Bovine serum albumin (BSA) can markedly increase the number of size of erythropoietic bursts produced by mononuclear cells from human bone marrow and peripheral blood, and reduce the threshold amount of erythropoietin (Epo) required for initial burst formation. The purpose of this study was to determine a possible burst-promoting activity (BPA) of BSA. The experiments were performed in a miniaturized agar system, in which the addition of sheep Epo to cultures with or without BSA was delayed for five days. The results obtained have shown that, with or without BSA, Epo deprivation of up to five days (an epoprival state) did not markedly decrease the number of bursts produced by unfractionated peripheral mononuclear cells compared to the number produced in the presence of Epo from the beginning of culture. Similar results were found whether the fetal calf serum (FCS) concentration was 15% or 2%. The preservation of potential BFU-e formation during the epoprival state has therefore been attributed to the ability of T-lymphocytes and/or monocytes to supply BPA. In order to reduce the endogenous amount of BPA, a nonadherent, E-rosette-negative cell fraction was cultured in the presence of Epo, with or without BSA, in serum-free medium containing transferrin (TF). Under these conditions, an equal number of bursts was obtained in FCS and in serum-free medium containing Epo, BSA and TF, whereas no BFU-e growth was found in the presence of Epo and TF, but without BSA. If Epo was withheld for up to five days, the capacity to form erythroid colonies was still retained by the monocyte- and T-lymphocyte-depleted cell fraction in the continuous presence of BSA. However, BPA could not be detected in the BSA. This observation was further supported by experiments in serum-free medium using human recombinant Epo, in which no BFU-e colony formation could be detected in the presence of BSA. From our investigations carried out at limited cell density and in serum-free medium, it could be concluded that the crude Epo preparation was the source of BPA.

Reinfusion of undetected tumour cells is a possible cause of relapse after autologous bone-marrow transplantation. In this paper, a system for in-vitro purging of bone marrow is presented which involves the B-cell neoplasia-associated monoclonal antibody anti-Y 29/55 and complement. Five patients with Burkitt's lymphoma were transplanted with purged marrow, demonstrating the clinical feasibility of the method. The pretransplant regimen included vincristine 2 mg/m2, adriamycin 60 mg/m2, four doses of cyclophosphamide 45 mg/kg and total body irradiation with 6 Gy. Tumour control appears to be better in patients with purged bone marrow as compared to an earlier patient group with unpurged marrow.

Thirty-seven patients with chronic cytopenia were studied using a CFU-gm assay in agar. Cell proliferation was evaluated on days 2, 3, 5, 7, and 10 of incubation. Growth patterns were different in cultures of hematologically healthy persons versus patients with preleukemic syndrome (PL) and aplastic anemia (AA). Three types of PL syndrome and two types of AA (C1 and C2) were distinguished. Bone marrow dysfunction was evaluated further using cytochemistry and electron microscopy to morphologically study cell proliferation in vitro. Cytochemical staining performed in agar demonstrated well-defined maturation defects in myelopoietic precursor cells from the bone marrow of PL patients. Electron microscopic findings of Auer-body-like inclusions in "statu nascendi" in the vacuoles of preleukemic cells supported our results. PL patient groups at high risk for development of overt leukemia and patients with grave prognosis in AA were distinguished. Our results are relevant for the clinical diagnosis and prognosis of patients with cytopenia.

S ummary Cellular interactions responsible for regulating in vitro megakaryocytopoiesis were studied using a microagar culture system which permits the simultaneous proliferation of human megakaryocytic progenitor cells (CFU‐M) and T‐lymphocytic colonies (CFU‐TL). The proliferation of these colony types depends mainly on two factors: phytohaemagglutinin (PHA) and erythropoietin (EP). The direct addition of increasing PHA concentrations to the liquid overlayer resulted in a parallel increase of CFU‐M and CFU‐TL. If the T‐lymphocytes were removed by an E‐rosetting technique a marked diminution of CFU‐M and CFU‐TL numbers was observed. However, monocyte depletion resulted in a marked augmentation of CFU‐M proliferation compared to unfractionated mononuclear cells. In order to confirm that the reduction of CFU‐M proliferation observed after T‐depletion was primarily mediated by the absence of T‐lymphocytes, we have co‐cultured different concentrations of previously removed autologous T‐lymphocytes with a constant number of T‐depleted bone marrow cells. A parallel increase of CFU‐TL and CFU‐M was found if 0.75‐7.5 × 10 4 T‐lymphocytes were added to the culture. In conclusion, our results indicate that activated T‐lymphocytes augment proliferation of human bone marrow CFU‐M and that monocytes are less important for the growth of megakaryocytic colonies.

Nineteen patients with advanced malignant tumors, less than 20 years old were treated with intensive chemotherapy (vincristine 2 mg/m2 i.v. and adriamycin 60 mg/m2 i.v. on day - 7; cyclophosphamide 45 mg/kg i.v. on days -6 to -3), total body irradiation (TBI, 600 rads on day -1) and autologous bone marrow transplantation (ABMT, day 0). Prior to this procedure induction of complete or partial remission by conventional therapy was attempted. Ten patients had intra-abdominal non-Hodgkin's lymphoma (NHL); three, yolk sac tumor; three, Ewing's sarcoma; and three, neuroblastoma. The supportive care included reverse isolation, immunoglobulin 400 mg/kg i.v. q 2 weeks, cotrimoxazole per os, and cell support as needed. No correlation between the bone marrow dose and the time of hematological reconstitution could be established. Five of seven patients with intra-abdominal NHL stage III (transplanted in first remission) are surviving disease-free for 5+, 5+, 20+, 23+, and 35+ months after ABMT. None of three patients with intra-abdominal NHL stage IV is surviving (two of them were transplanted in second remission). One of three patients with yolk sac tumor is surviving disease-free for 27+ months. There are no survivors among the patients with Ewing's sarcoma and neuroblastoma. Only one of 19 patients was lost due to therapeutic complications, while 12 died due to tumor. Regarding treatment results for advanced intra-abdominal NHL, the procedure described here is comparable to the best conventional regimens. In vitro methods for tumor cell eradication in the collected bone marrow might further improve the results of ABMT.

The micro-agar-culture technique for cloning early and late erythropoietic progenitor cells (BFU-E and CFU-E) was further modified and miniaturized in order to study the optimal growth conditions with a minimal consumption of erythropoietin (EP). Using microtiter plates, the total incubation volume was lowered from 0.5 to 0.1 ml and thus reduced the necessary amount of EP and cells by a factor of 5. The method consists of a 50 microliter agar layer, in which the mononuclear cells are suspended, and a 50 microliter liquid overlayer containing bovine serum albumin (BSA), transferrin (TF), and EP. After a seven- or 14-day incubation, the whole agar layers were fixed, transferred to microscopic slides, dried, stained using the Pappenheim method, and permanently preserved. The influences of FCS, BSA, and TF in the presence of EP were studied on the proliferation of human bone marrow CFU-E and BFU-E. The variation of FCS concentration showed an optimum at 10%. The addition of BSA in the presence of optimal concentrations of EP markedly increased the number of BFU-E, but not CFU-E. Furthermore, the threshold concentration of EP required for the initial burst formation could be reduced by half in the presence of BSA. By the addition of TF, a further increase in the number of BFU-E was obtained.

Human bone marrow cells were grown in a micro-agar culture system in the presence of human placenta (HPCM) and giant cell tumor conditioned media (GCT). The effects of HPCM and GCT conditioned media on linearity, growth dynamics, and morphological composition of colonies were studied after 7 and 14 days of incubation. Under the described conditions the dose-response curves for HPCM and GCT were different: on day 7 maximal stimulation was obtained with 2.5% HPCM and 20% GCT; on day 14 a maximal response was reached with 1.25% HPCM and 5% GCT. Both stimuli produced maximal growth after 7 days of incubation, followed by a rapid decrease in the number of formed colonies up to day 14. The morphological study of aggregates showed that after 7 days of incubation 80% pure granulocyte and 20% mixed granulocyte-macrophage colonies were found in the presence of both stimuli. However, on day 14 the incidence of granulocyte-macrophage colonies increased to 60%, whereas the percentage of pure granulocyte colonies decreased to 20%. The frequency of eosinophil colonies was relatively low (median 15%) with both stimuli. The described system can be applied successfully for studies of myelopoiesis in vitro. Both sources of colony stimulating activitites (CSA) employed had no significant difference in their ability to stimulate myelopoiesis.

A 70-year-old woman developed typical clinical symptoms of pure red cell anemia (PRCA) following a history of rheumatoid arthritis (RA). The patient's bone marrow erythropoietic progenitors cells were cloned in a micro agar culture system several times over a period of 11 months, revealing a diminished frequency of bone marrow erythroblasts paralleled by a markedly reduced number of CFU-e and BFU-e in vitro. No inhibitory activity in the patient's IgG fraction could be detected either by preincubation with IgG and/or rabbit complement, or in the continuous presence of IgG. Depletion of T lymphocytes from the patient's bone marrow cells led to an improved in vitro erythroid proliferation. Cytostatic therapy with cyclophosphamide clinically induced a marked increase in the bone marrow erythroblast and reticulocyte number, correlated in vitro by normalization of CFU-e levels and increase in the number of BFU-e. Nevertheless, BFU-e values never attained normal levels, which could be attributed to a reduced stem cell pool resulting from previous therapy with cyclophosphamide and/or antirheumatic drugs. Two independent factors, a reduced pool of committed stem cells as well as an autoimmune cell-mediated suppression, may both contribute to the pathomechanism of the disease in this patient.

A simple and reproducible micro-agar culture technique for cloning human CFU-M is described. Human bone marrow mononuclear cells were suspended in agar and incubated for 12 days. Stimulation was provided by the direct addition of phytohemagglutinin-P (PHA-P), erythropoietin (Epo) and 2-mercaptoethanol (2-ME) to the liquid overlayer. A shift from BFU-E and CFU-C proliferation to CFU-M and CFU-TL was observed with increasing PHA concentrations. Under optimal conditions (PHA 50 micrograms, Epo 1.2 IU, 2-ME 2 x 10(-4) M, 1% purified BSA, 0.04% human transferrin, saturated with Fe C13) a linear relationship between colonies formed and plated cell number were observed. For the routine morphological analysis, the whole agar layers were stained using the Pappenheim method. For further characterization of CFU-M, cytochemical stainings and immunofluorescence tests with rabbit-antihuman factor VIII-related antigen were performed on the whole agar layers.

In vitro myelopoiesis in a group of ten patients with acute leukemia (6 AML, 1 APML, 3 ALL) in long-term remission has been studied. The patients remained in first stable remission for at least 4 years and maintenance therapy had been completed in all patients except one. In the patients, colony formation of bone marrow cells after 7 days of incubation was significantly increased compared to a representative control group. The proliferation of microclusters after 3 incubation days was also markedly enhanced, and accelerated proliferation of all aggregates (microclusters, macroclusters and colonies) could be demonstrated in culture with the patients' bone marrow cells. The use of autologous feeder layers and autologous serum showed no inhibitory effect on colony formation. The proportion of eosinophil colonies formed with the patients' bone marrow cells was in the normal range. In contrast to the high proliferation capacity of bone marrow precursor cells the CFU-c number of peripheral blood was significantly decreased in the patients' group. No significant correlation between CFU-c number of bone marrow and blood cells could be found. The colony stimulating activity of the patients' peripheral mononuclear cells was normal compared to healthy controls. We conclude from this study that even in long-term remission of acute leukemia certain in vitro abnormalities exist in myelopoietic proliferation and regulation.

Thirty-seven patients with chronic cytopenia were studied using a CFU-c assay in agar. On the basis of the growth pattern three types of preleukaemic syndrome (PL) and two types of aplastic anaemia were distinguished. Further evaluation of the bone marrow dysfunction was attempted with a combined application of cytochemistry and electron microscopy for the morphologic study of cells proliferating in vitro. Well-defined maturation defects in the growing cells from the bone marrow of patients with PL were demonstrated with cytochemical stainings performed in agar. These results were supported by electron microscopic findings of Auer-body-like inclusions in "statu nascendi" in the vacuoles of preleukaemic cells. On the basis of our results a high risk group of PL for development of overt leukaemia and a group of patients with a grave prognosis in aplastic anaemia were distinguished. The data obtained are relevant for the clinical diagnosis and prognosis of patients with cytopenias.

A microagar-culture system for the in vitro study of myelopoiesis committed stem cells has been reported. Stimulation was induced by a commercially available conditioned medium obtained from culture of histiocytoma cells. The effect of different concentrations of conditioned medium and a linear relation between the seeded cells and the formed colonies has been established. The granulocytic-monocytic character of cell aggregates has been shown by morphologic studies. Further possibilities for the use of the described method have been briefly discussed.