Analytical Comparison of Three Commercial Lysing Reagents for Full-Spectrum Flow Cytometric Immunophenotyping

Introduction: Flow cytometry (FC) enables rapid identification of cell lineages based on their fluorescent and light-scattering properties, playing a critical role in diagnosing and monitoring oncohematological diseases. Standardization of procedures in spectral FC is essential for method validation studies. Methods: We designed a comparative study using 21 blood samples from healthy donors. Three commercial lysing solutions used in spectral FC immunophenotyping were evaluated. Samples were stained with a panel of eight antibody-conjugated fluorochromes and aliquoted for assessment with each lysing solution (Excellyse I, Excellyse Easy, and Tombo RBC Lysis Buffer), following the manufacturers' protocols. Data acquisition was performed using a full-spectrum flow cytometer. Results: An average cell loss of 31.4% was observed, with no statistically significant differences between lysing solutions, although Excellyse I showed lower cell loss under the no-wash protocol. This reagent also achieved superior resolution in distinguishing lymphocytes from debris. It demonstrated the lowest coefficients of variation in both scatter and fluorescence parameters of leukocyte populations, highlighting its ability to preserve the optical characteristics of stained cells. RBC-T performed well in specific channels such as APC and PE-Cy7, whereas Excellyse Easy produced a resolution comparable to Excellyse I, albeit with greater signal dispersion. Conclusion: Excellyse I demonstrated the best overall performance, with greater cell preservation, clearer population resolution, and lower variability in scatter and fluorescence. Nonetheless, Excellyse Easy may still be considered a viable alternative.