Whole blood flow cytometry is commonly used to detect in vivo
platelet activation in health and disease[9
]. Technical variation in blood collection methods, delays in specimen transport and processing, and heterogeneity in clinical features may confound these measurements and impede accurate comparisons between patient groups and across studies. In our prospectively studied cohort of critically-ill patients, levels of PMA were significantly higher in both unstimulated and TRAP-activated arterial blood compared to venous blood. In contrast, PAC-1 binding and P-SEL expression were unaffected by differences in vascular sampling sites.
Although there is limited published data on how vascular sampling site influences measurements of platelet activation, our data are consistent, in part, with prior observations. Rubens and colleagues examined platelet activation in eight patients undergoing elective cardiac surgery[19
]. Overall, levels of GP IIbIIIa (i.e. PAC-1) and GMP-140 (i.e. P-SEL) did not differ significantly between arterial and venous whole blood. However, when these assays were performed in triplicate on two patients, levels of GP IIbIIIa and GMP-140 were higher in arterial whole blood[19
Our study extends this published work to a larger cohort of patients not previously studied in this setting. The underlying mechanism(s) responsible for the increased levels of PMA in arterial blood from critically-ill patients with sepsis and ALI/ARDS is not entirely clear but several explanations are plausible. Although not well characterized, during septic syndromes, platelets have been suggested to undergo sequestration in venous vascular beds and thus may not as readily interact with and bind to monocytes to form heterotypic PMAs[20
An alternative hypothesis that is supported with both in vitro
and in vivo
data is that the septic milieu alters normal platelet reactivity to make them more susceptible to activation from shear forces. Shear forces in arteries are often greater than forces in veins [21
] and play a pivotal role in platelet activation, adhesion, and aggregation[22
]. For example, in vitro
studies demonstrate that platelet-leukocyte aggregation and P-SEL expression are increased in whole blood subject to shear stress [23
]; an effect that may be reversed by blocking P-SEL[24
]. Similarly, in stable patients with coronary artery disease, intracoronary shear stress induced platelet activation as evidenced by up-regulation of PMA formation without platelet activation of glycoprotein IIb/IIIa [25
], which can be detected through PAC-1 binding. Our findings are consistent with these published in vitro
and in vivo
observations as we found that PMA formation, but not PAC-1 binding, was increased in arterial blood. Although we did not observe increased P-SEL expression in arterial whole blood, in patients with sepsis, platelet P-selectin may be rapidly translocated to the cell surface and shed even though platelets continue to circulate and function[6
]. This rapid shedding of P-SEL, which likely occurred early during the onset of sepsis (and perhaps before patients were admitted to the ICU), may have prevented us from detecting differences.
processing time delays greater than 45 minutes resulted in levels of PMA that were 2-fold higher compared to levels in whole blood that was processed within 15 minutes of collection. In comparison, delays in processing time did not affect PAC-1 binding or P-SEL expression in either unstimulated or TRAP-activated whole blood. These data parallel published reports in healthy subjects. For example, Harding et al[17
] observed a 2.8% increase in levels of PMA for every 10 minute delay prior to processing for whole blood flow cytometry, a trend very similar to our observations in critically-ill subjects (i.e. 1.7% absolute increase for every 10 minute delay prior to processing). Our findings build on Harding and colleague’s report by demonstrating that delays in processing time also influence TRAP-stimulated levels of PMA. Additionally, our data support the importance of timely processing of whole blood for flow cytometry when measuring PMA formation in critically-ill patients[15
Since platelets undergo auto-activation in whole blood ex vivo
, once drawn, blood should be promptly utilized for measurements of platelet activation by whole blood flow. PMAs, suggested to be a very sensitive marker of platelet activation[7
], may offer advantages for the detection of in vivo
platelet activation. Nevertheless, since PMAs are also influenced by the source of blood, processing time, and platelet counts, any studies using PMAs as a marker of platelet activation should follow strict procedures when drawing and processing blood to minimize confounding. In situations where facilities for the flow cytometry preparation are not located in close proximity to clinical care units and thus delays in processing are likely, measuring PAC-1 binding or P-SEL expression may be advantageous as these markers of platelet activation appear to be less sensitive to processing delays.
Finally, these data also demonstrate that in unstimulated whole blood from critically ill patients, PMA formation, but not PAC-1 binding or P-SEL expression, increased with greater numbers of circulating platelets. Not surprisingly, upon stimulation with TRAP, levels of PMA also increased in patients with higher platelet counts, perhaps reflecting the presence of a larger population of platelets responding to ex vivo activating signals.
The strengths of our study include the large number of patients, the prospective study design, and rigorous data collection methods. Potential confounders were well controlled for in multivariate regression analyses, minimizing bias and reducing the likelihood that our findings were due solely to chance. In addition, as variability in vascular sampling site, processing time, and platelet counts selectively influenced levels of PMA but not PAC-1 binding or P-SEL expression, our findings are unlikely to be due solely to chance. Rather, they suggest intrinsic differences between these indices of platelet activation in response to agonists commonly present during sepsis.
Although we could not collect blood from all patients simultaneously from both an arterial and venous blood vessel, we did measure levels of PMA, PAC-1 binding, and P-SEL expression, in unstimulated arterial and venous blood drawn simultaneously from septic patients who were prospectively studied (n=4). Consistent with the findings of our larger study cohort (), we found that levels of PMA were higher in arterial whole blood (Supplemental Figure 2A
), although the sample size was too small to demonstrate significant differences. Also consistent with our observations in the larger cohort (), there was no difference in PAC-1 binding or P-SEL expression (Supplemental Figure 2B
We also did not study a group of healthy, control subjects in parallel and this may be considered a limitation. Nevertheless, the objective of our study was to prospectively examine how variability in blood collection and processing influences measurements of in vivo platelet activation. Additional studies are necessary to determine if our findings are also true in healthy subjects as well as hospitalized patients without ALI/ARDS (e.g. elective or urgent surgery, heart failure, acute myocardial infarction, etc).
Heparin, often used to maintain the patency of arterial catheters, may inadvertently cause platelet activation[26
]. However, this did not influence the findings of our study as all the arterial and venous lines were maintained with normal saline rather than heparin. Furthermore, we also performed subgroup analyses to confirm that levels of PMA, PAC-1 binding, and P-SEL expression did not differ between septic patients receiving heparinoids and those not receiving heparinoids (data not shown). Thus, ex vivo
activation due to heparinoids cannot account for our observations. Anti-platelet agents, such as aspirin, may attenuate platelet activation responses and confound measurements. Nevertheless, published and unpublished studies have demonstrated that aspirin does not influence platelet-leukocyte aggregation or P-SEL expression([28
] and Rondina et al, unpublished data). Furthermore, only a small number of patients in our study were taking aspirin at the time of ICU admission (6%, n=7). In pre-specified analyses where these patients were excluded, differences in levels of PMA (but not PAC-1 binding or P-SEL expression) between arterial and venous whole blood persisted. Moreover, these differences were also apparent in analyses restricted to only the patients taking aspirin at the time of admission. Thus, based on these secondary analyses, we do not think that concomitant aspirin treatment affected the results of our study.
Finally, FACS lysis buffer, which we used to measure PMA levels, may increase P-SEL expression by lysing red blood cells[29
]. However, as our incubation times with lysis buffer remained constant, regardless of the vascular sampling site, processing times, or platelet counts, this was unlikely to have contributed to the findings of our study.