Erythropoiesis In Patients Research Articles

Effect of stem cell factor on in vitro erythropoiesis in patients with bone marrow failure syndromes

Stem cell factor (SCF) enhances normal hematopoiesis. We examined its effect in vitro on bone marrow and blood progenitors from patients with inherited bone marrow failure syndromes, including 17 patients each with Diamond-Blackfan anemia (DBA) and Fanconi's anemia (FA), 3 with dyskeratosis congenita (DC), and 1 each with amegakaryocytic thrombocytopenia (amega) and transient erythroblastopenia of childhood (TEC). Mononuclear cells were cultured with erythropoietin (Ep) alone or combined with SCF or other factors. SCF increased the growth of erythroid progenitors in cultures from 50% of normal controls, 90% of DBA, 70% of FA, 30% of DC, and the amega and TEC patients; normal numbers were reached in 25% of DBA studies. Improved in vitro erythropoiesis with SCF in all types of inherited marrow failure syndromes does not suggest a common defect involving kit or SCF, but implies that SCF may be helpful in the treatment of hematopoietic defects of varied etiologies.

Effect of Stem Cell Factor on In Vitro Erythropoiesis in Patients With Bone Marrow Failure Syndromes

Stem cell factor (SCF) enhances normal hematopoiesis. We examined its effect in vitro on bone marrow and blood progenitors from patients with inherited bone marrow failure syndromes, including 17 patients each with Diamond-Blackfan anemia (DBA) and Fanconi's anemia (FA), 3 with dyskeratosis congenita (DC), and 1 each with amegakaryocytic thrombocytopenia (amega) and transient erythroblastopenia of childhood (TEC). Mononuclear cells were cultured with erythropoietin (Ep) alone or combined with SCF or other factors. SCF increased the growth of erythroid progenitors in cultures from 50% of normal controls, 90% of DBA, 70% of FA, 30% of DC, and the amega and TEC patients; normal numbers were reached in 25% of DBA studies. Improved in vitro erythropoiesis with SCF in all types of inherited marrow failure syndromes does not suggest a common defect involving kit or SCF, but implies that SCF may be helpful in the treatment of hematopoietic defects of varied etiologies.

Effect of subcutaneously administered human recombinant erythropoietin on erythropoiesis in patients with myelodysplasia

In a phase II study, 12 patients with a myelodysplastic syndrome (MDS) and anaemia (nine transfusion-dependent) were treated with recombinant human erythropoietin (rHuEpo) to assess the therapeutic effect on erythropoiesis and on transfusion requirement. Patients with a low risk of developing acute leukaemia were included, i.e. refractory anaemia (RA), RA with ringed sideroblasts (RARS) and RA with excess blasts (RAEB), providing the percentage of myeloblasts in the bone marrow did not exceed 10%. Recombinant HuEpo treatment was initiated at a dose of 50 units/kg body weight and administered subcutaneously three times weekly. At 3-week intervals the dose was increased with 50 units/kg per injection, until after 15 weeks a maximum dose of 250 units/kg three times weekly was reached. All patients completed the study. Recombinant HuEpo was well tolerated and no serious side effects were seen. There was no evidence of the emergence of a new malignant clone in response to rHuEpo as shown by sequential karyotyping. In none of the patients was an increase in haemoglobin level or a diminished red blood cell transfusion requirement seen. In four out of 10 evaluable sequential bone marrow smears, an increase in erythropoiesis was seen, suggesting stimulation of ineffective red cell production. One of these patients also showed a rise in reticulocyte count. The number of erythroid progenitor cells (BFU-E and CFU-E) in blood and bone marrow was not affected by rHuEpo treatment. Also no change in the number of myeloid progenitor cells (CFU-GM) in blood and bone marrow was noted. In conclusion, subcutaneous treatment with rHuEpo at dosages up to 250 units/kg body weight (three times weekly) fails to increase the haemoglobin level or to diminish the transfusion requirement in patients with MDS and anaemia. It is unclear whether higher doses of rHuEpo are effective or whether patients with less severe anaemia who are transfusion independent, have a higher likelihood of response.

Erythropoietin for the treatment of anemia of malignancy associated with neoplastic bone marrow infiltration.

This clinical trial was performed to study the effects of intravenously (IV) administered recombinant human (rh) erythropoietin (EPO) at escalating doses (150, 300, and 450 U/kg, administered as an IV bolus injection, twice weekly, for 6, 4, and 4 weeks, respectively) in five patients with low-grade non-Hodgkin's lymphoma (Ig NHL) and bone marrow involvement and one patient with multiple myeloma (MM). All patients were anemic due to underlying disease. None of the patients had a history of bleeding, hemolysis, renal insufficiency, or other disorders causing anemia in addition to bone marrow infiltrating malignancy. Endogenous EPO serum levels were significantly increased in all patients (74 to 202 mU/mL). Five patients (one MM, four small-cell lymphocytic [SCLC] NHL) showed a dramatic increase of hemoglobin (Hb), hematocrit (Hk) and RBC count becoming obvious on the second EPO dose level. Initial ferritin serum values, which were high mostly due to polytransfusion, were significantly reduced in responding patients. Erythropoiesis of one patient with extensive follicular mixed (fm) NHL did not respond to EPO treatment. Platelet (PLT) count increase (greater than 75% above starting levels) during and following EPO therapy was observed in one patient with MM. Adverse events due to EPO therapy have not been recorded. These findings point out a previously unrecognized capacity of EPO given at pharmacologic doses to stimulate erythropoiesis in patients with anemia due to bone marrow infiltration by neoplastic lymphocytes in spite of enhanced endogenous EPO expression.

Serum erythropoietin and erythropoiesis in patients with myelodysplastic syndromes

Ineffective erythropoiesis is an early feature of the myelodysplastic syndromes (MDS), usually accompanying an hypercellular marrow. In a previous study, concentrations of serum erythropoietin (EPO) in MDS have been shown to correlate inversely both with haemoglobin concentration and with % bone marrow erythroblasts. We have measured erythroid production using a radioisotopic technique in 20 patients with MDS. Although haemoglobin concentration shows a weak inverse relationship with serum EPO concentration there is considerable variation in EPO concentration at a given haemoglobin level. There is no correlation between serum EPO and total erythroid production, though there is a weak correlation with effective erythropoiesis. The data suggests that control mechanisms of erythropoiesis in patients with MDS are complex.

Ferrokinetic evaluation of erythropoiesis in patients with myelodysplastic syndromes.

Erythropoietic activity in patients with myelodysplastic syndrome (MDS) was evaluated by ferrokinetic measurements. Since the conventional plasma iron turnover of MDS patients increased with plasma iron levels after multiple blood transfusions, erythron transferrin uptake was chosen as a parameter of erythroid marrow activity. Although a correlation was shown between plasma iron level and plasma iron turnover (r = 0.50, 0.01 less than p less than 0.02), no correlation existed between the plasma iron level and erythron transferrin uptake (r = 0.25, p greater than 0.1). Erythron transferrin uptake, independent of plasma iron, was significantly higher in MDS patients than in normal subjects (110.6 +/- 67.6 and 67.6 +/- 18.8 mumol/l/dl, respectively; 0.01 less than p less than 0.02). An increased erythropoiesis occurring concomitantly with morphologically normal or increased erythroid cellularity was demonstrated in patients with MDS. The measurement of erythron transferrin uptake might be valuable as an accurate expression of erythroid activity in the hyperferremic state.

Separation of an inhibitor of erythropoiesis in "middle molecules" from hemodialysate from patients with chronic renal failure.

A "middle molecule" inhibitor of erythropoiesis in patients with chronic renal failure was separated from a large amount of hemodialysate. Hemodialysate was passed through Amberlite XAD-4 resin, Sephadex G-50 gel, and DEAE-Sephadex A-25, followed by reversed-phase liquid chromatography. The erythroid colony assay (CFU-E) was used to detect the inhibitory effect of the sample solution. Certain fractions from the DEAE-Sephadex A-25 column showed a dose-related inhibition of CFU-E formation as great as that of standard spermine. The inhibitory effect of these fractions decreased to the control value after proteolytic digestion. The inhibitor was eluted from the liquid-chromatographic column by a solvent gradient containing 390 to 425 mL of methanol per liter. These results suggest there is an inhibitor of CFU-E with a relative molecular mass of 1000 to 10 000 and an active site composed of peptide. This technique may prove useful for separation of the inhibitor of erythropoiesis in uremic body fluid.

Serum Erythropoietin levels and inhibitors of erythropoiesis in patients with chronic renal failure.

The role of various factors in erythropoiesis was studied in 13 predialysis patients and 41 hemodialysis patients. Serum erythropoietin (ESF) was measured by the fetal mouse liver cell bioassay in vitro. The effect of uremic sera on heme synthesis and erythroid progenitor cell (CFU-E) formation was examined using normal human bone marrow cultures. Serum ESF levels in both predialysis (99.9 +/- 45.0 mU/ml) and dialysis (141.2 +/- 109.7 mU/ml) patients were significantly higher than those in normal controls (42.0 +/- 25.8 mU/ml), although the titers were not sufficiently increased to correct the anemia. Serum ESF concentrations did not correlate with hemoglobin level or inhibition of both heme synthesis and CFU-E formation. Bone marrow CFU-Es in uremic patients were normal in the number, the responsiveness to ESF and the percentage in S-phase. Significant decrease in heme synthesis was observed in dialysis patients. The degree of inhibition of CFU-E formation showed a relationship to hemoglobin levels in uremic patients. By the CFU-E formation assay, the difference in inhibitory effects of the sera obtained from dialysis patients immediately before and after a hemodialysis was significant only under a low ESF concentration (0.125 U/ml) but not under a high concentration (1.0 U/ml). In conclusion, inhibition of heme synthesis and CFU-E formation, in addition to inadequate ESF production, plays an important role in renal anemia.

STUDIES ON THE PROLIFERATIVE ACTIVITY OF ERYTHROBLASTS IN VARIOUS TYPES OF ANEMIA USING MITOTIC INDEX AND LABELING INDEX OF 3H-TdR

Mitotic index (MI) and 3H-TdR labeling index (LI) were determined in hematologically normal subjects and patients with various types of anemia. The results obtained were as follows : 1) In patients with hemolytic anemia, iron deficiency anemia, or acquired sideroblastic anemia, there was no significant difference in both MI and LI of basophilic, polychromatic or total erythroblasts as compared with those in normal subjects. No significant difference was also observed in these values in patients with aplastic anemia or pernicious anemia except for significant decrease in LI or MI of total erythroblasts, respectively.2) The lower value of LI or MI of total erythroblasts in aplastic anemia or pernicious anemia, respectively, was presumed to have been caused by the increase in number of orthochromatic erythroblasts.3) In patients with pernicious anemia, treatment with parenteral Vit. B12 resulted in a significant increase in both MI and LI of polychromatic erythroblasts to normal levels.4) In cases of erythroleukemia, both LI and MI of basophilic or polychromatic erythroblasts were significantly lower as compared with those in normal subjects. From these results it is suggested that proliferation activity of erythroblasts is obviously reduced even in an earlier stage of the maturation.5) Mitotic duration of the bone marrow erythroblasts was determined using stathmokinetic method in one case of pernicious anemia and 2 cases of hemolytic anemia. Among these patients, no significant difference was observed in mitotic duration and mean value was 43.6 ± 3.1 minutes. From this value the generation time of total erythroblasts was calculated as 26.3 ± 2.3 hours.6) In all patients with above mentioned anemias and normal subjects there was significant correlation between MI and LI of total or polychromatic erythroblasts. Significant correlation was also observed between these values, both in patients with increased ineffective erythropoiesis such as pernicious anemia, erythroleukemia, acquired sideroblastic anemia and in those without increased ineffective erythropoiesis such as iron deficiency anemia, hemolytic anemia and aplastic anemia. There was no significant correlation between MI and LI in basophilic erythroblasts. The main reason is surmised to be low cell counts. Therefore using MI, it seems to be possible to presume the proliferative activity of erythroblasts similarly to using LI. MI seems to be less accurate than LI but easy to obtain from observation of usual bone marrow smears. In the case of MI, neither3H-labeled thymidine nor specialized technique of radioautography is required.7) There was no significant correlation between PIT in ferrokinetics and LI or MI in these patients, but in patients with high MI and LI, the RCIT tended to be high. In cases without increased ineffective erythropoiesis, MI was found to have significant correlation with both PIT and RCIT. Further investigation was required in order to be able to evaluate these results and draw a definite conclusion as to the correlation of MI and LI to ferrokinetics.From the results above described, the changes in proliferative activity of erythropoiesis in patients with various types of anemia were discussed.

In-vitro studies of ineffective erythropoiesis in rheumatoid arthritis.

Ineffective erythropoiesis was assessed in a series of 32 patients with rheumatoid arthritis by means of a new in-vitro method which measures the release of haem from a labelled cohort of erythroblasts in culture. Haem release was significantly increased in patients with the anaemia of chronic disorders but was normal in those who were not anaemic or who had an iron-deficiency anaemia. In 2 patients with anaemia of chronic disorders haem release returned to normal after successful antirheumatic therapy. The increased ineffective erythropoiesis in patients with rheumatoid arthritis and anaemia of chronic disorders appeared to be unrelated to functional iron deficiency and was not attributable to a serum factor.

Identification of spermine as an inhibitor of erythropoiesis in patients with chronic renal failure.

Fetal mouse liver and normal human bone marrow cell cultures were used for studies on the inhibition of erythroid colony formation (CFU-E) by sera from anemic patients with end-stage renal failure and the polyamine spermine. Sera from each of eight predialysis uremic anemic patients with end-stage renal failure produced a significant (P < 0.001) inhibition of erythroid colony formation in the fetal mouse liver cell cultures when compared to sera from normal human volunteers. In vivo or in vitro dialysis of the uremic sera with a 3,500-dalton exclusion limit membrane removed the inhibitor from uremic sera. The uremic serum dialysate provided by the membrane fractionation was significantly inhibitory in the erythroid cell cultures. When this dialysate was applied to gel filtration chromatography (Bio-Gel P-2) the inhibitor was found to be in the same molecular weight range as [(14)C]spermine. The polyamine spermine produced a dose-related inhibition of erythroid colony formation (CFU-E) in fetal mouse liver and normal human bone marrow cultures. Thus, the following evidence is provided that the in vitro inhibitor of erythropoiesis found in chronic renal failure patients' sera is identical with the polyamine spermine: (a) the inhibitor and radiolabeled spermine appeared in identical Bio-Gel P-2 effluent fractions; (b) when spermine was added to normal human sera at concentrations reported in sera of uremic patients, and studied in both the fetal mouse liver cell culture and normal human bone marrow cultures, a dose-related inhibition of erythroid colony (CFU-E) formation was noted; and (c) the inhibitory effects of crude uremic serum, uremic serum dialysate, and fractions of uremic serum dialysate from a Bio-Gel column, on erythroid colony formation were completely abolished by the addition of a specific rabbit antiserum to spermine.

Quantification of ineffective erythropoiesis in megaloblastic anaemia by determination of endogenous production of 14CO after administration of glycine-2-14C.

The determination of the early peak of 14CO production that results from haem turnover after administration of glycine-2-14C was studied in patients with pernicious anaemia. The accuracy of this technique in measuring ineffective erythropoiesis was assessed by comparison with other measurements of the severity of the disease. 6 patients were studied before specific treatment and 2 of these also after treatment. The endogenous production of 14 CO was calculated from determinations of the production of CO by a rebreathing technique and of the specific activity of CO washed out from the body CO store by oxygen breathing. The early labelled peak was increased 7-39 times compared to that in healthy control subjects and represented 35-86% of the total production of 14CO as compared to 13% in controls. The early labelled peak was significantly correlated to the Hb concentration, mean red cell volume and endogenous production of CO. 2 patients, twice examined, showed a moderately increased early labelled peak also after specific treatment. This was attributed to persistence of some ineffective erythropoiesis in 1 patient and possibly increased hepatic haem turnover in the other. The magnitude of the early labelled peak and the maximal activity of 14CO found in the samples of CO collected from the body CO store were significantly correlated. In conclusion, with the present method the early peak of 14CO reflected the ineffective erythropoiesis in patients with pernicious anaemia. The early labelled peak could be predicted from the maximal activity of 14CO found in the samples of CO washed out from the body CO store.

Quantitative studies of splenic erythropoiesis in polycythaemia vera and myelofibrosis.

A quantitative scanning method employing cyclotron-produced 52Fe has been developed to assess splenic erythropoiesis in patients with myeloproliferative disorders. In 12 patients with myelofibrosis splenic uptake of 52Fe was from 5.0% to 48% of the injected dose. Although a single patient with classical polycythaemia vera had a minor uptake of 2.8% of six other patients with this diagnosis showed no concentration of isotope in the splenic area. The fraction of 52Fe in the spleen of four patients with 'transitional' myeloproliferative disorders characterized by a high red cell mass, hypercellular bone marrow and a leucoerythroblastic blood film varied from 5% to 41%. No clear relationship was noted between the degree of splenic erythropoiesis as defined by this technique and the level of haemoglobin, the degree of splenomegaly, the effectiveness of erythropoiesis of traditional 59Fe surface counting. If splenectomy is considered in patients with myelofibrosis splenic 52Fe quantitation will provide more precise data on the contribution of splenic erythropoiesis than 59Fe surface counting alone.

Studies on erythropoiesis in patients with renal failure and after kidney transplantation.

Ferrokinetic studies were made in 16 patients before and after kidney transplantation. The Fe clearance decreased in 9 of 11 patients, the plasma iron turnover showed no variation in six patients, while it increased in three and decreased in one, the production of hemoglobin increased significantly after transplantation. The nonnephrectomized patients showed more erythropoiesis after transplantation, manifested by higher hemoglobin level, shorter Fe clearance, and lower serum iron. Uremic patients, including the anephric, had significant responses in erythropoiesis both to stimulation by phlebotomy and to inhibition by blood transfusion. After transplantation, however, the response to phlebotomy was greater and the blockade by blood transfusion smaller, than during uremia.

Inhibitors of erythropoiesis in patients with aplastic anemia.

An inhibitor of erythropoiesis was found in the urine of patients with aplastic anemia. The inhibitor was concentrated with kaolin and separated from erythropoietin by subsequent fractionation with alcohol and gel filtration on Sephadex G-100 and DEAE-Sephadex A-50. The inhibitor blocked the effect of erythropoietin (standard C) when injected into mice with hypoxic polycythemia 3 H before or along with exogenous erythropoietin; partial neurtralization of standard C also was observed on incubation with the inhibitor.

Androgen Therapy in Patients with and Without Kidneys Undergoing Hemodialysis.

Excerpt Androgens increase renal but not extrarenal erythropoietin production in mice. They also increase erythropoiesis in patients undergoing hemodialysis. This study compares the effects of andr...

Clinical studies on iron kinetics. II. Iron-kinetics studies in patients with malignant neoplasms. With special references to ferrokinetics and 59Fe labeled iron-dextran studies.

Although extensive studies on the anemia and iron metabolism in patients with malignant neoplasms have been made, the pathogenesis of the anemia ob· served in cancer patients remains obscure. The first purpose of this paper is to elucidate the functional impairment in red cell production in patients with malignant neoplasm by means of ferrokinetic studies. The second one· is to clarify how the reticulo-endothelial cells partici· pate in alterations of iron metabolism in malignancy by means of 59Fe labeled iron-dextran. The third one is to enumerate sideroblasts in bone marrow smears of cancer patients for assessing stainable iron in erythroblasts. The results obtained were as follows: 1) Ferrokinetic studies with 59Fe-citrate showed the increased rate of erythropoiesis in patients with malignant neoplasm. However, studies on cases with infections gave results very similar to those in malignant neoplasms, suggesting the· existence of a mechanism common in both conditions. 2) The studies with 59 Fe labeled iron-dextran revealed a significant decrease of utilization of radioiron for hemoglobin synthesis from iron-dextran captured in reticulo-endothelial cells in malignant diseases. 3) The percentage of bone marrow sideroblasts in patients with malignant neoplasms showed a significant decrease with the mean value of 28 percent. The evidences obtained above demonstrated that the erythropoietic activity in patients with malignant neoplasms is maintained above normal, while there is an evident impairment of iron release from reticulo-endothelial cells. However, the mechanism responsible for the defect of iron release in malignancy is not clear and required further study.

Erythropoiesis in patients with renal failure undergoing chronic dialysis.

THE anemia of renal insufficiency is associated with both increased red-cell destruction and impaired erythropoietic response to anemia. Variations in the relative importance of these 2 mechanisms and in the degree of the anemia observed may be due, at least in part, to the severity of renal disease in the patients studied. The purpose of the present report is to characterize red-cell production and destruction in a special group of patients with severe renal failure who have been stabilized at a similar degree of azotemia by repeated dialysis. An additional finding emerging from this study is the improvement in erythroid . . .

Effect of androgenic hormone in myelofibrosis.

Androgenic hormones have stimulated erythropoiesis in patients with breast cancer and those with refractory anemia, as well as in castrated and in normal experimental animals. Observation of this erythropoietic effect by several investigators has led to use of androgens in treatment of myelofibrosis. Six of 8 such patients treated by the author for prolonged periods with massive doses of androgenic hormone showed improvement. Two women, aged 55 and 75, and 1 man of 64 responded with increased hemoglobin, reticulocytosis, cessation of transfusion requirements, and hypercellular marrow. Three women, aged 56 to 62, required fewer or no transfusions and showed reticulocytosis. Corticosteroids were found to potentiate the effect in some instances. A prolonged remission persisted after cessation of androgen therapy in one case but could not be sustained. Therapeutic levels have in some cases led to polycythemia.

Alterations in osmotic and mechanical fragility related to in vivo erythrocyte aging and splenic sequestration in hereditary spherocytosis.

Increased osmotic and mechanical fragility of the erythrocytes is a consistent finding in patients with hereditary spherocytosis, although in a few individuals it is necessary to incubate the cells at 370 C. for 24 hours before the otherwise latent abnormality becomes demonstrable. Splenectomy will correct the hemolytic process of hereditary spherocytosis despite the continuing production of abnormally fragile cells by the patient. The spleen is, therefore, essential in the destruction of the intrinsically defective erythrocytes. In the peripheral blood of some individuals with this disease a double population of cells exists. One component consists of more markedly fragile cells that are not present after splenectomy and must, therefore, have been related to the presence of the spleen. The present study was undertaken to investigate the changes in osmotic and mechanical fragility accompanying in vivo erythrocyte aging and splenic sequestration and to elucidate the source and fate of the more markedly fragile group of red cells found in some patients. Advantage was taken of the increased erythropoiesis in patients with hereditary spherocytosis prior to splenectomy to label in vivo with Fe59 the new red cells produced during a short time span, thereby obtaining an identifiable group of cells of known age. Using techniques that will be described, it was possible to differentiate these isotopically-labeled cells from the other cells in the peripheral circulation and to compare their char