Whole blood flow cytometry measurements of in vivo platelet activation in critically-Ill patients are influenced by variability in blood sampling techniques

Abstract

Introduction: Flow cytometry is often used to measure in vivo platelet activation in critically-ill patients. Variability in blood sampling techniques, which may confound these measurements, remains poorly characterized.

Materials and methods: Platelet activation was measured by flow cytometry performed on arterial and venous blood from 116 critically-ill patients. We determined how variability in vascular sampling site, processing times, and platelet counts influenced levels of platelet-monocyte aggregates (PMA), PAC-1 binding (for glycoprotein (GP) IIbIIIa), and P-selectin (P-SEL) expression.

Results: Levels of PMA, but not PAC-1 binding or P-SEL expression, were significantly affected by variability in vascular sampling site. Average PMA levels were approximately 60% higher in whole blood drawn from an arterial vessel compared to venous blood (16.2±1.8% vs. 10.7±1.2%, p<0.05). Levels of PMA in both arterial and venous blood increased significantly during ex vivo processing delays (1.7% increase for every 10 minute delay, p<0.05). In contrast, PAC-1 binding and P-SEL expression were unaffected by processing delays. Levels of PMA, but not PAC-1 binding or P-SEL expression, were correlated with platelet count quartiles (9.4±1.6% for the lowest quartile versus 15.4±1.6% for the highest quartile, p<0.05).

Conclusions: In critically-ill patients, variability in vascular sampling site, processing times, and platelet counts influence levels of PMA, but not PAC-1 binding or P-SEL expression. These data demonstrate the need for rigorous adherence to blood sampling protocols, particularly when levels of PMA, which are most sensitive to variations in blood collection, are measured for detection of in vivo platelet activation.

Figure 1

Platelet-monocyte aggregates are higher in arterial whole blood from critically-ill patients. Platelet-monocyte aggregates were assessed in (A) baseline (e.g. unstimulated) or (B) TRAP-activated arterial or venous whole blood. Panel C shows a representative flow cytometric analysis of unstimulated and TRAP-activated platelet-monocyte aggregates measured in arterial (left) and venous (right) whole blood samples (*p<0.05 versus venous whole blood samples in multivariate comparisons after controlling for APACHE II score, processing time, ICU day, age, gender, and platelet count).

Figure 2

PAC-1 binding and P-SEL expression does not differ between (A) baseline or (B) TRAP-activated arterial and venous whole blood in critically-ill patients (p=NS all comparisons in multivariate comparisons after controlling for APACHE II score, processing time, ICU day, age, gender, and platelet counts).

Figure 3

Delayed processing times were associated with increased levels of platelet-monocyte aggregates (PMA), but not PAC-1 binding or P-SEL expression. Whole blood was left alone (baseline, Panel A) or stimulated with TRAP (Panel B) for 15 minutes. (*p<0.05 for trend in multivariate comparisons after controlling for APACHE II score, age, gender, vascular sampling site, ICU day, and platelet counts).

Figure 4

Levels of PMA correlate with the number of circulating platelets. Whole blood was left alone (baseline, Panel A) or stimulated with TRAP (Panel B) for 15 minutes (*p<0.05 for trend in multivariate analyses after controlling for APACHE II score, age, gender, ICU day, vascular sampling site, and processing time).