josé-enrique o’connor, ph.d. centro de citometría y citómica … · 2003-07-13 · centro de...

STANDARDIZATION IN FLOW CYTOMETRY

STANDARDIZATION IN STANDARDIZATION IN FLOW CYTOMETRYFLOW CYTOMETRY

JoséJosé--Enrique O’Connor, Ph.D.Enrique O’Connor, Ph.D.Centro de Citometría y CitómicaCentro de Citometría y Citómica

Universitat de ValènciaUniversitat de València

II have looked upon those bright creatureshave looked upon those bright creaturesW.B.W.B. YeatsYeats,,

The Swans at CooleThe Swans at Coole, 1919, 1919

Flow CytometryFlow Cytometry: A: A SelfSelf--DefinitionDefinition

Flow Cyto- -Metry

•Cells or microscopic particlesin appropriate suspension

• Hydrodynamically focusedby pressure-driven flow sheath

• Single-particle analysis• Sample volume: > 150 µL• Sample flow: 10-60 µL/min• Manual or automatic load• Bio-hazard safety

• Large range of cell types• Living or fixed cells• Subcellular elements• Soluble analytes• Fluorescent markers• Intrinsic properties

•Multiple fluorescences•Light Scatter properties•Thousands of events/second•Direct signals•Derived signals•Different types of amplification•Kinetic measurements•On-line and off-line analysis

Flow CytometryFlow Cytometry:: The ApplicationsThe Applications

FCM is applied to a vaste range of cellular and molecular fields,but the relevance of immunological and onco-hematological issueshas turned FCM a significant clinical methodology.


Currently, the trend for routine applications of FCM, both diagnostic and prognostic, is to become partly or entirely automatized.

Analysis:• Direct transfer of carousel• Push AUTO button• Walkaway sample analysis

Analysis:Analysis:•• Direct transfer of carouselDirect transfer of carousel•• Push AUTO buttonPush AUTO button•• WalkawayWalkaway sample analysissample analysis

STANDARDIZATION IN FLOW CYTOMETRYSTANDARDIZATION IN FLOW CYTOMETRYSTANDARDIZATION IN FLOW CYTOMETRY

WhatWhat dodo you makeyou make, so, so fair and brightfair and bright??W.B.W.B. YeatsYeats,,

The CloakThe Cloak,, the Boat and the Shoesthe Boat and the Shoes, 1889, 1889

The need for StandardizationThe need for Standardization

•Because of the clinical implications of FCM, technical development

has been accompanied by strong urge for standardization.

•This effort can be deduced from the number of dedicated committees

and working groups that establish guidelines for standardization

and quality control in FCM, as well as from the increasing

availability of biological and synthetic standards.

Working Groups for StandardizationWorking Groups for Standardization•The USA National Committee for Clinical Laboratory Standards

• The International Federation of Clinical Chemistry:

•Standardization Committee on Clinical Flow Cytometry

•The European Working Group on Clinical Cell Analysis (EWGCCA)

• The UK NEQAS for Leukocyte Immunophenotyping

• The International Society for Analytical Cytology

• The Iberian Society for Cytometry

• Other Working Groups and Cytometric Societies

Working Groups for StandardizationWorking Groups for Standardization

Particles for StandardizationParticles for Standardization

The need for StandardizationThe need for Standardization

While the concept of standardization is esential to all analytical procedures, it is paramount for most clinical applications of FCM, due to:• Automation: routine walk-away multicolor phenotyping • Special sensitivity : rare event detection • Need for calibration:

• Absolute cell count • Antigen quantitation

Integrated Quality Control and StandardizationIntegrated Quality Control and Standardization(QCS)(QCS)

in the Flow Cytometry Laboratoryin the Flow Cytometry Laboratory

Based upon BeckmanBased upon Beckman--Coulter systemsCoulter systems

BeckmanBeckman--Coulter QCS elementsCoulter QCS elements

• QCS Reagents from Beckman Coulter

• Quality Module of SYSTEM II v3 Software for

EPICS® XL™ / XL-MCL™ Flow Cytometer

Daily QCS Flow Chart in EPICS XL/MCLDaily QCS Flow Chart in EPICS XL/MCL

Run Patient Run Patient

Obtain Obtain

Results Results

Generate Generate

ReportReport

PASSPASS

Troubleshooting Troubleshooting MaintenanceMaintenance

FAILFAIL

Call Call ServiceService

Solve Solve ProblemProblem

Lot to Lot Lot to Lot ComparisonComparison

Function & Function & Maintenance Maintenance

ChecksChecks

QC Data QC Data MonitoringMonitoring

QC Data QC Data CollectionCollection

AlignmentAlignment

StandardizationStandardization

CompensationCompensation

VerificationVerification

FAILFAIL

According to NCCLS HAccording to NCCLS H--4242--A guidelinesA guidelines

System II AutostandardizationSystem II Autostandardization

• The Autostandardization Panels in SYSTEM II v3 software automatically record QC data into files for monitoring and reporting on a daily, weekly or monthly basis.

• QC data should be collected daily or whenever application is run.

System II System II AutostandardizationAutostandardization

• Specific QC protocols are linked to create QC panels that automatically

standardize the XL / XL-MCL flow cytometer

• When necesary, System II v3 retrieves the QC files for:

• Maintaining the system standardized

• Recording cytosettings for QCS monitoring


• QC Templates:• Define QC parameters for calibrating, monitoring and reporting

• Include voltages, compensations & region statistics

• Only use regions with “QC” in the name

• Are associated with the protocol

• Can be modified for user specific needs

• Can also be created for new protocols


• QC Templates can be created from:• _A Protocols: provide QC templates to monitor the voltages and gains

of calibration protocols

• _C Protocols: provide QC templates to monitor compensation values

used with fluorescence compensation reagent systems

• _Q Protocols: provide QC templates to monitor statistics, such as CVs,

% positive, peak position, etc., of biological standards and test samples

through the preparation method


• Levey-Jennings Graphs:• Automatically generated for parameters chosen in a QC Template• Can be viewed by Day, Month, 3-Month or 6-Month displays• Use limit criteria either calculated from the data or from user defined

means and SDs• Data Point Information Box displays the information for the highlighted

data point: Red data points are outliers.• WESTG button displays the Westgard analysis rules.

Outliers in Red

Parameters being monitored


System II AutostandardizationSystem II Autostandardization• Lot Number Pop-Up Box:• Lot to lot comparison of reagents to ensure new lots of reagents compare

with old lots of reagents before placing new lots in service.

• Displayed statistics: mean, SD, and CV of compared data

• Compare Screen:• Compare two different QC files.Two QCS for two QCC files.• The RD REF button uses the selected runs in each file to calculate the

stats for comparison. • Compare two different lots of QC beads when performing new lot

validation procedures.• Compare QC data over extended periods of time.

Beads forBeads for QCSQCS

• Flow-CheckTM Fluorospheres:• Verification of instrument alignment and fluidics


• Flow-SetTM Fluorospheres:• Automatic adjustment of high voltage and gains

J =Target Region I = Capture Region

Target Region JTarget Region J

FlowFlow--Set AutostandardizationSet Autostandardization

_A 4color Flow_A 4color Flow--SetSet QCS File: QCS File: Voltages & gains Voltages & gains --check or adjustcheck or adjust

QCS PanelQCS PanelQCS Panel


• Immuno-Brite™ Fluorospheres:• Monitor fluorescence resolution• Used to assess instrument linearity • According to NCCLS Guidelines H-42-A section 10.2.2

Cells forCells for QCSQCS

• CYTO-COMP™ Cell Kit & Reagent Kit:• Automatic Color Compensation with mutually exclusive

markers for each dye combination

• Monitor fluorescence compensation according to NCCLS Guidelines H-42-A section 10.1.2.2[3]

• Freshly prepared specimen stained with relevant fluorochrome with acceptance values established under appropriate compensation settings

_C1 FITC/RD1 Cyto-Comp_C2 RD1/ECD Cyto-Comp_C3 ECD/PC5 Cyto-Comp_C4 RD1/ECD Cyto-Comp

Read Baseline QCS file and write compensations back to it

CytoCyto--Comp Autostandardization Comp Autostandardization

_A 4color Flow-SetQCS File: Voltages & gains

-check or adjust

QCS PanelQCS Panel

Cells forCells for QCSQCS• Immuno-TROLTM Control Cells:• Process and method control• According to NCCLS Guidelines H-42-A section 10.2.4• Normal specimen prepared using laboratory’s standard

test protocol and reagents with established laboratory reference values

Whole Blood Immuno-TROL

_C_C11 FITC/RD1FITC/RD1 CytoCyto--CompComp_C_C22 RD1/ECD RD1/ECD CytoCyto--CompComp_C_C33 ECD/PC5 ECD/PC5 CytoCyto--CompComp_C_C44 RD1/ECD RD1/ECD CytoCyto--CompComp

Read Baseline QCS Read Baseline QCS file and write file and write compensations back compensations back to itto it

ImmunoImmuno--TROL AutostandardizationTROL Autostandardization

_A CD34 Flow_A CD34 Flow--SetSetQCS File: QCS File: Voltages & gains Voltages & gains

--check or adjustcheck or adjust

QCS PanelQCS Panel

_V_V ImmunoTrolImmunoTrolVerifies Verifies automated setautomated set--upup

Cells forCells for QCSQCS

• Stem-Trol™ Control Cells• Verify instrument and staining performance for stem cell

enumeration.• Provides percent and absolute number Quality Control for

stem cells.

Summary of Automated QCSSummary of Automated QCS• Verifies instrument alignment and linearity• Standardizes light scatter and fluorescence

intensities• Adjusts color compensation• Verifies antibody function• Verifies lysing process• Determines direct absolute count results• Creates Data Base • Creates Levey-Jennings Charts

Total Automation in EPICS XL/MCLTotal Automation in EPICS XL/MCL

Run Patient Run Patient sample with Tetra sample with Tetra

One SystemsOne Systems

Obtain Obtain ResultsResults

Generate Generate ReportReport

PASPASSS

TroubleshootTroubleshootMaintenanceMaintenance

FAILFAIL

Call Call ServiceService

Fix Fix ProblemProblem

FAILFAIL

FlowFlow--CheckCheck

FlowFlow--SetSet

CytoCyto--CompComp

ImmunoImmuno--TROLTROLStemStem--TROLTROL

SystemSystem IIII

SystemSystem IIII

SystemSystem IIII

SystemSystem IIII

CALIBRATION IN FLOW CYTOMETRYCALIBRATION IN FLOW CYTOMETRYCALIBRATION IN FLOW CYTOMETRY

II saw ninesaw nine--andand--fifty swansfifty swansW.B.W.B. YeatsYeats,,

The Swans at CooleThe Swans at Coole, 1919, 1919

Cell count CalibrationCell count Calibration

• Flow-Count™ Fluorospheres:• Monitoring absolute count values, as in peripheral blood

cell populations in HIV disease or in blood Stem Cell harvests

• According to NCCLS Guidelines H-42-A section 10.2.4

gpIIb/IIIa (MFI)

Cel

l Cou

nt

MAb Molec / platelet

100

1 000

10 000

100 000

0,1 1 10 100 1000

A (500)

B (7 000)

C (23 000)

D (60 000)

Fluorescence intensity (arbitrary units)

4014.2

13,00039,000

Normal Donor

Anti-GpIIb/IIIa treated

GpIIb/IIIa occupancy CalibrationGpIIb/IIIa occupancy Calibration

Occupancy Ratio: 66.7% Occupancy Ratio: 66.7%

Intracellular pH CalibrationIntracellular pH Calibration

NH4ClNa Propionate

Changes in fluorescence ratio Fl1/FL3 in platelets stained with BCECF

pH6.6 pH7.1 pH7.6

FL1/FL3

Calibration curve for pHi changesin whole blood platelets stained with BCECF

7,9 7,4 6,9 6,4 70

80

90

100

pH

FL1/FL3

Changes in platelet ratio FL1/FL3 in K+HEPESbuffer containing nigericine, at different pHs

Platelet pHi: 7.2 ∆ sodium propionate: - 0.6 pH units∆ ammonium chloride: + 0.25 pH units

Enzyme Kinetics CalibrationEnzyme Kinetics CalibrationRhodamine 123

Uptake rate

More on Flow Cytometry?More on Flow Cytometry?

This presentation and other material related to basic and clinical applications of flow cytometry can be downloaded from our Web site at:

http://www.uv.es/cytomics

Centro de Citometría y CitómicaDepartamento de Bioquímica y Biología MolecularFacultad de Medicina, Universidad de Valenciae-mail: [email protected]: +34 963 864186

josé-enrique o’connor, ph.d. centro de citometría y citómica … · 2003-07-13 · centro de...

Documents