1. Quality Assurance in a Flow Cytometry Lab
Dr Kunal Sehgal, M.D
Associate Consultant
Hematopathology,
Department of Laboratory Medicine,
P.D.HINDUJA Hospital and MRC

2. 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

3. 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

4. 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

5. Quality Assurance 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

6. Flow Cytometry Assay

7. 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

8. Sample Storage Peripheral Blood and Bone Marrow
Unprocessed bone marrow and peripheral blood can be stored as is, at room temperature overnight.
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
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.

9. 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

10. 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.

11. Viability
7AAD
CD45 vs. SSC plot and FSC vs. SSC Plot
Cell Counters

12. 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

13. 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

14. Choosing a Fluorochrome

15. Reagents Purchase policy Stock maintenance Labeling policy Storage
Titration of Antibodies
Antibody Cocktails

17. Titration of Antibodies

18. 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

21. Instrument What all is to be monitored How do you monitor Optics
Fluidics
Electronics
How do you monitor
Manual methods
Automated methods
QC beads are very useful for monitoring and evaluating instrument performance

22. 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

23. 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

24. 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

25. Know your beads Seven colour setup beads
- Instrument monitoring with compensation
CS&T beads
- Instrument monitoring without compensation

26. SEVEN COLOUR SETUP BEADS REPORT

28. 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

29. Compensation How to do it How often to do compensation
Using Cells
Antibody Capture Beads
How often to do compensation
Is it important to keep a check on the same

30. 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

31. 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?”

32. Verification and Validation
Lot to Lot Antibody VERIFICATION
Setting up a new permeabilisation and fixing procedure using home brewed reagents -
Assay validation

33. 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

34. Sample processing
Stain lyse wash
Lyse wash stain
Ficoll Hypaque

35. Required Concentration: 0.1 -1 x 106 cells/ tube
Cell counting
Required Concentration: 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 cells
1 x 106 cells
Per ml
1 x 106 cells/ml
10 x 106 cells/ml
CBC Count
1x 103/ul
10x103/ul

36. Stain-Lyse-Wash
100 ul of whole blood (0.1-1 million cells)+ Antibodies
Vortex
Incubate at RT in the dark for 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.

37. 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

38. FCM Reporting Consolidated Report Clinical History in short
Morphology findings if Available
FCM data
Cytogenetic and Molecular correlations
Advise

39. FCM Report Format

40. 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

41. 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

42. Proficiency Testing
Participation in any PT program is ideal and valuable
Inter lab Comparison program - TMH
CAP
RCPA
Wet samples or Dry challenges

43.             
Thank You