Flow cytometer applications Flashcards
What can a flow cytometer be used in?
Transplantations, transfusion, immunophenotyping, DNA analysis, cell cycle analysis, immunology (disease diagnosis).
What type of flow cytometry can be used for transplantation?
FlowPRA and CD34 analysis to detect HLA ANTIBODIES (IgM and IgG)
How does flowPRA (One lambda) work?
FlowPRA analyses HLA class 1 and 2, and antibody specificity to allow CROSS-MATCHING OF ORGANS for transplantation. Beads are COATED WITH HLA and mixed with the patient’s anti-HLA then INCUBATED in the dark at 22-25C to facilitate binding. WASHING is performed to remove unbound antibodies. IgG-PHYCOERYTHRIN CONJUGATE is added and incubated in the dark at 22-25C, and washing is done again. Beads are then SEPARATED via phycoerythrins fluorescence signals where 8 beads are analysed at a time.
What is CD34 analysis via flow cytometry for?
CD34 analysis is for HLA TYPING to assist Hematopoietic stem cell (HSC) transplantation.
What is the complement-dependent cytotoxicity (CDC) test in flow cytometry used for? and how does it work?
The CDC test is an ANTI-HLA TYPING to detect complement IgG and IgM using the patient’s HLA via the MEMBRANE ATTACK COMPLEX (MAC) to trigger LYSIS, this aids organ cross-matching. The patient’s HLA is added to wells coated with anti-HLA and then incubated at 22C for 30 mins. Then complement is added and the MAC forms for lysis to occur. Propidium iodide (PI) is added to STAIN THE DEAD CELLS and incubated at 22C for 60 mins. Ink and EDTA are added to overcome the PROZONE EFFECT then the ANALYSIS is done.
How does the 3-colour lymphocyte immunofluorescence test (LIFT) work?
The 3-colour LIFT detects the B and T lymphocytes using ANTIBODIES CONJUGATED with 3 fluorochromes and an ARGON LASER to emit fluorescence. The patient’s ANTIBODIES are incubated with TYPED CELLS then the antibody-fluorophores are added and incubated together. Then the argon laser is applied to emit fluorescence and detect the specific lymphocytes with different colours.
How does Luminex perform HLA typing?
Luminex uses PCR-SSO (sequence-specific oligonucleotide) to amplify the HLA locus of interest with biotinylated primers and then denatures it to hybridize with oligonucleotide probes bound to beads. Then the streptavidin/PE fluorophore is added for identification. Computer analysis of the fluorescence from amplicons is performed.
What are the important blood transfusion antigens?
ABO, HLA class 1, and HPA (human platelet antigen)
How is DNA analysis conducted using flow cytometry?
DNA analysis will analyse the DNA content of a tumour cell in relation to a normal cell and express it as DNA INDEX. Propidium iodide (PI) will bind to all DNA molecules with help from Triton-X (detergent) to disrupt the cell membrane then cell count is measured against the fluorescence signal. The graph will show aneuploids (tumours) and diploids.
