Flow Cytometry Flashcards
What is flow Cytometry?
Study of cells in a heterogenous population, e.g. A blood sample
Measures different parameters or characteristics of a single particle within a mixed population
Using FACS – Fluorescent Activated Cell Sorting
What is the role of FACS?
Identify cell populations, function and expression via:
Cell surface markers
Intracellular markers
Cell sorting by population
They are set up to separate individual cells into droplets.
There is an additional step involving electrostatic charges to separate individual cell populations.
They check each population for purity and reanalyse by flow Cytometry.
Collected cells can be used for further work, eg cell cultures.
Though they are mainly used in research labs.
How is the sample prepared?
Protocols vary, but all have same outcome – allowing analysis of cell suspension
- Sample collection: EDTA/citrate sample - prevents clotting/clumping so you can see individual cells. Fresh sample and not aged so there is no reduced function/cell death
- For cell culture or tissue samples, use enzymes/homogeniser and filter
- Lysis of red blood cells, without centrifugation
How are samples stained?
Direct – incubate sample with antibody-fluorochrome conjugate in the dark to prevent fluorochrome quenching or degradation
- wash to remove debris - (fix) resuspend using a buffer for example
Indirect – use secondary labelled antibody
Intracellular (eg protien inside cell) – requires fixation or permeabilisation so antibody can get inside cell
How is analysis performed?
Analysis is automated in bench top analysers.
The process is Hydrodynamic focusing, which involves cells passing a light source 1 by 1. This can assess the cell structure;
- Size - FSc (more forward scatter = larger cells)
- Granularity/complexity - SSc (more side scatter = more granular cells)
Analyse marker expression or identification of target;
2 signals – scatter for information about each event
- 1 dot = 1 event (not one cell, which is why you don’t want clumping)
- fluorescence - detectors (fluorescent signal = target cell is present - usually measuring the presence of fluorescently labelled antibodies in the cell)
What is hydrodynamic focussing?
Inject cell suspension into the system at higher pressure than the sheath fluid which is flowing through system all the time. This uses Benoli’s principle, whereby there are two layers of liquid flowing at different rates so have different pressures. So when they enter the narrow nozzle of the flow cytometer the cells in the cell suspension are aligned, so the laser at the side can interrogate each cell one at a time, measuring its size, granularity and antibodies present. Means you collect lots of accurate data.
By injecting into flowing stream you can measure
- Fluid dynamics
- Laminar flow
How does the instrumentation allow hydrodynamic focussing?
In the flow cell the sample is injected and the (blue) laser comes in at the side, interacting with each cell one by one. Light is refracted or scattered at all angles as it passes through the laser beam. On the opposite side of the nozzle to where the laser is focussing is a detector measuring the forward scatter - light refracted in a forward direction from the light source – size measuring scattering between 2 and 10 degrees angle from the laser direction. This records cell size, larger cell = more forward scatter. Perpendicular to the laser injection is the sideways scatter detector, which recognises granularity. The blue laser hits the cells scattering them in different directions. There are different sideways scatter detectors for the different wavelengths of light that are used to measure the fluorescence given off during sideways scattering. Scattered light splits into wavelengths through filters and mirrors – optics
Focussed towards detector (photomultiplier tubes). Each detector has a mirror that allows only a certain wavelength through, and reflects other wavelength, which will be detected by another detector. This allows you to use only certain wavelength detectors depending on which fluorophore you are using.
How are the results of flow Cytometry plotted on a graph?
In forwards scatter all populations of different cells overlap, where as in side scatter each population has different fluorescence intensities. This means that you can plot forwards scatter vs side scatter, giving information on both what the cells look like and what their characteristics should be from the ganuoles they have.
If cells are not fully listed they will not show well defined populations on the graph.
Lymphocytes have low side and forwards scatter as they are small and non-granular.
Monocytes have slightly more side and forward scatter.
Neutrophils have similar forwards scatter to monocytes, but significant side scatter due to its abundance of granuoles.
How is fluorescence used in identification?
Fluorescence involves excitation of a fluorophore to a higher excitation state. Where the fluorophore absorbs red light and reflects blue light. In the wavelength spectrum of colour blue light has a shorter wavelength and red has a longer wavelength.
Fluorochromes absorb and emit photons of light at different wavelengths. Flourochromes can be synthetic, organic dyes, eg FITC and Cy dyes (various colours); or proteins, eg PE (phycoerythrin) and APC (allophycocyanin).
If you want to measure two different things you can use two fluorophores that have wavelength emissions that are distinct. You then need to set up the flow cytometer so that the signals don’t overlap. This is called Compensation. Use compensation to avoid crossover. Used during set up, takes a particularly wavelength out of detector to avoid crossover. Crossover occurs because, as described, sideways scatter is detected by different detectors depending on which wavelength of light the fluorophore sideways scatters. This means you can only use specific detectors. However, each fluorophore has crossover with others. So if you want to use two to measure two different cell types, it is best to use ones that detect wavelengths that are far apart to avoid doing lots of compensation due to crossover, for example use FITC (green) and APC (red).
What does gating involve?
Process by which you isolate population based on one characteristic. Then apply a further requirement to identify population of interest.
Fluorochrome conjugated antibodies to various CD (cluster differentiation) markers can be used to determine various cell populations, eg all lymphocytes have CD 45.
This is useful in looking for immune deficiency – under/over expression and absence of certain cells. It can also be very useful for determining the lineage and degree of differentiation of each cell population, eg of leukaemia
- Presence / absence of differing CD markers (diff markers at diff stages of B cell differentiation - are there B memory cells?)
What are the lymphocyte CD subset markers used in gating?
CD45 = lymphocyte marker CD3 = T cells CD4 = T helper cells CD8 = cytotoxic T cells CD19 = B cells CD56 = NK cells
Specific to only these cell subsets
What are the controls used in flow Cytometry?
Flow set - light scatter and fluorescence intensity (should be within set limits)
Flow check - optical alignment and fluidics (should also be in set limits)
Synthetic (blood) sample controls (eg. Immunotrol - beads with different sixes and markers - they know what it should be! Couldn’t use an actual sample as it is unstable)
Fluorochrome controls
- Isotype control - looks at background fluorescence, some patients have autofluorescence - naturally have different fluorescence that show up in results - Positive control - tell you that you're picking up at the correct antigen with the correct antibody - Negative control - show there is no crossover if results
Healthy normal controls (for functional assays)
What if a specific control is not available?
- functional tests (how cells work) are difficult to get controls for. - Usually ask for a health sample from a donor. This tracks the transportation of the sample and that the test has worked as it should give you a normal result. Requires ethical approval (usually comes from a scientist or nurse that provides blood)
How has the assay been developed for ALPS?
Autoimmune Lymphoproliferative Syndrome (ALPS)
- Very rare autoimmune genetic disorder
- Very generic symptoms or asymptomatic - Enlarged spleen (splenomegaly), lymph nodes (lymphadenopathy) and liver (hepatomegaly)
- Susceptibility to malignancy
- Tested because of expression of symptoms - Anaemia, eosinophilia, monocytosis, neutropenia, or family member with disease.
Caused by defective apoptosis
- Accumulation of lymphocytes and autoreactive cells
Required criteria:
- Patient should have had chronic Lymphadenopathy and/or splenomegaly (for more than 6 months)
- Genetics-wise they should have a lymphocyte apoptosis defect (FAS/FAS ligand)
- Increased CD3+TCRαβ+CD4-CD8- (>2.5% of T cells) - double negative T cells expressed alpha beta - so we need a marker for CD4,3 and 8 double negative T cells, but also alpha beta expression, so needs 4 markers.
- Requires a new assay: the original assay only estimated DNT from total lymphocyte gate from subset analysis (only had CD3 as the initial marker and then TCR alpha/beta or TCR gamma/delta, but no way of knowing if cells were double negative, so not fit for purpose!)
Problem with inaccuracy and possibility of increased referral / reflex testing unnecessarily.
To investigate why the prev didn’t work, set up a series of tubes. Running individual antigens to find the route of the problem. When CD3 wasn’t put in the tube, we could see G/D and A/B, so we know it’s something to do with the interaction of antibodies in the tube. So from this we know the antibodies can bind, therefore that the fluorochromes are not being quenched.
Spectral overlap (compensation)?
Interference of fluorescence between fluorochromes with emission spectra that are very close, or overlap. Is CD3 quenching signal from alpha beta?
Steric hindrance?
Molecular spatial structure prevents or retards inter- or intramolecular interactions - large molecules are blocking the signals from smaller ones
More likely to be steric hindrance : PC5 is very large, so since CD3 and alpha beta are very close together n the molecule PC5 could be blocking the active site of alpha beta.
Alternative suggested approach:
Try using different combinations of fluorochromes
Use FITC on CD3 means opening up for binding on αβ TCR
Need to consider which other fluorochromes to use for other structures
Validation:
Prove the assay is fit for purpose
Analyse samples from 18 anonymised healthy donor volunteers
From the guidance document, we know the normal range is less than 2.5%, so we can decide upon a reference range according to these normal results and performing statistical analysis
97.5th centile 2.35% TCRαβ DNT
Stability assessed – analyse samples post bleed at room temperature and 4oC (to account for samples that haven’t been analysed on the same day as bleed, are they stabile over 24 hours and do what do they need to be stored at?)
