CyFlow
™
Space | Operating Manual | March 2021
17
6 True Volumetric Absolute Counting – Overview
Analysis of concentrations of cells and particles, loosely called Absolute Counting, can be
of significant interest for various studies e.g. monitoring the number of cells in cell cultures
or biotechnological processes.
6.1 Non-volumetric Flow Cytometric Techniques for Absolute
Counting
i) Dual Platform Technique
To determine the concentration of a sample flow cytometers (FCM) are frequently used in
combination with Haematology Counters.
Disadvantages:
Two instruments required
This technique relies on the assumption that the number of cells seen by the FCM and
counted by the Haematology Counter are identical.
ii) Single Platform Technique with Reference Beads
An alternative method employs reference beads of known concentration, which are added
to the sample of unknown concentration.
Disadvantages:
Depends on the accuracy of the specified beads concentration
Constant running costs for the reference beads
6.2 True Volumetric Absolute Counting
To overcome the drawbacks of the Dual- and Single-Platform Technique with reference
beads, Sysmex Partec instruments additionally offer an alternative way of absolute cell
counting, which is based directly on the basic definition of a concentration by precisely
counting the number N of cells suspended in a purely mechanically defined volume V.
𝑐
𝑁
𝑉
i) Precise Counting: Determination of N
For True Volumetric Counting the precise detection of cells is an essential. This requires
fast recognition and analysis of the events by electronics and computer. All Sysmex Partec
instruments are specifically designed to minimize counting losses by providing direct
connection between computer and electronics, which avoids dead-times involved in
traditional FCM designs and instrument interfaces. This reduces the probability of a
counting loss for typical event rates below 2%.
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