Operating Basics
Rev. 007 Date 2010-01-13
CyFlow
®
space Instrument Operating Manual
9/26
True Volumetric Absolute Counting
– Overview
Analysis of concentrations of samples, loosely
called Absolute Counting, can be of significant
interest for medical diagnostics or monitioring the
status of cells in cell cultures or biotechnological
processes.
Traditional 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 Hematology Counters.
Disadvantages:
Two instruments required
This technique relies on the assumption that
the number of cells seen by the FCM and
counted by the Hematology Counter are
identical.
ii) Single Platform Technique with Reference
Beads
An alternative method employes 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
Partec True Volumetric Absolute Counting
To overcome the drawbacks of the Dual- and
Single-Platform Technique with reference beads,
Partec intruments 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
.
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 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%.
ii) Electrode-Principle: Determination of
V
The method for True Volumetric Absolute
Counting supported by the CyFlow
®
space is
based on the precise measurement of a fixed
sample volume by means of two electrodes.
Fig. 1: True Volumetric Absolute Counting by the
Electrode Principle.
During analysis by the CyFlow
®
space, the
sample liquid loses contact first from the upper
and then from the lower electrode. These events
trigger a START and a STOP signal, respectively
which is stored by software.
The volume
V
of sample liquid analysed between
the START and STOP signal is physically defined
by the distance of the two electrodes and the
diameter of the sample tube. The analysed
volume is exactly 200 µl (= counting volume).
Effects of the sample meniscus in the tube are
eliminated because the START and STOP
electrodes are arranged symmetrically. The
sample conductivity does not influence the
volumetric measurement, as long as the fluids can
be detected electrically.
In some cases, larger or smaller counting volumes
V
are advantageous, either to improve the
counting statistics (e.g. for rare events < 10/ml) or
to decrease the analysis time. This can be
accomplished by replacing the standard 200 µl
volumetric sample port by one with a different
volume (Please contact Partec or your local
distributor for details).
c
=
N
/
V
,
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