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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
Beckman Coulter 47%
53%
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
More automated differentials per day are performed using the science of Coulter VCS technology than any other method
Why? - Because it is the best tool for the job
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 es 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 from cells they are insulator
Current does not from cells they are insulator
Electrolytes solutions allow current to from them
Electrolyte Like ISOTON III
The Coulter principle Red Blood Cell
A red cell es through RBC aperture
Sensing Zone
Oscilloscope Oscilloscope
The Coulter principle Neutrophil
A White cell es 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 AP1(4.21)
AP2(4.14)
2. Result is NOT reported = ----AP1(4.21) AP2(3.82)
Benefits: - Eliminate imprecision - Prevents data error - Flags possible problems
AP3(5.01)
AP3(5.01)
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 ing 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 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
RBC’s are eliminated by an advanced, lytic reagent Cells below 35 fl are excluded from the WBC count Fragile WBC’s 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 / µl reduces repeats
Coulter WBC histogram
Mono
Lymp
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 band filter centered @ 525nm
Multiple reagent blanks
Hgb Measurement In Beckman Coulter Instruments
WBC Bath
ADC
Signal Processor
HGB Lamp
Derivation Absorbance= log 10 ( VR / Vs ) Where VR = Reference voltage Vs = Sample voltage
Sample
HGB Sensor & PRE-AMP
Broad Spectrum HGB Readings Narrow band Fileter
Broad band 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 MCV’s 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/µL
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
age 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 / µl.
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 technology showed a better
0 COULTER Log Fit PLT Count
overall correlation coefficient.
1,000,000
Laboratory Hematology 4:80-87 © 1998 Carden Jennings Publishing Co. Ltd.
0
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 4:264–268 “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® Gen•S ™ hematology analyzer E. GRIMALDI, F. SCOPACASA Laboratory Hematology 4:264–268 “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 Gen•S.”
Beckman Coulter’s VCS Technology Beckman Coulter’s 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 RBC’s Stabilyse returns the WBC’s to their near native size
Neut
Eo
Lymph
WBC Swelling
WBC Shrinkage
Lys is Nuclei
RBC Swelling
RBC ghos t, fragment, dis olve
ErythroLys e
StabiLys e
RBC
Blood
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 cell’s volume is determined in the electro-optical flow cell and for the first time in history, a cell’s near native state volume is measured. Coulter’s 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
V o l u m e
RF
Not good seperation of each cell population Cell’s size effect RF signal Due to this, LY, MO, GR population is not clear
Opacity
V o l u m e
Adjust RF signal by remove cells size data Opacity: Reflect the cell’s true nuclear density & internal Composition
Opacity
SCATTER MEASUREMENT As cells are ing 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
V o l u m e
Size of cell effects light scatter data especially severe at low angle detection Not clear separation of cell population
Scatter
Rotated Light Scatter (RLS)
V o l u m e
RLS
Median Angle Light Scatter (MALS) Signals by RLS measurement reflect true granularity & surface characteristics of cells,resulting good separation of each cell population.
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
Gen•S System 2
STKS
Primary Analysis Science(s) Native State WBC Chemistry
HmX
MAXM
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 Gen•S™
Intellikinetics
AccuGate™ software
New colour - coding system
Ongoing Flagging development
TM
Application
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
1. Reagent Temp 2. Reagent Vol 3. Reaction Time
VCS Measurement
Mixing Chamber
Intellikinetics Applied
Flow Cell
Intellikinetics 1. Reagent Temp
2. Reagent Volume
3. Reaction Time
TM
Intellikinetics
Diluted Blood to Mixing Chamber
TM
Intellikinetics
Intellikinetics Applied
TM
Application
Intellikinetics
VCS measurement in flow cell
TM
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 Gen•S™
Intellikinetics
AccuGate
New colour - coding system
Ongoing Flagging development
TM
TM
Application
software
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 Gen•S™
Intellikinetics
AccuGate
New colour - coding system
Ongoing Flagging development
TM
TM
Application
software
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 Gen•S™
Intellikinetics
AccuGate™ software
New colour - coding system
Ongoing Flagging development
TM
Application
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) 3 Immature Granulocytes
2 1 4
4 Band Neutrophils
3
5 Lympho-Blasts (LY Blast Flag)
5 6
7 8
9
10
6
Variant Lymphocytes
7
Low Volume Lymphocytes
8
PLT Clumps
9
Giant Platelets
10
RBC Parasites (Malaria, etc)
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 RBC’s, WBC’s, PLT’s and NRBC’s Reticulocytes are stained with a supravital stain (NMB)
Prior to flow cytometric analysis, the RBC’s 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%
Source: Reticulocyte performance on the COULTER System:Lab Hematol. 3:41-47 1997
Gen·S
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 WBC’s & NRBC’s
MI = HLR count
Total Retics Mature RBC’s Retics
Mature Young Platelets
PBSC Transplantation in NHL
MI MFR + HFR
0
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
Sickle Thalassemia
Interpretive Messages Laboratory Parameters
HLR % and # (High Light Scatter Retics) MSCV (Mean Sphered Cell Volume)
Monitor Marrow Recovery
More Diagnostic Information
4
4
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 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 - 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
Condition
Suspect
Definitive
WBC
Normal WBC Pop Abnormal WBC Pop No message
NE Blasts Imm NE 1 Imm NE 2 Variant Lymphs Review Slide
Leukopenia Leukocytosis Neutropenia Neutrophilia Lymphopenia Lymphocytosis Monocytosis Eosinophilia Basophilia
RBC
Normal RBC Pop Abnormal RBC Pop No message
NRBCs Dimorphilc RBC Pop Micro RBCs/RBC Fragments RBC Agglutination
Anemia Anisocytosis Microcytosis Marocytosis Hypochromia Poikilocytosis Erythrocytosis Pancytopenia
PLT
Normal WBC Pop Abnormal WBC Pop No message
Platelate Clumps Giant Platelates
Thrombocytopenia Thrombocytosis Small Platelates Large Platelae
Automated RBC Morphology
Anisocytosis +, ++, +++ Microcytosis +, ++, +++ Macrocytosis +, ++, +++ Hypochromia +, ++, +++ Poikilocytosis +, ++, +++ Dimorphic RBC Population Micro RBC’s/RBC Fragments RBC Agglutination
Flag- (3) Codes -Software define Code flagging
< Code> ----
Total Vote out
R
Review
P
Partial Aspiration
H/L High/Low …… ……
No Diff
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 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 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?
Gen•S™ gives the control over sensitivity they wish to obtain
• Provide a variety of
•
• •
sensitivity settings based on performance in NCCLS tests Give s 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 Definable
Age, Gender & Location
Reference Intervals Action Limits Critical Limits Definitive Message Limits Graded RBC Morphology
Excellence in Decision
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 Defined “Review by Exception” rules-based system
What is Decision ?
A programmed environment that automates the review of every sample analyzed:
Normal, or Expected results are ed through for reporting
Abnormal, or Unexpected results are held for operator review and validation
Why Automate Decision ?
Consistency in handling all results
Appropriate follow-up can be pre-programmed
Eliminates
Automates Slide Making & Review Protocols Differences in personnel experience Procedural differences when staff work at more than one site
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 Gen•S
SE 9500
STKS
257
257
257
False Positive False Negative True Positive True Negative
6.23% 0.39% 3.50% 89.88%
28.02% 0.00% 3.89% 68.09%
8.95% 0.00% 3.89% 87.16%
FP/TOTAL #
Rate Review Rate
90.27% 9.73%
68.09% 31.91%
87.16% 12.84%
(TN + FN)/TOTAL #
Sensitivity (% TP) Specificity (% TN)
90.00% 93.52%
100.00% 70.85%
100.00% 90.69%
TP/(TP + FN)
Predictive Value Of Neg Predictive Value Of Pos
99.57% 36.00%
100.00% 12.20%
100.00% 30.30%
TN/(TN + FN)
Efficiency
93.39%
71.98%
91.05%
n
Central US - Publication Pending
FN/TOTAL # TP/TOTAL # TN/TOTAL #
(TP + FP)/TOTAL #
TN/(TN + FP)
TP/(TP + FP) (TP + TN)/TOTAL #
...
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 ?
Results from External (Customer) Evaluations Gen•S vs Competition Difference False Negative (FN) rate
3.7 vs 5.4%
32% lower
False Positive (FP) rate
12 vs 23%
48% lower
Review rate
23 vs 32 %
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) 3 Immature Granulocytes
2 1 4
4 Band Neutrophils
3
5 Lympho-Blasts (LY Blast Flag)
5 6
7 8
9
10
6
Variant Lymphocytes
7
Low Volume Lymphocytes
8
PLT Clumps
9
Giant Platelets
10
RBC Parasites (Malaria, etc)
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
Suspect flags: • Lymphoblasts
Retics
• Low Volume Lymphocytes
RBCs
Blood Film
Plts.
Small Lymphoblasts
Case 1 Instrument: HmX
Instrument Discovery: AML - M1
13.2 3.3 7.7 87.4
Case 2 Instrument: Gen•S
0.1 1.5
121.2
0.4
94.7
1.0
4.8
11.6
0.3
0.0
0.1
0.2
Suspect: Blasts
0.1 114.8 5.8 0.4
Suspect: NE Blasts
0.1 0.1
Clinical Flow Diagnostic : AML - M1
NTIAL 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 *
Instrument: XL / MCL
Case 1 Instrument: HmX
Instrument Discovery: AML - M5
78.3 17.0 5.8 76.2
Case 2 Instrument: Gen•S
1.0 0.0
64.2
13.3
5.7
4.5
15.6
59.7
78.3
0.8 0.0
0.1
Suspect: Blasts
0.3 3.7 10.0 50.2
Suspect: MO Blasts
0.1 0.2
Clinical Flow Diagnostic : AML - M5
ENTIAL
TES
4 7 47 0 0 2 6 34
CD MARKER CD2 CD3 CD5 CD7 CD10 CD19 CD20 HLA-DR CD13
CD14 CD33 CD34
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 *
MONOCYTIC EARLY MYELOCYTIC; MONOCYTIC PRECURSORS
Instrument: XL / MCL
81.6 * 91.2 * 44.1 *
Case 1 Instrument: HmX
Instrument Discovery: CLL
96.4 2.9 93.4 3.1
Case 2 Instrument: Gen•S
0.1 0.5
31.3
2.8
14.6
90.0
80.9
3.0
3.5
0.1
0.8
0.5
Suspect: Blasts Suspect: Atyp. Lymph
0.2 4.6 25.3 1.1
Suspect: Var. Lymph
0.3 0.1
ENTIAL 16 84 0 0 0
Clinical Flow Diagnostic : 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
Instrument: XL / MCL
Hematology
Discovers and Monitors
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 s 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 Coulter’s 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
External Quality Assurance Schemes
national commercial
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
Distributor has direct access to COULTER product, laboratory and service
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 ?
Manufacturer’s technical
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
External
CAP IQAP, Others, QC Program
Moving average : Bull’s Algorithm
* * * *
MCV, MCH, MCHC 20 patient specimen 1 Batch mean 20 Batch mean Acceptable limits: +３％
＋３％ ８９．５ －３％
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 s 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 s
IQAP
Country
Hong Kong China Singapore Taiwan Thailand Total
No. of Participants by hospitals
19 22 6 33 78 158
Certificate
Thank You for your kind Attention !
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
Beckman Coulter 47%
53%
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
More automated differentials per day are performed using the science of Coulter VCS technology than any other method
Why? - Because it is the best tool for the job
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 es 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 from cells they are insulator
Current does not from cells they are insulator
Electrolytes solutions allow current to from them
Electrolyte Like ISOTON III
The Coulter principle Red Blood Cell
A red cell es through RBC aperture
Sensing Zone
Oscilloscope Oscilloscope
The Coulter principle Neutrophil
A White cell es 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 AP1(4.21)
AP2(4.14)
2. Result is NOT reported = ----AP1(4.21) AP2(3.82)
Benefits: - Eliminate imprecision - Prevents data error - Flags possible problems
AP3(5.01)
AP3(5.01)
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 ing 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 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
RBC’s are eliminated by an advanced, lytic reagent Cells below 35 fl are excluded from the WBC count Fragile WBC’s 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 / µl reduces repeats
Coulter WBC histogram
Mono
Lymp
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 band filter centered @ 525nm
Multiple reagent blanks
Hgb Measurement In Beckman Coulter Instruments
WBC Bath
ADC
Signal Processor
HGB Lamp
Derivation Absorbance= log 10 ( VR / Vs ) Where VR = Reference voltage Vs = Sample voltage
Sample
HGB Sensor & PRE-AMP
Broad Spectrum HGB Readings Narrow band Fileter
Broad band 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 MCV’s 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/µL
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
age 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 / µl.
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 technology showed a better
0 COULTER Log Fit PLT Count
overall correlation coefficient.
1,000,000
Laboratory Hematology 4:80-87 © 1998 Carden Jennings Publishing Co. Ltd.
0
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 4:264–268 “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® Gen•S ™ hematology analyzer E. GRIMALDI, F. SCOPACASA Laboratory Hematology 4:264–268 “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 Gen•S.”
Beckman Coulter’s VCS Technology Beckman Coulter’s 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 RBC’s Stabilyse returns the WBC’s to their near native size
Neut
Eo
Lymph
WBC Swelling
WBC Shrinkage
Lys is Nuclei
RBC Swelling
RBC ghos t, fragment, dis olve
ErythroLys e
StabiLys e
RBC
Blood
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 cell’s volume is determined in the electro-optical flow cell and for the first time in history, a cell’s near native state volume is measured. Coulter’s 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
V o l u m e
RF
Not good seperation of each cell population Cell’s size effect RF signal Due to this, LY, MO, GR population is not clear
Opacity
V o l u m e
Adjust RF signal by remove cells size data Opacity: Reflect the cell’s true nuclear density & internal Composition
Opacity
SCATTER MEASUREMENT As cells are ing 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
V o l u m e
Size of cell effects light scatter data especially severe at low angle detection Not clear separation of cell population
Scatter
Rotated Light Scatter (RLS)
V o l u m e
RLS
Median Angle Light Scatter (MALS) Signals by RLS measurement reflect true granularity & surface characteristics of cells,resulting good separation of each cell population.
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
Gen•S System 2
STKS
Primary Analysis Science(s) Native State WBC Chemistry
HmX
MAXM
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 Gen•S™
Intellikinetics
AccuGate™ software
New colour - coding system
Ongoing Flagging development
TM
Application
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
1. Reagent Temp 2. Reagent Vol 3. Reaction Time
VCS Measurement
Mixing Chamber
Intellikinetics Applied
Flow Cell
Intellikinetics 1. Reagent Temp
2. Reagent Volume
3. Reaction Time
TM
Intellikinetics
Diluted Blood to Mixing Chamber
TM
Intellikinetics
Intellikinetics Applied
TM
Application
Intellikinetics
VCS measurement in flow cell
TM
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 Gen•S™
Intellikinetics
AccuGate
New colour - coding system
Ongoing Flagging development
TM
TM
Application
software
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 Gen•S™
Intellikinetics
AccuGate
New colour - coding system
Ongoing Flagging development
TM
TM
Application
software
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 Gen•S™
Intellikinetics
AccuGate™ software
New colour - coding system
Ongoing Flagging development
TM
Application
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) 3 Immature Granulocytes
2 1 4
4 Band Neutrophils
3
5 Lympho-Blasts (LY Blast Flag)
5 6
7 8
9
10
6
Variant Lymphocytes
7
Low Volume Lymphocytes
8
PLT Clumps
9
Giant Platelets
10
RBC Parasites (Malaria, etc)
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 RBC’s, WBC’s, PLT’s and NRBC’s Reticulocytes are stained with a supravital stain (NMB)
Prior to flow cytometric analysis, the RBC’s 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%
Source: Reticulocyte performance on the COULTER System:Lab Hematol. 3:41-47 1997
Gen·S
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 WBC’s & NRBC’s
MI = HLR count
Total Retics Mature RBC’s Retics
Mature Young Platelets
PBSC Transplantation in NHL
MI MFR + HFR
0
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
Sickle Thalassemia
Interpretive Messages Laboratory Parameters
HLR % and # (High Light Scatter Retics) MSCV (Mean Sphered Cell Volume)
Monitor Marrow Recovery
More Diagnostic Information
4
4
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 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 - 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
Condition
Suspect
Definitive
WBC
Normal WBC Pop Abnormal WBC Pop No message
NE Blasts Imm NE 1 Imm NE 2 Variant Lymphs Review Slide
Leukopenia Leukocytosis Neutropenia Neutrophilia Lymphopenia Lymphocytosis Monocytosis Eosinophilia Basophilia
RBC
Normal RBC Pop Abnormal RBC Pop No message
NRBCs Dimorphilc RBC Pop Micro RBCs/RBC Fragments RBC Agglutination
Anemia Anisocytosis Microcytosis Marocytosis Hypochromia Poikilocytosis Erythrocytosis Pancytopenia
PLT
Normal WBC Pop Abnormal WBC Pop No message
Platelate Clumps Giant Platelates
Thrombocytopenia Thrombocytosis Small Platelates Large Platelae
Automated RBC Morphology
Anisocytosis +, ++, +++ Microcytosis +, ++, +++ Macrocytosis +, ++, +++ Hypochromia +, ++, +++ Poikilocytosis +, ++, +++ Dimorphic RBC Population Micro RBC’s/RBC Fragments RBC Agglutination
Flag- (3) Codes -Software define Code flagging
< Code> ----
Total Vote out
R
Review
P
Partial Aspiration
H/L High/Low …… ……
No Diff
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 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 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?
Gen•S™ gives the control over sensitivity they wish to obtain
• Provide a variety of
•
• •
sensitivity settings based on performance in NCCLS tests Give s 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 Definable
Age, Gender & Location
Reference Intervals Action Limits Critical Limits Definitive Message Limits Graded RBC Morphology
Excellence in Decision
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 Defined “Review by Exception” rules-based system
What is Decision ?
A programmed environment that automates the review of every sample analyzed:
Normal, or Expected results are ed through for reporting
Abnormal, or Unexpected results are held for operator review and validation
Why Automate Decision ?
Consistency in handling all results
Appropriate follow-up can be pre-programmed
Eliminates
Automates Slide Making & Review Protocols Differences in personnel experience Procedural differences when staff work at more than one site
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 Gen•S
SE 9500
STKS
257
257
257
False Positive False Negative True Positive True Negative
6.23% 0.39% 3.50% 89.88%
28.02% 0.00% 3.89% 68.09%
8.95% 0.00% 3.89% 87.16%
FP/TOTAL #
Rate Review Rate
90.27% 9.73%
68.09% 31.91%
87.16% 12.84%
(TN + FN)/TOTAL #
Sensitivity (% TP) Specificity (% TN)
90.00% 93.52%
100.00% 70.85%
100.00% 90.69%
TP/(TP + FN)
Predictive Value Of Neg Predictive Value Of Pos
99.57% 36.00%
100.00% 12.20%
100.00% 30.30%
TN/(TN + FN)
Efficiency
93.39%
71.98%
91.05%
n
Central US - Publication Pending
FN/TOTAL # TP/TOTAL # TN/TOTAL #
(TP + FP)/TOTAL #
TN/(TN + FP)
TP/(TP + FP) (TP + TN)/TOTAL #
...
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 ?
Results from External (Customer) Evaluations Gen•S vs Competition Difference False Negative (FN) rate
3.7 vs 5.4%
32% lower
False Positive (FP) rate
12 vs 23%
48% lower
Review rate
23 vs 32 %
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) 3 Immature Granulocytes
2 1 4
4 Band Neutrophils
3
5 Lympho-Blasts (LY Blast Flag)
5 6
7 8
9
10
6
Variant Lymphocytes
7
Low Volume Lymphocytes
8
PLT Clumps
9
Giant Platelets
10
RBC Parasites (Malaria, etc)
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
Suspect flags: • Lymphoblasts
Retics
• Low Volume Lymphocytes
RBCs
Blood Film
Plts.
Small Lymphoblasts
Case 1 Instrument: HmX
Instrument Discovery: AML - M1
13.2 3.3 7.7 87.4
Case 2 Instrument: Gen•S
0.1 1.5
121.2
0.4
94.7
1.0
4.8
11.6
0.3
0.0
0.1
0.2
Suspect: Blasts
0.1 114.8 5.8 0.4
Suspect: NE Blasts
0.1 0.1
Clinical Flow Diagnostic : AML - M1
NTIAL 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 *
Instrument: XL / MCL
Case 1 Instrument: HmX
Instrument Discovery: AML - M5
78.3 17.0 5.8 76.2
Case 2 Instrument: Gen•S
1.0 0.0
64.2
13.3
5.7
4.5
15.6
59.7
78.3
0.8 0.0
0.1
Suspect: Blasts
0.3 3.7 10.0 50.2
Suspect: MO Blasts
0.1 0.2
Clinical Flow Diagnostic : AML - M5
ENTIAL
TES
4 7 47 0 0 2 6 34
CD MARKER CD2 CD3 CD5 CD7 CD10 CD19 CD20 HLA-DR CD13
CD14 CD33 CD34
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 *
MONOCYTIC EARLY MYELOCYTIC; MONOCYTIC PRECURSORS
Instrument: XL / MCL
81.6 * 91.2 * 44.1 *
Case 1 Instrument: HmX
Instrument Discovery: CLL
96.4 2.9 93.4 3.1
Case 2 Instrument: Gen•S
0.1 0.5
31.3
2.8
14.6
90.0
80.9
3.0
3.5
0.1
0.8
0.5
Suspect: Blasts Suspect: Atyp. Lymph
0.2 4.6 25.3 1.1
Suspect: Var. Lymph
0.3 0.1
ENTIAL 16 84 0 0 0
Clinical Flow Diagnostic : 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
Instrument: XL / MCL
Hematology
Discovers and Monitors
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 s 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 Coulter’s 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
External Quality Assurance Schemes
national commercial
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
Distributor has direct access to COULTER product, laboratory and service
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 ?
Manufacturer’s technical
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
External
CAP IQAP, Others, QC Program
Moving average : Bull’s Algorithm
* * * *
MCV, MCH, MCHC 20 patient specimen 1 Batch mean 20 Batch mean Acceptable limits: +３％
＋３％ ８９．５ －３％
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 s 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 s
IQAP
Country
Hong Kong China Singapore Taiwan Thailand Total
No. of Participants by hospitals
19 22 6 33 78 158
Certificate
Thank You for your kind Attention !
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