Identification of leukaemic cells in bone marrow and blood samples by a new cytofluorometric assay

Abstract

Summary The expression of thymidine kinase -an enzyme of the DNA precursor pathway -is strictly regulated during the normal cellular cycle, but is much higher and permanently expressed in malignant growing cells. We used this fact to detect neoplastic cells in samples freshly taken from leukaemia patients and kept frozen in liquid nitrogen until analysis. Using a new cytofluorometric assay for thymidine kinase in single cells, we were able to identify leukaemic cells in a surplus of normal ones. Our results demonstrate the benefits of this assay for leukaemia diagnosis.Keywords: tumour diagnosis; thymidine kinase; fluorescent deoxynucleoside analogue Thymidine kinase (TK) catalyses the ATP-dependent phosphorylation of thymidine and deoxyuridine. The activity of TK is strictly regulated during the normal cell cycle, peaking at the onset of DNA synthesis but remaining extremely low in resting cells (Bello, 1974;Wawra et al., 1981). In the past, we have established a cytofluorometric method based on the uptake of a fluorescent nucleoside that is subsequently phosphorylated by the cells in the same manner as thymidine (Hengstschlager and Wawra, 1993a (Hengstschlager et al., 1994b). Moreover, this method enabled us to identify a few transformed cells mixed with a 10 000-fold excess of normal ones (Hengstschlager and Wawra, 1993b isolated from heparinised peripheral blood of leukaemia patients and from two normal subjects using the FicollHypaque gradient method. After washing three times in phosphate-buffered saline (PBS), the leucocytes were resuspended in RPMI-1640 medium containing 20% human AB plasma and 10% dimethylsulphoxide. Cells were slowly cooled to -70°C and then stored in liquid nitrogen. In addition, normal lymphocytes were stimulated with 5 pg phytohaemagglutinin per ml of medium for about two cell doubling times (40 h). Buffy coat, obtained by leukapheresis, stimulated lymphocytes, and bone marrow samples from patients were treated and frozen as described above.Non-stimulated leucocytes from leukaemia patients and normal controls, phytohaemagglutinin-stimulated normal lymphocytes and bone marrow cells were all thawed as fast as possible in a 37°C water bath and transferred into 5 ml of RPMI-1640 medium containing 20% fetal calf serum. After 30 min, cells were washed with RPMI-1640 medium without serum and incubated with the fluorescent thymidine analogue as described below. For analysis, we used between 106 and 107 cells per sample.Determination of intracellular TK activity simultaneously with DNA distribution Synthesis and purification of the fluorescent thymidine analogue N-dansyl-amino-uracil-deoxyriboside (DAUdR, formerly called AUdR/DANS) have been published previously, and cytofluorometric measurement of TK activity in living cells has also generally been performed as described (Hengstschlager and Wawra, 1993a). We just added some minor revisions at the DNA staining part of the procedure mainly to improve the resolution of DNA analysis. Cells were exposed in RPMI-1640 medium without serum for 30 m...

Fetal cells in the peripheral blood of pregnant women express thymidine kinase: a new marker for detection

“…(1) It has been demonstrated that circulating cells of fetal origin include trophoblasts, fetal lymphocytes, nucleated red blood cells and hematopoetic stem cells [3]. We showed that trophoblasts and fetal cord blood cells express high intracellular levels of TK activity, whereas normal adult peripheral blood is totally TK-negative (this study and [17]). (2) The small cell population with high TK activity specifically occurs in peripheral blood samples of pregnant women and not in the circulation of non-pregnant individuals.…”

“…Analysing peripheral blood cells (about 30 million per individual) of 24 non-pregnant women (without any previous pregnancy) and 13 male individuals, we could not detect TK-positive cells (Fig. 2, see also [17]). Strikingly, using the same approach to analyse 14 pregnant women we observed a very small cell population with high TK activity (Fig.…”

“…Only those experiments with positive PCR signals using the control oligonucleotides for the oncogene HERS are presented. [11,12,16,17]: The cells are incubated with the fluorescent thymidine analogue AUdR/DANS. Intracellular accumulation of this analogue reflects TK activity.…”