Analytical and biological assessment of circulating human erythroferrone.

BACKGROUND: Erythroferrone (ERFE) is an erythroid hormone putatively involved in stress erythropoiesis. Its regional clearance and circulating form in humans, as well as levels in normal health and coronary disease remain unclear. METHODS: To establish a reference interval, ERFE was measured in 155 healthy volunteers using the Intrinsic LifeSciences ELISA. To identify trans-organ gradients in ERFE, regional blood sampling was undertaken in patients (n = 13) undergoing clinically indicated cardiac catheterisation. The Intrinsic ELISA was assessed for reproducibility, stability, linearity and possible cross-reactivity, interference and anticoagulant effects. Circulating forms of ERFE were evaluated by HPLC. RESULTS: In healthy individuals, the median concentration of ERFE was 0.51 ng/mL (IQR: 0.12-1.25), with men (n = 78) having higher levels than women (n = 77) (0.67 vs 0.32 ng/mL, p = 0.0001). ERFE concentrations in trans-organ sampling revealed no clear organ of clearance or production. Samples with high endogenous ERFE levels were suppressed by haemoglobin (≥2 g/L), bilirubin (≥200 µmol/L), lipaemia (>1 g/L), and freeze thawing (≥2 cycles), but this was not observed with low ERFE concentrations. Endogenous ERFE immunoreactivity was 46% higher in EDTA plasma compared with serum and lithium heparin plasma. On SE-HPLC, ERFE eluted as intact and cleaved forms. CONCLUSION: We provide a useful reference range for ERFE in EDTA plasma. We found no specific site of secretion or clearance. The Intrinsic ELISA performed adequately but is limited by interference and stability when endogenous levels are high. Circulating forms are multiple and complex.

Comparison and optimization of SAR-PAGE tests for erythropoietins in doping analysis.

Erythropoietins (EPOs) are substances listed in S2 of the World Anti-Doping Agency (WADA) Prohibited List and are used commonly by athletes to increase endurance performance. According to the current WADA Technical Documents, sarcosyl-polyacrylamide gel electrophoresis (SAR-PAGE) followed by western blotting to differentiate erythropoietins based on their molecular weights is the only method that can be used for both screening and confirmation of all types of erythropoietins. The efficiency of immunopurification and protein transfer is crucial for ensuring the selectivity and sensitivity of erythropoietin detection. Several comparisons and optimization of the SAR-PAGE tests were conducted in this study. We optimized the first blotting conditions and then compared different immunopurification methods based on their selectivity, repeatability, and sensitivity for both urine and blood analysis. Additionally, rapid procedures for both urine and blood analysis were established and compared. The two-step procedure at 1.0 mA/cm2 for 60 min followed by 1.56 mA/cm2 for 20 min increased the blotting efficiency compared with the commonly used constant current approach. Comparison of immunopurification revealed no significant difference in selectivity and sensitivity between the different methods. For other factors, such as operation complexity, time and cost, a StemCell® purification kit followed by single blotting and magnetic beads followed by double blotting are recommended for urine screening and confirmation, respectively. While magnetic beads and a MAIIA® kit followed by double blotting are recommended for both screening and confirmation of blood samples, respectively. To ensure high sensitivity and selectivity, double blotting is recommended for a rapid procedure for both urine and blood analysis.

Effect of a Quality Improvement Program to Improve Guideline Adherence and Attainment of Clinical Standards in Dialysis Care: Report of Outcomes in Year 1.

BACKGROUND: Best practice in dialysis is synthesised in clear international guidelines. However, a large gap remains between the international guidelines and the actual delivery of care. In this paper, we report outcomes for the first year of a multifaceted dialysis improvement programme in our network. METHODS: One year collaborative involving 3 haemodialysis units and a peritoneal dialysis (PD) programme involving 299 dialysis patients. Each unit addressed a different indicator (unit A - catheter-related bloodstream infection [CRBSI], unit B - pre-dialysis blood pressure [BP], unit C - dialysis dose, unit D - anaemia) with a shared aim to match the top 10% in the UK. Tailored multifaceted approaches include a modified collaborative methodology with an aim, framework, driver diagram, learning sessions, facilitated meetings, plan-do-study-act cycles and continuous measurement. Analysis of outcomes, costings, erythropoietin stimulating agent and iron use, and safety culture attributes. RESULTS: Unit A reduced CRBSI from 2.65 to 0.5 per 1,000 catheter days (p = 0.02). Unit B improved attainment of target BP from 37.5 to 67.2% (p = 0.003). Unit C improved attainment of target urea reduction ratio from 75.8 to 91.4% (p = 0.04). PD unit D improved attainment of target haemoglobin from 45.5 to 62.7% (p = 0.01), with no significant change in the indicators in a non-intervention unit. Safety culture attributes improved. Costs associated with admission for fluid overload and infection, erythropoietin, iron and thrombokinase use decreased 36% (£415,620-£264,143). CONCLUSIONS: Units that took part in this collaborative improved guideline adherence compared both to their own pre-intervention performance and a non-intervention unit. Such multifaceted interventions are a useful methodology to improve dialysis care.

Fetal limb soft tissue assessment for prediction of birth weight and umbilical cord blood analytes in gestational diabetes.

OBJECTIVE: To evaluate the value of third trimester ultrasound (estimated fetal weight, cheek-to-cheek diameter, sectional Wharton's jelly area, sectional areas and fractional volumes in extremities) to predict birth weight and cord biochemical markers at birth (leptin, insulin, c-peptide, IGF1, erythropoietin and ferritin) in diabetic pregnancies. METHOD: Prospective study in 49 patients with gestational diabetes. An ultrasound was performed between 32 and 34 weeks. Clinical data were collected, and a blood sample was obtained from cord after birth. ROC curve models were evaluated for 75(th) and 90(th) birth weight percentile. Univariate and multivariate models were used to assess the association between ultrasound and neonatal outcomes. RESULTS: Sectional areas and fractional volumes showed significant differences and highest AUC values for predicting birth weight. A significant association was found for extremities measurements with total birth weight and its percentile. The only marker which showed a significant association to estimated fetal weight was erythropoietin. Sectional areas and fractional volumes related to cord leptin, erythropoietin, insulin and c-peptide. CONCLUSION: Sectional areas and fractional volumes improve the predictive value of estimated fetal weight in diabetic pregnancies. They also show a predictive association to biochemical changes in cord (leptin, insulin and erythropoietin) related to increased adiposity and risk of fetal hypoxia. © 2015 John Wiley & Sons, Ltd.

Maternal inflammation at delivery affects assessment of maternal iron status.

Pregnant adolescents (aged ≤ 18 y, n = 253) were followed from ≥ 12 wk of gestation to delivery to assess longitudinal changes in anemia and iron status and to explore associations between iron status indicators, hepcidin, and inflammatory markers. Hemoglobin, soluble transferrin receptor (sTfR), ferritin, serum iron, erythropoietin (EPO), hepcidin, C-reactive protein, interleukin-6 (IL-6), folate, and vitamin B-12 were measured, and total body iron (TBI) (milligrams per kilogram) was calculated using sTfR and ferritin values. Anemia prevalence increased from trimesters 1 and 2 (3-5%, <28 wk) to trimester 3 (25%, 33.2 ± 3.7 wk, P < 0.0001). The prevalence of iron deficiency (sTfR > 8.5 mg/L) doubled from pregnancy to delivery (7% to 14%, P = 0.04). Ferritin and hepcidin concentrations at delivery may have been elevated as a consequence of inflammation because IL-6 concentrations at delivery were 1.6-fold higher than those obtained at 26.1 ± 3.3 wk of gestation (P < 0.0001), and a positive association was found between IL-6 and both hepcidin and ferritin at delivery (P < 0.01). EPO was consistently correlated with hemoglobin (r = -0.36 and -0.43, P < 0.001), ferritin (r = -0.37 and -0.32, P < 0.0001), sTfR (r = 0.35 and 0.25, P < 0.001), TBI (r = -0.44 and -0.37, P < 0.0001), and serum iron (r = -0.22 and -0.16, P < 0.05) at mid-gestation and at delivery, respectively. EPO alone explained the largest proportion of variance in hemoglobin at 26.0 ± 3.3 wk of gestation (R(2) = 0.13, P = 0.0001, n = 113) and at delivery (R(2) = 0.19, P < 0.0001, n = 192). Pregnant adolescents are at high risk of anemia. EPO is a sensitive indicator of iron status across gestation, is not affected by systemic inflammation, and may better predict risk of anemia at term. The trial was registered at clinicaltrials.gov as NCT01019902.

Running economy and maximal oxygen consumption after 4 weeks of oral Echinacea supplementation.

The purpose of this investigation was to determine the effects of 4 weeks of oral Echinacea (ECH) supplementation on erythropoietin (EPO), red blood cell (RBC) count, running economy (RE), and VO2max. Twenty-four men aged 24.9 ± 4.2 years, height 178.9 ± 7.9 cm, weight 87.9 ± 14.6 kg, body fat 19.3 ± 6.5% were grouped using a double-blind design and self-administered an 8,000-mg·d(-1) dosage of either ECH or placebo (PLA) in 5 × 400 mg × 4 times per day for 28 days. Blood samples were collected and analyzed for RBCs and EPO using automated flow cytometery and enzyme-linked immunosorbent assay. Maximal graded exercise tests (GXTs) were administered to measure VO2max, RE, and heart-rate responses. Analysis of variance was used to determine statistically significant differences (P ≤ 0.05). The EPO increased significantly in ECH at 7 days (ECH: 15.75 ± 0.64, PLA: 10.01 ± 0.73 mU·ml(-1)), 14 days (ECH: 18.88 ± 0.71, PLA: 11.02 ± 0.69 mU·ml(-1)), and 21 days (ECH: 16.06 ± 0.55, PLA: 9.20 ± 0.55 mU·ml(-1)). VO2max increased significantly in ECH (ECH: 1.47 ± 1.28, PLA: -0.13 ± 0.52%). Running economy improved significantly in ECH as indicated by a decrease in submaximal VO2max during the first 2 stages of the GXT (stage 1: ECH -1.50 ± 1.21, PLA 0.60 ± 1.95%; stage 2: ECH -1.67 ± 1.43, PLA 0.01 ± 1.03%). These data suggest that ECH supplementation results in significant increases in EPO, VO2max, and running economy.

Population pharmacokinetic and pharmacodynamic model-based comparability assessment of a recombinant human Epoetin Alfa and the Biosimilar HX575.

The aim of this study was to develop an integrated pharmacokinetic and pharmacodynamic (PK/PD) model and assess the comparability between epoetin alfa HEXAL/Binocrit (HX575) and a comparator epoetin alfa by a model-based approach. PK/PD data-including serum drug concentrations, reticulocyte counts, red blood cells, and hemoglobin levels-were obtained from 2 clinical studies. In sum, 149 healthy men received multiple intravenous or subcutaneous doses of HX575 (100 IU/kg) and the comparator 3 times a week for 4 weeks. A population model based on pharmacodynamics-mediated drug disposition and cell maturation processes was used to characterize the PK/PD data for the 2 drugs. Simulations showed that due to target amount changes, total clearance may increase up to 2.4-fold as compared with the baseline. Further simulations suggested that once-weekly and thrice-weekly subcutaneous dosing regimens would result in similar efficacy. The findings from the model-based analysis were consistent with previous results using the standard noncompartmental approach demonstrating PK/PD comparability between HX575 and comparator. However, due to complexity of the PK/PD model, control of random effects was not straightforward. Whereas population PK/PD model-based analyses are suited for studying complex biological systems, such models have their limitations (statistical), and their comparability results should be interpreted carefully.

Assessment of the safety and biodistribution of a regulated AAV2 gene transfer vector after delivery to murine submandibular glands.

Clinical gene transfer holds promise for the treatment of many inherited and acquired disorders. A key consideration for all clinical gene transfer applications is the tight control of transgene expression. We have examined the safety and biodistribution of a serotype 2, recombinant adeno-associated viral (AAV2) vector that encodes a rapamycin-responsive chimeric transcription factor, which regulates the expression of a therapeutic transgene (human erythropoietin [hEpo]). The vector, AAV2-TF2.3w-hEpo (2.5 × 10(7)-2.5 × 10(10) particles), was administered once to a single submandibular gland of male and female mice and mediated hEpo expression in vivo following a rapamycin injection but not in its absence. Control (saline treated) and vector-treated animals maintained their weight, and consumed food and water, similarly. Vector delivery led to no significant toxicological effects as judged by hematology, clinical chemistry, and gross and microscopic pathology evaluations. On day 3 after vector delivery, vector copies were not only abundant in the targeted right submandibular gland but also detected in multiple other tissues. Vector was cleared from the targeted gland much more rapidly in female mice than in male mice. Overall, our results are consistent with the notion that administration of the AAV2-TF2.3w-hEpo vector to salivary glands posed no significant risk in mice.

Prediction of long-term survival in chronic heart failure by multiple biomarker assessment: a 15-year prospective follow-up study.

BACKGROUND: In chronic heart failure (CHF), several plasma biomarkers identify subjects at risk of death over the midterm. However, their long-term predictive value in the context of other candidate predictors has never been assessed. This information may prove valuable in the management of a chronic disease with a long natural history, as CHF is today. HYPOTHESIS: We aimed to assess the very-long-term prognostic power of a set of biomarkers to identify CHF patients at highest risk for all-cause mortality. METHODS: A group of 106 consecutive outpatients with CHF (85 male and 21 female, median age 56 y) was followed for 15 years. Echocardiographic tracings and blood samples were collected at study entry to evaluate cardiac function, plasma atrial natriuretic peptide (ANP), aldosterone, and erythropoietin, and plasma renin activity. The relationships between biomarkers, clinical and echocardiographic variables, and mortality were assessed. RESULTS: After 15 years, 86 of the 106 patients (81%) had died. Multivariate analysis showed that ANP was the best independent predictor of survival over several clinical, echocardiographic, and humoral variables (hazard ratio: 5.62, 95% confidence interval: 3.37-9.39, P < 0.001 for plasma levels < median value of 71 pg/mL). Plasma renin activity and erythropoietin provided prognostic information in univariate analysis, but lost their predictive power when adjusted for covariates. CONCLUSIONS: The present study represents the longest available follow-up of patients with CHF evaluating the prognostic power of multiple biomarkers. It shows that a simple assessment of plasma ANP levels is the strongest long-term predictor of death in all stages of heart failure.

[Assessment of relationship between cord blood cotinine levels and some factors of perinatal hypoxia]. / Ocena zaleznosci miedzy stezeniem kotyniny we krwi pepowinowej a wybranymi wskaznikami niedotlenienia okoloporodowego.

OBJECTIVE: To determine the relationship between cord blood cotinine levels and some markers of perinatal hypoxia such as cord blood erythropoietin levels, parameters of umbilical arterial blood gas analysis and Apgar scores. METHODS: 150 women with uncomplicated, healthy singleton pregnancies were assessed by means of a patient questionnaire. Neonates born by the examined pregnant women were divided into 3 groups according to recorded maternal smoking status--active smoking: n = 51, passive smoking: n = 49, non smoking: n = 50. Immediately after birth umbilical venous (for cotinine and erythropoietin levels) and arterial blood (for pH, pO2, pCO2, BE) were collected. RESULTS: Cotinine levels were significantly higher (p < 0.00001) in active smoking group (Me = 19.3 ng/ml) than in passive smoking (Me = 0.75 ng/ml) and non smoking (Me = 0.72 ng/ml) ones. Cord blood erythropoietin, pH values and 1, 3 and 5-minute Apgar scores did not differ significantly between the study groups. No significant correlation between cotinine and erythropoietin, pH and Apgar scores results in all study material and in the compared groups was found. CONCLUSION: No correlation between cord blood cotinine and erythropoietin levels was detected. Cord blood cotinine concentration does not influence the condition of the newborn assessed by Apgar scores and umbilical arterial blood gas analysis.

Reticulocytes in untreated obstructive sleep apnoea.

BACKGROUND AND AIM: The short, repetitive hypoxaemic episodes observed in obstructive sleep apnoea (OSA) may determine small augmentations in mature red blood cells. It is unknown whether they affect reticulocyte release. This study explored whether the number and degree of maturation of circulating reticulocytes may be altered in OSA, possibly through the effect of erythropoietin. METHODS: Fifty male adult patients with suspected OSA, normoxic during wakefulness, were studied. After nocturnal polysomnography, a blood sample was withdrawn for blood cells count, erythropoietin, iron and transferrin determination. Reticulocyte concentration and degree of immaturity [high (H), medium (M), or low (L)] were also determined. Immature reticulocyte fraction (IRF) was calculated as (M+H) percentage of reticulocytes. RESULTS: A wide range of OSA severity was found [apnoea/hypopnoea index (AHI): 44.3 +/- 30.4, range 0.3-105; sleep time spent at oxyhaemoglobin saturation <90%: 18.1 +/- 22.2%, range 0-81%]. Both reticulocyte count and IRF slightly exceeded the normal range. Patients with a reticulocyte concentration > 2% had higher EPO levels (p < 0.05), but not worse nocturnal desaturations, than those with values < 2%. By contrast, subjects with IRF < 15% showed worse desaturations (p < 0.05), but similar EPO concentrations, when compared to subjects whose IRF was < 10%. At univariate analysis, reticulocyte count correlated to erythropoietin, while IRF to transferrin saturation, BMI and OSA severity. At multiple regression, only lowest nocturnal oxygen saturation remained a significant contributor to IRF (r2 0.223, p < 0.05). CONCLUSIONS: This data suggests that hypoxaemia due to OSA could influence the release of immature reticulocytes, but this effect is not mediated by erythropoietin.

An assessment of recombinant human erythropoietin effect on reticulocyte production rate and lifespan distribution in healthy subjects.

PURPOSE: An empirical pharmacodynamic model was developed to assess the effect of recombinant human erythropoietin (rHu-EPO) treatment on the reticulocyte production rate and lifespan distribution. MATERIALS AND METHODS: Single doses of rHu-EPO at levels 20, 40, 60, 90, 120, and 160 kIU were administered to healthy volunteers (n = 8 per dose level). Erythropoietin plasma concentrations as well as hematologic responses were measured up to 42 days. The hematological data were used to determine explicit relationships between reticulocyte and red blood cell counts (RBC) and the reticulocytes' production rate and lifespan distribution. RESULTS: The parameter estimates obtained by simultaneous fitting of the model to the reticulocyte and RBC data revealed that rHu-EPO transiently increased the reticulocyte lifespan from the baseline value of 1.7 days to 3.4 days and the effect lasted for 8.3 days. The dose dependent increase in the reticulocyte production had the maximal value of 77.5 10(9) cells/l/day and was followed by a rebound that was less than 9% of the baseline value. Both reticulocyte and RBC responses were preceded by a dose-independent lag time of 1.7 days. CONCLUSIONS: The effect of rHu-EPO on the reticulocyte production rate and lifespan distribution was characterized. The results of the present study can be further utilized in building more mechanistic pharmacodynamic models of rHu-EPO stimulatory effects.

Improvement of band segmentation in Epo images via column shift transformation with cost functions.

In recent years, the development of methodology and laboratory techniques for doping control (DC) of recombinant erythropoietin (rEpo) has become one of the most important topics pursued by doping control laboratories accredited by World Anti-Doping Agency (WADA). The software system GASepo has been developed within the international WADA project as a support for Epo doping control. Although a great number of functions for automatic image processing have been involved in this software, for Epo images with considerably distorted bands additional effort is required from the user to interactively correct the results of improper band segmentation. In this paper a problem of geometrically distorted bands is addressed from the viewpoint of how to transform the lanes in distorted Epo images in order to reach better band segmentation. A method of band straightening via column shift transformation has been proposed that is formulated as an optimization procedure with cost functions. The method involves several novel approaches: two-stage optimization procedure, four cost functions and selection of relevant columns. The developed band straightening algorithm (BSA) has been tested on real Epo images with distorted bands. Based on the evaluation scheme involving the GASepo software itself a recommendation is made for implementation of the method with the cost function based on correlation matrix. Estimates of computational complexity of the individual steps of BSA are also given.

Comparative analysis of methods for assessment of circulating endothelial progenitor cells.

The number and properties of endothelial progenitor cells (EPC) in disease states is of considerable interest due to the importance attributed to this distinct cell population. However, there has been no study comparing each of the methods employed in the same sampled individuals. Herein, we performed an analysis of several methods used for circulating EPC assessment and correlated them with humoral factors known to influence their numbers. Thirty-eight individuals (mean age of 34 +/- 9 years) were tested. Peripheral blood mononuclear cells were obtained and stained for FACS analysis with antibodies to CD34, CD45, CD133, and KDR and the remaining cells grown under endothelial cell conditions for assessment of colony-forming unit (CFU) numbers and adhesive properties. Levels of circulating vascular endothelial growth factor (VEGF), erythropoietin (EPO), and C-reactive protein (CRP) were determined and correlated with each of the EPC markers. CFU numbers did not correlate with CD34/KDR or CD34/CD133/KDR and negatively correlated with CD34/ CD133 numbers. CD34/KDR numbers correlated with CD34/CD133/KDR, but not with CD34/ CD133. Only CD34/KDR and CD34/CD133/KDR correlated with VEGF serum levels. The number of EPC adhering to fibronectin and endothelial cells correlated with CFU numbers and not with either of the EPC membrane markers. Current methods for quantitatively assessing numbers of circulating EPC are not correlated. VEGF serum levels are associated only with CD34/KDR and CD34/ CD133/KDR, whereas CFU numbers correlate with EPC functional properties. These findings may suggest that CD34/KDR is more appropriate for the definition of circulating EPC, whereas CFU numbers are more likely to reflect their ability to proliferate.

Comparison of cost effectiveness between measuring the serum erythropoietin level and reticulocyte count for monitoring thalassemic patients: a note in Thai beta thalassemia/Hb E subjects.

Iron deficiency anaemia and the thalassemia syndromes, a group of disorders resulting from inherited abnormality of globin chain production, are common causes of anaemia in Thailand, and in the Far East in general. Monitoring erythropoiesis in these patient is very important in evaluating the disease and the response to treatment. Recently, our group has just reported the feasibility in using serum erythropoietin (EPO) for monitoring purposes in thalassemia and demonstrated that the determination of serum EPO can be an alternative choice in the follow up of these conditions. This study reports a cost effectiveness analysis comparing the recently reported radioimmunoassay (RIA) for serum EPO determination and the previously used tool, the reticulocyte count. The study reports a higher detection rate for ineffective erythropoiesis when using serum EPO determination, however, the increased sensitivity is at balanced by a higher unit cost. In this analysis, the cost effectiveness for serum EPO and reticulocyte are 208.33 and 50 baht/detection, respectively. () Therefore, the reticulocyte count is more cost effective and is recommended for routine usage in our current medico-economic setting.