Mononuclear cells contaminating acute lymphoblastic leukaemic samples tested for cellular drug resistance using the methyl-thiazol-tetrazolium assay

Abstract

Summary The methyl-thiazol-tetrazolium (MTU) assay is a drug resistance assay which cannot discriminate between malignant and non-malignant cells. We previously reported that samples with > 80% leukaemic cells at the start of culture give similar results in the MTT assay and the differential staining cytotoxicity assay, in which a discrimination between malignant and non-malignant cells can be made. However, the percentage of leukaemic cells may change during culture, which might affect the results of the MTT assay. We studied 106 untreated childhood acute lymphoblastic leukaemia (ALL) samples with > 80% leukaemic cells at the start of culture. This percentage decreased below 80% in 28%, and below 70% in 13%, of the samples after 4 days of culture. A decrease below 70% occurred more often in case of 80-89% leukaemic cells (9/29) than in case of > 90% leukaemic cells at the start of culture (5/77, P = 0.0009). Samples with <70% leukaemic cells after culture were significantly more resistant to 6 out of 13 drugs, and showed a trend towards being more resistant to two more drugs, than samples with > 80% leukaemic cells. No such differences were seen between samples with 70-79% and samples with > 80% leukaemic cells after culture. We next studied in another 30 ALL samples whether contaminating mononuclear cells could be removed by using immunomagnetic beads. Using a beads to target cell ratio of 10:1, the percentage of leukaemic cells increased from mean 72% (s.d. 9.3%) to mean 87% (s.d. 6.7%), with an absolute increase of 2-35%. The recovery of leukaemic cells was mean 82.1% (range 56-100%, s.d. 14.0%). The procedure itself did not influence the results of the MTT assay in three samples containing only leukaemic cells. We conclude that it is important to determine the percentage of leukaemic cells at the start and at the end of the MTT assay and similar drug resistance assays. Contaminating mononuclear cells can be successfully removed from ALL samples using immunomagnetic beads. This approach may increase the number of leukaemic samples which can be evaluated for cellular drug resistance with the MTT assay or a similar cell culture drug resistance assay.

Elevated S100A8/S100A9 expression causes glucocorticoid resistance in MLL-rearranged infant acute lymphoblastic leukemia

“…3 Mononuclear cells were isolated and contaminating non-leukemic cells were removed using immunomagnetic beads as described before. 19 As a result, all leukemic samples used in this study contained 490% of leukemic blasts.…”

Cell proliferation is related to in vitro drug resistance in childhood acute leukaemia

“…After washing, the cells were resuspended into culture medium consisting of RPMI 1640 (Dutch modification without L-glutamine; Gibco BRL, Breda, The Netherlands), containing 20% fetal calf serum (FCS; Integro, Zaandam, The Netherlands), 2 mM L-glutamine (ICN Biochemicals, Costa Mesa CA, USA), ITS media supplement (5 mg ml À1 insulin, 5 mg ml À1 transferrin, 5 ng ml À1 sodium selenite) (Sigma, St Louis MO, USA), 80 IU ml À1 penicillin (Gibco BRL Breda, The Netherlands) 80 mg ml À1 streptomycin (Gibco BRL), 0.1 mg ml À1 fungizone (ICN Biomedicals), and 0.2 mg ml À1 gentamycin (Flow Laboratories, Irvine, Scotland). Contaminating lymphocytes were removed using immunomagnetic beads as described previously (Kaspers et al, 1994). This procedure was carried out at room temperature instead of at 371C in order to avoid phagocytosis of the beads by the myeloid leukaemia cells.…”

The modulating effect of PSC 833, cyclosporin A, verapamil and genistein on in vitro cytotoxicity and intracellular content of daunorubicin in childhood acute lymphoblastic leukemia

“…Contaminating normal cells were eliminated using monoclonal antibodies linked to magnetic beads as previously described. 13 All processed samples contained more than 80% leukemic cells which was determined on cytospin preparations stained with May-Grü nwald Giemsa (MGG; Merck, Darmstadt, Germany). The viability of cells was determined by trypan blue exclusion, and was Ͼ90% at the start of all experiments.…”

“…The assay conditions have been well-characterized and standardized as previously described. [13][14][15] In 96-well round-bottomed microculture plates (Greiner Labortechnik, Alphen aan den Rÿn, The Netherlands), leukemic cells (160 000 cells/well) were incubated with six different concentrations of DNR ranging from 0.002 to 2 g/ml with and without the indicated concentration of modulator in humidified air containing 5% CO 2 . Control leukemic cells from the sample were incubated with and without the modulator only.…”

“…14 Samples with more than 70% of leukemic cells in the control wells after 4 days of culture (without drug) and a control OD value higher than 0.050 arbitrary units (adjusted for blank values) were used to calculate the LC 50 value, ie the concentration of drug that is lethal to 50% of the cells (in g/ml). 13,16 The LC 50 of DNR in the presence of the modulator was corrected for the cell kill mediated by the modulator itself. The modulating effect on DNR cytotoxicity was expressed as the ratio between the LC 50 value of DNR with and without modulator: a ratio Ͼ1 indicates a sensitizing effect of the modulator, ie the LC 50 of single DNR was higher than the LC 50 of DNR in the presence of the modulator; a ratio Ͻ−1 indicates an adverse effect of the modulator, ie the LC 50 of DNR in the presence of the modulator is higher than the LC 50 of single DNR.…”