Abstract
Three experiments were carried out to investigate the myelotoxicity of busulphan in female B6C3F1 mice using the Technicon H*1 and the Sysmex R-1000 flow cytometers, instruments which produce a full blood count and a differential leucocyte count, and an automated reticulocyte count, respectively. In Experiment 1, a single dose of busulphan was administered at levels from 0 to 60 mg/kg and blood parameters measured at day 14. In Experiment 2, four doses of busulphan, from 0 to 40 mg/kg, were given at fortnightly intervals, and blood samples taken at days 14 and 42. In the third experiment, a single dose of busulphan was given at 0, 35 or 45 mg/kg and sequential blood, marrow and spleen samples examined up to day 10.
In the first experiment there was a dose-related depression in the numbers of all leucocyte types. Values for Hb, RBC and HCT were not affected, whereas MCV and percentage macrocytic erythrocytes were increased, and MCHC was decreased, at high dose levels. Platelet numbers showed marked dose-related decreases. There were dose-related decreases in the numbers of all leucocyte types in Experiment 2 at days 14 and 42. Large unstained cell (LUC) numbers were reduced, and the mean neutrophil peroxidase index (MPXI) was increased, at high busulphan levels. Hb, RBC and HCT were reduced, whereas MCV, MCH and percentage macrocytic and percentage hypochromic erythrocytes were increased, in a dose-related fashion. Reticulocyte numbers showed a dose-related upward trend, but platelet counts illustrated a dose-related decrease, at days 14 and 42. In Experiment 3, busulphan caused a depression with a ‘U’-shaped curve, in the numbers of monocytes, eosinophils, lymphocytes and neutrophils. Decreased values and ‘U’-shaped curves were also seen for Hb, RBC, HCT and reticulocyte counts. Reticulocyte fluorescence ratio analysis showed that the high fluorescence ratio (HFR) was affected first and most profoundly. Calculation of the reticulocyte maturation index also demonstrated a dose-related effect on the earliest reticulocytes, and a ‘rebound’ effect. Total nucleated cell counts of the spleen and femur showed decreasing cell numbers and ‘U’-shaped responses with 45 mg/kg busulphan.
This series of three experiments has established the use of a 6 week dosing regimen, with busulphan administered at fortnightly intervals, to induce myelotoxicity in a range of haematological parameters in female B6C3F1 mice. We consider the use of the newly-developed flow cytometers and associated software, and the measurement of ‘non-standard parameters’ such as LUC, HFR and MPXI, to be particularly effective in the charcterisation of these busulphan-induced haematological changes.
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Andrews CM, Turton JA, Spurting NW (1992) Complete automated evaluation of haematological parameters using the Technicon H*1 system: experimental studies with busulphan in the mouse. Human Exp Toxicol 11:583
Bentley SA, Pegram MD, Ross DW (1987) Diagnosis of infective and inflammatory disorders by flow cytometric analysis of blood neutrophils. Am J Clin Pathol 88:177–181
Bentley SA, Aysone LH, Watson JM et al. (1989) The clinical utility of discriminant functions for the differential diagnosis of microcytic anaemia. Blood Cells 15:575–582
Bessman D (1982) Prediction of platelet production during chemotherapy of acute leukaemia. Am J Haematol 13:219–227
Bessman JD, Gilmer PR, Gardner FH (1985) Use of mean platelet volume improves detection of platelet disorders. Blood Cells 11:127–135
Bizzaro N, Briani G, Boccato P (1988) Acquired myeloperoxidase deficiency of neutrophils in a patient with aplastic anaemia (idiopathic marrow aplasia). Acta Haematol 80:71–73
Boggs DR, Geist A, Chevenick PA (1969) Contribution of the mouse spleen to post-haemorrhagic erythropoiesis. Life Sci 8:587–599
Boyd RL, Caro J, Halka KG et al. (1986) Granulopoiesis in long-term culture by marrow from mice with busulphan-induced chronic latent aplasia. Int J Cell Cloning 4:357–367
Bozzini CE, Barrio Rendo ME, Devoto FCH et al. (1970) Studies on medullary and extramedullary erythropoiesis in the mouse. Am J Physiol 219:724–728
Cavill I (1990) Automated reticulocyte counting. Sysmex J 13:450–453
Coulet M, Bezou MJ (1990) Utilization of the automated reticulocyte counter Sysmex R-1000. Sysmex J 13:393–406
Davies DT, Fisher GV (1991) The evaluation and application of the Technicon H*1 for the complete automated evaluation of laboratory animal haematology. Comp Haematol Int 1:803–808
Davies SV, Cavill I, Bentley N et al. (1992) Elevation of erythropoiesis after bone marrow transplantation: quantitative reticulocyte counting. Br J Haematol 81:12–17
Davis BH, Bigelow N, Ball ED et al. (1989) Utility of flow cytometric reticulocyte quantification as a predictor of engraftment in autologous bone marrow transplantation. Am J Haematol 32:81–87
Eder H, Wiemer U (1987) Die Beeinflussung der Retikulozytenzahl und ihrer Reifegradverteilung durch Erythropoietin bei der Ratte. Folia Haematol 114:517–522
Elson LA (1955) A comparison of the effects of radiation and radiomimetic chemicals on the blood. Br J Haematol 1:104–116
Elson LA, Galton DAG, Till M (1958) The action of chlorambucil (GB1348) and busulphan (myleran) on the haemopoietic organs of the rat. Br J Haematol 4:355–374
Fitchen JH, Cline MJ (1980) The effect of granulopoictic stress in mice with ‘latent’ bone marrow failure. Exp Haematol 8:788–794
Freireich DJ, Grehan EA, Rall DP et al. (1966) Quantitative comparison of toxicity of anti-cancer agents in mouse, rat, hamster, dog, monkey and man. Cancer Chemother Rep 50:219–244
Froom P, Quitt M, Aghai E (1989) The mean leucocyte myeloperoxidase index in haematological patients. Am J Clin Pathol 92:791–793
Fuchs A, Eder H (1991) Automated reticulocyte analysis of blood in different species. Sysmex J Int 1:34–38
Gulley ML, Bentley SA, Ross DW (1990) Neutrophil myeloperoxidase measurement uncovers masked megaloblastic anaemia. Blood 76:1004–1007
Hayhoe FGJ, Flemans RJ (1992) Haematological cytology (3rd edn) Wolfe Medical Publications, London
Hewitt J, Reardon DM (1991) The measurement of neutrophil myeloperoxidase by flow cytometry is influenced by the pseudoperoxidase activity of haemoglobin. Blood 78:1891
Ideriha NM, Vugman I, Falcao RP (1984) Ectopic bone marrow development in experimental busulphan-induced hypoplastic anaemia in mice. Blut 48:277–284
Jelkmann W, Bauer C (1980) β2-Adrenergic stimulation of erythropoiesis in busulphan treated mice. Exp Haematol 8:742–748
Joyce RA, Hartmann O, Chervenick PA (1979) Splenic granulopoiesis in mice following administration of cyclophosphamide. Cancer Res 39:215–218
Kershaw GW, Robin H, Kronenberg H (1987) Evaluation of the Technicon H*1 haematology analyser. Pathology 19:305–309
Kinsey SE, Watts MJ (1988) Accurate automated leucocyte differential counts despite profound leucopenia. J Clin Pathol 41:1236–1239
Kinsey SE, Carter AB, Watts MJ et al. (1988) The use of the H*1 in predicting marrow recovery following ablative chemotherapy in leukaemia and lymphoma. Clin Lab Haematol 10:1–5
Kline A, Bird A, Adams L et al. (1989) Identification of blast cells in the peripheral blood of patients with acute leukaemia using the Technicon H*1. Clin Lab Haematol 11:111–116
Mechetner EB, Sedmak DD, Barth RF (1991) Heterogeneity of peripheral blood reticulocytes: a flow cytometric analysis with monoclonal antibody HAE9 and thiazole orange. Am J Haematol 38:61–63
Mohondas N (1991) Commentary on Marvel JS et al.: Accurate determination of mean cell volume by isotope dilution in erythrocyte populations with variable deformity. Blood Cells 17:513–515
Morley A, Blake J (1974a) An animal model of chronic aplastic marrow failure. I. Late failure after busulphan. Blood 44:49–56
Morley A, Blake J (1974b) Haemopoietic precursor cells in experimental hypoplastic marrow failure. Aust J Exp Biol Med Sci 52:909–914
Morley AA, Trainor KJ, Seshadri RS (1978) Chronic hypoplastic marrow failure and residual injury. Blood Cells 4:253–266
Nagao A, Nakamura S, Fukimoto K (1990) Application of the R-1000 automated reticulocyte analyser to research in in vitro reticulocyte maturation. Sysmex J 13:407–415
Nehls V, Drenckhahn D, Joshi R et al. (1991) Adducin in erythrocyte precursor cells of rats and humans: expression and compartmentalisation. Blood 78:1692–1696
d'Onofrio G, Manicini S, Leone G et al. (1987) Identification of blast cells in peripheral blood through automatic assessment of nuclear density. Br J Haematol 66:473–477
Pugsley CAJ, Forbes IJ, Morley AA (1978) Immunologic abnormalities in an animal model of chronic hypoplastic marrow failure induced by busulphan. Blood 51:601–610
Ross DW, Bentley SA (1986) Evaluation of an automated haematology system (Technicon H*1). Arch Pathol Lab Med 110:803–808
Santos GW, Tutschka PJ (1974) Marrow transplantation in the busulphan-treated rat: preclinical model of aplastic anaemia. J Natl Cancer Inst 53:1781–1785
Seligman PA, Allen RH, Kirchanski SJ et al. (1983) Automated analysis of reticulocytes using fluorescent staining with both acridine orange and an immunofluorescence technique. Am J Haematol 14:57–66
Skipper HE, Schabel FM, Wilcox WS (1964) Experimental evaluation of potential anticancer agents. XIII On the criteria and kinetics associated with ‘curability’ of experimental leukaemia. Cancer Chemother Rep 35:3–111
Takami T, Sakata T (1990) Fundamental studies on the automated measurement of reticulocyte count by Sysmex R-1000 (for animal specimens). Sysmex J 11 (Suppl II)
Tatsumi N, Tsuda I (1991) Reticulocyte maturation index as a useful diagnostic parameter. Sysmex J Int 1:23–28
Trainor KJ, Morley AA (1976) Screening of cytotoxic drugs for residual bone marrow damage. J Natl Cancer Inst 57:1237–1239
Tycko DH, Metz MH, Epstein EA et al. (1985) Flow cytometric light scattering measurement of red blood cell volume and haemoglobin concentration. Appl Optics 24:1355–1365
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Originally presented at ECCP 93.
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Andrews, C.M., Spurling, N.W. & Turton, J.A. Characterisation of busulphan-induced myelotoxicity in B6C3F1 mice using flow cytometry. Comp Haematol Int 3, 102–115 (1993). https://doi.org/10.1007/BF00368113
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DOI: https://doi.org/10.1007/BF00368113