double-click on
header to change
potency status from Sample
value to Standard
• Potency of Sample X calculated vs. In-house
Unknown potency value

Estimated as 31 IU/ml
against In-house

Double-Clicks…
Header: status
from Sample Cell: exclude or
to Standard include 1 result
Doses: exclude Dose: exclude

or include all or include a row
results of results
Remarks box: Add reason

for missing or excluded data

Doses: Explicit Notation
These notations are all equivalent

X-fold dilution Volume (mL) Content (IU)
Log-notations
-1log10 = 1/10
-2log10 = 1/100
1/10 of 0.1 mL of 0.04 IU
EQ. EQ. -3log10 = 1/1000
0.4 IU/mL 0.4 IU/mL in 1st well
1/1 = undiluted

Doses: Explicit Notation with Pre-dilution
These notations are NOT all equivalent

X-fold dilution Volume (mL) Content (IU) CombiStats converts any pre-
dilution and dose notation
Into a final content
If dose notation = final content,

then any pre-dilution
is ignored
Preferred notation for explicit

notation: X-fold dilution
1/10 of a half diluted solution 0.04 IU
= 0.02 IU in 1st well in 1st well

Doses: Explicit Notation with unknown Ass. pot.
Assigned potency of Standard known (required) Doses in IU with unknown Ass.pot.
Potency of Sample may be unknown
X-fold dilution Volume (mL) Content (IU) Calculation cannot be done
0.02 IU = 0.1 mL 0.1 mL ? mL

in 1st well in 1st well in 1st well Preferred notation for explicit
notation: X-fold dilution

Doses: Symbolic Notation
These notations are all equivalent

Explicit Symbolic
If
Increasing
Dose order & Dilution

1/10 of
EQ.
10-fold pre-dil.
EQ.
0.04 IU step must be specified
0.4 IU/mL of 0.4 IU/mL in 1st well
Applicable to equally
Symbolic notations are just labels distributed doses only
(Well 1 = dose A = low = …)
(Decreasing): first well = 1/1
(Increasing): last well = 1/1

Notation of Pre-Dilution Steps
Pre-dilutions must be entered in
a logical order…
1000 IU / vial
1 vial in 0.5 ml
1/10
0.5 ml in 10 ml
1.0 ml in 20 ml 1/20
0.1 ml in well 1/10

1 in 2 in 1st well 1/2
Units before the slash 1000 IU / (10×20×10×2)

must be the same as = 0.25 IU in first well
after the slash of
the previous step

Notation of Pre-Dilution Steps
Check: Final doses on the regression plot
3rd 2nd 1st

well well well

Experimental Design

Experimental Design
For reliable results & conclusion: identification and control of confounders

(e.g. cage, Petri dish, operator, instruments).
The design is defined and agreed before the experimental

phase (per protocol). It may be adapted, depending on
unexpected events occurring at the bench (operator’s
lab notebook).
Completely randomised No experimental factor effect
Randomised block
Possible experimental factor
(Latin) square effect

Completely Randomised Design
Example
Protocol: A test preparation is to be compared to a BRP using a multiple
dilution assay (3 doses x 2 rep. per prep.)
The 12 treatments can be carried out by 1 operator in 1 session,
using the same material, buffer solution and batch of each reagent.
Treatments to be carried out in random order.
Lab notebook: No deviation from protocol
=> Treatments can be compared all things otherwise being equal.

Randomised Bock Design (1)
Example: 3 doses of a Standard and a Test are applied in 6 replicates in 6 petri-dishes. Each dish
can accommodate 6 treatments.
Problem: Solution 1: Solution 2:

Effect of treatment cannot be Each treatment appears exactly The treatments are balanced
distinguished from the effect of once in each dish. Each dish can over all absolute positions (once
the dish. The effects are said to be regarded as a mini-assay. in each position) and relative
be confounded with each other. They are experimental blocks. positions (not always next to the
same treatment).
But, positions always the same.
Treatments positioned randomly within the blocks: randomised block design
Even with all positions randomized we must not neglect the effect of
order of administration:
• Were the treatments S1 added to all dishes before going to S2?
• Were all 6 treatments in dish 1 administered before going to dish 2?
• If so, in which order (clockwise, counter-clockwise, low to high dose?)
In practice, not all possible confounders may be important enough

to justify a complex design.
Practical considerations may outweigh the importance of (small) confounders, e.g. increased risk of
mistakes, time constraints, etc.
In general, a good compromise between theoretical and practical considerations has to be found.
In CombiStats we call designs in solutions 1 and 2 randomised block design, even if the treatments
are not truly randomised within the blocks
CombiStats assumes that the blocks

are identified by the number
between brackets.
All responses with the same number
are assumed to belong to the same
block, across all preparations

What to do when blocks are too small to accommodate all treatments?

Example of a balanced incomplete block design:
1 Standard and 2 Tests
Each tested at 3 doses
Each dose in 6 replicates
Dishes can only have 6 treatments
Each block
has its own
number.
The blocks are
incomplete,
but the design
is balanced

x|y|z: x is number of the table

y is number of the dose
z is number of the replicate The colors identify the same number in Observ.
preparations => same block

What About Latin Square Design (1)
A design aiming to control two possible factors that could influence the response
of specific groups of units in the same way
Typical case: antibiotic agar diffusion assay (Ph.Eur 5.1.2)
Agar Gradient of heterogeneity
plate
Antibiotic conc. in agar plate may
Gradient of heterogeneity
not be uniform…
Antibiotic
Latin square design allows controlling
concentration possible heterogeneity in two directions
(rows & columns), how?
 Each treatment occurs only once

per row and per column

What About Latin Square Design (2)
Antibiotic agar diffusion assay (Ph. Eur. 5.1.2)
Std. and Test prep. (S & T)
3 doses per prep.
Agar Gradient of heterogeneity
plate
S1 T1 T2 S3 S2 T3 Each treatment
Gradient of heterogeneity
T1 T3 S1 S2 T2 S3
(e.g. T2) occurs
once per row and
T2 S3 S2 S1 T3 T1 per column
S3 S2 T3 T1 S1 T2
S2 T2 S3 T3 T1 S1 The mean result

of each treatment
T3 S1 T1 T2 S3 S2 x|y: x is number of the table
shows limited bias
y is number of the dose
number of the replicate defined by
number in brackets

Further use of « Show design » option
Elisa 96-well plate: BRP and 3 samples in duplicate
Design: completely randomised

Negative
Positive
Number in brackets:
no particular meaning
Each sample different colour
Facilitated data entry

Statistical Analysis &
Interpretation of Results

Steps of a Statistical Analysis
Review of Raw Data
Fit Regression Model
Check FAIL Refine Model

Validity E.g. data transf.
PASS
Interpret Results
Summary tables & plots

Raw Data Review
Any problem to escalate?
Purpose
• Check/correct any typo error
• Detect outliers, pattern in
missing data
How
• Typo error & outliers: data table
and regression plot scrutiny

Statistical Analysis
Review of Raw Data

PASS
Interpret Results

Regression models
Common structure in CombiStats
• X = Several doses for one or more preparations

Y = f (X)
• Y = Single or repeated measurements
Quantal response Quantitative response

Y = Proportion of responder Y = continuous/discrete data
E.g. in-vivo & in-vitro assay E.g. ELISA (absorbance)
Raw data: pos./neg. Aggregated

Binary Proportions

Model Selection (1)
Binary Quantitative
or
Resp. Y
Proportions
Y linearly related Y ~ Ln(Dose)
to Dose (Y ~ Dose) (log scale on x-axis)
Y = f(X)
Linear Range + Asymptotes Linear Range

(6 to 12 dil. points) (3 to 5 dil. points)
Dil. pts
From assay development

to routine activities
Quantal Slope Sigmoid Parallel

Analysis Ratio Curves Lines
Spl Spl Spl Spl
Ref
Ref Ref Ref

Model Selection (2)
Slope Parallel Sigmoid Quantal
Ratio Lines Curves Analysis

Model Selection (3)
Wizard: options to select Don’t forget the
appropriate statistical model experimental design…
Run Statistical analysis

Review of Raw Data

PASS
Interpret Results

Model Validity Criteria – Model Assumption
Model assumption to verify for parallel line, sigmoid curve and slope ratio:
Data are normally distributed with constant variance
Criterion met

Model Validity Criteria – Model Assumption
Model assumption to verify for parallel line, sigmoid curve and slope ratio:
Data are normally distributed with constant variance
Criterion not met

Model Validity Criteria – Dose-Response Relationship
Slope Parallel Sigmoid

Ratio Lines Curves
Quantal
Analysis
Regression – Linearity – Intersection Regression – Linearity – Parallelism

Model Validity Criteria - ANOVA
Analysis of variance (ANOVA) table to check the validity criteria
CombiStats labels the Probabilities (p-values)

with stars according to the level of significance
p-value ≥ 0.05 No stars

p-value < 0.05 One star (*)
p-value < 0.01 Two stars (**)
p-value < 0.001 Three stars (***)
Total observed variation is split to difference sources of variation.
The variability for source of interest (e.g. Regression) is compared to the experimental variability
resulting from replications of assays (Residual Error).
Example Source of variation « Regression », probability of common slope being 0 is lower than
0.001. Conclusion, slope significantly different from 0.

Model Validity Criteria – Dose-Response Relationship
Parallel line assays, sigmoid curve and assays with quantal response:
• The p-value for regression is significant (at least one star).
• The p-value for non-parallelism is not significant (no stars).
• The p-value for non-linearity is not significant (no stars).
Slope ratio assays:

• The p-value for regression is significant (at least one star).
• The p-value for intersection is not significant (no stars).
• The p-value for non-linearity is not significant (no stars).
For linearity criterion, assay requires at least 3 dilutions of each preparation

Sigmoid curves and quantal responses: data transformation is applied prior to testing the validity criteria
Review of Raw Data

PASS
Interpret Results

Interpretation of Results
Calculator: run calculations and display tables of results
Output Input
Potency: 5456 IU/mg with 95% confidence limits [5093-5843]
Rel. to Ass.: relative to initial assumed potency 5600 IU/mg

5456 5092 5843
∗ 100 = 97.4% , 95% confidence limits ∗ 100 = 90.9% and ∗ 100 = 104.3%
5600 5600 5600
Rel. to Est.: relative to estimated potency 5456 IU/mg

5456 5092 5843
∗ 100 = 100.0% , 95% confidence limits ∗ 100 = 93.3% and ∗ 100 = 107.1%
5456 5456 5456

Regression Models
for Quantitative Response

Sigmoid Curves Model
Applicable to
• Quantitative results (y-axis)
• Doses on log-scale (x-axis)

RP
• Clear lower & upper plateaus
(min. of 6 dil.-points needed)
• Typical example: ELISA
4-parameter logistic regression model

• a and d as lower and upper asymptotes 𝑑𝑑 − 𝑎𝑎
𝑦𝑦 = 𝑎𝑎 + 𝑏𝑏 + 𝑒𝑒
• b and c as slope and inflection point 𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷
1+
𝑐𝑐
• e as experimental/residual error

Parallel Lines Model
Applicable to RP =(cTest - cRef) / b
• 3 to 5 dilution points usually RP
Linear regression model
• b and c as slope and intercept
bRef = bTest = b (common slope) 𝑦𝑦 = 𝑐𝑐 + 𝑏𝑏 � 𝑙𝑙𝑙𝑙 𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 + 𝑒𝑒

Sigmoid and Parallel Lines Models
Parallel lines as a special case of Sigmoid curves
• Limited to linear ranges

of sigmoid curves
• 96-well plate: from 8-12
dilution points to 4
(double capacity…)
• Linearity and parallelism
as validity criteria for
both models

Slope Ratio Model
Applicable to RP = bTest / bRef
• Dose units on x-axis

(arithmetic, additive scale)
• 3 to 5 dilution points usually
Linear regression model

• b and c as slope and intercept
cTest = cRef = c 𝑦𝑦 = 𝑐𝑐 + 𝑏𝑏 � 𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 + 𝑒𝑒

Regression Models
for Quantal Response

Model for Quantal Responses
Quantal Response
Applicable to
Binary data: +, – or 0,1
• Proportions of responder (y-axis)
• Clear lower & upper plateau (no response, 100% response)
• Typical example: in-vivo assay

Proportions
Regression analysis
RP
• After transformation
• Several transformation exist
• Probit transformation (default)

Model for Quantal Responses - Transformations
Five transformations of proportions available
• Probit (default) and Logit as most common
• Other: Gompertz (seldom used), Angular, Rectangular

EDXX Determination One Single Preparation
Applicable to
• Proportions of responder (y-axis)
• Clear lower & upper plateau (no response, 100%

response)
Analysis Effective Dose 50 (ED50):

• Same as Quantal Responses dose giving 50% response
• EDXX: estimate the amount of dose that induces
XX% of response

ED50 and Relative Potency
ED50 (T)
RP =
ED50 (S)
1 vial of Standard contains 56 doses

to introduce a response at 50%
𝟔𝟔𝟔𝟔.𝟏𝟏𝟏𝟏
RP = = 1.234 Potency (T) = RP x Potency (S)
𝟓𝟓𝟓𝟓.𝟎𝟎𝟎𝟎
ED50
= 1.234 x 132 = 162.86 Values

ED50 for Quantitative Assays
1 mL of Standard contains 74.2 doses to introduce a response at 50%
50% of response are introduced by 0.005 IU of Standard and by 0.0018 IU of Sample 1 (Ass.pot.)

ED50 for Quantitative Assays
50% of response are introduced by 0.005 IU of Standard and by 0.0018 IU of Sample 1
BUT if unknown potency for sample 1 ? IU/ml

then output given in ml, not IU for Sample 1
Dose in ml
0.10
0.05
0.025
0.0125
0.00625
0.003125
0.0015625
0.00078125
0.000390625
0.000195313

Data Transformation

Data Transformations
x-axis
• No transformation: doses on linear scale (slope ratio)
• Logarithms: doses on log-linear scale (sigmoid curves, PLA)
y-axis
• Model dependent transformation
Models: quantal response, EDXX determination and sigmoid curve
Purpose: linearization of sigmoid dose-response relationship
Example: probit default for quantal response, logit for sigmoid curve
• Measurement dependent transformation

Models: parallel line and slope ratio
Purpose: improve linearity and parallelism, stable variance over doses
Example: logarithm, square root

Data Transformations – Example 5.1.4. from Ph.Eur.
no transformation Log-transformed
y --> ln(y)
Data are Data are

normally normally
distributed distributed
with constant with constant
variance: variance:
not met met
Parallel line model cannot be applied Parallel line model can be applied

Combination of Assay
Results

Why
One estimate from several independent assays needed, i.e. to fulfil the requirements of Ph.Eur.
How
Open all assays and click on melting pot button
Results
CombiStats calculates three types of combinations: Unweighted combination, Weighted

Combination and Semi-Weighted Combination

Combination of Assay Results - Example
Three valid Erythromycin assays

To select one of the approaches
answer following questions
• Were assays performed independently?
Independent assays: Different runs: different
operators, days, working solutions, ...
• Are the assay results homogeneous?

Homogeneous results: Limited difference
between results of different runs (i.e. low inter-run
variability)
Formal test is performed (p-value = probability)
If p-value ≤ 0.10 (10%), then homogeneity is rejected

Flow Chart K assays
NO
E.g. linearity, parallelism Valid Remove assay(s)
E.g. different operators, NO

days, working solutions Independent Unweighted
Steep regression line NO

(parameter C in-between 1 Significant
and 1.1)
NO
Min. of 6 degrees of freedom
for the residual of each assay Precise
YES NO
Weighted Homogeneous Semi-Weighted

File Management

Quality Assurance (QA)
QA Controls: Version, date & time [time-zone] at printing, page X of Y
• Time-zone synchronization under user’s responsibility (IT-management)
• Same date & time on each page
Remarks box: important notes for later reference

• E.g. problems during assay, unusual observations, reason for missing or excluded data

QA Controls: license holder, optional: file name & size, hash code
• Repeated on each page, shown on printed version, not on screen
• Define the display of the file name & size, hash code in preferences
Print filename:
-1: full path & name
0: no print
1: file name
>1: file name + folders

Hash code
• “fingerprint” of the file
• Any modification to the file will result in a different hash code
• Allows matching printed version with electronic version (source file)
• Making changes and printing a QA controlled version without saving triggers a warning
message and ad-hoc footer
• Two files with same hash code are identical
• Visible on printed version only but can be checked

from Menu > File > Hash REPEMD-160

Title 21 CFR Part 11 and EU GMP Annex 11
Names are not intended as digital signatures (no legal status)
• Printed copy should be signed with hand-written signatures
• Acrobat Distiller may be used to add digital signatures to CombiStats file
• The link between the PDF version, the paper version, and the epa-file is
made by the le name, the le size and the hash code printed on each page.
Use hash code as Use hash code as

check for data integrity check for data integrity
Apply public algorithm RIPEMD-160 to produce hash

Creation of templates
Demo

Protection
Templates and data-sheets can be protected from accidental editing
CombiStats logo as black,

Logo blue, green and red for
Level 1 to Level 4
Password is optional

Thank you for your attention
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THE EUROPEAN

DIRECTORATE FOR THE

2 © EDQM, Council of Europe, 2021. All rights reserved.

Data Entry & Experimental Design

Analysis & Interpretation of Results

Combination of Assay Results

3 © EDQM, Council of Europe, 2021. All rights reserved.

4 © EDQM, Council of Europe, 2021. All rights reserved.

• Validation: numerical examples (Ph. Eur., D.J. Finney)

• User Manual: detailed presentation of CombiStats

First release in 1999 to OMCLs, in 2005 to non-OMCLs

Agreed reference in its domain, contributing to mutual recognition of statistical

5 © EDQM, Council of Europe, 2021. All rights reserved.

Bioassay An experiment that measures the biological response to a given stimulus

The observed response leads to an immediate

The observed response leads to a numerical

Measures the dose needed to produce

Measures the response obtained Example: in-vivo, in-vitro

6 © EDQM, Council of Europe, 2021. All rights reserved.

A guinea-pic antiserum is assayed against a standard serum Standard S

technique (ELISA). Dil. Obs.1 Obs.2 Dil. Obs.1 Obs.2

1/10 2.912 2.917 1/2.5 2.914 2.921

1/20 2.579 2.654 1/5 2.586 2.662

1/80 1.651 1.638 1/20 1.654 1.640

1/160 1.073 0.973 1/40 1.078 0.974

1/320 0.585 0.666 1/80 0.587 0.674

1/640 0.463 0.356 1/160 0.465 0.361

1/2560 0.228 0.197 1/640 0.232 0.200

7 © EDQM, Council of Europe, 2021. All rights reserved.

Ref. preparation Test preparation(s)

8 © EDQM, Council of Europe, 2021. All rights reserved.

• X = Several doses for one or more preparations

Quantal response Quantitative response

Raw data: pos./neg. Aggregated

9 © EDQM, Council of Europe, 2021. All rights reserved.

10 © EDQM, Council of Europe, 2021. All rights reserved.

File Menu Window Menu

11 © EDQM, Council of Europe, 2021. All rights reserved.

12 © EDQM, Council of Europe, 2021. All rights reserved.

13 © EDQM, Council of Europe, 2021. All rights reserved.

Assigned or assumed potency

14 © EDQM, Council of Europe, 2021. All rights reserved.

Unknown potency value

15 © EDQM, Council of Europe, 2021. All rights reserved.

Doses: exclude Dose: exclude

Remarks box: Add reason

16 © EDQM, Council of Europe, 2021. All rights reserved.

These notations are all equivalent

17 © EDQM, Council of Europe, 2021. All rights reserved.

These notations are NOT all equivalent

If dose notation = final content,

Preferred notation for explicit

18 © EDQM, Council of Europe, 2021. All rights reserved.

X-fold dilution Volume (mL) Content (IU) Calculation cannot be done

0.02 IU = 0.1 mL 0.1 mL ? mL

19 © EDQM, Council of Europe, 2021. All rights reserved.

These notations are all equivalent

Dose order & Dilution