Flow cytometry – HIV

Background

Lasers are used to count different cell populations based on the light that their specific labels emit

HIV replicates in, and destroys CD4 cells that are crucial to effective immune responses. It is therefore necessary to monitor the numbers of competent cells as this is a good indicator of disease progression, patient response to therapy and what infections the patient will be prone to catching. In the laboratory cells are “counted” using flow cytometry. This technique allows scientists to label specific blood cells with molecules that will emit light of a specific wavelength when exited by a laser. Different cell populations can be counted based on the light that their specific labels emit.

Science

Flow cytometry results are represented graphically; cells are recorded as having a specific signature based on size, granularity, and abundance of surface markers

Individual populations of immune cells, with specific roles in infection control, have different groups of molecules on their surface, this characteristic is useful for quantifying the cellular populations. As with many other techniques used in the immunology laboratory, commercially available antibodies that have specificity for single unique surface molecules are mixed with the patient’s blood so that they can bind to their targets. These antibody labels are connected to dyes (fluorochromes) that emit light at specific wavelengths when excited by light at a lower wavelength (curriculum), several different colours attached to antibodies with different specificities can be used in the same sample to identify different cell classes. Cells labelled with the different antibodies are passed rapidly in single file through a laser beam that excites the dye molecules. The light that is emitted at different at different wavelengths is detected through a series of finely aligned detectors. In addition the shadow of the body of the cell passing through the laser beam is recorded and these measurement are extrapolated to determine how big and granular they are. The results are represented graphically and by using this data every cell is recorded as having a specific signature based on size, granularity, and abundance of surface markers. CD4 T cells are the most significant cellular indicators of disease progression in HIV so a scientist can differentiate and quantify them based on their medium size (relative to other blood cells) low granularity and strong binding with light fluorochrome labelled antibodies targeted against the CD4 cell surface marker.

Clinical

The HIV virus infects and destroys CD4 T cells

The flow cytometry results, once validated by a senior healthcare scientist are passed to the clinician who will consider them in association with the viral load results and the patients current symptomatic status. An individual with HIV can have a healthy CD4 count for many years but during periods when the level drops they are prone to develop a range of infections that become more severe and unusual the lower the count gets. A normal CD4 count for a healthy adult individual is considered to be in the range 450-1660 cells/mm3or 0.45 to 1.7×109 cells per litre of blood. Once the CD4 count drops below this the patient becomes prone to a range of diseases that gradually become more life threatening. With only slightly low CD4 counts a patient is more likely to get less severe infections such as Gingivitis, herpes or Oral candidiasis. As the immune depletion becomes more severe, the infections also become more severe with patients contracting Toxoplasmosis and opportunistic pulmonary infections such as environmental mycobacteria. These later stage diseases are life threatening and fall into a group of conditions referred to as AIDS defining illnesses.