beckman coulter cases

General Chemistry

Immunodiagnostics

Centrifugation

Disease Management

Hematology

Hemostasis

Lab Automation

Data Management

Flow Cytometry

Primary Care

Bulletin 9165

Clinical Case Studies

COULTER® GEN•S™ SystemEnhanced VCS Technology

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)

ENHANCED VCS TECHNOLOGY ON THE COULTER GEN•S SYSTEM

COULTER GEN•S Clinical Case Study



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Beckman Coulter VCS Technology is the most powerful tool available for blood cellTechnology 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

Cell Stream

Flow Cell

Diagram 1

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)

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 2a

Diagram 2b

Diagram 2c

Diagram 3

VCS-Cube

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).

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™

and AccuGate™) are the result of eight years’ experience with the VCS

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



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VCS

IntelliKinetics™ AccuGate™

S c a t t e r S c a t t e r

S c a t t e r S c a t t e r

1

3

2

4

Vo

lum

e

Vo

lum

e

Vo

lum

e

Vo

lum

e

Diagram 4

Diagram 5

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.

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.



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Diagram 1

Diagram 3

Diagram 5

Diagram 2

Diagram 4

Diagram 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.

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).

Diagram 1:Platelets are classified first.

Diagram 2:The mature red cell population isdistinguished from the immaturereticulocytes.

Diagram 3:Once isolated, the level of Reticulocytematurity can be calculated.

DIAGNOSIS: NORMAL BLOOD


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GEN•S SYSTEM FLAGS

Suspect:None

Definitive:None

Manual Differential:Neutrophilis 65%Lymphocytes 24%Monocytes 8%Eosinophilis 2%Basophilis 1%

RBC Morphology:Normal

DIAGNOSIS: LEFT SHIFT


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Suspect:Imm NE 1

Definitive:None

Manual Differential:Segmented Neutrophils 80%Band Neutrophils 9%Lymphocytes 6%Monocytes 3%Eosinophils 1%Metamyelocytes 1%


DIAGNOSIS: SEVERE INFECTION, POST OPERATIVE


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Suspect:NE BlastsImm NE 1Imm NE 2

Definitive:LeukocytosisNeutrophilia #Eosinophilia #ErythrocytosisAnisocytosis 1+

Manual Differential:Segmented Neutrophils 79%Band Neutrophils 8%Lymphocytes 4%Monocytes 2%Eosinophils 2%Metamyelocytes 1%Myelocytes 4%

RBC Morphology:Anisocytosis 1+Platelet Anisocytosis

DIAGNOSIS: ACUTE MYELOID LEUKEMIA (M1) AT PRESENTATION


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Definitive:LeukocytosisNeutrophilia #Eosinophilia #AnemiaAnisocytosis 1+

Manual Differential:Blasts 95%Lymphocytes 5%

RBC Morphology:Anisocytosis 1+

DIAGNOSIS: ACUTE MYELOID LEUKEMIA (M4) IN RELAPSE


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Definitive:AnemiaAnisocytosis 1+

Manual Differential:Blasts 20%Myelocytes 1%Metamyelocytes 3%Band Neutrophils 1%Segmented Neutrophils 65%Lymphocytes 9%Monocytes 1%


DIAGNOSIS: VIRUS INFECTION


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Suspect:Variant LY

Definitive:Neutrophilia #Thrombocytopenia

Manual Differential:Segmented Neutrophils 41%Lymphocytes 34%Atypical Lymphocytes 8%Monocytes 12%Eosilnophils 5%

RBC Morphology:Platelet Anisocytosis

DIAGNOSIS: CHRONIC LYMPHOCYTIC LEUKEMIA


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Suspect:None

Definitive:Neutrophilia #Lymphocytosis #Macrocytosis 1+Aniscytosis 1+Thrombocytopenia

Manual Differential:Segmented Neutrophils 1%Lymphocytes 99%

RBC Morphology:Macrocytosis 1+Anisocytosis 1+

DIAGNOSIS: MYELODYSPLASTIC SYNDROME ON TREATMENT


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Suspect:None

Definitive:Neutropenia #PancytopeniaAnisocytosis 1+

Manual Differential:Lymphocytes 19%Basophils 1%


Manual Platelet Count:<10 x 109/L

DIAGNOSIS: NORMOCHROMIC ANEMIA


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Suspect:Imm NE1NRBC

Definitive:AnemiaAnisocytosis 1+

Manual Differential:Band Neutrophils 7%Segmented Neutrophils 0%Lymphocytes 9%Monocytes 13%Eosinophils 1%

Normoblasts: 9 per 100 WBC’s


Corrected WBC:6.0 x 109/L

DIAGNOSIS: EDTA INDUCED PLATELET AGGREGATION


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Suspect:Imm NE1Platelet Clumps

Definitive:LeukocytosisNeutrophilia #Monocytosis #Thrombocytopenia

Manual Differential:Band Neutrophils 10%Segmented Neutrophils 57%Lymphocytes 18%Monocytes 11%Eosinophils 4%

RBC Morphology:Platelet Clumped on Film

Corrected WBC:16.5 x 109/L

Platelet Count on citrated sample:25.3 x 109/L

DIAGNOSIS: MONOCYTOSIS


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Suspect:None

Definitive:Monocytosis #AnemiaAnisocytosis 1+ThrombocytopeniaReticulocytosis

Manual Differential:Band Neutrophils 3%Segmented Neutrophils 42%Lymphocytes 11%Monocytes 42%Eosinophils 1%Basophils 1%

RBC Morphology:Anisocytosis 2+Hypochromasia 1+Polychromasia 1+

DIAGNOSIS: EOSINOPHILIA (AGED SAMPLE)


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Suspect:Aged Sample(Research Screen)

Definitive:Eosinophilia %

Manual Differential:Band Neutrophils 1%Segmented Neutrophils 64%Lymphocytes 19%Monocytes 4%Eosinophils 11%Basophils 1%


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