Summary. The Brown Norwegian rat transplanted with promyelocytic leukaemic cells (BNML) has been used as a model for human acute myeloid leukaemia. We have previously shown that both the blood supply to the bone marrow and the metabolic rate decrease in relation to the leukaemic development in these rats. Here we have investigated how the development and progression of this leukaemia affect oxygenation, pH and proliferation of normal and leukaemic cells in vivo. Bone marrow pH was measured by a needle electrode. Nitroimidazol-theophylline (NITP) was used to identify hypoxic cells, and we applied bromodeoxyuridine (BrdUrd) to identify DNA replicating cells.The leukaemia progressed slowly until day 27 after which a rapid deterioration could be observed leading to severe changes over the following 5 d. In whole blood there was evidence of progressing metabolic acidosis. In bone marrow the fraction of leukaemic cells increased to > 90% and the pH dropped to about 6·5. The fraction of NITP þ cells increased to > 80% in bone marrow and to about 40% in blood. The fraction of BrdUrd þ cells was unchanged in blood, but decreased in bone marrow both for normal cells (from about 20% to 5%), and for leukaemic cells (from about 45% to 25%), evidently as a result of the severely changed microenvironment. In this study we have demonstrated in vivo the development of an acidic and hypoxic bone marrow hampering normal haemopoiesis during leukaemic growth. Our data support the notion of BNML as a valuable tool for studying leukaemogenesis.

The microenvironmental changes in the bone marrow, spleen and liver during progression of the transplantable promyelocytic leukaemia in the Brown Norwegian rat (BNML) have been studied. We used flow cytometry to estimate cellular hypoxia and proliferation based on in vivo pulse-labelling with a mixture of 2-nitroimidazole linked to theophylline (NITP) and bromodeoxyuridine (BrdUrd). The leukaemic cells were identified with the RM124 antibody. In rats inoculated with leukaemic cells the fraction of RM124+ cells was significantly increased from day 20 onwards in the spleen and from day 27 in the bone marrow and liver, reaching a level of 65-87% in these organs at day 32. At day 32, the NITP+ fraction of RM124+ cells had increased significantly in the bone marrow and spleen to 88% and 90%, respectively. The corresponding fractions of NITP+ normal cells reached 63% and 65%, respectively. From day 13 to day 32, the DNA-synthesizing (BrdUrd+) fraction of RM124+ cells in the bone marrow decreased significantly from 52% to 25%, and of normal cells from about 20% to 6%. In the bone marrow and spleen at day 27 and 32, the S-phase and G2/M-phase fractions according to DNA content were higher for the NITP+ than for the NITP- cells. This could partly be explained by an impaired cell cycle progression due to hypoxia. Nevertheless, we found indications of leukaemic cells that were simultaneously labelled with NITP and BrdUrd, in the bone marrow and spleen. These latter findings suggest that in contrast to normal cells some of the leukaemic cells can proliferate even during hypoxia, and this subpopulation may consequently renew and expand the leukaemic cell load.

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The changes in peripheral blood counts and number of colony forming cells (CFU-c) in the bone marrow after 131I treatment for thyroid cancer were followed in 24 patients. The median WBC declined to 78% and the median blood platelet count to 69% of the pretreatment values after 4 treatments. In 4 patients a moderate pancytopenia developed and in 1 patient additionally treated with chemotherapy and preleukaemia was suspected. In 19 bone marrow samples obtained from 13 patients no difference in number of CFU-c could be found related to cumulated 131I activities given to those patients. In 5 of 7 patients examined between the 9th and the 19th day afer a treatment the number of CFU-c had declined by 50% or more. It is concluded that radioiodine exerts a protracted suppressive effect on the bone marrow which is seldom severe. Agar culture of bone marrow cells with counting of CFU-c was not found to be more sensitive than the peripheral blood counts in predicting bone marrow damage and cannot be recommended for routine use.

Washless methods for double staining of nuclear antigen and DNA in unfixed nuclei were compared with established methods for staining of fixed cells. The methods were tested on phytohemagglutinin (PHA)-stimulated normal human blood lymphocytes for the double staining of 1) Ki-67 antigen and DNA and 2) bromodeoxyuridine (BrdUrd) and DNA, in continuously BrdUrd-labeled cells. With respect to the discrimination between antigen-positive and -negative subpopulations, there was no statistically significant differences between the results from direct (Ki-67) or indirect (Ki-67 or BrdUrd) washless staining of unfixed nuclei and the results from staining of fixed cells. Washless staining of unfixed nuclei was found to be rapid and simple and resulted in greater precision of the DNA analysis and in less aggregation and loss of cells.Key terms: Ki-67 antibody, anti-bromodeoxyuridine antibody, DNA synthesis, cell cycle, cell proliferation, lymphocyte activation, HL-60 cellsThe analysis of cell cycle distributions by flow cytometric DNA measurement (static parameter) may be extended with a cell kinetic (dynamic) parameter using simultaneous measurement of bromodeoxyuridine (BrdUrd). BrdUrd is incorporated into DNA-synthesizing cells and can be stained by use of anti-BrdUrd antibody, which recognizes BrdUrd, but only in singlestranded DNA (5,101. The Ki-67 antibody recognizes a nuclear antigen naturally expressed in cycling cells and may be more practical for clinical investigations than the anti-BrdUrd antibody (2,7,8). This is because its use is not dependent on complicated vital or supravital labeling and subsequent denaturation of the labeled molecule.For flow cytometric application of the Ki-67 antibody, several methods have been described, all using fixed cells and a large number of washings. Methods for combined analysis of surface markers and Ki-67 anti- In this study, we apply new methods for washless double staining of unfixed nuclear suspensions (13) to achieve a flow cytometric analysis of the correlation between the nuclear contents of DNA and a specific antigen. The feasibility of this methodological principle is demonstrated for two proliferation-associated nuclear antigens, by combined measurements in phytohemagglutinin (PHA)-stimulated lymphocytes of 1) Ki-67 antigen and DNA and 2) continuously incorporated BrdUrd and DNA. Furthermore, the washless methods for staining of unfixed nuclei are compared with established methods for staining of fixed cells (1,51.