Incomplete Red Blood Cell Lysis in Whole Blood (Flow Cytometry (CST Guide))

Question

Accepted Answer

Red blood cell debris persists in whole blood samples after lysis protocol, causing high background and interfering with target cell population analysis. Common causes: 1) Additional washes insufficient to eliminate red blood cell debris using Flow Alternate Protocol 2) 0.1% Triton X-100 solution not fresh or improperly prepared, reducing lysis efficiency 3) Deviation from optimized CST Flow protocol steps or reagent concentrations Solutions: 1) Add up to 3 additional washes between each step to thoroughly remove red blood cell debris 2) Use fresh reagents and prepare new 0.1% Triton X-100 solution immediately before use 3) Closely follow CST Flow Alternate Protocol derived from Chow et al. (2005) Cytometry Part A 67A:4–17, which has been extensively optimized for whole blood