What is the difference between reticulocytes and erythrocytes

The potential of Ret-He was demonstrated in a study with patients with chronic rheumatic disease and anemia. The predictive value of RetHe was tested in response to oral iron therapy and, according to the authors, the findings support the role of Ret-He as a marker for iron responsiveness.

Potential utility of the new Sysmex XE red blood cell extended parameters in the study of disorders of iron metabolism. Clin Chem Lab Med. Efficacy of advanced discriminant algorithms for screening on iron-deficiency anemia and -thalassemia trait: a multicentric evaluation. Am J Clin Pathol. The MHe index was first proposed and tested by Urrechaga et al.

The performance of this index was better sensitivity Serum transferrin receptor and its ratio to serum ferritin in the diagnosis of iron deficiency. The ratio of serum transferrin and serum ferritin in the diagnosis of iron status. Br J Haematol. Thus, the transferrin receptor synthesis is stimulated when there is a reduction of functional iron as shown by the results of this study. The role of serum transferrin receptor in the diagnosis of iron deficiency.

In the clinical practice this differentiation is important because iron supplementation is beneficial to ACD combi patients, but may be deleterious for ACD patients. In fact, the absolute iron deficiency associated to ACD increases the number of microcytic and hypochromic red cells, causing cell features similar to IDA.

ACD patients showed evidence of reduced iron availability for erythropoiesis, but the disturbance of the iron metabolism in functional iron deficiency was less remarkable than in the association of ACD with IDA.

Therefore, the challenge persists, and other studies are needed to identify a parameter with clinical decision value. Abrir menu Brasil. Revista Brasileira de Hematologia e Hemoterapia. Abrir menu. E-mail address: lenagrotto gmail. Objective: The aim of this study was to evaluate the effectiveness of mature red cell and reticulocyte parameters to identify three conditions: iron deficiency anemia, anemia of chronic disease, and anemia of chronic disease associated with absolute iron deficiency.

Results: There was no difference between the groups of iron deficiency and anemia of chronic disease associated with absolute iron deficiency for any of the parameters. Conclusion: The erythrocyte and reticulocyte indices are moderately good to identify absolute iron deficiency in patients with anemia of chronic disease. Automation; Anemia; iron-deficiency Erythrocyte indices Reticulocytes Erythropoiesis.

Introduction New automated blood cell analyzers can provide information about individual cell characteristics, such as hemoglobin content of reticulocytes, hemoglobin content of mature erythrocytes, and percentages of microcytic erythrocytes and hypochromic cells.

Table 1 Demographic characteristics and hematological data of patients and the control group. Table 2 Biochemical data.

Table 3 Reticulocyte and red cell indices for patients and the control group. The role of iron status markers in predicting response to intravenous iron in haemodialysis patients on maintance erythropoietin. An exponential drop of reticulocytes was observed in cultures. High fluorescence reticulocytes disappeared completely in These times significantly decreased in red blood cell units stored for more time. The in vitro half-life at 0. The possible explanation is that blood bank storage does not cause irreversible damage to the human reticulocyte maturational machinery.

Maturation to discocytes depends on energy and occurs during progressive stages in which RNA content decreases, protein synthesis becomes complete and the plasma membrane and cytoskeleton become remodeled.

In vitro maturation of nascent reticulocytes to erythrocytes. The maturation and fate of reticulocytes after in vitro labeling with tritiated amino acids. Maturation and destruction of transfused human reticulocytes, evaluation of reticulocyte experiments for the measurement of hemoglobin metabolism. J Clin Investig. The maturation rate of reticulocytes. In vitro , under culture conditions, reticulocyte maturation is characterized mainly by: 1 decreasing number of RNA-containing RBCs total reticulocyte count ; 2 progressive decrease of corpuscular RNA content; and 3 reductions in cell size.

In vitro and in vivo persistence of reticulocytes from donor red cells. Reticulocytes can be classified according to RNA content into subtypes reflecting successive stages during maturation: high fluorescence reticulocytes LFR mature to medium fluorescence reticulocytes MFR and these to low fluorescence reticulocytes LFR. Ztschr Klin Med.

Flow cytometric reticulocyte maturity index: a useful laboratory parameter of erythropoietic activity in anemia. Human reticulocytes isolated from peripheral blood: maturation time and hemoglobin synthesis. Lab Hematol. Reticulocyte count in red-blood-cell units stored in AS Vox Sang. A flow cytometric method for phenotyping recipient red cells following transfusion. Re-establishing physiological conditions temperature and pH , as occurs during culture conditions, may reflect in the restoration of the kinetics of cell metabolism or not.

This study aimed to describe the influence of previous blood bank storage on the kinetics of reticulocyte disappearance during in vitro culturing. This study was approved by the institutional ethics committee and followed current protocols for research involving humans and the handling of biological samples Declaration of Helsinki.

The resulting buffy coat-depleted RBC units in AS-1 were stored in standard blood bank conditions for six weeks. RBC units entered the study 72—96 h 0. For the experiments, 10 mL aliquots of each unit were obtained after 0. Considering that the reticulocyte count in blood bank RBC units is lower than in peripheral blood and the number decreases further during refrigerated storage as we have previously reported, 9 9 Urbina A, Palomino F.

Thus, before inoculation in culture bottles, reticulocyte-enriched fractions were obtained from RBC unit aliquots using Percoll density gradient separation Sigma—Aldrich, St.

Continuous density gradients were prepared using Percoll density marker beads Sigma—Aldrich, St. Flow cytometry with thiazole orange Retic-Count, Becton Dickinson Company, San Jose, CA, USA was used to measure the total reticulocyte count and reticulocyte subtype concentrations before and after separation as described below.

The yield of the reticulocyte-enrichment procedure is presented in the Results Section. Culture bottles were inoculated with a reticulocyte-enriched concentrated aliquot from each RBC unit obtained at the different sampling times. The pH was 7. Total RBC concentration in the bottles was 3. After incubation at 1- to 4-h intervals, one bottle was taken from each set for reticulocyte analysis.

RBC counts were determined by impedance cytometry and total hemoglobin by the sodium lauryl sulphate method in an automated hematology analyzer Sysmex-R, Sysmex America Inc.

Percentage hemolysis was calculated as free hemoglobin percentage regarding total hemoglobin. Reticulocyte percentage was determined by flow cytometry using thiazole orange staining. Precision profile changes according to reticulocyte concentration had Determination coefficient R 2 test—retest was 0.

The performance of the technique was thus close to that of fully-automated, clinical hematology analyzers 7. Five fully automated for performing immature reticulocyte fraction: comparison in diagnosis of bone marrow aplasia. Am J Clin Pathol. Proposal for standardization of flow cytometric reticulocyte maturity index RMI measurements. Light scatter and fluorescence intensity were plotted to analyze reticulocyte subtype size and fluorescence changes during culturing.

Active caspases-8 and -3 in circulating human erythrocytes purified on immobilized annexin-V: a cytometric demonstration. For estimates of kinetics, reticulocyte counts expressed as percentage of total red cells were used, as were the absolute reticulocyte counts calculated from percentages and RBC counts.

Figure 1 Reticulocyte analysis by flow cytometry using thiazole orange of a red blood cell unit stored for 0. Fluorescence scatterplot from the red blood cell gate after excluding autofluorescent cells. FL1: fluorescence intensity; FSC: forward light scattering. The main mechanism for mitochondrial clearance is mitophagy, a selective type of autophagy that allows the degradation of damaged mitochondria.

The importance of this process is highlighted by knowing that an impairment in mitochondrial function triggers an increase in reactive oxygen species production, which can in turn cause damage to cellular components proteins, nucleic acid, and lipids and trigger cell death Lee et al. One of these allows the assembly of the phagophore and involves several autophagy-related proteins Atg , such as Atg5 and Atg7.

Atg4 and Atg7 cooperate to conjugate LC3 onto phosphatidylethanolamine in the lipid bilayer of the membrane originated from the ER-mitochondria contact site Tooze and Yoshimori, ; Hamasaki et al. The elongated phagophore is then recruited to engulf targets via adaptor proteins, containing an LC3-interacting region LIR that forms a double-membrane autophagosome, which will fuse with a lysosome, initiating the degradation of the autophagosome components. Upon mitochondria damage or depolarization, the mitochondrial membrane proteins are exposed and act as a beacon to recruit the phagophore membranes Liu et al.

Unlike regular mitophagy induction, targeted mitochondria, during erythroblast maturation, are fully functional. This protein is upregulated during erythropoiesis and induces mitochondrial membrane depolarization and membrane conjugated LC3 recruitment to the mitochondria Aerbajinai et al.

Nix action is not mediated by its BH3 domain but rather seems to be due to a cytoplasmic short linear motif, acting as a cellular signal to recruit other proteins Zhang et al. However, whether Nix-induced mitochondrial depolarization activates the Parkin-dependent pathway is still unknown Yuan et al. It remains unknown whether they play a role in erythroid maturation.

Canonical Atg proteins also participate in terminal maturation. In human erythropoiesis, LC3 cleavage is under the control of the endopeptidase Atg4 and is needed for autophagosome maturation Betin et al.

In mice, Ulk1 Atg1 expression correlates with terminal differentiation and participates in mitochondria and ribosome elimination Chan et al. The ubiquitination-dependent pathway also plays a role in reticulocyte maturation but is not essential. However, Nix and Ulk1 activation appears to be essential Mortensen et al.

Autophagosomes, formed in a Ulk1-dependent pathway, fuse with Golgi-derived vesicles and late endosomes in a Rab9a-dependent manner before they are targeted to the lysosomes Wang et al. Interestingly, Rab proteins were also recently shown to be involved in mitochondria removal in a complete autophagy-independent pathway.

Depolarized mitochondria appear to be engulfed in Rab5-positive endosomes that mature into Rab7-positive late endosomes and then fuse with lysosomes Hammerling et al. Unlike canonical autophagy, which involves the surrounding of a ubiquitin-decorated target by a double membrane structure, the entire mitochondria appears to be engulfed by an early endosome membrane invagination through the ESCRT machinery.

Whether this might also occur in maturing erythroblasts is not known. Mitophagy also appears to be transcriptionally regulated. Indeed, hemin-dependent differentiation of an erythroid cell line shows features of mitophagy Fader et al.

The NF-E2 transcription factor involved in globin gene expression also regulates mitophagy through the regulation of Nix and Ulk1 genes Gothwal et al. In parallel to the autophagic pathway, cytosolic degradation seems to occur during reticulocyte maturation.

This mechanism is still controversial, as LOX might also act in the autophagy pathway as an OMM pH gradient disruptor that can induce mitophagy Vijayvergiya et al. In general, autophagy plays an essential role in the elimination of other organelles, such as lysosomes, peroxisomes and ER. However, the literature presents only very few studies in erythroid cells Table 1. While Nix is required for mitochondria removal, Ulk1 is involved in ribosome and mitochondria degradation Schweers et al.

These data suggest that non-autophagic or Atg7-independent autophagic pathways might exist for the elimination of other organelles Figure 1A.

In non-erythroid cells from mammals, it was proposed that peroxisomes are eliminated by three different pathways: macroautophagy Iwata, , LOX mediated Yokota et al. Furthermore, the autophagic degradation of lysosomes lysophagy was recently identified in HeLa cells where it is mediated by ubiquitination and involves p62 protein Hung et al.

After enucleation, reticulocytes mature in the bone marrow R1 and then exit in the blood stream R2 to complete the process. While the degradation of organelles starts at the time of enucleation, the elimination of mRNA occurs in the blood stream and is mediated by ribonucleases, generating nucleotides that are degraded by the erythroid pyrimidine nucleotidase. This elimination is crucial, as the deficiency in this enzyme causes hemolytic anemia Valentine et al.

This supports the importance of the exosome pathway for the final maturation into RBCs with an active elimination of other subcellular components. Exosomes are small vesicles that are secreted into the extracellular medium from various kind of cells. PM invaginations form early endosomes that engulf various targets forming multivesicular bodies MVB, late endosomes that eventually fuse with the PM and release exosomes. In reticulocytes, this pathway is thought to be involved in cell volume and membrane remodeling to reduce volume and remove unwanted membrane proteins.

This was first discovered in sheep reticulocytes where transferrin receptor TfR is first internalized into small vesicles of — nm before being engulfed into the MVBs Pan et al. The internalization step is clathrin-dependent, and the degradation is lysosome-independent and occurs by exocytosis after the fusion of the MVBs with the PM as shown in Figure 1B Killisch et al.

This process is required for the final elimination of other membrane proteins that are essential for the reticulocyte but are absent in the mature cell. Proteins such as aquaporin-1 AQP1 Blanc et al. While plenty of evidence notes the role of autophagy in removing organelles during terminal maturation, the degradation step itself shows discrepancies with canonical proteolysis involving lysosomal proteins because of the disappearance of the lysosomal compartment during the maturation and removal of LAMP2 by exocytosis Barres et al.

Recently, GPA-positive endosomes were found to express LC3 at the endosome membrane, suggesting the cooperation of both autophagy and exocytosis in the removal of remnant organelles in R2 reticulocytes. These hybrid vesicles contain mitochondria, Golgi and lysosomes might be formed by the fusion of the outer-membrane of the autophagosome and the PM derived endosome Griffiths et al.

The exocytosis of this vesicle might be favored by the spleen, as splenectomized patients present large vacuoles inside reticulocytes Holroyde and Gardner, It should be pointed out the importance of lipids domain such as cholesterol and sphingomyelin-enriched domains in the PM remodeling, as they were find both in membrane vesiculation specific sites Leonard et al. Even if all the animal models used to identify the molecular players involved during terminal differentiation exhibit maturation defects and anemia, links between organelle clearance and human hematological diseases are still mostly unknown.

Impaired autophagy is involved in cytosolic toxic Lyn accumulation and mitochondria and lysosome degradation delay in chorea-acanthocytosis Lupo et al.

Moreover, anemia in Pearson's syndrome was recently linked to incomplete mitochondrial clearance from reticulocytes Palis, and an asynchronization of iron loading Ahlqvist et al. Unraveling the molecular mechanisms and interplays ruling erythroblast terminal maturation would be priceless in hematological disease therapy. Great care should be applied when interpreting results, considering the important differences between mouse and human erythropoiesis as well as the in vivo and in vitro environments, as highlighted in the extensive transcriptome analysis across a terminal erythroid differentiation study An et al.

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We thank I. Marginedas-Freixa and C. Hattab for helpful discussions. Adolfsson, J. Cell , — Aerbajinai, W.

The proapoptotic factor Nix is coexpressed with Bcl-xL during terminal erythroid differentiation. Blood , — Ahlqvist, K.

MtDNA mutagenesis impairs elimination of mitochondria during erythroid maturation leading to enhanced erythrocyte destruction. An, X. Global transcriptome analyses of human and murine terminal erythroid differentiation.

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