Effects of thermal exposure on immunophenotyping combined with in situ PCR, measured by flow cytometry.

Background: The combination of in situ PCR and cell phenotyping by antibody labeling (ISPCR/Flow) allows for the identification of cell subsets carrying a particular genetic sequence. ISPCR utilizes thermal cycling for genetic amplification, which can reduce the effectiveness of surface antibody labeling. This study explored and characterized the effects of thermal exposure on antibody labeling using CD4 and CD45.

Methods: Single temperature incubations and thermal cycling exposures were performed on leukocytes labeled with either direct antibody conjugates or with biotinylated antibodies and PE-streptavidin.

Results: Fluorescence emission decreased above 70 degrees ( )C when cells were stained with directly conjugated antibodies or a biotinylated antibody and PE-streptavidin prior to high heat exposure. If counter stained with PE-streptavidin after heat, fluorochrome fluorescence was detectable. We tested a second CD4 clone, that provided poor results under similar labeling conditions, suggesting the combination of fixation and heat may have an epitope specific effect for the same cellular antigen.

Conclusions: Immunophenotyping can be combined with ISPCR, but each antibody must be tested to determine its efficacy. The denaturation of protein above 70 degrees C appears to be the main reason for loss of fluorescence. The best procedure is to first stain cells with a biotinylated antibody to an epitope that survives fixation and thermocycling. The cells are then subjected to the desired PCR procedure. Finally they are stained with a fluorochrome conjugated streptavidin.