Flow cytometry is mainly applied in the monitoring of AIDS patients.
The frequency of various T cell subsets during HIV-1 infection is a subject of intense investigation these days. Flow cytometry is currently being done in the CFAR laboratory to analyze the different T cell subsets in HIV patients. These include CD8+ activation, CD8+ effector memory, central memory, CD8+ terminally differentiated and CD4+ Effecter memory, Central memory and the terminally differentiated effecter cells.
Specimens for flow can be prepared from whole blood or tissue, frozen PBMCs (from liquid nitrogen). Flow cytometry using the BD FACScaliber is done to determine the frequency of lymphocytes expressing of the following sets of surface markers: CD3, CD4, CD8, CCR7 (CD197), CD38, CD45RO, CD45RA, and HLA-DR.
PBMC isolation from whole blood
Cryopreserved PBMCs are a common specimen source for studies of immunological responses to vaccines, immunotherapies, etc. The health and viability of cells recovered post-cryopreservation are of course critical to the success and accuracy of immunological assays performed on them. The separation of PBMC (peripheral blood mononuclear cells) is essential for all subsequent analyses in immune monitoring. Thus effective and careful isolation, freezing and thawing of the cells is required.
For separation of mononuclear cells from whole blood or bone marrow the density gradient centrifugation is the most widely used robust method. The technique of separating peripheral blood mononuclear cells (PBMCs) from whole blood is not new, having been developed over 30 years ago. This technique has been used worldwide to collect and study lymphocytes and monocytes from a variety of subjects and in a variety of diseases
Modern advances in biochemistry and molecular biology now allow researchers and clinicians to investigate diseases at the molecular level. In a recent finding, a novel protein has been identified in patients with a subset of Chronic Fatigue Syndrome. The current assay for this protein requires that PBMCs be separated from whole blood within hours after drawing, and the subsequent PBMC “pellet” be frozen at –70°C until assay
Lymphocyte isolation from other body fluids
Lymphocyte isolation from tissues
Viral load determination from different body fluids and tissues
Low frequency polymorphism determination using OLA
Coreceptor usage determination assays prior to initiation of coreceptor antagonists for HIV treatment.
For further information on all the above, please contact us