Interplay between liver and blood stages of Plasmodium infection dictates malaria severity via γδ T cells and IL-17-promoted stress erythropoiesis.

Plasmodium replicates within the liver prior to reaching the bloodstream and infecting red blood cells. Because clinical manifestations of malaria only arise during the blood stage of infection, a perception exists that liver infection does not impact disease pathology. By developing a murine model where the liver and blood stages of infection are uncoupled, we showed that the integration of signals from both stages dictated mortality outcomes. This dichotomy relied on liver stage-dependent activation of Vγ4+ γδ T cells. Subsequent blood stage parasite loads dictated their cytokine profiles, where low parasite loads preferentially expanded IL-17-producing γδ T cells. IL-17 drove extra-medullary erythropoiesis and concomitant reticulocytosis, which protected mice from lethal experimental cerebral malaria (ECM). Adoptive transfer of erythroid precursors could rescue mice from ECM. Modeling of γδ T cell dynamics suggests that this protective mechanism may be key for the establishment of naturally acquired malaria immunity among frequently exposed individuals.

Functional and immunological mapping of domains of the reticulocyte binding protein, Plasmodium vivax PvRBP2a.

We previously described a novel Plasmodium vivax invasion mechanism into human reticulocytes via the PvRBP2a-CD98 receptor-ligand pair. We assessed the PvRBP2a epitopes involved in CD98 binding and recognised by antibodies from infected patients using linear epitope mapping. We identified two epitope clusters mediating PvRBP2a-CD98 interaction. One cluster named cluster B (PvRBP2a431-448, TAALKEKGKLLANLYNKL) was the target of antibody responses in P. vivax-infected humans. Peptides from each cluster were able to prevent live parasite invasion of human reticulocytes. These results provide new insights for development of a malaria blood stage vaccine against P. vivax.

A comparative study of two routinely used protocols for ex vivo erythroid differentiation.

BACKGROUND: Erythropoiesis is a complex developmental process in which a hematopoietic stem cell undergoes serial divisions and differentiates through well-defined stages to give rise to red blood cells. Over the last decades, several protocols have been developed to perform ex vivo erythroid differentiation, allowing investigation into erythropoiesis and red cell production in health and disease. RESULTS: In the current study, we compared the two commonly used protocols by assessing the differentiation kinetics, synchronisation, and cellular yield, using molecular and cellular approaches. Peripheral blood CD34+ cells were cultured in a two-phase (2P) or a four-phase (4P) liquid culture (LC) and monitored for 20 days. Both protocols could recapitulate all stages of erythropoiesis and generate reticulocytes, although to different extents. Higher proliferation and viability rates were achieved in the 4P-LC, with a higher degree of terminal differentiation and enucleation, associated with higher levels of the erythroid-specific transcription factors GATA-1, KLF-1, and TAL-1. Although the 2P-LC protocol was less efficient regarding terminal erythroid differentiation and maturation, it showed a higher yield of erythroid progenitors in the erythropoietin (EPO)-free expansion phase. CONCLUSIONS: We provide data supporting the use of one protocol or the other to study the biological processes occurring in the early or late stages of erythroid differentiation, depending on the physiological process or pathological defect under investigation in a given study.

The Gárdos Channel and Piezo1 Revisited: Comparison between Reticulocytes and Mature Red Blood Cells.

The Gárdos channel (KCNN4) and Piezo1 are the best-known ion channels in the red blood cell (RBC) membrane. Nevertheless, the quantitative electrophysiological behavior of RBCs and its heterogeneity are still not completely understood. Here, we use state-of-the-art biochemical methods to probe for the abundance of the channels in RBCs. Furthermore, we utilize automated patch clamp, based on planar chips, to compare the activity of the two channels in reticulocytes and mature RBCs. In addition to this characterization, we performed membrane potential measurements to demonstrate the effect of channel activity and interplay on the RBC properties. Both the Gárdos channel and Piezo1, albeit their average copy number of activatable channels per cell is in the single-digit range, can be detected through transcriptome analysis of reticulocytes. Proteomics analysis of reticulocytes and mature RBCs could only detect Piezo1 but not the Gárdos channel. Furthermore, they can be reliably measured in the whole-cell configuration of the patch clamp method. While for the Gárdos channel, the activity in terms of ion currents is higher in reticulocytes compared to mature RBCs, for Piezo1, the tendency is the opposite. While the interplay between Piezo1 and Gárdos channel cannot be followed using the patch clamp measurements, it could be proved based on membrane potential measurements in populations of intact RBCs. We discuss the Gárdos channel and Piezo1 abundance, interdependencies and interactions in the context of their proposed physiological and pathophysiological functions, which are the passing of small constrictions, e.g., in the spleen, and their active participation in blood clot formation and thrombosis.

Accessory-cell-free differentiation of hematopoietic stem and progenitor cells into mature red blood cells.

BACKGROUND AIMS: The culture and ex vivo engineering of red blood cells (RBCs) can help characterize genetic variants, model diseases, and may eventually spur the development of applications in transfusion medicine. In the last decade, improvements to the in vitro production of RBCs have enabled efficient erythroid progenitor proliferation and high enucleation levels from several sources of hematopoietic stem and progenitor cells (HSPCs). Despite these advances, there remains a need for refining the terminal step of in vitro human erythropoiesis, i.e., the terminal maturation of reticulocytes into erythrocytes, so that it can occur without feeder or accessory cells and animal-derived components. METHODS: Here, we describe the near-complete erythroid differentiation of cultured RBCs (cRBCs) from adult HSPCs in accessory-cell-free and xeno-free conditions. RESULTS: The approach improves post-enucleation cell integrity and cell survival, and it enables subsequent storage of cRBCs for up to 42 days in classical additive solution conditions without any specialized equipment. CONCLUSIONS: We foresee that these improvements will facilitate the characterization of RBCs derived from gene-edited HSPCs.

Genetic diversity and natural selection of Plasmodium vivax reticulocyte invasion genes in Ecuador.

BACKGROUND: Knowledge of the diversity of invasion ligands in malaria parasites in endemic regions is essential to understand how natural selection influences genetic diversity of these ligands and their feasibility as possible targets for future vaccine development. In this study the diversity of four genes for merozoite invasion ligands was studied in Ecuadorian isolates of Plasmodium vivax. METHODS: Eighty-eight samples from P. vivax infected individuals from the Coast and Amazon region of Ecuador were obtained between 2012 and 2015. The merozoite invasion genes pvmsp-1-19, pvdbpII, pvrbp1a-2 and pvama1 were amplified, sequenced, and compared to the Sal-1 strain. Polymorphisms were mapped and genetic relationships between haplotypes were determined. RESULTS: Only one nonsynonymous polymorphism was detected in pvmsp-1-19, while 44 nonsynonymous polymorphisms were detected in pvdbpII, 56 in pvrbp1a-2 and 33 in pvama1. While haplotypes appeared to be more related within each area of study and there was less relationship between parasites of the coastal and Amazon regions of the country, diversification processes were observed in the two Amazon regions. The highest haplotypic diversity for most genes occurred in the East Amazon of the country. The high diversity observed in Ecuadorian samples is closer to Brazilian and Venezuelan isolates, but lower than reported in other endemic regions. In addition, departure from neutrality was observed in Ecuadorian pvama1. Polymorphisms for pvdbpII and pvama1 were associated to B-cell epitopes. CONCLUSIONS: pvdbpII and pvama1 genetic diversity found in Ecuadorian P. vivax was very similar to that encountered in other malaria endemic countries with varying transmission levels and segregated by geographic region. The highest diversity of P. vivax invasion genes in Ecuador was found in the Amazonian region. Although selection appeared to have small effect on pvdbpII and pvrbp1a-2, pvama1 was influenced by significant balancing selection.

Associations of obesity and body shape with erythrocyte and reticulocyte parameters in the UK Biobank cohort.

BACKGROUND: Obesity is associated with type 2 diabetes mellitus and chronic low-grade inflammation. Although chronic inflammatory conditions and diabetes are associated with anaemia, less is known about associations of obesity and body shape, independent of each other, with erythrocyte and reticulocyte parameters. METHODS: We investigated the associations of body mass index (BMI) and the allometric body shape index (ABSI) and hip index (HI), which are uncorrelated with BMI, with erythrocyte and reticulocyte parameters (all continuous, on a standard deviation (SD) scale) in UK Biobank participants without known metabolic, endocrine, or major inflammatory conditions (glycated haemoglobin HbA1c < 48 mmol/mol, C-reactive protein CRP < 10 mg/L). We examined erythrocyte count, total reticulocyte count and percent, immature reticulocyte count and fraction (IRF), haemoglobin, haematocrit, mean corpuscular haemoglobin mass (MCH) and concentration (MCHC), mean corpuscular and reticulocyte volumes (MCV, MRV), and red cell distribution width (RDW) in multivariable linear regression models. We additionally defined body shape phenotypes with dichotomised ABSI (≥ 73 women; ≥ 80 men) and HI (≥ 64 women; ≥ 49 men), including "pear" (small-ABSI-large-HI) and "apple" (large-ABSI-small-HI), and examined these in groups according to BMI (18.5-25 normal weight; 25-30 overweight; 30-45 kg/m2 obese). RESULTS: In 105,853 women and 100,854 men, BMI and ABSI were associated positively with haemoglobin, haematocrit, and erythrocyte count, and more strongly with total reticulocyte count and percent, immature reticulocyte count and IRF. HI was associated inversely with all, but least with IRF. Associations were comparable in women and men. In groups according to obesity and body shape, erythrocyte count was ~ 0.6 SD higher for obese-"apple" compared to normal-weight-"pear" phenotype (SD = 0.31*1012/L women, SD = 0.34*1012/L men), total reticulocyte count was ~ 1.1 SD higher (SD = 21.1*109/L women, SD = 23.6*109/L men), immature reticulocyte count was ~ 1.2 SD higher (SD = 7.9*109/L women, SD = 8.8*109/L men), total reticulocyte percent was ~ 1.0 SD higher (SD = 0.48% women and men), and IFR was over 0.7 SD higher (SD = 5.7% women and men). BMI but not ABSI or HI was associated more weakly inversely with MCV, MRV, and MCH, but positively with MCHC in men and RDW in women. CONCLUSIONS: In obesity uncomplicated with diabetes, larger BMI and ABSI are associated with increased erythropoiesis and reticulocyte immaturity.

Immunological impact of the CD71+ RBCs: A potential immune mediator in transfusion.

Donor - recipient sex - mismatched transfusion is associated with increased mortality. The mechanisms for this are not clear, but it may relate to transfusion-related immunomodulation. Recently, CD71+ erythroid cells (CECs), including reticulocytes (CD71+ RBCs) and erythroblasts, have been identified as potent immunoregulatory cells. The proportion of CD71+ RBCs in the peripheral blood is sufficient to play a potential immunomodulatory role. Differences in the quantity of CD71+ RBCs are dependent on blood donor sex. The total number of CD71+ RBCs in red cell concentrates is also affected by blood manufacturing methods, and storage duration. As a component of the total CECs, CD71+ RBCs can affect innate and adaptive immune cells. Phagocytosed CECs directly reduce TNF-α production from macrophages. CECs can also suppress the production of TNF-α production from antigen presenting cells. Moreover, CECs can suppress T cell proliferation thorough immune mediation and / or direct cell-to-cell interactions. Different in their biophysical features compared to mature RBCs, blood donor CD71+ RBCs may be preferential targets for the macrophages. This report summarizes the currently literature supporting an important role for CD71+ RBCs in adverse transfusion reactions including immune mediation and sepsis.

Cultured CD71+ erythroid cells modulate the host immune response.

OBJECTIVE: The study aimed to determine the impact of Red Blood Cells (RBCs) generated from peripheral blood mononuclear cells (PBMCs) on T cell proliferation and host response following whole blood stimulation. BACKGROUND: Culturing RBCs is a potential solution for donor shortage. The impact of immature cultured RBCs which express CD71+ on host immune response is not known. METHODS/MATERIALS: PBMCs were seeded in an erythroid expansion medium. CD71+ cells were isolated at days 14 and 21 of culture and incubated with either purified T cells or with LPS-stimulated whole blood. Controls were incubated with medium. RESULTS: At day 9, the percentage of cells that expressed CD45 and CD71 reached to the highest level (32.9%, IQR; 26.2-39.05) while the percentage of cells that expressed CD71 and CD235a reached to the highest level on day 17 (70.2%, IQR; 66.1-72.8). Incubation of T cells with days 14 CD71+ cells and day 21 CD71+ cells increased T cell proliferation. In a whole blood stimulation assay, day 21 CD71+ cells, but not day 14 CD71+ cells, inhibited the production of IL-6 and TNFα. CONCLUSION: Cultured erythroid cells can modulate the immune response by promoting T cell proliferation and inhibiting cytokine secretions following whole blood stimulation.

Varying aerobic capacity in relation to breeding stage and reproductive success in giant petrels (Macronectes spp.).

Reproduction, and parental care in particular, are among the most energy-demanding activities within the annual cycle of adult birds. Parents that cannot meet the metabolic demands and other physiological costs of raising offspring may opt to abandon chicks in favour of self-maintenance and future reproduction. Recent work examining reproductive trade-offs in birds revealed an important role of oxygen carrying capacity in mediating variation in parental effort. This study explores the aerobic factors underlying the success or failure of parental care in two closely-related petrel species during their breeding season on Bird Island, South Georgia: northern giant petrels (Macronectes halli) and southern giant petrels (M. giganteus). Failed breeders of both sexes and species had significantly lower hematocrit levels (by 5.48 ± 0.64%) than successful breeders, and reticulocyte counts also tended to be lower in failed males, consistent with the hypothesis that parental care and workload depend on aerobic capacity. We discuss these results in relation to differences in the foraging ecology of both species and sexes.

Assessment of iron metabolism disorders and adequate treatment of anemic syndrome in patients with breast cancer on the background of adjuvant chemotherapy.

Early and adequate correction of the anemic syndrome (AS) of cancer patients can prevent deterioration in the quality of life and be considered as a reserve for increasing the effectiveness of treatment for breast cancer (BC). The aim of the study was to assess the status of iron using modern methods of ferrokinetics in breast cancer patients on the background of adjuvant chemotherapy for early diagnosis and adequate treatment of AS. The object of the study included 21 breast cancer patients with a relatively favorable prognosis, with luminal types A and B (Her 2 / neu positive or negative), three times negative type. The examination was carried out in the postoperative period, against the background of adjuvant chemotherapy. The main metabolites of ferrokinetics were studied: hepcidin 25 (GP25); ferritin (FR); soluble transferrin receptors (rRTP); transferin (TRF); iron (Fe); erythropoietin (EPO); CRP and IL-6 indicators. AC correction was performed (ferinject, epotin-alpha, B12). 10 (47.6%) patients with breast cancer had AS. Most of them were diagnosed with IDA with microcytic, hypochromic characteristics of erythrocytes, low concentration of FR, Fe, GP25, IL-6, CRP, and high levels of TRP and rRTP. Functional iron deficiency (FDF) was established in some patients. In contrast to patients with IDA, they had a high concentration of FR, CRP and significant production of GP25, IL-6. The EPO level was not optimal for the majority of patients with AS. In isolated cases, during treatment with recombinant erythropoietins, a deficiency of vitamin B12 (cyanocobalamin) was revealed. The rational use of iron preparations, vitamins, and recombinant forms of EPO made it possible to restore Fe metabolism, stabilize the hemoglobin level, and also improve the condition of most breast cancer patients. The obtained data on IL-6, GP25, CRP indicate a certain relationship between them in the development of anemia with VDF in breast cancer patients and the need for further study of the characteristics of iron metabolism in cancer patients.

Measurement of Immature Reticulocytes in Dried Blood Spots by Mass Spectrometry.

BACKGROUND: Immature reticulocytes (IRC) are the first cells to respond to changes in erythropoiesis. For antidoping applications, measurement of IRC may improve detection of blood doping practices. Unfortunately, this small cell population has limited stability in liquid blood samples and is difficult to measure with optimal precision. We developed a method to measure 3 IRC membrane proteins in dried blood spots (DBS) to monitor changes in erythropoiesis. METHODS: DBS spots were washed with buffers to remove soluble proteins, membrane proteins remaining in the spot were digested with trypsin, and one peptide for each protein was measured by LC-MS/MS. IRC protein concentration was determined using a DBS single point calibrator. RESULTS: Intraassay precision for IRC proteins was between 5%-15%. IRC proteins were stable in DBS for 29 days at room temperature. In a longitudinal study of 25 volunteers, the mean intraindividual variation for 3 IRC proteins was 17%, 20%, and 24% from capillary blood DBS. In comparison, the mean longitudinal variation for IRC counts measured on an automated hematology analyzer was 38%. IRC protein concentration from capillary blood DBS correlated well with venous blood DBS protein concentrations. CONCLUSIONS: Measurement of IRC proteins in DBS samples provides a method to measure changes in erythropoiesis with improved analytical sensitivity, stability, and precision. When combined with the inherent advantages of capillary blood collection in the field, this method may substantially improve the detection of blood doping practices.

Systemic deficiency of mouse arachidonate 15-lipoxygenase induces defective erythropoiesis and transgenic expression of the human enzyme rescues this phenotype.

Arachidonic acid 15-lipoxygenases (ALOX15) are lipid peroxidizing enzymes, which has previously been implicated in the maturational breakdown of intracellular organelles and plasma membrane remodeling during reticulocyte-erythrocyte transition. Conventional Alox15-/- mice are viable, develop normally but do not exhibit a major defective erythropoietic phenotype. To characterize the putative in vivo relevance of Alox15 for red blood cell development, we explored the impact of systemic inactivation of the Alox15 gene on mouse erythropoiesis. We found that Alox15-/- mice exhibited reduced erythrocyte counts, elevated reticulocyte counts and red cell hyperchromia. The structure of the plasma membrane of Alox15-/- erythrocytes is altered and a significant share of the red cells was present as echinocytes and/or acanthocytes. An increased share of the Alox15-/- erythrocytes cells were annexin V positive, which indicates a loss of plasma membrane asymmetry. Erythrocytes of Alox15-/- mice were more susceptible to osmotic hemolysis and exhibited a reduced ex vivo life span. When we transgenically expressed human ALOX15 in Alox15-/- mice under the control of the aP2 promoter the defective erythropoietic system was rescued and the impaired osmotic resistance was normalized. Together these data suggest the involvement Alox15 in the maturational remodeling of the plasma membrane during red cell development.

Dynamics of Deformation Properties of Erythrocytes in Wistar Rats during Postnatal Ontogeny.

We performed a detailed analysis of changes in the profiles of osmotic deformability using the method of gradient ektacytometry. Changes in all determinants that form the deformation properties of red blood cells in Wistar rats in the juvenile period and before puberty were determined. The dynamics of the formation of the rheological properties of the blood after birth is characterized by a wave-like change in the studied determinants. The changes are explained by adaptive reactions to extrauterine life as a result of hematopoiesis activation and the transition of the red bone marrow to a new level of functioning with the predominant replacement of physiological reticulocytosis in newborns with mature erythrocytes. The most critical period is from 10 days to 1 month after birth. Starting from the second month, the deformation parameters of erythrocytes are stabilized.

Features of modern clinical research blood analysis in the assessment of erythropoiesis in deep-premature newborns.

One of the urgent problems of nursing premature babies is the timely prediction, diagnosis and treatment of anemia, the frequency of which reaches 90%. Of particular importance is the study of reticulocytic blood parameters in deep-premature newborns, since the correct assessment of hemograms is crucial in the management of this category of children. To determine the characteristics of red blood cells and reticulocyte parameters of venous blood hemogram in premature newborns 24-32 weeks of gestation in the dynamics of the neonatal period. 111 newborns were examined at 24-32 weeks of gestation in the early neonatal period (on day 3-7) and at the age of 1 month of life. Along with standard diagnostic procedures, in accordance with current clinical recommendations and standards, 28 parameters of erythrocyte and reticulocyte hemogram parameters were determined for children. Venous blood was examined using an automatic hematological analyzer ADVIA 2120i, Siemens, USA. In deep-premature newborns in the early neonatal period, there is a high activity of erythropoiesis, respectively, the severity of respiratory and metabolic disorders with rejuvenation of reticulocytic subpopulations. Negative values of Delta hemoglobin were found against the background of a decrease in the average amount of hemoglobin in reticulocytes in children 24-27 weeks of gestation, which characterizes the lowest values of iron availability for erythropoiesis in this category of newborns. It is shown that a decrease in reticulocyte counts and normochromia in all examined newborns by the age of one month are accompanied by high levels of immature reticulocyte fraction, while a third of children still have limited iron availability for erythropoiesis.

Suppression of compensatory erythropoiesis in hemolytic disease of the fetus and newborn due to intrauterine transfusions.

BACKGROUND: Infants with severe hemolytic disease of the fetus and newborn often require 1 or multiple intrauterine transfusions to treat fetal anemia. Intrauterine transfusions may have an inhibiting effect on fetal and neonatal erythropoiesis. OBJECTIVE: To quantify the effect of 1 or multiple intrauterine transfusions on the fetal erythropoiesis by assessing the fetal reticulocyte counts in a population with severe hemolytic disease of the fetus and newborn. STUDY DESIGN: This was an observational cohort study in infants admitted to the Leiden University Medical Center who received 1 or multiple intrauterine transfusions for hemolytic disease of the fetus and newborn caused by (Rh)D or Kell antibodies and were born between January 2005 and December 2018. RESULTS: A total of 235 patients were included, of whom 189 were patients with D-mediated hemolytic disease of the fetus and newborn and 46 with Kell-mediated hemolytic disease of the fetus and newborn. Absolute fetal reticulocyte count in D-mediated hemolytic disease of the fetus and newborn declined exponentially over the course of consecutive intrauterine transfusions, with a 62% decline after 1 intrauterine transfusion (95% confidence interval, 56-67). A similar exponential decline was observed in Kell-mediated hemolytic disease of the fetus and newborn, with 32% (95% confidence interval, 19-45) decline after 1 intrauterine transfusion. This decline was not associated with the varying gestational age at the time of the first intrauterine transfusion or the total number of intrauterine transfusions. The number of red blood cell transfusions for postnatal anemia was greater for infants with D and Kell-mediated hemolytic disease of the fetus and newborn with >2 intrauterine transfusions (median of 3 [interquartile range, 2-3] vs 2 [interquartile range, 1-3], P=.035, in D-mediated disease and median of 2 [interquartile range, 1-2] vs 1 [interquartile range, 1-1], P<.001, in Kell-mediated disease). Infants born after >2 intrauterine transfusions less often required exchange transfusion in D-mediated hemolytic disease of the fetus and newborn (19/89 [21%] vs 31/100 [31%], P=.039), compared with infants with 1-2 intrauterine transfusions. CONCLUSION: Treatment with intrauterine transfusions causes an exponential decrease in fetal reticulocyte counts in both D- and Kell-mediated hemolytic disease of the fetus and newborn. Suppression of the compensatory erythropoiesis leads to prolonged postnatal anemia and an increased requirement of red blood cell transfusions after birth.

Receptor-ligand and parasite protein-protein interactions in Plasmodium vivax: Analysing rhoptry neck proteins 2 and 4.

Elucidating receptor-ligand and protein-protein interactions represents an attractive alternative for designing effective Plasmodium vivax control methods. This article describes the ability of P. vivax rhoptry neck proteins 2 and 4 (RON2 and RON4) to bind to human reticulocytes. Biochemical and cellular studies have shown that two PvRON2- and PvRON4-derived conserved regions specifically interact with protein receptors on reticulocytes marked by the CD71 surface transferrin receptor. Mapping each protein fragment's binding region led to defining the specific participation of two 20 amino acid-long regions selectively competing for PvRON2 and PvRON4 binding to reticulocytes. Binary interactions between PvRON2 (ligand) and other parasite proteins, such as PvRON4, PvRON5, and apical membrane antigen 1 (AMA1), were evaluated and characterised by surface plasmon resonance. The results revealed that both PvRON2 cysteine-rich regions strongly interact with PvAMA1 Domains II and III (equilibrium constants in the nanomolar range) and at a lower extent with the complete PvAMA1 ectodomain and Domains I and II. These results strongly support that these proteins participate in P. vivax's complex invasion process, thus providing new pertinent targets for blocking P. vivax merozoites' specific entry to their target cells.

Measurement of iron status in chronic kidney disease.

Anemia is a common complication of chronic kidney disease (CKD) in children, and dysregulation of iron homeostasis plays a central role in its pathogenesis. Optimizing iron status is a prerequisite for effective treatment of anemia. Insufficient iron can lead to inappropriate escalation of the erythropoiesis-stimulating agent (ESA) dose, which is associated with adverse outcomes. Excess iron supplementation also has negative sequelae including free radical tissue damage and increased risk of systemic infection. Notwithstanding the importance of optimizing bioavailable iron for erythropoiesis for children with advanced CKD, achieving this remains challenging for pediatric nephrologists due to the historical lack of practical and robust measures of iron status. In recent years, novel techniques have come to the fore to facilitate accurate and practical assessment of iron balance. These measures are the focus of this review, with emphasis on their relevance to the pediatric CKD population.

Assessment of iron-restricted erythropoiesis in chronic renal disease: evaluation of Abbott CELL-DYN Sapphire mean reticulocyte hemoglobin content (MCHr).

We studied the performance of mean reticulocyte hemoglobin content (MCHr), measured on Abbott CELL-DYN Sapphire analyzer for the detection of functional iron deficiency. Patients with anemia and with renal disease were prospectively selected from the outpatient population of our hospital, 512 subjects were studied. Diagnoses and other medical data were retrieved from the hospital information system. MCHr was measured using a CELL-DYN Sapphire hematology analyzer (Abbott Diagnostics). Standard laboratory and statistical tests were used. Receiver operating characteristic (ROC) analysis was used to establish the diagnostic performance of MCHr for detecting iron-restricted erythropoiesis, with transferrin saturation <20% as the gold standard. Patients in the iron deficiency anemia group all had a state of iron depletion and iron-restricted erythropoiesis (median MCHr 25.4 pg). Patients with anemia of chronic disease showed also low MCHr, median 27.6 pg, but not statistically different from the iron deficient group (p = .0585). Renal patients with iron restriction (n = 66) had significantly lower MCHr (p < .0001) than those receiving adequate iron supply (n = 100): median MCHr were 27.9 and 32.5 pg, respectively. ROC analysis gave sensitivity 84.4% and specificity 80.1% with area under curve 0.863 (95% CI 0.823-0.902) at an MCHr cut-off 30.0 pg. MCHr on CELL-DYN Sapphire has equivalent clinical performance for detecting absolute or functional iron deficiency in patients with chronic kidney disease as previously published for another type of analyzer.

Quantitative mass spectrometry of human reticulocytes reveal proteome-wide modifications during maturation.

Erythropoiesis is marked by progressive changes in morphological, biochemical and mechanical properties of erythroid precursors to generate red blood cells (RBC). The earliest enucleated forms derived in this process, known as reticulocytes, are multi-lobular and spherical. As reticulocytes mature, they undergo a series of dynamic cytoskeletal re-arrangements and the expulsion of residual organelles, resulting in highly deformable biconcave RBCs (normocytes). To understand the significant, yet neglected proteome-wide changes associated with reticulocyte maturation, we undertook a quantitative proteomics approach. Immature reticulocytes (marked by the presence of surface transferrin receptor, CD71) and mature RBCs (devoid of CD71) were isolated from human cord blood using a magnetic separation procedure. After sub-fractionation into triton-extracted membrane proteins and luminal samples (isobaric tags for relative and absolute quantitation), quantitative mass spectrometry was conducted to identify more than 1800 proteins with good confidence and coverage. While most structural proteins (such as Spectrins, Ankyrin and Band 3) as well as surface glycoproteins were conserved, proteins associated with microtubule structures, such as Talin-1/2 and ß-Tubulin, were detected only in immature reticulocytes. Atomic force microscopy (AFM)-based imaging revealed an extended network of spectrin filaments in reticulocytes (with an average length of 48 nm), which shortened during reticulocyte maturation (average spectrin length of 41 nm in normocytes). The extended nature of cytoskeletal network may partly account for increased deformability and shape changes, as reticulocytes transform to normocytes.