In vitro growth of myeloid colonies from bone marrow of patients with acute leukemia in remission.

By JULEs HARRIS AND EMIL J. FREIREICH T HE TREATMENT OF LEUKEMIA is guided by histologic examination of bone marrow specimens. Morphologic criteria alone may be insufficient for determining whether therapy has produced remission of disease. For cxample, in chronic myelogenous leukemia the marrow may appear morphologically normal but cytogenetic examination of bone marrow samples will show the persistence of the abnormal Philadelphia chromosome. Functional criteria to distinguish between leukemic and normal myeloid cell populations might provide a better means of evaluating response to treatment. Normal human bone marrow will form colonies in soft agar culture.’ In contrast, bone marrow from patients with acute leukemia will not produce such colonies.’ This investigation was conducted to determine whether marrow from patients with chemotherapeutically induced remission of leukemia would form soft agar colonies. Its ability to do so would provide a useful functional parameter for defining remission of disease. We report here that bone marrow obtained from patients with acute leukemia in complete hematologic remission will produce colonies when grown in agar. Marrow specimens were aspirated from the posterior iliac crest. 2-3 cc. of marrow were drawn into a syringe previously moistened with heparin, (1 cc. = 1000 U.S.P. units). The sample was immediately transferred to 20 cc. of Tris-buffered isotonic ammonium chloride and washed several times in this solution to remove red blood cells by hemolysis.2 5 X 10#{176}-4X 10 nucleated bone marrow cells were obtained from aspirates in this manner. The technique of bone marrow culture was a modification of the method of Bradley and Metcalf.3 Bone marrow was cultured in 35 X 10-mm. plastic petri dishes (Falcon Plastics). Human urine contains a factor capable of stimulating the growth of mouse and human bone marrow.45 Urine from a number of patients was screened for such activity and a urine with high activity was selected for use in these experiments. The urine was prepared and stored as described by Robinson et al.4 Each culture dish received 0.15 cc. of the standard urine, together with 1 cc. of culture solution containing CMRL 1066 (Grand Island Biological Co.) 0.3 per cent agar, 10 per cent pooled human serum (Philadelphia Blood Center), and the appropriate dilution of nucleated bone marrow cells. Dilutions over the range 2 X 10 -2 X 106 were plated