Applications Of Flow Cytometry Flashcards
Name 4 applications of flow cytometry (FC).
- Transplantation
- Immunophenotyping
- DNA analysis
- Immunology
Define what transplantation is.
It is the movement of material from one individual to another (related or not).
I.e. donation of a kideny from one individual to another individual.
What is HLA?
Human Leucocyte Antigen. They are antigens that are found on human cells, and is a way of self-ID self from non-self. They have two subtypes/classes: HLA type I and HLA type II. Need to be able to detect pre-existing antibodies (Abs)/antigens in recipient’s before they get transplants to prevent HAR (hyperactive rejection).
How do we test compatibility of a donor with a recipient?
Do cross-match test. This can be done using FC and/or looking at CD34 (a rare marker that is only found in progenitor cells for bone marrow- BM- and the hematopoietic system) analysis (to give understanding on whether the patient will have cells that can re-populate the cells that have been destroyed (I.e. from chemotherapy treatment in cancer patients).
HLA Detection.
Use flow methods that use microsphere beads that ate coated in the HLA antigens/HLA typed cells. However, there is a limitation to how many tests can do at once (~4-8 different antigen beads/1 cell). This means that would have to repeat tests over several times which could increase error. If were to use Luminex technology instead, can do batch testing using 1000s of beads. Likewise, could just use cells that recognise which antigens exist in the panel (like in astrology assay using a 96 well plate).
HAR caused by HLA Ab IgG.
If having a transplant, and have pre-formed HLA Ab of IgG in the body which is complementary to the HLA antigens on the donated organ tissue, then the organ will be destroyed through complement fixing IgG.
IgM presence in relation to HLA- Abs in transplantation.
Having pre-existing allo-IgM Abs against mis-matched antigens of donors can be detrimental (this is because it’s associated with naive cytotoxic lymphocytes- CyA sensitive). Patients who have HLA IgM, may also have HLA IgG of the same specificity.
Ensure no rejection occurs, what are things we need to ensure the recipient has/shouldn’t have?
- Recipient has NO HLA Abs present
- Recipient has NO CURRENT HLA Abs present
- Recipient has HLA Abs present, but they don’t react with the antigens that are present on the organ tissue ect.
Flow PRA (One Lambda).
Flow PRA = Flow Panel Reacting Antibodies.
Have small set of beads that can be separated based on their varying levels of pychoerythryin (an fluorophore that is tagged on the ends of Abs). Can place HLA antigens on surface of the beads and then take the patients serum and mix the two together. From this, will be able to see whether the patients serum contains the complementary Abs that will bind the antigens on the beads/not. The set of beads are washed in a series of wash steps (to remove unbound Abs), and anti-human IgG tagged with FITC is added. FITC is a powerful fluorophore that will give off a green fluorescent if it binds to the IgG antigens on the beads.
What is the limitation with Flow PRA?
There is a low max amount of beads that can be tested at one time, which means that repeats would have to be done, which increases the risk of error each time.
Principle of Luminex HLA Ab Detection.
- Have 3 bead types: HLA A1, HLA A2, HLA A3. They all have different ratio of red:IF dye. These beads are then incubated with patients serum containing anti-HLA A3, at room temperature in the dark with slight agitation. This allows for binding of the Abs (of present) to the complementary bead.
- The beads then undergo a series of wash steps to remove unbound Abs and reduce background ‘noise’.
- The beads are then incubated, in the same condition, with anti-human IgG tagged with PE.
- They are then washed again to remove unbound Abs and transferred to the lasers of the Luminex machine.
- The Luminex has 2 lasers: 1st one is the REPORTER LASER = ID the bead by detecting the IF dye, the 2nd one is the MICROSPHERE ID LASER = detects the presence of the HLA antigen/Ab complex on the bead surface.
A more modern version of Complement Dependent Cytotoxic test?
Three Colour Lymphocyte Immunofluoresence (LIFT).
What is the aim of LIFT?
To ID T cells (which have only HLA class I antigens on their surface), and B cells (which have both classes of HLA antigens on their surface) in order to see if any action needs to take place.
What is the principle behind LIFT?
Takes bloods/blood related sample and run using FC. Need to take sample from known types of cells, and take serum from patient and incubate them together with 3 Abs: anti-CD3-PE (for T cells), anti-CD19-PerCP (for B cells), and anti-IgG-FITC.
Would be able to get forward scatter (FS) and side scatter (SS) and then look at the fluorescent signal that is given off to ID whether it is a T/B cell? Lymphocytes have a low FS and SS (not very big and don’t have much cellular content).
Presence of PE signal should indicate the type of lymphocyte that is present.
T CELLS = laser light is one colour, PE is the second colour, and FITC is the third colour = LIFT!
B CELLS = laser light is one colour, PerCP is the second colour, and FITC is the third colour = LIFT!
HLA typing using PCR.
Take patient DNA which has HLA locus of interest and let it undergo PCR (to amplify the HLA locus of interest). Can then denature the PCR product and add oligonucleotide probe that is attached to the bead, to re-hybridise the DNA.
ONLY SEQUNECE MATCHING CAN OCCUR TO MAKE DNA RE-HYBRIDISE.