Leucocyte cytokines dominate platelet cytokines overtime in non-leucoreduced platelet components

Abstract

Background: Leucoreduction of blood components, including platelet components, is strongly encouraged but not yet universal, especially outside high income countries. As both leucocytes and platelets secrete copious amounts of pro-inflammatory cytokines/chemokines under various conditions and during storage, we investigated the potential of the respective secretory programmes of these cells in order to evaluate their subsequent pathophysiological effects.

Material and methods: A total of 158 individual non-leucoreduced platelet components were obtained from Tunisian donors and tested for characteristic biological response modifiers (BRM) of leukocytes (IL-1β, IL-8), platelets (sCD62P, sCD40L) and both cell types (TNF-α, RANTES) in the presence or absence of thrombin stimulation and after different periods of storage (up to 5 days). BRM levels were determined using enzyme-linked immunosorbent assays and Luminex technology. Platelet-leucocyte aggregate formation during storage was analysed using flow cytometry.

Results: Leucocyte- and platelet-associated BRM had clearly distinct profiles both at the onset (day 0) and termination (day 5) of the observation period but altered during the intermediate period so that their respective importance was inverted; in fact, the profiles were merged and indistinguishable on days 2-3. The leucocyte-derived BRM largely dominated over platelet-derived ones and further altered the BRM platelet secretion programme.

Discussion: This study contributes substantial, new information on leucocyte/platelet interactions and their likely role in transfusion when leucodepletion cannot be performed or is only partially achieved.

Figure 1

Kinetics of BRM release depending on the day of storage. * or ^≠: p<0.05; ** or ^≠≠: p<0.001; *** or ^≠≠≠: p<0.0001. Asterisks and hashtags represent significant differences against D0 and D1, respectively, using the Kruskal-Wallis test with Dunn’s correction for multiple comparisons. Data are presented as mean ± SEM; n=158, 29, 65 and 64 for D0, D1+2, D3+4 and D5, respectively. D: day; TNF: tumour necrosis factor; RANTES: regulated on activation, normal T cell expressed and secreted; IL: interleukin; SEM: standard error mean.

Figure 2

A two-dimensional correlation monoplot of the coefficients of the first two principal components, showing relationships between the BRM during storage. The correlation monoplot has vectors pointing away from the origin to represent the original variables. The angle between the vectors is an approximation of the correlation between the variables. A small angle indicates that the variables are positively correlated, an angle of 90 degrees indicates the variables are not correlated, and an angle close to 180 degrees indicates the variables are inversely correlated. The length of the line and its closeness to the circle indicate how well the variable is represented in the plot. A, B, C and D illustrate correlations at D0, D1+2, D3+4 and D5, respectively. D: day; TNF: tumour necrosis factor; RANTES: regulated on activation, normal T cell expressed and secreted; IL: interleukin.

Figure 3

Leucocyte-platelet aggregate formation during PC storage. Complexes were detected by flow cytometry using allophycocyanin (APC)-conjugated anti-CD45 (BD Biosciences) and fluorescein isothiocyanate (FITC)-conjugated anti-CD41 (R&D system). Data are presented as mean ± SEM; n=6. PC: platelet concentrate; D: day; SEM: standard error mean.

Figure 4

Secretory profiles of BRM in response to TRAP stimulation. Comparisons used the mean of the Wilcoxon sign ranked test for paired samples. White boxes represent the unstimulated condition and grey boxes represent the TRAP-stimulated condition. Data are presented as mean ± SEM; n=158, 29, 65 and 64 for D0, D1+2, D3+4 and D5, respectively. * p<0.05; ** p<0.001; *** p<0.0001. BRM: biological response modifier; TRAP: thrombin receptor activator peptide; TNF: tumour necrosis factor; RANTES: regulated on activation, normal T cell expressed and secreted; IL: interleukin.

Figure 5

Fold-changes of BRM levels in response to TRAP stimulation depending on storage time. Data are presented as mean ± SEM; n=158, 29, 65 and 64 for D0, D1+2, D3+4 and D5, respectively. BRM: bilogical response modifier; TRAP: thrombin receptor activator peptide; RANTES: regulated on activation, normal T cell expressed and secreted; TNF: tumour necrosis factor; IL: interleukin; D: day; SEM: standard error mean.