Reactive Oxygen Species Generated by CD45- Cells Distinct from Leukocyte Population in Platelet Concentrates Is Correlated with the Expression and Release of Platelet Activation Markers during Storage

Abstract

Background: Platelet stimulation with agonists is accompanied by the generation of reactive oxygen species (ROS) which promotes further platelet activation and aggregation. Considering different cell populations in platelet concentrates (PCs), this study investigates the correlation of ROS generation with the expression and release of platelet activation markers during storage. Methods: Samples obtained from 6 PCs were subjected to flow cytometry and ELISA to evaluate the expression and shedding of platelet P-selectin or CD40L during storage. Intracellular ROS were detected in either CD45- or CD45+ population by flow cytometry using dihydrorhodamine 123, while ROS production was analyzed in both P-selectin+ or P-selectin- and CD40L+ or CD40L- populations. To further evaluate the correlation between ROS generation and release function, TRAP-stimulated platelets were also subjected to flow cytometry analysis. Results: ROS detected in the CD45- population (leukocyte-free platelets) was significantly increased by fMLP and PMA. P-selectin- or CD40L- platelet did not show significant amount of ROS. Total ROS generation was significantly increased during platelet storage (day 0 vs. day 5; p = 0.0002) while this increasing pattern was directly correlated with the expression of P-selectin (r = 0.72; p = 0.0001) and CD40L (r = 0.69; p = 0.0001). ROS generations were significantly correlated with ectodomain shedding of these pro-inflammatory molecules. Conclusion: Our data confirmed increasing levels of intracellular ROS generation in both platelets (CD45-) and platelet-leukocyte aggregates (CD45+) during PC storage. The amount of detected ROS is directly correlated with platelet activation and release in each population while platelet-leukocyte aggregates generate higher levels of ROS than single platelets.

ROS scavenger, N‐acetyl‐l‐cysteine and NOX specific inhibitor, VAS2870 reduce platelets apoptosis while enhancing their viability during storage

“…The flow cytometric settings were optimized for the acquisition of platelets by logarithmic signal amplification in all four detectors (forward and side scatter channels and fluorescence channels FL1 and FL2). For analysis, the gate was set around an intact PLT population with the lowest level of leukocyte contamination detected by CD45 staining (as already described except that here DHE was used instead of DHR123). The percentage of platelets expressing intracellular DHE fluorescence in comparison with the background (negative control) was recorded.…”

Flow Cytometric Analysis of Leukocyte Populations in the Lung Tissue of Dromedary Camels

“…Further studies can be planned using this methodology to investigate the antimicrobial functions of camel lung phagocytes under inflammatory conditions (infection or injury). Although dihydrorhodamine is among the most frequently used dyes for measuring ROS by flow cytometry [63][64][65], the method is limited by the reduced sensitivity to ROS and non-specific reaction of DHR with other oxidants [65]. Therefore, DHR may be replaced with other stains, such as DCFDA, in future studies.…”

Prophylactic platelet transfusion at higher thresholds was associated with increased risk of death or major bleeding in neonates

“…It is unclear why infants who received platelet transfusion at high threshold are at increased risk of death and major bleeding. Infants who receive platelet transfusion are at risk of transfusion-related immunological and inflammatory effects,5 unbalanced neonatal platelet homeostasis from adult platelets, haemodynamic shifts due to platelet transfusion volume resulting in disturbed cerebral blood flow,6 platelet-derived reactive oxygen species7 aggravating aberrant angiogenesis of lung blood vessels and compromised blood flow from platelet microthrombi.…”