No lyse no wash flow cytometry for maximizing minimal sample preparation.

Red blood cell lysis is an integral part of many flow cytometry protocols. It's potential to cause artifacts has been known for decades, but lysis free sample preparation has failed to replace lysis in most applications. Studies of various lysing protocols on cell losses and effects on phenotypic markers and cell function began early in the history of immunophenotyping and continue to this day. Opportunities to combine live cell response and functional assessment with phenotyping have sparked increasing interest in no lyse no wash protocols, with minimizing sample preparation effects on the cell biology as the primary goal. No lyse no wash protocols reduce sample handling and are procedurally less complex than lysis protocols, but the impact of keeping intact red blood cells that grossly outnumber the target white blood cells, must be understood to fully take advantage of this simplicity. Presented here are theories and methods for executing and interpreting no lyse no wash assays in whole blood. Methods for distinguishing white blood cells and platelets from red blood cells and improving scatter data by combining 405 nm and 488 nm side scatter are shown. Methods for assessing white blood cell light scattering profiles for individual instruments and sample treatments are discussed within the context of example profiles for no lysis and hypotonic and ammonium chloride lysis treatments. The utility of overcoming no lyse no wash scatter and fluorescence background limitations using alternate scatter and fluorescence thresholding strategies is also discussed in the context of application examples.