Comparative study of five commercial reagents for preparing normal and leukaemic lymphocytes for immunophenotypic analysis by flow cytometry

The flow cytometric analysis of leucocytes in whole blood is usually perfor med on samples in which the erythrocytes have been lysed and the leucocytes fixed. Because lysis and fixation reagents have the potential to introduce artefacts, several commercially available reagents were used to prepare no rmal and leukaemic lymphocytes for immunophenotypic analysis by flow cytome try, and the results were compared with those obtained from live whole bloo d. The reagents tested were the ImmunoPrep system and Optilyse C (Coulter), LF-1000-Lyse and Flow (Harlan), Uti-Lyse (Dako) and FAGS Lysing Solution ( Becton Dickinson). The effect of each reagent on the apparent expression of CD3, CD5, GD11b, CD45, FMC7, kappa and lambda antigens was determined on l ymphocytes from six normal controls and from six patients with chronic lymp hocytic leukaemia (CLL). The following observations were made: (i) the time in minutes for each procedure varied markedly and was 1.5, 15, 20, 30 and 30 for the ImmunoPrep system, Optilyse C, Uti-Lyse, FAGS Lysing Solution, a nd LF-1000, respectively, but only 0.5 min for live whole blood. (ii) The f orward and side scatter characteristics were affected by all of the lysis a nd fixation procedures, and this was most marked for LF-1000-Lyse and Flow. (iii) OptiLyse G gave preparations with poor forward and side scatter reso lution due to the presence of residual red cell fragments. (iv) Lysis and f ixation procedures did not affect the apparent expression of the GD3, CD45, or FMG7 antigens on normal or CLL samples, but gave highly variable result s for the expression of the CD5, CD11b, kappa, and lambda antigens on the C LL samples. We conclude that lysis and fixation procedures can introduce di fferent artefacts in the analysis of normal and leukaemic samples that are best avoided by analysing live whole blood. (C) 1999 Wiley-Liss, Inc.