Leucocyte viability and de-novo synthesis of cytokines in platelet concentrates

Purpose: White blood cells (WBC) in platelet concentrates (PC) are a potent source of inflammatory cytokines and chemokines responsible for side effects such as febrile non-hemolytic transfusion reactions (FNHTR). It remained questionable which WBC content can be tolerated to avoid the accumulation of cytokines and whether these mediators originate from degranulating WBC or de-novo synthesis during storage. Methods: We investigated the secretion of IL-1β, IL-2, IL-6, IL-8, TNF α and Interferon γ and quantified the corresponding mRNA in PCs with regard to different WBC contamination and storage times. The viability of WBCs in PC during storage was determined by flow cytometry using 7AAD staining. To investigate the ability of WBC in PC to effect active cytokine synthesis, we performed a superantigen stimulation of PC samples at the end of storage and determined the content of IL-lβ, IL-6 and IL-8. Results: We detected a significant increase of cytokines in PCs with > 10⁸ WBCs during storage for IL-lβ, IL-6, IL-8 and TNF α. The mRNA quantification of these cytokines showed increasing mRNA copy numbers depending on the level of WBC contamination. WBC viability was still 82.7 +/- 0.8% on day 5 of storage. At the end of storage PC samples with a WBC contamination of 10⁹ were stimulated with LPS and TSST-1 with and without addition of mRNA transcription inhibitor actinomycin D (AD). After 24 hours of stimulation, the content of IL-1β, IL-6 and IL-8 in stimulated samples was 22 to 45-fold higher than in unstimulated samples or samples with addition of AD. Conclusion: In PCs with 10⁷WBC or lower, we hardly found any cytokine contamination. In contrast, PCs with a WBC contamination of 10⁹ contained IL-lβ, IL-6, IL-8 and TNF α, with highest levels on day 5. PCs with 10⁸ WBC had an intermediate position. The results of the cytokine mRNA quantification corresponded to these findings. To prove the hypothesis of de-novo cytokine synthesis in PCs by WBC, we analysed the WBCs viability after 5 days and found more than 80% are 7-AAD negative and in addition did a superantigen stimulation of PC samples which clearly showed that WBCs in PCs are able to produce high amounts of cytokines and chemokines by active mRNA transcription and protein synthesis even at the end of storage. Therefore, there is great evidence that cytokine accumulation in PCs is due to de-novo synthesis by activated WBCs.