In Vitro Characterization and Metabolomic Analysis of Cold-Stored Platelets

Platelet transfusion has become an integral part of modern blood cell transfusion therapy. Currently, platelets are stored under room temperature with constant agitation for 5-7 days. Unfortunately, platelets when incubated at room temperature risk bacterial growth and significantly lowering their shelf life. The shortened shelf life leads to platelet wastage and increased cost.

Cold-stored platelets are considered a lasting solution by decelerating bacterial growth and extending the shelf life of room temperature platelets. However, previous in vivo transfusion studies showed that cold platelets were hastily cleared from the recipient’s circulation. Platelets losing their discoid morphology to become spherical was thought to be the reason behind their rapid clearance.

Efforts have been put towards improving their in vivo survival and recovery by preserving their discoid morphology. Unfortunately, other studies showed that in vivo survival and recovery were unaffected by platelet shape when cold-stored platelet surface glycoproteins GP1b clusters and glycosylates exposing β-N-acetylglucosamine. However, efforts to re-glycosylate refrigerated platelets have been slowed down by the realization that re-glycosylation cannot prevent clearance of human platelets refrigerated for 2 days.

Studies also have shown that storing platelets in cold temperatures helps to reduce the platelet’s cellular metabolism, maintain their ability to clot, and limit bacterial growth that may cause infections. Platelets stored at room temperature expend energy more quickly and, as a result, may struggle to form clots.

In recognition of the unique needs of bleeding patients, there’s an urgent need to look into the storage features of refrigerated platelets using up-to-date assay procedures. Moreover, it is important to understand the details of temperature-related biological alterations in platelets. In light of these facts and observations, University of British Columbia researchers: HanQi Wayne Zhao, Dr. Katherine Serrano, and Professor Dana Devine, together with Dr. Davide Stefanoni and Professor Angelo D’Alessandro from the University of Colorado, explored in vitro features and metabolic phenotypes of agitated and non-agitated refrigerated platelets and what distinguishes them from the standard room-temperature platelets. They adopted an untargeted metabolic procedure in examining the biochemical changes in platelets stored at 4 °C. The original research article is now published in the Journal of Proteome Research.

The research team collected blood samples from donors and made buffy coat platelet assemblages suspended in plasma. The buffy coat units were split into three units. They stored the first unit (RP) at 22 °C under constant agitation, the second unit (CPA) at 4 °C in a platelet agitator, and the third unit (CP) at 4 °C without agitation. The authors sampled the units between 1 and 14 days of storage. They counted the platelets, performed biochemical analysis, and compared in vitro data for any statistical significance.

Through careful  in vitro characterization, the authors observed that cold-stored units, both CPAs and CPs, had decreased platelet count, stable pH, lactate synthesis, and reduced glucose uptake compared to room-temperature units. The units also had a swift increase in P-selectin expression and elevated lactadherin binding compared to room-temperature units.

Aggregometry data in this study shows that cold-stored platelets had an improved aggregation response than room-temperature platelets. It’s this rapid response that makes cold-stored platelets beneficial for patients in need of immediate coagulation.

The authors carefully looked at the metabolomic analysis and showed that CPA and CP registered relatively stable metabolite rates than RPs. Slower enzyme kinematics due to lower storage temperature is the most likely reason behind this. In addition, higher α-ketoglutarate and fumarate levels in RPs suggested elevated mitochondrial activity. Consequently, higher mitochondrial activity results in reactive oxygen species production, detrimental to aggregation and general platelet function. This is why room temperature platelets are associated with high mitochondrial dysfunction, which impairs platelet activation and hemostatic function. On the contrary, having preserved mitochondrial function reference to lower storage temperature, CPAs and CPs become extremely useful for bleeding patients.

The authors also found that CPAs and CPs had lower oxidate stress, as shown by their preserved pentose and phosphate and glutathione pools. They also registered decreased markers of amino acid catabolism and beta-oxidation, implying a low energy demand. Agitation in the cold didn’t yield much impact on both metabolite levels and in vitro platelets functions. The observed decrease in pH of RPs indicated oxidative stress and uptake of sugar molecules. Decreased pH is usually an indication of platelet storage lesion.

The findings of UBC scientists pave the way for increasing the availability of platelets, a key blood component that is often in high demand to advance our understanding of in vivo functions of transfused cold-stored platelets.