Fluorescence spectrophotometry as applied in BD FACSPresto™ Near-Patient CD4 Counter

Luminescence = is production of light through excitation by means other than increasing temperature

Fluorescence is a short-lived type of luminescence created by electromagnetic excitation /light /photons

Fluorescence occurs when an photons (light energy) raises an electron’s energy state (excitation) and the electron undergoes rapid thermal energy loss (vibration) to a singlet energy state and light (photon) is emitted as a by-product of it returning to its ground state

The inside of the cartridge is coated with the following antibodies:

• CD4 PE -Cy™5
• CD3 APC
• CD45RA APC
• CD14 PE

The CD4 is a molecule found on a subset of immune cells within blood.

It acts as a receptor for human immunodeficiency virus (HIV) to gain entry into cells.

CD3 APC and CD45RA APC = total lymphocytes.

• CD3+CD4+ molecules are present on 28% to 58% of lymphocytes in normal peripheral blood

CD4 PE-Cy5 = CD4 lymphocytes.

• CD14 PE identifies monocytes, which are excluded from the analysis.

Binding of secondary antibody to the primary antibody

Secondary antibody is tagged with a fluorochrome

Spectrophotometry is the measurement of the intensity of light at selected wavelength

The method depends on the light absorbing property of the substance being analysed

The light absorption is directly related to the concentration of the compound in the sample.

As Concentration increases, light absorption increases linearly and light transmission decreases, exponentially

• Evaluate and monitor the immune status of patients diagnosed with, or suspected of developing, immune deficiencies such as acquired immune deficiency syndrome (AIDS).

• The number of CD4 T lymphocytes declines in HIV infection. Moreover, absolute number and percentage of CD4 T lymphocytes are the cellular parameters most closely associated with HIV disease progression and patient prognosis