General Chemistry

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Clinical Case Studies
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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

ENHANCED VCS TECHNOLOGY ON THE COULTER GEN•S SYSTEM

Coulter VCS Technology

IntelliKinetics™ Process

AccuGate™ Software

Reticulocyte Analysis

CASE STUDIES
Normal Blood

Left Shift

Severe Infection, Post Operative

Acute Myeloid Leukemia (M1) at Presentation

Acute Myeloid Leukemia (M4) in Relapse

Virus Infection

Chronic Lymphocytic Leukemia

Myelodysplastic Syndrome on Treatment

Normochromic Anemia

EDTA Induced Platelet Aggregation

Monocytosis

Eosinophilia (aged sample)

COULTER GEN•S Clinical Case Study

ENHANCED VCS TECHNOLOGY ON THE COULTER GEN•S SYSTEM

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.

The analysis begins with a properly prepared sample using a specialized

reagent system. Two reagents (Erythrolyse™ and Stabilyse™) are mixed
with the blood sample in an orbital mixing chamber to remove red blood
cells while leaving WBCs in an unaltered or “near native” state. First,
Erythrolyse reagent is used to lyse RBCs. Stabilyse reagent is then added
to stop the lytic reaction, leaving the Leukocytes ready for analysis.

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

ENHANCED VCS TECHNOLOGY ON THE COULTER GEN•S SYSTEM

is subdivided into 256 channels, giving over 16,700,000 possible data

locations within which cells with similar characteristics will form distinct
clusters. The operator may visualize the cube either in 3D or as a two
dimensional dataplot that shows Volume (Y axis) and Light Scatter (X axis).

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

ENHANCED VCS TECHNOLOGY ON THE COULTER GEN•S SYSTEM

Diagram 1 Diagram 2

Diagram 3 Diagram 4

Diagram 5 Diagram 6

1. First, Eosinophils are separated from all other populations.

2. Monocytes are identified as a separate population and
classified.
3. Neutrophils are separated from Lymphocytes and Basophils.
4. Basophils and Lymphocytes are classified.
5. Completed separation (2-dimensional).
6. Actual 3-dimensional display, illustrating color separation of each white
cell subtype.

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.

Within the analyzer, a small segment of the blood sample is incubated in a

heated chamber with a special new methylene blue solution, precipitating any
residual RNA within the erythrocytes. A portion of the stained blood sample is
then transferred to a second chamber together with a hypotonic clearing
solution that will remove erythrocyte hemoglobin but preserve the stained
RNA within the cell. Almost immediately, the sample is processed through the
VCS flow cell for analysis by the same three independent probes used for
differential analysis.

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.

Diagram 1: Diagram 2: Diagram 3:

Platelets are classified first. The mature red cell population is Once isolated, the level of Reticulocyte
distinguished from the immature maturity can be calculated.
reticulocytes.

Contour gating is of particular benefit in reticulocyte analysis because the

cell population under study is a gradual continuum of increasing maturity. The
non-linear separation techniques and multiresolution analysis used by the
AccuGate algorithm allow the GEN•S System to provide improved precision
and accuracy of results particularly in the presence of abnormal RBC types.
New parameters, such as Immature Reticulocyte Fraction (IRF) and Mean
Reticulocyte Volume (MRV), are now available. “Research only” parameters
can also be provided, including Mean Sphered Cell Volume (MSCV) and
High Light scatter Reticulocyte percent and absolute number (ALR%,
HLR absolute).
COULTER GEN•S Clinical Case Study

DIAGNOSIS: NORMAL BLOOD

GEN•S SYSTEM FLAGS

Suspect:
None
Definitive:
None
Manual Differential:
Neutrophilis 65%
Lymphocytes 24%
Monocytes 8%
Eosinophilis 2%
Basophilis 1%
RBC Morphology:
Normal

8
COULTER GEN•S Clinical Case Study

DIAGNOSIS: LEFT SHIFT

GEN•S SYSTEM FLAGS

Suspect:
Imm NE 1
Definitive:
None
Manual Differential:
Segmented Neutrophils 80%
Band Neutrophils 9%
Lymphocytes 6%
Monocytes 3%
Eosinophils 1%
Metamyelocytes 1%
RBC Morphology:
Normal

10
COULTER GEN•S Clinical Case Study

DIAGNOSIS: SEVERE INFECTION, POST OPERATIVE

GEN•S SYSTEM FLAGS

Suspect: Manual Differential:
NE Blasts Segmented Neutrophils 79%
Imm NE 1 Band Neutrophils 8%
Imm NE 2 Lymphocytes 4%
Monocytes 2%
Definitive:
Eosinophils 2%
Leukocytosis
Metamyelocytes 1%
Neutrophilia #
Myelocytes 4%
Eosinophilia #
Erythrocytosis RBC Morphology:
Anisocytosis 1+ Anisocytosis 1+
Platelet Anisocytosis

12
COULTER GEN•S Clinical Case Study

DIAGNOSIS: ACUTE MYELOID LEUKEMIA (M1) AT PRESENTATION

GEN•S SYSTEM FLAGS

Suspect: Manual Differential:
NE Blasts Blasts 95%
Imm NE 1 Lymphocytes 5%
Imm NE 2 RBC Morphology:
Definitive: Anisocytosis 1+
Leukocytosis
Neutrophilia #
Eosinophilia #
Anemia
Anisocytosis 1+

14
COULTER GEN•S Clinical Case Study

DIAGNOSIS: ACUTE MYELOID LEUKEMIA (M4) IN RELAPSE

GEN•S SYSTEM FLAGS

Suspect: Manual Differential:
NE Blasts Blasts 20%
Imm NE 1 Myelocytes 1%
Imm NE 2 Metamyelocytes 3%
Band Neutrophils 1%
Definitive:
Segmented Neutrophils 65%
Anemia
Lymphocytes 9%
Anisocytosis 1+
Monocytes 1%
RBC Morphology:
Anisocytosis 1+

16
COULTER GEN•S Clinical Case Study

DIAGNOSIS: VIRUS INFECTION

GEN•S SYSTEM FLAGS

Suspect: Manual Differential:
Variant LY Segmented Neutrophils 41%
Lymphocytes 34%
Definitive:
Atypical Lymphocytes 8%
Neutrophilia #
Monocytes 12%
Thrombocytopenia
Eosilnophils 5%
RBC Morphology:
Platelet Anisocytosis

18
COULTER GEN•S Clinical Case Study

DIAGNOSIS: CHRONIC LYMPHOCYTIC LEUKEMIA

GEN•S SYSTEM FLAGS

Suspect: Manual Differential:
None Segmented Neutrophils 1%
Lymphocytes 99%
Definitive:
Neutrophilia # RBC Morphology:
Lymphocytosis # Macrocytosis 1+
Macrocytosis 1+ Anisocytosis 1+
Aniscytosis 1+
Thrombocytopenia

20
COULTER GEN•S Clinical Case Study

DIAGNOSIS: MYELODYSPLASTIC SYNDROME ON TREATMENT

GEN•S SYSTEM FLAGS

Suspect: Manual Differential:
None Lymphocytes 19%
Basophils 1%
Definitive:
Neutropenia # RBC Morphology:
Pancytopenia Anisocytosis 1+
Anisocytosis 1+ Manual Platelet Count:
<10 x 109/L

22
COULTER GEN•S Clinical Case Study

DIAGNOSIS: NORMOCHROMIC ANEMIA

GEN•S SYSTEM FLAGS

Suspect: Manual Differential:
Imm NE1 Band Neutrophils 7%
NRBC Segmented Neutrophils 0%
Lymphocytes 9%
Definitive:
Monocytes 13%
Anemia
Eosinophils 1%
Anisocytosis 1+

Normoblasts: 9 per 100 WBC’s

RBC Morphology:
Anisocytosis 1+
Corrected WBC:
6.0 x 109/L

24
COULTER GEN•S Clinical Case Study

DIAGNOSIS: EDTA INDUCED PLATELET AGGREGATION

GEN•S SYSTEM FLAGS

Suspect: Manual Differential:
Imm NE1 Band Neutrophils 10%
Platelet Clumps Segmented Neutrophils 57%
Lymphocytes 18%
Definitive:
Monocytes 11%
Leukocytosis
Eosinophils 4%
Neutrophilia #
Monocytosis # RBC Morphology:
Thrombocytopenia Platelet Clumped on Film
Corrected WBC:
16.5 x 109/L
Platelet Count on
citrated sample:
25.3 x 109/L

26
COULTER GEN•S Clinical Case Study

DIAGNOSIS: MONOCYTOSIS

GEN•S SYSTEM FLAGS

Suspect: Manual Differential:
None Band Neutrophils 3%
Segmented Neutrophils 42%
Definitive:
Lymphocytes 11%
Monocytosis #
Monocytes 42%
Anemia
Eosinophils 1%
Anisocytosis 1+
Basophils 1%
Thrombocytopenia
Reticulocytosis RBC Morphology:
Anisocytosis 2+
Hypochromasia 1+
Polychromasia 1+

28
COULTER GEN•S Clinical Case Study

DIAGNOSIS: EOSINOPHILIA (AGED SAMPLE)

GEN•S SYSTEM FLAGS

Suspect:
Aged Sample
(Research Screen)
Definitive:
Eosinophilia %
Manual Differential:
Band Neutrophils 1%
Segmented Neutrophils 64%
Lymphocytes 19%
Monocytes 4%
Eosinophils 11%
Basophils 1%
RBC Morphology:
Normal

30
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