1 Introduction

Reliable fluid responsiveness monitoring for hemodynamically unstable patients has been sought for decades. Traditionally used preload estimates such as central venous pressure (CVP) are unreliable [1]. This has led to favouring more functional approaches such as passive leg raising methods and dynamic variables in relation to controlled mechanical ventilation [e.g. pulse pressure variation (PPV)]. Dynamic variable monitoring is a concept that repeatedly has provided convincing results [1]. The monitoring has several advantages: It is continuous and based on already acquired minimally invasive data. Unfortunately, dynamic variables are limited to controlled mechanically ventilated patients and it is even limited to an increasing extent due to influence of tidal volume [2] and respiratory frequency [3]. Tidal volume needs to be at least 8–10 ml/kg for dynamic variables to be reliable [1, 2] and the recommended tidal volume for most patients is around 6 ml/kg [4, 5]. Concerning respiratory frequency, it should not exceed more than approximately one fourth of the heart rate (HR) for PPV to be reliable [3]. In addition, weaning from mechanical ventilation should be considered as soon as possible [6] and even support ventilation mode renders dynamic variables unreliable [7]. Dynamic variables are today mainly useful in the operating rooms, in the post-operative hours following major surgery and in the initial hours after intubation of critically ill patients. Hence, no reliable continuous fluid responsiveness monitoring technique exists for the vast majority of critically ill patients and it has never existed for spontaneously breathing patients. Hence, the struggle of finding reliable fluid responsiveness prediction methods for these patients continues.

Yet, the fundamental physiological idea of dynamic variables [8]—a varying preload—appears useful. In lack of ventilator treatment, different interventional approaches such as passive leg raising methods [911] have been investigated with reasonable results. However, the interventional nature of this approach and the need for reliable cardiac output (CO) monitoring are clinically relevant obstacles for passive leg raising use. It would therefore be preferable if reliable minimally invasive or non-invasive monitoring that did not require an intervention was available for these patients in line with dynamic variables.

Arrhythmias such as frequent extra systoles are a limitation regarding ventilation induced dynamic variables because they interfere with the induced cycling changes in preload and attempts have been made to reject extra systoles from analysis when calculating dynamic variables [12]. However, in the absence of a ventilator, an extra systole in itself induces an intermittent preload shift: The post ectopic beat represents a heart beat with increased preload [13]—potentially a standardised reversible fluid challenge. Thus, analysis of the post-ectopic beat could be a useful method for predicting fluid responsiveness in spontaneously breathing patients (and perhaps also during different mechanical ventilation modes).

With the present study, we propose that the occurrence of an extra systole may be a convenient preload varying mechanism. In a porcine experimental model, we investigated the hemodynamic effect of the post-ectopic beat and we hypothesised that the difference between cardiac performance at the post-ectopic beat and cardiac performance at preceding normal sinus beats would be able to predict fluid responsiveness.

2 Materials and methods

The experiment was approved by the Danish National Animal Ethics Committee (journal number 2012/561-195). The study included 10 Danish female landrace pigs.

2.1 Anaesthesia, analgesia, ventilator settings and fluid management

The animals (weight range: 38–40 kg) were premedicated intramuscularly with midazolam (0.5 mg/kg), ketamine (5 mg/kg), and atropine (0.5 mg). Anaesthesia was induced with propofol (3 mg/kg) and fentanyl (1 μg/kg) and maintained with propofol (10 mg/kg/h) and fentanyl (0.5 mg/kg/h). The animals were intubated and mechanically ventilated. We significantly reduced respiratory blood pressure variations by setting tidal volume low (always <5.5 ml/kg) and respiratory frequency high (approximately 30 breaths/min) titrated to keep arterial pH at 7.4. Positive end-expiratory pressure was 5 cm H2O. With these ventilator settings, dynamic variables were comparable to values from spontaneously breathing pigs exposed to a similar experimental protocol [14]. Ringer acetate was infused (10 ml/kg) during the instrumentation period. At the end of the experiment, the animals were euthanized with pentobarbital.

2.2 Instrumentation and monitoring

Four venous sheets were placed in left (2) and right (2) external jugular veins. One sheet was used for drug and fluid administration as well as for pulmonary artery catheterisation (CCOmboV/SvO2, 7.5 F, Edwards Lifesciences, Irvine, USA). Another sheet was used for blood sampling and volume expansion. The final two sheets were used for two 5-French four-pole pacing catheters (St. Jude Medical, Minnesota, USA), of which one was placed in the right atrium, the other at the right ventricular apex for induction of supraventricular and ventricular extra systoles, respectively. Placement was guided by X-ray and validated from ECG throughout the experiment.

An arterial catheter was placed through a sheet in a femoral artery and used for arterial blood sampling as well as continuous monitoring of arterial pressure (AP). A pulse oximeter was placed and securely fixed on the animal’s tale. Three lead ECG was monitored and a bladder catheter was placed for urine output monitoring.

ECG (lead II), AP, and plethysmographic (Pleth) curves were continuously sampled at 300 Hz throughout the experiment and were offline upsampled to 1,000 Hz (see signal processing section below). Continuous CO, HR and mean arterial pressure (MAP) was sampled every minute by monitor dedicated software (S/5 Collect, Datex-Ohmeda Division, Instrumentarium Corp., Helsinki, Finland).

2.3 Experimental protocol

Following baseline data registration, a pacing protocol was executed (see below). Then, four intravascular volume shifts were performed, of which each was followed by the same pacing protocol. The first volume shift was a controlled bleeding of 25 % estimated blood volume (660 ml for all pigs) performed during 15–20 min (we refer to this step as blood depletion). The blood was collected in a bag with anticoagulants (ACD-A, Fenwal Inc., Illinois, USA). In the second volume shift we re-transfused 500 ml blood. In an attempt to reach a more clinically relevant state, only 19 % (500 ml) and not 25 % was re-transfused because pigs auto-transfuse from the spleen following bloodletting. In the following, we refer to this volaemic level as retransfusion. The two last volume shifts were each done as a volume expansion with 500 ml hydroxyethyl starch 130/0.4 in 0.9 % NaCl (Voluven, Fresenius Kabi, Germany) and we refer to them as volume expansion 1 and volume expansion 2.

2.4 Pacing protocol

At baseline and after each volume shift, atrial pacing was initiated in the following way: The pacer (Biotronik Universal Heart Stimulator, UHS 20, BIOTRONIK SE & Co.KG, Berlin, Germany) determined RR intervals and was manually set to stimulate at an initial coupling interval 50–100 ms less than the observed RR interval. After each paced extra systole, the coupling interval was automatically decreased by 10 ms for the subsequently paced extra systole, which was induced 10 heart beats later. This pacing regimen was continued until the coupling interval became so low that refractoriness was reached or the aortic valve did not open at the ectopic beat for approximately 10 consecutive induced extra systoles. At that point, we changed the electrode leads and performed ventricular pacing following the same pacing scheme.

2.5 Outcomes and statistics

The primary aim was to evaluate the fluid responsiveness predictive value of different variables (see below and Table 1) from both supraventricular and ventricular extra systoles.

Table 1 Abbreviations and corresponding explanations for the variables used for fluid responsiveness prediction

Fluid responsiveness was defined as an increase in SV of 15 % or more following volume expansion.

Hemodynamic characteristics at baseline and the four intravascular volume levels were analysed with ANOVA for repeated measures using Stata (StataCorp LP, College Station, USA). Assumptions for the model (normality, equal variance as well as normality of model residuals) were tested with QQ plot inspection and Bartlett test. Bonferroni corrected post hoc testing was used to identify differences between volaemic levels (10 tests performed, thus, p < 0.005 was considered significant after correction). If model assumptions were not met, we refrained from making further post hoc testing and reported only summary statistics.

Extra systoles from the blood depletion level, the retransfused level, and the level after volume expansion 1 were used to predict the hemodynamic effect of the subsequent volume expansion. The predictive value of the post-ectopic beat induced changes in the different variables (Table 1) was determined with receiver operating characteristics (ROCs) curves. Characteristics from each eligible post-ectopic beat entered this analysis. Thus, ROC analysis assumptions are violated (independence assumption) and we merely used the analysis to report sensitivity and specificity and to find optimal thresholds, not to compute comparative statistics. Sensitivity, specificity, and corresponding thresholds are reported for ROC areas >0.65.

Data are presented as mean (SD), p < 0.05 was considered significant.

2.6 Signal processing of ECG, blood pressure and Pleth curves

The 300 Hz curves (ECG, AP, and Pleth curves) were digitally upsampled offline to 1,000 Hz as previously described [15]. The upsampling was performed in order to obtain a temporal resolution of 1 ms in the curves, which is a prerequisite regarding some of the investigated hemodynamic variables (see below). The upsampled ECG was high-pass filtered with cut-off frequency at 10 Hz to reduce T spike amplitude. After that, R spike detection was done with a simple threshold algorithm with subsequent maximal value search for exact R spike position. Blood pressure and curves were low-pass filtered with cut-off frequency at 25 Hz. When performing derivative calculations, however, the cut-off frequency was 10 Hz (see below) [16].

All signal processing and subsequent data analysis was carried out in Matlab (Mathworks Inc., Natick, USA).

2.7 Detection of post ectopic beats

We visually inspected the detected RR interval time series to identify all useful post-ectopic beats. They were considered useful, if all heart beats since the previously induced extra systole were sinus beats. Investigating pilot data, we saw that coupling intervals close to the baseline RR interval did not change cardiac performance much at the post-ectopic beat. This led to the decision that only extra systoles with coupling intervals reduced 20 % or more compared to the baseline HR were used for fluid responsiveness prediction. Additionally, we subdivided the analyses of extra systoles in two scenarios: Those extra systoles where any ejection was observed in blood pressure at the ectopic beat and those, where no ejection occurred at the ectopic beat.

2.8 Detection of hemodynamic variables

The pre-ejection period (PEP) is a systolic time interval and is defined as the duration of the isovolumetric contraction phase of the left ventricle, i.e. the time interval between ECG Q spike occurrence (beginning of ventricular contraction) and occurrence of aortic valve opening. PEP is preload dependent and shortens when a fluid responsive subject encounters a preload increase [17]. For each post-ectopic beat, we detected several hemodynamic variables: PEP from the AP curve (using the maximal 2nd derivative of the curve [16]), PEP from the Pleth curve (with the same method), pulse pressure (PP), systolic pressure (SP), and maximal slope of the blood pressure upstroke (dP/dt).

For PEP and SP, both relative and absolute changes were derived. Only relative changes were calculated for PP and dP/dt. Each of these variables was compared with their averaged reference value: Median value of the four sinus heart beats preceding the ectopic beat leading to the variable definitions shown in Table 1.

A decrease in PEP indicates an improvement in cardiac contraction as opposed to decreases in SP, PP, and dP/dt. For the sake of eased interpretation, we choose to calculate all the ∆PEP-values after the scheme PEPsinus beat−PEPpost-ectopic beat, whereas the other variables (e.g. SP changes) were calculated as SPpost-ectopic beat−SPsinus beat; in short: a reversal of the operational sign for the ∆PEP-values. In this way, a positive value of all variables listed in Table 1 are associated with an improvement in cardiac contraction.

Stroke volume (SV) was calculated as CO/HR. CVP and pulmonary artery occlusion pressure (PAOP) was obtained prior to each volume expansion.

3 Results

Table 2 presents the overall hemodynamic characteristics from the experiment. All tested hemodynamic variables were significantly altered by the controlled bleeding and all variables but MAP returned to baseline level following retransfusion. HR data did not comply with statistical model assumptions (variance was not equal across volaemic levels) but nine of ten pigs increased HR with controlled bleeding and for nine of ten pigs, HR fell following retransfusion. Fluid responsiveness was encountered for all but one pig following blood retransfusion and for one pig following the first colloid volume expansion.

Table 2 Hemodynamic variables during the experiment

The usefulness of post-ectopic beats varied considerably. This was mainly caused by the occurrence of spontaneous extra systoles but also—for ventricular extra systoles—because the refractory period at low coupling intervals was not always long enough to prevent the underlying sinus node rhythm from inducing QRS complexes, i.e. no sinus beats fell out despite an induced ventricular extra systole between the sinus beats. In fact, in six of the ten animals, this phenomenon was encountered before the pacing protocol had reached the “no-ejection-at-ectopic beat” level, mostly, throughout the experimental protocol. Therefore, we refrained from analysing the data, where no ejection occurred. In Table 3, we report how many post-ectopic beats (with preceding ectopic beat ejection) were eligible from each pig at each volaemic level for both atrial and ventricular extra systoles.

Table 3 Number of eligible post-ectopic beats from each pig

Figure 1 shows PEP changes (derived from AP curve) related to the ectopic beat and the post-ectopic beat from a representative pig in which ventricular extra systoles were induced. PEP was improved (shortened) considerably more at the post-ectopic beat, when blood had been depleted (and in this case a fluid responsive stage) compared to the two subsequent volume shift stages (after volume expansion 1 and 2—cases not associated with fluid responsiveness).

Fig. 1
figure 1

Pre-ejection period changes related to the ectopic beat and the post-ectopic beat from a representative pig in which ventricular extra systoles were induced. PEP is derived from the arterial blood pressure curve. The three larger markers (around RR interval of 900–1,000 ms) indicate the median RR interval of the normal sinus beats surrounding the extra systoles. The markers to the left of these sinus beat markers represent the ectopic beats induced by the pacer and the markers to the left represent the subsequent post-ectopic beat. As indicated by the latter markers, PEP was improved (shortened) considerably more at the post ectopic beat, when the pig had been blood depleted (indicated by circles) compared to the other stages following retransfusion and volume expansion 1. AP arterial pressure, BD blood depletion, RT retransfusion, VE1 volume expansion 1

Figure 2 shows, how ∆absPEPAP (defined in Table 1) from ventricular extra systoles from all animals was related to SV changes following volume expansion. The vertical line indicates the global optimal ∆absPEPAP threshold (6 ms) for fluid responsiveness prediction. Figure 3 shows the corresponding ROC curve presenting an area under the ROC curve of 0.84. The optimal 6 ms threshold corresponded to a sensitivity of 71 % and specificity of 77 %. Sensitivity and specificity values of the variables in Table 1 from both atrial as well as ventricular extra systoles are reported in Table 4, which shows that post-ectopic beat characteristics from ventricular extra systoles were generally superior to those from atrial extra systoles in predicting fluid responsiveness. Classification characteristics for CVP were: Area under ROC curve: 0.89, sensitivity of 100 %, specificity of 65 % at a threshold of 4.5 mmHg. Classification characteristics for PAOP were: Area under ROC curve: 0.90, sensitivity of 100 %, specificity of 65 % at a threshold of 7.5 mmHg.

Fig. 2
figure 2

Relation between SV changes and ∆absPEPAP (absolute PEP changes derived from the AP curve)

Fig. 3
figure 3

ROC curve for ∆absPEPAP

Table 4 Areas under the ROC curve (AUC) for the variables listed in Table 1

4 Discussion

Based on Frank–Starling curve considerations, the present study hypothesised that cardiac contraction performance is generally more improved at the ventricular extra systolic post-ectopic beat when the heart is fluid responsive as compared to when it is fluid unresponsive. The hypothesised physiologic mechanism exists for ventricular extra systoles but it was not as clear for supraventricular extra systoles.

Whether derived from the AP curve or from the Pleth curve, the family of ∆PEP variables (defined in Table 1) from ventricular extra systoles provided an approximate 70–75 % sensitivity and specificity and reasonable ROC areas around 0.8, whereas the post-ectopic beat changes in “pressure variables” offered good specificity (around 90 %) on the expense of sensitivity (around 55 %) with lower ROC areas around 0.7. Due to the independence violation for ROC analysis, the current study is considered hypothesis confirming regarding physiology and encourages further research on ventricular extra systoles in both the clinical and experimental setting.

∆PP poorly predicted fluid responsiveness. This was surprising, considering that PPV is one of the most useful dynamic variables. However, being defined as systolic pressure minus diastolic pressure, PP is strongly influenced by the declining diastolic pressure in the compensatory pause which may explain ∆PP’s poor predictive value. Both ∆absSP and ∆relSP had a fair area under the ROC curve but the optimal threshold was 0 (% and mmHg) and even associated with a low sensitivity. Again, this may be attributed to the diastolic pressure characteristics of the post-ectopic beat. On the other hand, ∆dP/dt was considerably larger with optimal threshold at 27 %. CVP and PAOP offered high sensitivity and specificity values which is typical for these variables under experimental conditions. However, preload variables, in particular CVP, are not valid fluid responsiveness predictors in the clinical setting [18].

The reason why variables derived from supraventricular extra systoles were not useful for fluid responsiveness prediction was difficult to interpret from the data. The compensatory pause is longer for ventricular extra systoles compared to supraventricular extra systoles and could theoretically create a “stronger signal” that could better separate responders from non responders. However, the classification performance differences between the two types of extra systoles appeared to be explained by differences in cardiac performance at post-ectopic beats during fluid unresponsiveness: A variable like ∆absPEPAP was generally higher at supraventricular extra systoles compared to ventricular extra systoles under that condition. This appeared not to be explained by the differences in eligible post-ectopic beats between the two types of extra systoles.

An important issue for the clinical applicability of the presented extra systole method is prevalence of spontaneous extra systoles. Extra systoles are generally considered benign and prevalent in both diseased and healthy subjects but prevalence increases with age [1921] and heart disease [22]. 76 % of non-hospitalised elderly women (aged 65 or more) and 88 % of non-hospitalised elderly men have one or more ventricular extra systoles during 24 h ECG recordings, and nearly 40 % of men aged >80 years have more than 15 ventricular extra systoles hourly [19]. To the author’s knowledge, their prevalence is not well investigated among critically ill patients but we speculate that prevalence is higher for this patient group compared to healthy volunteers. As such, analysing extra systoles appears a feasible semi-continuous monitoring method but for a part of (particularly young) patients, the method is probably limited/not useful due to a low prevalence of extra systoles.

There are some limitations regarding the current study.

First, the current study was in young, healthy animals not comparable to the usual critically ill patient. For instance, we observed a relatively low HR in the study not comparable to the clinical setting of critically ill patients. This was the main reason for not analysing the data where no ejection occurred at the ectopic beat.

Second, the volume shifts were larger than would be performed in the clinical setting.

Third, the induced extra systoles were artificial and always originating from the same area. This is probably most important for ventricular extra systoles because supraventricular extra systoles are associated with a normal ventricular contraction. It is difficult to theoretically argue how ventricular extra systoles of different anatomic origin will affect the cardiac performance at the subsequent post-ectopic beat.

Fourth, our experiment gave more missing data than anticipated, especially regarding paced ventricular extra systoles. The pig is generally prone to arrhythmia and in this study, spontaneous arrhythmias were more prevalent during ventricular pacing. Yet, we do not believe that this affected our results significantly.

Fifth, we violated the independence assumptions for ROC analysis and different animals provided different number of eligible post-ectopic beats for the analysis (Table 3). Future data will reveal if the hypotheses can be confirmed in the clinical setting.

Sixth, the present method will theoretically have much the same limitations known for dynamic variables such as atrial fibrillation, very frequent extra systoles, and right heart failure.

Even though some limitations suggest that post-ectopic beat cardiac performance changes may not be more reliable in the clinical setting than presented here, there are also settings from this experimental study that suggests possible classification improvements in a subsequent clinical study. First, the pigs were mechanically ventilated. Despite the efforts to reduce ventilator induced dynamic variables, we did see blood pressure swings (largest the blood depleted step) that affected all investigated variables. This is probably partly explaining the “vertical variance” in Fig. 2, largest at fluid responsive levels. Second, we have only investigated post-ectopic beat characteristics and only classified with single variables. Utilising cardiac performance information from e.g. the ectopic beat or the post–post ectopic beat and combining variables may significantly improve fluid responsiveness prediction based on ventricular extra systoles.

In previous studies, pigs were generally fluid unresponsive when reaching the step following retransfusion of all depleted blood [14, 15]. The attempt to reach a more clinically relevant level after retransfusion was somewhat achieved. Inspecting mean value differences between “baseline” and “after retransfusion” from a previous study in piglets [15] with complete retransfusion of depleted blood, SV increased 13 %, PAOP increased 1.7 mmHg (significant), CVP increased 1.6 mmHg, and MAP fell 4 mmHg from baseline to the retransfused level. In this study, mean SV increased 3 %, PAOP increased 0.3 mmHg, CVP increased 0.3 mmHg, and MAP fell 8.5 mmHg (significant). Yet, only one pig was fluid responsive at the retransfused level in the present study, which was the same for the previous study in eight piglets. We believe, that the present study’s SV, CVP, and PAOP levels indicate similar circulating blood volume at baseline and after retransfusion but considering MAP, an optimal fluid responsiveness animal model is not completely settled with the attempt of only partly retransfusing depleted blood.

When considering the extra systole as a potential preload varying mechanism, atrial fibrillation could also be interesting to investigate. In a previous study, Muntinga et al. [23] showed that the preceding RR interval is related to the ejection fraction (EF) in atrial fibrillation patients. When scatter plotting preceding RR intervals against EF, a “blurred Frank–Starling curve” appeared in that study. Such a plot might be useful to predict fluid responsiveness but at least two predictable complicating issues arise. First, atrial fibrillation continuously causes preload (and SV) changes in both right and left ventricular SV. As a consequence, the varying preload depends not only on the preceding RR interval but also how the right ventricle produced flow through the pulmonary circuit during several of the preceding RR intervals. This may be the reason why the plot of preceding RR and EF was blurred in the previous study compared to what we showed in Fig. 1. Second, since atrial fibrillation is not associated with a certain working position of the Frank–Starling curve (a working range could be a more precise term), it is more complex to predict how a right shift in working range (i.e. a volume expansion) will affect cardiopulmonary hemodynamics. Presumably, SV will increase for those heart beats with the lowest preceding RR intervals but the heart beats with longer preceding RR intervals the heart may be challenged (and increase the risk of e.g. edema). Atrial fibrillation could be another interesting way of looking at fluid responsiveness but it appears more complex than using extra systoles because extra systoles are (generally) preceded by sinus beats.

In conclusion, analysis of cardiac performance at ventricular extra systolic post-ectopic beat in pigs is a technique with which it is possible to predict fluid responsiveness with reasonable accuracy. The relevant monitoring technology is readily available: Extra systoles are detected by today’s monitors and e.g. PP, SP and PEP are (easily) calculated, too. This technique may contribute to predicting fluid responsiveness in minimally or even non-invasively monitored spontaneously breathing (and perhaps also mechanically ventilated) patients where other reliable hemodynamic monitoring is not available and where ventricular extra systoles are prevalent. However, experimental studies are needed to clarify a number of issues and limitations arising from this hypothesis confirming experimental study and clinical studies are needed to address the feasibility of using extra systoles to predict fluid responsiveness in patients.