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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 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

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 Diagram 2b

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 Diagram 2c

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

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). On the GEN•S System, VCS technology has been enhanced by the addition

VCS

of two major modifications that allow even better differential results with an IntelliKinetics™

AccuGate™

Diagram 4

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

Volume

Volume

laboratory environment. Using the IntelliKinetics process, the GEN•S System

3 Diagram 5

4

2

Scatter

Volume

Scatter

Volume

1

Scatter

4

Scatter

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.

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.

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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).

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

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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

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COULTER GEN•S Clinical Case Study DIAGNOSIS: SEVERE INFECTION, POST OPERATIVE

GEN•S SYSTEM FLAGS Suspect: NE Blasts Imm NE 1 Imm NE 2 Definitive: Leukocytosis Neutrophilia # Eosinophilia # Erythrocytosis Anisocytosis 1+

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Manual Differential: Segmented Neutrophils 79% Band Neutrophils 8% Lymphocytes 4% Monocytes 2% Eosinophils 2% Metamyelocytes 1% Myelocytes 4% RBC Morphology: Anisocytosis 1+ Platelet Anisocytosis

COULTER GEN•S Clinical Case Study DIAGNOSIS: ACUTE MYELOID LEUKEMIA (M1) AT PRESENTATION

GEN•S SYSTEM FLAGS Suspect: NE Blasts Imm NE 1 Imm NE 2 Definitive: Leukocytosis Neutrophilia # Eosinophilia # Anemia Anisocytosis 1+

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Manual Differential: Blasts 95% Lymphocytes 5% RBC Morphology: Anisocytosis 1+

COULTER GEN•S Clinical Case Study DIAGNOSIS: ACUTE MYELOID LEUKEMIA (M4) IN RELAPSE

GEN•S SYSTEM FLAGS Suspect: NE Blasts Imm NE 1 Imm NE 2 Definitive: Anemia Anisocytosis 1+

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

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COULTER GEN•S Clinical Case Study DIAGNOSIS: VIRUS INFECTION

GEN•S SYSTEM FLAGS Suspect: Variant LY Definitive: Neutrophilia # Thrombocytopenia

Manual Differential: Segmented Neutrophils 41% Lymphocytes 34% Atypical Lymphocytes 8% Monocytes 12% Eosilnophils 5% RBC Morphology: Platelet Anisocytosis

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COULTER GEN•S Clinical Case Study DIAGNOSIS: CHRONIC LYMPHOCYTIC LEUKEMIA

GEN•S SYSTEM FLAGS Suspect: None Definitive: Neutrophilia # Lymphocytosis # Macrocytosis 1+ Aniscytosis 1+ Thrombocytopenia

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Manual Differential: Segmented Neutrophils 1% Lymphocytes 99% RBC Morphology: Macrocytosis 1+ Anisocytosis 1+

COULTER GEN•S Clinical Case Study DIAGNOSIS: MYELODYSPLASTIC SYNDROME ON TREATMENT

GEN•S SYSTEM FLAGS Suspect: None Definitive: Neutropenia # Pancytopenia Anisocytosis 1+

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Manual Differential: Lymphocytes Basophils

19% 1%

RBC Morphology: Anisocytosis 1+ Manual Platelet Count: <10 x 109/L

COULTER GEN•S Clinical Case Study DIAGNOSIS: NORMOCHROMIC ANEMIA

GEN•S SYSTEM FLAGS Suspect: Imm NE1 NRBC Definitive: Anemia Anisocytosis 1+

Manual Differential: Band Neutrophils 7% Segmented Neutrophils 0% Lymphocytes 9% Monocytes 13% Eosinophils 1% Normoblasts: 9 per 100 WBC’s RBC Morphology: Anisocytosis 1+ Corrected WBC: 6.0 x 109/L

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COULTER GEN•S Clinical Case Study DIAGNOSIS: EDTA INDUCED PLATELET AGGREGATION

GEN•S SYSTEM FLAGS Suspect: Imm NE1 Platelet Clumps Definitive: Leukocytosis Neutrophilia # Monocytosis # Thrombocytopenia

Manual Differential: Band Neutrophils Segmented Neutrophils Lymphocytes Monocytes Eosinophils

RBC Morphology: Platelet Clumped on Film Corrected WBC: 16.5 x 109/L Platelet Count on citrated sample: 25.3 x 109/L

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10% 57% 18% 11% 4%

COULTER GEN•S Clinical Case Study DIAGNOSIS: MONOCYTOSIS

GEN•S SYSTEM FLAGS Suspect: None Definitive: Monocytosis # Anemia Anisocytosis 1+ Thrombocytopenia Reticulocytosis

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Manual Differential: Band Neutrophils 3% Segmented Neutrophils 42% Lymphocytes 11% Monocytes 42% Eosinophils 1% Basophils 1% RBC Morphology: Anisocytosis 2+ Hypochromasia 1+ Polychromasia 1+

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 Segmented Neutrophils Lymphocytes Monocytes Eosinophils Basophils RBC Morphology: Normal

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1% 64% 19% 4% 11% 1%

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