Clonogenic cultures support the proliferation of haemopoietic cells into colonies of differentiated progeny. Using these techniques it has been established that normal haemopoietic progenitors are dependent upon growth factors for their survival, proliferation and differentiation in vitro. However, progenitors from patients with polycythaemia vera (PV) generate erythroid colonies in cultures deprived of exogenous erythropoietin. These have been termed endogenous erythroid colonies (EEC). Recently, endogenous megakaryocytic colonies (EMC), those arising in assays without megakaryocyte growth factors, have been reported, particularly in patients with primary thrombocythaemia (PT). Many investigators have found an EEC positive culture to have 100% diagnostic specificity and sensitivity for PV. Similarly, EMC have been shown by some to be an unequivocal marker of PT. Accordingly, clonogenic assays for EEC and EMC have been advocated as diagnostic markers of PV and PT. However, the specificity of these assays is not universally attested to as there are some reports of EEC in patients with secondary polycythaemia and of EEC and EMC in normal subjects. Thus, for diagnostic use, EEC and EMC assays must be exhaustively validated for specificity using clinically appropriate controls. Furthermore, clonogenic culture techniques are not amenable to external quality assurance, are technically demanding and are unlikely to be available in most haematology laboratories. These considerations must be taken into account when assigning weighting to EEC and EMC assays as diagnostic criteria in myeloproliferative disorders.

Diagnostic applications of haemopoietic progenitor culture techniques in polycythaemias and thrombocythaemias.

Responsiveness of bone marrow erythropoietic stem cells (CFU-E and BFU-E) to recombinant human erythropoietin (rh-Ep) was examined in vitro in 23 patients with aplastic anemia and 14 with myelodysplastic syndrome (MDS) to investigate the clinical use of rh-Ep for these diseases. Bone marrow mononuclear cells were cultured by methylcellulose methods for CFU-E and BFU-E assays. In normals, the CFU-E numbers reached a plateau of increase at Ep doses of almost 2-5 units, and no further increase was observed with the addition of larger Ep doses. In aplastic anemia, the responses of CFU-E to Ep were relatively good in nonsevere type and generally poor in severe type. However, the CFU-E numbers increased with increasing doses of Ep in some of the patients with aplastic anemia. Among the patients with MDS, the responses of CFU-E to Ep were relatively good in primary acquired refractory anemia (PARA) and primary acquired sideroblastic anemia. On the other hand, the responses of CFU-E to Ep were poor in refractory anemia with an excess of blasts (RAEB) and RAEB in transformation among the MDS patients. BFU-E responses to Ep were poor in severe aplastic anemia, RAEB, and RAEB-T. However, there are Ep responsive patients in some of aplastic anemia and PARA. High titers of rh-Ep were suggested to be effective clinically in some patients with aplastic anemia and those with PARA.

Responsiveness of bone marrow erythropoietic stem cells (CFU‐E and BFU‐E) to recombinant human erythropoietin (rh‐Ep) in vitro in aplastic anemia and myelodysplastic syndrome

Publisher Summary This chapter discusses the chemistry and physiology of erythropoietin. Erythropoietin is the primary regulator of erythropoiesis in mammals. It is a glycoprotein hormone produced primarily in the kidney and secreted into the circulation, acting on the bone marrow to induce the differentiation of hematopoietic stem cells along erythroid lines. Mammalian erythropoietins show relatively little species specificity with regard to biologic activity. Anemic plasma and serum can stimulate erythropoiesis in polycythemic hosts of either the same or unrelated mammalian species, thereby allowing the use of a standardized assay animal—the polycythemic mouse or the fasted rat. Likewise, tissue and circulating erythropoietins from several mammalian species react with an antibody to the human urinary hormone and inhibit binding of the pure human urinary erythropoietin tracer in the radioimmunoassay. Species differences have been observed, both in biologic and immunologic activity. For example, in a series of studies, preparations of sheep erythropoietin did not give dose-response curves parallel to those for human material assayed in the polycythemic mouse system.

The chemistry and physiology of erythropoietin.

The existence of erythropoietin (epo) —then termed "hemopoietine" was postulated by Carnot and Dellandre, as long ago as 1906, but there were questions whether the experimental evidence was sufficient to indicate a humoral factor that could regulate the rate of red cell formation [I]. For more than 30 years the field of study of epo was largely in eclipse. Of the 40 years between the reemergence of the field in the 50s and the present, the years until 1987 could be described as a lag period, preceding the present-day exponential rise in both widespread interest and publications on epo. This later period, the time after the cloning and expression of the human epo gene, has seen the field become full-fledged with respect to therapeutic use, marketing, and patent litigation. It also has been a time of important advances in the study of the molecular and cellular biology of epo, its receptor, and of erythropoiesis. While this notable expansion in research is still in process, it seemed appropriate for me to agree with the suggestion of the editor of Perspectives to write a personal account of my experience that spans the period when epo was considered to be a laboratory curiosity (if it had any existence at all) to the present.

Erythropoietin: A Somewhat Personal History

On the mechanism of erythropoietin-induced differentiation. XV. Induced transcription restricted by cytosine arabinoside.

White et al.1 developed a process for preparing erythropoietin concentrate from the plasma of sheep which were made anemic with phenylhydrazine. They obtained an average yield of approximately 2,000 units of concentrate per sheep. We have been able to increase this yield to over 10,000 units per sheep by instituting a number of modifications in the procedures for preparing and treating animals and for processing the concentrate. The improvements make it possible to reduce the cost of preparing the concentrate and allow the preparation of quantities in the order of millions of units. Since it had been shown by White et a2.l that the erythropoietin concentration in blood rises steeply when the hematocrit falls below 15%, it seemed important to us to achieve conditions for reproducibly lowering the hematocrit below this level. To achieve this, we introduced a number of modifications in their method. Animals were rendered anemic by phenylhydrazine (PH) which was injected subcutaneously on alternate days as before. However, the hematocrit of each animal was determined at least every other day and the dose of PH, initially in the range of 16 mg/kg bodyweight, was adjusted as the anemia progressed so as to lower the hematocrit steadily to the desired level. In this way each animal was, in effect, “titrated” to a severe state of anemia. Once the hematocrit fell below lo%, the animal was exsanguinated. Our early results with normal sheep treated in this manner were erratic. Many of the animals died abruptly before becoming anemic, as was observed previously by White et a2.I Since the deaths were frequently associated with gastric and intestinal parasite infections, we turned to animals from a specific pathogen-free (SPF) flock which was free of parasites. With these animals we had no unexpected deaths and all animals had profound anemia when bled out. A further modification in our preparation of anemic sheep was prompted by recent experiments on the production of erythropoietin by rats. Hogson2 has found that total body radiation, which inhibited the erythropoietic response, facilitated the preparation of highly anemic animals and thus increased the yield of plasma erythropoietin. For our experiments, therefore, sheep were irradiated with 800 rads 6oCo y-rays (midline air dose given bilaterally at a dose rate of 15 rads/min. A further increase in the yield of plasma was achieved by infusing Ringer’s-Lactate solution during the bleed-out so as to flush the residual blood from the animal. Each animal was bled out from the carotid artery under local anesthesia and it was a simple matter at this time to infuse 1-2 of Ringer’s-Lactate solution into the jugular vein before the animal expired. The result of the modified procedure was a significant increase in yield of erythropoietin per sheep. The values of hematocrits for two typical SPF sheep treated as described above are shown in FIGURE 1. These animals were bled out at seven and nine days when their hematocrits had reached 8%. Each animal yielded about 10,000 units. TABLE 1 gives the yield of erythropoietin in the plasma of

The commercial production of erythropoietin from anemic sheep plasma

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The commercial production of erythropoietin from anemic sheep plasma