Block B - Flow cytometry for monitoring progression of HIV to AIDS Flashcards
what does HIV infect and what is it associated with ?
HIV infects CD4+ T cells, the infection is associated with a decline in the percentage and absolute number of CD4+ T cells
why is it important to monitor CD4+ T cells ?
Monitoring the number of CD4+ T cells in an individual it can be used to follow the progression of disease towards AIDS. It can also therefore be used to monitor the effectiveness of chemotherapy.
what is the correlation of CD4+ T cells during infection ?
the peak of the virus correlates with the decrease in the number of CD4 T cells as they are being killed by the virus. Then the CD4 T cells recover slightly before declining as the virus is brought under control , there is a lull in the viral load but the virus is not eliminated. Then when the CD4 T cell numbers are very low the virus begins to replicate.
acute infections ?
Acute infection is associated with fever
chronic lymphadenopathy ?
chronic lymphadenopathy is when there is a peak in the virus
subclinical immune dysfunction ?
Subclinical immune dysfunction occurs as there is an inadequate number of CD4 T cells.
when does skin and mucous membrane defects occur ?
Skin and mucous membrane defects occur before system immune deficiency occurs and at this point it is AIDS.
how are the levels of CD4+ T cells monitored ?
flow cytometry
how are lymphocytes split into different populations ?
Lymphocytes can be split into different populations identified by surface markers.
Lymphocytes can be categorized into B and T cells. The T cells are CD3+ and they can be further categorized into T helper cells which are C3+/CD4+ and cytotoxic T cells which are CD3+/CD8+. These markers are displayed on their surface and due to this they can be identified using antibodies.
an antibody can contain a fluorescent label for CD3+ , what will this recognise ?
An antibody can contain a fluorescent label for CD3+ which will recognise all T cells: helper and cytotoxic T cells.
what will the other CD4+ fluorescent label recognise ?
Another antibody can be labelled with fluorescent CD4+ will recognise the CD3+/CD4+ double positive cells which will appear both green and red.
fluorescent microscopy ?
Take blood sample and quantify number of lymphocytes in a known volume using markers described above. Make smears (or ‘cytospins’) of blood on slides. Carefully examine using a fluorescent microscope. Count the number of CD3 positive cells. Count the number of CD3 and CD4 positive cells. Therefore you can calculate number and percentage of CD4 positive cells.
problems with this ?
However Problems occur as it is time consuming , can only count a small number of cells and it is dependent on operator interpretation of each cell.
what is flow cytometry ?
this analyses cell suspensions , cells are passed through a laser beam. Light emitted from each individual cell and it is collected and analysed using a computer
Forward Scatter is the light emitted from cells at narrow angles to the axis of the laser is proportional to cell size.
Side Scatter: Light emitted from cells at right angles to axis of laser is proportional to cell granularity and complexity of surface topography.
filter/mirror allow ?
The filter/mirror will allow the flowcytometry to be able to send data to the detector. For example, the FPMT1. This allows the generation of data from red light. FMPT2 will allow the detection of green light using certain filters.
