Flow-cytometric detection of terminal deoxynucleotidyl transferase and other intracellular antigens in combination with membrane antigens in acute lymphatic leukemias

Abstract

Development of a new fixation procedure allowed flow-cytometric analysis of nuclear and other intracellular antigens in acute lymphatic leukemia (ALL). A short fixation of the cells with buffered formaldehyde acetone (BFA) rendered the cell membrane permeable, allowing the monoclonal antibodies (MoAbs) to penetrate the cell. Through this method, a rapid analysis of intracellular antigens, specific for acute lymphatic leukemia [such as terminal deoxynucleotidyl transferase (TdT), immunoglobulin M (IgM) heavy chain, and antigens recognized by the CD22 or CD3 MoAbs) was performed by flow cytometry. The surface antigens remained intact after this fixation procedure, enabling simultaneous detection of membrane and intracellular antigens. The binding of biotinylated antibodies against several B- and T-lymphoid membrane antigens was detected with streptavidin-phycoerythrin (red fluorescence), whereas the intracellular antigens were stained with FITC-labeled polyclonal antibodies, or indirectly with FITC-labeled goat anti-mouse IgG (green fluorescence). Through this combination of markers, minor cell populations can be detected and a rapid and quantitative immunodiagnosis can be performed.

Sequential paraformaldehyde and methanol fixation for simultaneous flow cytometric analysis of DNA, cell surface proteins, and intracellular proteins

“…The paraformaldehyde-induced decrease in propidium iodide fluorescence was greater for diploid cells 22.5% methanol at 4°C for 1 h; C, 0.25% w/v paraformaldehyde at 4' C for 15 min, plus 0.1% Triton X-100 for 2 min a t 4"C, and D, 50% methanol at 4°C for 1 h. than for aneuploid cells within the same sample, as indicated in Table 6. In Table 6, methanol-fixed diploid cells in tumor samples are compared with a methanolfixed external normal lymphocyte standard, and paraformaldehyde/methanol-fixed diploid cells in tumor samples are compared with paraformaldehydeimethanol-fixed normal lymphocytes.…”

“…Occasionally, this paraformaldehyde fixation-induced effect is useful in identifying biologically distinct near-diploid subpopulations in tumors. 0 1992 Wiley-Liss, Inc.There are many published studies in which specific intracellular proteins have been studied quantitatively by means of flow cytometric immunofluorescent techniques (1,(15)(16)(17)(18)(19)(20)(21)(22)(23)25), often in combination with cellular DNA content. Unfortunately, no clear consensus has emerged from these studies with regard to optimal cell fixation conditions for multiparameter flow cytometric analysis.…”

Membrane cell permeabilisation with saponin and multiparametric analysis by flow cytometry

“…Recently, three techniques have been described that are suitable for intracellular antigen detection without altering membrane antigen expression. The first uses lysolecithin as a permeabilizing agent ( l l ) , the second is a fixation procedure with buffered formaldehyde acetone (12), and the third is a fixation procedure using paraformaldehyde and ethanol (9). Compared to the above techniques, saponin permeabilization provided many advantages: 1) It is a simple and fast method that does not require any additional step during the experiments; 2) it does not induce any increase in autofluorescence and only a minimal increase in non-specific background fluorescence with irrelevant antibodies; 3) it does not result in cell aggregation as shown by microscopic examination; 4) while slightly modifying the cell morphology, it still allows detection of cell subsets differing on the basis of light scattering characteristics; 5 ) it permeabilizes cytoplasm as well as nuclear membranes; and 6) it does not alter the expression of most membrane antigens.…”

Detection of intracellular lymphoid differentiation antigens by flow cytometry in acute lymphoblastic leukemia

“…Several intracellular markers, particularly terminal deoxynucleotidyl transferase ( TdT; a nuclear DNA polymerase) and cytoplasmic mu immunoglobulin heavy chain, have also gained an accepted place in the classification of ALL ( 1,6,14). These markers have traditionally been detected by staining of cytocentrifuge preparations of leukemic cells, although flow cytometric methods for analysis of TdT and cytoplasmic mu expression have been reported (7,11,12,15 ). Two further useful cytoplasmic markers for establishing lineage in ALL are CD3 and CD22 (4,9,10,13).…”