Erythropoietic Activity Research Articles

In-depth validation of acridine orange staining for flow cytometric parasite and reticulocyte enumeration in an experimental model using Plasmodium berghei

Flow cytometry is potentially an effective method for counting malaria parasites, but inconsistent results have hampered its routine use in rodent models. A published two-channel method using acridine orange offers clear discrimination between the infected and uninfected erythrocytes. However, preliminary studies showed concerns when dealing with Plasmodium berghei-infected blood samples with high numbers of reticulocytes. In hyperparasitemic or chronic P. berghei infection, enhanced erythropoietic activity results in high numbers of circulating immature reticulocytes. We show that even though the protocol offered good discrimination in newly infected animals, discrimination between infected erythrocytes and uninfected reticulocytes became difficult in animals with hyperparasitemia or chronic infections maintained with subcurative treatment. Discrimination was especially hampered by increased nucleic acid content in immature uninfected reticulocytes. Our data confirms that though flow cytometry is a promising analytical tool in malaria research, care should still be taken when analysing samples from anemic or chronically infected animals.

Sex-dependent regulation of hypoxic ventilation in mice and humans is mediated by erythropoietin

Acclimatization to hypoxic exposure relies on an elevated ventilation and erythropoietic activity. We recently proposed that erythropoietin (Epo) links both responses: apart from red blood cell production, cerebral and plasma Epo interact with the central and peripheral respiratory centers. Knowing that women cope better than men with reduced oxygen supply (as observed at high altitude), we analyzed the hypoxic ventilatory response in Epo-overexpressing transgenic male and female mice with high Epo levels in brain and plasma (Tg6) or in wild-type animals injected with recombinant human Epo (rhEpo). Exposure to moderate and severe hypoxia as well as to hyperoxia and injection of domperidone, a potent peripheral ventilatory stimulant, revealed that the presence of transgenic or rhEpo extensively increased the hypoxic ventilatory response in female mice compared with their corresponding male siblings. Alterations of catecholamines in the brain stem's respiratory centers were also sex dependent. In a proof-of-concept study, human volunteers were intravenously injected with 5,000 units rhEpo and subsequently exposed to 10% oxygen. Compared with men, the hypoxic ventilatory response was significantly increased in women. We conclude that Epo exerts a sex-dependent impact on hypoxic ventilation improving the response in female mice and in women that most probably involves sexual hormones. Our data provides an explanation as to why women are less susceptible to hypoxia-associated syndromes than men.

Soluble transferrin receptor and depth of bone marrow suppression following high dose chemotherapy

Soluble transferrin receptor (sTfR) reflects erythropoietic activity in the bone marrow. We have sought to identify whether serial measurement of sTfR may predict depth or duration of myelosuppression in patients receiving high dose chemotherapy. We prospectively measured serial sTfR in nine consecutive patients with haematologic malignancy admitted for various high dose regimens. The sTfR nadir was associated with white cell nadir, and sTfR nadir and day 5 sTfR were inversely associated with the number of red cell transfusions required. Our data suggest that sTfR may be helpful in predicting the degree of bone marrow suppression in patients receiving intensive chemotherapy.

Assessment of Urinary Concentrations of Hepcidin Provides Novel Insight into Disturbances in Iron Homeostasis during Malarial Infection

Disturbances in iron homeostasis are frequently observed in individuals with malaria. To study the effect of malaria and its treatment on iron homeostasis and to provide a mechanistic explanation for observed alterations in iron distribution, we studied the course of the iron regulatory hormone hepcidin in anemic Tanzanian children with febrile Plasmodium falciparum malaria. Before initiation of antimalarial treatment, urinary concentrations of hepcidin were strongly elevated and were associated with iron maldistribution, as was suggested by the presence of hypoferremia and high serum concentrations of ferritin. Antimalarial treatment resulted in a rapid decrease in urinary concentrations of hepcidin and reversal of the hypoferremia. Exploration of regulatory pathways of hepcidin production by analysis of iron, erythropoietic, and inflammatory indices suggested that reduced erythropoietic activity and inflammation stimulated hepcidin production. We conclude that high concentrations of hepcidin explain the observed disturbances in host iron homeostasis associated with malaria and may contribute to malarial anemia and an impaired erythropoietic response to iron supplementation.

Short report: soluble transferrin receptor and depth of bone marrow suppression following high dose chemotherapy

Aim The transferrin receptor (TFR) mediates the transport of iron into cells. Circulating TFR can be measured as soluble transferrin receptor (sTfR) which has been shown to reflect erythropoietic activity in the bone marrow (Beguin 1992, Huebers et al 1990). We have sought to identify whether serial measurement of sTfR may predict depth or duration of bone marrow suppression in patients receiving high dose chemotherapy. We prospectively measured serial sTfR in nine consecutive patients with haematological malignancy admitted for various high dose regimens. Methods Samples for sTfR were collected from surplus patient specimens, centrifuged at 1000×g and frozen at -70°C, thawed and assayed as a batch following patient recovery. sTfR was measured by an automated turbidimetric method on an Abbott Architect ci8200 (Tina quant, Roche) that has been previously evaluated (Pfeiffer et al 2007). Haemoglobin and WCC were measured on Abbott Cell-Dyne 4000 analysers (Abbott Diagnostics). Results The sTfR nadir was associated with white cell nadir, and sTfR nadir and Day 5 sTfR were inversely associated with the number of red cell transfusions required. Conclusion Our preliminary data suggest that sTfR may be helpful in predicting the degree of bone marrow suppression in patients receiving intensive chemotherapy.

Analysis of manual reticulocyte counts in the clinical laboratories of Ponta-Grossa and Campos Gerais, PR, Brazil

Reticulocyte counts are widely used in laboratories to evaluate bone marrow erythropoietic activity and have great diagnostic and prognostic importance in the treatment of anemias. Reticulocytes are supravitally stained with new methylene blue or brilliant cresyl blue, which highlight the characteristic aspect of the reticulum visible by light microscopy. Known criteria were observed for good manual reticulocyte counting with special attention being paid to the preparation of reticulocyte slides, to counting fields without overlapping cells, and to the number of evaluated cells. The aim of this study was to evaluate the inter-observer variation and also to analyze the statistical error of manual reticulocyte counting. The Intraclass correlation coefficient (ICC) according to Bartko was used to evaluate the level of agreement among 12 laboratory technicians who evaluated the same 25 blood smears. The results of statistical analyses showed that the amount of random error, defined as (1-r2), varied from 4% to 60% among the technicians. Although imprecision occurred, the overall profiles were quite similar, and intraclass correlation coefficients indicated that the results obtained have clinical significance.

Accuracy of serum transferrin receptor levels in the diagnosis of iron deficiency among hospital patients in a population with a high prevalence of thalassaemia trait

The diagnosis of iron deficiency in hospital patients can be difficult in the presence of inflammation. A raised serum transferrin receptor (sTfR) level is useful as a marker of iron deficiency as it is unaffected by inflammation. However, diseases that cause an increase in erythropoietic activity can also result in a raised sTfR level. In South-East Asia, the prevalence of thalassaemia trait is high. As thalassaemia trait is associated with ineffective erythropoiesis and therefore an increase in the sTfR level, we studied the influence of thalassaemia trait on the diagnosis of iron deficiency in hospital patients. Among 431 patients with different combinations of iron deficiency, alpha- and beta-thalassaemia trait, we found that the sTfR level is an excellent diagnostic test for iron deficiency only in patients without thalassaemia trait. alpha-Thalassaemia trait worsened its diagnostic accuracy and beta-thalassaemia trait rendered it a non-diagnostic test. We conclude that in populations with a high prevalence of thalassaemia trait, the sTfR level is not useful in diagnosing iron deficiency unless the patient's thalassaemia status is known.

The effect of cold and haemorrhagic stress on reticulocytes in male albino Wistar rats

Background: In this study, the effect of cold and haemorrhagic stressors in male albino Wistar rats will be investigated. Methods: Cold stress was induced by placing the animals in thermostatic chamber containing ice cold water (0-10oC) and well aerated. Rats were allowed to stay for 5, 10, 15 and 20 minutes before they were bled from the tail vein at 2, 4, 6, 8 and 24 hours for reticulocyte counts. For the haemorrhagic stress, 30% of blood was withdrawn from the vein after which reticulocytes were counted at 2, 4, 6, 8 and 24 hours. Results: Cold and haemorrhagic stress caused increased reticulocyte counts in male albino Wistar rats. The controls were not subjected to any condition. In cold stress, the maximum percentage reticulocyte count was (at zero hour) 6.21 + 0.21% while the control was 1.02 + 0.12%. In the case of the haemorrhagic stressor, the percentage reticulocyte was seen to peak at the 6th hour (6.19 + 0.12%) and subsequently decrease to 2.59 + 0.02%. The probable causes of this rise in reticulocytes are discussed. Conclusion: In cold and haemorrhagic stress, reticulocyte count peaked at 2 and 6 hours respectively, depicting when maximum erythropoietic activity was reached in blood forming organs. Keywords: vPort Harcourt Medical Journal Vol. 3 (1) 2008: pp. 72-76

Adrenergic Regulation of Erythropoiesis during Cytostatic-Induced Myelosuppressions

The role of central adrenergic structures in the regulation of the erythroid hematopoietic stem was studied during administration of cyclophosphamide and 5-fluorouracil. The central nervous system contributed to suppression of erythropoiesis during cytostatic treatment. The suppressive effect of brain adrenergic structures on the erythron after treatment with cyclophosphamide and 5-fluorouracil was related to dysfunction of adherent cells in the hemopoiesis-inducing microenvironment (formation of hemopoietic islets and secretion of erythropoietic activity) and production of growth factors by myelokaryocytes, respectively. Brain norepinephrine had an inhibitory effect on proliferative activity and differentiation of erythroid precursors that were associated with the erythropoietin and peripheral alpha-adrenergic structures. However, stimulation of beta-adrenergic structures was followed by an increase in the rate of erythroid cell maturation.

Effects of p38 MAP Kinase Inhibitors on the Differentiation and Maturation of Erythroid Progenitors

In rodents, p38 MAP kinase inhibitors (p38is) induce bone marrow hypocellularity and reduce reticulocyte and erythrocyte counts. To identify target cell populations affected, a differentiating primary liquid erythroid culture system using sca-1(+)cells from mouse bone marrow was developed and challenged with p38is SB-203580, SB-226882, and SB-267030. Drug-related alterations in genes involved at different stages of erythropoiesis, cell-surface antigen expression (CSAE), burst-forming unit erythroid (BFU-E) colony formation, and cellular morphology (CM), growth (CG), and viability were evaluated. CSAE, CM, and decreases in BFU-E formation indicated delayed maturation, while CG and viability were unaffected. Terminal differentiation was delayed until day 14 versus day 7 in controls. CSAE demonstrated higher percentages of sca-1(+)cells after day 2 and reduced percentages of ter119(+) cells after day 7 in all treated cultures. Real-time reverse transcriptase polymerase chain reaction revealed a transient delay in expression of genes involved at early, intermediate, and late stages of erythropoiesis, followed by rebound expression at later time points. Results demonstrate p38is do not irreversibly inhibit erythrogenesis but induce a potency-dependent, transient delay in erythropoietic activity. The delay in activity is suggestive of effects on sca-1(+)bone marrow cells caused by alterations in expression of genes related to erythroid commitment and differentiation resulting in delayed maturation.

Serum hepcidin level and erythropoietic activity after hematopoietic stem cell transplantation

The relationship between serum hepcidin, a key regulator of body iron homeostasis, and erythropoiesis was investigated before and after stem cell transplantation in 31 patients with hematopoietic malignancies. Serum hepcidin-25 was monitored using a liquid chromatography-tandem mass spectrometry-based assay system. Other iron- and erythropoiesis-related parameters and known hepcidin regulators, such as interleukin-6 and growth differentiation factor-15, were also monitored. The serum hepcidin level peaked one week after stem cell transplantation, followed by a gradual decrease with a parallel change in interleukin-6 and a reciprocal change in reticulocyte count. Multivariate regression analysis demonstrated that the serum hepcidin level at four weeks after stem cell transplantation showed significant inverse correlations with erythropoietic activity markers, such as the soluble transferrin receptor, but not with growth differentiation factor-15. These results indicate the existence of an unknown functional erythropoiesis-associated circulating factor, other than growth differentiation factor-15, that negatively regulates hepcidin production in stem cell transplantation settings.

The Potency of Erythropoietin-Mimic Antibodies Correlates Inversely with Affinity

Preclinical animal studies have shown that Ab12.6, an agonistic human Ab targeting the erythropoietin receptor (EPOR), exhibits several potential dosing and safety features that make it an attractive clinical candidate for the treatment of anemia. Ab12.6 was derived by yeast display affinity maturation of parental Ab12, a strategy initially intended to improve off-rate and affinity for EPOR, thereby enhancing erythropoietic activity. Analysis of full-length IgGs derived from yeast clones identified sequences within Ab12 CDRH2 that independently influenced both affinity and potency. The Ab12.6 derivative displayed improved in vitro potency and in vivo efficacy, although its binding affinity to the EPOR was lower than that of the parent Ab12. Additional Ab12 derivatives also exhibited an inverse correlation between affinity and potency. These results suggest that for this class of agonistic Abs, faster off-rates may permit continuous receptor stimulation and more efficient erythropoiesis.

Serum erythropoietin levels for predicting bone marrow recovery following chemotherapy in leukaemia

Summary To investigate whether serum erythropoietin (EPO) levels are influenced by the intensity of bone marrow erythroid activity, we used a radioimmunoassay (Nippon DPC Co., Japan) to study EPO levels in three acute leukaemia patients during treatment with intensive chemotherapy. We also measured the reticulocyte count and reticulocyte maturity using an automated reticulocyte analyser (Sysmex R-2000) to detect erythropoietic activity. From the day after the initiation of chemotherapy, EPO levels increased markedly without any change in Hb levels, suggesting that some other mechanism was regulating serum EPO. A decrease in EPO after chemotherapy was accompanied by an increase in high fluorescence ratio, an index of immature reticulocvtes, but the actual increase in the reticulocyte count was delayed for 3-4 days after this. These findings suggest that the decrease in serum EPO levels was closely related to the early stage of red cell production and that EPO levels may be a useful marker for the recovery of erythropoietic activity after chemotherapy.

Reticulocyte maturity pattern analysis as a predictive marker of erythropoiesis in paediatrics. Part II: Pilot study for clinical application.

Reticulocyte quantification in peripheral blood samples is a commonly used diagnostic indicator of erythropoietic activity. A methodology based on flow cytometry additionally separates reticulocytes into 3 groups by fluorescence staining of the residual RNA. This identifies cells as 'high (HFR), medium (MFR) and low (LFR) fluorescence intensity' reticulocytes. In part II of the study we looked for the clinical applicability in paediatrics. Selected groups of patients with ineffective erythropoiesis, i.e. suffering from renal failure, oncologic patients with suppressed bone marrow activity caused by chemotherapy and anaemic new-born infants have been observed longitudinally for their reticulocyte maturity profiles. Data were compared to the commonly used parameters RBC, Hb and Hct. In all cases in which effective erythropoiesis returned documented by a normalization of standard blood parameters, HFR cells reacted significantly earlier than the traditional markers. These preliminary observations suggest the reticulocyte maturity pattern analysis can be used as an additional aid in diagnosis and as a helpful parameter for the monitoring of any anaemic situation.

Reticulocyte maturity pattern analysis as a predictive marker of erythropoiesis in paediatrics. Part I: Evaluation of age-dependent reference values.

Reticulocyte quantification in peripheral blood samples is a commonly used diagnostic indicator of erythropoietic activity. A methodology based on flow cytometry additionally separates reticulocytes into three groups by fluorescence staining of the residual RNA. This identifies cells as high (HFR), medium (MFR) and low (LFR) fluorescence intensity reticulocytes. In the present study an automated counter was evaluated and tested for its clinical applicability in paediatrics. In part I, reference intervals for different periods of childhood were determined. Except for the neonatal period there was no age-dependence so that children aged one week to 16 years have been summarized in one group. The wide variations we found in preterm children can be explained by different erythropoietic stimuli as the result of anaemia in infants with very low birthweight. No significant differences could be found between the sexes, not even at the onset of puberty. When using the reticulocyte maturity pattern analysis in clinical practice, the data give a helpful indication of the efficiency of the erythropoietic system.

The effect of cytokines on the in-vitro erythropoietin bioassay.

The effects of various concentrations of granulocyte macrophage colony stimulating factor (GM-CSF), gamma interferon (gamma IFN) and interleukins 1 alpha (IL-1 alpha), 1 beta (IL-1 beta) and 3 (IL-3) on the anaemic mouse spleen cell bioassay for erythropoietin (EPO) were investigated. Addition of IL-3 and GM-CSF at various concentrations had no effect on EPO stimulated 3H thymidine incorporation. However the addition of IL-1 alpha, IL1-beta and gamma IFN (3.3 x 10(-8) gl-1) caused a significant (P < 0.01) inhibition of EPO stimulated thymidine incorporation. This suggests that the EPO bioassay may be influenced by variable levels of some inflammatory cytokines in serum. Previous studies have shown that the bioassay is influenced by serum transferrin levels and thus serum immunoassays remain the technique of choice for specific estimates of EPO. Since EPO bioassays are not specific, they should be reserved for situations in which an estimate of the total erythropoietic activity of serum is required.

Influence of inflammation as measured by α-1-acid glycoprotein on iron status indicators among HIV-positive postpartum Zimbabwean women

The acute phase response (APR) influences indicators of iron status. A recent WHO/CDC consultation recommended concurrent measurement of alpha-1-acid-glycoprotein (AGP) in surveys to control for the APR, and aid in interpreting iron status. They proposed further exploratory analyses using AGP. We examined whether the APR (measured by AGP) influences the expected relationships between iron status indicators in an HIV-infected population. We measured hemoglobin (Hb), serum ferritin (SF), transferrin receptor (TfR), erythropoietin (EPO) and AGP in a cross-sectional survey of 643 HIV-positive Zimbabwean women. SF was significantly higher in APR-positive (AGP>1 g/l) women (P<0.001), in whom there was no association between SF and Hb. TfR was inversely associated with Hb, in both APR-positive and APR-negative women (P<0.001). However, among anemic women (Hb<110 g/l), APR-positive women had marginally lower TfR concentrations (P=0.053). There was no difference in EPO response to decreasing Hb among APR-positive and APR-negative women. AGP captured the influence of the APR on iron indicators and their relationships with each other. The APR influenced SF and its relationship with Hb as expected. TfR behaved unexpectedly. Although TfR has been promoted as an iron indicator that is uninfluenced by the APR, TfR concentrations were depressed among anemic APR-positive women. Because TfR reflects iron deficiency and erythropoietic activity, pro-inflammatory cytokines associated with the APR may be inhibiting erythropoiesis, which is reflected by lower TfR concentrations. We support the WHO/CDC recommendation that AGP is a useful indicator to assess the influence of the APR on iron status indicators.

Requirement for erythroblast-macrophage protein (Emp) in definitive erythropoiesis

Emp, erythroblast-macrophage protein was initially identified as a mediator of erythroblast-macrophage interactions during erythroid differentiation. More recent studies have shown that targeted disruption of Emp leads to abnormal erythropoiesis in the fetal liver, and fetal demise. To further address the activity of Emp in the hematopoietic lineage in adult bone marrow, we conducted fetal liver HSC reconstitution assay. Emp null fetal liver cells were transplanted into lethally irradiated wild-type sibling mice, and assessed the erythropoietic activity. We found that Emp null cells rescued lethally irradiated mice with efficiency comparable to that of wild-type cells. However, the recipients of Emp null cells showed abnormal erythropoiesis as indicated by the presence of persistent anemia, extensive extramedullary erythropoiesis, and increased apoptosis of erythroid precursors. Extramedullary erythropoiesis suggests perturbed interactions between the Emp-deficient hematopoietic cells and the wild-type niche. Furthermore, in spleen colony-forming unit assays, proliferation rates of the Emp null cells were greater than those of the wild-type cells. Similarly, in vitro burst-forming unit-erythroid and colony-forming unit-erythroid assays showed increased erythroid colony numbers from Emp null livers. Morphologic examination showed that Emp null CFU-E-derived erythroblasts were immature compared to those derived from wild-type CFU-Es, suggesting that loss of Emp function in erythroid cells results in impaired proliferation and terminal differentiation. These results demonstrate that Emp plays a cell intrinsic role in the erythroid lineage.

Iron regulation and erythropoiesis

The peptide hormone hepcidin regulates iron metabolism in response to erythropoietic demand, iron stores and inflammation. Major advances have been made in understanding the regulation of hepcidin production, and consequently the availability of iron for erythropoiesis. It is becoming clear that the bone morphogenetic protein (BMP) pathway plays a major role in setting the baseline hepcidin level and, with the assistance of BMP2/4 and hemochromatosis-related proteins hemojuvelin, HFE and transferrin receptor 2, also regulates hepcidin expression in response to iron. Regulation of hepcidin in anemias has now been linked to increased erythropoietic activity and is likely mediated by factor(s) secreted by erythroid precursors. GDF-15 was identified as a candidate for one of the erythroid factors suppressing hepcidin. Tissue hypoxia may also directly contribute to hepcidin suppression in anemias. Regulation of hepcidin by inflammation may include multiple cytokines and the Toll-like receptors pathways. Although it has not yet been shown that increased hepcidin is indispensible for the development of anemia of inflammation, transgenic overexpression of hepcidin was sufficient to replicate its key features. Regulation of hepcidin and iron availability for erythropoiesis has revealed unexpected pathways and much complexity. The renaissance of the study of iron regulation continues to reward researchers with interesting biology and potential therapeutic targets.

Erythropoietin ameliorates chemotherapy‐induced fibrosis of the lungs in a preclinical murine model

Organ toxicity induced by chemotherapeutic drugs is a serious obstacle in the effective treatment of patients suffering from cancer and autoimmune disease. A strong association exists between pulmonary toxicity, particularly fibrosis, and chemotherapeutic drugs. Attempts have been made to identify compounds capable of suppressing fibrosis. In addition to its erythropoietic activity, erythropoietin (EPO) has been shown to have effects on nonhemopoietic cells. Therefore, we postulated that EPO may exert beneficial effects on lung tissue during chemotherapy. To test our hypothesis, we investigated pulmonary changes caused by bleomycin, a fibrosis-inducing agent, in animals treated with the drug alone and in combination with EPO. Fibrosis, cellular alterations and structural changes were assayed by blind analysis of the lung sections. A 6-fold decrease in the number of prominent endothelial cells--suspected to be indicative of cellular activation and inflammatory response--was observed in lung sections derived from mice treated with bleomycin and EPO compared to animals injected with bleomycin alone (p < 0.008). Additionally, there was twice the number of ICAM1-positive endothelial cells in animals treated with bleomycin alone compared with the number in the bleomycin and EPO-treated group (p < 0.05). Alveolar mononuclear phagocytic hyperplasia was reduced by as much as 100% in animals treated with bleomycin and EPO compared to animals treated with bleomycin alone (p < 0.03). Finally, a 5-fold decrease in interstitial fibrosis was observed in lung sections obtained from animals treated with bleomycin and EPO (p < 0.02). We conclude that EPO can ameliorate drug-induced fibrosis and endothelial damage caused by chemotherapeutic agents.