No effect of lysis solutions on absolute CD19+ lymphocytes count and CD45 index in chronic lymphocytic leukemia

The absolute CD19+ lymphocytes count is essential for chronic lymphocytic leukemia (CLL) management. At the present time, no standardized flow cytometry (FCM) protocol to measure B-lymphocytes counts is established. The aims of the present study were first to evaluate the effect of different lysis solutions and of red blood cell lysis per se on CLL lymphocytes count and B-lymphocytes CD45 expression and second to compare absolute B-lymphocytes counts obtained by single (SP) and dual platforms (DP). Absolute CD19+ B-lymphocytes counts and CD45 expression in 35 whole-blood CLL samples were determined by FCM using either different lysis solutions or using a no wash no lyse (NWNL) protocol. Single platform using microbeads was also evaluated for absolute quantification. The absolute CD19+ B-lymphocytes counts using different red blood cell lysis solutions correlated with NWNL method without any effect on CD45 expression. Bland and Altman plot showed homogenous distribution of bias; mean bias was less than 1% for all lysing solutions. Moreover, no statistically significant difference between SP and DP was observed. The type of lysis solution influences neither the CD19+ B-lymphocytes count nor the CD45 expression. The two systems, SP and DP, yield comparable values with excellent agreement. However, the tendency of slightly lower results with SP showed the requirement of larger studies before standardization of B-lymphocytes count in CLL patients.