Clinical Case Studies COULTER® GEN•S™ System Enhanced VCS ...

General Chemistry 

Clinical Case Studies 

COULTER ® GEN•S System 

Enhanced VCS Technology 

Immunodiagnostics 

Centrifugation 

Disease Management 

Hematology 

Hemostasis 

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)



Beckman Coulter 

Technology 

VCS Technology is the most powerful tool available for blood cell 

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

Diagram 2a 

Diagram 2b 

Diagram 2c 

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 

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 

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 

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



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 

IntelliKinetics 

AccuGate 

improved level of flagging efficiency. These two new features (Intellikinetics 

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 

1 

Scatter 

2 

Scatter 

Volume 

Volume 

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 applications. 

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.



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: 

Platelets are classified first. 

Diagram 2: 

The mature red cell population is 

distinguished from the immature 

reticulocytes. 

Diagram 3: 

Once isolated, the level of Reticulocyte 

maturity can be calculated. 

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).


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


DIAGNOSIS: LEFT SHIFT 


Suspect: 

Imm NE 1 

Definitive: 

None 


Segmented Neutrophils 80% 

Band Neutrophils 9% 


Monocytes 3% 

Eosinophils 1% 

Metamyelocytes 1% 


Normal 

10


DIAGNOSIS: SEVERE INFECTION, POST OPERATIVE 


Suspect: 

NE Blasts 

Imm NE 1 

Imm NE 2 

Definitive: 

Leukocytosis 

Neutrophilia # 

Eosinophilia # 

Erythrocytosis 

Anisocytosis 1+ 





Monocytes 2% 



Myelocytes 4% 



Platelet Anisocytosis 

12


DIAGNOSIS: ACUTE MYELOID LEUKEMIA (M1) AT PRESENTATION 


Suspect: 

NE Blasts 

Imm NE 1 

Imm NE 2 

Definitive: 

Leukocytosis 


Eosinophilia # 

Anemia 



Blasts 95% 




14


DIAGNOSIS: ACUTE MYELOID LEUKEMIA (M4) IN RELAPSE 


Suspect: 

NE Blasts 

Imm NE 1 

Imm NE 2 

Definitive: 

Anemia 



Blasts 20% 

Myelocytes 1% 





Monocytes 1% 



16


DIAGNOSIS: VIRUS INFECTION 


Suspect: 

Variant LY 

Definitive: 


Thrombocytopenia 




Atypical Lymphocytes 8% 

Monocytes 12% 

Eosilnophils 5% 


Platelet Anisocytosis 

18


DIAGNOSIS: CHRONIC LYMPHOCYTIC LEUKEMIA 


Suspect: 

None 

Definitive: 


Lymphocytosis # 

Macrocytosis 1+ 

Aniscytosis 1+ 






Macrocytosis 1+ 


20


DIAGNOSIS: MYELODYSPLASTIC SYNDROME ON TREATMENT 


Suspect: 

None 

Definitive: 

Neutropenia # 

Pancytopenia 




Basophils 1% 



Manual Platelet Count: 


DIAGNOSIS: NORMOCHROMIC ANEMIA 


Suspect: 

Imm NE1 

NRBC 

Definitive: 

Anemia 






Monocytes 13% 


Normoblasts: 9 per 100 WBC’s 



Corrected WBC: 

6.0 x 10 9 /L 

24


DIAGNOSIS: EDTA INDUCED PLATELET AGGREGATION 


Suspect: 

Imm NE1 

Platelet Clumps 

Definitive: 

Leukocytosis 


Monocytosis # 






Monocytes 11% 



Platelet Clumped on Film 

Corrected WBC: 

16.5 x 10 9 /L 

Platelet Count on 

citrated sample: 

25.3 x 10 9 /L 

26


DIAGNOSIS: MONOCYTOSIS 


Suspect: 

None 

Definitive: 

Monocytosis # 

Anemia 



Reticulocytosis 





Monocytes 42% 


Basophils 1% 



Hypochromasia 1+ 

Polychromasia 1+ 

28


DIAGNOSIS: EOSINOPHILIA (AGED SAMPLE) 


Suspect: 

Aged Sample 

(Research Screen) 

Definitive: 

Eosinophilia % 





Monocytes 4% 


Basophils 1% 


Normal 

30

I MPROVE THE PROCESS—AT EVERY LEVEL 

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www.beckmancoulter.com © Copyright 2000 Beckman Coulter, Inc. PRINTED IN U.S.A.

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