Pseudoreticulocytosis by the ADVIA 2120 Hematology Analyzer and Other Hematologic Changes in a Cynomolgus Macaque (Macaca fascicularis) With Malaria.

Important hematologic changes can be observed in nonhuman primates with malaria, including inaccurate reticulocyte counts by the ADVIA 2120 hematology analyzer. A 5-year-old male purpose-bred cynomolgus macaque (Macaca fascicularis) imported from a commercial source in Cambodia was enrolled in a nonclinical toxicity study investigating the effects of an immunomodulatory pharmaceutical agent. On study day 22, an increase in large unstained cells (LUCs), due to increased monocytes (2.20 × 103/µl, reference interval: 0.17-0.76 × 103/µl), was reported by the analyzer during a scheduled hematologic evaluation, which prompted blood smear review and revealed that the macaque had a high burden of Plasmodium spp.. The macaque did not have clinical signs for the infection at this time point. Progressively higher parasite burdens and persistently increased monocytes (markedly increased by study day 56, 10.38 × 103/µl) were observed at subsequent hematologic evaluations. New Methylene Blue stain manual reticulocyte counts were performed on study day 43 and at later time points, and showed that the analyzer reported erroneous higher reticulocyte counts (study day 43: +6.7%, +266.2 × 109/L; study day 50: +18.9%, +409.8 × 109/L) compared with the manual reticulocyte counts (pseudoreticulocytosis). The magnitude of regenerative response was considered inadequate for the severity of anemia at these time points. Atypical reticulocyte scatter plot distributions from the analyzer were also observed at time points with high parasite burdens, and combined with increased LUCs, may suggest high burden parasitemia. Verification of automated reticulocyte counts is important in cases with high malarial parasite burdens and the recognition of pseudoreticulocytosis is prudent in assessing appropriateness of the regenerative response. Increases in monocytes correlated with higher parasite burdens and marked increases may be an indicator of advanced disease.

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.

Cell Mechanics Based Computational Classification of Red Blood Cells Via Machine Intelligence Applied to Morpho-Rheological Markers.

Despite fluorescent cell-labelling being widely employed in biomedical studies, some of its drawbacks are inevitable, with unsuitable fluorescent probes or probes inducing a functional change being the main limitations. Consequently, the demand for and development of label-free methodologies to classify cells is strong and its impact on precision medicine is relevant. Towards this end, high-throughput techniques for cell mechanical phenotyping have been proposed to get a multidimensional biophysical characterization of single cells. With this motivation, our goal here is to investigate the extent to which an unsupervised machine learning methodology, which is applied exclusively on morpho-rheological markers obtained by real-time deformability and fluorescence cytometry (RT-FDC), can address the difficult task of providing label-free discrimination of reticulocytes from mature red blood cells. We focused on this problem, since the characterization of reticulocytes (their percentage and cellular features) in the blood is vital in multiple human disease conditions, especially bone-marrow disorders such as anemia and leukemia. Our approach reports promising label-free results in the classification of reticulocytes from mature red blood cells, and it represents a step forward in the development of high-throughput morpho-rheological-based methodologies for the computational categorization of single cells. Besides, our methodology can be an alternative but also a complementary method to integrate with existing cell-labelling techniques.

A low-cost, accurate method for detecting reticulocytes at different maturation stages based on changes in the mitochondrial membrane potential.

In the clinical setting, reticulocytes are used as an index for the hematopoietic function of the bone marrow. Different maturation stages of reticulocytes are early markers for bone marrow hematopoietic stem cell transplantation and bone marrow regeneration after chemotherapy. Therefore, we aimed to establish a method for detecting the different reticulocyte maturation stages. Based on the decreases in mitochondrial membrane potential during reticulocyte maturation, we used MitoTracker Green (MTG)/tetramethylrhodamine, ethylester (TMRE) to identify the different reticulocyte maturation stages and used Hoechst33342 to exclude nucleated cells. The results show that this method was universal and could be applied to detect the proportions of reticulocytes in different samples. Their proportion in normal peripheral blood, a blood deficiency model, bone marrow, and spleen were (6 ± 2)%, (38 ± 4)%, (14 ± 4)%, and (3 ± 1)%, respectively. The results obtained using this method were similar to those obtained using the manual counting method (methylene blue); the correlation was good (R = 0.817; p < .01) and the coefficient of variation was lower for the method established. Moreover, reticulocytes in peripheral blood could be further divided into three distinct maturation stages: R1 (MTGneg/TMREhigh), R2 (MTGhigh/TMREhigh), and R3 (MTGhigh/TMREneg). Reticulocytes in the bone marrow and spleen could be further divided into four distinct maturation stages: R1 (MTGneg/TMREhigh), R2-1 (MTGhigh/TMREhigh/FSbig), R2-2 (MTGhigh/TMREhigh/FSsmall), and R3 (MTGhigh/TMREneg). Based on changes in mitochondrial membrane potential, MTG/TMRE/Hoechst33342 staining could be used to detect reticulocytes in different samples and at different maturation stages with low cost and high accuracy.

Reticulocyte and red blood cell deformation triggers specific phosphorylation events.

The capacity to undergo substantial deformation is a defining characteristic of the red blood cell (RBC), facilitating transit through the splenic interendothelial slits and microvasculature. Establishment of this remarkable property occurs during a process of reticulocyte maturation that begins with egress through micron-wide pores in the bone marrow and is completed within the circulation. The requirement to undertake repeated cycles of deformation necessitates that both reticulocytes and erythrocytes regulate membrane-cytoskeletal protein interactions in order to maintain cellular stability. In the absence of transcriptional activity, modulation of these interactions in RBCs is likely to be achieved primarily through specific protein posttranslational modifications, which at present remain undefined. In this study, we use high-throughput methods to define the processes that underlie the response to deformation and shear stress in both reticulocytes and erythrocytes. Through combination of a bead-based microsphiltration assay with phosphoproteomics we describe posttranslational modification of RBC proteins associated with deformation. Using microsphiltration and microfluidic biochip-based assays, we explore the effect of inhibiting kinases identified using this dataset. We demonstrate roles for GSK3 and Lyn in capillary transit and maintenance of membrane stability following deformation and show that combined inhibition of these kinases significantly decreases reticulocyte capacity to undergo repeated deformation. Finally, we derive a comprehensive and integrative phosphoproteomic dataset that provides a valuable resource for further mechanistic dissection of the molecular pathways that underlie the RBC's response to mechanical stimuli and for the study of reticulocyte maturation.

Genetic manipulation of cell line derived reticulocytes enables dissection of host malaria invasion requirements.

Investigating the role that host erythrocyte proteins play in malaria infection is hampered by the genetic intractability of this anucleate cell. Here we report that reticulocytes derived through in vitro differentiation of an enucleation-competent immortalized erythroblast cell line (BEL-A) support both successful invasion and intracellular development of the malaria parasite Plasmodium falciparum. Using CRISPR-mediated gene knockout and subsequent complementation, we validate an essential role for the erythrocyte receptor basigin in P. falciparum invasion and demonstrate rescue of invasive susceptibility by receptor re-expression. Successful invasion of reticulocytes complemented with a truncated mutant excludes a functional role for the basigin cytoplasmic domain during invasion. Contrastingly, knockout of cyclophilin B, reported to participate in invasion and interact with basigin, did not impact invasive susceptibility of reticulocytes. These data establish the use of reticulocytes derived from immortalized erythroblasts as a powerful model system to explore hypotheses regarding host receptor requirements for P. falciparum invasion.

Protein Synthesis and Translational Control: A Historical Perspective.

Protein synthesis and its regulation are central to all known forms of life and impinge on biological arenas as varied as agriculture, biotechnology, and medicine. Otherwise known as translation and translational control, these processes have been investigated with increasing intensity since the middle of the 20th century, and in increasing depth with advances in molecular and cell biology. We review the origins of the field, focusing on the underlying concepts and early studies of the cellular machinery and mechanisms involved. We highlight key discoveries and events on a timeline, consider areas where current research has engendered new ideas, and conclude with some speculation on future directions for the field.

In-depth phenotypic characterization of reticulocyte maturation using mass cytometry.

Progress towards an in-depth understanding of the final steps of the erythroid lineage development is paramount for many hematological diseases. We have characterized the final stages of reticulocyte maturation from bone marrow to peripheral blood using for the first time single-cell Mass Cytometry (CyTOF). We were able to measure the expression of 31 surface markers within a single red blood cell (RBC). We demonstrate the validity of CyTOF for RBC phenotyping by confirming the progressive reduction of transferrin receptor 1 (CD71) during reticulocyte maturation to mature RBC. We highlight the high-dimensional nature of mass cytometry data by correlating the expression of multiple proteins on individual RBCs. We further describe a more drastic reduction pattern for a component of the alpha4/beta1 integrin CD49d at the very early steps of reticulocyte maturation in bone marrow and directly linked with the mitochondria remnants clearance pattern. The enhanced and accurate RBC phenotyping potential of CyTOF described herein could be beneficial to decipher RBC preferences, as well as still not well understood receptor-ligand interaction of some hemotropic parasites such as the malaria causing agent Plasmodium vivax.

Plasmodium vivax Infection Impairs Regulatory T-Cell Suppressive Function During Acute Malaria.

The balance between pro- and antiinflammatory mechanisms is essential to limit immune-mediated pathology, and CD4+ forkhead box P3 (Foxp3+) regulatory T cells (Treg) play an important role in this process. The expression of inhibitory receptors regulates cytokine production by Plasmodium vivax-specific T cells. Our goal was to assess the induction of programmed death-1 (PD-1) and cytotoxic T-lymphocyte antigen (CTLA-4) on Treg during malaria and to evaluate their function. We found that P. vivax infection triggered an increase in circulating Treg and their expression of CTLA-4 and PD-1. Functional analysis demonstrated that Treg from malaria patients had impaired suppressive ability and PD-1+Treg displayed lower levels of Foxp3 and Helios, but had higher frequencies of T-box transcription factor+ and interferon-gamma+ cells than PD-1-Treg. Thus malaria infection alters the function of circulating Treg by triggering increased expression of PD-1 on Treg that is associated with decreased regulatory function and increased proinflammatory characteristics.

A regenerative erythropoietic response does not increase the frequency of Pig-a mutant reticulocytes and erythrocytes in Sprague-Dawley rats.

The in vivo rodent Pig-a mutation assay is a sensitive test to identify exposure to mutagenic substances, and has been proposed as an assay for the identification of impurities for pharmaceuticals. Red blood cells (RBCs) and reticulocytes (RETs) are analyzed by flow cytometry after exposure to potentially mutagenic chemicals for cells deficient in the cell surface anchored protein CD59, representing mutation in the X-linked Pig-a gene. The full potential of the assay as well as its limitations are currently being explored. The current study investigated the effects of regenerative erythropoietic bone marrow responses on the frequency of Pig-a mutated reticulocytes (RETCD59- ) and erythrocytes (RBCCD59- ). We hypothesized that a robust regenerative erythropoietic response would not increase the basal frequency of RETCD59- or RBCCD59- cells. Two groups of six male Sprague-Dawley rats either had 2 mL of blood sampled each day via an indwelling catheter over a period of 5 days or were minimally sampled for hematology and used as controls. Blood was also then collected and evaluated 5, 18, and 49 days after the initial bleed period for the number of Pig-a mutant cells in either the RET or RBC population. Despite the expected decrease in hematocrit and the correlative increase in reticulocytes after bleeding, no increase in the number of Pig-a mutant cells was observed in male Sprague-Dawley rats that were bled for five consecutive days. These results indicate that changes in erythropoiesis and hematology parameters in rats appear to have no effect on the background levels of Pig-a mutated RETs and RBCs. Environ. Mol. Mutagen. 59:91-95, 2018. © 2017 Wiley Periodicals, Inc.

Diagnostic performances of manual and automated reticulocyte parameters in anaemic cats.

Objectives The objective of this study was to evaluate the diagnostic performances of manual and instrumental measurement of reticulocyte percentage (Ret%), reticulocyte number (Ret#) and reticulocyte production index (RPI) to differentiate regenerative anaemia (RA) from non-regenerative anaemia (NRA) in cats. Methods Data from 106 blood samples from anaemic cats with manual counts (n = 74; 68 NRA, six RA) or instrumental counts of reticulocytes (n = 32; 25 NRA, seven RA) collected between 1995 and 2013 were retrospectively analysed. Sensitivity, specificity and positive likelihood ratio (LR+) were calculated using either cut-offs reported in the literature or cut-offs determined from receiver operating characteristic (ROC) curves. Results All the reticulocyte parameters were significantly higher in cats with RA than in cats with NRA. All the ROC curves were significantly different ( P <0.001) from the line of no discrimination, without significant differences between the three parameters. Using the cut-offs published in literature, the Ret% (cut-off: 0.5%) was sensitive (100%) but not specific (<75%), the RPI (cut-off: 1.0) was specific (>92%) but not sensitive (<15%), and the Ret# (cut-off: 50 × 10³/µl) had a sensitivity and specificity >80% and the highest LR+ (manual count: 14; instrumental count: 6). For all the parameters, sensitivity and specificity approached 100% using the cut-offs determined by the ROC curves. These cut-offs were higher than those reported in the literature for Ret% (manual: 1.70%; instrumental: 3.06%), lower for RPI (manual: 0.39; instrumental: 0.59) and variably different, depending on the method (manual: 41 × 10³/µl; instrumental: 57 × 10³/µl), for Ret#. Using these cut-offs, the RPI had the highest LR+ (manual: 22.7; instrumental: 12.5). Conclusions and relevance This study indicated that all the reticulocyte parameters may confirm regeneration when the pretest probability is high, while when this probability is moderate, RA should be identified using the RPI providing that cut-offs <1.0 are used.

Antagonists of the system L neutral amino acid transporter (LAT) promote endothelial adhesivity of human red blood cells.

The system L neutral amino acid transporter (LAT; LAT1, LAT2, LAT3, or LAT4) has multiple functions in human biology, including the cellular import of S-nitrosothiols (SNOs), biologically active derivatives of nitric oxide (NO). SNO formation by haemoglobin within red blood cells (RBC) has been studied, but the conduit whereby a SNO leaves the RBC remains unidentified. Here we hypothesised that SNO export by RBCs may also depend on LAT activity, and investigated the role of RBC LAT in modulating SNO-sensitive RBC-endothelial cell (EC) adhesion. We used multiple pharmacologic inhibitors of LAT in vitro and in vivo to test the role of LAT in SNO export from RBCs and in thereby modulating RBC-EC adhesion. Inhibition of human RBC LAT by type-1-specific or nonspecific LAT antagonists increased RBC-endothelial adhesivity in vitro, and LAT inhibitors tended to increase post-transfusion RBC sequestration in the lung and decreased oxygenation in vivo. A LAT1-specific inhibitor attenuated SNO export from RBCs, and we demonstrated LAT1 in RBC membranes and LAT1 mRNA in reticulocytes. The proadhesive effects of inhibiting LAT1 could be overcome by supplemental L-CSNO (S-nitroso-L-cysteine), but not D-CSNO or L-Cys, and suggest a basal anti-adhesive role for stereospecific intercellular SNO transport. This study reveals for the first time a novel role of LAT1 in the export of SNOs from RBCs to prevent their adhesion to ECs. The findings have implications for the mechanisms of intercellular SNO signalling, and for thrombosis, sickle cell disease, and post-storage RBC transfusion, when RBC adhesivity is increased.

Nucleoside-Based Ultrasensitive Fluorescent Probe for the Dual-Mode Imaging of Microviscosity in Living Cells.

Microviscosity changes of living cells have a far-reaching influence on diffusion and movement capacity of RNA and, more seriously, could modify RNA functions in living cells. Fluorescent rotor, whose fluorescence responds to different environmental viscosities, holds great potential for the imaging of viscosity in biosystem. Although many fluorescent rotors have been reported for viscosity, the fluorogenic rotor with ultrasensitivity for the determination of microviscosity (<10 cP) was rarely reported. Herein, we report a nucleoside-based two-photon fluorescent rotor (dABp-3) that can selectively and ultrasensitively image microviscosity in RNA region of living cells for the first time. 2'-Deoxyadenosine is selected as an electron donor to permit energy transfer via the acetylenic bond to acceptor, a typical boron dipyrromethene moiety. Another highlight, dABp-3 is based on 2'-deoxyadenosine, which result in its recognition capacity for RNA. dABp-3 with ultrasensitivity provides a varied linear response to the microrange viscosity (1.8-6.0 cP) in RNA region of living cells on dual-mode-two-photon ratio mode and fluorescence lifetime mode. After screening and optimization, advantageously, dABp-3 can be used to screen reticulocytes from mature blood cells of thrombosis models in vitro and in vivo because of targeting RNA, while simultaneously image microviscosity changes in these cells. So, dABp-3 as an analytical tool holds considerable promise for bioimaging and monitoring of microviscosity changes in complex biological systems.

Monoclonal gammopathy-associated pure red cell aplasia.

Pure red cell aplasia (PRCA) is a rare disorder characterized by inhibition of erythroid precursors in the bone marrow and normochromic, normocytic anaemia with reticulocytopenia. Among 51 PRCA patients, we identified 12 (24%) patients having monoclonal gammopathy, monoclonal gammopathy of undetermined significance or smouldering multiple myeloma, with presence of monoclonal protein or abnormal serum free light chains and atypical bone marrow features of clonal plasmacytosis, hypercellularity and fibrosis. Thus far, three patients treated with anti-myeloma based therapeutics have responded with reticulocyte recovery and clinical transfusion independence, suggesting plasma cells play a key role in the pathogenesis of this specific monoclonal gammopathy-associated PRCA.

Hoxa10 null animals exhibit reduced platelet biogenesis.

The transcription factor HOXA10 is an important regulator of myelopoiesis. Engineered over-expression of Hoxa10 in mice results in a myeloproliferative disorder that progresses to acute myeloid leukaemia (AML) over time, and in humans over-expression is associated with poor outcomes in AML. Here, we report that loss of Hoxa10 expression in mice results in reduced platelet count and platelet production, but does not affect clotting efficiency. About 40% fewer platelets were found in Hoxa10 null animals in comparison to wild type littermates. We found a nearly 50% reduction in the percentage of reticulated platelets in Hoxa10 null mice, suggesting deficient platelet production. Furthermore, Hoxa10 null animals recovered less efficiently from induced thrombocytopenia, supporting our hypothesis of defective platelet production. This also correlated with reduced colony formation potential of stem and progenitor cells seeded in megakaryocyte-enhancing conditions in vitro. Together, our results indicate that HOXA10 is important for megakaryopoiesis and platelet biogenesis.

A Chemical Screening Approach to Identify Novel Key Mediators of Erythroid Enucleation.

Erythroid enucleation is critical for terminal differentiation of red blood cells, and involves extrusion of the nucleus by orthochromatic erythroblasts to produce reticulocytes. Due to the difficulty of synchronizing erythroblasts, the molecular mechanisms underlying the enucleation process remain poorly understood. To elucidate the cellular program governing enucleation, we utilized a novel chemical screening approach whereby orthochromatic cells primed for enucleation were enriched ex vivo and subjected to a functional drug screen using a 324 compound library consisting of structurally diverse, medicinally active and cell permeable drugs. Using this approach, we have confirmed the role of HDACs, proteasomal regulators and MAPK in erythroid enucleation and introduce a new role for Cyclin-dependent kinases, in particular CDK9, in this process. Importantly, we demonstrate that when coupled with imaging analysis, this approach provides a powerful means to identify and characterize rate limiting steps involved in the erythroid enucleation process.

Reticulocyte hemoglobin content does not differentiate true from functional iron deficiency in dogs.

BACKGROUND: True and functional iron deficiency can result in anemia. Current tests to assess iron status often do not allow differentiation between these entities, which can affect optimal treatment. Previous work suggested low reticulocyte hemoglobin content (CHr) may be an early indicator of iron deficiency. OBJECTIVE: This study aimed to correlate several inflammation markers with CHr values in dogs. We hypothesize that dogs with low CHr values have hematologic and biochemical evidence of inflammation. METHODS: Animals with CHr values below the reference interval were included in the low CHr group, while dogs with normal or increased CHr were included in the control group. HCT, MCV, CHr, reticulocyte mean cell volume (MCVr), concentrations of serum iron, C-reactive protein (CRP), ferritin, and ceruloplasmin, and total iron-binding capacity (TIBC), percent transferrin saturation (% sat), and total WBC, neutrophil, and monocyte counts were determined. Nonparametric tests were performed; median values and percentage of abnormalities between each group were compared. RESULTS: Relative to control dogs, animals in the low CHr group had higher median values for CRP, ferritin, ceruloplasmin, and WBC concentration (P ≤ .05), and lower median values for HCT and MCV (P ≤ .0001). Higher frequencies of abnormalities for CRP, ferritin, WBC, neutrophil, and monocyte concentrations (P ≤ .02) were present in the low CHr group. CONCLUSIONS: Dogs with low CHr values often have evidence of inflammation, but low CHr did not reliably predict Fe deficiency. Additional diagnostic tests are needed to differentiate true and functional iron deficiency.

Mean reticulocyte volume enhances the utility of red cell mean sphered cell volume in differentiating peripheral blood spherocytes of hereditary spherocytosis from other causes.

CONTEXT: Mean sphered cell volume (MSCV) and mean reticulocyte volume (MRV) are additional reticulocyte parameters generated while processing the blood samples on Beckman coulter LH 755 in the reticulocyte mode using the volume, conductivity and scatter technology. It has been observed that the difference between mean corpuscular volume (MCV) and MSCV is higher in the cases of hereditary spherocytosis (HS) and this difference is increasingly being utilized as a screening tool for spherocytes. In addition now there have been new observations that reticulocyte volume in cases of HS is less as compared to normal reticulocyte. AIMS: Our aim was to test the usefulness of reticulocyte parameters like MSCV and MRV in distinguishing cases of HS and autoimmune hemolytic anemia (AIHA). MATERIALS AND METHODS: This is a retrospective and partly prospective study where peripheral blood ethylenediaminetetraacetic acid samples from cases of HS (n = 57) and AIHA (n = 29) were processed on LH 755 in both the differential and the reticulocyte mode. The data generated were analyzed and compared with data from normal healthy donors (n = 46). RESULTS: Using an algorithm of MCV - MSCV >10 and MRV - MSCV <25, a sensitivity of 84.2% and specificity of 94.7% was observed in cases of HS. CONCLUSIONS: With the reticulocyte analysis, we may now have a simple and cheap additional tool for screening of HS.

Bone marrow reticulocytes: a Plasmodium vivax affair?

In this issue of Blood, Malleret and colleagues show the importance of the bone marrow in Plasmodium vivax biology by proving the preferential infection of young reticulocytes (generally restricted to the bone marrow), which then experience accelerated maturation postinvasion.