Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.

In the 1980s, exosomes were described as vesicles of endosomal origin secreted from reticulocytes. Interest increased around these extracellular vesicles, as they appeared to participate in several cellular processes. Exosomes bear proteins, lipids, and RNAs, mediating intercellular communication between different cell types in the body, and thus affecting normal and pathological conditions. Only recently, scientists acknowledged the difficulty of separating exosomes from other types of extracellular vesicles, which precludes a clear attribution of a particular function to the different types of secreted vesicles. To shed light into this complex but expanding field of science, this review focuses on the definition of exosomes and other secreted extracellular vesicles. Their biogenesis, their secretion, and their subsequent fate are discussed, as their functions rely on these important processes.

The unravelling of the ubiquitin system.

Today, many scientific discoveries are made using a top-down experimental approach. The ubiquitin system was discovered using a 'classic' bottom-up approach to tackle the question: 'how are cellular proteins selectively degraded?' A simple proteolytic assay, which used a crude cell-extract, was all that was required to address this question; it was followed by fractionation and reconstitution experiments to decipher the role of the components in this multi-step process. This 'biochemistry at its best' approach, which was published in a periodical that today would not be regarded as highly visible, provided magnificent findings.

The direct binding of Plasmodium vivax AMA1 to erythrocytes defines a RON2-independent invasion pathway.

We used a transgenic parasite in which Plasmodium falciparum parasites were genetically modified to express Plasmodium vivax apical membrane antigen 1 (PvAMA1) protein in place of PfAMA1 to study PvAMA1-mediated invasion. In P. falciparum, AMA1 interaction with rhoptry neck protein 2 (RON2) is known to be crucial for invasion, and PfRON2 peptides (PfRON2p) blocked the invasion of PfAMA1 wild-type parasites. However, PfRON2p has no effect on the invasion of transgenic parasites expressing PvAMA1 indicating that PfRON2 had no role in the invasion of PvAMA1 transgenic parasites. Interestingly, PvRON2p blocked the invasion of PvAMA1 transgenic parasites in a dose-dependent manner. We found that recombinant PvAMA1 domains 1 and 2 (rPvAMA1) bound to reticulocytes and normocytes indicating that PvAMA1 directly interacts with erythrocytes during the invasion, and invasion blocking of PvRON2p may result from it interfering with PvAMA1 binding to erythrocytes. It was previously shown that the peptide containing Loop1a of PvAMA1 (PvAMA1 Loop1a) is also bound to reticulocytes. We found that the Loop1a peptide blocked the binding of PvAMA1 to erythrocytes. PvAMA1 Loop1a has no polymorphisms in contrast to other PvAMA1 loops and may be an attractive vaccine target. We thus present the evidence that PvAMA1 binds to erythrocytes in addition to interacting with PvRON2 suggesting that the P. vivax merozoites may exploit complex pathways during the invasion process.

The Plasmodium vivax MSP1P-19 is involved in binding of reticulocytes through interactions with the membrane proteins band3 and CD71.

The parasite Plasmodium vivax preferentially invades human reticulocytes. Its merozoite surface protein 1 paralog (PvMSP1P), particularly the 19-kDa C-terminal region (PvMSP1P-19), has been shown to bind to reticulocytes, and this binding can be inhibited by antisera obtained by PvMSP1P-19 immunization. The molecular mechanism of interactions between PvMSP1P-19 and reticulocytes during P. vivax invasion, however, remains unclear. In this study, we analyzed the ability of MSP1P-19 to bind to different concentrations of reticulocytes and confirmed its reticulocyte preference. LC-MS analysis was used to identify two potential reticulocyte receptors, band3 and CD71, that interact with MSP1P-19. Both PvMSP1P-19 and its sister taxon Plasmodium cynomolgi MSP1P-19 were found to bind to the extracellular loop (loop 5) of band3, where the interaction of MSP1P-19 with band3 was chymotrypsin sensitive. Antibodies against band3-P5, CD71, and MSP1P-19 reduced the binding activity of PvMSP1P-19 and Plasmodium cynomolgi MSP1P-19 to reticulocytes, while MSP1P-19 proteins inhibited Plasmodium falciparum invasion in vitro in a concentration-dependent manner. To sum up, identification and characterization of the reticulocyte receptor is important for understanding the binding of reticulocytes by MSP1P-19.

Functional and Immunologic 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. Using linear epitope mapping, we assessed the PvRBP2a epitopes involved in CD98 binding and recognized by antibodies from patients who were infected. We identified 2 epitope clusters mediating PvRBP2a-CD98 interaction. Cluster B (PvRBP2a431-448, TAALKEKGKLLANLYNKL) was the target of antibody responses in humans infected by P vivax. 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.

Characterization of immortalized bone marrow erythroid progenitor adult (imBMEP-A)-The first inducible immortalized red blood cell progenitor cell line derived from bone marrow CD71-positive cells.

BACKGROUND AIMS: Ex vivo production of red blood cells (RBCs) represents a promising alternative for transfusion medicine. Several strategies have been described to generate erythroid cell lines from different sources, including embryonic, induced pluripotent, and hematopoietic stem cells. All these approaches have in common that they require elaborate differentiation cultures whereas the yield of enucleated RBCs is inefficient. METHODS: We generated a human immortalized adult erythroid progenitor cell line derived from bone marrow CD71-positive erythroid progenitor cells (immortalized bone marrow erythroid progenitor adult, or imBMEP-A) by an inducible expression system, to shorten differentiation culture necessary for terminal erythroid differentiation. It is the first erythroid cell line that is generated from direct reticulocyte progenitors and demonstrates robust hemoglobin production in the immortalized state. RESULTS: Morphologic analysis of the immortalized cells showed that the preferred cell type of the imBMEP-A line corresponds to hemoglobin-producing basophilic erythroblasts. In addition, we were able to generate a stable cell line from a single cell clone with the triple knockout of RhAG, RhDCE and KELL. After removal of doxycycline, part of the cells differentiated into normoblasts and reticulocytes within 5-7 days. CONCLUSIONS: Our results demonstrate that the imBMEP-A cell line can serve as a stable and straightforward modifiable platform for RBC engineering in the future.

Cryo-EM structure of an essential Plasmodium vivax invasion complex.

Plasmodium vivax is the most widely distributed malaria parasite that infects humans1. P. vivax invades reticulocytes exclusively, and successful entry depends on specific interactions between the P. vivax reticulocyte-binding protein 2b (PvRBP2b) and transferrin receptor 1 (TfR1)2. TfR1-deficient erythroid cells are refractory to invasion by P. vivax, and anti-PvRBP2b monoclonal antibodies inhibit reticulocyte binding and block P. vivax invasion in field isolates2. Here we report a high-resolution cryo-electron microscopy structure of a ternary complex of PvRBP2b bound to human TfR1 and transferrin, at 3.7 Å resolution. Mutational analyses show that PvRBP2b residues involved in complex formation are conserved; this suggests that antigens could be designed that act across P. vivax strains. Functional analyses of TfR1 highlight how P. vivax hijacks TfR1, an essential housekeeping protein, by binding to sites that govern host specificity, without affecting its cellular function of transporting iron. Crystal and solution structures of PvRBP2b in complex with antibody fragments characterize the inhibitory epitopes. Our results establish a structural framework for understanding how P. vivax reticulocyte-binding protein engages its receptor and the molecular mechanism of inhibitory monoclonal antibodies, providing important information for the design of novel vaccine candidates.

Assessment of myeloid response and iron status among Sudanese pediatric ESKD on hemodialysis through reticulocyte parameters and ß-globin mRNA expression.

BACKGROUND: Sudanese children with End-Stage Kidney Disease (ESKD) often show limited improvement in hemoglobin levels despite treatment with recombinant human erythropoietin (rHuEPO). This study aims to assess the response to rHuEPO therapy by analyzing ß-globin mRNA expression and reticulocyte parameters. Additionally, it classifies anemia among Sudanese pediatric patients based on iron status, considering age and gender as biological markers for evaluating treatment response. METHODS: A prospective observational cohort study was conducted from January 2019 to February 2020 in Khartoum, Sudan, involving 45 anemic children aged 2 to 15 years diagnosed with ESKD. The treatment protocol included rHuEPO injections and maintenance hemodialysis. Laboratory assessments consisted of complete blood count (CBC), absolute reticulocyte count, ferritin, and transferrin measurements. ß-globin mRNA expression was quantified using reverse transcription polymerase chain reaction (RT-PCR), and reticulocyte parameters, including Reticulocyte Hemoglobin Content (CHr), percentage of hypochromic reticulocytes (HYPO%), and Immature Reticulocyte Fraction (IRF), were measured via flow cytometry. RESULTS: Significant variations in hemoglobin levels were observed across different age groups (p = 0.011). Gender analysis revealed a significant association with IRF, showing a lower IRF in male patients (p = 0.017). However, there were no significant differences in hemoglobin levels between genders (p = 0.999). ß-globin mRNA expression showed considerable variability, with a strong positive correlation with hemoglobin levels (r = 0.875, p < 0.0001). CONCLUSION: Age and gender significantly influence treatment responses in children with ESKD, highlighting the need to consider growth physiology in anemia management. This study underscores the variability in ß-globin mRNA expression and its association with Flow Cytometry parameters, demonstrating their effectiveness in evaluating iron status and guiding rHuEPO dosage.

The role of reticulocyte hemoglobin equivalent on the evaluation of iron deficiency and iron deficiency anemia in pediatric cyanotic heart disease: a diagnostic study in Indonesia.

BACKGROUND: In light of prolonged hypoxia, children with cyanotic heart disase (CHD) are at a high risk of developing iron deficiency iron deficiency (ID) and iron deficiency anemia (IDA). Reticulocyte hemoglobin equivalent (Ret-He) is a novel and dependable indicator for assessing iron status. However, there has been no previous study regarding cut-off value in pediatric CHD group. The purpose of this study is to assess the role of Ret-He and to establish cut-off points in the diagnosis of iron deficiency and IDA in pediatric cyanotic heart disease. METHOD: This study was conducted in two tertiary hospitals in Jakarta, Indonesia. 59 children with CHD, aged 3 months to 18 years, were enrolled consecutively. To determine iron status, hematological parameters (hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin) and biochemical parameters for iron status (serum ferritin, transferrin saturation) were analysed and compared to Ret-He levels. The receiver operating characteristic (ROC) analysis was performed for the Ret-He cut-off points for ID and IDA. Sensitivity, specificity, positive and negative predictive value were calculated for each cut-off point. RESULT: Normal iron status was identified in 27 (45.8%) subjects, ID in 8 (13.5%) subjects, and IDA 24 (40.7%) subjects. The ID cut-off value for Ret-He is 28.8 pg (sensitivity 75%, specificity 85.2%, PPV 60%, NPV 92%, and AUC 0.828) and the Ret-He cut-off point for IDA is 28.15 pg (sensitivity 75%, specificity 88.9%, PPV 85.7%, NPV 80%, and AUC 0.824). Hemoglobin should be used in conjunction with Ret-He. ID might be detected in this cohort with Ret-He 28.8 pg and hemoglobin > 16,5 g/dL. While Ret-He 28.15 pg or Ret-He 28.15-28.8 pg with hemoglobin 16.5 g/dL could be used to diagnose IDA. CONCLUSION: The reticulocyte hemolgobin equivalent could be utilised as an iron status parameter in pediatric CHD, with a cut-off value of 28.8 pg for ID and 28.15 pg for IDA.

Genotoxic mode of action and threshold exploration of 2-methyl furan under 120-day sub-chronic exposure in male Sprague-Dawley rats.

2-Methylfuran (2-MF) is an important member of the furan family generated during food thermal processing. An in-vivo multiple endpoint genotoxicity assessment system was applied to explore the genotoxic mode of action and threshold of 2-MF. Male Sprague-Dawley rats received 2-MF by oral gavage at doses of 0.16, 0.625, 2.5, and 10 mg/kg.bw/day for 120 days. An additional 15 days were granted for recovery. The Pig-a gene mutation frequency of RET and RBC showed significant increases among the 2-MF groups on day 120. After a 15-day recovery period, the Pig-a gene mutation frequency returned to levels similar to those in the vehicle control. The tail intensity (TI) values of peripheral blood cells at a dose of 10 mg/kg.bw/day significantly increased from day 4 and remained at a high level after the recovery period. No statistical difference was found in the micronucleus frequency of peripheral blood between any 2-MF dose group and the corn oil group at any timepoint. 2-MF may not induce the production of micronuclei, but it could cause DNA breakage. It could not be ruled out that 2-MF may accumulate in vivo and cause gene mutations. Hence, DNA, other than the spindle, may be directly targeted. The mode of action of 2-MF may be that it was metabolized by EPHX1 to more DNA-active metabolites, thus leading to oxidative and direct DNA damage. The point of departure (PoD) of 2-MF-induced genotoxicity was derived as 0.506 mg/kg bw/day.

Resolution of RHCE Haplotype Ambiguities in Transfusion Settings.

Red blood cell (RBC) transfusion, limited by patient alloimmunization, demands accurate blood group typing. The Rh system requires specific attention due to the limitations of serological phenotyping methods. Although these have been compensated for by molecular biology solutions, some RhCE ambiguities remain unresolved. The RHCE mRNA length is compatible with full-length analysis and haplotype discrimination, but the RHCE mRNA analyses reported so far are based on reticulocyte isolation and molecular biology protocols that are fastidious to implement in a routine context. We aim to present the most efficient reticulocyte isolation method, combined with an RT-PCR sequencing protocol that embraces the phasing of all haplotype configurations and identification of any allele. Two protocols were tested for reticulocyte isolation based either on their size/density properties or on their specific antigenicity. We show that the reticulocyte sorting method by antigen specificity from EDTA blood samples collected up to 48 h before processing is the most efficient and that the combination of an RHCE-specific RT-PCR followed by RHCE allele-specific sequencing enables analysis of cDNA RHCE haplotypes. All samples analyzed show full concordance between RHCE phenotype and haplotype sequencing. Two samples from the immunohematology laboratory with ambiguous results were successfully analyzed and resolved, one of them displaying a novel RHCE allele (RHCE*03 c.340C>T).

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.

Single-cell profiling of ineffective erythropoiesis in a mouse model of ß-thalassaemia intermedia.

Beta-thalassaemia is an inherited haemoglobin disorder characterised by ineffective erythropoiesis (IE). The detailed pathogenesis of IE remains unclear. In this study, we used single-cell RNA sequencing (scRNA-seq) to examine IE in Th3/+ ß-thalassaemic mice. The results showed that the erythroid group was remarkably expanded, and genes involved in biological processes such as iron metabolism, haeme synthesis, protein folding, and response to heat were significantly upregulated from erythroid progenitors to reticulocytes in ß-thalassaemic mice. In particular, we identified a unique cell population close to reticulocytes, named ThReticulocytes, characterised by a high level of heat shock protein 70 (Hsp70) expression and dysregulation of iron metabolism and haeme synthesis signalling. Treatment of ß-thalassaemic mice with the haeme oxygenase inhibitor tin-mesoporphyrin effectively improved the iron disorder and IE, and the ThReticulocyte population and Hsp70 expression were significantly suppressed. This study revealed in detail the progression of IE at the single-cell level and possibly provided clues to find therapeutic targets in thalassaemia.

Membrane skeleton modulates erythroid proteome remodeling and organelle clearance.

The final stages of mammalian erythropoiesis involve enucleation, membrane and proteome remodeling, and organelle clearance. Concomitantly, the erythroid membrane skeleton establishes a unique pseudohexagonal spectrin meshwork that is connected to the membrane through junctional complexes. The mechanism and signaling pathways involved in the coordination of these processes are unclear. The results of our study revealed an unexpected role of the membrane skeleton in the modulation of proteome remodeling and organelle clearance during the final stages of erythropoiesis. We found that diaphanous-related formin mDia2 is a master regulator of the integrity of the membrane skeleton through polymerization of actin protofilament in the junctional complex. The mDia2-deficient terminal erythroid cell contained a disorganized and rigid membrane skeleton that was ineffective in detaching the extruded nucleus. In addition, the disrupted skeleton failed to activate the endosomal sorting complex required for transport-III (ESCRT-III) complex, which led to a global defect in proteome remodeling, endolysosomal trafficking, and autophagic organelle clearance. Chmp5, a component of the ESCRT-III complex, is regulated by mDia2-dependent activation of the serum response factor and is essential for membrane remodeling and autophagosome-lysosome fusion. Mice with loss of Chmp5 in hematopoietic cells in vivo resembled the phenotypes in mDia2-knockout mice. Furthermore, overexpression of Chmp5 in mDia2-deficient hematopoietic stem and progenitor cells significantly restored terminal erythropoiesis in vivo. These findings reveal a formin-regulated signaling pathway that connects the membrane skeleton to proteome remodeling, enucleation, and organelle clearance during terminal erythropoiesis.

Reticulocyte Hemoglobin Equivalent has Comparable Predictive Accuracy as Conventional Serum Iron Indices for Predicting Iron Deficiency and Anemia in a Nonhuman Primate model of Infantile Iron Deficiency.

BACKGROUND: Infantile iron deficiency (ID) causes anemia and compromises neurodevelopment. Current screening relies on hemoglobin (Hgb) determination at 1 year of age, which lacks sensitivity and specificity for timely detection of infantile ID. Low reticulocyte Hgb equivalent (RET-He) indicates ID, but its predictive accuracy relative to conventional serum iron indices is unknown. OBJECTIVES: The objective was to compare diagnostic accuracies of iron indices, red blood cell (RBC) indices, and RET-He for predicting the risk of ID and IDA in a nonhuman primate model of infantile ID. METHODS: Serum iron, total iron binding capacity, unsaturated iron binding capacity, transferrin saturation (TSAT), Hgb, RET-He, and other RBC indices were determined at 2 wk and 2, 4, and 6 mo in breastfed male and female rhesus infants (N = 54). The diagnostic accuracies of RET-He, iron, and RBC indices for predicting the development of ID (TSAT < 20%) and IDA (Hgb < 10 g/dL + TSAT < 20%) were determined using t tests, area under the receiver operating characteristic curve (AUC) analysis, and multiple regression models. RESULTS: Twenty-three (42.6%) infants developed ID and 16 (29.6%) progressed to IDA. All 4 iron indices and RET-He, but not Hgb or RBC indices, predicted future risk of ID and IDA (P < 0.001). The predictive accuracy of RET-He (AUC = 0.78, SE = 0.07; P = 0.003) for IDA was comparable to that of the iron indices (AUC = 0.77-0.83, SE = 0.07; P ≤ 0.002). A RET-He threshold of 25.5 pg strongly correlated with TSAT < 20% and correctly predicted IDA in 10 of 16 infants (sensitivity: 62.5%) and falsely predicted possibility of IDA in only 4 of 38 unaffected infants (specificity: 89.5%). CONCLUSIONS: RET-He is a biomarker of impending ID/IDA in rhesus infants and can be used as a hematological parameter to screen for infantile ID.

Translation initiation on mammalian mRNAs with structured 5'UTRs requires DExH-box protein DHX29.

Eukaryotic protein synthesis begins with assembly of 48S initiation complexes at the initiation codon of mRNA, which requires at least seven initiation factors (eIFs). First, 43S preinitiation complexes comprising 40S ribosomal subunits, eIFs 3, 2, 1, and 1A, and tRNA(Met)(i) attach to the 5'-proximal region of mRNA and then scan along the 5' untranslated region (5'UTR) to the initiation codon. Attachment of 43S complexes is mediated by three other eIFs, 4F, 4A, and 4B, which cooperatively unwind the cap-proximal region of mRNA and later also assist 43S complexes during scanning. We now report that these seven eIFs are not sufficient for efficient 48S complex formation on mRNAs with highly structured 5'UTRs, and that this process requires the DExH-box protein DHX29. DHX29 binds 40S subunits and hydrolyzes ATP, GTP, UTP, and CTP. NTP hydrolysis by DHX29 is strongly stimulated by 43S complexes and is required for DHX29's activity in promoting 48S complex formation.

eIF3j facilitates loading of release factors into the ribosome.

eIF3j is one of the eukaryotic translation factors originally reported as the labile subunit of the eukaryotic translation initiation factor eIF3. The yeast homolog of this protein, Hcr1, has been implicated in stringent AUG recognition as well as in controlling translation termination and stop codon readthrough. Using a reconstituted mammalian in vitro translation system, we showed that the human protein eIF3j is also important for translation termination. We showed that eIF3j stimulates peptidyl-tRNA hydrolysis induced by a complex of eukaryotic release factors, eRF1-eRF3. Moreover, in combination with the initiation factor eIF3, which also stimulates peptide release, eIF3j activity in translation termination increases. We found that eIF3j interacts with the pre-termination ribosomal complex, and eRF3 destabilises this interaction. In the solution, these proteins bind to each other and to other participants of translation termination, eRF1 and PABP, in the presence of GTP. Using a toe-printing assay, we determined the stage at which eIF3j functions - binding of release factors to the A-site of the ribosome before GTP hydrolysis. Based on these data, we assumed that human eIF3j is involved in the regulation of translation termination by loading release factors into the ribosome.

Purinergic signaling is essential for full Psickle activation by hypoxia and by normoxic acid pH in mature human sickle red cells and in vitro-differentiated cultured human sickle reticulocytes.

Paracrine ATP release by erythrocytes has been shown to regulate endothelial cell function via purinergic signaling, and this erythoid-endothelial signaling network is pathologically dysregulated in sickle cell disease. We tested the role of extracellular ATP-mediated purinergic signaling in the activation of Psickle, the mechanosensitive Ca2+-permeable cation channel of human sickle erythrocytes (SS RBC). Psickle activation increases intracellular [Ca2+] to stimulate activity of the RBC Gardos channel, KCNN4/KCa3.1, leading to cell shrinkage and accelerated deoxygenation-activated sickling.We found that hypoxic activation of Psickle recorded by cell-attached patch clamp in SS RBC is inhibited by extracellular apyrase, which hydrolyzes extracellular ATP. Hypoxic activation of Psickle was also inhibited by the pannexin-1 inhibitor, probenecid, and by the P2 antagonist, suramin. A Psickle-like activity was also activated in normoxic SS RBC (but not in control red cells) by bath pH 6.0. Acid-activated Psickle-like activity was similarly blocked by apyrase, probenecid, and suramin, as well as by the Psickle inhibitor, Grammastola spatulata mechanotoxin-4 (GsMTx-4).In vitro-differentiated cultured human sickle reticulocytes (SS cRBC), but not control cultured reticulocytes, also exhibited hypoxia-activated Psickle activity that was abrogated by GsMTx-4. Psickle-like activity in SS cRBC was similarly elicited by normoxic exposure to acid pH, and this acid-stimulated activity was nearly completely blocked by apyrase, probenecid, and suramin, as well as by GsMTx-4.Thus, hypoxia-activated and normoxic acid-activated cation channel activities are expressed in both SS RBC and SS cRBC, and both types of activation appear to be mediated or greatly amplified by autocrine or paracrine purinergic signaling.

Screening for hereditary spherocytosis in daily practice: what is the best algorithm using erythrocyte and reticulocyte parameters?

Hereditary spherocytosis (HS) is the most common inherited chronic haemolytic anaemia in Northern Europe. During the last decade, additional erythrocyte and reticulocyte parameters have been developed on last-generation haematology analysers, leading to many publications about their effectiveness as a HS screening tool. For the first time on an independent cohort, we evaluated and compared the effectiveness of six published algorithms for the screening of HS using the UniCel DxH800 (Beckman-Coulter) and the XN-9000 (Sysmex) and determined which algorithm could be the most suitable in our daily clinical practice. A total of 95 EDTA samples were analysed prospectively on both haematology analysers. These included 11 confirmed HS patients and 84 non-HS patients. The specific reticulocyte parameters used on the DxH800 were mean reticulocyte volume, immature reticulocyte fraction and mean sphered cell volume, and on the XN-9000 were hypohaemoglobinised erythrocytes, microcytic erythrocytes and immature reticulocyte fraction. The three algorithms using parameters specific to Beckman-Coulter analysers provided a sensitivity of 100% with various specificities, ranging from 7.1 to 73.8%. The three algorithms published based on the parameters specific to Sysmex showed much lower performances, i.e. out of the 11 patients with HS, between one to five patients were screened as negative for HS. However, 100% sensitivity and specificity were reached using the EMA binding test concomitantly with those three algorithms. The algorithms using reticulocyte and erythrocyte parameters offered by the recent analysers are promising options as a HS first-tier screening tool. Nevertheless, they must be evaluated by each laboratory on their own analyser before implementation.

Utility of reticulocyte haemoglobin content and immature reticulocyte fraction in early diagnosis of latent iron deficiency in whole blood donors.

BACKGROUND AND OBJECTIVES: The present study was planned to assess the clinical utility of reticulocyte haemoglobin content (CHr) and immature reticulocyte fraction (IRF) in the early detection of latent iron deficiency in blood donors. MATERIALS AND METHODS: The prospective longitudinal observational study was conducted using the purposive sampling method. Written informed consent was obtained and donors were allocated into the first-time (FTD) and regular donor (RD) group. The enrolled blood donors (n = 205 in each group) were followed up for two subsequent whole blood donations. Haemoglobin (Hb), CHr, IRF and serum ferritin values were recorded at enrolment and two follow-ups. RESULTS: The sensitivity of CHr in detecting iron-deficient erythropoiesis (serum ferritin values ≤ 26 µg/dl) was 45% and 56.7%, specificity 96.7%, positive predictive value (PPV) 85.6% and 90.8% and negative predictive value (NPV) 80.1% and 78.7%, respectively in FTD and RD cohorts. The sensitivity of IRF was 45.1% and 44.8%, specificity 93.4% and 97.1%, PPV 74.8% and 90.4% and NPV 79.6% and 74.5%, respectively in both the cohorts. The sensitivity of CHr in detecting absent iron stores (serum ferritin values ≤ 15 µg/dl) was 66.2% and 74.4%, specificity 92% and 90.6%, PPV 56.7% and 68.7% and NPV 94.5% and 92.8% among FTD and RD cohort, respectively. The sensitivity of IRF was 72.7% and 65.3%, specificity 90.3% and 94.3%, PPV 54.4% and 76% and NPV 95.4% and 90.8%, respectively in both the cohorts. CONCLUSION: Reticulocyte hemoglobin content and IRF can be used along with complete blood count for early detection of iron deficiency in blood donors using the same blood sample at no extra cost.