Immature Red Cells Research Articles

Mitochondrial ferritin expression in erythroid cells from patients with sideroblastic anemia

The sideroblastic anemias are characterized by ring sideroblasts, that is, red cell precursors with mitochondrial iron accumulation. We therefore studied the expression of mitochondrial ferritin (MtF) in these conditions. Erythroid cells from 13 patients with refractory anemia with ring sideroblasts (RARS) and 3 patients with X-linked sideroblastic anemia (XLSA) were analyzed for the distribution of cytoplasmic H ferritin (HF) and MtF using immunocytochemical methods. We also studied 11 healthy controls, 5 patients with refractory anemia without ring sideroblasts (RA), and 7 patients with RA with excess of blasts (RAEB). About one fourth of normal immature red cells, mostly proerythroblasts and basophilic erythroblasts, showed diffuse cytoplasmic positivity for HF, but very few were positive for MtF (0%-10%). Similar patterns were found in anemic patients without ring sideroblasts. In contrast, many erythroblasts from patients with sideroblastic anemia (82%-90% in XLSA and 36%-84% in RARS) were positive for MtF, which regularly appeared as granules ringing the nucleus. Double immunocytochemical staining confirmed the different cellular distribution of HF and MtF. There was a highly significant relationship between the percentage of MtF(+) erythroblasts and that of ring sideroblasts (Spearman R = 0.90; P <.0001). Reverse transcription-polymerase chain reaction studies demonstrated the presence of MtF mRNA in circulating reticulocytes of 2 patients with XLSA but not in controls. These findings suggest that most of the iron deposited in perinuclear mitochondria of ring sideroblasts is present in the form of MtF and that this latter might be a specific marker of sideroblastic anemia.

Historical Perspectives: The Underpinnings of Neonatal/Perinatal Medicine

The accompanying article by Dr. John Bowman was contributed in response to a request to place his important paper (1) into context. Dr. Bowman is the former Medical Director of the Winnipeg Rh Laboratory and a Distinguished Professor Emeritus at the University of Manitoba. Although not the first to describe the use of Rh immune globulin, the clinicians in Winnipeg were leaders in the use of antenatal prophylaxis. At present, Rh isoimmunization is extremely rare, in large measure because of the pioneers described by Dr. Bowman. Antenatal administration of anti-D immune globulin now is “standard operating procedure,” but this was not always the case. It is important to understand how the problem of Rh isoimmunization has been virtually eliminated from neonatal practice. 1. ↵ Bowman JM, Chown B, Lewis M, Pollock JM. Rh isoimmunization during pregnancy: antenatal prophylaxis. Can Med Assoc J. 1978;118:623–627 [OpenUrl][1][Abstract][2] # Historical Perspectives: The Underpinnings of Neonatal/Perinatal Medicine {#article-title-2} Rh hemolytic disease was reported initially by a French midwife in 1609. She described the birth of twins: the first, who was pale and bloated with fluid (hydrops fetalis), died within minutes of birth; the second, in much better condition, became deeply jaundiced in the first 3 days after birth, developed rigidity, lay in a position of opisthotonus (kernicterus), and died on the fourth day after birth. Hydrops fetalis and kernicterus were not linked again until 323 years later. In 1932, Diamond and associates determined that they were simply different aspects of a spectrum representing the same disorder. The more severe, hydrops, was characterized by hemolysis of fetal red blood cells that produced severe anemia, followed by hepatosplenomegaly, extramedullary erythropoiesis, and the outpouring of immature nucleated fetal red cells, from which Diamond coined the diagnostic term “erythroblastosis fetalis.” Uncovering the cause of the fetal red blood cell hemolysis had to await the discovery of the Rh blood group system by Landsteiner and Wiener in 1940 and the subsequent determination in 1941 by Levine that the cause of hemolytic … [1]: {openurl}?query=rft.jtitle%253DCanadian%2BMedical%2BAssociation%2BJournal%26rft.stitle%253DCMAJ%26rft.issn%253D0008-4409%26rft.aulast%253DBowman%26rft.auinit1%253DJ.%2BM.%26rft.volume%253D118%26rft.issue%253D6%26rft.spage%253D623%26rft.epage%253D627%26rft.atitle%253DRh%2Bisoimmunization%2Bduring%2Bpregnancy%253A%2Bantenatal%2Bprophylaxis%26rft_id%253Dinfo%253Apmid%252F77714%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Ajournal%26ctx_ver%253DZ39.88-2004%26url_ver%253DZ39.88-2004%26url_ctx_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%253Actx [2]: /lookup/ijlink?linkType=ABST&journalCode=cmaj&resid=118/6/623&atom=%2Fneoreviews%2F3%2F11%2Fe223.atom

Changes in the G Gamma- and A Gamma-Globin mRNA Components of Fetal Hemoglobin during Human Development

To determine the postnatal developmental changes in ^Gγ- and ^Aγ-globin mRNAs of HbF, blood samples were obtained from 28 pre-term infants born at ≤ 33 weeks of gestation (27.9 ± 2.5), 9 term-born infants and 26 adults. Adult samples were used to determine the relationship between the levels ^Gγ- and ^Aγ-mRNAs of HbF and the–158 (C→T) variation in the promoter region of the ^Gγ-globin gene. The results showed that ^Gγ-globin mRNA to total γ-globin mRNAs remained around 66% (66.2 ± 4) until the 44 week of postconceptual age when a change in the ^Gγ- and ^Aγ-globin mRNA proportions occurred. The immature red cells of adults had a range of ^Gγ-globin mRNA to total γ-globin mRNAs varying from 20 to 74% (53.1 ± 16.9). The high levels of ^Gγ-globin mRNA to total γ-globin mRNAs are associated with the presence of a –158 (C→T) polymorphism. The gene frequency of polymorphism was 0.32.

Haematological Parameters and Altered Erythrocyte Metabolism in Anaemic Dogs

In previous studies, an increase in the activity of pyruvate kinase (PK) and glucose-6-phosphate dehydrogenase (G6PDH), an increase in the concentration of 2,3-diphosphoglycerate (2,3DPG), and alterations in osmotic fragility were found in dogs with haemolytic anaemia. These changes were mainly caused by the presence of immature red cells found in regenerative anaemias. In the present study, the same parameters were evaluated in dogs with different types of anaemia. The haematological patterns of 40 anaemic dogs were analysed to define the pathogenesis and the haematological features of each case. Non-regenerative anaemias could be attributed principally to chronic diseases and to the haemolysis that accompanies the early stages of canine babesiosis. Regenerative anaemias were mainly due to haemolysis, in some cases with an immune-mediated pathogenesis. PK activity was higher in regenerative than in non-regenerative anaemias, but G6PDH activity and 2,3DPG concentration increased in both types of anaemia. This suggests that PK activity is influenced by the presence of immature red cells, but the requirements for reducing compounds and oxygen are not dependent on the type of anaemia. Abnormalities in osmotic fragility were detected in haemolytic anaemias and in those non-regenerative anaemias in which reticulocyte percentage, but not reticulocyte production index (RPI), increased. The osmotic fragility could be used as an early indicator of erythrocyte regeneration.

Iron deficiency in children: detection and prevention.

Iron deficiency in children: detection and prevention.

HbF synthesis during stress erythropoiesis as determined by gamma-mRNA/non-alpha-mRNA quantification.

To determine whether a quantitative relationship exists between globin mRNAs and their translation product during stress erythropoiesis in infants with increased production of fetal hemoglobin (HbF), we measured and compared the relative amounts of the mRNAs of alpha-, beta-, and gamma-globins and their protein synthesis. The synthesis of globin in immature red cells was determined by the incorporation of [3H]leucine, followed by separation and quantification of the polypeptides by C4-reverse phase HPLC. The relative proportions of the mRNAs of the globins were determined by RNase protection assay. A comparison of blood samples from 17 infants expected to have increased production of HbF in relation to their developmental age (five infants of diabetic mothers, two infants with intrauterine growth retardation, one infant with bronchopulmonary dysplasia, and seven infants with cyanotic heart disease) revealed a very significant correlation (r2 = 0.994; p < 0.001) between the ratio of globin mRNAs encoding HbF ([gamma/(gamma + beta)] mRNAs) and the ratios of the de novo synthesis of HbF [gamma/(gamma + beta)]. When only the 10 infants who had increased HbF synthesis are included, the correlation remains unchanged (r2 = 0.997, p < 0.001). The data demonstrated that under conditions of erythropoietic stress, when HbF production is increased, there is a close relationship between the quantification of gamma-globin mRNA and gamma-globin synthesis. The usual methods of determining HbF synthesis can be replaced by globin mRNA determination, which can be performed rapidly with a minimal amount of blood.

Index of Suspicion

Index of Suspicion

Cleavage of the transferrin receptor by human granulocytes: Preferential proteolysis of the exosome-bound TFR

In contrast with other mammalian granulocytes, human granulocytes rapidly cleave the transferrin receptor (TFR) from sheep exosomes. Proteolysis of TFR from exosomes is more rapid and more extensive than that from the sheep reticulocyte cell surface itself, although little difference in cleavage is seen when immunoprecipitates or when immobilized, solubilized receptors from the two sources are compared. Circulating exosomes but not the plasma membrane fraction from seven species of immature red cells or erythropoietic cells show the presence of a peptide of approximately 18 kD recognized by an antibody to the cytoplasmic domain of the TFR. This 18 kD peptide is virtually absent from the corresponding cellular plasma membranes including human reticulocyte membranes. Taken together, the data are consistent with the conclusion that the exosomes released to the circulation from maturing red cells are the principal source of the soluble, circulating, truncated TFR. The granulocyte protease appears to be present on the cell surface and not released into the medium after short (30 min) periods of incubation at 37 degrees C. The protease is inhibited by PMSF but only at high (1 mM) concentrations. Using sheep exosome bound-TFR as substrate, human granulocytes exceed other granulocytes in their capacity to cleave TFR, suggesting that this may be a key factor for the prominent amount of circulating, soluble receptor found in human sera during periods of elevated reticulocyte levels. Human exosomes, unlike those from other species, contain little native size TFR. Truncated receptor containing the cytoplasmic domain being predominant in human exosomes.

Cleavage of the transferrin receptor by human granulocytes: Preferential proteolysis of the exosome‐bound TFR

In contrast with other mammalian granulocytes, human granulocytes rapidly cleave the transferrin receptor (TFR) from sheep exosomes. Proteolysis of TFR from exosomes is more rapid and more extensive than that from the sheep reticulocyte cell surface itself, although little difference in cleavage is seen when immunoprecipitates or when immobilized, solubilized receptors from the two sources are compared. Circulating exosomes but not the plasma membrane fraction from seven species of immature red cells or erythropoietic cells show the presence of a peptide of ∼18 kD recognized by an antibody to the cytoplasmic domain of the TFR. This 18 kD peptide is virtually absent from the corresponding cellular plasma membranes including human reticulocyte membranes. Taken together, the data are consistent with the conclusion that the exosomes released to the circulation from maturing red cells are the principal source of the soluble, circulating, truncated TFR. The granulocyte protease appears to be present on the cell surface and not released into the medium after short (30 min) periods of incubation at 37°C. The protease is inhibited by PMSF but only at high (1 mM) concentrations. Using sheep exosome bound-TFR as substrate, human granulocytes exceed other granulocytes in their capacity to cleave TFR, suggesting that this may be a key factor for the prominent amount of circulating, soluble receptor found in human sera during periods of elevated reticulocyte levels. Human exosomes, unlike those from other species, contain little native size TFR. Truncated receptor containing the cytoplasmic domain being predominant in human exosomes. © 1996 Wiley-Liss, Inc.

Hsp-70 is closely associated with the transferrin receptor in exosomes from maturing reticulocytes.

The presence of the heat shock protein (hsp-70) has been detected in exocytosed vesicles (which are named exosomes) from mammalian and avian immature red cells (i.e. reticulocytes) as well as from a differentiating avian erythroleukaemic cell line. The close, but non-covalent, association of hsp-70 with the transferrin receptor (TFR) in exosomes is demonstrated by: (1) the ability to cross-link hsp-70 to TFR; (2) the co-immunoprecipitation of hsp-70 and TFR with an antibody against TFR, and the co-immunoprecipitation of TFR and hsp-70 with antibody against hsp-70; and (3) the retention of TFR by hsp-70 bound to ATP-agarose and the simultaneous elution of both proteins by excess ATP. Semi-quantitative analysis of the relative efficiency of cross-linking of these proteins in exosomes versus plasma membranes shows that TFR in exosomes is preferentially bound to hsp-70. From an analysis of the relative amounts of hsp-70 and TFR regenerated from the cross-linked complex, the ratio of TFR monomer bound to hsp-70 is approximately 1.5 to 1. Given the presence of hsp-70 in exosomes from several species and its close association with TFR (the major protein lost during reticulocyte maturation) it is proposed that hsp-70 plays a role in exosome formation and/or release in immature red cells.

Flow cytometric immunophenotyping of lymphocyte subsets in samples that contain a high proportion of non‐lymphoid cells

Flow cytometric (FCM) immunophenotyping of peripheral blood from thalassemia patients presents technical difficulties because of the high proportion of immature red cells. The combination of forward scatter (FSC) and side scatter (SSC) with fluorescence associated with human leukocyte antigen/monocyte antigen (CD45/CD14) was unable to identify the lymphocyte population in thalassemia patients; therefore, it was necessary to exclude immature red cells to analyze lymphocyte subsets in these patients. A simultaneous three-color FCM method was developed, with the basis that transferrin receptor (CD71) or glycophorin A (glyco A) is present on all immature red cells, but is not expressed on CD45 positive leukocytes. In this study, the lymphocyte population was identified by gating out unwanted cell populations using the FSC/CD71-fluorescein isothiocyanate (FITC), FSC/glyco A-FITC, or FSC/CD45-peridinin chlorophyll protein (PerCP) profiles. The CD71-FITC negative cells, glyco A-FITC negative cells, or CD45-PerCP positive cells were identified, then analyzed on the basis of FSC/SSC to allow any remaining non-lymphocyte cells in FSC/SSC gate to be excluded. The cells in FSC/SSC gate were then analyzed using other irrelevant two-color antibodies. Of the three gating strategies, CD45-PerCP and glyco A-FITC methods are better than the CD71-FITC gating method. Both methods markedly increase the purity of lymphocytes in the analysis gate. Either method is easy, straightforward, requires a six-tube set of reagent tubes, and provides a reliable method for immunophenotyping lymphocyte subsets in preparations containing a large percentage of non-lymphoid cells.

Control of cell pH in immature primitive red cells from chick embryo

1. 1. The intracellular pH in primitive red cells from 4 day chick embryos was measured with the digitonin null-point method and the fluorescent indicator SNARF-1. At physiological pH e of 8.0 red cell pH is 7.39 at day 4. 2. 2. The calculated proton equilibrium potential of − 38 mV is in good agreement with previous measurements of E m (Engelke et al., 1988) and supports the conclusion that the E m is dominated by a proton conductance. 3. 3. The sodium-proton exchanger is present in primitive red cells but quiescent under physiological conditions. 4. 4. The results indicate that the bicarbonate-chloride exchange via Band 3 protein is impaired.

Haematological response to reduced oxygen‐carrying capacity, increased temperature and hypoxia in goldfish, Carassius auratus L.

Goldfish were exposed to three qualitatively distinct forms of respiratory stress: phenylhydrazine HCl‐induced reduction in blood oxygen‐carrying capacity; transient (3 h) hypoxia; and transient (1 or 2 h) temperature‐forced increases in oxygen demand. Response embodied three common features: (1) the relative number of immature red cells increased, and these were 3H‐thymidine positive; (2) the abundance of apparently karyorrhectic or degenerating cells rose; (3) cells undergoing a form of division became prevalent. Neither of the latter two cell types evidenced thymidine uptake. These observations suggest that respiratory distress triggers the proliferation of new erythrocytes, and that this is accompanied by elimination of some portion of the preexisting red cell population. It is hypothesized that the latter process limits viscosity‐related elevation of cardiac work costs. The significance of division by circulating erythrocytes is not clear.

Reticulocytosis, increased mean red cell volume, and greater blood viscosity in altitude susceptible compared to altitude resistant rats

We have identified two strains (H and M) of Sprague-Dawley rat with markedly different susceptibilities and cardiopulmonary responses to chronic hypobaria. To further characterize factors responsible for these differing cardiopulmonary responses to chronic hypobaria, the present study examined differences in hematologic responses between the strains and assessed the contribution of differences in blood viscosity to differences in pulmonary vascular resistance. Following a 4–5 week exposure to simulated high altitude (0.5 atm), hemoglobin, hematocrit, mean red cell volume, and reticulocyte count were all increased in the susceptible H compared to the resistant M rats, whereas red blood cell counts were similar. Sea level controls manifested no differences. Blood viscosity, measured in a capillary viscometer, was 53% greater in chronically hypoxic H than in M rats, and plasma viscosities were similar. Blood from high altitude H rats increased pulmonary vascular resistance more than blood from high altitude M rats when perfused into lungs isolated from high rats of either strain. In conclusion, high altitude H rats have an increased population of immature red cells, leading to a greater mean red cell volume and hematocrit than in high altitude M rats. These hematologic differences contribute to the increased blood viscosity and greater pulmonary vascular resistance of H compared to M rats after 4 weeks' high altitude exposure.

Action of erythropoietin in vitro on rabbit reticulocyte membrane Ca2+-ATPase activity.

The mechanism of action of erythropoietin is thought to require specific interaction with the target cell surface and involve alteration of cellular calcium metabolism. Using the rabbit reticulocyte membrane as a model of the immature red cell membrane, we investigated the effects of human recombinant erythropoietin on membrane Ca2+-ATPase (calcium pump) activity in vitro. Erythropoietin in a concentration range of 0.025 to 3.0 U/ml progressively decreased membrane Ca2+-ATPase activity by up to 64% (P less than 0.01). These concentrations have been shown by others to stimulate in vitro erythroid growth. The action of erythropoietin on reticulocyte Ca2+-ATPase required an incubation time of 1 h before enzyme assay for maximum effect and was neutralized by antierythropoietin antiserum. Other nonhemopoietic growth factors (epidermal growth factor, insulin) had no effect in this assay. Ca2+-ATPase activity of membranes prepared from rabbit mature red blood cells was not inhibited by erythropoietin. The novel effect of erythropoietin on reticulocyte membrane Ca2+-ATPase activity is a mechanism by which erythropoietin can influence cellular Ca2+ metabolism.

Erythrocyte Porphobilinogen Deaminase Activity in Lives Disease

The activities of erythrocyte porphobilinogen deaminase were studied in patients with various liver diseases and in control groups. The lowest enzyme activities were found in patients with acute intermittent porphyria, and the highest ones in those with increased hemopoietic activity. Patients with liver cirrhosis or chronic active hepatitis had porphobilinogen deaminase activities that were significantly higher than in normal subjects and did not depend on disease activity. In patients with acute hepatitis, porphobilinogen deaminase activities varied depending on the phase of disease, being normal at onset and after 3-4 mo, and elevated to the values observed in chronic liver disease between 2 and 4 wk of hospitalization. The differences in porphobilinogen deaminase activities between patients with liver disease and controls did not relate to red cell age as determined by density gradient centrifugation. Therefore, although the mechanism responsible for the increase in porphobilinogen deaminase activities in liver disease is not clear, the results of this study suggest that it is independent of the presence of immature red cells in the circulation.

Reticulocytosis, increased mean red cell volume, and greater blood viscosity in altitude susceptible compared to altitude resistant rats

We have identified two strains (H and M) of Sprague-Dawley rat with markedly different susceptibilities and cardiopulmonary responses to chronic hypobaria. To further characterize factors responsible for these differing cardiopulmonary responses to chronic hypobaria, the present study examined differences in hematologic responses between the strains and assessed the contribution of differences in blood viscosity to differences in pulmonary vascular resistance. Following a 4–5 week exposure to simulated high altitude (0.5 atm), hemoglobin, hematocrit, mean red cell volume, and reticulocyte count were all increased in the susceptible H compared to the resistant M rats, whereas red blood cell counts were similar. Sea level controls manifested no differences. Blood viscosity, measured in a capillary viscometer, was 53% greater in chronically hypoxic H than in M rats, and plasma viscosities were similar. Blood from high altitude H rats increased pulmonary vascular resistance more than blood from high altitude M rats when perfused into lungs isolated from high altitude rats of either strain. In conclusion, high altitude H rats have an increased population of immature red cells, leading to a greater mean red cell volume and hematocrit than in high altitude M rats. These hematologic differences contribute to the increased blood viscosity and greater pulmonary vascular resistance of H compared to M rats after 4 weeks' high altitude exposure.

950 ADENOVIRUS ASSOCIATED HEMOPHAGOCYTIC SYNDROME IN A BONE MARROW TRANSPLANT PATIENT

Virus-associated hemophagocytic syndrome (VANS) has been linked most commonly to herpes group viruses. It has not previously been reported in a bone marrow transplant (BMT). VAHS occurred in a 5 y/o girl who underwent an autologous BMT for metastatic Wilms tumor. Prior to BMT she received doxorubicin, high dose melphalan, and local radiation to her chest and abdomen. Adenovirus was isolated consistently throughout the course of the transplant from urine and stool, but not from buffy coats or respiratory tract secretions. Bone marrow exam (BME) 2 wks post BMT showed hematopoietic recovery with all three cell lines present. At +4 wks she developed hepatomegaly with elevated liver enzymes, intermittent fever and pancytopenia. BME at that time was hypocellular with marked histiocyte phagocytosis of immature and mature red cells, white cells and platelets. The histiocytes were mature with abundant vacuolated cytoplasm and inconspicuous nucleoli. A BME at +7 wks showed prominent histiocytes and hemophagocytosis and aplasia of all hematopoietic cells. A culture of this marrow grew adenovirus. Nine wks after BMT she developed fatal interstitial pneumonia. Cultures of pleural fluid and lung tissue from open lung biopsy grew adenovirus. Post mortem cultures from lung, heart, spleen and GI tract grew adenovirus. This is the first case of culture proven adenovirus VAHS in a BMT patient.

Differentiation of human erythroid cells is associated with increased O-glycosylation of the major sialoglycoprotein, glycophorin A.

Glycophorin A, the major human erythrocyte sialoglycoprotein, is found exclusively on cells of the erythroid lineage. The amino acid sequence is known, and glycophorin A isolated from mature erythrocytes contains a single N-glycosidic and 15 O-glycosidic oligosaccharides. Monoclonal antibodies against erythrocyte glycophorin A reacted weakly with erythroid precursors while a monospecific rabbit antiserum reacted strongly with immature and mature red cells. Glycophorin A was isolated from cells representing various stages of erythropoiesis in normal bone marrow, from blood cells of neonates with erythroblastosis fetalis, and from the erythroleukemic cell lines K562 and HEL before and after induced differentiation. Analysis of the oligosaccharides showed less O-glycosylation of glycophorin A in erythroid precursors. The degree of glycosylation increased concomitantly with differentiation.

Identification of two types of immature antigenic molecules on chicken red cells

Surface-labelled antigen analysis of circulating embryonic erythrocytes shows that the population of 48 000 D antigenic molecules involves an agerelated antigenic complex referred to as E 1 48 K component and, in addition, an antigenic molecule also expressed on immature red cells from anemic adult chickens referred to as Im 48 K component. A second immature antigenic molecular species of 140 000 D was detected on red cells from anemic birds and is referred to as Im 140 K component. The Im 48 K and Im 140 K components have no common determinants, although they are both found on immature red cells of anemic animals. This work suggests that these two immature antigens could be involved in the differentiation process, possibly at different levels of erythroid progenitors.