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

Abstract

Background: Results 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 methods: We performed an integrated mass spectrometry-based metabolomics and proteomics time-course investigation on SAGM-stored red blood cells. In parallel, structural changes during storage were monitored through scanning electron microscopy.

Results: We 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, anti-oxidant 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.

Conclusions: Biochemical 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.

Figure 1.

In (A) Two-dimensional gel electrophoresis (extraction protocol without incubation with the alkylating agent N-ethyl maleimide -NEM) of day 0 (A1) versus day 14 (A2) and 35 (A3) RBC. First dimension IEF pI values linearly span between 3 and 10, while molecular weights are indicated on the left. Spots characterized by different photodensities in the low molecular weights region were individuated and further identified through mass spectrometry (Table 1A). (B) Two-dimensional gel electrophoresis (extraction protocol including incubation with NEM of day 0 (B1) versus day 35 (B2) RBC. First dimension IEF pI values linearly span between 3 and 10, while molecular weights are indicated on the left. Spots displaying different photodensities in the low molecular weight region were individuated and further identified through mass spectrometry (Table 1B).

Figure 2.

Scanning electron images (JEOL JSM 5200 scanning electron microscope) of long-stored RBC. A detail of a 42-day echinocyte (7,500x; scale bar = 1 μm) (A). A panoramic view of a 28-day RBC sample (2,000x; scale bar = 10 μm) (B). A 2,000x field of 42-day RBC (scale bar = 10 μm) (C).