Between February 2006 and February 2009, 102 severe sepsis subjects were enrolled, and 95 had adequate brachial artery reactivity measurements (Figure ). Seven enrolled patients were not analyzed because 2-D images were inadequate (n = 4), Doppler images were inadequate (n = 2), or images were lost (n = 1). Fifty-two control subjects without acute illness were recruited. In general, the procedure was well tolerated. Several subjects noted mild discomfort during the stagnant forearm ischemia that rapidly resolved after cuff deflation. No adverse events occurred.
Enrollment algorithm for severe sepsis patients.
Clinical characteristics of the study subjects are shown in Table . Brachial artery reactivity was measured 41 (30 to 57) hours after patients met severe sepsis diagnostic criteria. As shown in Table , 85% of our severe sepsis patients were in septic shock at the time of diagnosis. However, most patients had recovered normal blood pressure (mean arterial pressure = 80 (72 to 90) mm Hg), and only 28% required vasopressor infusions when brachial artery reactivity was measured (Table ), indicating some degree of cardiovascular stabilization by the time measurements were performed.
Clinical characteristics of study subjectsa
Severe sepsis versus control subjects
Compared with control subjects, severe sepsis patients had significantly lower FMD (controls = 4.11 (3.06 to 6.78)%, severe sepsis = 2.65 (0.81 to 4.79)%; P < 0.001) and HV (controls = 63 (52 to 81) cm/cardiac cycle, severe sepsis = 34 (25 to 48) cm/cardiac cycle; P < 0.001; Table Figure ). Lower HV in severe sepsis versus control subjects was not explained by differences in the duration of the cardiac cycle because the baseline velocity-time integral was similar in the two groups (Table ).
Brachial artery reactivity measurements
Figure 2 Brachial artery reactivity in severe sepsis patients versus control subjects: hyperemic velocity (a) and flow-mediated dilation (b). Box plots show the median (horizontal line), 25th, and 75th percentiles (lower and upper limits of the box). The dots (more ...)
Stratified analyses showed that the association between severe sepsis and FMD depends on age category. For subjects younger than 60 years (the median age of cases and controls combined), FMD was lower in severe sepsis patients (2.65 (0.91 to 4.13)%; n
= 45) than control subjects (4.82 (3.76 to 8.33)%; n
= 30; P
< 0.001). For subjects older than 60 years, FMD was similar in severe sepsis (2.80 (0.77 to 5.31)%; n
= 50) and control subjects (3.56 (1.80 to 5.92)%; n
= 22; P
= 0.34). The test for interaction was significant (P
< 0.02), confirming that the relation between FMD and severe sepsis depended on age category. Among the other covariables, no confounding or effect modification was identified, although only one smoking control subject was tested, so tobacco use could not be fully evaluated (for complete results of this stratified analyses, see Tables E1 to E2 of Additional file 1
online data supplement).
Moderate correlation was found between FMD and HV in the combined study sample (Spearman rho = 0.38; P < 0.001; n = 147). This was primarily accounted for by the control subjects (Spearman rho = 0.44; P = 0.001; n = 52) because the correlation in severe sepsis subjects alone was poor (Spearman rho = 0.18; P = 0.08; n = 95).
Multivariable analyses assessing the independent relations between brachial artery reactivity and severe sepsis began with all specified covariables except smoking. The final logistic regression model included age, gender, history of hypertension, mean arterial pressure at the time of measurements, and Charlson comorbidity index. This model had excellent discrimination (C statistic = 0.91) but poor calibration (Hosmer-Lemeshow χ2
= 119; P
< 0.001). We performed two multivariable analyses to assess the independent relation between FMD and severe sepsis because of the aforementioned age-FMD interaction. In subjects 60 years or younger, lower FMD was independently associated with severe sepsis (odds ratio (OR) for severe sepsis per 1% decrease in FMD = 1.64; 95% confidence interval (CI) = 1.15 to 2.35; P
< 0.01). In contrast, FMD was not independently associated with severe sepsis in subjects older than 60 years (OR for severe sepsis per 1% decrease in FMD = 1.07, 95% CI = 0.90 to 1.26; P
= 0.45). Hyperemic velocity was independently associated with sepsis in the multivariable model (OR for severe sepsis per 1 cm/cardiac-cycle decrease in HV = 1.05; 95% CI = 1.02 to 1.08; P
= 0.001). For complete results of these multivariable analyses, see Tables E3 and E4 of Additional file 1
online data supplement.
Relation of brachial artery reactivity to outcomes and severity of illness in severe sepsis
Seventeen of the enrolled severe sepsis patients died before hospital discharge, 14 of the original sepsis episode, one during a subsequent sepsis episode, and two of stroke after sepsis resolution. FMD tended to be lower in nonsurvivors, but the difference was not statistically significant (survivors = 2.96 (0.91 to 4.86)%; nonsurvivors = 1.90 (0.68 to 3.41)%; P = 0.12; Figure , Table ). In contrast, HV was significantly lower in nonsurvivors (survivors = 39 (30 to 50) cm/cardiac cycle; nonsurvivors = 25 (16 to 28) cm/cardiac cycle; P < 0.001; Figure , Table ). The change in velocity (HV minus baseline velocity) was also significantly lower in nonsurvivors versus survivors (Table ), indicating that the lower HV in nonsurvivors was indeed reflecting lower RH (and not simply a reflection of the marginally lower baseline velocity). In stratified analysis, HV remained lower in nonsurvivors within subgroups of all prespecified covariables (Table ). The time interval from sepsis diagnosis to brachial artery measurements was similar in survivors and nonsurvivors (survivors = 41 (29 to 56) hours; nonsurvivors = 44 (36 to 71) hours; P = 0.26).
Figure 3 Brachial artery reactivity in severe sepsis survivors versus nonsurvivors: hyperemic velocity (a) and flow-mediated dilation (b). Box plots show the median (horizontal line), 25th, and 75th percentiles (lower and upper limits of the box). The dots represent (more ...)
Hyperemic velocity in survivors versus nonsurvivors: stratified analysisa
In multivariable analysis beginning with all of the specified covariables, the final model included age and medical history of diabetes mellitus. This model had very good discrimination and calibration (C statistic = 0.77; H-L χ2
= 5.2; P
= 0.78). When controlling for these covariables, HV was an independent predictor of hospital mortality: the odds ratio for hospital mortality per 1-cm/cardiac cycle decrease in HV was 1.11 (95% CI = 1.04 to 1.19; P
= 0.003; see Table E5 of Additional file 1
online data supplement).
Secondary outcome measures
HV was significantly negatively correlated with maximum and median SOFA scores from days 0 through 7, and significantly positively correlated with the number of organ failure-, ICU-, and ventilator-free days from days 0 to 28 (Table ). FMD was not correlated with any of these variables. Quartiles of HV, but not FMD, predicted survival over the 6 months after severe sepsis diagnosis (Figure ).
Correlations of brachial artery reactivity with severity of illness/secondary outcomes
Figure 4 Kaplan-Meier survival probability plots for quartiles of hyperemic velocity (a) and flow-mediated dilation (b). No subjects were lost to follow-up. The log-rank test was used to evaluate the statistical significance of the trend in survival per quartile (more ...)
Receiver operator characteristics analysis
The area under the curve (AUC) was higher (P = 0.03) for HV (0.82; 95% CI = 0.71 to 0.93) than for FMD (0.62; 95% CI = 0.48 to 0.77). The optimal HV cut-point for predicting mortality was 29 cm/cardiac cycle, with sensitivity of 88% and specificity of 77%. The optimal FMD cut-point for predicting mortality was 1.98%, with a sensitivity of 59% and a specificity of 68%.
Repeated measurements performed by the same sonographer blinded to the first measurement were highly correlated (intraclass correlation coefficient was 0.80 for FMD and 0.97 for HV). However, the paired FMD measurements appeared to stray from the line of identity (see Figure E1 of Additional file 1
online data supplement). Intraobserver repeatability was assessed by using the methods of Bland and Altman [45
] (see Figure E2 of Additional file 1
online data supplement). The coefficient of repeatability (the expected difference between repeated measurements for 95% of paired observations) was 4.1% for FMD and 10 cm/cardiac cycle for HV. Next, we assessed whether agreement existed between paired values when classifying subjects. Measurements were dichotomized into "normal" or "septic" categories based on the median values in control subjects. Excellent agreement was found between the paired values for HV (kappa = 0.89) but only fair agreement for FMD (kappa = 0.45). Further analysis indicated that the paired measurements of baseline and hyperemic brachial artery diameter were precise (intraclass correlation coefficients were 0.99, and the coefficients of repeatability were 0.02 cm for both baseline and hyperemic brachial artery diameter; see Figures E1 and E3 of Additional file 1
online data supplement).
This analysis indicates that although arterial diameter measurements were precise and repeatable, the repeatability of percent FMD was poor, even when performed by the same highly trained sonographer. As a result, FMD-based patient classification was prone to error. In contrast, HV was highly repeatable, and paired measurements had excellent agreement in classifying patients.