The effect of near-infrared low-level light on the in vitro quality of platelets during storage.

BACKGROUND AND OBJECTIVES: Near-infrared (NIR) light has been successfully applied to improve the quality of mouse platelets during storage. Because it is suspected that the mitochondria contain the primary photon acceptor, we hypothesized that human platelets for transfusion may be affected similarly and could benefit from NIR light treatment. MATERIALS AND METHODS: The optimal light dose was determined using portions of platelet concentrates (PCs) in PAS-E. A pool-and-split design was used to prepare PCs in PAS-E or plasma (n = 6). On day 1, one unit of both pairs was illuminated with 830 nm light (light-emitting diodes, 15 J/cm2). PCs were stored at 22°C and sampled regularly for analysis. Data were compared with their corresponding controls with a paired two-sided t-test. RESULTS: Illuminated platelets in PAS-E were less activated with significantly lower CD62P expression (day 8: 10.8 ± 1.8 vs. 12.2 ± 2.6, p < 0.05) and lower Annexin A5 binding (day 8: 11.8 ± 1.9 vs. 13.1 ± 2.4, ns). They produced significantly less lactate resulting in a higher pH (days 6-10). ATP content and mitochondrial membrane potential were not affected. Although these trends were also observed for PCs in plasma, the differences did not reach statistical significance as compared with the control group. CONCLUSION: Our study demonstrates that the glycolysis rate of human platelets can be modulated through the use of NIR, possibly through mitochondrial aerobic metabolism, but this requires confirmation. If NIR illumination can be further optimized, it may potentially become a useful tool in situations in which glycolysis and platelet activation are exacerbated.

Why might cord blood be a better source of platelets for transfusion to neonates?

Thrombocytopenia (defined as a platelet count <150×109/L) is a common condition in preterm neonates and may occur in 18-35% of all infants admitted to the Neonatal Intensive Care Unit (NICU). Neonatal platelet functionality in terms of reactivity is often described as reduced compared to adults, even in healthy, term neonates. However, this platelet "hyporeactivity" does not correspond to a global functional impairment of the normal delicately balanced neonatal hemostatic system. The extent to which neonatal thrombocytopenia and platelet hyporeactivity contribute to the bleeding risk in preterm neonates remains unknown. Prophylactic platelet transfusions are often administered to them to reduce the risk of bleeding. However, recent literature indicates that adopting a higher platelet transfusion threshold than a lower one results in significantly higher death rates or major bleeding and can be harmful. Although the mechanism by which this occurs is not entirely clear, a mismatch between adult transfused platelets and the neonatal hemostatic system, as well as volume overload, are speculated to be potentially involved. Therefore, future research should consider novel transfusion products that may be more suitable for premature neonates. Blood products derived from umbilical cord blood (UCB) are promising, as they might perfectly match neonatal blood features. Here, we discuss the current knowledge about UCB-derived products, focusing on UCB-derived platelet concentrates and their potential for future clinical application. We will discuss how they may overcome the potential risks of transfusing adult-derived platelets to premature infants while maintaining efficacy.

Red blood cells as oxygen carrier during normothermic machine perfusion of kidney grafts: Friend or foe?

Renal ex vivo normothermic machine perfusion (NMP) is under development as an assessment tool for high-risk kidney grafts and as a means of achieving more physiologically accurate organ preservation. On-going hemolysis has been reported during NMP, as this technique relies on red blood cells for oxygen delivery. In this study, we confirm the occurrence of progressive hemolysis during 6-hour kidney NMP. NMP-associated erythrostasis in the glomeruli and in peri-glomerular vascular networks points to an interaction between the red blood cells and the graft. Continuous hemolysis resulted in prooxidative changes in the perfusate, which could be quenched by addition of fresh frozen plasma. In a cell-based system, this hemolysis induced redox stress and exhibited toxic effects at high concentrations. These findings highlight the need for a more refined oxygen carrier in the context of renal NMP.

Efficacy and safety of platelet additive solution-E stored platelet concentrates.

INTRODUCTION: In 2018, platelet (PLT) additive solution-E (PAS-E) was introduced. The implementation of PAS-E was expected to diminish the number of allergic reactions in recipients following a PLT transfusion. Here, we evaluated the efficacy and safety of transfusions with PLTs stored in PAS-E. STUDY DESIGN AND METHODS: After implementation of PAS-E, data were collected from 2 cohorts of patients with hematological disorders as well as oncology patients, receiving PLTs in PAS-E. A similar patient group in a recent RCT, receiving PLTs in plasma, was used as a historical control group for both cohorts. Endpoints were corrected count increments (CCIs), bleeding scores (only reported in cohort 1), and the incidence of adverse reactions. RESULTS: In cohort 1, the mean 1-h CCI was 14.3 ± 6.9, and the 24-h CCI was 8.7 ± 5.6. In cohort 2, the 1-h CCI was 11.6 ± 7.8 and the 24-h CCI was 7.0 ± 6.1. In the control group, the 1-h CCI was 15.4 ± 5.5 and 24-h CCI 8.7 ± 4.8. Bleeding complications of WHO grade ≥2 occurred in 40% of patients in cohort 1 compared to 44% in plasma PCs. The incidence of adverse reactions was 1.2% in the two PAS-E cohorts, compared to 3.0% in plasma PCs. National hemovigilance data showed a significant reduction in allergic reactions with PAS-E PC transfusions as compared to plasma PCs with an odds ratio of 0.46 (CI 95% 0.37-0.58). CONCLUSION: The CCIs of PLTs in PAS-E were decreased compared to plasma PCs, but clinically acceptable. Allergic transfusion reactions were decreased in PAS-E PCs compared to plasma PCs.

Allogeneic Serum Eye Drops: A Randomized Clinical Trial to Evaluate the Clinical Effectiveness of Two Drop Sizes.

INTRODUCTION: Allogeneic serum from blood donors is starting to be used to treat patients with dry eye disease (DED). However, the optimal dose is not known. We therefore aimed to evaluate the clinical efficaciousness and user-friendliness of micro-sized versus conventional-sized allogeneic serum eye drops (SEDs). METHODS: In a randomized trial, patients with DED first receive micro-sized SEDs (7 µl/unit) for 1 month, followed by a 1-month washout, before receiving conventional-sized SEDs (50 µl/unit) for 1 month; or vice versa. The primary endpoint was the Ocular Surface Disease Index (OSDI) score. Secondary endpoints were tear break-up time (TBT), tear production (TP), and presence of corneal punctate lesions (CP). The user-friendliness of both application systems was also compared. A linear mixed model for cross-over design was applied to compare both treatments. RESULTS: Forty-nine patients completed the trial. The mean OSDI score significantly improved from 52 ± 3 to 41 ± 3 for micro-sized SEDs, and from 54 ± 3 to 45 ± 3 for conventional-sized SEDs. Non-inferiority (margin = 6) of micro-sized SEDs was established. We demonstrate a significant improvement for TBT in case of conventional-sized SEDs and for CP in both treatment groups. TP trended towards an improvement in both treatment groups. The user-friendliness of the conventional drop system was significantly higher. CONCLUSIONS: For the first time, non-inferiority of micro-sized allogeneic SEDs was established. The beneficial effect of both SED volumes was similar as measured by the OSDI score. Although user-friendliness of the micro drop system was significantly lower, it is an attractive alternative as it saves valuable donor serum. TRIAL REGISTRATION: ClinicalTrials.gov (NCT03539159).

Recommendations for in vitro evaluation of blood components collected, prepared and stored in non-DEHP medical devices.

BACKGROUND AND OBJECTIVES: DEHP, di(2-ethylhexyl) phthalate, is the most common member of the class of ortho-phthalates, which are used as plasticizers. The Medical Device Regulation has restricted the use of phthalates in medical devices. Also DEHP has been added to the Annex XIV of REACH, "Registration, Evaluation, Authorisation and Restriction of Chemicals" due to its endocrine disrupting properties to the environment. As such, the sunset date for commercialisation of DEHP-containing blood bags is May 27th 2025. There are major concerns in meeting this deadline as these systems have not yet been fully validated and/or CE-marked. Also, since DEHP is known to affect red cell quality during storage, it is imperative to transit to non-DEHP without affecting blood product quality. Here, EBA members aim to establish common grounds on the evaluation and assessment of blood components collected, prepared and stored in non-DEHP devices. MATERIALS AND METHODS: Based on data as well as the input of relevant stakeholders a rationale for the validation of each component was composed. RESULTS: The red cell components will require the most extensive validation as their quality is directly affected by the absence of DEHP, as opposed to platelet and plasma components. CONCLUSION: Studies in the scope of evaluating the quality of blood products obtained with non-DEHP devices, under the condition that they are carried out according to these recommendations, could be used by all members of the EBA to serve as scientific support in the authorization process specific to their jurisdiction or for their internal validation use.

Donor variation in stored platelets: Higher metabolic rates of platelets are associated with mean platelet volume, activation and donor health.

BACKGROUND: Platelets (PLTs) differ in glycolytic activity, resulting in rapid acidification of 'poor' storing PLT concentrates (PCs) in plasma, or depletion of glucose when stored in PLT additive solution (PAS). We aimed to understand why PLT glycolysis rates vary between donors and how this affects storage performance. STUDY DESIGN AND METHODS: Buffy coats from donors <45, 45-70 and >70 years were selected and single-donor PCs in plasma or PAS-E were prepared. PCs were stored for 8 days at 22 ± 2°C and sampled regularly for analysis. Mitochondrial activity was analyzed with an Oroboros oxygraph. Age groups, or subgroups divided into quartiles based on glucose consumption, were analyzed with ANOVA. RESULTS: In each comparison, PCs of the different groups were not different in volume and cellular composition. PLTs with the highest glucose consumption had a higher initial mean platelet volume (MPV) and developed higher CD62P expression and Annexin A5 binding during storage. Higher glycolytic activity in these PLTs was not a compensation for lower mitochondrial ATP production, because mitochondrial ATP-linked respiration of fresh PLTs correlated positively with MPV (R2  = 0.71). Donors of high glucose-consuming PLTs had more health-related issues. Storage properties of PCs from donors over 70 were not significantly different compared to PCs from donors younger than 45 years. CONCLUSIONS: High glucose-consuming PCs developing higher activation levels, not only displayed enhanced mitochondrial activity but were also found to contain larger PLTs, as determined by MPV. Storage performance of PLTs was found to be associated with donor health, but not with donor age.

Clinical and in vitro evaluation of red blood cells collected and stored in a non-DEHP plasticized bag system.

BACKGROUND AND OBJECTIVES: Di-ethyl-hexyl-phthalate (DEHP) is currently the main plasticizer used for whole blood collection systems. However, in Europe, after May 2025, DEHP may no longer be used above 0.1% (w/w) in medical devices. DEHP stabilizes red cell membranes, thereby suppressing haemolysis during storage. Here we compared in vitro quality parameters of red cell concentrates (RCCs) collected and stored in DEHP-, DINCH- or DINCH/BTHC-PVC hybrid blood bags with saline-adenine-glucose-mannitol (SAGM) or phosphate-adenine-glucose-guanosine-saline-mannitol (PAGGSM) storage solution. Last, we performed haemovigilance surveillance for RCC collected in DINCH-PVC and stored in PAGGSM/BTHC-PVC. MATERIALS AND METHODS: In vitro quality parameters of RCC were determined during 42 days of storage. Haemovigilance surveillance was conducted to compare the frequency and type of transfusion reaction. RESULTS: Haemolysis levels were increased in SAGM/BTHC-PVC as compared to SAGM/DEHP-PVC (0.66% ± 0.18% vs. 0.36% ± 0.17%). PAGGSM storage solution was able to adequately suppress haemolysis to levels observed during storage in SAGM/DEHP-PVC, both in BTHC-PVC (0.38% ± 0.12%), and to a slightly lesser extent in DINCH-PVC (0.48% ± 0.17%). A total of 1650 PAGGSM/BTHC-PVC and 5662 SAGM/DEHP-PVC RCC were transfused yielding a transfusion reaction frequency of 0.24% (95% CI 0.0000-0.0048) and 0.44% (95% CI 0.0027-0.0061) respectively. CONCLUSION: The in vitro quality of RCC stored in PAGGSM/BTHC-PVC and SAGM/DEHP-PVC is comparable. There is no indication that transfusion of erythrocytes stored in PAGGSM/BTHC-PVC results in increased transfusion reaction frequency. These initial results provide a basis for further clinical evaluation to narrow down the confidence interval of transfusion reaction frequency.

The history of buffy coat platelet concentrates: The Dutch story.

The buffy coat method as a source for platelet concentrates was developed in the 1970s and is still used in many blood centres around the world. Development of the method sparked various technological advances in blood collection, processing and storage. At the time, the need for platelet concentrates sharply increased because of better treatment regimens for (onco)haematological diseases, which forced blood centres to standardize and automate their production processes as much as the technology would allow. In this review, a historical overview of the Dutch experiences is provided in the context of the international developments.

Malaria-associated adhesion molecule activation facilitates the destruction of uninfected red blood cells.

Severe malarial anemia (SMA) is the main cause of malaria-associated infant mortality in malaria endemic countries. One major factor that contributes to SMA is the accumulation of uninfected red blood cells (uRBCs) in the spleen. We report the activation of adhesion molecules Lutheran/basal cell adhesion molecule (Lu/BCAM) and CD44 on uRBCs from Plasmodium falciparum in vitro cultures and patients with malaria that mediates adherence to the splenic extracellular matrix (ECM) components laminin-α5 and hyaluronic acid (HA), respectively. This tight ECM-adhesion molecule interaction was associated with elevated intracellular Ca2+ levels, increased shedding of microvesicles, and Lu/BCAM clustering on altered uRBCs. Moreover, we observed that a soluble parasite-derived factor promoted the adhesive phenotype of uRBCs, as the incubation of RBCs with filtered malaria-conditioned medium reproduced the same adhesive effect in malaria culture-derived uRBCs. Eventually, Lu/BCAM and CD44 activation facilitate the adherence to ECM components of the red pulp, resulting in the enhanced splenic retention of uRBCs. Our results suggest a novel adhesion molecule-dependent mechanism that augments malaria-induced anemia.

The Gardos effect drives erythrocyte senescence and leads to Lu/BCAM and CD44 adhesion molecule activation.

Senescence of erythrocytes is characterized by a series of changes that precede their removal from the circulation, including loss of red cell hydration, membrane shedding, loss of deformability, phosphatidyl serine exposure, reduced membrane sialic acid content, and adhesion molecule activation. Little is known about the mechanisms that initiate these changes nor is it known whether they are interrelated. In this study, we show that Ca2+-dependent K+ efflux (the Gardos effect) drives erythrocyte senescence. We found that increased intracellular Ca2+ activates the Gardos channel, leading to shedding of glycophorin-C (GPC)-containing vesicles. This results in a loss of erythrocyte deformability but also in a marked loss of membrane sialic acid content. We found that GPC-derived sialic acid residues suppress activity of both Lutheran/basal cell adhesion molecule (Lu/BCAM) and CD44 by the formation of a complex on the erythrocyte membrane, and Gardos channel-mediated shedding of GPC results in Lu/BCAM and CD44 activation. This phenomenon was observed as erythrocytes aged and on erythrocytes that were otherwise prone to clearance from the circulation, such as sickle erythrocytes, erythrocytes stored for transfusion, or artificially dehydrated erythrocytes. These novel findings provide a unifying concept on erythrocyte senescence in health and disease through initiation of the Gardos effect.

CALIBER: a phase II randomized feasibility trial of chemoablation with mitomycin-C vs surgical management in low-risk non-muscle-invasive bladder cancer.

OBJECTIVES: To evaluate the activity of intravesical mitomycin-C (MMC) to ablate recurrent low-risk non-muscle-invasive bladder cancer (NMIBC) and assess whether it may enable patients to avoid surgical intervention for treatment of recurrence. PATIENTS AND METHODS: CALIBER is a phase II feasibility study. Participants were randomized (2:1) to treatment with four once-weekly MMC 40-mg intravesical instillations (chemoablation arm) or to surgical management. The surgical group was included to assess the feasibility of randomization. The primary endpoint was complete response to intravesical MMC in the chemoablation arm at 3 months, reported with exact 95% confidence intervals (CIs). Secondary endpoints included time to subsequent recurrence, summarized by Kaplan-Meier methods. RESULTS: Between February 2015 and August 2017, 82 patients with visual diagnosis of recurrent low-risk NMIBC were enrolled from 24 UK hospitals (chemoablation, n = 54; surgical management, n =28). The median follow-up was 24 months. Complete response at 3 months was 37.0% (20/54; 95% CI 24.3-51.3) with chemoablation and 80.8% (21/26; 95% CI 60.6-93.4) with surgical management. Amongst patients with complete response at 3 months, a similar proportion was recurrence-free by 12 months in both groups (84%). Amongst those with residual disease at 3 months, the 12-month recurrence-free proportion was lower in the surgical management group (40.0%) than in the chemoablation group (84%). Recruitment stopped early as chemoablation did not meet the prespecified threshold of 45% complete responses at 3 months. CONCLUSION: Intravesical chemoablation in low-risk NMIBC is feasible and safe, but did not demonstrate sufficient response in the present trial. After chemoablation there may be a reduction in recurrence rate, even in non-responders, that is greater than with surgery alone. Further research is required to investigate the role and optimal schedule of neoadjuvant intravesical chemotherapy prior to surgery for NMIBC.

Properties and Function of the Flexor Hallucis Capsularis Interphalangeus Tendon.

INTRODUCTION: The fibrocartilagenous plantar plates of the forefoot are biomechanically important, forming the primary distal attachment for the plantar aponeurosis. They are integral to the function of the windlass mechanism in supporting the arches of the foot in gait. Dissection of the cadaveric hallux revealed an organised sagittal thickening of the dorsal side of the flexor hallucis longus (FHL) sheath, which attached the interphalangeal plantar plate to the metatarsophalangeal (MTP) plantar plate. A description of a similar structure was made in 1984 when it was termed the flexor hallucis capsularis interphalangeus (FHCI) - however, it has not been researched since, and we aim to study it further and identify its characteristics. METHOD: Eight specimens were dissected from four cadavers. Two were stained and examined under magnification in both polarized and non polarized light. The remaining 6 were subjected to micrometer testing of their tensile properties. RESULTS: Both the histological features and mechanical properties were consistent with tendon; with cross sectional area, ultimate tensile strength and stiffness varying between specimens. CONCLUSIONS: Based on its location and properties, the FHCI tendon may be involved in limiting dorsiflexion of the first MTP joint and could have clinical relevance in pathological processes around both the first and second MTP joints. Clin. Anat., 33:689-695, 2020. © 2019 Wiley Periodicals, Inc.

Does physiological tolerance to acute hypoxia and salinity change explain ecological niche in two intertidal crab species?

Intertidal biota is subjected to significant fluctuations in environmental parameters such as salinity and dissolved oxygen (DO). In the current study, the effects of salinity and DO on metabolic rate, critical oxygen partial pressure (P crit), heart rate and osmoregulation in two intertidal crab species commonly found on New Zealand coastlines, Hemigrapsus crenulatus and Hemigrapsus sexdentatus, were measured. Based on its habitation of burrows in the lower intertidal zone, H. crenulatus was predicted to be more resilient to these environmental stressors than H. sexdentatus, which is distributed in the mid to high tidal zone. However, relative to the full-strength seawater control, there were no consistent salinity-dependent changes in respiratory or cardiovascular endpoints in either species following acute 6-h exposures mimicking a tidal cycle. Analysis of haemolymph osmolality and ions determined that both crab species were strong osmotic and ionic regulators over the 6-h exposure period. However, the threshold salinities at which significant changes in osmotic and ionic regulation occurred did differ and generally indicated that H. crenulatus was the better regulator. Respiratory and cardiovascular responses to DO were prominent, with a strong bradycardia observed in both species. Changes in osmolality and sodium ion regulation were also seen as DO declined. The effect on sodium ion levels had its onset at a higher oxygen partial pressure in H. sexdentatus than in H. crenulatus, indicative of a relatively poorer hypoxia tolerance in the former species. The relative resilience of respiratory, cardiovascular and osmoregulatory processes to salinity and DO variations likely contribute to distinct habitat distributions of the two crab species on New Zealand shorelines, although behaviour and inter-specific interactions may also play important roles. Environmental change, in the form of coastal erosion and anthropogenic contamination of estuaries, has the potential to disturb the delicate niche separation that exists between these species.

IDF Diabetes Atlas: A review of studies utilising retinal photography on the global prevalence of diabetes related retinopathy between 2015 and 2018.

AIMS: The purpose of this study is to assess the prevalence of diabetic retinopathy (DR) world-wide from articles published since 2015 where the assessment of the presence and severity of DR was based on retinal images. METHODS: A total of 4 databases were searched for the MESH terms diabetic retinopathy and prevalence. Of 112 publications 32 studies were included and individual data pooled for analysis. The presence of any DR or diabetic macular edema (DME) was recorded and severity as mild, moderate or severe non-proliferative DR (NPDR), proliferative DR (PDR) and DME and/or clinically significant macular edema (CSME). The level of severity of DR reported refer to persons with diabetes and not individual eyes. RESULTS: The global prevalence of DR and DME, for the period 2015 to 2019 were 27.0% for any DR comprising of 25.2%, NPDR, 1.4% PDR and 4.6% DME. The lowest prevalence was in Europe at 20.6% and South East Asia at 12.5% and highest in Africa at 33.8%, Middle East and North Africa 33.8%, and the Western Pacific region at 36.2%. CONCLUSIONS: This study illustrated difficulties in deriving a meaningful global prevalence rate for DR and DME due to the lack of uniformity in defining the study populations, methodological differences, retinal image capture and grading criteria. Therefore, international consensus is required using a minimal data set for future studies.

A Homozygous Mutation on the HBA1 Gene Coding for Hb Charlieu (HBA1: c.320T>C) Together with ß-Thalassemia Trait Results in Severe Hemolytic Anemia.

A 4-year-old boy, a ß-thalassemia (ß-thal) carrier, with an unexplained severe chronic microcytic anemia was referred to us. Sequencing of the α-globin genes revealed a Hb Charlieu [α106(G13)Leu→Pro, HBA1: c.320T>C, p.Leu107Pro] mutation present on both HBA1 genes. Quantitative polymerase chain reaction (qPCR) confirmed αCharlieu mRNA in the proband and his parents, showing that the mutation does not affect mRNA stability. However, we were unable to detect the Hb Charlieu protein by capillary electrophoresis (CE), reverse phase electrophoresis, cation exchange electrophoresis or isoelectric focusing. Mass spectrometry (MS) allowed us to confirm the presence of the Hb Charlieu peptide in erythrocyte progenitors. These findings suggest that the mutation affects the stability of αCharlieu. As hemoglobin (Hb) heat stability tests showed no abnormalities in erythrocytes, we speculated that αCharlieu is already degraded during red blood cell (RBC) development. The clinical severity in the proband and the presence of new methylene blue-stained aggregates in his reticulocytes indicates that incorporation of αCharlieu destabilizes Hb. This, combined with an excess of unstable free α-globins as the result of ß-thal minor, results in severely impaired erythropoiesis and, as a consequence, severe and chronic microcytic anemia in the proband.

Endotoxemia Results in Trapping of Transfused Red Blood Cells in Lungs with Associated Lung Injury.

BACKGROUND: Red blood cell (RBC) transfusion is associated with organ failure, in particular in the critically ill. We hypothesized that endotoxemia contributes to increased trapping of RBCs in organs. Furthermore, we hypothesized that this effect is more pronounced following transfusion of stored RBCs compared with fresh RBCs. METHODS: Adult male Sprague-Dawley rats were randomized to receive injection with lipopolysaccharide from E coli or vehicle and transfusion with fresh or stored biotinylated RBCs. After 24 h, the amount of biotinylated RBCs in organs was measured by flow cytometry, as well as the 24-h post-transfusion recovery. Markers of organ injury and histopathology of organs were assessed. RESULTS: Endotoxemia resulted in systemic inflammation and organ injury. Following RBC transfusion, donor RBCs were recovered from the lung and kidney of endotoxemic recipients (1.2 [0.8-1.6]% and 2.2 [0.4-4.4]% of donor RBCs respectively), but not from organs of healthy recipients. Trapping of donor RBCs in the lung was associated with increased lung injury, but not with kidney injury. Stored RBCs induced organ injury in the spleen and yielded a lower 24-h post-transfusion recovery, but other effects of storage time were limited. CONCLUSION: Endotoxemia results in an increased percentage of donor RBCs recovered from the lung and kidney, which is associated with lung injury following transfusion.

Residual pyruvate kinase activity in PKLR-deficient erythroid precursors of a patient suffering from severe haemolytic anaemia.

OBJECTIVE: Here, we present a 7-year-old patient suffering from severe haemolytic anaemia. The most common cause of chronic hereditary non-spherocytic haemolytic anaemia is red blood cell pyruvate kinase (PK-R) deficiency. Because red blood cells rely solely on glycolysis to generate ATP, PK-R deficiency can severely impact energy supply and cause reduction in red blood cell lifespan. We determined the underlying cause of the anaemia and investigated how erythroid precursors in the patient survive. METHODS: PK activity assays, Western blot and Sanger sequencing were employed to determine the underlying cause of the anaemia. Patient erythroblasts were cultured and reticulocytes were isolated to determine PK-R and PKM2 contribution to glycolytic activity during erythrocyte development. RESULTS: We found a novel homozygous mutation (c.583G>A) in the PK-R coding gene (PKLR). Although this mutation did not influence PKLR mRNA production, no PK-R protein could be detected in the red blood cells nor in its precursors. In spite of the absence of PK-R, the reticulocytes of the patient exhibited 20% PK activity compared with control. Western blotting revealed that patient erythroid precursors, like controls, express residual PKM2. CONCLUSIONS: We conclude that PKM2 rescues glycolysis in PK-R-deficient erythroid precursors.

From the Cradle to the Grave: The Role of Macrophages in Erythropoiesis and Erythrophagocytosis.

Erythropoiesis is a highly regulated process where sequential events ensure the proper differentiation of hematopoietic stem cells into, ultimately, red blood cells (RBCs). Macrophages in the bone marrow play an important role in hematopoiesis by providing signals that induce differentiation and proliferation of the earliest committed erythroid progenitors. Subsequent differentiation toward the erythroblast stage is accompanied by the formation of so-called erythroblastic islands where a central macrophage provides further cues to induce erythroblast differentiation, expansion, and hemoglobinization. Finally, erythroblasts extrude their nuclei that are phagocytosed by macrophages whereas the reticulocytes are released into the circulation. While in circulation, RBCs slowly accumulate damage that is repaired by macrophages of the spleen. Finally, after 120 days of circulation, senescent RBCs are removed from the circulation by splenic and liver macrophages. Macrophages are thus important for RBCs throughout their lifespan. Finally, in a range of diseases, the delicate interplay between macrophages and both developing and mature RBCs is disturbed. Here, we review the current knowledge on the contribution of macrophages to erythropoiesis and erythrophagocytosis in health and disease.