Flow cytometry Flashcards
How is CD34 enumeration performed?
o HSCs identified by their CD45, forward and side scatter characteristics. The CD34+ cells are then selected.
- PE is the recommended fluorochrome; bright, CD34+ cells are relatively rare events
- Only want to measure viable cells: use 7AAD as a viability marker and gait the 7AAD negative, CD34+ cells.
o Fluorescent beads of a known concentration are then added.
o The absolute number of CD34+ can then be derived.
No washing needed (no Fc gamma receptor on CD34+ cells)
No ficol: may reduce the amount of CD34+ cells.
How is flow cytometry used to quantify FMH?
- IgG monoclonal anti-D reagent used that reacts with all D phenotypes capable of stimulating the production of anti-D
- ≥500 000 events
- Not affected by presence of F cells
- More sensitive than anti-F method
- Can only be used when there is a D neg mother and D pos foetus
- Signal weakened by passive anti-D
• BCSH suggests bleeds >2mL as detected by Kleihauer (0.08%) should be confirmed by flow
What is fluorescence spill over?
• Emission spectra of multiple fluorochromes being detected by a single detector
What is compensation?
What are some clues that the data is poorly compensated?
- Process of mathematically correcting for fluorescence spill over between detectors.
- Removes false negatives and false positives.
- Becomes more complicated as more fluorochromes are added and requires expertise to do it properly.
- Compensation is often performed at the instrument using an auto-compensation wizard.
• Clues of poor compensation
- Different MFIs between the positive and neg populations
- ‘Streaking’ of a cell population at 45deg on the scatter plots.
What methods can be used as compensation control?
• Beads or cells
- Single stained compensation beads or “Antibody Capture Beads”
- Homogenous polystyrene particles coated in antibodies that can bind antibodies via the Fc region.
- One drop of negative beads and one drop of positive beads placed in each test tube.
- Single colour antibody conjugates are added into individual, labelled test tubes at the antibody concentration used for the application
- Fluorescent signals are detected and plotted on FS and SC plots
- Single stained cells
What are the three rules of compensation controls?
1) Must Be Bright:
- Positive single colour controls must be at least as bright as the signal expected on the experimental samples.
- If you are using compensation microspheres (i.e. beads) these must be brighter than your stained sample
2) Like With Like: Background (autofluorescence) must match between the positive population and negative population for any given parameter.
- If you use beads for positive controls, you must use unstained beads as a negative.
- If you use cells for positive stains, you must use the same cell as unstained.
3) Matched Fluorophores: The fluorochrome used for the control must exactly match the fluorochrome used in the experiment (FITC cannot be used instead of GFP, tandem dyes must be from the same vial as the experimental stain.
What are some of the benefits of using beads as compensation controls?
- Reproducibility. Using beads, you will always know that you’ll get a bright signal. You can work out what dose to stain your beads (i.e. some beads can be too bright when saturated) and use this for all future experiments.
- Low frequency: If the frequency of the positive signal (i.e. rare cells) will not be adequate to get sufficient events for statistical significance (i.e. compensation wizard suggests 5000 events).
- Too dim: If the marker of choice is dim, or poorly resolved. In addition, if the marker of interest is modulateable (i.e. expressed upon activation) it will be more reliable to use beads.
- Sample availability: tissue, clinical or animal samples are often precious, or not in plentiful supply to consider “wasting” on compensation controls. In most cases beads will always be the control of choice in this situation
What are some of the disadvantages of using beads?
- Cellular samples will require further refinements within the compensation matrix (physical and staining properties of beads are always different from cells. It gives a rough rather than exact estimate of the compensation matrix)
- Autofluorescence can result in cellular overcompensation
What are some of the advantages of using cells as compensation controls?
- Give a better representation of the compensation required in the assay
- Disadvantage: can be difficult to find samples with negative and positive populations
What factors affect compensation?
- Fluorochrome/ dye spectral overlap
- Laser and filter configurations
- PMT voltages
- Hardware/ software compensation
- Autofluorescence
- Panel design: Even with proper compensation, if an overly bright signal spills over into a neighbouring detector where a dim signal is to be detected, resolution sensitivity will likely be affected and the populations may not be resolved accurately. This results in ‘data spread’.
List preanalytical factors that are important in flow cytometry?
- Specimen collection: clotted or haemolysed sample.
- First vs subsequent draw
- Sample volume (dependent on assay (diagnostic vs MRD)/ sample (PB vs BM) and white cell count) 1-4mL PB generally required.
- Age of sample: should be processed fresh. Reliable identification of cell surface antigens is questionable in samples >48rs old.
- Transport and storage: controlled room temperature if processed fresh. Can be refriderated if delay expected.
What analytical factors are important in flow cytometry?
○ Validated method (i.e 8-10 colour preferred for MRD)
○ Interference: primarily from unlysed red cells (increased nucleated red cells and target cells) and haemodilution assess numbers of haematogones, myeloblasts, NRBCs or mast cells). Also consider monoclonal antibody therapy, normal populations
○ Panel design (selection of cellular targets, assignment of fluorochromes to each target.)
○ Reagents
○ Instrumentation (detector voltages, sample acquisition rate/ coincidence detection, compensation)
Discuss how you would set up a flow panel?
1) Determine the objective of the panel
2) Determine the antigens that should be targeted to meet the objective
3) Design the panel
○ May involve a literature search- Are there any validated panels/ methods reported?
○ Consider using combinations of fluorochromes that minimise spectral overlap
○ Couple low density or weak antigens with strong fluorochromes and vice versa
4) Validate/ verify assay and refine the assay
○ Develop a validation/ verification protocol
○ Perform compensation experiments
○ Voltage optimisation
○ Antibody titration
○ Defining sensitivity, lower limit of detection, lower limit of quantification, limit of blank, specificity, accuracy, precision, CV, measurement uncertainty
○ Establish the most appropriate denominator to use
○ Test 20- 30 normals
○ Testing often needs to be performed in conjunction with a reference laboratory
5) Ensuring on-going quality of the assay following validation/ verification
○ Standard operating procedures/ guidelines/ troubleshooting documents
○ Staff training and competency
○ Machine and PMT voltage calibration
○ QC (Rainbow beads, internal controls, fluorescence minus one)
○ Participation in external QC where available
○ Ensure minimum number of tests are performed to maintain ongoing proficiency (30- 50 tests per year for MRD assessment.)
○ Certification/ Accreditation
Discuss quality control in flow MRD assays
• Reproducibility is extremely important in MRD assays
○ Assay needs to be highly stable
○ Regular QC required to maintain consistency between runs and machines over a long period of time.
○ Rainbow beads
- Fluoresce in every channel.
- Measure fluorescence in each channel daily or prior to each MRD assessment.
- Have targets for fluorescence in each channel. This ensures consistency.
What should be included in an MRD flow report?
Antibody combinations used, LLOQ, LLOD, number of events, % MRD, denominator used, sample quality, conclusion
