Routine storage of red blood cell (RBC) units in additive solution-3: a comprehensive investigation of the RBC metabolome.
Author(s): D'Alessandro A, Nemkov T, Kelher M, West FB, Schwindt RK, Banerjee A, Moore EE, Silliman CC, Hansen KC
Publication: Transfusion, 2015, Vol. 55, Page 1155-68
PubMed ID: 25556331 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to assess potential effects of leukocyte reduction (leukoreduction) and refrigerated storage on the red blood cell (RBC) metabolome.
Conclusion of Paper
Hierarchical clustering analysis revealed that metabolic profiles of RBCs and supernatants collected pre-leukoreduction, during leukoreduction, and shortly after leukoreduction (immediately, 3 h and 1 day post-leukoreduction) clustered together. Metabolite-specific trends (that included a progressive increase or decrease in expression) were observed after 7 to 14 days of storage in leukoreduced samples; however, longer storage durations elicited a shift in metabolic profiles, such that samples collected at 14 d post-leukoreduction served as an inflection point. Among the metabolites affected by storage were amino acids, metabolites related to oxidative stress, urea cycle intermediaries, transamination/one-carbon metabolism products and energy metabolism metabolites.
The purpose of this paper was to assess potential effects of leukoreduction and refrigerated storage on the RBC metabolome. Approximately 500 ml of whole blood was collected from five healthy donors into bags containing citrate phosphate dextrose and centrifuged to obtain RBCs, which were then suspended in additive solution 3 (AS-3). RBCs were sampled before, during and after leukocyte reduction by filtration. Post-leukoreduction samples were stored at 1-6˚C and sampled after 3 h, and after 1, 7 14, 21, 28, 35 and 42 days. All samples underwent two centrifugation steps to isolate RBCs from supernatant, an initial spin at 5,000g for 7 min at 4°C at followed by a second spin at 12,500g for 6 min at 4°C. Metabolites were isolated immediately using ice-cold lysis extraction buffer from both RBCs and supernatants. RBC and supernatant metabolite extracts were then frozen at -80˚C until analysis by ultra–high performance liquid chromatography (UPLC) mass spectrometry (MS).
Summary of Findings:
A total of 229 and 200 metabolites were unambiguously identified and quantified in RBC and RBC supernatants, respectively.
Hierarchical clustering analysis of metabolic profiles of RBCs and supernatants revealed distinct clustering of pre-leukoreduction samples, but overlapping clustering of samples collected during and shortly after leukoreduction (post-leukoreduction: 3 h, 1 d). Results of partial least square discriminant analysis (PLS-DA) revealed that metabolic profiles were comparable among extracts from RBCs as well as supernatants stored for 1 day pre-leukoreduction and post-leukoreduction . Both extracts from RBCs and supernatants also displayed storage duration-dependent clustering of post-leukoreduction sample, although the degree of overlap was somewhat greater among supernatant samples. Trends in storage-induced changes to metabolomic profiles were observed among RBC and supernatant extracts. The direction of change with sample storage post-leukoreduction was specific to the metabolite, with both increases and decreases observed. Interestingly, samples stored for 14 d served as an inflection point, after which a change in trend was observed for the majority of metabolites.
Leukoreduction resulted in lower levels of amino acids, potentially due to a higher incidence of contaminating sources in pre-leukoreduced samples. Refrigerated storage of leukoreduced RBCs resulted in increases in metabolites related to oxidative stress, urea cycle intermediaries and transamination/one-carbon metabolism products including lactate, alanine, sarcosine, asparagine, poly-gamma-glutamate, uracil, nicotinamide, adenosyl homocysteine, homocysteine, oxoglutaramate, 5-oxoproline; but also decreases in metabolites involved in energy metabolism that included phosphoglycerate, NAD+, cyclic adenosine monophosphate (cAMP), oxalomalate, hexose phosphate, and orthophosphate. In the first 3 h to 7 days of storage of leukoreduced RBCs glycolytic precursors and pentose phosphate pathway intermediates increased, although they then decreased after 7 days of storage.
- Other Preservative
Analyte Technology Platform Small molecule HPLC-MS
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood and blood products Red blood cells
Non-leukoreduced red blood cells
Leukoreduced red blood cells
Red blood cell supernatant
Storage Storage duration 0 h
Biospecimen Aliquots and Components Blood processing method Pre-leukoreduction