Cellular pathology: Flow Cytometry - Introduction Flashcards
What is flow cytometry?
- A technique which simultaneously measures several physical characteristics belonging to a single cell in suspension
What things are used during flow cytometry to measure the physical charcteristics of a cell?
- Light scatter and fluorescence
What is flow sorting (Fluorescence-Activated Cell Sorting (FACS)?
- Sorting (separating) cells based on properties measured in flow
What can a flow cytometer tell us about a cell?
- Its Relative Size
- Its Relative Granularity/Internal Complexity
- Its Relative Fluorescence Intensity
What aspects of a cell can be measured using flow cytometry?
- Cell surface receptors
- Adhesion molecules
- Cytokines
- Enzymes
- DNA - look at cell cycle, apoptosis, cell viability
Apart from flow cytometry what are some other techniques that use fluorescenec to view cells?
- Fluorescence Microsocopy
- Flow Cytometry
What are the advantages of flow cytometry compared with flourescent microscopy?
- Flow cytometry can look at thousands of cells simultaneously while fluorescent microscopy can only look at a limited no. of cels in each field of view of microscope (difficult) to view lots of cells
- Flow cytometry can look for rare cells very easily while with fluorescent microcospy rare cells hard to find
- Flow cytometry is a quantative process while fluorescent microscopy isn’t a very quantative process (can only look at around 20 cells per field)
- Intensity of fluorescence in flow cytometry is very accurate while with fluorescent microscopy te instensity is variable
What are the major components of a flow cytometry machine?
- Fluidics - Cells in suspension flow in single file
- Optics - Cells flow through an illuminated volume where a laser hits each cell and causes them to scatter light and emit fluorescence
- Electronics - Fluorescence is collected, filtered and converted to digital values that are stored on a computer so they can then be analysed
In a flow cytometer how are cells put in suspension and made sure that they flow in single file?
- This is done by injecting the sample fluid contianing the cells into a nozzle tip containing sheath fluid as it passes through a small (50-300 µm) orifice (hole)
- Sample fluid flows in a central core that does not mix with the sheath fluid
What type of flow does the sample fluid experience since it does not mix with the sheath fluid in the flow cytometer?
- Laminar flow
What is the term used to describe the introduction of a large volume into a small volume?
- Hydrodynamic focusing
Describe the lasers used as part of the optics component of a flow cytometer
- Lasers used are a single wavelength of light (a laser line) or (more rarely) a mixture of wavelengths
- They can:
- Provide anywhere between milliwatts and watts of light
- Be inexpensive, air-cooled units or expensive, water-cooled units
- Provide coherent light (Single frequency)
What happens when light from a laser hits a cell?
- Light scatters in the foward direction (foward light scatter) which is proportional to the size of the cell
- Light also scatters at a 90º angle (side scatter) which is proportional to the granularity of the cell
What information about a population of cells can be obtained from just the light scatter of that population?
- From light scatter patterns of a population a dot plot showing “increase in foward scatter” on the X-axis and “increase in side scatter” on the y-axis can be produced
- This dot plot can be used to group cells into distinct types based on amount of foward and side scatter
- E.g. graph below groups white blood cells from a blood sample into their different types with each dot representing a cell
Describe the basic concept of laser-based flow cytometry using antibodies works
- Cells flow out of the nozzle tip of the flow cell
- Cells have been labelled with 4 different antibodies which each have a different colour fluoresent marker attaced to them
- The laser then hits each cell which causes fluorescence to be emitted
- The fluorescence produced goes through different dichroic filters
- This allows a specific photo multiplier tube (PMT) to pick up a specific wavelength of light (fluoresence)
- At each photo multiplier tube light (fluorescence) is converted into data
Describe the elctronics componets of a flow cytometer?
- This is where the processing of signals from detectors occurs - done by photo multiplier tubes (PMTs)
- Analog-Digital Conversion also occurs so the data can be analysed on a computer
What is a fluorochrome (fluorophore)?
- A fluorochrome is a chemical that is able to absorb light and then re-emit it when it is excited by light
Explain how a fluorochrome produces fluoresence
- When a fluorochrome is hit with light from a laser it absorbs the energy from the light and becomes excited
- Eventually the fluorochrome emits the same amount of energy that it absorbed, as light but it emits it at higher wavelength than the light that it absorbed
What is stokes shift?
- It is the energy difference between the lowest energy peak of absorbance and the highest energy of emission
Give some examples of common fluorochromes and the colours of light they emit when they fluoresce
- Fluorescein isothiocyanate (FITC) - Green
- Phycoerythrin (PE) - Orange
- Peridinin Chlorophyll Protein (PerCP) - Red
Why can FITC, PE and PerCP fluorochromes be used at the same time? CP
- It’s because they emit at different wavelengths which means they can be detected at the same time
- This means all 3 can be used to make 3 different antibodies fluorsecent and can then be added to a group of cells
- This allows you to obtain diferent information about the cells within that population
Why are the dichroic filters needed in laser-guided flow cytometry ?
- The dichroic filters are needed because light emitted from different flouorophores may be of a similar wavelength and so they allow the photo multiplier tubes to pick up light of a specific wavelength from a particular fluorophore
- They do this by refelecting light of a specific wavelength to one of the photo multipliers
Give some examples of single cells in suspension that are analysed using in flow cytometry
- Peripheral blood
- Bone marrow
- Fine Needle Aspirate
- CSF and other fluids
- Fresh Tissue
What are the 2 ways that cells can be labelled in flow cytometry?
- Direct: Monoclonal antibodies (MoAbs) are labelled with fluorochromes
- Indirect: Unlabelled primary monoclonal antibodies are joined to a secondary antibody which is labelled with a fluorochrome
When would the indirect method of labelling a cell be used instead of the direct method?
- Indirect method used when you are unable to label yoyr primary antibody with a fluorochrome
In what ways can the data obtained from flow cytometry be displayed?
- Dot plot
- Histogram - X-axis = Fluorescence intensity, Y-axis = No. of cells
What is gating?
- Gating is a data analysis technique used that allows you to hone in on a particular area of a dot plot that may represent a particular type of cell and show the data fro that group in more detail
