Time-course investigation of SAGM-stored leukocyte-filtered red bood cell concentrates: from metabolism to proteomics

Abstract

The online version of this article has a Supplementary Appendix. BackgroundResults from recent, highly debated, retrospective studies raised concerns and prompted considerations about further testing the quality of long stored red blood cells from a biochemical standpoint. Design and MethodsWe performed an integrated mass spectrometry-based metabolomics and proteomics timecourse investigation on SAGM-stored red blood cells. In parallel, structural changes during storage were monitored through scanning electron microscopy. ResultsWe detected increased levels of glycolytic metabolites over the first 2 weeks of storage. From day 14 onwards, we observed a significant consumption of all metabolic species, and diversion towards the oxidative phase of the pentose phosphate pathway. These phenomena coincided with the accumulation of reactive oxygen species and markers of oxidation (protein carbonylation and malondialdehyde accumulation) up to day 28. Proteomics evidenced changes at the membrane protein level from day 14 onwards. Changes included fragmentation of membrane structural proteins (spectrin, band 3, band 4.1), membrane accumulation of hemoglobin, antioxidant enzymes (peroxiredoxin-2) and chaperones. While the integrity of red blood cells did not show major deviations at day 14, at day 21 scanning electron microscope images revealed that 50% of the erythrocytes had severely altered shape. We could correlate the scanning electron microscopy observations with the onset of vesiculation, through a proteomics snapshot of the difference in the membrane proteome at day 0 and day 35. We detected proteins involved in vesicle formation and docking to the membrane, such as SNAP alpha. ConclusionsBiochemical and structural parameters did not show significant alterations in the first 2 weeks of storage, but then declined constantly from day 14 onwards. We highlighted several parallelisms between red blood cells stored for a long time and the red blood cells of patients with hereditary spherocytosis.Key words: red blood cell, storage, mass spectrometry, proteomics, metabolomics. Haematologica 2012;97(1):107-115. doi:10.3324/haematol.2011 Citation: D'Alessandro A, D'Amici GM, Vaglio S, and Zolla L. Time-course investigation of SAGM-stored leukocyte-filtered red blood cell concentrates: from metabolism to proteomics.

Biochemical and Cellular Changes in Leukocyte-Depleted Red Blood Cells Stored for Transfusion

“…The decreased concentration of extracellular Na + can be associated with water influx to the cytosol. This process and the absence of a selective mechanism of functionality, as performed in the spleen, could explain the increase of MCV in vitro with consecutive morphological RBC changes [5,14,15,16,17]. …”

An Efficient Apparatus for Rapid Deoxygenation of Erythrocyte Concentrates for Alternative Banking Strategies

“…The glass slides were kept in a moist atmosphere for 1 h, washed in phosphate buffer, postfixed in 1% osmium tetroxide for 1 h, rinsed in distillated water, and dehydrated in graded ethanol (50%–70%–90%–100%). After critical-point drying with liquid CO 2 in a vacuum apparatus and covering with a gold-palladium layer, the samples underwent scanning electron microscopic analysis and classification between reversible and irreversible phenotypes, as in the work of D'Alessandro et al [9]. The percentages of discocytes, echinocytes, spheroechinocytes, stomatocytes, spherostomatocytes, and spherocytes were evaluated by counting 1000 to 1500 cells in randomly chosen fields.…”

“…Storage lesions include alterations to either morphology (shape changes leading from a discoid to a spherocytic phenotype) or functionality (metabolism and oxygen delivery capacity through an increase in oxygen affinity mediated by a rapid fall in 2,3-diphosphoglycerate concentrations [8–13]). Further lesions occur in stored RBCs which are reversible to some extent, such as potassium leakage to the supernatant and depletion of ATP and DPG stores, while others are not, such as the alteration of lipids and membrane proteins (membrane protein fragmentation and migration to the membrane and/or vesiculation of subsets of structural or antioxidant proteins [9]), which results in more rigid cell structures, increased osmotic fragility, higher haemolytic rates, phosphatidylserine exposure to the outer membrane leaflet, increased vesiculation rate, and reduced oxygen off-loading capacity [8–12, 14, 15]. …”

“…Membrane protein fragmentation [9, 13], storage time-dependent migration of cytosolic proteins to the membrane [9, 14, 15], and increased oxidative stress-related parameters [9, 16] have been also reported to correlate with storage duration. Biochemical studies explicitly suggested that there is considerable room for improvement in the field of RBC biopreservation, especially when considering that the lifespan of RBCs in vivo is approximately 120 days [17].…”

“…Biochemical studies explicitly suggested that there is considerable room for improvement in the field of RBC biopreservation, especially when considering that the lifespan of RBCs in vivo is approximately 120 days [17]. One major phenomenon seems to lie at the root of storage lesions to RBCs: oxidative stress [9, 13, 16]. In order to cope with oxidative stress triggering phenomena, alternative RBC storage strategies have been recently proposed, such as the addition of higher loads of antioxidants (vitamin E, C, and beta-carotene) in additive solutions for storage purposes [18] or anaerobic storage [13, 19–21].…”