No lyse no wash flow cytometry for maximizing minimal sample preparation

Area of Science:

• Flow cytometry
• Immunophenotyping
• Cellular analysis

Background:

• Red blood cell lysis is standard in flow cytometry but can cause artifacts.
• Lysis-free methods offer simpler sample preparation but require understanding red blood cell interference.
• Minimizing sample preparation effects on cell biology drives interest in no lyse no wash protocols.

Purpose of the Study:

• To present theories and methods for executing and interpreting no lyse no wash flow cytometry assays.
• To demonstrate methods for distinguishing white blood cells and platelets from red blood cells.
• To discuss strategies for overcoming limitations in no lyse no wash assays.

Main Methods:

• Combining 405 nm and 488 nm side scatter to improve data quality.
• Assessing white blood cell light scattering profiles for different sample treatments (no lysis, hypotonic lysis, ammonium chloride lysis).
• Utilizing alternate scatter and fluorescence thresholding strategies to address background limitations.

Main Results:

• Methods for distinguishing cell populations in the presence of intact red blood cells are presented.
• Comparative analysis of light scattering profiles under different lysis conditions.
• Demonstration of effective thresholding strategies for improved data interpretation.

Conclusions:

• No lyse no wash protocols offer procedural simplicity but require careful consideration of red blood cell interference.
• Optimized scatter and fluorescence thresholding are crucial for successful no lyse no wash assays.
• This approach enables combining functional assessments with phenotyping while minimizing sample preparation artifacts.