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Immunodiagnostics
Centrifugation
Clinical Case Studies
Disease Management
Hematology
COULTER® GEN•S™ System Hemostasis
Enhanced VCS Technology Lab Automation
Data Management
Flow Cytometry
Primary Care
Bulletin 9165
COULTER GEN•S Clinical Case Study
IntelliKinetics™ Process
AccuGate™ Software
Reticulocyte Analysis
CASE STUDIES
Normal Blood
Left Shift
Virus Infection
Normochromic Anemia
Monocytosis
Beckman Coulter VCS Technology is the most powerful tool available for blood cell
Technology analysis. An acronym for Volume, Conductivity and Scatter, this proprietary
technology offers the greatest sensitivity, specificity and efficiency of any
cell analysis system available today.
VCS technology uses a specially constructed flow cytometer that has been
modified to provide more information on unstained cells than is possible
using light scatter alone. The system contains a quartz crystal flow cell
through which the Leukocytes are passed using hydrodynamic focusing to
ensure that they appear in front of the detection system one at a time.
(Diagram 1)
VCS is the only single channel analysis that uses three independent energy
sources to probe up to 8,192 cells within the flow cell. This combination of
volume, conductivity and light scatter allows the direct measurement of all
five normal Leukocyte classes.
(Diagrams 2 a,b,c)
HYDRODYNAMIC FOCUSING
Sheath Stream
Flow Cell
Cell Stream
Diagram 1
2
Volume
VCS utilizes the Coulter principle of electrical impedance to measure the
volume that the entire cell displaces in an isotonic diluent. This method
accurately sizes all cell types regardless of their orientation in the light
path. Because VCS technology uses such a highly accurate measure of
cell volume, this information can be used to correct the conductivity and
Diagram 2a
scatter signals to give a pair of measurements that are very powerful, and
unique to Coulter. (See diagram 2a)
Conductivity
Alternating current in the radiofrequency (RF) range passes through a cell’s
membrane, penetrating the cell. This powerful probe is used to collect
information about cell size and internal structure, including chemical
composition and nuclear volume. By correcting the conductivity signal so
that it is no longer influenced by cell size, we obtain a measurement that is
related only to the internal structure of the cell. This new measure, called
Diagram 2b
opacity, allows VCS technology to separate cells of similar size, but
different internal composition. It also allows the instrument to calculate
the Nuclear/cytoplasmic ratio - a feature useful in distinguishing variant
lymphocytes from normal lymphs. (See diagram 2b)
Scatter
Within the VCS system a focused elliptical light beam from a Helium-Neon
Laser is used to give information about cellular granularity, nuclear
lobularity and cell surface characteristics. Coulter eliminates the size
Diagram 2c
component of the light scatter signals to give a new measurement called
Rotated Light Scatter (RLS). In doing so we are able to determine the
optimum angle of scatter for each cell type and design the scatter detector
to cover this range (10-70 degrees). This allows VCS technology to
accurately separate what would normally be mixed cell types (such as
Neutrophils and Eosinophils) into distinct clusters without mathematical
manipulation. It also enhances the separation between the non granular
cell types. (See diagram 2c)
VCS-Cube
Results from each of the 8,000 analyzed
white cells, (or 32,000 red cells in the
Reticulocyte analysis) are assigned X,Y & Z
co-ordinates in a 3-dimensional array based
respectively on their RLS, volume and
opacity (Diagram 3). Each axis of the cube
Diagram 3
COULTER GEN•S Clinical Case Study
VCS On the GEN•S System, VCS technology has been enhanced by the addition
of two major modifications that allow even better differential results with an
improved level of flagging efficiency. These two new features (Intellikinetics™
IntelliKinetics™ AccuGate™
and AccuGate™) are the result of eight years’ experience with the VCS
Diagram 4 technology. Their addition allows the GEN•S System to provide reduced
levels of False Positive and False Negative differential flagging, thus saving
the laboratory from performing unnecessary film reviews. Enhanced VCS
technology can also improve differential results with respect to aged sample
stability, elimination of interferences and comparison to reference methods.
The two systems are also applied to the Reticulocyte analysis on the GEN•S
System, providing improved separation and reduced flagging levels in these
samples as well. (See Diagram 4)
IntelliKinetics™ Process The IntelliKinetics application is a hardware and software management tool
that has been developed to assist in the control of fluctuations within the
laboratory environment. Using the IntelliKinetics process, the GEN•S System
Volume
Volume
1 Scatter 2 Scatter
Volume
Volume
3 Scatter 4 Scatter
Diagram 5
4
monitors and reacts to variables such as the ambient temperature,
optimizing the instrument system to provide consistent reaction kinetics. This
is achieved using intelligent management of reagent reaction temperature,
exposure time, and reagent delivery volume which are controlled through
automatic system adjustments. The combination of this new application with
improved electronics provides a high quality data signal to the VCS probes
and to the analysis algorithms, resulting in cell populations that are in a
consistent location in multidimensional space.
The two examples demonstrate the benefits that the IntelliKinetics process
provides in terms of improved population separation. Plots 1 & 2 represent a
fresh blood sample with (Plot. 2) and without (Plot. 1) the use of reaction
control, Plots 3 & 4 repeat the experiment this time for a day old blood
sample. Plot 3 shows the scatterplot without the use of the IntelliKinetics
process, while Plot 4 has the reaction management added. (See Diagram 5)
AccuGate™ Software List Mode data collected from the VCS probes is analyzed by the AccuGate
software. This new computer algorithm uses advanced contour gating to
identify and classify the WBC sub-populations in a tailored, sample specific
way. AccuGate software contains adaptive statistical tools that assist in
delineating overlapping populations, such as clusters of variant lymphocytes
and monocytes, that are difficult to distinguish using linear gating applica-
tions. The algorithm is also capable of adapting to shifts in populations which
are often manifested when morphologic abnormalities are present.
The benefits of adaptive gating include new suspect flags and additional
“research only” parameters, increased precision and accuracy of results,
color enhancement of population density and cell category dataplots. White
cell differentials on the GEN•S System can be presented in multidimensional
formats; additionally an operator can hide or display any series of individual
cell populations within the display.
The six diagrams on the following page illustrate an example of white cell
differential analysis using AccuGate software. Although shown two
dimensionally, separation actually occurs in three dimensions, and we have
attempted to visualize how contour gating works using the bending line in the
DC (x-axis) vs. RLS (y-axis) two-dimensional views.
COULTER GEN•S Clinical Case Study
Diagram 1 Diagram 2
Diagram 3 Diagram 4
Diagram 5 Diagram 6
6
Reticulocyte Analysis The Reticulocyte analysis combines the established methodology of the New
Methylene Blue procedure with the standardized analysis and greater
precision of flow cytometry utilizing the VCS system. In doing so, it provides
high quality results without the need for fluorescent dyes and expensive
argon ion laser systems, and opens up to the user the potential for providing
improved reproducible data to their clinician customers.
The AccuGate algorithm system is also used for the analysis of reticulocyte
samples. Again, we have illustrated the separation two dimensionally although
separation actually occurs in three dimensions.
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DIAGNOSIS: MONOCYTOSIS
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