Instrumentation & Sample Preparation
Transcript of Instrumentation & Sample Preparation
Instrumentation & Sample Preparation
Amr Rajab BSc, SCYM (ASCP)CM
Flow cytometry Technical Specialist
LifeLabs, Toronto, Canada
TOPICS
✓Instruments Qualification (IQ, OQ, PQ)✓Review of Instrument Quality Control.✓Understand how to optimize your Instrument including PMT
settings.✓How to Setup Compensation.✓Reagents QC✓Optimization of Specimen Preparation.✓Result QC
DEFINITION OF FLOW CYTOMETRY...
FLOW CYTOMETRY is defined as the measurement of cells in a flow system that has been designed to deliver particles (cells) in single file past a point of measurement.
A basic flow cytometer consists of a light source (laser), a lens system to focus the light into the flow cell, a flow cell, optical components to direct light to the detectors, electronic components to covert the light signals and a computer to analyse the data.
Application of Flow cytometry…✓ Antipolymorphonuclear neutrophils (PMN) titer✓ Antigen-specific T-cell detection (multimer staining)✓ Apoptosis and viability analysis✓ CD34 stem cell counts✓ Chronic granulomatous disease and other genetic causes of PMN dysfunction detection✓ Fetal maternal hemoglobin detection✓ Functional assays such as intracellular cytokine detection✓ HLA-B27 measurement✓ Immunoplatelet counts (CD61, CD42, CD41)✓ Infection/sepsis (CD64 expression levels) detection✓ Leukemia/lymphoma/myelodysplasia detection✓ Lymphocyte enumeration (T cells, B cells, NK cells)✓ Lymphocyte HLA crossmatches for solid organ transplants✓ MRD monitoring✓ Phosphoflow assays✓ PNH evaluation✓ Proliferation assays✓ RO assays✓ Reticulated platelets or immature platelet fraction measurement✓ Reticulocytes, including immature reticulocyte fraction measurement✓ T- and B-cell immune profiling (eg, naïve, memory, proliferation, activation, exhaustion) CLSI H62
Question 1: For those performing L/L analysis, what instrument is used?
A. BD FACSCalibur
B. BD FACSCanto
C. BD FACSLyric
D. BD LSRII or Fortessa
E. Coulter FC500
F. Coulter Gallios/Navios
G. Coulter Cytoflex
H. Other
I. Don’t know5
Questions to lab Director/Manager
What is the purpose of assay/
assays to be developed?
Sample availability?
Which instrument to acquire?
Do we have the required Medical
and Technical expertise?
Is there demand for the intended
assay/assays?
Instrument Validation (IQ, OQ & PQ)
Objective: The objective of the validation is to provide documented evidence that the instrument is installed, operates and performs according to the Laboratory and manufacturer specifications and requirements.
Study Outline:
✓ Required validation steps
✓ Activities
✓ Responsibilities
✓ Timelines
✓ Acceptance criteria
✓ Approval
Installation Qualification (IQ)✓Pre-installation
1. Site fulfillment of the manufacturer’s recommendations (e.g.: electrical requirements, environmental conditions, vibration level and safety features)
2. Sufficient space
✓Physical installation
Parts and accessories Manuals
Computers and software Filters
LasersConsumables
Operational Qualification (OQ)Verify that the instrument operates according to the vendor’s specifications and the lab’s requirements
✓ Daily Start-up & Shutdown procedures
✓ Instrument maintenance (daily, weekly & monthly)
✓ Loading and unloading of reagents
✓ Monitoring expiration dates of reagents
✓ Instrument Quality Control procedures
✓ Running samples using different modes
✓ System alarms and error codes
✓ LIS interface
✓ Reporting results
✓ Security limits
Operational Qualification (OQ)Documents were developed as part of the OQ:
✓ SOPs and forms
✓ Training protocol
✓Competency assessment
✓ Preventative maintenance protocol
Performance Qualification (PQ)Confirm that the instrument functions according to laboratory, regulatory and accrediting agency requirements and consistently with the manufacturer’s claims.
✓Instrument’s Precision and stability study
✓Instrument Sensitivity and Linearity
✓Assessment of Carryover
✓Instrument’s PMTs optimization and compensation
✓ Precision Study
✓Accuracy
Validation References1. CLSI EP12-A2: User Protocol for Evaluation of Qualitative Test Performance - Second Edition – Approved Guideline. Wayne,
PA:National Committee for Clinical Laboratory Standards, 2008.
2. CLSI Document H43-A2, Volume 27, No.11. Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved Guideline--Second Edition, April 2007.
3. CLSI. A Framework for using CLSI documents to evaluate clinical laboratory measurement procedures. 2d ed. CLSI report EP19. Wayne, PA: CLSI, 2015.
4. CLSI. Laboratory Instrument Implementation, Verification, and Maintenance; Approved Guideline. CLSI GP31-A. Wayne, PA:CLSI; 2009
5. Owens MA, Vall HG, Hurley AA, Wormsley SB. Validation and quality control of immunophenotyping in clinical flow cytometry. J Immunol Meth 2000;243:33–50.
6. Cunliffe J, Derbyshire N, Keeler S, Coldwell R. An Approach to the validation of flow cytometry methods. Pharm Res 2009;26:2551.
7. O’Hara D, Xu Y, Lianz E, Reddy M, Wu D, Litwin V. Recommendations for the Validation of Flow Cytometric Testing During Drug Development: II Assays. J Immunol Meth 2011;363:120.
8. Brent Wood, Dragan Jevremovic, Marie C. Bene, Ming Yan, Patrick Jacobs, Virginia Litwin; on behalf of ICSH/ICCS Working Group. Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS – Part V – Assay Performance Criteria. Cytometry Part B (Clinical Cytometry) 84B:315–323 (2013).
9. Amr Rajab, Afaf Erfaei, Laith Hatahet & Miranda Wozniak. Validation of a Flow Cytometer Instrument for Leukemia/Lymphoma Immunophenotyping. ICCS e-Newsletter VOL. VIII No.2, Spring 2017
http://www.cytometry.org/public/newsletters/eICCS-8-2/article6.php
Published Guidelines
Pre-analytic
Analytic
Post analytic
Performance specifications
Instrument Quality Control
14
Why QC?
• Regulatory Requirements
FLO.24300 QC Confirmation of Acceptability
FLO.24475 Monthly QC Review
FLO.23800 QC - Single/Dual Platform Tests
FLO.23925 QC Range Verification
FLO.24230 QC Corrective Action
FLO.24250 QC Handling
FLO.24300 QC Confirmation of Acceptability
FLO.24475 Monthly QC Review
FLO.24650 Comparability of Instrument/Method
FLO.25100 Function Checks
FLO.25150 Optical Alignment
FLO.25250 Instrument/Equipment Service Records
FLO.25300 Instrument/Equipment Maintenance Review
FLO.30250 Fluorochrome Standards
FLO.30260 Color Compensation Settings
FLO.30270 Laser Current
FLO.30280 Calibration/Laser Review
Step 1 - Initial Instrument Setup
Getting instrument “Bead Ready”
• Start up and Check the Fluidics.
• Perform a Clean Procedure.
• Run Daily QC Beads:
✓CS &T and 7-Color Setup Beads (BD)
✓Flow Check and Flow Set (Beckman Coulter)
Daily Beads – Flow Check Pro▪ Purpose: To verify instrument optical alignment and fluidics
▪ Problem: Increased CV of population signals the need for alignment
17
QC: Fluidics and Flow Rate
✓Fluidics performance has a direct impact on quality results.
✓Poor fluidics performance effects resolution, linearity, and sensitivity.
✓Most QC Beads include indicators that ascertain fluidics performance (flow rate, pressure, etc.)
Daily Beads – FlowSet▪ Purpose: To verify instrument’s performance and standardization
19
Lacombe F,et al. Harmonemia: a universal strategy for flow cytometry immunophenotyping. A European
LeukemiaNet WP10 study. Leukemia. 2016, 30(8):1769-72.
◼Compensation LifeLabs
Standardization
◼Red events: Lab1
◼Blue events: Lab2
Standardization
DAILY COMPENSATION VERIFICATION
DAILY COMPENSATION VERIFICATION
Assessment of Carryover
24
Total events % Carryover
Flow Check Pro 9851 -
Iso Flow 1 71 0.72%
Iso Flow 2 51 0.51%
Final Result PASS
Maximum Carryover %= 1%
25
Instrument’s Sensitivity and linearity
26
27
Proficiency Testing Programs
✓ IQMH: FLOW-HD, FLOW-HV
✓ CAP: FL3, FL5, PNH, FL1, LN22
Instrument QC Schedule
✓Verify laser output – Daily (beads)
✓Verify optical alignment – Daily (beads)
✓Monitor analysis ranges - Daily(beads)✓Light scatter
✓Fluorescence
✓Verify compensation - Daily (cells)✓Set up: initial optimization & after major maintenance
✓Verify multiple instrumentation so they give the same results – Bi-annually
✓Assessment of carryover - Bi-annually
✓Verify sensitivity and linearity – Annually
Instrument Optimization
Step 2 – Instrument Optimization
⚫ Getting instrument “Patient Ready”
⚫ Optimizing each PMT using Biological controls.
⚫ Setting up Compensation for each fluorochrome.
⚫ Fine-Tuning Compensation Using Fully Stained Samples
Voltage Adjustment for FS & SS
31
FS too low Increased FS
• Set discriminator to eliminate majority of platelets and debris
• Some debris should be left to allow for appropriate debris gate setting
• Cells below discriminator are lost forever
Andrea Illingworth. Instrument Optimization – Adjusting PMT voltages and compemsation on a Beckman Coulter Sysytem. Module# 2, https://www.cytometry.org/web/modules/module2.pdf
Instrument Optimization
Goal: Establish PMT Settings that place the positive and negative populations in an optimal area of the scale and allow detection of weak positives.
PMT Optimization Methods
1. (Good) Run an Unstained Sample• Okay as a starting point.
• Suboptimal for fluorochromes with longer wavelengths such as APC-A700, etc.
2. (Better) Run a Fully-Stained Sample for each PMT. • Use what you assay.
3. (Also Better) Use Fluorescent Beads with multiple intensities.
Unstained Sample Method 11. Run an unstained, lysed sample.
2. Gate on the lymphocytes and adjust the PMT settings to set the edge of the noise signal on the edge of the 1st decade of the log scale for each detector.
3. Repeat step 2 for each detector.
4. Record the final PMT settings for each detector on the worksheet.
Method 2: Optimization with Stained Samples
✓ Pick an antibody that gives signals throughout the intensity range expected of your panel. Good markers for this are CD33, CD8, and CD4.
✓ Run the single-labeled specimen and adjust your detectors to demonstrate each population present.
✓ Navios users: Turn off "baseline offset" to avoid the negative population pushed on scale artificially.
Andrea Illingworth. Instrument Optimization – Adjusting PMT voltages and compemsation on a Beckman Coulter Sysytem. Module# 2, https://www.cytometry.org/web/modules/module2.pdf
Example of 10C PMT Adjustment
36
Dot plots of SS vs all PMTs allow for assessment of s/n ratio as well as separation between all subpopulations
Andrea Illingworth. Instrument Optimization – Adjusting PMT voltages and compemsation on a Beckman Coulter Sysytem. Module# 2, https://www.cytometry.org/web/modules/module2.pdf
37
Question 2: For those performing L/L analysis, how many colors are used?
A. 5 or fewerB. 6 to 8C. 9 or 10D. >10E. Don’t know
Compensation
Fluorescence Spillover
• Fluorescence spillover from other channels:
• Directly and irreversibly reduces the resolution sensitivity of that channel. Contributes to background
• This “background” is subtracted in the process called Compensation.
405nm
488nm
Intralaser Compensation
638nm
488nm
405nm
638nm
488nm
Interlaser Compensation
488nm
Compensation
• The goal of good compensation is for the Mean Fluorescence Intensity (MFI) of the negative cells is equal to the MFI of the positive cells (+/- 10)
Compensation with Beads
✓Antibody capture beads
✓Label with your Ab/fluorochrome
✓Pros:✓Always positive events available
✓Can be prepared ahead
✓No precious sample wasted
✓Cons:✓May not be as bright as sample & smaller than cells
✓Cannot be used with non-mAb reagents (7AAD, Syto-6).
Run Verify tube after Autocompensation
• Should see all populations “on-scale
• Check compensation matrix
• Minor compensation adjustment can be made at this time
• Subsequent later changes can be saved in “settings protocol”
44
Logic versus Logicle scale
45
Salima Janmohamed-Anastasakis, Amr Rajab and Andrea Illingworth. Compensation Tips for Beckman Coulter 10-Color Navios Platform. Module# 8, Quality and Standards Committee, International Clinical Cytometry Society (ICCS) 2018. http://www.cytometry.org/web/modules/Module%208.pdf
Specimen Processing
Antibodies QC
• Antibodies titration.
• New lot number/ shipment verification
• Antibodies cocktail verification
What is your lab specimen processing procedure?
1. RBC Lysis
▪ Tube lysing (SLW or SLNW)
▪ Bulk lysing (LWS)
2. Using blocking agent.
3. Incubation
4. Polyclonal or monoclonal antibodies
How many washes?
◼Module # 6: Flow Cytometric Testing for Kappa and Lambda light chains.
◼By Melanie O’Donahue, Laura Johnson, Ben Hedley and Erin Vaughan.
Question 3: For those performing B-cells LC study, how many times do you wash cells prior to labelling with Anti-Kappa and Anti-Lambda?
A. One wash
B. Two washes
C. Three washes
D. Four washes
E. Don’t wash cells.
50
Effect of lysing solution
◼Module #1 (http://www.cytometry.org/web/modules/module1.pdf): Lysing Methods and Reagents for Flow cytometric Immunophenotyping. By Melanie O’Donahue and Laura Johnson.
Monoclonal vs. Polyclonal Antibodies
Horna P, Olteanu H, Kroft SH, Harrington AM. Flow cytometric analysis of surface light chain expression patterns in B-cell lymphomas using monoclonal and polyclonal antibodies. Am J Clin Pathol. 2011 Dec;136(6):954-9
Specimen processing
At LifeLabs
• Wash cells x 2 with PBS (RT or 37c)
• Re-suspend cells in 0.5% BSA in PBS
• Add Ab cocktail to 100ul (5 x105-106 cells) of sample
• Incubate for 15 minutes in dark at RT.
• Lyse with 1mL VersaLyse (BC ref. IM3648) plus 25 uL IOTest3 Fixative (BC ref. IM3515)
• Wash and suspend in 1 mL PBS and 12.5 uL IOTest 3 Fixative (BC ref. IM3515)
• Acquired on the Navios Flow cytometer
RESULT QC
Result QC
Evaluate Sample Quality
Suboptimal Compensation
Inadequate mixing of cells and Abs
Smearing of populations
Wrong Antibody Added
Flow vs. Morphology
Flow vs. CBC
CBC
WBC 50.8 x10^9/L
Hb 120g/L
PLT 225 x10^9/L
Neutrophil: 34%
Lymphocyte: 60%
Monocyte: 4%
Eosinophil: 2%
Basophil: 0%
Wash with warm PBS (37C)
Washing with RT PBS
Washing with warm PBS
◼PB sample with cold agglutination
ICCSClinical Cytometry Resource Center
http://www.cytometry.org/Resource-Center//
Thank you for your attention
Amr Rajab BSc, MLT, SCYM (ASCP)CMFlow cytometry Technical Specialist
LifeLabs, Toronto, CanadaE [email protected]