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

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].

sufficient (G6PD þ ) 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 Nacetylcysteine 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]. &

Value of the data
The different storage profile when additional oxidative stimuli are added, is of value for the understanding of G6PD deficient red blood cells' physiology and its clinical relevance to the transfusion's outcomes.
Cell fragilities profiles and microparticles' characteristics might help elucidating the storage capacity ("storability") of G6PD deficient red blood cells.
Our data contribute to the clarification of donor-variation and N-acetylcysteine supplementation effects on red blood cell storage lesion

Data
We assessed the storage quality of red blood cells (RBCs) donated by glucose-6-phosphate dehydrogenase (G6PD) deficient, yet eligible, donors compared to control (G6PD sufficient) red blood cells [1]. Intracellular reactive oxygen species (ROS) accumulation was similar in energy depleted (24 h/37°C) G6PD À and G6PD þ stored RBCs while stimulation by tert-Butyl hydroperoxide (tBHP) and diamide oxidants resulted in statistically significant increase in ROS accumulation in the G6PD À group (n ¼6) compared to the G6PD þ group (n¼ 3) (Fig. 1). RBC fragility (both mean corpuscular fragility, MCF and mechanical fragility index, MFI) ( Fig. 2) and the characteristics of the microparticles (accumulation, pro-coagulant activity and protein carbonylation index, PCI) ( Fig. 3) were equal between the groups under examination throughout the storage period, while only slight differences were observed in the antioxidant capacity of the supernatant (Fig. 4).
Malate levels decreased faster in G6PD À RBCs than in control, G6PD þ RBCs during the storage period in CPD-SAGM (Fig. 5). Finally, N-acetylcysteine (NAC) supplementation (at the concentration used) induced similar changes in both stored RBCs and supernatant (Fig. 6).

Blood collection and processing
Blood samples from 9 regular male donors 22-30 years old (n ¼6 for Mediterranean variant grade II WHO G6PD À donors and n ¼ 3 for donors with normal levels of G6PD activity) were collected into EDTA or citrate vacutainers. The quality of RBC concentrates prepared from the same donors was evaluated in pre-storage leukoreduced (RC High efficiency leukocyte removal filters, Haemonetics, MA, USA), citrate-phosphate-dextrose (CPD)/saline-adenine-glucose-mannitol (SAGM) units throughout a 42 days storage in a controlled environment at 4°C as previously described [1]. Samples were taken after 2 days of storage and weekly thereafter or on days 21 and 42 (middle and maximal storage duration, respectively).
The ferric reducing antioxidant power (FRAP) assay was used for the estimation of Total Antioxidant Capacity (TAC) of the plasma [3]. Briefly, small aliquots of plasma were mixed with freshly prepared working FRAP solution (containing acetate buffer (pH 3.6, 300 mM), TPTZ (2,4,6-tripyridyl- s-triazine, 10 mM) in HCl (40 mM) and FeCl 3 (20 mM) in 10:1:1 ratio) and incubated for 4 min at 37°C in a water bath. Absorbance was measured at 593 nm. Uric acid-dependent and -independent antioxidant capacity were determined after uricase treatment of the samples (for 20 min at 25°C) [4]. Redox status estimation tests were also performed after N-acetylcysteine (NAC) supplementation (2.5 mM) or incubation for 24 h at 37°C.

Hemolysis and cellular fragility assays
Plasma free hemoglobin was calculated following a method first described by Harboe [5]. Blood samples were centrifuged at 1000 Â g for 10 min. Plasma was collected and centrifuged again under the same conditions. Cell free supernatants were diluted in distilled water and incubated at 20°C for 30 min. Hb absorbance was measured versus blank at 380, 415 and 450 nm. The final OD was calculated as follows: 2 Â OD415 ÀOD380 À OD450.
In vitro osmotic fragility behavior of erythrocytes was determined in solutions of increasingly saline concentration (0.0-0.9% w/v NaCl) [6]. 10 ml of blood samples were added in 1.0 ml of each saline solution, incubated for 15 min at 20°C and then centrifuged at 1500 rpm for 5 min. Hb released in the supernatant was measured at 540 nm, plotted against saline concentration and the mean corpuscular fragility (MCF), which corresponds to the saline concentration causing 50% of hemolysis, was calculated.
Mechanical fragility of RBCs was evaluated as previously described [7]. Briefly, blood from each donor was mixed with stainless steel beads, rocked in a rocker platform for 1 h and free Hb was measured in the plasma by using both Harboe's method against an un-rocked control. The mechanical fragility index (MFI) was calculated using the formula:  where Hb rocked is the mean free Hb concentration in the supernatants of the rocked specimens, Hb control is the average free Hb concentration in the supernatants of the control samples, and Hb aliquot is the average Hb concentration of the RBC aliquots at a Hct of 40%. Hemolysis and cellular fragility tests were also performed after NAC supplementation (2.5 mM) or incubation for 24 h at 37°C.

Analysis of microparticles
Enumeration and characterization of supernatant and circulating microparticles was performed by flow cytometry in aliquots of supernatant or plasma produced after two "light" spins of citrated blood at 20°C. Microparticles were identified by size (o1 μm), RBC origin (FITC-conjugated anti-CD235) and PS exposure (AnnexinV-positive, AnnV þ ) as previously described [8].  6. NAC effect on physiological characteristics of stored G6PD À (n ¼6) and control (G6PD þ , n ¼3) 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 o 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 þ , supernatant potassium; PS, phosphatidylserine exposure at RBC surface; data shown as mean7 standard deviation.