Stored red blood cell susceptibility to in vitro transfusion-associated stress conditions is higher after longer storage and increased by storage in saline-adenine-glucose-mannitol compared to AS-1

Abstract

Background: Biochemical changes induced in red blood cells (RBCs) during storage may impair their function upon transfusion. Transfusion-associated stresses may further amplify storage lesion effects including increased phosphatidylserine (PS) exposure at the RBC membrane, microparticle (MP) release, and adhesion to endothelial cells (ECs). RBC stress susceptibility in vitro was investigated in relation to storage time and additive solution.

Study design and methods: Leukoreduced whole blood donations (n = 18) were paired, mixed, and resplit before separating the RBCs for storage in saline-adenine-glucose-mannitol (SAGM) or AS-1. Samples were taken after 3, 21, or 35 days. For oxidative stress treatment, RBCs were exposed to 0.5 mmol/L tert-butylhydroperoxide. Transfusion-associated stress was simulated by overnight culture at 37 °C with plasma containing inflammatory mediators. PS exposure and MPs were measured by flow cytometry and adhesion to ECs was tested under flow conditions. PS specificity of adhesion was tested by blocking with PS-containing lipid vesicles.

Results: Oxidative stress induced significantly higher PS exposure and adhesion to ECs in RBCs stored for 35 days compared to 3 days (p < 0.04). PS-containing vesicles blocked RBC-EC adhesion. After overnight culture with or without plasma, PS exposure and EC adhesion were significantly increased (p < 0.05). MP numbers increased with longer RBC storage and after RBC culture with plasma. Culture conditions influenced MP numbers from Day 35 RBCs. RBCs stored in SAGM had significantly higher PS exposure after stress treatment than AS-1 RBCs (p < 0.02).

Conclusion: Storage for 35 days significantly increased RBC susceptibility to oxidative and in vitro transfusion-associated stresses and was higher for RBCs stored in SAGM compared to AS-1.

Figure 1. Longer-stored RBCs had increased PS-exposure and decreased GSH

(A) Flow cytometry plots of day 35 stored RBCs showing the gating strategy and PS-exposure detected by staining with FITC-labelled lactadherin. Log-scale light scatter plot (left) was used to set the RBC gate, which was then displayed on a fluorescence plot (right). 20,000 events were collected. (B) Frequency of PS⁺ RBCs during 35 days storage of SAGM RBCs (n = 9) and AS-1 RBCs (n = 7). (C) Concentration of RBC reduced glutathione (GSH) in SAGM RBCs (n = 9) and AS-1 RBCs (n = 7) after 3, 21 and 35 days of storage. SAGM RBCs (○), AS-1 RBCs (●) Significance defined as *p < 0.05 and **p < 0.01.

Figure 2. Longer-stored RBCs were more susceptible to oxidative stress

(A) Flow cytometry plots of tBuOOH-induced PS exposure on day 35 stored RBCs detected by FITC-labelled lactadherin. Control, no tBuOOH (left plot); treated with 0.5 mM tBuOOH for 30 min (right plot). Representative of five independent experiments. (B) RBCs after 3, 21 and 35 days of storage were treated with 0.5 mM tBuOOH for 30 min and then tested for adhesion to ECs under continuous flow perfusion conditions at 37 °C. Adhesion of tBuOOH-treated RBCs to ECs was significantly higher after 35 days of storage compared to 21 days of storage for SAGM RBCs (○) (p = 0.039) and AS-1 RBCs (●) (p = 0.016). Control, no tBuOOH SAGM RBCs (△) AS-1 RBCs (▲) are shown for comparison. Results are for 7 pairs. (C) RBCs at different storage ages from separate donors were treated with or without 0.5 mM tBuOOH for 30 min or 90 min as indicated and then tested for adhesion to ECs under continuous flow perfusion conditions at 37 °C. Day 6 (▽, donor 1); Day 6 (△, donor 2); Day 23 (■); Day 31 RBCs (●).

Figure 3. PS-containing lipid vesicles blocked adhesion of oxidized RBCs to ECs

Adhesion of RBCs to ECs was measured under continuous flow perfusion conditions at 37 °C, before and after tBuOOH-treatment of RBCs and with or without pre-incubation of ECs with PS-containing or PC-only (control) lipid vesicles as indicated. Horizontal bars represent the median of six individual SAGM-RBC units tested at median storage time of 34 days (range 23 to 42 days). Significance defined as *p < 0.05.

Figure 4. In vitro transfusion-associated stress increased PS-exposure and RBC-EC adhesion

(A) PS-exposure on stored RBCs before and after overnight culture at 37 °C in the presence of inflammatory plasma or RBC supernatant. PS-exposure was detected by staining with FITC-labelled lactadherin and flow cytometry. Culture induced significantly increased PS-exposure, which increased with longer storage of RBCs. Culture in RBC supernatant, Day 3 to 35, SAGM RBCs (○) (p = 0.004), AS-1 RBCs (●) (p = 0.016). Cultured in inflammatory plasma, Day 3 to 35, SAGM RBCs (▲) (p = 0.004), AS-1 RBCs (●) (p = 0.031). There was no significant difference between culture in inflammatory plasma or RBC supernatant. SAGM RBCs had significantly higher levels of PS-exposure induced by overnight culture compared to AS-1 RBCs at day 21 and day 35 of storage, p = 0.016 for both time-points and culture in RBC supernatant or inflammatory plasma (n = 7 pairs). Controls (‘no culture’, meaning stored samples were not incubated overnight) are also shown, SAGM RBCs (*), AS-1 RBCs (▲). (B) RBC adhesion to ECs before and after overnight culture at 37°C in the presence of plasma. RBC adhesion was measured under continuous flow perfusion conditions. After overnight culture, adhesion of RBCs stored for 35 days was significantly increased compared to RBCs stored for 3 days in SAGM (△) (p = 0.004) or AS-1 (▲) (p = 0.016). Uncultured RBCs had lower adhesion compared to cultured RBCs, but similarly showed increased adhesion with longer storage time; Day 3 to 35, SAGM RBCs (○) (p = 0.014); AS-1 RBCs (●) (strong trend, p = 0.075). After overnight culture, the number of adherent day 35 RBCs was significantly increased compared to no culture for AS-1 RBCs (p = 0.031) and almost significant for SAGM RBCs (p = 0.055). Results are from 9 SAGM RBCs and 7 AS-1 RBCs.

Figure 5. Number of MPs after in vitro transfusion-associated stress was influenced by RBC storage duration and presence of plasma

Stored RBCs were cultured overnight at 37 °C in the presence of inflammatory plasma or RBC supernatant. The numbers of total MPs (A), RBC-derived CD235a⁺ MPs (B) and lactadherin-binding PS⁺ MPs (C) released into the culture supernatant was quantified by flow cytometry. The numbers of MPs following culture of RBCs in plasma significantly increased with longer storage duration of SAGM RBCs (▲) (total MPs, p = 0.027; CD235a⁺ and PS⁺ MPs, p = 0.008) and AS-1 RBCs (○) (total MPs, not significant; CD235a⁺ and PS⁺ MPs, p = 0.016). There was no significant storage effect following culture of SAGM RBCs (○) or AS-1 RBCs in RBC supernatant. The differences in numbers of MPs following culture in the presence of plasma compared to RBC supernatant were significant at day 35 of storage for SAGM RBCs (total MPs, p = 0.004; CD235⁺ MPs, p = 0.008; PS⁺ MPs, p = 0.004) and AS-1 RBCs (total MPs, p = 0.031; CD235⁺ MPs, p = 0.031; PS⁺ MPs, p = 0.047). The difference in the number of PS⁺ MPs released by day 35 SAGM RBCs cultured in plasma was significantly higher than their AS-1 counterparts (p = 0.047). Results are from 9 SAGM RBCs and 7 AS-1 RBCs.