Purified granulocyte concentrates from buffy coats with extended storage time

Background

Granulocyte concentrates (GC) have limited clinical applicability due to rapid quality deterioration caused by excessive lactate production from erythrocytes, leading to decreased pH within 24 hours. Buffy coat-derived granulocytes are an alternative to apheresis donations. We have developed a system compatible with standard blood banking technologies to remove erythrocytes and platelets and extend the storage time.

Methods

Five to six AB0 blood group-matched buffy coats were combined to form a pool preparation. Subsequently, the erythrocytes were sedimented using HES solution. The resulting leucocyte-rich supernatant was washed twice with saline to remove the majority of platelets and then resuspended in AB0-matched donor plasma. This leucocyte concentrate was transferred to a platelet storage bag and stored for 72 hours. Cell count and viability, pH, blood gases, phagocytosis and oxidative burst activity were monitored daily.

Results

Applying this approach, erythrocyte and platelet concentrations were reduced to 0.4 % and 6.1 % of the baseline levels. A total of 49.8% of the originally present leucocytes could be extracted. This corresponds to an absolute granulocyte count of about 3.37 x 10⁹ cells. In the course of 72 hours of storage, there were no significant changes in cell counts. The viability exceeded 98% during the entire period. The rate of granulocytes performing phagocytosis and oxidative burst remained above 95% anytime.

Conclusion

Designed to obtain a leukocyte concentrate from buffy coats, our technique provides a precious alternative source to obtain granulocytes. Purification extends the maximum shelf life of GC from 24 h to 72 h and may therefore facilitate the availability for clinical purposes. For a clinically usable GC with > 1 x 10¹⁰ granulocytes about 15 to 20 buffy coats need to be pooled.

Offenlegung Interessenkonflikt:

Fanny Doss, Steffen Mitzner, Jens Altrichter are employees or shareholders of Artcline GmbH.

All others have no conflict of interest.