PURPOSE: Whole blood flow cytometry is commonly used to detect in vivo platelet activation during critical illness. Heterogeneity in blood sampling techniques, processing time, and platelet counts may significantly influence measurements and confound comparisons yet have not been adequately characterized.
METHODS: We prospectively collected whole blood from 116 critically-ill patients and measured platelet-monocyte aggregates (PMA), platelet surface P-selectin expression (P-SEL), and PAC-1 binding (for integrin αIIb activation) using flow cytometry.
RESULTS: The average age (±SD) of patients was 51.4 (±19.1) years and 58.6% were female. The average APACHE II score was 18.1 (±6.8) and 80% met consensus criteria for severe sepsis or septic shock. The in-hospital mortality rate was 18.1%. Levels of unstimulated and thrombin-receptor activating peptide (TRAP)-stimulated PMA were significantly higher in arterial vs. venous whole blood (16.2±1.8% vs. 10.7±1.2% and 59.4% vs. 52.9% respectively, p<0.05) and increased with longer delays ex vivo in the time between collection from patients and fixation for flow cytometry (1.7% increase for every 10 minute delay in processing, p<0.05). Levels of PMA were significantly lower in patients with platelet counts in the lowest quartile compared to the highest quartile (9.4±1.6% vs. 15.4±1.6%, p<0.05). In contrast, P-SEL expression and PAC-1 binding did not differ between arterial and venous blood samples and were not affected by delays in processing times or differences in platelet counts. Levels of PMA, P-SEL, and PAC-1 did not differ in blood drawn from central venous catheters compared to peripheral venipunctures.
CONCLUSIONS: To our knowledge, this is the first prospective study in a large, critically-ill cohort demonstrating that variability in vascular sampling site, processing time, and platelet count significantly influence levels of PMA but not P-SEL or PAC-1. Differences between arterial and venous whole blood may be due to vascular sequestration.
CLINICAL IMPLICATIONS: Knowledge of these potential confounders is important for investigators characterizing platelet activation in critical illness. These data are also consistent with prior observations supporting PMA as a more sensitive marker of in vivo platelet activation than P-SEL or PAC-1.
DISCLOSURE: The following authors have nothing to disclose: Matthew Rondina, Estelle Harris, Saritha Kalva, Shaohua Men, Diana Kastendieck, Guy Zimmerman, Andy Weyrich, Colin Grissom
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