Reticulocyte haemoglobin equivalent (RET-He) as an early marker of responsiveness to oral iron supplementation.

AIM: We investigated the potential of reticulocyte haemoglobin equivalent (RET-He) as an early marker of responsiveness to iron supplementation. METHODS: Data were obtained from a randomised controlled trial of daily iron supplementation in 356 Cambodian women (18-45 y) who received 60 mg elemental iron for 12 weeks. A fasted venous blood specimen was collected at baseline, 1-week and 12-week timepoints. Whole blood haemoglobin (g/L) and RET-He (pg) were measured using a Sysmex haematology analyser. RET-He measures were evaluated for their predictive ability on haemoglobin response to iron supplementation (defined as ≥10 g/L at 12 weeks). Receiver operating characteristic (ROC) curves were used to assess discrimination performance, and the area under the ROC curve (AUCROC) served as a measure of the ability of each predictor to discriminate between women likely or unlikely to elicit a haemoglobin response. RESULTS: Predictive ability (AUCROC (95% CI)) of baseline, 1-week, and change from baseline to 1-week RET-He on haemoglobin response was 0.70 (0.63 to 0.76), 0.48 (0.41 to 0.56) and 0.81 (0.75 to 0.87), respectively. Based on the Youden index, an absolute increase in RET-He of ~1.1 pg or a percentage increase of ~4.4% over 1 week were optimal thresholds to predict responsiveness to iron supplementation. CONCLUSION: Single timepoint RET-He measures have poor predictive ability; however, change in RET-He after 1 week was a strong predictor of haemoglobin response among Cambodian women receiving 60 mg elemental iron and can be measured easily and quickly after only 1 week of iron therapy.

Reticulocytes in donor blood units enhance red blood cell alloimmunization.

Although red blood cell (RBC) transfusions save lives, some patients develop clinically-significant alloantibodies against donor blood group antigens, which then have adverse effects in multiple clinical settings. Few effective measures exist to prevent RBC alloimmunization and/or eliminate alloantibodies in sensitized patients. Donor-related factors may influence alloimmunization; thus, there is an unmet clinical need to identify which RBC units are immunogenic. Repeat volunteer blood donors and donors on iron supplements have elevated reticulocyte counts compared to healthy non-donors. Early reticulocytes retain mitochondria and other components, which may act as danger signals in immune responses. Herein, we tested whether reticulocytes in donor RBC units could enhance RBC alloimmunization. Using a murine model, we demonstrate that transfusing donor RBC units with increased reticulocyte frequencies dose-dependently increased RBC alloimmunization rates and alloantibody levels. Transfusing reticulocyte-rich RBC units was associated with increased RBC clearance from the circulation and a robust proinflammatory cytokine response. As compared to previously reported post-transfusion RBC consumption patterns, erythrophagocytosis from reticulocyte-rich units was increasingly performed by splenic B cells. These data suggest that reticulocytes in a donated RBC unit impact the quality of blood transfused, are targeted to a distinct compartment, and may be an underappreciated risk factor for RBC alloimmunization.

The effects of iron injection on blood doping biomarkers in dried blood spots.

Iron supplementation is not considered as a doping method; however, it can affect the levels of several biomarkers of the hematologic module of the athlete biological passport (ABP), such as the reticulocyte percentage (%RET) and hemoglobin (HGB) level. Thus, iron injection could be a confounding factor in antidoping analyses. Previous studies have suggested that the HGB level and the expression levels of reticulocyte-related-mRNAs, such as 5'-aminolevulinate synthase 2 (ALAS2) and carbonic anhydrase 1 (CA1), could be promising biomarkers for the ABP and detectable in dried blood spots (DBSs). Therefore, in this study, we examined the impact of iron injection on the levels of these potential biomarkers in DBSs. Reticulocyte-related-mRNAs analyses were performed by RT-qPCR. Ferritin level in DBS was measured with enzyme-linked immunosorbent assay (ELISA) method. Notably, there were no significant effects of iron supplementation on the levels of ALAS2 and CA1 mRNAs but by contrast, the %RET and immature reticulocyte fraction (IRF) measured in whole blood increased significantly following iron injection. As expected, iron supplementation increased the ferritin level significantly in both serum and DBS samples. In conclusion, these findings reinforce the specificity of reticulocyte-related mRNAs in DBSs as biomarkers of blood doping to target in antidoping analyses.

Prevalence and Implications of Low Reticulocyte-Hemoglobin Levels among Extreme Preterm Neonates: A Single-Center Retrospective Study.

This retrospective cohort study aims to determine the epidemiology of iron deficiency among extreme preterm neonates and the association of iron-deficient status during the NICU stay with neurodevelopmental outcomes at 18−24 months. Neonates ≤29 weeks gestational age (GA) born between June 2016 and December 2019, who received routine iron supplementation were enrolled. Iron deficiency was defined as reticulocyte−hemoglobin (Ret-Hb) levels ≤ 29 pg at 36 weeks corrected age. A subcohort of neonates completed standardized developmental assessment at 18−24 months corrected age. Significant neurodevelopmental impairment (sNDI) was defined as either Bayley Scales of Infant Development score < 70 or cerebral palsy or blindness or hearing aided. Among a cohort of 215 neonates [GA 25.8 (1.7) weeks, birthweight 885 (232) g], prevalence of iron deficiency was 55%, 21%, 26%, and 13%, in neonates <24 weeks, 24−25 + 6 weeks, 26−27 + 6 weeks, and ≥ 28 weeks GA, respectively. Male sex and receipt of corticosteroid therapy were associated with iron-deficiency. In the subcohort analysis (n = 69), there was no statistically significant association between Ret-Hb levels at 36 weeks corrected age and the risk of sNDI [OR 0.99 (95% CI 0.85−1.2)]. Male infants and those who received postnatal corticosteroids are likely to have iron-limited erythropoiesis at corrected term despite routine iron-supplementation; however, low Ret-Hb levels during the neonatal period were not associated with significant neurological disability in early childhood.

Reticulocyte hemoglobin content changes after treatment of anemia of prematurity.

BACKGROUND: Iron deficiency during infancy is associated with poor neurological development, but iron overload causes severe complications. Appropriate iron supplementation is therefore vital. Reticulocyte hemoglobin content (RET-He) provides a real-time assessment of iron status and chracterezes hemoglobin synthesis in preterm infants. However, the existing literature lacks detailed reports assessing chronological changes in RET-He. The aim of this study was to assess the chronological changes in RET-He during oral iron dietary supplementation, and concomitant therapy with recombinant human erythropoietin (rHuEPO) in preterm very low birthweight infants. METHODS: Very low birthweight infants, admitted to our neonatal intensive care unit were analyzed retrospectively. Hemoglobin (Hb), reticulocyte percentage (Ret), mean corpuscular volume, RET-He, serum iron (Fe), and serum ferritin were recorded. Data at birth (T0), the initial day of rHuEPO therapy (T1), the initial day of oral iron supplementation (T2), 1-2 weeks (T3), 3-4 weeks (T4), 5-6 weeks (T5), and 7-8 weeks (T6) from the initial day of oral iron supplementation were extracted, and their changes over time were examined. RESULTS: Reticulocyte hemoglobin content was highest at birth and declined rapidly thereafter, especially after starting rHuEPO therapy. There was no upward trend in RET-He after the initiation of oral iron supplementation, with a slower increase during 5-6 weeks after the initiation of iron therapy. CONCLUSIONS: During the treatment of anemia of prematurity, low RET-He levels may be prolonged. Anemia of prematurity should therefore be assessed and treated on a case-by-case basis, while considering the iron metabolic capacity of preterm infants.

Reticulocyte hemoglobin content as a function of iron stores at 35-36 weeks post menstrual age in very premature infants.

BACKGROUND: Premature infants are born with lower iron stores and are at risk for iron deficiency during early infancy. To prevent iron deficiency, premature infants are routinely supplemented with 2 mg/kg/day oral elemental iron. Reticulocyte hemoglobin content (RET-He), a measure of iron deficiency, has not been well evaluated prior to discharge in premature infants. OBJECTIVES: Our objectives were to evaluate RET-He and its correlation with serum ferritin (SF), an index of iron stores, at 35-36 weeks postmenstrual age (PMA) in ≤32 weeks gestational age (GA) infants. METHODS: We performed a prospective nested study involving 24-32 weeks GA infants who were receiving 2 mg/kg/day oral elemental iron with full enteral feedings at 35-36 weeks PMA. Infants with the following conditions were excluded: craniofacial malformation, chromosomal disorders, TORCH (toxoplasmosis, other infections, rubella, cytomegalovirus, and herpes simplex), culture-proven sepsis, C-reactive protein >5 mg/l within 10 days of iron status assessment, or erythropoietin therapy. SF and RET-He were measured at 35-36 weeks PMA using chemiluminescence immunoassay and Sysmex XN hematology analyzer, respectively. RET-He <27 pg was deemed indicative of iron deficiency. RESULTS: Ninety-eight infants were studied, of which 21 infants had RET-He <27 pg. There was a positive correlation between RET-He and SF (coefficient 0.22, p = .03) that remained significant after controlling for GA (coefficient 0.21, p = .03) and frequency of prior erythrocyte transfusions (coefficient 0.21, p = .03). On stratified analysis, there was a positive correlation between SF and RET-He in females (N = 52, coefficient 0.23, p = .02), but not in males (N = 46, coefficient 0.05). CONCLUSIONS: Most premature infants receiving 2 mg/kg/day oral elemental iron are iron replete for erythropoiesis at 35-36 weeks PMA. RET-He increases with an increase in iron stores, suggesting that additional iron supplementation prior to discharge to very premature infants with borderline low RET-He may help prevent iron deficiency during early infancy.

Fluctuations in hematological athlete biological passport biomarkers in relation to the menstrual cycle.

The interpretation of athlete biological passport (ABP) is strengthened by understanding the natural fluctuations in its biological parameters. Here we have assessed the influence of the menstrual cycle on the hematological module of the ABP. Seventeen women with regular menses were included. Blood samples were collected once a week for two consecutive cycles and analyzed for hematological parameters. Menstrual phases were hormonally determined. The intra-individual variation in the hematological parameters was similar between the two cycles. Reticulocyte percentage was significantly lower in the follicle phase (median 0.95%) than in the ovulatory (median 1.10%) and luteal phases (median 1.16%), P = 0.006, whereas no differences were found in hemoglobin concentration, hematocrit, red blood cell count, or red blood cell indices. When the values were entered into the ABP model, findings outside the program-calculated individual thresholds were identified in two participants. One woman showed an atypical low OFF-score in the last sample collected, mainly because of increased reticulocyte percentage. This was likely a response to treated insufficient iron stores. One woman displayed an atypical hemoglobin value at the lower limit 2 weeks after ovulation, which was likely due to fluctuations in plasma volume. In conclusion, the ABP parameters in general are stable throughout the menstrual cycle. Significant differences between the menstrual phases were found in reticulocytes; however, the variation was not related to findings outside the individual thresholds, except in one individual. Moreover, our results highlight the importance of having information about iron supplementation available when evaluating hematological passports.

Efficacy and safety of a low monthly dose of intravenous iron sucrose in peritoneal dialysis patients.

PURPOSE: Scientific data regarding intravenous iron supplementation in peritoneal dialysis (PD) patients are scarce. In attempting to administer the minimum monthly IV iron dose that could improve erythropoiesis, we wanted to assess the safety and efficacy of monthly maintenance intravenous administration of 100 mg iron sucrose in PD patients. METHODS: In a 9-month prospective study, all clinically stable PD patients received intravenously 200 mg of iron sucrose as a loading dose, followed by monthly doses of 100 mg for five consecutive months. Levels of hemoglobin (Hb), ferritin, transferrin saturation (TSAT), reticulocyte hemoglobin content (CHr) and C-reactive protein (CRP) were measured before each administration and 3 months after the last iron infusion. Also, doses of concurrent erythropoietin administration were recorded. RESULTS: Eighteen patients were eligible for the study. Mean levels of Hb and ferritin increased significantly (from 10.0 to 10.9 mg/dL, p = 0.01 and from 143 to 260 ng/mL, p = 0.005), as well as the increase in TSAT levels approached borderline significance (from 26.2 to 33.1%, p = 0.07). During the 6 months of iron administration, the erythropoietin dose was reduced in five patients and discontinued in one. During the 3 months following the last iron infusion, three of them again raised the erythropoietin dose to previous levels. None of the patients experienced any side effects related to IV iron administration. CONCLUSIONS: A monthly maintenance intravenous dose of 100 mg iron sucrose may be a practical, effective, and safe in the short term, treatment of anemia in PD patients resulting in improved hemoglobin levels, iron indices, and erythropoietin response.

Glutathione peroxidase 4 and vitamin E control reticulocyte maturation, stress erythropoiesis and iron homeostasis.

Glutathione peroxidase 4 (GPX4) is unique as it is the only enzyme that can prevent detrimental lipid peroxidation in vivo by reducing lipid peroxides to the respective alcohols thereby stabilizing oxidation products of unsaturated fatty acids. During reticulocyte maturation, lipid peroxidation mediated by 15-lipoxygenase in humans and rabbits and by 12/15-lipoxygenase (ALOX15) in mice was considered the initiating event for the elimination of mitochondria but is now known to occur through mitophagy. Yet, genetic ablation of the Alox15 gene in mice failed to provide evidence for this hypothesis. We designed a different genetic approach to tackle this open conundrum. Since either other lipoxygenases or non-enzymatic autooxidative mechanisms may compensate for the loss of Alox15, we asked whether ablation of Gpx4 in the hematopoietic system would result in the perturbation of reticulocyte maturation. Quantitative assessment of erythropoiesis indices in the blood, bone marrow (BM) and spleen of chimeric mice with Gpx4 ablated in hematopoietic cells revealed anemia with an increase in the fraction of erythroid precursor cells and reticulocytes. Additional dietary vitamin E depletion strongly aggravated the anemic phenotype. Despite strong extramedullary erythropoiesis reticulocytes failed to mature and accumulated large autophagosomes with engulfed mitochondria. Gpx4-deficiency in hematopoietic cells led to systemic hepatic iron overload and simultaneous severe iron demand in the erythroid system. Despite extremely high erythropoietin and erythroferrone levels in the plasma, hepcidin expression remained unchanged. Conclusively, perturbed reticulocyte maturation in response to Gpx4 loss in hematopoietic cells thus causes ineffective erythropoiesis, a phenotype partially masked by dietary vitamin E supplementation.

Reticulocyte Hemoglobin Content (Ret He): A Simple Tool for Evaluation of Iron Status in Childhood Cancer.

BACKGROUND: Cancer-related anemia is a common complication of cancer and its treatment that may be mediated by nutritional deficiency or inflammatory cytokines inhibiting erythropoiesis. AIM: We evaluated the value of reticulocyte hemoglobin content (Ret He) as a marker of iron availability for erythropoiesis in childhood cancer and the impact of oral iron supplementation on hematologic parameters in patients with low Ret He. MATERIALS AND METHODS: This prospective study included 100 pediatric patients with cancer on chemotherapy who were screened for the presence of anemia. Patients with anemia underwent testing for complete blood count including Ret He on Sysmex XE 2100 and assessment of reticulocyte count, serum iron, serum ferritin, transferrin saturation, total iron-binding capacity, and C-reactive protein. Patients were classified according to their level of Ret He into normal or low Ret He using a cutoff level of 28 pg. Patients with low Ret He were subjected to 6 weeks' treatment with oral ion and were followed up with complete blood count and iron profile. RESULTS: Thirty-one (77.5%) patients had normal Ret He, and 9 (22.5%) had low Ret He. Ret He was positively correlated with red cell indices, but not with iron parameters. After oral iron supplementation, a significant increase in hemoglobin, reticulocyte count, and iron was found. CONCLUSIONS: We suggest that Ret He could be used as an easy and affordable tool for the assessment of iron deficiency anemia in childhood cancer during chemotherapy treatment. A trial of oral iron in patients with low Ret He may be useful to correct the associated anemia.

The Role of Reticulocyte Hemoglobin Content for Diagnosis of Iron Deficiency and Iron Deficiency Anemia, and Monitoring of Iron Therapy: a Literature Review.

BACKGROUND: The diagnosis of iron deficiency anemia is still complicated and most of the tests have drawbacks. Bone marrow examination, the gold standard for the diagnosis of iron deficiency and iron deficiency anemia, is a painful, invasive, and costly procedure. Other methods are also used to diagnose iron deficiency and iron deficiency anemia; soluble transferrin receptor, serum iron, serum ferritin, and transferrin saturation are most common biomarkers of iron status that are frequently affected by inflammation, chronic diseases, and in the normal aging process (except soluble transferrin receptor). All are less available compared to complete blood count with reticulocyte hemoglobin content (CHr). Reticulocytes have a normal life span of one or two days in the circulation. CHr is a good indication of iron availability and an early marker of iron deficient erythropoiesis which can be obtained readily using automated blood cell analyzers. Therefore, the main objective of the current review is to assess the role of CHr for diagnosis of iron deficiency, iron deficiency anemia, and monitoring of iron therapy. METHODS: Studies published in English were searched using the National Library of Medicine, PubMed, and Google scholar databases. RESULTS: According to this review, CHr has a moderate sensitivity and specificity for diagnosing iron deficiency, and is less affected by inflammation than serum iron, transferrin saturation, and ferritin and is an early predictor of treatment response. It is used in screening of iron deficiency, diagnosis of iron deficiency anemia, and diagnosis of functional iron deficiency anemia in acute or chronic diseases or inflammation. CHr is also important in treatment monitoring. It is useful for early measurement of response to iron therapy, increasing within days of the initiation of iron therapy. It helps monitoring of intravenous iron supplementation, recombinant human erythropoie¬tin therapy, and oral iron therapy in hemodialysis and non-hemodialysis patients, and children. CONCLUSIONS: It is easy to analyze, less time consuming, and less expensive than bone iron examination and iron biochemical tests. However, there is no standardized cutoff point and different researchers use varying cutoff values which affects its accuracy in diagnosing iron deficiency and it should therefore be standardized. Moreover, since CHr can be affected with any conditions that cause iron restricted erythropoiesis, further analysis may be needed.

Effect of L-Leucine Therapy on Hematopoietic Function in Elderly Myelodysplastic Syndrome Patients.

Patients with myelodysplastic syndrome (MDS) often require blood transfusion and anticancer therapy; however, elderly patients are intolerant to the associated side effects of anticancer therapy. Because L-leucine can be used to treat Diamond-Blackfan anemia, which is caused by defects in ribosomal protein (RP) genes, resulting in increased in vivo hemoglobin synthesis, it is possible that some MDS patients who have aberrations in their RP genes could also be effectively treated with L-leucine. In the present study, we investigated the effects of L-leucine on hematopoietic function (reticulocyte count), red blood cell count, and hemoglobin level in MDS patients. We administered L-leucine (1.8 g, twice daily, 3 d/week) with oral vitamin B6 supplements to a final cohort of eight MDS patients for 15 (interquartile range: 11-18) weeks. We assessed the patients at 10 ± 2 weeks after therapy initiation. Only the absolute reticulocyte count was affected, improving in 6/8 (75%) patients. The median absolute reticulocyte count was 3.5 × 104 (range: 2.7-6.4 × 104) cells/µL, an increase of 0.5 × 104 (range: 0.2-0.7 × 104) cells/µL. At 10 weeks, there was only one case of an improved hemoglobin level. Non-hematological adverse events of grade 3 were observed one raised triglycerides. These data suggest that L-leucine has little effect on MDS. However, it may contribute to the recovery of hematopoietic function, futher study be desired.

Trends in reticulocyte hemoglobin equivalent values in critically ill neonates, stratified by gestational age.

OBJECTIVE: The reticulocyte index reticulocyte hemoglobin equivalent (Ret-He) was evaluated as a marker of iron status. STUDY DESIGN: This is a retrospective cohort study of all infants admitted to the University of Washington Neonatal Intensive Care Unit, who received Ret-He measurements as part of routine care within the first 120 days of life. RESULT: A total of 730 Ret-He measurements from 249 infants were analyzed (median gestational age at birth 32.1 weeks; 49 infants <28 weeks and 200 ≥28 weeks). Initial Ret-He measurements were lower in infants <28 weeks (28.24 vs. 33.34 pg). Ret-He values initially decreased, then slowly increased. Infants received an average of 3.9, 6.5, and 8.2 mg/kg/day of enteral iron sulfate at 30, 60, and 90 days, respectively. CONCLUSION: Ret-He values showed a slow uptrend with enteral iron supplementation following an initial decrease, suggesting that neonates are able to improve their iron sufficiency status with supplementation.

The effect of lycopene supplementation on radiation-induced micronuclei in mice reticulocytes in vivo.

Lycopene (LYC) is a natural pigment present in tomatoes and other red fruits and vegetables including red carrots, red peppers, watermelons, pink grapefruits, apricots, pink guavas, and papaya. There is some evidence that LYC may provide protection against mutations induced by ionizing radiation. The study aimed to investigate whether the genetic material of reticulocytes (RET) could be protected from radiation-induced damage by LYC. Mice were treated with LYC [0.15 mg/kg bodyweight (bw), 0.30 mg/kg bw], acute and fractionated irradiation (0.5 Gy, 1 Gy applied daily), or with both agents (0.5 Gy + 0.15 mg/kg bw LYC, 0.5 Gy + 0.30 mg/kg bw LYC, 1 Gy + 0.15 mg/kg bw LYC, 1 Gy + 0.30 mg/kg LYC). LYC supplementation was started at 24 h or 1 week after the first irradiation. Irradiation significantly enhanced the frequency of micronuclei (MN) in RET. LYC treatment at a dose of 0.15 mg/kg bw 24 h after starting fractionated radiation at 1 Gy significantly decreased (41-68%, p < 0.0125) the level of MN in peripheral blood and bone marrow RET. LYC supplementation at 0.30 mg/kg bw did not significantly alter the frequency of MN in peripheral blood, but significantly increased the frequency of bone marrow RET MN. LYC treatment on day 8 following the first radiation exposure showed results similar (92-117%, p > 0.24) to those obtained with irradiation alone. Lycopene may act as a radiomitigator but must be administered at low doses and as soon as possible after irradiation. Contrary, combined exposure with high doses of irradiation and LYC may enhance the mutagenic effect of irradiation.

Adherence to iron prophylactic therapy during pregnancy in an urban regional hospital in South Africa

Background: Iron and folic acid supplementation plays a major role in the prevention and control of iron-deficiency anaemia in pregnancy. Therefore, this study assesses adherence to prophylactic iron supplementation during the antenatal period in South Africa. Methods: An observational study was conducted in a regional hospital from January to December 2016. HIV-uninfected(n= 100) and HIV-infected (n= 100)] women were enrolled and subdivided into three groups: (a)≤34 weeks (n= 33), (b)34­36 weeks (n= 34) and (c)≥37 weeks (n= 33) gestational age respectively. A structured questionnaire was used for data collection. Data were coded and statistically analysed using SPSS software. Pill count and self-reported data from women (n= 24) at≤34 weeks and 34­36 weeks reflected < 50% adherence and 46% non-adherence, being higher in the HIV-infected women (75%). Nausea was the commonest side effect across all trimesters (79. 2%). Adherence (27.8%) and non-adherence (72.1%) to iron, folic acid and calcium supplementation were found in 88% of women. Conclusion: This study found that adherence to micronutrient supplementation is low in pregnancy, albeit higher in HIV-infected women receiving antenatal care at a regional hospital in Durban, South Africa

Zinc Supplementation Stimulates Red Blood Cell Formation in Rats.

In rats, mice, and humans, it is known that zinc deficiency may be related to anemia, and zinc supplementation influences hemoglobin production. Our previous studies indicate that in fish, zinc supplementation stimulates red blood cell (RBC) formation (erythropoiesis). However, it is not clear whether the mechanism of zinc-induced erythropoiesis stimulation in fish also occurs in rats. We induced anemia in rats using phenylhydrazine (PHZ) and injected either saline or ZnSO4 solution. We found that an appropriate amount of zinc stimulated erythropoiesis in the PHZ-induced anemic rats. The effects of ZnSO4 injection were dose-dependent. When the concentration of ZnSO4 was higher than 2.8 mg zinc/kg body weight, the RBC level of the anemic rats increased from 60 ± 7% to 88 ± 10% that of the normal rats in two days. Rat bone marrow cells with or without ZnCl2 supplementation were cultured in suspension in vitro. In the cell culture when the zinc concentration was at 0.3 mM, a 1.6-fold proliferation of nascent immature reticulocytes (new RBCs) was observed after one day. In the rat blood, zinc was combined with serum transferrin to induce erythropoiesis. The stimulation of RBC formation by zinc appears to be common among different animals.

Applying newer parameter Ret-He (reticulocyte haemoglobin equivalent) to assess latent iron deficiency (LID) in blood donors-study at a tertiary care hospital in India.

BACKGROUND: It is important to detect Latent Iron Deficiency (LID) to prevent development of an overt iron deficiency anemia. Early detection is difficult by using conventional hematological and biochemical parameters. Soluble transferrin receptor (sTfR) is presently the gold standard for diagnosing LID. We evaluated the utility of Reticulocyte Hemoglobin Equivalent (Ret-He), a newer hematological parameter, to predict LID in blood donors as compared to sTfR. METHODS: This was a randomized prospective study performed on 501 donor samples over a period of three-months. All donors were included after administering medical history questionnaire and a brief physical examination in accordance with national guidelines (Hb ≥12.5). Additional samples were collected during donation according to the institutional standard operating procedure (SOP). All hemograms were performed on the Sysmex XE-2100 analyzer which included Ret-He. sTfR was measured in batch assays by ELISA (Biovendor, Czech Republic). Ret He <28 pg and sTfR≥3µg/ml were used to diagnose LID. Serum Iron, Total Iron Binding Capacity (TIBC) and Serum Ferritin were also measured simultaneously. RESULTS: Of the 501 blood donors, sTfR and Ret-He detected LID in 148 and 135 donors respectively. In comparison to sTfR, Ret-He had sensitivity of 92.7%, a specificity of 97.16%, PPV of 93.1% and NPV of 96.3%. Serum Ferritin, TIBC and serum Iron had comparatively lower sensitivity of 87.16%, 79.7% and 77.7% respectively. CONCLUSION: Ret-He can be used as a routine screening test to detect LID in blood donors. This could provide an opportunity to make appropriate and timely interventions like dietary changes or drug supplementation.

Oral administration of the LSD1 inhibitor ORY-3001 increases fetal hemoglobin in sickle cell mice and baboons.

Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD). More effective strategies to increase HbF are needed because the current standard of care, hydroxyurea, is not effective in a significant proportion of patients. Treatment of the millions of patients projected worldwide would best be accomplished with an orally administered drug therapy that increased HbF. LSD1 is a component of corepressor complexes that repress γ-globin gene expression and are a therapeutic target for HbF reactivation. We have shown that subcutaneous administration of RN-1, a pharmacological LSD1 inhibitor, increased γ-globin expression in SCD mice and baboons, which are widely acknowledged as the best animal model in which to test the activity of HbF-inducing drugs. The objective of this investigation was to test the effect of oral administration of a new LSD1 inhibitor, ORY-3001. Oral administration of ORY-3001 to SCD mice (n = 3 groups) increased γ-globin expression, Fetal Hemoglobin (HbF)-containing (F) cells, and F reticulocytes (retics). In normal baboons (n = 7 experiments) treated with ORY-3001, increased F retics, γ-globin chain synthesis, and γ-globin mRNA were observed. Experiments in anemic baboons (n = 2) showed that ORY-3001 increased F retics (PA8695, predose = 24%, postdose = 66.8%; PA8698: predose = 13%, postdose = 93.6%), γ-globin chain synthesis (PA8695: predose = 0.07 γ/γ+ß, postdose = 0.20 γ/γ+ß; PA8698: predose = 0.02 γ/γ+ß, postdose = 0.44 γ/γ+ß), and γ-globin mRNA (PA8695: predose = 0.06 γ/γ+ß, postdose = 0.18 γ/γ+ß; PA8698: predose = 0.03 γ/γ+ß, postdose = 0.33 γ/γ+ß). We conclude that oral administration of ORY-3001 increases F retics, γ-globin chain synthesis, and γ-globin mRNA in baboons and SCD mice, supporting further efforts toward the development of this drug for SCD therapy.

Influence of combined iron supplementation and simulated hypoxia on the haematological module of the athlete biological passport.

The integrity of the athlete biological passport (ABP) is underpinned by understanding normal fluctuations of its biomarkers to environmental or medical conditions, for example, altitude training or iron deficiency. The combined impact of altitude and iron supplementation on the ABP was evaluated in endurance-trained athletes (n = 34) undertaking 3 weeks of simulated live-high: train-low (14 h.d-1 , 3000 m). Athletes received either oral, intravenous (IV) or placebo iron supplementation, commencing 2 weeks prior and continuing throughout hypoxic exposure. Venous blood was sampled twice prior, weekly during, and up to 6 weeks after altitude. Individual ABP thresholds for haemoglobin concentration ([Hb]), reticulocyte percentage (%retic), and OFF score were calculated using the adaptive model and assessed at 99% and 99.9% specificity. Eleven athletes returned values outside of the calculated reference ranges at 99%, with 8 at 99.9%. The percentage of athletes exceeding the thresholds in each group was similar, but IV returned the most individual occurrences. A similar frequency of abnormalities occurred across the 3 biomarkers, with abnormal [Hb] and OFF score values arising mainly during-, and %retic values mainly post- altitude. Removing samples collected during altitude from the model resulted in 10 athletes returning abnormal values at 99% specificity, 2 of whom had not triggered the model previously. In summary, the abnormalities observed in response to iron supplementation and hypoxia were not systematic and mostly in line with expected physiological adaptations. They do not represent a uniform weakness in the ABP. Nevertheless, altitude training and iron supplementation should be carefully considered by experts evaluating abnormal ABP profiles.