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

Matthew T Rondina, Colin K Grissom, Shaohua Men, Estelle S Harris, Hansjorg Schwertz, Guy A Zimmerman, Andrew S Weyrich, Matthew T Rondina, Colin K Grissom, Shaohua Men, Estelle S Harris, Hansjorg Schwertz, Guy A Zimmerman, Andrew S Weyrich

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.

Copyright © 2011 Elsevier Ltd. All rights reserved.

Figures

Figure 1
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

Figure 2

PAC-1 binding and P-SEL expression…

Figure 2

PAC-1 binding and P-SEL expression does not differ between (A) baseline or (B)…

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…

Figure 3

Delayed processing times were associated with increased levels of platelet-monocyte aggregates (PMA), but…

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

Figure 4

Levels of PMA correlate with…

Figure 4

Levels of PMA correlate with the number of circulating platelets. Whole blood was…

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
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References
    1. Schwertz H, Weyrich AS, Zimmerman GA. Cellular interactions of platelets, leukocytes and endothelium in systemic inflammatory responses and sepsis. In: Castro Faria Neto H, Marcus D, editors. Sepsis: From Bench to Bedside. Rio de Janerio: Revinter Press; 2007. pp. 107–120.
    1. Gawaz M, Dickfeld T, Bogner C, Fateh-Moghadam S, Neumann FJ. Platelet function in septic multiple organ dysfunction syndrome. Intensive care medicine. 1997;23(4):379–385. - PubMed
    1. Gawaz M, Fateh-Moghadam S, Pilz G, Gurland HJ, Werdan K. Platelet activation and interaction with leucocytes in patients with sepsis or multiple organ failure. Eur J Clin Invest. 1995;25(11):843–851. - PubMed
    1. Michelson AD, Furman MI. Laboratory markers of platelet activation and their clinical significance. Curr Opin Hematol. 1999;6(5):342–348. - PubMed
    1. Michelson AD. Flow cytometry: a clinical test of platelet function. Blood. 1996;87 (12):4925–4936. - PubMed
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Figure 2
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
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

Figure 4

Levels of PMA correlate with…

Figure 4

Levels of PMA correlate with the number of circulating platelets. Whole blood was…

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
Similar articles
Cited by
References
    1. Schwertz H, Weyrich AS, Zimmerman GA. Cellular interactions of platelets, leukocytes and endothelium in systemic inflammatory responses and sepsis. In: Castro Faria Neto H, Marcus D, editors. Sepsis: From Bench to Bedside. Rio de Janerio: Revinter Press; 2007. pp. 107–120.
    1. Gawaz M, Dickfeld T, Bogner C, Fateh-Moghadam S, Neumann FJ. Platelet function in septic multiple organ dysfunction syndrome. Intensive care medicine. 1997;23(4):379–385. - PubMed
    1. Gawaz M, Fateh-Moghadam S, Pilz G, Gurland HJ, Werdan K. Platelet activation and interaction with leucocytes in patients with sepsis or multiple organ failure. Eur J Clin Invest. 1995;25(11):843–851. - PubMed
    1. Michelson AD, Furman MI. Laboratory markers of platelet activation and their clinical significance. Curr Opin Hematol. 1999;6(5):342–348. - PubMed
    1. Michelson AD. Flow cytometry: a clinical test of platelet function. Blood. 1996;87 (12):4925–4936. - PubMed
Show all 29 references
Publication types
MeSH terms
[x]
Cite
Copy Download .nbib .nbib
Format: AMA APA MLA NLM
Figure 4
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

References

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