Lymphocyte Survival and Activation in Stored Platelet Concentrates After Gamma-Irradiation or Pathogen Reduction Technology Treatment.

Abstract 2113

Poster Board II-90

The presence of white blood cells, particularly lymphocytes, in blood products has been shown to contribute to the development of a variety of adverse events including both donor anti-recipient and recipient anti-donor responses. Therefore leukoreduction of blood products or inactivation of leukocytes is necessary. We are currently using gamma-irradiation (Radiator Gammacell 3000 Elan) to inactivate leukocytes in blood components. This study shows the comparison of irradiation and PRT treatment (Mirasol*^®) on lymphocyte survival and inactivation in non-leukoreduced platelet concentrates (PCs). PRT treatment is a pathogen reduction technology that targets nucleic acids after exposure to riboflavin and UV-light. We analyzed 7 untreated (C), 7 PRT treated (M) and 7 irradiated PCs (RD) in our study. Non-leukoreduced buffy coats (mean volume 65 ml) were obtained from the Regional Blood Center, Warsaw. PCs were prepared by pooling 15 buffy coats (ABO identical) suspended in 3 plasma units, and dividing the pool into 3 equal-weight units in bags made of the same material. Following addition of 35 ml of riboflavin solution, M units were illuminated. The same volume of saline solution, was added to the C group and RD units. All PCs were then stored at 22°C with agitation for 5 days. Samples were removed on days 1, 3 and 6 for analysis. The lymphocyte survival rate was determined by 7AAD (7-amino-actinomycin D staining of dead cells, Becton Dickinson) and their activation by anti-CD69-APC staining (Becton Dickinson). Samples were also stained with anti-CD45-PE antibodies to identify and gate on lymphocytes. Samples were analyzed on the Becton Dickinson Cytometer FACSCanto I.

No increase in the number of dead cells was observed during 6 days of storage in the C group. After 3 days of storage however, in the M group the percentage of dead cells was significant higher than in C and RD groups (Student t-test, p=0.004 and p=0.03, respectively). After 6 days, the percentage of dead cells in the M samples was 72% vs. 30% following irradiation. The percentage of 7AAD-positive cells was significantly higher compare to C samples, both in M (p=0,001) and in RD samples (p=0,004). The percentage of dead lymphocytes was also observed to be statistically higher in M samples than in RD (p= 0,001).

Analysis of lymphocyte activation was performed on live (7AAD-negative, CD45-positive) cells only. CD69 expression ranged between 20% and 40% in all tested samples (C, M, RD) during 6 days of storage. On days 1 and 3 of storage, Mirasol treatment significantly reduced lymphocyte activation as shown by the ratio (test/control) of %CD69-positive cells (p=0,004 and p=0,001, respectively) and mean fluorescence expression intensity of CD69. Interestingly, after 6 days of storage, RD samples showed significant higher lymphocyte activation then C and M samples (p=0,03 and p=0,02, respectively).

In summary, a significant increase of dead lymphocytes after 6 days of storage was observed in PRT-treated PCs. This increase was two-fold higher than in gamma-irradiated PCs. At the same time a decrease in lymphocyte activation during 6 days of storage was observed in PRT-treated PCs. Overall, the use of PRT achieves better leukocyte inactivation than gamma-irradiation.