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

Abstract

The flow cytometric analysis of leucocytes in whole blood is usually performed 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 normal and leukaemic lymphocytes for immunophenotypic analysis by flow cytometry, and the results were compared with those obtained from live whole blood. The reagents tested were the ImmunoPrep system and OptiLyse C (Coulter), LF‐1000‐Lyse and Flow (Harlan), Uti‐Lyse (Dako) and FACS Lysing Solution (Becton Dickinson). The effect of each reagent on the apparent expression of CD3, CD5, CD11b, CD45, FMC7, κ and λ antigens was determined on lymphocytes from six normal controls and from six patients with chronic lymphocytic 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, FACS Lysing Solution, and LF‐1000, respectively, but only 0.5 min for live whole blood. (ii) The forward and side scatter characteristics were affected by all of the lysis and fixation procedures, and this was most marked for LF‐1000‐Lyse and Flow. (iii) OptiLyse C gave preparations with poor forward and side scatter resolution due to the presence of residual red cell fragments. (iv) Lysis and fixation procedures did not affect the apparent expression of the CD3, CD45, or FMC7 antigens on normal or CLL samples, but gave highly variable results for the expression of the CD5, CD11b, κ, and λ antigens on the CLL samples. We conclude that lysis and fixation procedures can introduce different artefacts in the analysis of normal and leukaemic samples that are best avoided by analysing live whole blood. Cytometry (Comm. Clin. Cytometry) 38: 153–160, 1999. © 1999 Wiley‐Liss, Inc.

Comparative analysis of minimal residual disease detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH PCR in CLL after allogeneic and autologous stem cell transplantation

“…The relative specificity threshold is particularly dependent on debris and unspecific antibody binding. 38 It is influenced by conjugation, clone, and target of the moabs used, by lysing procedures, [39][40][41] by gating strategies, and by the immunophenotype of nonmalignant cells present in the sample. 42 From our analysis of 23 healthy donors, we calculated a relative specificity threshold of 9.7 false positive cells per 100 000 leukocytes measured.…”

Comparison of sample fixation and the use of LDS-751 or anti-CD45 for leukocyte identification in mouse whole blood for flow cytometry

“…However, studies indicate that erythrocyte lysing reagents cause increased leukocyte expression of glycoprotein CD11b and shedding of granulocyte glycoprotein L-selectin (McCarthy, Macey, Cahill, and Newland, 1994;Macey, et al, 1995;Macey, et al, 1999;Alvarez-Larran, Toll, Rivas, and Estella, 2005). Erythrocyte lysing also causes other blood cells, including leukocytes and platelets, to lyse (Terstappen, Meiners, and Loken, 1989;Alvarez-Larran, et al, 2005), creating cell microparticles and debris that interact with and activate leukocytes (Repo, Jansson, and Leirisalo-Repo, 1993).…”

“…An example of a nuclear stain used historically for flow cytometry to label leukocytes is Laser dye styryl-751 (LDS-751) (Hokama, et al, 2000;McDonagh, Hokama, Copeland, and Reynolds, 1997;McCarthy, Macey, Cahill, and Newland, 1994;Macey, et al, 1999;Simon, Chambers, and Sklar, 1990;Terstappen, et al, 1991;Terstappen, Meiners, and Loken, 1989;Repo, Jansson, and Leirisalo-Repo, 1993;Himmelfarb, Hakim, Holbrook, Leeber, and Ault, 1992). Although LDS-751 has been used extensively to discriminate leukocytes in whole blood samples, it has been reported to indiscriminately stain both live and dead cells (O'Brien, and Bolton, 1995) and has been used to identify platelets and nucleated erythrocytes in whole blood (Terstappen, 1991).…”

“…Studies that have examined fixation on sample preparation have produced conflicting results. A number of studies determined that cell fixation decreases the mean fluorescence of several surface antigens, including leukocyte CD11b and CD18, regardless of erythrocyte lysing agents, anticoagulant, and time of fixation (McCarthy, Macey, Cahill, and Newland, 1994;Macey, McCarthy, Milne, Cavenaugh, and Newland, 1999). Conversely, other groups demonstrate that granulocyte CD11b expression and platelet-leukocyte conjugate formation are increased with sample fixation after antibody staining (Repo, Jansson, and Leirisalo-Repo, 1993).…”

Harmonization of light scatter and fluorescence flow cytometry profiles obtained after staining peripheral blood leucocytes for cell surface‐only versus intracellular antigens with the Fix & Perm™ reagent

“…This was mainly due to the lack of reproducible intracellular staining procedures that could be universally applied for the detection of the most relevant nuclear and cytoplasmatic proteins (5). During the 90s, several new intracellular staining procedures based on sequential fixation and permeabilization of nucleated cells, have become available (6)(7)(8). Once compared, these intracellular staining approaches show significantly different sensitivities for the detection of intracellular proteins, due to a variable effect on increasing cellular background autofluorescence and decreasing specific antigen-associated fluorescence of different cell populations coexisting in bloodcontaining samples (9,10).…”