In vitro measures of membrane changes reveal differences between red blood cells stored in saline-adenine-glucose-mannitol and AS-1 additive solutions: a paired study

Abstract

Background: Saline-adenine-glucose-mannitol (SAGM) and a variant solution, AS-1, have been used for more than 30 years to preserve red blood cells (RBCs). Reputedly these RBC components have similar quality, although no paired study has been reported. To determine whether differences exist, a paired study of SAGM RBCs and AS-1 RBCs was conducted to identify membrane changes, including microparticle (MP) quantitation and in vitro RBC-endothelial cell (EC) interaction.

Study design and methods: Two whole blood packs were pooled and split and RBCs were prepared (n=6 pairs). One pack was suspended in SAGM and one in AS-1. Samples were collected during 42 days of refrigerated storage. RBC shape and size and glycophorin A (GPA)(+) and phosphatidylserine (PS)(+) MPs were measured by flow cytometry. RBC adhesion to ECs was determined by an in vitro flow perfusion assay. Routine variables (pH, hemolysis) were also measured.

Results: Compared to SAGM RBCs, AS-1 RBCs had lower hemolysis (p<0.04), lower GPA(+) MPs (p<0.03), and lower PS(+) MPs (p<0.03) from Day 14 onward. AS-1 RBCs had higher (p<0.02) side scatter from Day 28 onward compared to SAGM RBCs. SAGM RBCs were more adherent to ECs on Day 28 of storage compared to AS-1 RBCs (p=0.04), but reversed on Day 42 (p=0.02).

Conclusion: SAGM RBCs lose more membrane during storage. SAGM RBCs had increased adherence to ECs on Day 28 of storage, while AS-1 RBCs were more adherent on Day 42. The effect of these differences on the function and survival of SAGM RBCs and AS-1 RBCs after transfusion remains to be determined.

Figure 1

Routine quality parameters of paired RBC components stored refrigerated in SAGM or AS-1 additive solutions for 42 days. (A) pH and (B) % hemolysis. SAGM (closed symbol); AS-1 (open symbol). * Significant difference (p < 0.04) between SAGM RBCs and AS-1 RBCs, by paired t-test. # Significant change (p < 0.001) across storage period, by RM-ANOVA. Results are mean ± SD (n = 6 pairs).

Figure 2

Size and shape of RBCs stored in SAGM or AS-1 additive solutions. RBC mean cell volume (MCV) was calculated by an automated hematology analyzer (A). The change in arbitrary size of the RBCs during storage was measured by flow cytometric forward light scatter (FSC) (B); and the change in unevenness of the RBC surface membrane during storage was measured by flow cytometric side scatter (SSC) (C). SAGM (closed symbol); AS-1 (open symbol). * Significant difference (p < 0.02) between SAGM RBCs and AS-1 RBCs, by paired t-test. # Significant change (p < 0.001) across storage period, by RM-ANOVA. Results are mean ± standard error of the mean (n = 6 pairs).

Figure 3

Accumulation of MPs in the supernatant of RBCs stored in SAGM or AS-1 additive solutions. MPs were quantitated by flow cytometric absolute bead count assay. Glycophorin A⁺ (GPA) MPs were detected by staining with PE-conjugated anti-CD235a (A); and MPs with exposed phosphatidylserine were determined by binding of APC-conjugated annexin V (B). SAGM (closed bars); AS-1 (open bars). *Significant difference (p < 0.03) between SAGM RBCs and AS-1 RBCs, by paired t-test. # Significant change (p < 0.001) across storage period, by RM-ANOVA. Results are mean ± standard error of the mean (n = 6 pairs).

Figure 4

Adhesion of RBCs stored in SAGM or AS-1 additive solutions to ECs under continuous flow conditions. RBCs were perfused across ECs at a shear stress of 0.5 dyne/cm (A). Strength of adhesion of RBCs to ECs was determined by increasing the shear stress to 3 dyne/cm and the number of remaining adherent RBCs was scored (B). SAGM (closed symbol); AS-1 (open symbol). * Significant difference (p < 0.04) between SAGM RBCs and AS-1 RBCs, by paired t-test. # Significant change (p < 0.002) across storage period, by RM-ANOVA. Results are mean ± standard error of the mean (n = 6 pairs).