Quality Assurance in a Flow Cytometry Lab

Dr Kunal Sehgal, M.DAssociate Consultant

Hematopathology,Department of Laboratory Medicine,

P.D.HINDUJA Hospital and MRCdrkunalsehgal@gmail.com

Diagnosis of Leukemias and Lymphomas

Multidisciplinary Approach

• Adequate Clinical History and Examination

• Laboratory work up - CBC, ESR, LDH, B2 microglobulin etc.

• Radiological Evaluation - PET Scan, CT scan etc.

• Molecular Studies

• Morphology - H & E

• Immunophenotyping - for diagnosis, subtyping, prognosis

Quality Assurance in FCM

Why should we care?

Assurance that flow cytometry results you are generating are due to the properties of the sample and not changes in:

• Instrument

• Assay performance

• Reagents

• Operator variation

How do I achieve good QA in a FCM Lab?

• Plan It !

Make A SOP and QC manual

• Implement It !

Systematically follow the procedures laid down by you in your manual

• Document It !

Prove that you have done what you planned to do

Preanalytical Analytical Postanalytical

•Staff•Safety policies•Waste Disposal•Sample transport•Sample storage•Sample handling•Sample viability•Sample Cell Count•Reagents •Instrument maintenance•Internal Quality Controls•Pipette calibration•Temperature charting

•Sample processing•Acquisition•Data templates•Analysis•Interpretation

•Result Reporting•Data Backup•Record retention•Clerical error policies•EQAS and Proficiency testing•Quality improvement program

Quality Assurance

Flow Cytometry Assay

What sample do I need to collect for doing a FCM assay?

• Clinical History• CBC• Bone Marrow slides – Air Dried• Bone Marrow Biopsy and Imprint smears

Sample for Flowcytometry - EDTA/Heparin

Sample for Cytogenetics - Heparin

Sample Storage

Peripheral Blood and Bone Marrow

1. Unprocessed bone marrow and peripheral blood can be stored as is, at room temperature overnight.

2. Suspected Burkitt's Lymphoma should be refrigerated at 2-8°C. Because of an increase in proliferation of these cells, they die quickly.

Fluids and Fine Needle Aspirates

3. Samples should be set up the same day, as cells die quickly.

2. Samples can be refrigerated (2-8°C)

Storage Media:

RNG = RPMI 1640 with 10% Newborn calf serum, glutamine, and Gentamicin.

OPERATING TECHNICIAN

• A basic science graduate is a must

• One year training/work experience in a busy flow cytometry lab is ideal

• Exposure to workshops and CME

• Participation in quality assurance program of the lab is essential

Viability

Trypan blue

• Add one part of 0.4% Trypan Blue to one part cell suspension (Example 50ul : 50ul). Incubate for 1-2 minutes.

• Place one drop (20ul) on hemacytometer.

• Count one out of a total of 9 large squares on hemacytometer. The viable cells are refractile; dead cells will take up the blue stain.

• % viability = live cells x 100 (live + dead cells)

Notify consultant/supervisor of any specimens with poor viability. Report may indicate that results may be affected by low viability.

• NCCLS. Clinical Applications of Flow Cytometry: Immunophenotyping of Leukemic Cells; Approved Guideline. NCCLS document H43-A, 1998.

• Bauer, Duke, Shankey, Clinical Flow Cytometry Principles and Application, Williams and Wilkins, Baltimore, 1993.

Viability

• 7AAD

• CD45 vs. SSC plot and FSC vs. SSC Plot

• Cell Counters

Slide Morphology and Panel Selection

It is ideal to make slides from all samples received in the laboratory for a given patient

Morphology based panels -– Acute leukemia panel– CLPD panel

Antibody Panel Design

• Weak Antibody - Strong Fluorochrome• Strong Antibody - Weak Fluorochrome

• Weak Antigens - B antigens and Myeloid antigens• Strong Antigens - T cell markers

• Strong Fluorochromes - PE, APC, PE-Cy7• Weak Fluorochromes - FITC, PerCP

Choosing a Fluorochrome

Reagents

• Purchase policy• Stock maintenance • Labeling policy• Storage • Titration of Antibodies• Antibody Cocktails

6 Colour Panel - Abs. Stock as on Sep. 1, 2009 - Two

P ECD # Cat. # Clone Batch # Exp. Dt. Vial # Vial #

11c 347 637 S-HCL-3 41523 31.03.2011 1 2

13 347 837 L138 45893 28-02-2011 1 x

347 837 L138 37956 30.11.2010 1 x

20 346 595 L27 39126 30.04.2010 1 x

346 595 L27 45891 30-04-2010 1 x

22 347 577 S-HCL-1 42801 3.11.2010 1 2

33 347 787 P67.6 40643 28.02.2011 1 2

34 348 057 8G12 39209 28.02.2011 1 2

45 555 483 H130 11684 30.092012 1 x

38452 30.09.2012 1 x

117 340 529 104D2 42553 31.10.2010 1 x

340 529 104D2 33020 31.10.2010 1 x

138 347 192 MI15 43994 31.12.2009 1 2

235a 340 947 GA-R2(HIR2) 44317 31.08.2010 1 2

Bcl-2 346 595 6C8 9051 31.07.2011 1 2

Ig D 555 779 IA6-2 40125 27.01-2015 1 2

Ki-67 556 027 B56 35807 31.10.2014 1 2

Kappa 346 601 TB28-2 40016 31.11.2010 1 2

TCRab 555 548 T10B9.1A-31 40077 31.08.2013 1 2

Titration of Antibodies

INSTRUMENT

• Basic Knowledge about the configuration of the machine is essential

• It is important to know basic start up and shut down procedures along with routine maintenance processes

• MAINTENANCE log has to be maintained

Instrument

• What all is to be monitored – Optics – Fluidics – Electronics

• How do you monitor– Manual methods– Automated methods

QC beads are very useful for monitoring and evaluating instrument performance

Flow Cytometry Beads

• Spherotech Beads• Cytometer Setup and Tracking (CS&T) beads• Seven colour setup beads• Calibrite Beads• Flow-Check™ Fluorospheres • IMMUNO-BRITE™ Standards Kit • Flow-Check™ Pro Fluorospheres• Cell Counting Beads• Antibody Capture beads

Key Performance Factors in High Quality Flow Cytometry Data

Relative measured values of fluorescence

Linearity and accuracy

Resolution of subpopulations, including dim subpopulations

Sensitivity

Reproducibility of results and cytometer performance

Tracking

Comparison of results across time and among laboratories

Standardization

CS&T

Functions of the CS&T beads

Fully characterize the cytometer’s performance – Linearity,– Detector efficiency (Qr);– Background fluorescence (Br);– Electronic noise (SDEN); and – laser alignment (rCV)

Optimize cytometer settings– Laser delays; – Area scaling factors;– PMT voltages

Track cytometer performance and Detect component failures and alterations

Provide graphical representations of performance trends over time

Know your beads

• Seven colour setup beads

- Instrument monitoring with compensation

• CS&T beads

- Instrument monitoring without compensation

SEVEN COLOUR SETUP BEADS REPORT

Why monitoring is important

Changes in parameters can indicate cytometer problems

– Increases in Electronic Noise (SDEN) • bad PMT connections or other electronic problem

– Decreases in Detector Efficiency (Q) • low laser power, dirty flowcell, alignment or filter issue

– Increases Optical Background (B) • fluorescent contaminant, failing laser or filter problems

Compensation

• How to do it– Using Cells– Antibody Capture Beads

• How often to do compensation

• Is it important to keep a check on the same

Optimisation

Compensation settings established with beads need to be optimized to the cells used in the experiment.

Eg: The 7 colour setup compensation settings may not always be the most accurate for your experiment and will require optimization and fine tuning for the cells used in the experiment

Verification and Validation

Wikipedia definition

Verification and Validation, in engineering or quality management systems, it is the act of reviewing, inspecting or testing, in order to establish and document that a product, service or system meets regulatory or technical standards.

Validation – “Are you building the right thing?”

Verification – “Are you building it right?”

Verification and Validation

• Lot to Lot Antibody VERIFICATION

• Setting up a new permeabilisation and fixing procedure using home brewed reagents -

Assay validation

Flowcytometry Assay

Verification

Confirm specifications established by the manufacturer

• Accuracy• Precision• Reportable range of results• Verify of manufacturer’s

normal range

Validation

In-house, home brewed assay

• Accuracy• Precision• Analytical sensitivity• Analytical specificity• Reportable range• Reference range• Calibration procedures• Control procedures• Sample stability

Sample processing

Stain lyse wash

Lyse wash stain

Ficoll Hypaque

Cell counting

Required Concentration: 0.1 -1 x 106 cells/ tube

Eg: If your target is 0.5 million (0.5 x 106) cells per tube (per 100ul sample volume)

0.5 x 106 / 100ul (x10) = 5 x 106 / 1000ul = 5 x 106 / 1 ml

You require a concentration of 5 x106 /ml or a CBC count of 5 x 103/ul ( 5000/cmm)

In clinical scenario, Antibody staining is volume dependent and time dependent

Target (100ul/tube)

0.1 x 106

cells1 x 106

cells

Per ml 1 x 106

cells/ml10 x 106

cells/ml

CBC Count

1x 103/ul 10x103/ul

Stain-Lyse-Wash

100 ul of whole blood (0.1-1 million cells)+ Antibodies

Vortex

Incubate at RT in the dark for 15-20 minutes.

2.0 ml Lysing Reagent- vortex- let sit in dark for 6-10 minutes at RT

Vortex and centrifuge at 1400 rpm for 5 minutes.

Carefully discard supernatant from cell pellet and vortex pellet

Wash with 2 ml of PBS-BSA. Centrifuge at 1400 rpm for 5 min.

Carefully aspirate supernatant and resuspend pellet in 0.5 ml of PBS-BSA and vortex.

Acquisition and Analysis

– No of events to acquire

– Gating Strategies

– Provision for checking internal controls using normal cells

– Doublet exclusion is ideal

– Ideal to have all possible permutations and combination plots

– Fixed templates help in reproducibility and consistency of data

– Data backup

FCM Reporting

• Consolidated Report– Clinical History in short– Morphology findings if Available– FCM data– Cytogenetic and Molecular correlations– Advise

FCM Report Format

Signatory Authority

• MD/DNB/DM Pathology or Hematology

• Six months – one year training in a busy hematology-flow cytometry lab is ideal

• Exposure to workshops and CME

• Participation in quality assurance program of the lab is essential

Quality Improvement Indicators

• TAT of FCM reports

• Number of samples requiring Repeat processing

• Number of samples requiring additional markers and evaluation of these markers

• Error reporting

Proficiency Testing

Participation in any PT program is ideal and valuable

• Inter lab Comparison program - TMH• CAP• RCPA

Wet samples or Dry challenges