Data on how several physiological parameters of stored red blood cells are similar in glucose 6-phosphate dehydrogenase deficient and sufficient donors

Abstract

This article contains data on the variation in several physiological parameters of red blood cells (RBCs) donated by eligible glucose-6-phosphate dehydrogenase (G6PD) deficient donors during storage in standard blood bank conditions compared to control, G6PD sufficient (G6PD(+)) cells. Intracellular reactive oxygen species (ROS) generation, cell fragility and membrane exovesiculation were measured in RBCs throughout the storage period, with or without stimulation by oxidants, supplementation of N-acetylcysteine and energy depletion, following incubation of stored cells for 24 h at 37 °C. Apart from cell characteristics, the total or uric acid-dependent antioxidant capacity of the supernatant in addition to extracellular potassium concentration was determined in RBC units. Finally, procoagulant activity and protein carbonylation levels were measured in the microparticles population. Further information can be found in "Glucose 6-phosphate dehydrogenase deficient subjects may be better "storers" than donors of red blood cells" [1].

Keywords: AnnV, annexin V; CPD, citrate-phosphate-dextrose; Cell fragility; FRAP, ferric reducing antioxidant power; FSC, forward scatter; G6PD deficiency; G6PD, glucose-6-phosphate dehydrogenase; G6PD−, G6PD deficiency; Hb, hemoglobin; Hct, hematocrit; K+, potassium; MCF, mean corpuscular fragility; MFI, mechanical fragility index; MP, micoparticles, microvesicles; MPPA, microparticles pro-coagulant activity; Microparticles; NAC, N-acetylcysteine; NS, non-stored; Oxidative stress; PBS, phosphate buffer saline; PCI, protein carbonylation index; PS, phosphatidylserine; RBC, red blood cell; RFU, relative fluorescence units; ROS, reactive oxygen species; Red blood cell storage lesion; SAGM, saline-adenine-glucose-mannitol; SSC, side scatter; TAC, total antioxidant capacity; UA-dep AC, uric acid dependent antioxidant capacity; UA-ind AC, uric acid independent antioxidant capacity; tBHP, tert-Butyl hydroperoxide.

Fig. 1

ROS generation in energy depleted G6PD-deficient (G6PD⁻ n=6) and control (G6PD⁺n=3) RBCs. RBCs stored for variable periods of time in CPD-SAGM preservative/additive solution were incubated for 24 h at 37 °C and intracellular ROS accumulation was estimated by fluorometry with or without stimulation by oxidants (100 μM tBHP for 20 min at 20 °C and 2 mM diamide for 45 min at 37 °C). Stimulated ROS were normalized to the pre-treatment levels (dashed lines). RFU, relative fluorescence units. *P<0.05 versus control (G6PD⁺) donors; data shown as mean±standard deviation.

Fig. 2

RBC fragilities profiles. Variation in mean corpuscular fragility (MCF) (A) and mechanical fragility index (MFI) (B) of G6PD⁻ (n=6) and control (G6PD⁺) (n=3) RBCs before (NS, non-stored) and during storage in CPD-SAGM. Left panels: MCF and MFI measurements in situ (no treatment of RBCs). Right panels: MCF and MFI measurements following incubation of non-stored and stored RBCs for 24 h at 37 °C (iMCF, iMFI). *P<0.05 versus control; data shown as mean±standard deviation.

Fig. 3

Microparticles characterization. Left panel: (A) Flow cytometry estimation of RBC-derived Annexin V-positive microparticles in control (n=3) and G6PD⁻ (n=6) samples before (non-stored, NS) and during the storage. (B) After normalization to the second day of storage (dashed line) the G6PD⁻ RBCs exhibited a trend for higher microvesiculation rate compared to the control RBCs. (C) Microparticles-associated procoagulant activity measured by Elisa was similar in the two groups under examination. *P<0.05 versus control; data shown as mean±standard deviation. Right panel: (D) relative percentage of carbonylated proteins in microparticles collected from G6PD⁺ (individuals # 1 and 2) and G6PD⁻ (individuals # 3–5) supernatant on the day 42 of storage and (E) representative immunoblot analysis by using anti-DNP antibody. Stomatin was used as internal control.

Fig. 4

Estimation of the antioxidant capacity in fresh plasma and supernatant collected from the stored RBC units. Total, uric acid-dependent and uric acid-independent antioxidant capacity in G6PD⁻ (n=6) and control (G6PD⁺) (n=3) samples in vivo (non-stored, NS) and during storage. *P<0.05 versus control. Data is shown as mean±standard deviation.

Fig. 5

Malate variation in G6PD⁻ (n=6) and control (G6PD⁺, n=3) RBCs during storage in CPD-SAGM. Metabolomics analysis showed a faster decrease of malate levels in G6PD⁻ RBCs than in controls. Blue line: control; solid and dashed red lines: median+SD for G6PD⁻ cells.

Fig. 6

NAC effect on physiological characteristics of stored G6PD⁻ (n=6) and control (G6PD⁺, n=3) RBCs. Packed RBC units were treated with 2.5 mM NAC (from day 21 to day 42) and samples were collected on the last day of storage. All data are normalized to pre-treatment levels (dashed lines). *P<0.05 versus control. TAC, total antioxidant capacity; UA-ind AC, uric acid independent antioxidant capacity; UA-dep AC, uric acid dependent antioxidant capacity, Sup K⁺, supernatant potassium; PS, phosphatidylserine exposure at RBC surface; data shown as mean±standard deviation.