Comparative analysis of different permeabilization methods for the flow cytometry measurement of cytoplasmic myeloperoxidase and lysozyme in normal and leukemic cells

Abstract

Using a direct one-color (fluorescein isothiocyanate; FITC) staining method with a Facscan flow cytometer, we evaluated the intracellular expression of two granular constituents of myeloid cells [myeloperoxidase (MPO) and lysozyme] on leukemic cells from 21 patients with acute myeloid leukemia (AML), and 6 patients with acute lymphoblastic leukemia (ALL). Three different permeabilization techniques were used [FACS Lysing Solution (FLy), B.Dis; Ortho-PermeaFix (OPF); Fix and Perm (F&P), Caltag] prior to monoclonal antibody (McAb) staining, in order to verify the specificity and the sensitivity of the three labelling methods towards the two model antigens. Peripheral blood cells from 15 healthy subjects and Ortho Absolute Control served as controls. Data were expressed as percentage of positivity, net fluorescence intensity, ratio between mean fluorescence intensity (MFI) of positive cells and that of isotypic controls (P/N ratio; evaluated in both geometric and arithmetic scale), and, in 12 representatives cases (7 AML, 5 normal samples), in the form of both molecules of equivalent soluble fluorochromes (MESF) and antibody binding capacities (ABC). As far as the antigenic expression of MPO and lysozyme in normal samples is concerned, F&P resulted, in our hands, in the most specific and sensitive staining, followed by FLy solution and OPF, which showed positivity for MPO, and, to lesser extent, for lysozyme in a considerable manner of lymphocytes (means 64% and 54%, respectively, for OPF and FLy; range of ABC/cell: 0.9-5.2 x 10(3)) obtained from healthy subjects. With the reference F&P permeabilizing solution, 90% and 80% of FAB M1-M5 cases were found to be positive for MPO and lysozyme, respectively. However, M1, M2, and M3 AML FAB (French-American-British) subvarieties were characterized by a brighter expression for MPO (mean ABC/cell: 89 x 10(3)) than that of lysozyme (mean ABC/cell: 12.5 x 10(3D)), whereas blast cells from patients with M5a FAB subtypes showed higher levels of lysozyme (mean ABC/cell: 65 x 10(3)) than that of MPO (mean ABC/cell: 0.1 x 10(3)). One of five cases of FAB MO AML showed a dull positivity for MPO-7 McAb. Patients with ALL were MPO and lysozyme negative using both F&P and FLy reagents, although a certain degree of positivity was documented in some cases with OPF. Taking these data together, it can be stated that the use of anti-MPO McAbs may be of great value for the diagnosis and monitoring of acute leukemia and, along with lysozyme McAb, can provide useful information in the distinction of myeloid from monocytic leukemias and in the lineage assignment of apparently biphenotypic forms. However, the methodology used for the detection of these myeloid-associated antigens is critical for a correct interpretation of cytofluorimetric data and should be taken into account when evaluating data coming from multicenter trials dealing with leukemias. A standardization of cytofluorimetric analysis of intracellular antigens is needed in order to improve the reproducibility and comparability of results in multicenter studies.