ABSTRACT Background: Reticulocytes are immature red blood cells containing RNA remnants. Their population kinetics has been documented under various in vivo and in vitro conditions, including after storage of red blood cells in blood banks. The purpose of this study was to describe the influence of blood bank storage on the kinetics of reticulocyte disappearance by in vitro culturing. Method: Samples of reticulocyte-enriched fractions (Percoll density-gradient) were obtained over different storage times from six red blood cell units stored in additive solution-1 (AS-1). Reticulocyte fractions were then cultured in enriched media at 37 ºC and analyzed by flow cytometry with thiazole orange taking into account hemolysis. Results: Density-gradient enriched reticulocyte fractions were obtain from standard red blood cell units with <1% of reticulocytes. An exponential drop of reticulocytes was observed in cultures. The time taken for reticulocyte disappearance in cultures was shorter with increased blood bank storage time (144 ± 46 h at 0.5 weeks of storage and 15 ± 14 h in the sixth week). High fluorescence reticulocytes disappeared completely in 42.5 ± 8.5 h, medium fluorescence reticulocytes in 73.4 ± 20.8 h and low fluorescence reticulocytes in 269.9 ± 98.8 h in red blood cell units stored for half a week. These times significantly decreased in red blood cell units stored for more time. Conclusion: In vitro reticulocyte disappearance was significantly faster after prolonged storage of red blood cell units at 4 ºC. The in vitro half-life at 0.5 weeks of storage was not significantly different from the values reported for fresh venous blood, but after the sixth week of storage, the half-lives were shorter. The possible explanation is that blood bank storage does not cause irreversible damage to the human reticulocyte maturational machinery.

ABSTRACT Background: Recent evidence shows a selective destruction of the youngest circulating red blood cells (neocytolysis) trigged by a drop in erythropoietin levels. Objective: The aim of this study was to evaluate the effect of recombinant human erythropoietin beta on the red blood cell storage lesion and apoptosis indices under blood bank conditions. Methods: Each one of ten red blood cell units preserved in additive solution 5 was divided in two volumes of 100 mL and assigned to one of two groups: erythropoietin (addition of 665 IU of recombinant human erythropoietin) and control (isotonic buffer solution was added). The pharmacokinetic parameters of erythropoietin were estimated and the following parameters were measured weekly, for six weeks: Immunoreactive erythropoietin, hemolysis, percentage of non-discocytes, adenosine triphosphate, glucose, lactate, lactate dehydrogenase, and annexin-V/esterase activity. The t-test or Wilcoxon's test was used for statistical analysis with significance being set for a p-value <0.05. Results: Erythropoietin, when added to red blood cell units, has a half-life >6 weeks under blood bank conditions, with persistent supernatant concentrations of erythropoietin during the entire storage period. Adenosine triphosphate was higher in the Erythropoietin Group in Week 6 (4.19 ± 0.05 µmol/L vs. 3.53 ± 0.02 µmol/L; p-value = 0.009). The number of viable cells in the Erythropoietin Group was higher than in the Control Group (77% ± 3.8% vs. 71% ± 2.3%; p-value <0.05), while the number of apoptotic cells was lower (9.4% ± 0.3% vs. 22% ± 0.8%; p-value <0.05). Conclusions: Under standard blood bank conditions, an important proportion of red blood cells satisfy the criteria of apoptosis. Recombinant human erythropoietin beta seems to improve storage lesion parameters and mitigate apoptosis.

Introducción: La oxidación, al igual que la hemólisis asociadas con el ejercicio tanto moderado como extenuante han sido condiciones objeto de múltiples estudios, pero sólo en algunos recientes se evaluó cómo las variaciones en la capacidad de los mecanismos antioxidantes enzimáticos eritrocitarios específicos y genéticamente determinados, condicionan distintos grados de susceptibilidad a la hemólisis inducida por el ejercicio, pero esos trabajos se han hecho sobre todo en individuos no sedentarios y con deficiencia franca de la enzima G6PD. Objetivo: El propósito de este trabajo fue determinar si existe o no relación entre el nivel de actividad de la enzima eritrocitaria G6PD y el grado de hemólisis inducida por el ejercicio en hombres adultos sedentarios en apariencia sanos, luego de un tiempo experimental de ejercicio moderado pre-extenuante y equivalente en modalidad y magnitud. Metodología: Se evaluaron 25 voluntarios hombres, sedentarios, en apariencia sanos; se les aplicó una sesión de ejercicio, sobre un cicloergómetro, de una hora de duración, a 70% del VO2pico previamente hallado. Sobre la base de la concentración de hemoglobina plasmática se calculó el porcentaje de hemólisis y la haptoglobina consumida, tomando en cuenta la variación en el plasma de la haptoglobina y la hemólisis intravascular inmediatamente después y hasta tres horas luego del ejercicio. Resultados: Se pudo determinar que los individuos presentaron, con base en la determinación de Hb plasmática libre, % de hemólisis y consumo de haptoglobina, hemólisis intravascular durante y hasta por lo menos tres horas post-ejercicio. Conclusiones: La magnitud de la hemólisis presentada, por un lado, tuvo una relación inversa significativa con la actividad de la G6PD, y por otro, que tal magnitud tuvo un comportamiento significativamente distinto por debajo del percentil 40 de la actividad hallada para G6PD y por encima de la misma, o sea, la respuesta hemolítica oxidativa en individuos sedentarios sanos con niveles más bajos de actividad de G6PD frente a una sesión de ejercicio a 70% del VO2pico presenta una gran variabilidad.

Introduction: Oxidation and hemolysis associated with physical exercise, moderate and severe, have been studied by several investigations, but only in the most recent have been evaluated how the variations of the genetic determined mechanisms for antioxidative enzymatic erytrocytic capacity, contribute to establish different levels of susceptibility to the hemolysis induced trough exercise, however, those studies have mainly been made on non sedentary subjects with deficiency of enzyme G6PD. Purpose: The objective of this study was to establish if there is either yes or not a relationship between the activity level of erythrocytic activity of the enzyme G6PD and the hemolysis grade, induced by exercise in adult healthy sedentary men and after an experimental bout of moderate exercise pre-exhaustive and equivalent in modality and intensity. Methods: Twenty five men, sedentary and seemingly healthy, were evaluated after an exercise session of 1 hour over a cycloergometer, with 70% of VO2peak previously founded. On the basis of plasmatic Hb, the proportion of hemolysis and haptoglobin consumed, taking in account the variation of plasma haptoglobin and intravascular hemolysis after and at least three hours post-exercise, were calculated. Results: The experimental subjects presented laboratory evidence (free plasma hemoglobin, percentage of hemolysis and haptoglobin) of intravascular hemolysis immediately after and at least past three hours of exercise bouts. Conclusion: The grade of hemolysis had an inversely significative correlation with the G6PD activity and, this intensity had a significative different behaviour below the percentil 40 media founded for G6PD and above of it. Thus, the oxidative hemolytic response in sedentary and healthy individuals who had lower levels G6PD activity with one bout of exercise with 70% of VO2peak was highly variable.