Synthesis of 1,25-dihydroxyvitamin D3 by spleen cells isolated from two patients with myelofibrosis and a normal subject.

The ability to synthesise [3H]1,25(OH)2D3 was studied in spleen cells incubated in short-term primary culture with [3H]25(OH)D3 which were isolated from two patients with idiopathic myelofibrosis and from one normal subject. Formation of a metabolite co-chromatographing with authentic 1,25(OH)2D3 on three different high-performance liquid chromatography systems was observed for cells from all three patients. [3H]1,25(OH)2D3 synthesis was 0.37 and 1.6 fmol/10(6) cells/incubation for cells with a density below that of lymphocyte separating media (1.077 g/ml) for the two myelofibrotic patients respectively and 0.15 fmol/10(6) cells/incubation for the normal subject. The most likely cell type capable of this synthesis were those of the monocyte-macrophage lineage which would have been present in abnormally high numbers in the patients with myelofibrosis. However, the exact identity of the cell-type responsible could not be determined because of the heterogeneity of cell types present. The observation that spleen cells from two patients with myelofibrosis and from a normal accident victim could metabolise [3H]25(OH)D3 to its active form [3H]1,25(OH)2D3 suggests a possible role for this metabolite in spleen haematopoiesis.

Simple method for granulocyte-macrophage cell culture and staining in soft agar: comparison with a standard research technique.

A modified, small volume, two phase, disc culture system for CFU-GM (seven and 14 days of incubation) was compared with a standard single layer system. The 1 ml single layer cultures were counted unstained in situ before both sets of cultures were transferred to glass slides for staining. Bone marrows were cultured from forty eight subjects, including normal controls and patients with acute non-lymphoblastic leukaemia, acute lymphoblastic leukaemia, and myelodysplastic syndrome. Observer error was least with the disc cultures, whereas variation between replicate cultures was similar for both methods. A high degree of correlation was found between the two methods for both day 7 (r = 0.90) and day 14 (r = 0.91) cultures. The number of colonies and clusters was higher with the disc system, indicating better cloning efficiency. Analysis of subsets of clinical groups showed similar patterns of abnormality with both systems. The simplicity of the method makes the use of this technology possible in most laboratories, and the superior morphological resolution may increase the clinical usefulness of such studies.