Prolonging neutrophil room temperature storage with clinically-approved solutions: Implications for granulocyte transfusion

Introduction: Granulocyte concentrates (GC) are leukocyte preparations enriched in neutrophils that can potentially save neutropenic patients from life-threatening, antimicrobial-resistant infections. The main challenge of GC transfusions is preserving the viability and antimicrobial activity of neutrophils beyond 24 h to reduce the logistical burden on collection centers and increase the availability of this cell therapy. Thus, the aim of this study was to explore extending the ex vivo viability and antimicrobial activity of GC neutrophils up to 72 h with a unique combination of the clinically-approved additives Plasma-Lyte, SAGM, AS-3 and Alburex.

Methods: Neutrophils isolated from healthy donors were resuspended in autologous plasma (AP) at the same concentration as in GCs and diluted with various combinations of Plasma-Lyte, SAGM, AS-3 and/or Alburex with or without the addition of buffers, and stored at room temperature for up to 72 h. During storage, neutrophil viability, phagocytosis and intracellular reactive oxygen species production were measured by flow cytometry. Extracellular reactive oxygen species production was measured by spectrophotometry and chemotaxis by the number of calcein stained neutrophils that migrated towards the chemotactic peptide, N-Formyl-Met-Leu-Phe (fMLF). The same assays were performed on pooled, residual leukocyte units generated by the Reveos system, after storage in the additive combination that most effectively preserved the viability and function of isolated neutrophils.

Results: The additive combination that best performed in the majority of the assays contained Plasma-Lyte, buffers and AS-3. Neutrophil viability was preserved for a maximum of 48 h and phagocytosis of opsonized bacteria and reactive oxygen species production up to 72 h of storage at room temperature. In contrast, fMLF-induced chemotaxis decreased by 20 % after 24 h storage while extracellular reactive oxygen species production increased significantly within the same time period. Supplementing GCs prepared from pooled, residual leukocyte units with this storage solution after the standard 16-24 h processing period as per the blood center guidelines, did not significantly improve the preservation of neutrophil viability and function.

Conclusion: Our findings provide proof of concept that mixtures of clinically-approved additives can be tailored to significantly prolong the viability and function of freshly isolated neutrophils during room temperature storage. The unique additive composition of this storage solution that we developed for freshly isolated neutrophils, requires further optimization for use with pooled, residual leukocyte units as well as the time point at which the solution is added during processing to prolong the viability and functions of neutrophils in this blood product.