In this study of brachial artery reactivity in severe sepsis patients, our main findings were that lower HV was independently associated with severe sepsis and hospital mortality. Lower brachial artery HV was also associated with higher severity of illness and lower 6-month survival. In contrast, although FMD was lower in sepsis patients than in control subjects, it did not predict severity of illness or adverse outcome.
Implications of low HV in severe sepsis
The physiologic mechanisms responsible for RH originate in the microvasculature and are both endothelium dependent and endothelium independent [
3‐
7]. They include a direct physical myogenic response [
6], opening of ATP-sensitive potassium channels [
3], and production of vasodilator substances, including prostaglandins [
6], nitric oxide [
5], and adenosine [
3‐
5,
7]. Microvascular obstruction from microthrombi, leukocyte adhesion, or sepsis-associated erythrocyte dysfunction may also contribute to impaired RH in sepsis [
8‐
11]. Our HV measurements do not allow us to decipher which of these physiological mechanisms are most disrupted or most lethal in severe sepsis. However, they do imply that dysregulation of one or more of these microvascular mechanisms, in concert, is responsible for the abnormal HV we observed in severe sepsis patients and nonsurvivors.
Our findings are consistent with previous studies showing that other indices of RH are reduced in sepsis [
8‐
11,
18,
19], associated with illness severity [
20‐
22], and associated with ICU mortality [
23]. Our study adds to these reports by uniquely demonstrating that HV remains independently associated with hospital mortality when specifically controlling for comorbidity, vasopressor use, or blood pressure (Table
4), and also in multivariable analysis (Table
5).
The finding that HV independently predicts severe sepsis mortality supports the concept that microvascular dysfunction is a central pathophysiologic process responsible for organ dysfunction and poor outcomes in sepsis [
25,
47]. In this context, treatments designed to improve microvascular function and clinical outcomes should be evaluated. For example, previous studies suggest that antioxidants may have benefit in critical illness [
36,
48], and they improve indices of RH and reduce inflammatory markers in other patient groups [
49]. It seems logical to investigate this same possibility in severe sepsis. In addition, NO donors improve microcirculatory flow in sepsis patients [
50]. Current efforts are under way to harness the beneficial effects of NO without causing the hypotension induced by organic nitrates [
25]. Indices of RH could serve as physiological biomarkers in trials of such agents, ensuring that appropriate severe sepsis patients with abnormal microvascular function are enrolled in studies designed to affect this mechanistic pathway. This type of study design is now recommended for clinical trials in critical care to overcome the patient heterogeneity that dilutes precise measurement of therapeutic effectiveness [
51,
52]. Along the same lines, indices of RH could be used to test whether the therapeutic intervention is having the intended physiologic effects and clinical benefits, thereby substantiating a clinically relevant mechanistic pathway.
Previous measures of RH in sepsis
Several techniques have been used to assess RH and microvascular function in patients with severe sepsis. Previous studies of RH in sepsis by using plethysmography are limited by small sample size [
9‐
11,
53]. Nevertheless, because plethysmography was the primary noninvasive method for determining the physiological mechanisms of RH in humans, it most closely approaches a gold-standard noninvasive measurement technique. Therefore, if other noninvasive methods are being used to investigate RH in sepsis patients, they should closely correlate with plethysmography.
A previous report showed that skin blood flow after stagnant ischemia, estimated by using transcutaneous laser Doppler measurements of erythrocyte velocity, was reduced in sepsis [
19]. However, a variable relation exists between the laser Doppler measurements and plethysmographic forearm blood-flow measurements, and these measurements vary with slight changes in skin-probe location [
54].
Reactive hyperemia peripheral arterial tonometry (RH-PAT) volumetrically measures digital pulse-wave amplitude in response to stagnant ischemia [
55]. Sepsis-associated reductions in RH-PAT that are correlated with severity of illness have been observed [
20]. Advantages of RH-PAT are that the computer-generated results are user independent, minimal training is involved, and the results are repeatable. Disadvantages are that the relation between RH-PAT and plethysmography is unknown, and it requires specialized and costly equipment.
Near-infrared spectroscopy plethysmography (NIRS) measures the change in microvascular hemoglobin levels and oxygen saturation during RH [
22]. Blood flow estimated by NIRS was tightly correlated with plethysmography in normal subjects at rest, although the correlation was weaker after exercise [
56]. NIRS-derived tissue oxygen consumption and tissue reoxygenation rate (or slope) after stagnant ischemia have been associated with sepsis, severity of illness, and clinical outcomes [
18,
22‐
24]. Disadvantages of NIRS are that it requires specialized equipment and disposable yet costly probes, and tissue fat and edema can produce interference that can impair accuracy.
Technical considerations of brachial artery reactivity measurement
Optimal comprehensive ultrasound measurement of brachial artery reactivity parameters (including FMD) requires extensive technical expertise, particularly the quantification of brachial artery diameter [
26]. We therefore required that all our studies be performed by experienced, registered sonographers. This requirement often delayed our measurements and limits the widespread clinical application of comprehensive brachial artery analysis. Conversely, previous studies in ICU patients demonstrate that accurate brachial artery blood-velocity measurements are easily learned by clinicians with minimal clinical experience [
57]. Because our study demonstrates that HV is the brachial artery reactivity parameter that predicts outcomes, future studies can focus exclusively on this measurement, eliminating the need for vessel diameter measurements and specialized expertise. Importantly, previous studies demonstrate that Doppler blood velocity is tightly correlated with plethysmographic blood flow, even over a wide range of arterial flow rates [
58,
59]. It is therefore likely that our HV measurements reflect the physiological mechanisms ascribed to RH and determined by plethysmography. The use of point-of-care, portable ultrasound in the intensive care unit has grown dramatically [
60], so the required equipment for HV measurement already exists in many ICUs, avoiding the additional equipment costs. Finally, HV has recently been measured in large cohort studies to quantify RH and to estimate microvascular function [
12‐
15]. These studies demonstrate that HV is an independent predictor of inflammatory markers, cardiovascular risk factors, and adverse events. For these reasons, HV is an attractive method for measuring RH and assessing microvascular function in future critical care studies.
FMD in sepsis: comparison with previous studies and measurement challenges
Brachial artery FMD was independently associated with sepsis after controlling for all covariables in subjects younger than 60 years, but not in older individuals. Preexisting age-related endothelial dysfunction or loss of arterial compliance, or both, may explain these findings [
61]. Contrary to our original hypothesis, we found no associations between FMD and hospital mortality or severity of illness.
Our findings contrast with those of Vaudo
et al. [
31]. These investigators found that sepsis patients with lower FMD at hospital admission experienced worsening severity of illness (SOFA score) over time. Differences in the patient samples probably account for these conflicting findings. Vaudo
et al. selected patients with Gram-negative sepsis and did not include patients with preexisting diabetes mellitus, hypertension, smoking, hyperlipidemia, or obesity. In addition, their patients were 41 ± 8 years of age and had no organ dysfunction at enrollment. In contrast, we included unselected consecutive patients with severe sepsis who were older, had greater comorbidity, and had greater severity of illness than did those of Vaudo
et al. These characteristics probably blunted FMD in our patients, decreasing the measurement signal, and making potential relations between FMD and severity of illness or mortality difficult to detect. Indeed, our FMD results (Table
3) are much lower than those reported by Vaudo
et al. (8.7 ± 3.6% in sepsis patients and 9.9 ± 1.1% in controls). Although small methodologic differences existed between our study (200 mm Hg cuff inflation for 5 minutes) and Vaudo
et al. (230 to 250 mm Hg cuff inflation for 4 minutes), it seems unlikely that they contributed substantially to our discrepant findings.
FMD predominantly reflects conduit artery endothelial NO production, although it can be influenced by sympathetic activation [
26,
27,
62]. We found no relation between lower FMD and adverse outcome. It is tempting to conclude that impairments in endothelial function/NO production are not associated with adverse outcomes in patients with severe sepsis. However, the analysis of intraobserver variability suggests the possibility that our FMD measurements were simply unable to detect greater endothelial dysfunction in nonsurvivors. Although measurement of brachial artery diameter was highly repeatable, the FMD measurement error was substantial. This is explained by difficulty quantifying the very small ischemia-induced change in vessel diameter. Importantly, the magnitude, dispersion, and intraobserver variability of our FMD measurements are comparable to those observed in relatively healthy people of similar age in the Framingham cohort studies [
63].
We induced stagnant ischemia with the arterial occlusion cuff placed below the antecubital fossa. Upper-arm cuff placement produces a greater FMD magnitude, so it could reduce measurement error [
26]. However, the upper-arm occlusion technique is not currently recommended for assessment of conduit artery endothelial function because it may also reflect several additional physiological mechanisms in addtion to endothelial NO production [
32]. A recent investigation of an alternative measure of endothelium-dependent vasodilation (change in aortic augmentation index after salbutamol inhalation) demonstrated a strong association with critical illness mortality [
64]. This supports the possibility that our FMD measurements were not sufficiently sensitive or precise to detect an association between impaired endothelial function and mortality.
Relation between HV and FMD in sepsis
To our knowledge, this is the first study comparing the predictive value of FMD and RH in human sepsis. We found that HV and FMD were only weakly correlated, consistent with previous studies [
41] and supporting the concept that these two indices of brachial artery reactivity are mediated by different mechanisms [
17]. These two indices of brachial artery reactivity were compared in the Framingham Offspring Study [
15], showing significant independent relations between inflammatory markers and HV, but not between inflammatory markers and FMD. The authors postulated that inflammation predominantly impairs microvascular function (measured with HV) instead of conduit artery endothelial function (measured with FMD). This conclusion resonates with our findings that HV alone is a powerful predictor of outcomes in the inflammatory milieu of severe sepsis. Again, the negative FMD findings could instead be explained by imprecise quantification of the small change in brachial artery diameter.
Strengths and limitations
The methodologic strengths of our study are that it included a large, consecutive sample of severe sepsis patients, measurements were made by using a standardized method with trained sonographers, and measurements were analyzed and quantified by a single sonographer blinded to patient outcomes.
Our study has a number of limitations. First, the observational design precludes establishment of a causal relation between low HV and adverse outcome. Proof of causality will require clinical trials testing interventions designed to improve both microvascular function and clinical outcomes. We hope our study will contribute to the rationale for such trials.
Another limitation is that we measured HV and FMD at only one time point during the acute illness, and measurements were often delayed because vascular-access devices were present in the target upper extremity, or sonographers were not immediately available. As shown in Table
2 although most patients initially presented with septic shock, most had recovered normal blood pressure at the time of brachial artery reactivity measurement. It is unknown whether earlier measurements, or serial measurements, would predict clinical outcomes more accurately. That said, it is striking that HV remains impaired and predicts mortality even when systemic blood pressure is normal, suggesting that microvascular dysfunction is pathologically important and persists even after global hemodynamics have stabilized. Our stratified analyses (Table
4) showing that HV remains lower in nonsurvivors, independent of blood pressure or vasopressor use, supports this conclusion as well.
We also must emphasize that HV and FMD measurements were performed by expert sonographers to ensure optimal precision. Therefore, our study is unable to address the feasibility of making similar measurements without registered sonographers. Previous literature provides optimism that accurate HV measurements can be made by other clinicians and investigators [
57], but this requires confirmation. Despite this technical expertise, we found substantial intraobserver variability in FMD, as noted earlier. In this context, it is important to note that we manually measured brachial artery boundaries, and we limited our postdeflation 2-D imaging to 30 to 90 seconds after cuff deflation, consistent with original studies and guidelines [
26,
39,
41]. However, more recent guidelines recommend the use of automated edge-detection software to reduce intraobserver variation, and extension of postdeflation 2-D imaging for 3 minutes to identify some patients with delayed peak FMD [
32]. These factors together increase the chance of a type II statistical error when we conclude that no association exists between FMD and mortality in severe sepsis.
Another limitation is that we did not measure blood or urine indices of inflammation, oxidative stress, or nitric oxide metabolism at the time of the brachial artery measurements in the severe sepsis cohort, so we are unable to analyze relations between these processes. We also did not routinely measure global hemodynamic variables (for example, central venous pressure or cardiac output), so we are unable to assess their relation with brachial reactivity.
Finally, it is likely that a number of unmeasured differences existed between our relatively healthy control group and the severe sepsis cohort (for example, other comorbidities, sedation, and analgesia). It is therefore possible that such unmeasured covariables confounded the association we observed between brachial artery reactivity and severe sepsis. In a similar way, we cannot determine from this study whether reduced HV is associated with poor outcomes in severe sepsis patients exclusively, or whether it is a more widely applicable prognostic indicator in other critically ill patients.