Donor-dependent aging of young and old red blood cell subpopulations: Metabolic and functional heterogeneity

Abstract

Background: Characteristics of red blood cells (RBCs) are influenced by donor variability. This study assessed quality and metabolomic variables of RBC subpopulations of varied biologic age in red blood cell concentrates (RCCs) from male and female donors to evaluate their contribution to the storage lesion.

Study design and methods: Red blood cell concentrates from healthy male (n = 6) and female (n = 4) donors were Percoll separated into less dense ("young", Y-RCCs) and dense ("old", O-RCCs) subpopulations, which were assessed weekly for 28 days for changes in hemolysis, mean cell volume (MCV), hemoglobin concentration (MCHC), hemoglobin autofluorescence (HGB), morphology index (MI), oxygen affinity (p50), rigidity, intracellular reactive oxygen species (ROS), calcium ([Ca²⁺ ]), and mass spectrometry-based metabolomics.

Results: Young RCCs having disc-to-discoid morphology showed higher MCV and MI, but lower MCHC, HGB, and rigidity than O-RCCs, having discoid-to-spheroid shape. By Day 14, Y-RCCs retained lower hemolysis and rigidity and higher p50 compared to O-RCCs. Donor sex analyses indicated that females had higher MCV, HGB, ROS, and [Ca²⁺ ] and lower hemolysis than male RBCs, in addition to having a decreased rate of change in hemolysis by Day 28. Metabolic profiling indicated a significant sex-related signature across all groups with increased markers of high membrane lipid remodeling and antioxidant capacity in Y-RCCs, whereas O-RCCs had increased markers of oxidative stress and decreased coping capability.

Conclusion: The structural, functional, and metabolic dissimilarities of Y-RCCs and O-RCCs from female and male donors demonstrate RCC heterogeneity, where RBCs from females contribute less to the storage lesion and age slower than males.

Keywords: RBC metabolomics; RBC morphology; RBC senescence; RBC storage lesion; young and old RBCs.

FIGURE 1

Changes in the structural characteristics of the RBCs during hypothermic storage. Changes in, A, MCV, and B, MCHC were assessed using a hematology analyzer. C, HGB was assessed using an IFC assay. Data are shown as means ± SD. *Significant difference (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) between groups calculated using paired t test (for normal distribution) and Wilcoxon test (for nonnormal distribution). Symbols (☉, ○, Δ, □) represent distribution of donor samples

FIGURE 2

Classification of RBCs of different morphology and changes in MI of RBCs during hypothermic storage. A, Morphology subclasses of RBCs, and B, RBC MI (means ± SD) was assessed using bright-field images from an imaging flow cytometer (60× magnification). Data are shown as means ± SD. *Significant difference (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) between groups calculated using paired t test (for normal distribution) and Wilcoxon test (for nonnormal distribution). Symbols (☉, ○, Δ, □) represent distribution of donor samples

FIGURE 3

Metabolomics analysis of RBCs after separation. Multivariate analyses were performed on metabolomics data, including, A, partial least-square discriminant analysis (PLS-DA), and B, hierarchical clustering analysis to identify unique metabolic signature in RBCs from unseparated RCCs of female or male donors (P-RCC, average/middle density) and separated young (Y-RCC) and old (O-RCC) RBCs. C, Changes in S-adenosylmethionine and glutathione disulfide, two of the top metabolites sorted by a two-way ANOVA, depending on RBC subpopulation and sex

FIGURE 4

Qualitative and functional properties of RBCs during hypothermic storage. Changes in, A, hemolysis, and B, p50 of different RCCs. Data are shown as means ± SD. *Significant difference (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) between groups calculated using paired t test (for normal distribution) and Wilcoxon test (for nonnormal distribution). Symbols (☉, ○, Δ, □) represent distribution of donor samples

FIGURE 5

Deformability characteristics of RBCs during hypothermic storage. Changes in, A, rigidity, and B, elongation of different RCCs. Data are shown as means ± SD. *Significant difference (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) between groups calculated using paired t test (for normal distribution) and Wilcoxon test (for nonnormal distribution). Symbols (☉, ○, Δ, □) represent distribution of donor samples. K_EI, shear stress required to achieve half of the EI_max

FIGURE 6

Intracellular content of ROS and Ca²⁺ in RBCs during hypothermic storage. Changes in, A, intracellular content of ROS, and B, [Ca²⁺] of different RCCs. Data are shown as a means ± SD. *Significant difference (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) between groups calculated using paired t test (for normal distribution) and Wilcoxon test (for nonnormal distribution). Symbols (☉, ○, Δ, □) represent distribution of donor samples