Assessment of purified granulocyte preparations in multiple environmental settings

Background

Dysfunctional immune cells contribute to sepsis-related immune paralysis. Transfusing healthy donor immune cells, like granulocyte concentrates (GC), can cause tissue damage via neutrophil activity. Prior trials utilized standard GC in an extracorporeal therapy system, separating patient and donor cells with plasma filters, resulting in positive effects with reduced side effects. Our ex vivo study demonstrates the efficacy of purified granulocyte preparations in diverse environmental conditions.

Methods

Standard GCs (sGCs) were sedimented, washed twice with 0.9% sodium chloride (NaCl), and resuspended in blood group-identical fresh frozen plasma. The resulting purified GCs (pGCs) were then stored in platelet bags at a cell concentration of about 5 × 10⁷ ± 1.8 × 10⁷ leukocytes/ml without agitation at room temperature for multiple days. Cell count and viability, pH, blood gases, phagocytosis, and oxidative burst were monitored daily. Furthermore, we simulated prolonged circulation times by subjecting the pGC to extended extracorporeal therapy simulations up to 24 h. Following the prolonged circulation, we analyzed the cytokine profile to determine any alterations in cytokine activity.

Results

Cells were viable throughout the study period and exhibited well-preserved functionality and efficient metabolic activity. Course of lactate dehydrogenase and free hemoglobin concentration yielded no indication of cell impairment. The capability of the cells to secret various cytokines was preserved after prolonged circulation. Of particular interest is preservation in performance of the cells after 5 days of storage, demonstrating the sustained shelf life and performance of the immune cells in the purified GCs.

Conclusion

Results demonstrate the suitability of a simplified extracorporeal system. Furthermore, granulocytes remain viable and highly active even beyond a 6 hour treatment even after storage of over 3 days supporting the treatment of septic patients with this system in advanced clinical trials.

Offenlegung Interessenkonflikt:

Steffen Mitzner, and Jens Altrichter are employees or shareholders of ARTCLINE GmbH. All others have no conflict of interest.