Reticulocyte Production Research Articles

Genetic variation in interleukin-7 is associated with a reduced erythropoietic response in Kenyan children infected with Plasmodium falciparum

BackgroundSevere malarial anemia (SMA) is a leading cause of malaria-related morbidity and mortality in children. The genetic factors that influence development of SMA and inefficient erythropoiesis, a central pathogenic feature of SMA, are only partially understood.MethodsWe performed a pilot Genome-wide Association Study (GWAS) on children with Plasmodium falciparum. The GWAS was performed using the Illumina® Infinium® HD Super Assay in conjunction with Illumina’s® Human Omni2.5-8v1 BeadChip (with > 2.45 M markers). Data were analyzed using single SNP logistic regression analysis with an additive model of inheritance controlling for covariates. Results from our pilot global genomics study identified that variation in interleukin (IL)-7 was associated with enhanced risk of SMA. To validate this finding, we investigated the relationship between genotypes and/or haplotypes of two single nucleotide polymorphisms (SNPs) in IL7 [72194 T/C and − 2440 A/G] and susceptibility to both SMA and inefficient erythropoiesis [i.e., reticulocyte production index (RPI) < 2.0 in anemic children (Hb < 11.0 g/dL). Children presenting with P. falciparum malaria (< 3 years, n = 883) were stratified into two groups: Uncomplicated malaria (UM, n = 718) and SMA (n = 165).ResultsRegression modeling, controlling for anemia-related confounders, revealed that carriage of the TC genotype at position 72194 T/C was associated with enhanced susceptibility to inefficient erythropoiesis (OR = 1.90; 95% CI 1.09–3.30; P = 0.02) as was homozygous CC (OR 5.14; 95% CI = 1.20–21.99; P = 0.03). Consistent with this finding, individuals with the CA (72194C/−2440A) haplotype had an increased risk of inefficient erythropoiesis (OR = 1.90; 95% CI = 1.10–3.30; P = 0.02), whereas TA haplotype carriers had marginal protection against inefficient erythropoiesis (OR = 0.24; 95% CI = 0.06–1.21; P = 0.05). These observations were supported by Cochran-Armitage trend test for inefficient erythropoiesis (CA > TA > CG; P < 0.01). Although none of the genotype and/or haplotypic variants were significantly associated with SMA, the direction of the risk profiles were consistent with the erythropoiesis results.ConclusionTaken together, variation in IL7 is associated with erythropoietic responses in children with falciparum malaria, a central physiological feature contributing to development of SMA.

Dengue Hemorrhagic Fever: Past, Present, and Future

Abstract: Dengue viral infection is a global disease with a spectrum of clinical manifestations mild fever to severe disease both dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DHF is severe form of dengue fever (DF), which can be life-threatening. Climate changes is not the only factor that affects dengue transmission, but also globalization changes includes travel and trade. The pathogenesis of dengue infection is complex. The mechanism involved antibody-dependent enhancement, NS1 and its antibodies, T cells, and DENV genomics. There are several novel methods to detect the presence of dengue virus in the body of infected patients. These include ELISA-specific IgM and IgG detection, detection of monoclonal antibodies and mosquito cell strains, and PCR reverse transcriptase detection. Several trials found novel methods to predict the severity of dengue hemorrhagic fever earlier. These include platelet count, Aspartate aminotransferase / platelet count index (APRI) Index, serum chymase level, serum cytokine/chemokine profile, Tropomyosin-alpha 1 (TPM 1), Reticulocyte Production Index (RPI), and Immature Platelet Fraction (IPF). Several pharmacological therapies are known to have potential antidengue effect. Some of these are corticosteroids, antimalarial drugs, doxycycline and tetracycline, anticholesterol drugs, IVIG, celgosivir, balapiravir, pentoxifylline and calcium supplementation. Some natural products are known to have activity against Aedes aegypti through antiviral mechanisms, larvacidal activity, mosquitocidal, and mosquito repellants. It can be developed as the latest therapy of dengue hemorrhagic fever on the future. The objective of this paper is to provide new insight about the development of dengue hemorrhagic fever related to the history of its distribution, pathogenesis, and the latest developments related to detection methods, severity prediction methods, and the management of dengue hemorrhagic fever on the future. Keywords: globalization changes, novel detection methods, severity prediction methods, latest development in DHF therapy

Hepcidin and diabetes are independently related with soluble transferrin receptor levels in chronic dialysis patients

Background: Soluble transferrin receptor (sTfR) is a biomarker of erythropoiesis, which is often impaired in dialysis patients. The aim of our study was to evaluate sTfR levels in chronically dialyzed patients and assess potential determinants of its levels.Methods: We performed a cross-sectional study by evaluating 246 end-stage renal disease patients undergoing dialysis and 32 healthy controls. Circulating levels of interleukin (IL)-6, C-reactive protein (CRP), tumor necrosis factor (TNF)-α, hepcidin, sTfR, growth differentiation factor 15 (GDF15), and traditional iron metabolism markers were measured, as well as hemogram parameters. Clinical data was obtained from all patients.Results: Compared to controls, patients presented similar values of sTfR, reticulocytes and reticulocyte production index (RPI), and significantly higher levels of IL-6, CRP, ferritin, hepcidin, TNF-α, and GDF15. Iron, transferrin, hemoglobin levels, erythrocyte count, mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC) values were significantly lower in dialysis group. Within patients, sTfR values were higher in diabetic patients and were positively and significantly correlated with reticulocytes and erythrocytes, RPI, and therapeutic doses of erythropoiesis stimulating agents (ESA) and intravenous iron; and inversely and significantly correlated with circulating iron, ferritin, transferrin saturation, hepcidin, MCH, and MCHC. In multiple linear regression analysis, ESA dose, RPI, serum iron, diabetes, and hepcidin levels were independently associated with sTfR levels in dialysis patients and, thus, with erythropoiesis.Conclusion: Our data suggest that, besides RPI and ESA dose, diabetes and hepcidin are closely related to erythropoiesis in dialysis patients. The influence of diabetes on sTfR levels deserves further investigation.

Cobalamin deficiency presenting with thrombotic microangiopathy (TMA) features: A systematic review

IntroductionCobalamin deficiency may result in hematologic characteristics similar to thrombotic microangiopathy (TMA). To facilitate diagnosis, we reviewed reported cases of acquired cobalamin deficiency presenting with TMA features (c.def-TMA). MethodsA literature search identified reports of c.def-TMA. Deficiency was defined as B12 levels of <118 pmol/L. Corrected reticulocyte counts and reticulocyte production indexes were calculated. Clinical features were presented as proportion abnormal and results summarized as medians and interquartile ranges (IQR). ResultsPatient level data was extracted from 41 identified cases. Median age (years) was 43 (30–55) with 21/41 (51%) being female. Cobalamin deficiency was noted in 35/40 (87.5%) but fold increases in MMA and HC were 30 and 6, respectively. The etiology was pernicious anemia in 28/41 (68%) cases. Anemia was both universal and severe, with hemoglobin levels of 55 g/L (4.7–6.6). Hypersegmented neutrophils were noted in 23/37 (62%), schistocytes in 29/38 (76%) and median LDH levels 3981 U/L (2004–5467). The RPI was <3.0% in all patients. Thrombocytopenia occurred in 33/41 (80.5%) with a median platelet count of 91 × 109/L (42–112). Plasma infusion or exchange was initiated in 14/41 (34%) with associated complications in 2 cases. ConclusionReticulocytopenia (RPI of <3.0%) was a universal finding that aids in differentiating c.def-TMA from other causes of hemolysis. C.def-TMA was associated with severe anemia, generally mild-moderate thrombocytopenia, and significant elevations in LDH.

Особенности показателей красной крови у пожилых пациентов с ишемической болезнью сердца и патологией клапанного аппарата до и после хирургической коррекции

To study indexes of red blood in elderly patients with ischemic heart disease (IHD) and pathology of heart valves before and after surgical correction, including the relationship with position and type of the prosthesis. 79 patients aged 61 [53;67] were evaluated. The patients had acquired heart valve disease of rheumatic origin, manifestations of heart connective провоtissue dysplasia, and IHD and were admitted to a cardiosurgical hospital. Blood tests were performed using a hematological analyzer, Cell-Dyn 3700 (USA), before the surgery and on days 5 and 10 following the surgical correction. Reticulocytes were counted using a unified manual method in a blood smear stained with brilliant cresyl blue and expressed per 1000 red cells. Reticulocyte indexes (corrected reticulocyte count, CRC, and reticulocyte production index, RPI) were computed. Elderly patients with a combination of acquired heart disease and mitral valve disease were characterized by preoperative morphological heterogenicity of red blood cells as evidenced by RDW. Postoperative anemia associated with significant decreases in mean hemoglobin concentration, red cell count, and hematocrit, and increased morphological heterogenicity of red cell pool were observed in patients with IHD and heart valve pathology on days 5 and 10 of the postoperative period. During the early postoperative period, more pronounced changes in red cell heterogenicity were observed in patients with implanted disk mechanical prosthesis, particularly in mitral position. In the early postoperative period following the surgical correction, elderly patients with IHD and valve disease were characterized with a low reticulocyte production index, which indicated reduced proliferative activity of bone marrow. The fastest stabilization of red blood indexes occurred in patients after coronary bypass.

High serum adiponectin is associated with anemia development in chronic kidney disease: The results from the KNOW-CKD study

BackgroundAdiponectin is an adipokine secreted by adipocytes. A low adiponectin level is a significant risk factor of diabetes mellitus and cardiovascular disease. Recent studies have shown that adiponectin is negatively associated with hematopoiesis and predicts the development of anemia in the general population. In chronic kidney disease (CKD) patients, circulating adiponectin level is paradoxically elevated and the role of adiponectin is complex. Therefore, we evaluated the relationship between adiponectin and anemia in these patients. MethodsThis prospective longitudinal study included 2113 patients from the KNOW-CKD study (KoreaN cohort study for Outcome in patients With CKD), after excluding 125 without data on adiponectin levels. Hemoglobin levels were measured yearly during a mean follow-up period of 23.7 months. Anemia was defined as hemoglobin levels of <13.0 and 12.0 g/dL for men and women, respectively. ResultsMean patient age was 53.6 ± 12.2 years, and 1289 (61%) were men. The mean estimated glomerular filtration rate (eGFR) was 50.4 ± 30.2 mL min−1 1.73 m−2. Serum adiponectin level was inversely associated with body mass index, eGFR, log-transformed C-reactive protein, and positively with Charlson comorbidity index, urine protein to creatinine ratio, and high density lipoprotein cholesterol. In addition, serum adiponectin level was also negatively correlated with hemoglobin level and reticulocyte production index in both men and women. In multivariable linear regression analysis after adjustment of multiple confounders, adiponectin was negatively associated with hemoglobin (men, β = −0.219, P < .001; women, β = −0.09, P = .025). Among 1227 patients without anemia at baseline, 307 newly developed anemia during the follow-up period. In multivariable Cox regression analysis after adjustment of confounders, high adiponectin level was significantly associated with an increased risk of incident anemia (per 1 µg/mL increase, hazard ratio, 1.02; 95% confidence interval 1.01–1.04; P = .001). ConclusionsA high serum adiponectin level is independently associated with a low hemoglobin level and predicts the development of anemia in patients with CKD. These findings reveal the potential role of adiponectin in CKD-related anemia.

In a Mouse Model of Sepsis, Hepcidin Ablation Ameliorates Anemia More Effectively than Iron and Erythropoietin Treatment.

Intensive care unit (ICU) anemia is an extreme version of anemia of inflammation that occurs commonly in critically ill patients and is associated with increased morbidity and mortality. Currently available therapies for ICU anemia have shown inconsistent efficacies in clinical trials. We conducted a systematic study of the effects of early versus delayed iron (Fe) and/or erythropoietin (EPO) therapy in our previously characterized mouse model of ICU anemia based on an injection of heat-killed Brucella abortus. To study the effects of ongoing inflammation on the response to therapy, inflamed wild-type (WT) and hepcidin knockout (HKO) mice were treated at either early (days 1 and 2) or delayed (days 7 and 8) time points after the inflammatory stimulus. In the early treatment group, Fe and/or EPO therapy did not increase hemoglobin (Hgb) levels or reticulocyte production in either the inflamed WT or HKO groups. In the delayed treatment group, combination Fe + EPO therapy did increase Hgb and reticulocyte production in WT mice (mean ΔHgb in WT saline group -9.2 g/dL vs. Fe/EPO -5.5 g/dL; P < 0.001). The HKO mice in the delayed treatment group did not improve their Hgb, but HKO mice in all treatment groups developed a milder anemia than the WT mice. Our findings indicate that combination Fe + EPO therapy is effective in partially reversing ICU anemia when administered after the phase of acute inflammation. Hepcidin ablation alone was more effective in attenuating ICU anemia than Fe + EPO therapy, which indicates the potential of antihepcidin therapeutics in treating ICU anemia.

Reticulocytic indices in assessing erythropoiesis effectiveness in multiple myeloma.

e19510 Background: The purpose of the study was to analyze changes in reticulocytic indices and their role in assessing the effectiveness of erythropoiesis in patients with multiple myeloma. Methods: Thestudy included 10 patients with primary multiple myeloma (MM) and 16 patients with recurrent MM, stage II-III. Parameters of the peripheral blood were studied: Hb, RBC, MCV, MCH, RBC-He (red cell hemoglobin content), RET (number and percentage), IRF (immature reticulocyte fraction), high (HFR), median (MFR) and low (LFR) fluorescence reticulocytes, RET-He (reticulocyte hemoglobin content), RPI (reticulocyte production index). Results: Anemia was found in21 (80.8%) patients before treatment: grade I in 30.8%, II in 46.1%, III in 3.8%; normocytic hypochromic anemia – 6, normocytic normochromic – 15. Anemia detection rate in primary MM was 60.0%, recurrent – 93.7%. RET levels in grade I-II anemia were close to the norm, but IRF was increased due to MFR increase by 3.7 times and HFR – by 4.7 times. Grade III anemia: RET number decreased by 1.6 times due to IRF reduction by 2 times, in particular MFR and HFR fractions. As a result, RET-He was decreased to varying degrees in all cases. Patients with complete or partial remission showed no significant changes in erythrocyte parameters, but had RET level increased by 2.5 times, IRF – by 9 times due to MFR increase by 6.5 times and HFR by 30.8 times, RPI - by 2.0 times. The data showed the effectiveness of erythropoiesis during anticancer therapy. We did not observe signs of increasing anemia syndrome in patients with stabilization or patients resistant to treatment, as well as in patients with initial grade III anemia. Hb, RBC, RET and RPI were unchanged, but the ratio of fractions changed: the number of mature ones - LFR increased by 1.4 times and young ones - HFR decreased by 2.5 times, RBC-He were increased and RET-He doubled compared to the levels before treatment, which indicated the activation of processes of erythrokaryocyte hemoglobinization. Conclusions: RET, IRF, RET-He and RPI indices adequately reflect the process of recovery of erythropoiesis and its effectiveness in real time which is extremely important in tumor therapy monitoring.

Rituximab Subcutaneous Formulation (SC-R) Is Safe, Effective and Cost-Saving in Patients Suffering from Autoimmune Hemolytic Anemia (AIHA)

Rituximab, a monoclonal antibody directed against the CD20 antigen expressed on B cells, has been shown to be effective in AIHA, both in idiopathic and secondary including those associated with autoimmune and lymphoproliferative disorders as well as in Evans syndrome. At standard dose of 375 mg/mq weekly for a median of 4 weeks , overall survival (OS), complete response (CR), desease free survival (DFS) were respectively OR 83-87%,CR 54-60% DFS 72% at one and 56% at two years. Moreover rituximab re-treatment is effective and some patients responded to re-treatment more than once. Because is available today a Rituximab solution for subcutaneous injection, we have used this formulation for the treatment of 6 patients (pts) suffering from AIHA.Methods6 pts (M 2, F 4 ) were enrolled in this study. Median age was 58.3 yr (range, 52-82) 2 out of 6 pts were idiopathic and the remaining 4 were associated with chronic lymphoproliferative syndromes. 2 out of 6 pts had relapsed after a first-line treatment with intravenous rituximab and steroids.For 2 pts mean Hb value and Ht at presentation were 6.2 g/dL ± 1.2, and 26 mL/dL, Median reticulocyte percentage was 10%, and median reticulocyte production index was 2.9 times basal. 24 % of cases had an initial reticulocyte count less than 4%, and 40% had an initial reticulocyte production index less than 2.0 times basal. These reticulocytopenic patients were prevalent in secondary cases. Pts had altered hemolysis markers and direct antiglobulin test (DAT) was positive for both complement and IgG ( IgA 1 pts). All cases had a bone marrow examination during hospitalization Erythroid hypoplasia was seen only in CLL pts.6/6 pts had serial reticulocyte measurements,Rituximab subcutaneous formulation was administered at a fixed dose of 1400 mg weekly for 4 weeks; Before starting MabThera subcutaneous injections, all patients received beforehand, a full dose of Rituximab by intravenous infusion. Premedication consisting of an anti-pyretic and an antihistaminic was given orally in the evening before and the morning of the subcutaneous administration in order to reduce the time to stay in day hospital . All pts received prednisone 1 mg/Kg/day /for 30 days ; for these reason premedication with glucocorticoids was avoided.ResultsAll pts completed treatment. No major infusion related side effects to subcutaneous Rituximab SC-R) were observed. Response criteria were defined as follows: Complete Response (CR): Hb >10 g/dl or Hb increase >1.5 g/dl, resolution of symptoms of anemia, transfusion independent; Partial Response (PR): Hb > 9 g/dl or Hb increase of 1-1.5 g/dl. improvement in symptoms of anemia, transfusion independent; NR (failure to meet CR/PR). 100% were eligible for response.Complete Responses were seen in 6/6 pts .At the end of treatment DAT became negative in 4/6 pts , concentration of lactic dehydrogenase , total bilirubin and indirect bilirubin began to decrease at 12 days after the first dose of rituximab, and decreased to normal range after 22 days. 3 patients required packed red cell transfusions before starting SC-R and all became transfusion-free. A moderate hemolysis still persisted only in one patient. rh-Epo has been administered in three pts initially reticulocytopenic. The reticulocyte production index rapidly increased, indicating a marrow erythropoietic response to rh-EpoConclusionOur experience demonstrates that SC- Rituximab is an effective and safe alternative to IV formulation both in the first line and in the relapsed pts. SC-Rituximab shortens the treatment time significantly, enabling administration over approximately 5 minutes compared with 3 hours during IV infusion. The ready-to-use SC formulation significantly reduce pharmacy time and the impact on hospital resources as medicine preparation time and hospital staff time per administration are significantly reduced also in our hands. DisclosuresNo relevant conflicts of interest to declare.

Effect of Lead Exposure on the Status of Reticulocyte Count Indices among Workers from Lead Battery Manufacturing Plant.

Earlier studies conducted on lead-exposed workers have determined the reticulocyte count (RC) (%), but the parameters of Absolute Reticulocyte Count (ARC), Reticulocyte Index (RI), and Reticulocyte Production Index (RPI) were not reported. This study assessed the effect of lead (Pb) exposure on the status of reticulocyte count indices in workers occupied in lead battery plants. The present cross-sectional study was carried out on 391 male lead battery workers. The blood lead levels (BLL) were determined by using an Atomic Absorption Spectrophotometer. The RC (%) was estimated by using the supravital staining method. The parameters, such as ARC, RI, and RPI, were calculated by using the RC (%) with the red cell indices (RBC count and hematocrit). The levels of RBC count and hematocrit were determined by using an ABX Micros ES-60 hematology analyzer. The levels of reticulocyte count indices - RC (%), ARC, RI, and RPI significantly increased with elevated BLL. The association between BLL and reticulocyte count indices was positive and significant. The results of linear multiple regression analysis showed that the reticulocyte count (β = 0.212, P < 0.001), ARC (β = 0.217, P < 0.001), RI (β = 0.194, P < 0.001), and RPI (β = 0.208, P < 0.001) were positively associated with BLL. The variable, smoking habits, showed a significant positive association with reticulocyte count indices: RC (%) (β = 0.188, P < 0.001), ARC (β = 0.174, P < 0.001), RI (β = 0.200, P < 0.001), and RPI (β = 0.151, P < 0.005). The study results revealed that lead exposure may cause reticulocytosis with an increase of reticulocyte count indices.

Dysregulated Erythropoietin, Hepcidin, and Bone Marrow Iron Metabolism Contribute to Interferon-Induced Anemia in Hepatitis C.

Anemia is a complication of interferon-containing hepatitis C treatments. We characterized effects of interferon-based therapy on hepcidin and erythropoietin (EPO) production, iron metabolism, hemolysis, and hematopoiesis. Standard hemopoiesis [reticulocyte hemoglobin (Hb), reticulocyte production index (RPI), free Hb, and haptoglobin], iron biochemistry, hepcidin, and EPO levels were measured in 10 subjects over 12 weeks. There was a rapid decline in Hb during treatment, from a mean pretreatment (t = 0 weeks) Hb of 158.6 to 125.2 g/L at week 4 (P = 0.003) and 122.8 g/L at week 12 (P = 0.005). Paradoxically, the RPI (a measure of bone marrow responsiveness to EPO) decreased on initiation of hepatitis C virus treatment from 0.78% to 0.53% (P = 0.04). Despite worsening anemia, there was no significant increase in EPO levels. Hepcidin levels increased to >20 nM in 3 subjects from 5.8 to 27.5 nM (P = 0.009) compared with 9.6 to 12.3 nM (P = 0.5) for the remainder of subjects. Hepcidin levels peaked at week 1 before returning to baseline levels at week 4. Subjects who responded with a rise in serum hepcidin levels to >20 nM had a significantly greater drop in Hb (27.2 g/L, P = 0.008) and reticulocyte Hb (-1.4 g/L, P = 0.013) compared with the subjects who did not exhibit any change in hepcidin production. In conclusion, 30% of subjects treated with interferon exhibited significant transient increase in serum hepcidin levels, which was associated with more extreme anemia and decreased iron availability as evidenced by decreased reticulocyte Hb. In addition, there was a failure to upregulate EPO production in response to anemia and hemolysis ( https://clinicaltrials.gov trial NCT01726400).

Prognostic significance of endogenous erythropoietin in long-term outcome of patients with acute decompensated heart failure.

Although previous reports suggest that an elevated endogenous erythropoietin (EPO) level is associated with worse clinical outcomes in chronic heart failure (HF) patients, the prognostic implication of EPO in patients with acute decompensated HF (ADHF) and underlying mechanisms of the high EPO level in severe HF patients who have a poor prognosis remain unclear. We examined 539 consecutive ADHF patients with EPO measurement on admission from our registry. During a median follow-up period of 329 days, a higher EPO level on admission was independently associated with worse clinical outcomes [hazard ratio (HR) 1.25, 95% confidence interval (CI) 1.06-1.48, P = 0.008], and haemoglobin level was the strongest determinant of EPO level (P < 0.001), whereas estimated glomerular filtration rate (eGFR) was not significant in multivariate regression analysis. In the anaemic subgroup of 318 patients, a higher EPO level than expected on the basis of their haemoglobin level was related to increased adverse events (HR 1.63, 95% CI 1.05-2.49, P = 0.028). Moreover, estimated plasma volume excess rate was positively associated with EPO level (P = 0.003), and anaemic patients with a higher than expected EPO level tended to have a higher estimated plasma volume excess rate and plasma lactate level, and lower systemic oxygen saturation level with the preservation of the reticulocyte production index than those with a lower than expected EPO level. A high EPO level predicts long-term worse clinical outcomes in ADHF patients, independent of anaemia and impaired renal function. Anaemia and hypoxia due to severe congestion may synergistically contribute to a high EPO level in high-risk HF patients.

Impaired renal endothelial nitric oxide synthase and reticulocyte production as modulators of hypertension induced by rHuEPO in the rat

Our aim was to study the effect of a broad range of recombinant human erythropoietin (rHuEPO) doses on hematological and biochemical parameters, blood pressure (BP), renal function and damage in the rat, focusing on endothelial nitric oxide synthase (eNOS) and hypoxia-inducible factors (HIFs).Male Wistar rats were divided in 5 groups receiving different doses of rHuEPO (100, 200, 400 and 600IU/kg body weight (BW)/week) and saline solution (control), during 3weeks. Blood and 24h urine were collected to perform hematological and biochemical analysis. BP was measured by the tail-cuff method. Kidney tissue was collected to mRNA and protein expression assays and to characterize renal lesions.A dose-dependent increase in red blood cells count, hematocrit and hemoglobin levels was found with rHuEPO therapy, in rHuEPO200, rHuEPO400 and rHuEPO600 groups. Increased reticulocyte count was found in rHuEPO400 and rHuEPO600 groups. BP raised in all groups receiving rHuEPO. The rHuEPO200 and rHuEPO600 groups presented increased kidney protein levels of HIF2α, a reduction in kidney protein levels of eNOS, and the highest grade of vascular and tubular renal lesions.Our study showed that rHuEPO-induced hypertension is present before significant hematological changes occur and, therefore, might involve direct (renal) and indirect (hematological) effects, which varies according to the dose used. The presence of renal hypoxia reduces eNOS activity. Excessive erythrocytosis increases blood hyperviscosity, which can be modulated by an increase in reticulocytes. Hypertension leads to early renal damage without alterations in traditional markers of renal function, thus underestimating the serious adverse effects and risks.

Reduced Hsp70 and Glutamine in Pediatric Severe Malaria Anemia: Role of Hemozoin in Suppressing Hsp70 and NF-κB activation.

Severe malarial anemia [SMA, hemoglobin (Hb) <5.0 g/dL] is a leading cause of global morbidity and mortality among children residing in Plasmodium falciparum transmission regions. Exploration of molecular pathways through global gene expression profiling revealed that SMA was characterized by decreased HSPA1A, a heat shock protein (Hsp) 70 coding gene. Hsp70 is a ubiquitous chaperone that regulates Nuclear Factor-kappa B (NF-κB) signaling and production of pro-inflammatory cytokines known to be important in malaria pathogenesis (e.g., IL-1β, IL-6 and TNF-α). Since the role of host Hsp70 in malaria pathogenesis is unexplored, we investigated Hsp70 and molecular pathways in children with SMA. Validation experiments revealed that leukocytic HSP70 transcripts were reduced in SMA relative to non-severe malaria, and that intraleukocytic hemozoin (PfHz) was associated with lower HSP70. HSP70 was correlated with reticulocyte production and Hb. Since glutamine (Gln) up-regulates Hsp70, modulates NF-κB activation, and attenuates over-expression of pro-inflammatory cytokines, circulating Gln was measured in children with malaria. Reduced Gln was associated with increased risk of developing SMA. Treatment of cultured peripheral blood mononuclear cells (PBMCs) with PfHz caused a time-dependent decrease in Hsp70 transcripts/protein, and NF-κB activation. Gln treatment of PBMCs overcame PfHz-induced suppression of HSP70 transcripts/protein, reduced NF-κB activation, and suppressed over-expression of IL-1β, IL-6 and TNF-α. Findings here demonstrate that SMA is characterized by reduced intraleukocytic HSP70 and circulating Gln, and that PfHz-induced suppression of HSP70 can be reversed by Gln. Thus, Gln supplementation may offer important immunotherapeutic options for futures studies in children with SMA.

Congenital Dyserythropoietic Anemias: Molecular Diagnosis and Diagnostic Approach in a Cohort of Italian Patients

Introduction. Congenital dyserythropoietic anemias (CDAs) are hereditary rare erythropoietic disorder characterized by distinct morphological abnormalities of the bone marrow cells, ineffective erythropoiesis and systemic iron overload. This conditions is characterized by a maturation arrest during erythropoiesis with a reduced reticulocyte production in contrast with erythroid hyperplasia in bone marrow. Three types of CDA are known: types 1, 2 and 3.The identification of their causative genes provided evidence that these conditions have different molecular mechanisms that induce abnormal cell maturation and division. We describe the clinical and laboratory manifestations, the diagnosis procedure, the therapeutic approaches and the clinical phenotype in some cases of congenital dyserythropoietic anemia (CDAs) in Italian patients. The molecular analysis allow us to identify several genetic variants some of which have never been described previously. This report highlights the importance of recognizing CDAI even in countries where thalassemia is common. Among the different tools genetic study of CDA-related genes remains the main approach for pursuing the right diagnosis.Methods. Peripheral blood samples from 100 normal adults and 20 patients with different types of anemia were collected. Blood samples were analyzed by EMA binding test and by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate ( SDS-PAGE). SDS PAGE was carried out in a 3.5-17% exponential gradient gel in Fairbanks buffer Genomic DNA was extracted from mononuclear cells of peripheral blood samples, by using a phenol-chloroform method. Polymerase chain reaction (PCR) products were sequenced directly using Big-Dye terminator 3.1 cycle sequencing kit and run on ABI PRISM 3130 DNA analyzer. The bone marrow of all patients was analysed with light or electron microscopy. The diagnosis of CDAs was based on the presence of mild to moderate anemia ineffective erythropoiesis and bone marrow erythroblasts morphological abnormalities. SEC23B, CDAN1, KLF1, BCL11A gene sequencing analysis was performed to highlight the presence of nucleotide variations and their relationship with the clinical presentation.Results and Discussion. We collected blood samples from 20 Italian patients with suspect of CDAs and 100 samples belonging to the healthy control subjects.None mutation in the genes analyzed was found in the samples controls.We identified SEC 23 B mutations in 4 out of 20 patients analysed. In two patients with suspect of CDAII we found two nucleotide variations; SDS PAGE in these patients identified the presence of abnormal band 3 and the EMA binding test resulted pathologic. Bone marrow (BM) light microscopy revealed more than 10% mature binucleated erythroblasts. In the patients suspected of CDA1, several gene variants were found in the CDAN1 gene, some described in the literature as mutations or polymorphisms, others of uncertain significance. In a patient diagnosed with CDA1 two novel mutations was found. EMA binding test resulted normal in all patients with CDA1. Light microscopy of the BM of CDA1 patients showed erythroid hyperplasia, presence of internuclear bridges between intermediate erythroblasts and characteristic pattern of spongy "swiss cheese" hetrochromatin in electron microscopy. None KLF1 and BCL11A mutations correlated with atypical CDAs was identified. In this last patients the EMA binding test resulted normal. CDAs are rare clinical hereditary disorders whose correct diagnosis is often delayed to adolescence or adulthood, when significant iron overload and end organ damage may have been occurred. Patients are often misdiagnosed with other congenital haemolytic anaemias. Inaccurate diagnosis can lead to inappropriate therapies, such as iron supplements, aggressive transfusion or splenectomy. However even if the gold standard for the CDAs diagnosis is the electronic microscopy, the identification of the mutated genes involved in the majority of CDAs patients made in recent years has improved the possibility of detecting these diseases. DisclosuresNo relevant conflicts of interest to declare.

Assessment of Bone Marrow Function in Sickle Cell Anaemia Patients Using Corrected Reticulocyte Counts

Introduction: Chronic hemolysis occurs in sickle cell anemia as a result of recurrent sickling and other abnormalities of the red blood cells including eryptosis. Exuberant reticulocytosis is anticipated to partially compensate for the resultant anemia. Sickle cell anemia patients may also have aplastic crisis, bone marrow (BM) infarction and erythropoietin deficiency which could lead to reticulocytopenia despite the anemia. High degree of reticulocytosis among asymptomatic infants with sickle cell anemia has been associated with an increased risk of death or stroke during childhood. Assessment of BM function in sickle cell anemia is important due to potential complications associated with both under-activity and hyperactivity. This study aimed at evaluating the erythropoietic function of the BM in steady state sickle cell anemia using corrected reticulocyte counts.Methods: This study was carried out at the hematology clinic in the University College Hospital, Ibadan. HbSS patients in steady state were recruited from the hematology clinic. Local ethical committee approval was obtained and all participants gave written informed consent. Patients with M. tuberculosis, Hepatitis B, HIV and P. falciparum infection were excluded. Peripheral blood samples were analyzed using Sysmex Ki-X21 for complete blood count (CBC) and standard point of care for serum electrolytes and liver function tests. The glomerular filtration rates were calculated using the Cockcroft-Gault formula. Reticulocyte counts were determined manually using fresh samples from K2 EDTA bottles and methylene blue stain. Two drops of stain were mixed with two to four volumes of anticoagulated blood and incubated at 37ºC for 15 minutes. Afterwards, the cells were re-suspended and blood films were made. Corrected reticulocyte count and reticulocyte production index were calculated. Participants were categorized according to corrected reticulocyte counts of greater than or less than 2.5%. Univariate and multivariate analyses were performed to determine variables associated with corrected reticulocyte count <2.5%.Results: 92 HbSS patients were recruited with a mean (SD) age of 19.6 (5.8) years. There was no correlation between age and eGFR (p-value: 0.227). Median (range) reticulocyte count, corrected reticulocyte count and reticulocyte production index were 5.5 (0.5 - 29.9), 3.3 (0.1 - 17.1) and 1.7 (0.2 - 8.6) respectively. 40 (43.5%) patients had corrected reticulocyte count <2.5% and 52 (56.5%) had a corrected count >2.5%. Those corrected reticulocyte count <2.5% were older (p: 0.013), taller (p: 0.041) and had higher aspartate transaminase (AST) levels (p: 0.006) than those with corrected counts >2.5% (Table 1). CBC parameters were not different when compared between both groups. Results of multivariate logistic regression analysis carried out showed that only AST was independently linked with corrected reticulocyte count <2.5% (R2: 0.172, p-value: 0.001) (Table 2).Table 1Factors Associated with Low Reticulocyte CountCorrected count<2.5%Corrected count>2.5%p-ValueAge (Mean, SD)21.4 (6.3)18.4 (5.0)0.013Gender (N, %)Male22 (55.0)28 (53.8)0.912Female18 (45.0)24 (46.2)Height (Mean, SD)1.6 (0.1)1.5 (0.1)0.041BMI (Mean, SD)18.7 (3.1)18.7 (3.0)0.753GFR (Mean, SD)64.3 (37.7)66.4 (29.3)0.453Bilirubin (Mean, SD)1.7 (1.1)1.9 (2.6)0.674AST (Mean, SD)22.5 (13.5)14.5 (6.6)0.006ALT (Mean, SD)13.4 (7.7)14.4 (11.1)0.876Table 2Independent Predictors of Corrected Reticulocyte Count <2.5%or95% CIp-ValueAge1.080.97 - 1.210.169Height19.80.11 - 366.100.259AST1.101.04 - 1.170.002Hemoglobin1.000.97 - 1.020.872R2: 0.172, p: 0.001Conclusion: Despite corrected reticulocyte count <2.5% in about half of the patients, there were similar hematological parameters and eGFR in both groups of patients. AST is a marker of hemolysis and low ALT rules out hepatic involvement. Since only 17.2% of the variability in BM response as assessed by corrected reticulocyte count could be accounted for by variables included in this study, there is a need to further evaluate the BM function of sickle cell patients to establish the causes of corrected reticulocyte count <2.5% in the setting of anemia, having ruled out erythropoietin as well as iron, folate or cobalamin deficiencies. This will aid the development of a functional algorithm for the individualized management of sickle cell disease patients with anemia. DisclosuresNo relevant conflicts of interest to declare.

Absolute Reticulocyte Count Acts as a Surrogate for Fetal Hemoglobin in Infants and Children with Sickle Cell Anemia.

Hemoglobin switching is largely complete in humans by six months of age. Among infants with sickle cell anemia (HbSS, SCA), reticulocytosis begins early in life as fetal hemoglobin (HbF) is replaced by sickle hemoglobin (HbS). The objective of this study was to determine if absolute reticulocyte count (ARC) is related to HbF levels in a cohort of pediatric SCA patients. A convenience sample of 106 children with SCA between the ages of 1 month and 20 years who were not receiving hydroxyurea or monthly blood transfusions were enrolled in this observational study. Hematologic data, including ARC and HbF levels, were measured at steady state. F-cells were enumerated by flow cytometry. Initial studies compared infants with ARC greater than or equal to 200 K/μL (ARC ≥ 200) based upon the previously reported utility of this threshold as a predictive marker for SCA severity. Mean HbF and F-cell levels were significantly lower in the ARC ≥ 200 group when compared to the ARC < 200 group. Both HbF and F-cell percentages were negatively correlated to ARC in infants and in children between the ages of 1 and 9 years. However, the inverse relationship was lost after the age of 10 years. Overall, decreased expression and distribution of HbF during childhood SCA is well-correlated with increased reticulocyte production and release into the peripheral blood. As such, these data further support the clinical use of reticulocyte enumeration as a disease severity biomarker for childhood sickle cell anemia.

Reticulocyte production index as a predictor of clinically significant anemia in chronic hepatitis C patients receiving pegylated interferon combination therapy

Summary Background This work was conducted to study the relationship of reticulocyte production index to clinically significant anemia in chronic hepatitis C patients receiving pegylated interferon combination therapy. Methods A total of 69 chronic hepatitis C patients receiving pegylated interferon combination therapy were included. Clinically significant anemia was defined as a hemoglobin level of p value Results Clinically significant anemia was observed in 30 patients (43.5%), and 39 patients (56.5%) never developed clinically significant anemia during the whole treatment course. On multivariate analysis, age > 60 years [odds ratio (OR), 2.94; 95% confidence interval (CI), 1.09–7.93], pretreatment hemoglobin level Conclusion Besides old age and low pretreatment hemoglobin level, our study showed that a reticulocyte production index

Nocturnal Hypoxemia (not sleep apnea) May Drive Reticulocytosis in Sickle Cell Disease

Introduction: Obstructive sleep apnea is a ventilatory defect that affects up to 10% of children with sickle cell disease (SCD). Sleep apnea has been implicated in cardiovascular disease in non-SCD adults and in cerebrovascular disease in children with SCD. In addition, prolonged hemoglobin oxygen desaturation from sleep disorderedbreathing would be predicted to increase sickle polymer formation and, possibly, activity of disease. We evaluated symptomatic adults from our SCD clinic for sleep apnea, in an effort to identify treatable disease modifying conditions.Methods: 46 adults with SCD were evaluated by polysomnography (PSG) between 2012 and 2014. Subjects were referred for symptoms or signs suggestive of sleep disordered breathing, including nighttime pain, snoring, early morning headache, narrow hypo-pharynx, and/or excessive reticulocytosis. Clinical characteristics of homozygous (HbSS, n=34) and compound heterozygous (HbSC and HbSBeta+Thalassemia, n=12) subjects are shown in Table 1.Results: Sleep apnea was diagnosed if the apnea/hypopnea index (AHI) was elevated: 5-15 (mild), 15-30 (moderate), or >30 (severe).Over half of our patients had sleep apnea by these criteria (16/34 HbSS and 7/12 compound heterozygotes). An elevated 3% oxygen desaturation index , i.e. >15 episodes per hour during which oxygen saturation dropped by 3% or more, was seen in one-third (14/43) of all evaluable subjects, and was 17 (median, range 1.2- 192.4) in subjects with, compared with 4.6 (median, range 1-23.7) in subjects without, sleep apnea (P<.001). Sleep apnea was moderate or severe in 5/34 (14.7%) and 3/12 (25%) of screened HbSS and compound heterozygote patients, respectively. However, the presence of sleep apnea, compared with the absence of sleep apnea, was not associated with differences in measured clinical variables including WBC, Hgb, absolute reticulocyte count, LDH, urinary albumin-to-creatinine ratio, or tricuspid regurgitant jet velocity.We then examined 8 subjects (7 HbSS, 1 SBeta+ thal) whose PSG showed >10% of sleep time afflicted by nocturnal hypoxemia. A median of 33.75% (range 16-98.6%) of sleep time was spent at <90% oxygen saturation in these subjects. Only 3 of 8 subjects with nocturnal hypoxemia had sleep apnea, based on an AHI >5, which was not different than the group as a whole (P=0.7). These 8 subjects had strikingly increased reticulocyte counts, compared with 38 subjects without nocturnal hypoxemia (median 460, range 100-923 (x103)/μL compared with median 303, range 76-733 (x103)/μL respectively, p=0.04), as well as a trend toward a higher LDH (median 488, range 140-752, compared with median 318, range 139-823, P=.08). Of note, hemoglobin levels were not different between subjects with and without nocturnal hypoxemia (mean 8.4±1.5 g/dL compared to mean 9.5±2.2 g/dL, respectively, p=0.17).Conclusions & Discussion: These studies suggest that, while sleep apnea is common in adults with SCD, greater physiological relevance may arise instead from prolonged nocturnal hypoxemia and associated reticulocytosis. We speculate that significant nighttime hypoxemia and hemoglobin desaturation may stimulate both endogenous erythropoietin (and therefore reticulocyte) production and HbS polymer formation and, possibly, hemolysis. This condition, while identifiable by conventional PSG, is not coincident with sleep apnea, and may be modulated by nighttime oxygen supplementation. These findings deserve confirmation in a larger cohort of patients. Table IClinical Characteristics of adults with SCD who underwent PolysomnographyHbSS(n=34)Compound Heterozygous (HbSC, n=8, HbSβ+Thal, n=4)P ValueAge (years)27 (19-50)29.5 (19-51)n.s.Body Mass Index23.8 (17.8-37.6)27.2 (20.1-59.8)n.s.Hgb (g/dL, mean±std dev)8.7±1.910.9±2.0P=.003ARC* (x103/μL),350 (93-923)200 (71-398)P=.004LDH (IU/L)398 (149-823)200 (140-632)P<.001TRV**(m/s)2.5 (n.d.-3.51)1.9 (n.d.-2.64)P<.001*Absolute reticulocyte count **Tricuspid regurgitant jet velocity ¦Median with range, unless otherwise indicated. DisclosuresNo relevant conflicts of interest to declare.

Hematopoietic stem/progenitor cell sources to generate reticulocytes for Plasmodium vivax culture.

The predilection of Plasmodium vivax (P. vivax) for reticulocytes is a major obstacle for its establishment in a long-term culture system, as this requires a continuous supply of large quantities of reticulocytes, representing only 1–2% of circulating red blood cells. We here compared the production of reticulocytes using an established in vitro culture system from three different sources of hematopoietic stem/progenitor cells (HSPC), i.e. umbilical cord blood (UCB), bone marrow (BM) and adult peripheral blood (PB). Compared to CD34+-enriched populations of PB and BM, CD34+-enriched populations of UCB produced the highest amount of reticulocytes that could be invaded by P. vivax. In addition, when CD34+-enriched cells were first expanded, a further extensive increase in reticulocytes was seen for UCB, to a lesser degree BM but not PB. As invasion by P. vivax was significantly better in reticulocytes generated in vitro, we also suggest that P. vivax may have a preference for invading immature reticulocytes, which should be confirmed in future studies.