A comparison of different methods of red blood cell leukoreduction and additive solutions on the accumulation of neutrophil-priming activity during storage

Abstract

Background: Three methods of leukoreduction (LR) are used worldwide: filtration, buffy coat removal (BCR), and a combination of the previous two methods. Additionally, there are a number of additive solutions (ASs) used to preserve red blood cell (RBC) function throughout storage. During RBC storage, proinflammatory activity accumulates; thus, we hypothesize that both the method of LR and the AS affect the accumulation of proinflammatory activity.

Study design and methods: Ten units of whole blood were drawn from healthy donors, the RBC units were isolated, divided in half by weight, and leukoreduced by: 1) BCR, 2) filtration, or 3) BCR and filtration (combination-LR); stored in bags containing AS-3 per AABB criteria; and sampled weekly. The supernatants were isolated and frozen (-80°C). RBC units drawn from healthy donors into AS-1-, AS-3-, or AS-5-containing bags were also stored and sampled weekly, and the supernatants were isolated and frozen. The supernatants were assayed for neutrophil (PMN)-priming activity and underwent proteomic analyses.

Results: Filtration and combination LR decreased priming activity accumulation versus buffy coat LR, although the accumulation of priming activity was not different during storage. Combination LR increased hemolysis versus filtration via proteomic analysis. Priming activity from AS-3 units was significant later in storage versus AS-1- or AS-5-stored units.

Conclusions: Although both filtration and combination LR decrease the accumulation of proinflammatory activity versus buffy coat LR, combination LR is not more advantageous over filtration, has increased costs, and may cause increased hemolysis. In addition, AS-3 decreases the early accumulation of PMN-priming activity during storage versus AS-1 or AS-5.

Figure 1. The supernatants of buffy-coat leukoreduced (BCR) samples accumulated more neutrophil priming activity and the combination LR does not decrease priming activity versus filtration

The maximal rate of SOD-inhibitable superoxide production is presented as a function of routine RBC storage. Compared to filtration (black bars), BCR (light gray bars) accumulated more PMN priming activity starting on D7 whereas there is no statistical difference versus combination LR (intermediate gray bars) on any of the storage days. * indicates p<0.05 from D1 within treatment and buffer-treated controls.

Figure 2. Lipids extracted from the supernatant of combination LR RBC units do not decrease PMN priming versus the lipids from filtered RBC units

The maximal rate of SOD-inhibitable superoxide anion production is presented as a function of routine storage. Lipids, known biological response modifiers, which prime PMNs, were isolated from both combination LR and filtration methods and used in PMN priming assays. No difference in priming was found comparing the two methods. * indicates p<0.05 from D1 within treatment and fMLF control and # indicates p <0.05 from filtration LR within day.

Figure 3. Arachidonic acid similarly accumulates in filtration and combination LR with storage

Concentrations of arachidonic acid, by ELISA, are presented as a function of routine storage. During storage, in both the filtered and combination LR RBC units there was an increase in arachidonic acid which was increased significantly (p<0.05) on D28 and D42 compared to D1 and D14 of storage. There were no statistical differences in AA concentration detected between the two leukoreduction methods. *= p<0.05 from D1 and D14 within LR groups

Figure 4. Peroxiredoxin-6 and α-enolase accumulate more in buffy-coat leukoreduced (BCR) samples during routine storage versus filtration LR

Panel A. A representative immunoblot which demonstrates increased amounts of peroxiredoxin-6 present in BCR supernatant versus filtration (LR) from D1 and again on D42. Panel B. * indicates p< 0.05 from D1 within treatment and fMLF control and # indicates p<0.05 from filtration LR within day. Panel C. Concentrations of α-enolase determined by ELISA show an increase from D1 to D42 in both methods, but there is increased accumulation of α-enolase present in the BCR supernatant on storage D28 and 42. The mean densitometry for panel A calculated with ImageJ software; * indicates p < 0.05 from D1 within treatment, and # indicates p <0.05 within day.

Figure 5. The effects of additive solutions on PMN priming activity in the supernatants of LR-RBCs through routine storage

The maximal rate of SOD-inhibitable superoxide anion production is shown as a function of routine storage of LR-RBCs in different additive solutions: AS-1, AS-3 and AS-5. Supernatants from AS-1 stored LR-RBCs (intermediate gray bars) had significant PMN priming activity by D14, which continued throughout storage. The priming activity from the supernatants from AS-3 stored LR-RBCs (Black bars) was significant on day 14, disappeared on D21 and reappeared in the supernatants from D28–D42. The priming activity from the supernatants from AS-5 stored LR-RBCs (light gray bars) became significant on day 14 which persisted throughout routine storage. * indicates p<0.05 within treatment groups and buffer-treated controls and †= p<0.05 for the supernatant priming activity of AS-1 RBCs vs. AS-3 and AS-5.