In vitro kinetics of reticulocyte subtypes: maturation after red blood cell storage in additive solution-1 (AS-1)

Abstract

BackgroundReticulocytes 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.MethodSamples 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 °C and analyzed by flow cytometry with thiazole orange taking into account hemolysis.ResultsDensity-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.ConclusionIn vitro reticulocyte disappearance was significantly faster after prolonged storage of red blood cell units at 4 °C. 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.

The protein organization of a red blood cell

“…However, we took additional steps to further eliminate contaminating plasma proteins and other cell types, namely leukocytes, reticulocytes, and platelets. Each blood unit was stored at 4 C for 5 days after receipt in order to allow reticulocytes to mature into RBCs before extracting proteins (Urbina and Palomino, 2018). To further reduce leukocytes and platelets, RBCs were washed with PBS pH 7.4 (Gibco, Thermo Scientific, CA, USA) at 200 3g for 15 minutes for 3 times.…”

Assessment of Reticulocyte and Erythrocyte Parameters From Automated Blood Counts in Vaso-Occlusive Crisis on Sickle Cell Disease

“…In Sysmex analysers, reticulocytes are fractionated in low (LFR), medium (MFR) and high fluorescence reticulocytes (HFR). Reticulocytes can be classified according to RNA content into subtypes reflecting successive stages during maturation: immature with HFR to MFR and mature with LFR ( 5 ). Whole reticulocyte fraction provides information on bone marrow recovery.…”

SIRPα controls CD47-dependent platelet clearance in mice and humans

“…2C), which demonstrates the hematopoietic system’s concerted efforts to compensate for the anemic and thrombocytopenic effects of anti-CD47 treatment. Based on maturation stage, reticulocytes can be classified into subtypes, where high fluorescence reticulocytes (HFR) and medium fluorescence reticulocytes (MFR) are immature and low fluorescence reticulocytes (LFR) are mature 28 . In the treated mice, immature reticulocyte fraction (IRF) significantly increased (p = 0.0011, Fig.…”