Flow cytometry is a technique that allows to obtain multiparametric data about the physical and chemical characteristics of cells in suspension. The development of monoclonal antibodies together with improvement of flow cytometers has allowed the use of flow cytometry in clinical diagnosis. From the study of CD4+ T lymphocytes to monitor the development of HIV, in the same way as the diagnosis of hematological pathologies and cellular cycle studies (ploidy), this is a technique of routine use in clinical laboratories. The possibility to use this technology to separate cells (sorting) according to their characteristics it is generally a reserved option to the research environment. Parallelly, most of the hematological counters also uses this technology.

Technical aspects

The flow cytometer has three main systems:

Hydraulic system

It is composed of a group of pneumatic controls and fluidic delivery system to establish a laminated flow that allows to the cellular suspension moves in single file through the flow chamber.

Optic system

It consists of: laser, filters, lents and detectors. The light source is usually produced by a laser (in some models, mainly in investigation, they combine several sources). The most commonly used light source is gas laser (commonly argon) refrigerated by air that produces a monochrome light of 488 nm. This light is used for the fluorochrome excitement and it causes the dispersion of light that informs us about cellular characteristics.

Computer system

This system receives the information from cytometer and processes it for their later analysis.

The perfect integration of these systems allows both accurate and fast analysis of a high number of cells in little time.
 

The cellular suspension is incubated previously with monoclonal antibodies conjugated with different fluorochromes (FITC, PE, PerCP, PECy5, APC,... ) and it is introduced in the hydraulic system so that the cells pass individually through the flow chamber, where the laser beam intercepts the cells. These contact produces two different dispersion patterns : frontal and side (90¼) dispersion . The frontal dispersion (Forward Scatter,A) gives us information about the cellular size , while lateral dispersion (Side Scatter,B) informs us about cellular complexity. The monoclonal antibodies linked to cellular surface is at the same time excited by the laser and generate fluorescences that are collected by photomultipliers (up to 4 at the moment) that transform the optic to electric signal, then cytometer send all information to the computer system for later analysis.

• Leukemias and lymphoma research
• Stem Cells
• Cellular activation
• CD4+ T lymphocytes monitoring in HIV
• Cellular immunity
• Cellular cycle (ploidy)
• Reticulocyte enumeration
• Platelet autoantibodies
• HLA B27
• Paroxismal Nocturnal Hemoglobinuria
• Cytogenetics