instrumentation & sample preparation

Instrumentation & Sample Preparation

Amr Rajab BSc, SCYM (ASCP)CM

Flow cytometry Technical Specialist

LifeLabs, Toronto, Canada

E: [email protected]

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

http://www.cytometry.org/public/newsletters/eICCS-8-2/article6.php

Published Guidelines

Pre-analytic

Analytic

Post analytic

Performance specifications

Instrument Quality Control

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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

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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

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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

Assessment of Carryover

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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%

Instrument’s Sensitivity and linearity

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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

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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.


Example of 10C PMT Adjustment

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Dot plots of SS vs all PMTs allow for assessment of s/n ratio as well as separation between all subpopulations


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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”

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Logic versus Logicle scale

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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.

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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]