Granulocytic Colony Stimulating Activity in Plasma and Leukocytes from Chronic Myelogenous Leukemia Patients

Colony stimulating activity present in plasma and in leukocytes from chronic myelogenous leukemia patients and from normal volunteers was determined by double layer bone-marrow tissue culture. Plasma and leukocytes incorporated into the bottom feeder layer stimulated the formation of colonies from human bone-marrow cells cultured in the upper semi-solid agar layer. The colony stimulating activity of plasma from chronic myelogenous leukemia patients was significantly increased over that of normal plasma (p<.01). The difference between the colony stimulating activity of chonic myelogenous leukemia and normal leukocytes was not statistically significant. After 14 days of incubation, all bone-marrow cell colonies were composed primarily of macrophages rather than granulocytes as had been previously reported. OHIO J. SCI. 76(6): 274, 1976 Colony stimulating factor (CSF), stimulates both proliferation and differentiation of granulocytic precursors in vitro. The stimulation obtained from this factor is referred to as colony stimulating activity (CSA). When human serum and plasma are added to murine bonemarrow cells cultured in vitro, the colony stimulating factor present in these substances stimulates hemopoietic stem cells in the cultured marrow to proliferate and to form colonies of up to 2000 leukocytes (Metcalf and Foster, 1967). Although human plasma and serum are capable of stimulating colony formation from murine bone-marrow cells, the stimulatory activity is not comparable to, or correlated with, the stimulation of cultured human bone-marrow cells (Lind et at, 1974). In fact, it has been repeatedly demonstrated that human plasma and serum are not capable of dramatically stimulating human bone-marrow colony growth. The most potent stimulus to colony formation by human bone-marrow cells has been observed with peripheral leukocytes (Robinson and Mangalik, 1972). Thus, in huManuscript received February 12, 1976, and in revised form August 5, 1976 (#76-15). Presently at Graduate School of Biomedical Sciences, University of Texas, Houston. Deceased September 5, 1975. man bone-marrow tissue culture, human leukocyte feeder layers have been used instead of serum or plasma as a source of CSF (Robinson and Pike, 1970). The present study was undertaken to determine if the levels of colony stimulating factor in chronic myelogenous leukemia patients differed significantly from levels present in normal persons. MATERIALS AND METHODS Peripheral blood from normal volunteers and from chronic myelogenous leukemia patients was collected by venipuncture using sterile 20 ml Vacutainer tubes containing two drops of preservative-free sodium heparin. After allowing cellular components of the blood samples to settle for two hours at room temperature, the plasma layer from each sample was collected and filtered through a sterile 0.22^ Millex filter. Each leukocyte layer, along with approximately 0.5 ml of plasma, was collected into separate sterile tubes. Leukocyte feeder layers were prepared by a modification of the procedure described by Pike and Robinson (1970). Tissue culture medium was heated to 40° and mixed 9:1 with boiled 5% Purified Agar (Difco) to give a final agar concentration of 0.5%. To this mixture human peripheral leukocytes were added to yield a final concentration of 1X10 cells per ml. For the preparation of plasma feeder layers, tissue culture medium heated to 40° was mixed 9:1 with boiled 6.5% agar. Human plasma was then added 1:3 to the medium yielding a final agar concentration of 0.5%. One ml aliquots of the feeder layer mixtures were pipetted into