Frontiers in immunology · 2025
Experimental conditions shape in vitro formation of murine platelet-leukocyte aggregates.
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Trivigno Silvia Maria Grazia, Assinger Alice, Schrottmaier Waltraud Cornelia
Аннотация
BACKGROUND: Platelets interact with leukocytes to fine-tune their functions, thus providing essential regulation of (patho-) physiologic immune responses in various diseases. Circulating platelet-leukocyte aggregates (PLAs) represent a sensitive biomarker to estimate disease severity both in patients and murine models. However, a limited understanding of the sensitivity of PLA measurements to methodological variables may undermine their accuracy and comparability.
OBJECTIVES: To elucidate how blood draw techniques, anticoagulation, processing delay and assay temperature affect murine platelet-leukocyte interactions.
METHODS: Murine blood was obtained via retro-orbital, vena cava or cardiac puncture, anticoagulated with heparin, citrate or acid-citrate-dextrose (ACD) +/- recalcification and stored for 30-120 min before stimulation at room temperature or 37°C with adenosine diphosphate (ADP), cross-linked collagen-related peptide (CRP-XL) and protease-activated receptor 4-activating peptide (PAR4-AP). PLA formation and leukocyte activation were analyzed by flow cytometry.
RESULTS: Basal PLAs were minimally affected by blood sampling and anticoagulant, though delayed processing significantly raised basal PLAs. Agonist-induced PLA formation was independent of anticoagulation, and sampling technique did not affect ADP- or PAR4-AP-induced PLA levels. However, CRP-XL sensitivity was elevated in blood obtained by cardiac puncture. Contrarily, both delayed processing and stimulation at 37°C impaired CRP-XL sensitivity, but augmented ADP and PAR4-AP responses. Regulation of leukocyte activation did not follow PLA patterns, with monocytes and neutrophils displaying distinct susceptibility to anticoagulation, storage and temperature.
CONCLUSIONS: Variations in preparing murine blood samples exert distinct influences on platelet-leukocyte interactions in vitro, underlining the critical need for fastidious assay optimization to support data reproducibility and comparability.
OBJECTIVES: To elucidate how blood draw techniques, anticoagulation, processing delay and assay temperature affect murine platelet-leukocyte interactions.
METHODS: Murine blood was obtained via retro-orbital, vena cava or cardiac puncture, anticoagulated with heparin, citrate or acid-citrate-dextrose (ACD) +/- recalcification and stored for 30-120 min before stimulation at room temperature or 37°C with adenosine diphosphate (ADP), cross-linked collagen-related peptide (CRP-XL) and protease-activated receptor 4-activating peptide (PAR4-AP). PLA formation and leukocyte activation were analyzed by flow cytometry.
RESULTS: Basal PLAs were minimally affected by blood sampling and anticoagulant, though delayed processing significantly raised basal PLAs. Agonist-induced PLA formation was independent of anticoagulation, and sampling technique did not affect ADP- or PAR4-AP-induced PLA levels. However, CRP-XL sensitivity was elevated in blood obtained by cardiac puncture. Contrarily, both delayed processing and stimulation at 37°C impaired CRP-XL sensitivity, but augmented ADP and PAR4-AP responses. Regulation of leukocyte activation did not follow PLA patterns, with monocytes and neutrophils displaying distinct susceptibility to anticoagulation, storage and temperature.
CONCLUSIONS: Variations in preparing murine blood samples exert distinct influences on platelet-leukocyte interactions in vitro, underlining the critical need for fastidious assay optimization to support data reproducibility and comparability.