Beckman Coulter Hematology

Corporate Mission

We will continue to be recognized as the world leader in cellular analysis by offering innovative products and solutions to simplify and automate cellular analysis processes

Large Hematology Analyzer Market Share

2001 Estimated Installed Base, Worldwide, of Large Hematology Analyzers

Others(Bayer,Sysmex,Abbott and etc)

6,940 units

53%

Beckman Coulter 47%

Earning Our Reputation

Coulter Science & Technology the cornerstone of the Cellular Analysis Industry

More CBCs are counted per day using the science of the Coulter Principle than any other method

Why? - Because it is the best tool for the job

More automated differentials per day are performed using the science of Coulter VCS technology than any other method

Why? - Because it is the most sensitive detector of true rare events

Leader in Clinical Flow Cytometry technology

1. CBC ANALYSIS
- The Coulter Principle - Enhancement to the Coulter Principle - Basic size and frequency Histogram - Parameter Deriviation

The Coulter Principle

A change occurs in electrical resistance between two electrodes when a nonconductive particle such as a blood cell passes between them - Wallace Coulter, 1947

CBC ANALYSIS - Impedance Principle

The Coulter method counts and sizes cells by detecting and measuring

Changes in electrical resistance when a particle(such as a cell) in a

Conductive liquid goes through a small aperture.
# of Pulse
Size of Pulase

Particle count
Cell Volume

Current does not pass from cells they are insulator

Current does not pass from cells they are insulator

Electrolytes solutions allow current to pass from them

Electrolyte Like ISOTON III

The Coulter principle

Red Blood Cell

A red cell passes through RBC aperture

Sensing Zone

Oscilloscope Oscilloscope

The Coulter principle

Neutrophil

A White cell passes through WBC aperture

Oscilloscope

Sensing Zone

Oscilloscope

Cell Volume or Size . . .

The Coulter Principle is used to accurately measure the size of the cell

Technology Choices

There are two basic approaches available for Cell Detection:

Electrical Current Visible Light

Neither approach is perfect

Both have limitations & interferences

Technology Choices

The Impedance Method remains the Gold Standard for Particle Counting & Sizing

Why?

Better Size Measurement

Volumetric Sizing - DC
DC measures total cell volume using the reference method of Direct Current impedance

Light Scatter estimates relative cell size based on forward scatter - what you really get is a measurement of cross-sectional diameter

Laser
O-3 deg (relative size)

Better Size Measurement

Volumetric Sizing - DC
A well known fact of light scatter is that size, refractive index, surface characteristics and internal components ALL contribute to ALL angles of light scatter DC remains the best tool for the right job

Laser
O-3 deg (relative size)

Better Size Measurement Leads to Better Counts

The ability to accurately size particles is key to the optimal separation of mixed cell populations

White Cell Sub-populations Red Cells from Platelets

Enhancement to Coulter Principle

Voting Sweep Flow Pulse Editing Coincidence Correction

Aperture Impedance System

Triplicate counting
Ensures Precision Reduces Repeats

Patented sweep-flow
Prevents RBC recirculation Ensures enhanced PLT linearity

Electronic pulse-editing

Provides accurate histograms and cell sizing for reliable RBC and PLT indices

Enhancement to Coulter Principle

Voting
- Process of analyzing the data from three aperture counts and rejecting any questionable data - In a Triple aperture system, the three counts are taken simultaneously

Voting

Example

1. Result is reported = 4.22 2. Result is NOT reported = ----AP1(4.21) AP2(3.82)

AP1(4.21) AP2(4.14) AP3(5.01)

AP3(5.01)

Benefits:
- Eliminate imprecision - Prevents data error - Flags possible problems

Enhancement to Coulter Principle

Sweep Flow
- Patented Technology - Sweeps cells away from the sensing zone

Sweep Flow

Enhancement to Coulter Principle

Pulse Editing
-Automatically eliminate atypical type
of pulse as it will influencing size measurement

Pulse Editing

Pulse Editing (Patented)

Cell flows through centre of aperture

Good Pulse

Diluent stream

Pulse editing

Non central cell flow

Pulse to be edited

Diluent stream

Pulse

Enhancement to Coulter Principle

Coincidence Correction
-Automatically corrects for occurrence of
two cells passing through the aperture at the same time

Coincidence Correction

Coincidence Correction

Pulse to be edited

Diluent stream

Basic Size and Frequency Histogram

To help you visualize numeric results To verify abnormal patterns To alert you to interfering particles

Threshold & Channel

THRESHOLD: An electronically set size limit CHANNEL: An area between 2 threshold

Applying Thresholds to separate Plt from RBC

20 fl

2 fl

Base line Platelets RBC of various sizes

Histogram - Resolution
Resolution = FEMTOLITER(fL)/CHANNEL Ex) 64 CHANNEL < 128 CHANNEL < 256 CHANNEL

Creating Histogram

Histogram Deriviation- Smoothing

Histogram

MODE CHANNEL Channel with most cells AVERAGE VOLUME(MEAN CHANNEL) Mean value of each channel

MCV(MEAN CELL VOLUME)

= Total Vol. Of all cell / Total channel = 813/10 = 81.3 fL RDW, PDW : Derived from HISTOGRAM

Basic Size and Frequency Histogram

CBC Parameter Derivation

Directly measured by the Coulter Principle Directly measured by Spectrophotometry Derived from size distribution Calculated from other parameters

WBC BATH - Triple Count

WBC BATH

LYSE reagent hemolyze RBC then measure WBC & HGB Dilution= 1 : 250 (ICSH REFERENCE)

WBC : >/= 36 fL
HGB : Cyanmethemoglobin method using 525nm Halogen light

WBC Counting

RBCs are eliminated by an advanced, lytic reagent Cells below 35 fl are excluded from the WBC count Fragile WBCs are included in the count Three independent counts / sample ensure precision Extended analysis for cytopenic samples ensures accuracy at critical levels Linear range expanded from 0 - 140,000 cells / l reduces repeats

Coulter WBC histogram

Lymp

Mono

Neut Baso Eos

256 channel high resolution WBC histogram display cell population data between 35 and 450 fl

WBC Histogram

Stable HGB Measurement

Industry standard spectrophotometry accurately and precisely measures:

Hemoglobin
MCH and MCHC

The method uses:

A modified hemoglobin reagent

A broad spectrum tungsten light source An infrared filter for heat absorption A broad bandpass filter centered @ 525nm

Multiple reagent blanks

Hgb Measurement In Beckman Coulter Instruments

WBC Bath HGB Lamp

ADC

Signal Processor

Derivation
Absorbance= log 10 ( VR / Vs ) Where VR = Reference voltage Vs = Sample voltage HGB Sensor & PRE-AMP

Sample

Broad Spectrum HGB Readings

Narrow bandpass Fileter

Broad bandpass Filter : Detect Shift of peak and adjustment

RBC Counting

Unique Coulter Features include:

3 independent Count/Best accuracy and precision Sweep flow eliminates RBC recirculation in an aperture and is patented feature Pulse editing eliminates skwed pulses and ensures the most accurate MCVs in the industry Extended Counting ensures RBC counts are linear to 0.0 High resolution histogram detect and flag the clinical abnormalities such as RBC fragments, RBC agglutination and etc.

RBC Counting
Anemia is one of the most common hematological disorders

Coulter impedance counting with high-resolution histograms and patented pulse editing circuitry provides the unique ability to:

Accurately measure MCV Grade RBC morphology Accurately measure RDW Detect dimorphic populations Extended linear range from 0 to 8.00 x 106 cells/L

RBC BATH - Triple Count

RBC BATH

Dilution = Blood (1.6 uL) : DILUENT (10 mL) = 1 : 6,250 (ICSH REFERENCE)

Measuring RBC & PLT

RBC : >/= 36 fL Cells PLT : 2 ~ 20 fL

Coulter RBC histogram

Main RBC population RBC doublets

256 channel high resolution RBC histogram display cell population data between 24 and 350 fl

PLT Counting

Unique Coulter Features include:

3 independent Count/Best accuracy and precision Sweep flow eliminates RBC recirculation in an aperture and is patented feature Pulse editing(another patented feature)ensures accurate counts Extended Counting ensures Plt counts are linear to 0.0

PLT Counting & Sizing

Three independent counts per sample

Uses sweep flow and pulse editing

Raw data is accumulated from 2 to 20 fL Particles between 2-20fL are incorporated into a least squares regression formular The formula defines a log normal curve

Passage of a RBC & Plt through aperture

RBCs

PLT

Cross section of RBC aperture (60x50 micron)

Oscilloscope screen

System with sweep flow

RBCs pushed away by diluent

RBC PLT

Oscilloscope screen Sensing Zone Diluent stream

Sweep Flow technology

RBCs swept away from sensing zone

Diluent stream

Platelet Raw Data

Lower limit of 2 fL
No noise No bacteria

Upper limit of 20 fL
No schistocytes No microcytes No White Cells

Platelet Fitted Curve

Uses least squares regression formula

Regression formula defines a log normal curve

Log normal curve is plotted from 0 - 70 fL to include giant platelets No floating thresholds

PLT Curve Integration

Integral calculus is used to find the area under the curve

The area under the curve is calibrated to a reference Platelet count Best overall correlation to the phase platelet count Highest Platelet Accuracy

Eliminates non-PLT Interference

Accurate Platelet Counts

Coulter log-fitting platelet technology provides the best overall correlation to the reference phase platelet count Non-platelet pulses between 0 and 70 fl. are excluded from the PLT count by the electronic curve The extrapolated area under the PLT curve includes giant platelets Linear range expanded to 1,500,000 cells / l.

PLT histogram and curve fitting

A fitted curve accurately reflecting the collected data is displayed between 2 and 35 fl

Raw data Fitted Curve displayed up to 35fl

PLT histogram is based on 256 channels

PLT curve fitting

The Platelet count is derived from the number of cells under the fitted curve beyond the 35fl display limit extending up to 70 fl

Raw data Fitted Curve extend up to 70 fl to report very large PLT count

PLT curve fitting

The curve fitting process allows more accurate counts when platelets of larger than 20 fl are present

PLT Histogram

Optical WBC and platelet counts are better . . .

The lower limit of linearity is still owned by impedance

This is where the vast majority of Clinical Decisions are made

Accurate Platelet counts across the full extended range of Zero to 3,000,000 Accurate WBC counts across the full extended range of Zero to 400,000

Accuracy confirmed by Reference Flow Cytometry

Optical Platelet Counts

CD4000 Optical PLT Count 1,000,000

In comparison to the reference phase platelet count, the Coulter log-fit platelet
0 COULTER Log Fit PLT Count 1,000,000

technology showed a better

overall correlation coefficient.

Laboratory Hematology 4:80-87 1998 Carden Jennings Publishing Co. Ltd.

CBC Parameters
1. DIRECT MEASUREMENT/IMPEDENCE

: RBC, WBC
2. SPECTROPHOTOMETRY /MODIFIED CYANMETHEMOGLOBIN : HGB 3. DIRECT MEASURMENT/ SIZE DISTRIBUTION (HISTOGRAM) : MCV, RDW, PLT, MPV 4. CALCULATION : HCT(%) (= RBC x MCV 10)

MCH(pg) (= (HGB RBC) x 10)

MCHC(g/dL) (= (HGB HCT) x 10)

Comments on Basic CBC Data

Evaluation of the Beckman-Coulter Gen-S hematology analyzer E. GRIMALDI, F. SCOPACASA Laboratory Hematology 4:264268
Linearity Linearity data show excellent correlation between expected and obtained values for all parameters sensitive to dilution (Table 2). Statistical analysis revealed a correlation coefficient >0.999 for all CBC parameters evaluated, also in a very low range. We also found that the unflagged percentage of WBC differential count results were reproducible and linear down to a level of 0.15 x109 /L WBC count (data not shown).

Comments on Basic CBC Data

Evaluation of the Beckman Coulter GenS hematology analyzer E. GRIMALDI, F. SCOPACASA Laboratory Hematology 4:264268
RBC and PLT counts in lipemic samples (Figs. 2 and 3) were overestimated on the H*2 system, but only for high lipid concentrations. No interference was found on the GenS.

Beckman Coulters VCS Technology

Beckman Coulters Electro Optical Flow Cell Technology - A Breakthrough Techcology

2. WBC Differential - V.C.S. Technology

Flow Cytometry

Generally Flow Cytometry Uses One Energy Source(Laser)

Enhanced Flow Cytometry

3-D VCS Technology: Advanced Multi-Parametric Cellular Analysis

COULTER VCS 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.

VCS + Flow Cytometry

V - VOLUME

C - CONDUCTIVITY S - SCATTER Measurement are taken: Independently

Simultaneously At the flow cell aperture

Laser System

Volume, Conductivity (Opacity),

Light Scatter Helium/Neon Laser Consume little electricity and lee heat produced

Stable alignment,Less Maintenance

The Triple Transducer Module

A major advance in technology An electro-optical flow cytometer Provides concurrent electronic and optical measurements

Advanced Multi-Angle Scatter Detector

100% solid state reliability Collects data on upper and lower median angle light scatter (MALS) Masks out unwanted signals

WBC Differential Analysis

2 reagents system

Erythrolyse : Hemolyze RBC Stabilyse : Near native state of WBC

Hydrodynamically focused flowcell.

Minimize Coincidence effects

VCS Principles High Resolution Scatter Plot (X,Y,Z )

256 x 256 x 256 = 16,777,216 of cells analyzed

WBC Differential

Unique dual reagent system

Erythrolyse osmotically lyses the RBCs Stabilyse returns the WBCs to their near native size

Neut

Eo

L ymph

W BC Swelling

W BC Shrinkage

L is Nuclei ys

RBC

Blood

RBC Swelling ErythroLys e

RBC ghos t, fragment, dis olve StabiLys e

C O U N T

Diff Flow

WBC 5 Part

One Cell at a Time . . .

The Multi-Parametric Flow Cell organizes the cells into single file for analysis

VOLUME MEASUREMENT

VCS utilises the Coulter Principle of counting and sizing to measure the volume of the cell by using Direct Current (DC) across the two electrode in a flow cell.

VOLUME MEASUREMENT

A true 3-D measurement of the cells volume is determined in the electro-optical flow cell and for the first time in history, a cells near native state volume is measured. Coulters research shows the impedence volume measurement to be more reliable than forward angle light scatter(FS) measurement. Both FS and impedence volume measurement are generated but only impedence is utilized due to its superiority in volume measurement

Better Size Measurement

Reference Impedance (DC)
DC measures total cell volume using the reference method of Direct Current impedance

Laser Light Scatter only estimates relative cell size based on forward scatter

Laser
O-3 deg (relative size)

CONDUCTIVITY MEASUREMENT
When a cell is exposed to high frequency current (RF) , it no longer remains insulator, the RF energy penetrate into cell and reveal information about its size and internal structure.

Conductivity

Conductivity High Signal Cell

-Internal Structure,complexity of nucleus,granuals

Conductivity Low Signal Cell -No grannuals, Simple nucleus

Intracellular Composition . . .

Intracellular Composition is examined using Radio Frequency

Better Internal Measurement

Intracellular Probe (RF)
RF measures internal cell structure using radiographic imaging similar to ultrasound

Low forward angle light scatter estimates cellular complexity - it is a relative measurement

Laser
7-1O deg (complexity)

Usual RF Scatterplot

Not good seperation of each cell population Cells size effect RF signal Due to this, LY, MO, GR population is not clear

V o l u m e

RF

Opacity

V o l u m e

Adjust RF signal by remove cells size data

Opacity: Reflect the cells true nuclear density & internal Composition

Opacity

SCATTER MEASUREMENT
As cells are passing through the flow cell in single stream they are exposed to a laser beam, when laser strike them they scatter light. The light scatter at angles between 10 and 70 deg is used by VCS instruments.(patented)

Specific Type of Granules . . .

Light Scatter is used to identify specific types of granules within the cells

Scatter

Scatter High Signal Cell

Scatter Low Signal Cell

Better Granularity Measurement

Multi-angle Light Scatter (MALS)
MALS measures granularity using a broad range of angles (10-70)

Standard cytometry measures granularity using a single angle

Laser

9O deg

Usual Light Scatter Sactterplot

Size of cell effects light scatter data especially severe at low angle detection Not clear separation of cell population

V o l u m e

Scatter

Rotated Light Scatter (RLS)

Median Angle Light Scatter (MALS)

Signals by RLS measurement reflect true granularity & surface characteristics of cells,resulting good separation of each cell population.

V o l u m e

RLS

VOLUME COMPENSATION

BEFORE VOLUME COMPENSATION

AFTER VOLUME COMPENSATION

3D Data Analysis

SIMULTANEOUS MEASUREMENTS

VCS is the only single channel analysis that uses 3 independent energy sources to probe approximately 8,192 cells in their near native state.Each providing 256 channels of resolution over 16,700,000 channels in all.

3D Data Analysis

Neuts
Monos Eos

Lymphs

Basos NRBCs

Beckman Coulter

Current 5-part Differential Products

GenS System 2 STKS HmX MAXM

Primary Analysis Science(s) Native State WBC Chemistry

Volumetric Impedance Radio Frequency Conductivity Multi-angle laser light scatter Helium/Neon

WBC Differential Parameters

1. Direct VCS Technology Measurement : LY%, MO%, NE%, EO%, BA%

2. Calculation
: LY# =(LY% x WBC) 100 MO# = (MO% x WBC) 100 NE# = (NE% x WBC) 100 EO# = (EO% x WBC) 100 BA# = (BA% x WBC) 100

Coffee Break ..

VCS Enhancements used on GenS

Intellikinetics

TM

Application

AccuGate software New colour - coding system Ongoing Flagging development

Improving VCS Technology

IntelliKineticsTM & AccuFlexTM

Two technologies proven to reduce false positive and false negative flagging to an all time low
THE PURPOSE:

Reduce False Flagging

Most Efficient Flagging

False Positive Rate Reduced By Up To 50%!

50%

False Negative Rate Reduced By Up To 10%!

10%

VCS sample flow

IntelliKinetics
Advanced Cell Preparation Technology

Prepares cells using advanced flow cytometry techniques Depending upon ambient conditions, the reagent volume, temperature and reaction time are automatically adjusted to optimize performance Provides customers with more consistent and reproducible performance in various ambient conditions

THE PURPOSE:

Enhance Flagging Performance

Diluted Blood

Mixing Chamber

1. Reagent Temp 2. Reagent Vol 3. Reaction Time

Intellikinetics Applied

VCS Measurement

Flow Cell

Intellikinetics
1. Reagent Temp

TM

2. Reagent Volume

3. Reaction Time

Intellikinetics

TM

Diluted Blood to Mixing Chamber

Intellikinetics

TM

Application

Intellikinetics Applied

Intellikinetics

TM

VCS measurement in flow cell

Benefits of Intellikinetics

VCS pattern

When Disabled

Benefits of Intellikinetics

VCS pattern

When Enabled

Unique 3D cube
256 x 256 x256 = 16 million data locations

Research positional parameters

List mode data files

VCS Enhancements used on GenS

Intellikinetics
AccuGate
TM

TM

Application

software

New colour - coding system Ongoing Flagging development

AccuGate

48 Hour Differential Stability

Live 3-Dimensional Analysis

Real-Time Cell Characterization

Characterization by Lineage and Frequency

Better Flagging Performance Increased Efficiency

Conventional VCS gates

Gates in AccuGate

TM

Software

VCS Enhancements used on GenS

Intellikinetics
AccuGate
TM

TM

Application

software

New colour - coding system Ongoing Flagging development

5 Sub Populations individually measured

Neutrophils Monocytes Eosinophils

Lymphocytes

Basophils

5 Sub Populations individually measured

Neutrophils Monocytes Eosinophils

Lymphocytes Basophils

5 Sub Populations individually measured

Neutrophils Monocytes

Lymphocytes

Basophils

5 Sub Populations individually measured

Monocytes

Lymphocytes Basophils

5 Sub Populations individually measured

Lymphocytes Basophils

5 Sub Populations individually measured

Basophils

Shading indicates population density

Least

Medium

Most

VCS Enhancements used on GenS

Intellikinetics

TM

Application

AccuGate software New colour - coding system Ongoing Flagging development

Comprehensive Suspect Flagging

NE Blasts MO Blasts Imm NE 2 Imm NE 1 LY Blasts Variant LY

NRBCs

Better Abnormal Cell Detection - Flagging

1 Mono-Blasts (MO Blast Flag) 2 Myelo-Blasts (NE Blast Flag)
2 1 4 3

3 Immature Granulocytes 4 Band Neutrophils 5 Lympho-Blasts (LY Blast Flag) 6 Variant Lymphocytes Low Volume Lymphocytes PLT Clumps Giant Platelets RBC Parasites (Malaria, etc) 7 8 9 10

5 6

7 8 9 10

Enhanced VCS delivers improved Retic counting

Use of Intellikinetics & AccuGate software Maturation Parameters

Reliable Reticulocyte Method

The method uses 3-D VCS Technology to separate reticulocytes from mature RBCs, WBCs, PLTs and NRBCs Reticulocytes are stained with a supravital stain (NMB)

Prior to flow cytometric analysis, the RBCs are cleared of their hemoglobin to enhance the separation
The cells labeled A, B & C represent various levels of reticulocyte maturity

On line Reticulocyte analysis

Flow cytometric analysis using VCS probes + Intellikinetics+Accugate Non fluorescent dye eliminates carcinogen hazard

3-Dimensional Discrimination

Over 32,000 cells are analyzed ensuring accuracy of the count Reticulocytes are identified based on their increased light scatter AccuGate clearly defines the reticulocyte population to minimize repeats even in the presence of gross abnormalities

Results are in percent and absolute number

THE PURPOSE:

Monitor Erythropoetic Status

Separation of Reticulocyte populations

Leukocytes
Mature Erythrocytes

Least Mature
Reticulocytes Most Mature

Non - RBC Interference

Retic Flow

Reticulocytes v Reference

Correlation to R-1000:

Correlation to Manual:
Flagging Agreement v R-1000:

r = 0.932 r = 0.872

85.4%
GenS

Source: Reticulocyte performance on the COULTER System:Lab Hematol. 3:41-47 1997

Reticulocyte Parameters
1.

VCS Technology : Retic% (Reticulocyte %) MRV (Mean Reticulocyte Volume) IRF (Immature Reticulocyte Fraction)

MSCV (Mean Sphered Cell Volume)

HLR% (High Light scatter Reticulocyte %) 2. Calculation : Retic# = (Retic% x RBC) 10

Enhanced VCS & Retic counting

Use of Intellikinetics & AccuGate Maturation Parameters Interpretation of RBC cluster shape

Derivation of MI Parameter
WBCs & NRBCs MI = HLR count

Total Retics
Mature RBCs Retics

Mature Young
Platelets

PBSC Transplantation in NHL

MI MFR + HFR

Days in Treatment

40

Enhanced VCS & Retic counting

Use of Intellikinetics & AccuGate Maturation Parameters Interpretation of RBC cluster shape

More Diagnostic Information

FDA Cleared Parameters

MRV (Mean Reticulocyte Volume) IRF (Immature Reticulocyte Fraction)

Suspect Flags

Interpretive Messages Laboratory Parameters

Sickle Thalassemia

HLR % and # (High Light Scatter Retics) MSCV (Mean Sphered Cell Volume)

Monitor Marrow Recovery

More Diagnostic Information

Research indicates:

Unique cluster shapes found in Thalassaemia/Sickle cell disease

Mean Volume indicates shape abnormalities such as Spherocytosis

Enhanced VCS Technology Provides

Improved long term & short term stability Better agreement to reference methods Better exclusion of interference's Reduced inaccurate flagging rates Better Reticulocyte counting Future developments

Questions???

FLAGGING SYSTEM
- Suspect flag - Definitive flag - Codes - AccuFlex - Decision rule & Criteria

Flag - (1)Suspect Flag

- Criteria from Software, Not user definable criteria

< SUSPECT FLAG >

BLAST (LY Blast, MO Blast, NE Blast)

Left Shift (Imm. NE1, Band) Imm. Grans (Imm. NE2) Variant Lymphocyte PLT Clumps Giant Plates NRBC Band form Promyelocyte, Metamyelocyte, Myelocyte Atypical lymphocyte

Flag- (2) Definitive Flag

- User definable criteria < Definitive flag > Anemia : RBC or HGB values are equal or lower than Low limit

-philia (Neutro-, Eosino-, Baso-) : Valus are equal or higher than

High limit -penia (Thrombo-, Leuko-, Lympho-, Mono-, Neutro-, Eosino-, Baso-, Pan-) : Low limit -cytosis (Erythro-, Aniso-, Macro-, Micro-, Thrombo-, Leuko-, Lympho-, Mono-, Reticulo-) : High limit

Parameter WBC

Condition Normal WBC Pop Abnormal WBC Pop No message

Suspect NE Blasts Imm NE 1 Imm NE 2 Variant Lymphs Review Slide

Definitive Leukopenia Leukocytosis Neutropenia Neutrophilia Lymphopenia Lymphocytosis Monocytosis Eosinophilia Basophilia Anemia Anisocytosis Microcytosis Marocytosis Hypochromia Poikilocytosis Erythrocytosis Pancytopenia Thrombocytopenia Thrombocytosis Small Platelates Large Platelae

RBC

Normal RBC Pop Abnormal RBC Pop No message

NRBCs Dimorphilc RBC Pop Micro RBCs/RBC Fragments RBC Agglutination

PLT

Normal WBC Pop Abnormal WBC Pop No message

Platelate Clumps Giant Platelates

Automated RBC Morphology

Anisocytosis +, ++, +++ Microcytosis +, ++, +++ Macrocytosis +, ++, +++ Hypochromia +, ++, +++ Poikilocytosis +, ++, +++ Dimorphic RBC Population Micro RBCs/RBC Fragments RBC Agglutination

Flag- (3) Codes

-Software define Code flagging

< Code> ---R P No Diff Total Vote out Review Partial Aspiration

H/L High/Low

AccuFlex Software

Your patient population is different, for optimum efficiency you need to select the sensitivity of the suspect flagging appropriate to your population AccuFlex software provides individual suspect flags with three user selectable sensitivities To meet the differing demands of global customers the IMM NE1 flag can be disabled

AccuFlex Software

For optimum efficiency labs need the ability to select the sensitivity of the suspect flagging appropriate to their population AccuFlex software provides user selectable sensitivities for individual suspect flags Three levels Highly Sensitive Very Sensitive Sensitive IMM NE1 flag can be disabled

Changing Requirements

Laboratory needs are changing Reduced standardisation in flagging requirements Shift to tailoring of flagging system in order to optimise analyser for individual site requirements

Example

NCCLS standard for Variant Lymph detection is >7% Some reference labs do not want to detect until level exceeds 10 -15% What's your criteria?

GenS gives user the control over sensitivity they wish to obtain

Provide a variety of

sensitivity settings based on performance in NCCLS tests Give users control over sensitivity they wish to obtain Treat each flag individually, i.e. High sensitivity for Blasts can be mixed with lower sensitivity to Left Shift

Reduced Manual Film Reviews

Lowest False Positive rate of any new generation system in independent evaluations Potential to reduce film review rate by up to 50% Flag selectivity offers opportunity for even greater savings

Excellence in Quantitative Flagging

All Quantitative Flags are User Definable Age, Gender & Location

Reference Intervals Action Limits Critical Limits Definitive Message Limits Graded RBC Morphology

Excellence in Decision Support

Decision Rule and Criteria

Reduce Slide Reviews with Automated Follow-Up Processing

Automate laboratory decisions Control handling procedures Patient result limits by: Age Gender Location User Defined Review by Exception rules-based system

What is Decision Support?

A programmed environment that automates the review of every sample analyzed:

Normal, or Expected results are passed through for reporting Abnormal, or Unexpected results are held for operator review and validation

Why Automate Decision Support?

Consistency in handling all results

Appropriate follow-up can be pre-programmed

Automates Slide Making & Review Protocols Differences in personnel experience Procedural differences when staff work at more than one site

Eliminates

Reduces Turnaround time

Increased productivity Improved service to clinicians

Who Defines the Criteria?

In virtually all instances, the laboratory establishes the criteria

The only exception is in the area of Suspect Messages ... . . . But even here, the laboratory can fine tune these flags for optimal sensitivity and specificity

using AccuFlex and decision rule/criteria

Case Example

...

Comparative Performance
GenS n 257 6.23% 0.39% 3.50% 89.88% 90.27% 9.73% 90.00% 93.52% 99.57% 36.00% 93.39% SE 9500 257 28.02% 0.00% 3.89% 68.09% 68.09% 31.91% 100.00% 70.85% 100.00% 12.20% 71.98% STKS 257 8.95% 0.00% 3.89% 87.16% 87.16% 12.84% 100.00% 90.69% 100.00% 30.30% 91.05%
FP/TOTAL # FN/TOTAL # TP/TOTAL # TN/TOTAL # (TN + FN)/TOTAL # (TP + FP)/TOTAL # TP/(TP + FN) TN/(TN + FP) TN/(TN + FN) TP/(TP + FP) (TP + TN)/TOTAL #

False Positive False Negative True Positive True Negative Pass Rate Review Rate Sensitivity (% TP) Specificity (% TN) Predictive Value Of Neg Predictive Value Of Pos Efficiency

Central US Account - Publication Pending

Coffee Break ..

Taking it to the Next Level

A Unique Combination of Technologies With One Goal . . .
Simultaneous Reduction in False Positives & False Negatives

Lower Morphologic False Positives

IntelliKinetics

AccuGate
AccuFlex

What does it mean to the User?

Results from External (Customer) Evaluations

GenS vs Competition Difference False Negative (FN) rate False Positive (FP) rate Review rate 3.7 vs 5.4% 12 vs 23% 23 vs 32 % 32% lower 48% lower 22% lower

(Sysmex SE-9500, Abbott CD3500 and CD4000, Bayer Advia 120) n = 1386 samples, 7 evaluations

From Discovery to Diagnosis

Case Examples

Histogram Case 1

ATYPICAL RBC HISTOGRAM

1. More than one population 2. High RDW

Patient with massive transfusion

Histogram Case 2

ATYPICAL PLT HISTOGRAM 1. Data curve show little population around > 20 fL base line 2. Fitted curve: 2-70fL

- Suspect: Large PLT or Giant PLT presence

Histogram Case 3
ATYPICAL PLT HISTOGRAM

Fitting Curve correct and give PLTresult.

Histogram Case 4
PLT POSITIVE CURVE Normal Raw data curve and Fitted curve

PLT NON-POSITIVE CURVE No, Normal Raw data curve was Present with no Fitted curve

Histogram Case 5

ATYPICAL WBC HISTOGRAM 1. Counting threshold, from 35 fL. 2. Small data curve on threshold. - Any particle like giant platelet or nRBC might present - If it is trigger algorithm, WBC interference, flag(*)

Histogram Case 6 - MCV, RDW

NORMAL MCV, RDW HISTOGRAM MCV (Mean Cell Volume) : Normocyte RDW (Red cell Distribution Width) : Coefficient of variation of all cells with the expected population

Histogram CASE 7- MCV, RDW

ABNORMAL MCV, RDW HISTOGRAM MCV : Increased. Macrocytosis RDW :Increased. Anisocytosis

Better Abnormal Cell Detection - Flagging

1 Mono-Blasts (MO Blast Flag) 2 Myelo-Blasts (NE Blast Flag)
2 1 4 3

3 Immature Granulocytes 4 Band Neutrophils 5 Lympho-Blasts (LY Blast Flag) 6 Variant Lymphocytes Low Volume Lymphocytes PLT Clumps Giant Platelets RBC Parasites (Malaria, etc) 7 8 9 10

5 6

7 8 9 10

5 Sub Populations individually measured

Neutrophils Monocytes Eosinophils

Lymphocytes
Basophils

Separation of Reticulocyte populations

Leukocytes
Mature Erythrocytes Least Mature Reticulocytes Most Mature Non - RBC Interference

CLL
Retics

Suspect flags: Lymphoblasts Low Volume Lymphocytes

RBCs

Plts.

Blood Film

Small Lymphoblasts

Case 1 Instrument: HmX

13.2 3.3 7.7 87.4 0.1 1.5

Instrument Discovery: AML - M1

Case 2 Instrument: GenS

121.2 94.7 4.8 0.3

0.4 1.0
11.6 0.0 0.2

Suspect: Blasts

0.1 0.1 114.8 5.8 0.4

Suspect: NE Blasts

0.1 0.1

Clinical Flow Diagnostic Support: AML - M1

1 13 0 1 1 84 CD MARKER CD2 CD3 CD5 CD10 CD19 CD20 HLA-DR CD13 CD14 CD33 CD34 CELL TYPE PERCENTAGES PAN T-CELL 8.1 IMMUNOCOMPETENT T-CELL 6.3 MATURE T; B SUBSET 5.5 COMMON ALL ANTIGEN 3.0 PAN B-CELL 7.2 PAN B-CELL 8.0 ACTIVATED T/B; MONO; MYELO 93.2 * MYELOCYTIC 94.6 * MONOCYTIC 2.5 EARLY MYELOCYTIC; MONOCYTIC 92.1 * PRECURSORS 59.1 *

NTIAL

Instrument: XL / MCL

Case 1 Instrument: HmX

78.3 17.0 5.8 76.2 1.0 0.0 13.3 4.5 59.7 0.8 0.0

Instrument Discovery: AML - M5

Case 2 Instrument: GenS

64.2 5.7 15.6 78.3

Suspect: Blasts

0.1 0.3 3.7 10.0 50.2

Suspect: MO Blasts

0.1 0.2

Clinical Flow Diagnostic Support: AML - M5

4 7 47 0 0 2 6 34 CD MARKER CD2 CD3 CD5 CD7 CD10 CD19 CD20 HLA-DR CD13 CELL TYPE PERCENTAGES PAN T-CELL 9.1 IMMUNOCOMPETENT T-CELL 5.5 MATURE T; B SUBSET 10.2 PAN T-CELL; T ALL 8.0 COMMON ALL ANTIGEN 6.6 PAN B-CELL 6.1 PAN B-CELL 4.7 ACTIVATED T/B; MONO; MYELO 96.1 * MYELOCYTIC 80.1 *

ENTIAL

TES

CD14 CD33 CD34

MONOCYTIC EARLY MYELOCYTIC; MONOCYTIC PRECURSORS

81.6 * 91.2 * 44.1 *

Instrument: XL / MCL

Case 1 Instrument: HmX

96.4 2.9 93.4 3.1 0.1 0.5 2.8 90.0 3.0 0.1 0.5

Instrument Discovery: CLL

Case 2 Instrument: GenS

31.3 14.6 80.9 3.5 0.8

Suspect: Blasts Suspect: Atyp. Lymph

0.2 4.6 25.3 1.1

Suspect: Var. Lymph

0.3 0.1

Clinical Flow Diagnostic Support: CLL

CD MARKER CD2 CD3 CD5/CD19 CD10 CD20 CD22/CD25 HLA-DR CD33 CD34 KAPPA LAMBDA IgD IgG IgM CELL TYPE PERCENTAGES PAN T-CELL 5.1 IMMUNOCOMPETENT T-CELL 3.7 CLL CELLS 75.8 * COMMON ALL ANTIGEN 4.6 PAN B-CELL 80.7 * HAIRY CELLS 1.8 ACTIVATED T/B; MONO; MYELO 90.6 * EARLY MYELOCYTIC; MONOCYTIC 6.8 PRECURSORS 0.4 SURFACE KAPPA 78.6 * SURFACE LAMBDA 3.3 SURFACE IgD 8.0 SURFACE IgG 89.5 * SURFACE IgM 10.6

ENTIAL 16 84 0 0 0

Instrument: XL / MCL

Discovers and Monitors

Hematology

Anemia Therapeutic Efficacy Morphologic Anomalies Infection & Inflammation Platelet Abnormalities Bleeding Tendencies Leukemia Lymphoma Immune Suppression

Discovery of Anemia

Anemia is the most common diagnosis in hematology Coulter systems accurately and precisely measures: Hemoglobin Concentration MCH MCHC Coulter impedance counting with high-resolution histograms and patented pulse editing circuitry provides a unique ability to: Accurately measure MCV Grade RBC Morphology Measure RDW Detect Dimorphic Populations

Monitoring for Therapeutic Efficacy

Reticulocyte Counts are used to monitor recovery from several conditions

Anemia Bone marrow transplantation EPO therapy

IRF now joins the Retic Count as a monitoring tool Automating and standardizing this labor- intensive test provides you improved accuracy and efficiency

Discovery of Morphologic Abnormality

Laboratories seldom use the same criteria for slide review Coulters proprietary AccuFlex technology allows optimized flagging sensitivity for your patient population

Maximizes discovery of rare events while maintaining high throughput

Discovery of Infection or Inflammation

Coulter hematology systems provide accurate WBC counts, differentials and sensitive detection of rare events An elevated WBC with neutrophilia may indicate a bacterial infection while a decreased WBC with lymphocytosis may indicate a viral infection

Screening for Bleeding Tendencies

Accurate counts are essential in the detection of low platelet numbers and the monitoring of transfusion therapy Patented log fit platelet technology detects and counts giant platelets ensuring patients results are reported accurately In the presence of a giant platelet flag, further studies by flow cytometry may aid in the enumeration of reticulated platelets

Screening for Leukemia

The unique combination of technologies that make up the VCS system provide the most accurate information on blast forms providing separate flags for:

Myeloblasts Lymphoblasts Monoblasts

This specific flagging indicates most appropriate reflex testing by Flow Cytometry for lineage determination

Screening for Lymphoma

VCS Technology has the ability to sub-classify the lymphocytes populations in into:

Lymphoblasts Low Opacity Lymphs

Flow Cytometric Analysis by monoclonal antibodies provides additional information

Taking the next step...

Once you have discovered an abnormality, the next step is to efficiently identify and classify the nature of the abnormality

Quality Assurance Reagents Calibrators Controls

Quality Assurance

The most important aspect of laboratory practice The one procedure which verifies that the end result of all your work is: an accurate patient result

Instrument:

Background check Voltages Latron Physical

COULTER Hematology Reagents

Coulter reagent are systems Continually tested with instruments Complex formulations

Biochemical reactions between reagents, cell membranes, cell contents

The balance of reagents give a system performance

Calibrators and Controls

Stabilized blood products Manufactured as part of the system Calibrators provide calibration using single values

traceable to ICSH and NCCLS reference methods

Controls provide quality control of the measurement process

confidence in reported result

S-CAL Calibration

Used to set calibratible parameters to recover whole blood values of ref. methods Daily use to zero bias instruments used in assay process Monitor value recovered vs value assigned Calibration factors must meet criteria prior to use of instrument Calibration factor applied to all results from that instrument , for that day

Control Recovery

Three levels of previously assayed control are run on each instrument

monitors day to day performance alerts need for service

Expected ranges used are tighter than published ranges Instrument considered qualified if

Calibration factor within set limits Control recovery within set limits

Quality Assurance

Controls

commercial laboratory patient means delta checks national commercial

External Quality Assurance Schemes

Why Quality Control ?

Quality patient care from accurate results

Proper diagnosis Proper treatment Good outcome Lower costs

Legal implications Regulatory requirements

Why COULTER Controls ?

Part of the System approach interlinking:

Reagents, Calibrators, Controls and Instruments

Comprehensive assay procedure with checks and balances Free entry to manufacturers IQAP Scheme for peer comparison

Why COULTER Controls ?

Manufacturers technical support

Distributor has direct access to COULTER product, laboratory and service support

other reports by lot number system information and access replacement product during investigation in-house retained lot- performance on system returned customer product- performance on system

Why COULTER Controls ?

Manufacturers technical support

Problem due to reagent, calibrator, control, or instrument? Reported as failed recovery of the control But investigation invariably shows that the control was doing its job by alerting the operator to a problem elsewhere e.g........

Why COULTER Controls ?

Report - 5C controls at all levels not giving a WBC differential Cause - incorrect chemistry due to non-optimised reagent pumps

Would not be shown by latex or strongly fixed white cell analogues

Why COULTER Controls ?

Report - 5C Abn I & II recovery OK but Normal RBC and Hgb recovering below expected range Cause - Instrument aspiration error due to poor preventative maintenance

shown by high viscosity of Normal control

Why COULTER Controls ?

Report - 5C Abn I & II recovery OK but Normal RBC and Hgb recovering below expected range Cause - Improper mixing of product prior to analysis

Why COULTER Controls ?

Report - All control levels recovering Hgb below expected range Cause - Lyse reagent stored above recommended temperature range or used past open expiry date

Why COULTER Controls ?

Stability claim vs size of expected range

if expected ranges are widened to accommodate longer stability claims the recovered values will not alert the operator to a problem affecting reported results. wider expected ranges for WBC and diff parameters decrease the value of the control in its ability to alert the operator to a problem

Why COULTER Controls ?

Biological sensitivity

Coulter products are of a similar biological nature to patient samples and respond to change in a similar way highly fixed cells have longer stability but will not alert operator to a problem affecting patient results Coulter controls are designed to be sensitive and respond to abnormal conditions

QC Method of Evaluation

1. Accuracy
2. Precision 3. Specificity 4. Sensitivity

Accuracy / Reproducibility(Precision)

Accuracy

Precision

Calibration
Zero bias instruments used in assay process Monitor value recovered vs. value assigned

* When to do Calibration? *Change of reagents lot * Out of Quality Control data * After replacing major part of system *Minimum twice a year

TWO TYPES:

Internal

Instrument QC program XB Reviewing QC data CAP IQAP, Others, QC Program

External

Moving average : Bulls Algorithm

* * * *

MCV, MCH, MCHC 20 patient specimen 1 Batch mean 20 Batch mean Acceptable limits: +

Review Control Data

2. Trend

+2SD
Target - 2SD

Reagent, Control, instrument change

3. Shift

+2SD Target - 2SD

Calibration factor change, Reference value check

Quality Assurance(External)

1. CAP(College of American Pathologists)Survey 2. IQAP(Interlaboratory Quality Assurance Program)

IQAP

Quality assurance program offered to users of Beckman Coulter hematology cell controls and calibrators worldwide.

IQAP

IQAP(Interlaboratory Quality Assurance Program)

IQAP

Has over 30,000 worldwide participants

Data could submitted via disk, hard copy Free to all Coulter control users

IQAP

Country

No. of Participants by hospitals

Hong Kong China Singapore Taiwan Thailand Total

19 22 6 33 78 158

Certificate

Thank You for your kind Attention !