Abstract 4724: No-lyse no-wash immunophenotyping.

Abstract

Abstract Immunophenotyping whole blood is a primary application in the study of blood cell populations and their functions. Red blood cells have traditionally been removed during sample preparation by lysis methods or density-gradient selection to enable the study of nucleated cell populations. This processing of whole blood has its draw backs due to the potential loss of cells of interest thru inadvertent lysis and unintentional selection of fragile and rare cells including blast cells, cancer cells, and cancer stem cells. The application of a No-Lyse No-Wash protocol in human whole blood samples benefit from a streamlined protocol that reduces cell loss. Testing of mouse whole blood presents additional challenges for cancer researchers due to the limited sample volume available (≤100 μL/day/animal), particularly in longitudinal studies. Specifically, these small volumes limit the ability to perform immunophenotyping experiments with required controls. This work describes a No-lyse No-wash method which takes advantage of acoustic focusing technology. Immunophenotyping panels are shown for both human and mouse whole blood. A total of 10μL of whole blood is stained in a 50 μL total volume and then diluted 400-fold in PBS. A fluorescence threshold is used to distinguish the white blood cell populations from the more abundant red blood cell population. In the mouse immunophenotyping example, CD45 is used to threshold on white blood cells. For the human immunophenotyping example, a cell permeable nucleic acid stain is used to threshold on all nucleated cells. At this dilution, the coincidence of the target population with red blood cells is reduced sufficiently so scatter signals are reliable for population identification. The significantly higher sample collection rates available with acoustic cytometry allows for the acquisition of high quality data utilizing a No-lyse No-wash protocol in a timely manner. Difficult-to-collect and precious samples like mouse blood can be stained and diluted without washing or performing RBC removal procedures. Loss of cells due to sample preparation is essentially eliminated, and ability to detect the cells of interest is enabled. Citation Format: Jolene A. Bradford, Rick Kerndt. No-lyse no-wash immunophenotyping. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4724. doi:10.1158/1538-7445.AM2013-4724