Improving materials and methods for storage of blood components

Project leader: Dirk de Korte PhD

The blood bank is a producer of blood products under GMP conditions and its needs to know the limits of the methods in use, for example with respect to temperature effects during collection and preparation. In 2010 some studies in this field were performed. Another aspect of this research line is the quality of blood components in relation to adverse effects of blood transfusion, like TRALI.

Active cooling of whole blood to room temperature improves blood component quality
Many countries use cooling plates to actively cool collected whole blood (WB) to room temperature. This was mainly introduced to standardize the temperature profiles and to allow component production during standard working hours. Until now, no paired comparison has been performed between active and not active cooling, and it was our aim to compare the effect of active versus no active cooling on the in vitro quality of WB and subsequently prepared blood components.

For this study, two units of WB were pooled and divided shortly after donation. One unit was placed under a butane-1,4-diol plate to obtain active cooling; the other was placed in an insulated box with other warm units to mimic worst-case holding conditions. WB was held overnight and processed into a white blood cell (WBC)-reduced red blood cells (RBCs), buffy coat (BC), and plasma. The BCs were further processed into platelet (PLT) concentrates. After overnight storage, ATP content of the RBCs was 4.9 ± 0.3 mmol/g Hb for actively cooled WB versus 4.5 ± 0.4 mmol/g Hb for not actively cooled WB (p < 0.001). On Day 42 of storage, RBCs prepared from this WB contained 3.1 ± 0.3 mmol ATP/g Hb with active cooling versus 2.6 ± 0.3 mmol/g Hb without (p < 0.001). Hemolysis on Day 42 was 0.35 ± 0.08% with active cooling and 0.67 ± 0.21% without (p < 0.001). No effect was observed on the in vitro quality of plasma, BC, or PLT concentrates. Active cooling of WB results in improved ATP levels and less hemolysis in WBC reduced RBCs, although the clinical implications are unclear. It has no effect on the in vitro quality of plasma or PLT concentrates.

Accumulation of bio-active lipids during storage of blood products is not cell but plasma-derived and temperature dependent
Bio-active lipids (lysophosphatidylcholines, (lysoPCs)) accumulating during storage of blood products are thought causative in onset of TRALI through activation of neutrophils. LysoPCs are thought to be derived from cell-membrane degradation products such as phosphatidylcholines (PC) by partial hydrolysis of PC, a process that is catalyzed by phospholipase A2 (PLA2). We investigated the underlying mechanisms of lysoPC generation and its contribution to in vitro neutrophil-priming capacity during storage of red blood cells (RBCs), platelet concentrates (PLTs) and cell-free plasma (see figure). Storage of RBCs in SAGM did not result in accumulation of lysoPCs or neutrophil-priming capacity. Replacement of SAGM by plasma as RBC storage medium caused elevated lysoPC levels at day 0, which did not further increase during storage. Cell-free plasma stored at 22°C showed accumulation of lysoPCs during storage, which was not present at 4°C. Addition of a soluble (s)PLA 2 or cytosolic (c)PLA2-inhibitor did not prevent accumulation of lysoPCs in plasma. In PLTs, lysoPC accumulation during storage was plasma dependent, but lysoPCs did not explain the observed neutrophil-priming effect as preventing accumulation of lysoPCs by removing the plasma fraction did not prevent the neutrophil-priming capacity. Based upon these results it was concluded that accumulation of lysoPCs during storage is not cell but plasma-derived and storage temperature-dependent and does not explain the neutrophil-priming effect of aged products. This research was performed in collaboration with Alexander Vlaar and Nicole Juffermans from Academic Medical Center.

Panel A shows H2O2-release when neutrophils were incubated with buffer solution (negative control) or E.Coli LPS/LPB 20ng/mL (positive control). Panel B shows H2O2-release when neutrophils were incubated with supernatant of cell depleted plasma obtained directly from whole blood donation, stored at 22˚C of different storage time points (day 0 and day 7). Panel C shows H2O2 -release when neutrophils were incubated with supernatant of platelet concentrates (PLTs) stored in 100 % plasma of different storage time points (day 0 and day 7). Panel D shows H2O2-release when neutrophils were incubated with supernatant of PLTs stored in 35% plasma: 65% SSP of different storage time points (day 0 and day 7). Panel E shows H2O2-release when neutrophils were incubated with supernatant of PLTs stored in 5% plasma: 95% SSP of different storage time points (day 0 and day 7). (n=3 batches for each measurement). *p<0.05, **p<0.01, paired t-test. Data are presented as Mean and SD.

Key publications

Bontekoe IJ, van der Meer PF, de Korte D. Effect of rate and delay of cooling during initial cooling process: in vitro effect on red cells. Vox Sang 2010; Dec 22 [Epub ahead of print].

Vlaar AP, Hofstra JJ, Kulik W, van Lenthe H, Nieuwland R, Schultz MJ, Levi MM, Roelofs JJ, Tool AT, de Korte D, Juffermans NP. Supernatant of stored platelets causes lung inflammation and coagulopathy in a novel in vivo transfusion model. Blood 2010; 116(8):1360-8.