The effect of ambient temperature on gross-efficiency in cycling

Time-trial performance deteriorates in the heat. This might potentially be the result of a temperature-induced decrease in gross-efficiency (GE). The effect of high ambient temperature on GE during cycling will be studied, with the intent of determining if a heat-induced change in GE could account for the performance decrements in time trial exercise found in literature. Ten well-trained male cyclists performed 20-min cycle ergometer exercise at 60% (power output at which VO2max was attained) in a thermo-neutral climate (N) of 15.6 ± 0.3°C, 20.0 ± 10.3% RH and a hot climate (H) of 35.5 ± 0.5°C, 15.5 ± 3.2% RH. GE was calculated based on VO2 and RER. Skin temperature (Tsk), rectal temperature (Tre) and muscle temperature (Tm) (only in H) were measured. GE was 0.9% lower in H compared to N (19.6 ± 1.1% vs. 20.5 ± 1.4%) (P < 0.05). Tsk (33.4 ± 0.6°C vs. 27.7 ± 0.7°C) and Tre (37.4 ± 0.6°C vs. 37.0 ± 0.6°C) were significantly higher in H. Tm was 38.7 ± 1.1°C in H. GE was lower in heat. Tm was not high enough to make mitochondrial leakage a likely explanation for the observed reduced GE. Neither was the increased Tre. Increased skin blood flow might have had a stealing effect on muscular blood flow, and thus impacted GE. Cycling model simulations showed, that the decrease in GE could account for half of the performance decrement. GE decreased in heat to a degree that could explain at least part of the well-established performance decrements in the heat.
Introduction
Performance decrements are widely observed during exercise in the heat compared to thermo-neutral circumstances (Febbraio et al. 1996; Gonzalez Alonso et al. 1999; Parkin et al. 1999; Romer et al. 2003; Tatterson et al. 2000; Tucker et al. 2004). It has been shown that fatigue at exhaustion is related to factors associated with thermoregulation and hyperthermia (Febbraio et al. 1996; Nielsen et al. 1990; Parkin et al. 1999). Time trial performance and fatigue (evidenced by decrements in power output over the race) and finish time have received much less attention in the literature. Accordingly, the present study will focus on the effect of a hot ambient temperature on thermal and cardio respiratory strain during the exercise that might contribute to the well-established decrease in power output.
Tatterson et al. (2000) measured time trial performance on a 30 min self-paced cycling time trial in 32°C, 60% RH versus 23°C, 60% RH. They observed that power output was reduced in the heat by 6.5% (345 ± 9 W vs. 323 ± 8 W). Tucker et al. (2004) compared 20 km time trials in 35 and 15°C and found a comparable reduction in average power output in the heat of about 6.3% (255 ± 47 W vs. 272 ± 45 W). This change in average power output led to a difference in final time on a 20 km time trial of about 48 s (29.6 ± 1.9 min vs. 28.8 ± 1.8 min), which equals about 2.8%. Possible causes for this deterioration in performance are associated with elevated body temperature (Tatterson et al. 2000) and anticipatory changes in pacing strategy to avoid hyperthermia (Tucker et al. 2004). Although most research has revolved around neuromuscular function, central drive and fatigue at the point of exhaustion, a plausible explanation for the deterioration in time trial performance in heat may also be found in a temperature-induced change in gross-efficiency (GE). GE is an important variable in cycling performance (Lucia et al. 2002; Moseley et al. 2004) and a linear relationship between body temperature and GE has been observed (Daanen et al. 2006).
A possible explanation for the decreased GE in the heat could be mitochondrial leakage. A temperature induced metabolic disruption caused by non-specific proton-leakage across the inner mitochondrial membrane has been shown to occur at high muscle temperatures (Brooks et al. 1971; Willis and Jackman 1994), resulting in a decrease in ADP:O ratio. Further, a heat induced skin vasodilatation could occur in heat. To prevent a resulting decrease in blood flow to exercising and respiratory muscles (Nielsen et al. 1990; Romer et al. 2003; Rowell et al. 1966) a higher cardiac output must exist to continue supplying the muscles with the same blood flow, but still sending extra blood to the skin for cooling.
In the present study we sought to determine the effect of heat on GE. GE can be assessed accurately during sub-maximal exercise at intensities as high as 60−80% VO2max by calculating the ratio between mechanical and metabolic (mainly dependent on aerobic energy metabolism) power output, as has been done in Daanen et al. (2006), Foster et al. (2003), Hettinga et al. (2006), Lucia et al. (2002), Moseley et al. (2001, 2004). The presence of a possible effect of ambient temperature on GE can serve as input for an energy flow model (De Koning et al. 1999) to quantify the impact of this effect on time trial performance.
Materials and methods
Subjects
Ten healthy, non-smoking, well-trained male subjects, familiar with cycling exercise at the club-level, participated in this study. They were informed of the nature of the experiment and provided written informed consent. Subject characteristics are presented in Table 1. The study was approved by the Medical Ethical Committee of the University Medical Centre Utrecht (The Netherlands).Table 1Subject characteristicsMean value ± SDAge (years)23.5 ± 4.4Height (cm)179.7 ± 9.1Body mass (kg)72.5 ± 7.2VO2max (l min−1)4.78 ± 0.41(W)354 ± 29Values are mean ± SD
Experimental design
Incremental test
The subjects first performed an incremental bicycle test to determine at which power output VO2max was attained  The incremental test was performed in the heat, under similar conditions as the constant intensity bouts, to make sure that  would not be overestimated in the hot condition. This incremental test was used solely to determine the relative intensities for the constant intensity cycling bouts. The test began at a power output (PO) of 150 W, after which PO was increased with 15 W every minute. Exercise was performed on an electronically braked cycle ergometer (Lode Excalibur, Lode NV, Groningen, The Netherlands) until exhaustion or until pedal frequency dropped below 80 rpm. Oxygen consumption (VO2) was measured breath by breath, using open circuit spirometry (Oxycon Pro-Delta, Jaeger, Hoechberg, Germany). The gas analyzer was calibrated using a Jaeger 2 l syringe, room air and a calibration gas mixture. Heart rate (HR) was monitored using radiotelemetry (Polar Electro, Kempele, Finland).
Constant intensity exercise bouts
On separate days, subjects performed a constant-intensity exercise bout at 60% of  in a thermo-neutral climate (N) and in a hot, dry climate (H). Temperature in the thermo-neutral climate was 15.6 ± 0.3°C, relative humidity (RH) was 20.0 ± 10.3%. In the hot, dry climate, the temperature was 35.5 ± 0.5°C and RH was 15.5 ± 3.2%. These temperatures were equivalent to the temperatures in the studies of Tatterson et al. (2000) and Tucker et al. (2004). All tests were performed in a climate-controlled room with a continuous airflow of 0.2 m s−1. RH was set low to increase the evaporative capacity of the environment. Further, all trials in the H-climate were performed with a simulated wind velocity of 1.72 m s−1 (6.2 km/h) to further increase evaporative heat loss, which is the most important form of heat loss in cycling in hot conditions (Saunders et al. 2005). Prior to the experiments, the subjects were asked to refrain from vigorous exercise for at least 48 h. They were also asked not to consume coffee, alcohol or drugs after 10 p.m. the day before the exercise and not to eat for two hours prior to the experiments. Subjects drank water ad libitum before the experiment.
Before the constant intensity bout in the H-condition, the subject stabilized for 50 min in the 35°C, 20% RH room. For the comfort of the subjects and to prevent them from shivering, the stabilizing period was reduced to 35 min for the N-condition in the 15°C, 20% RH room. During the test, oxygen consumption (VO2), respiratory exchange ratio (RER) and ventilation (VE) were measured breath by breath. Power output (PO) and heart rate were registered continuously.
After stabilization, a 5-min warm-up was performed at 100 W with a pedal frequency of ∼100 rpm. After 1 min of rest, the constant intensity bout was started (∼100 rpm). The subjects cycled for 20 min at a constant power output of 60% of the power output at which maximal VO2 was attained at the incremental test  in the H condition. Directly after the end of exercise, blood lactate concentration (BLC) was measured (Lactate Pro, Arkray, Kyoto, Japan).
Rectal temperature (Tre) was monitored every 5 s during the entire test using a thermistor temperature probe (YSI 701, Yellow Springs Instrument, Dayton, USA) inserted about ten centimeters in the rectum. Skin temperature (Tsk) was measured every 5 s at 14 different skin loci, conform ISO 9886, using thermocouples (YSI 709B, Yellow Springs Instrument, Dayton, USA). Data were recorded with a Data Translation acquisition board (DT2821, Viewdac, Keithley Instruments, Cleveland, USA). Further, muscle temperature (Tm) was measured at a minimum depth of two centimeters and recorded every 5 s.
Since this measurement was invasive, measurements were restricted to the constant intensity bout in H (n = 6). Tm was measured with a sterile thermal thermocouple-probe (type MAC08170A275SM, Ellab A/S, Rodovre, Denmark) in the right vastus lateralis muscle, inserted by a physician. One hour before inserting the temperature probe, a lidocaine plaster was attached to the skin, as a local anaesthetic.
Calculating gross-efficiency (GE)
Metabolic power (Pmet) was calculated by multiplying oxygen consumption with the oxygen equivalent: Pmet (W) = VO2 × [(4,940 × RER + 16,040)/60] according to Garby and Astrup (1987), assuming that respiratory exchange ratio (RER) equaled respiratory quotient (RQ) at sub-maximal intensities. Further, we assumed that respiratory exchange ratios in excess of 1.00 were attributable to buffering of lactate by bicarbonate. Ratios in excess of 1.00 were thus treated as if they equaled 1.00. The measured mechanical power output (PO) divided by the calculated Pmet defined GE. GE was calculated from 90 s after the start of exercise until the end of exercise.
Energy flow model
The energy flow model as described by De Koning et al. (1999) was used to calculate the effect of changes in GE on performance, by simulating a 20 km time trial as is studied in literature (Tatterson et al. 2000; Tucker et al. 2004). This model is based on power equations and has been reasonably successful in predicting performance in cyclic events as cycling (De Koning et al. 1999) and speed skating (De Koning et al. 2005). The energy flow model, also referred to as power balance model, relates to power production and power dissipation: Pprod = Plost + dE/dt, where Pprod is total power that can be produced, Plost power that is used to overcome frictional losses, and dE/dt rate of change of kinetic energy of the mass centre of the body. These terms can be calculated as described in De Koning et al. (1999), and the influence of changing one single variable, in this case GE, can be predicted.
Statistics
Paired Student t test’s were performed to test if data were significantly different between the H-condition and N-condition, P values less than 0.05 were accepted as statistically significant.
Results
Power output at 60%  was 211.5 ± 18.6 W. All subjects were able to complete the trials in both H and N.
VO2 was significantly higher in H than in N, resulting in GE being significantly lower in H compared to N. Further, HR and VE were higher in H compared to N. For RER and BLC, no main effect of temperature was found. Mean values are shown in Table 2.Table 2Gross-efficiency (GE), VO2, respiratory exchange ratio (RER), respiratory minute volume (VE), heart rate (HR) and blood lactate concentration (BLC) at 60%  in the thermo-neutral (N) and in the hot dry (H) climate60% NHGE (%)20.5 ± 1.419.6 ± 1.1*VO2 (ml min−1)3,002.8 ± 290.13,126.5 ± 268.3*RER0.89 ± 0.030.90 ± 0.01VE (l min−1)76.9 ± 7.782.0 ± 9.4*HR (bpm)145.1 ± 6.5155.4 ± 12.0*BLC (mmol l−1)2.6 ± 1.03.9 ± 2.1Values are mean ± SD* Significant differences with N (P < 0.05)
Mean GE over time is plotted for both conditions in Fig. 1. The difference in GE between conditions was 0.9% over the entire trial. From minute 5–8, the N − H difference was 0.6% ± 0.7%, from minute 15–18, the difference was 1.1 ± 1.3%, both significant. No significant change in GE within trials was observed comparing GE over the first half (min 5–8) with GE over the second half (min 15–18).Fig. 1Gross-efficiency (GE) plotted over time at 60%  in the thermo-neutral (N) climate (dashed line) and in the hot dry (H) climate (solid line) ±SD (dotted lines)
Tsk and Tre were significantly higher in H. Rectal and skin temperature changes combined, the difference in body heat content between N and H amounts to ∼138.5 ± 35.1 kJ, calculated assuming that the specific heat of the body tissue was 3.4 kJ kg−1°C−1. Maximal muscle temperature (Tmmax) in H was 38.7 ± 1.1°C. Mean temperature values are shown in Table 3. Figure 2 shows Tre, Tsk and Tm plotted over time for both conditions. Figure 3 shows the correlation between Tre and GE for both conditions. R2 in N was 0.04, R2 in H was 0.36.Table 3Rectal temperature (Tre), skin temperature (Tsk) and maximal muscle temperature (Tmmax) at 60%  in the thermo-neutral (N) and in the hot dry (H) climate60% NHTre (°C)37.03 ± 0.5837.35 ± 0.63*Tsk (°C)27.74 ± 0.7133.39 ± 0.57*Tmmax (°C)38.7 ± 1.1 (n = 6)Values are mean ± SD* Significant differences with N (P < 0.05)Fig. 2Top panel Rectal temperature (Tre) plotted over time at 60%  in the thermo-neutral (N) climate (dashed line) and in the hot dry (H) climate (solid line) ±SD (dotted lines). Center panel Skin temperature (Tsk) plotted over time at 60%  in the thermo-neutral (N) climate (dashed line) and in the hot dry (H) climate (solid line) ±SD (dotted lines). Bottom panel Muscle temperature (Tm) plotted over time at 60%  in the hot dry (H) climate (solid line) ±SD (dotted lines)Fig. 3Top panel Correlation between gross-efficiency (GE) and rectal temperature (Tre) for the thermo-neutral (N) climate. Bottom panel Correlation between gross-efficiency (GE) and rectal temperature (Tre) for the hot, dry (H) climate
To quantify the effect of the decrease in GE of 0.9%, a simulation with an energy flow model (De Koning et al. 1999) was performed. This simulation showed that a change in GE of 0.9% would result in a difference in final time of 25.6 s over 20 km. Thus, about half of the decrement in performance reported in the literature (a difference in final time of 48 s over 20 km, Tucker et al. 2004) could be accounted for by the measured decrease in GE. To explain the entire deterioration in time trial performance in the heat, the mean decrease in GE had to be 2%.
Discussion
GE during sub-maximal cycling exercise in the heat was significantly reduced compared to exercise in N by 0.9%. Values for GE corresponded to values found in literature, between 17 and 22% (Foster et al. 2003; Moseley et al. 2001). It has to be kept in mind that in calculating GE at 60%  anaerobic contribution was assumed to be negligible. But it might have been influenced by the different climates. Gonzalez-Alonso et al. (1999) reported that exercise in a hot environment results in a higher anaerobic contribution and found an increase in carbohydrate utilization and lactate accumulation during exercise. Though not significant, BLC in the present study was higher in H. According to di Prampero et al. (1999), an increase of BLC of 1 mmol l−1 of blood is equivalent to an O2 consumption of 3 ml kg−1 body weight. Differences in BLC in the present study between climates (2.6 mmol l−1 in N vs. 3.9 mmol l−1 in H) thus correspond with a difference in O2 consumption equivalent of ∼14.1 ml min−1. Accordingly, differences in GE between climates might be even larger then reported in the present study. If one adds this ‘anaerobic O2 equivalent’ to the VO2, then GE decreases about ∼0.19% in N compared to ∼0.27% in H, which means that the difference in GE between N and H increases with ∼0.07%.
Mean Tre was about 0.3°C higher in H compared to N. Mean Tsk was about 5.7°C higher in H than in N. Comparable differences of 0.2–0.4°C in Tre (Tatterson et al. 2000; Tucker et al. 2004) and ∼6.0°C in Tsk (Tatterson et al. 2000) were reported during cycling bouts of ∼30 min in climates comparable to the present study. Although the difference in rectal temperature between H and N was significant, it was not a large difference compared to literature. The measured absolute values for Tre were somewhat low, presumably due to the relatively short period of exercise in our protocol and the use of a fan to optimize losing heat to the environment, as was done in the studies of Tatterson et al. (2000) and Tucker et al. (2004). The fan was used to simulate competitive circumstances, and although wind velocity in this study was not as high as the cycling speed, combined with the relatively low RH, the effect would have been considerable (Saunders et al. 2005).
The differences in ambient temperature between H and N represent a considerable difference in thermal stress. Thermal strain is mainly visible in the increased skin temperature, but although the difference of 0.3°C in core temperature is small, it is significant. Rectal and skin temperature changes combined, the difference in body heat content between N and H amounts to 138.5 ± 35.1 kJ, which is considerable. Further, the differences between climates have been shown to be large enough to evoke a difference in GE of 0.9%, the main purpose of the present study.
A possible explanation for the lower GE in the heat may be the higher core temperature. Daanen et al. (2006) found a strong linear relationship between body temperature, mainly determined by Tre, and GE at 60%  in 30°C. Their study showed that our difference in Tre of 0.3°C could account for a reduction in GE of ∼0.2%, and thus was not large enough to explain the entire reduction in GE of 0.9%. In the study of Daanen et al. (2006), the skin temperature however was almost unchanged, while in the present study skin temperature in the H condition was about 5.6°C higher, indicating an increased blood flow to the skin. The present study did not find a strong correlation in heat (r2 = of 0.36) between Tre and GE. Further, remarkably, in the N-condition, no correlation between Tre and GE was found at all, even though Tre reached values which are also observed in the H-condition, only later in time (see Fig. 3). Apparently, Tre does not seem to be the main cause of the reduced GE. This is supported by the observation that even though Tre rose significantly during the time trial with about 1°C, GE did not increase significantly comparing the first half with the second half within the trials.
Two other explanations for the reduced GE in heat can be given. Firstly, an explanation for the temperature-induced reduction in GE might be a metabolic disruption that is known to occur at elevated muscle temperatures (Brooks et al. 1971; Willis and Jackman 1994). Though Ferguson et al. (2006) found no effect of increasing muscle temperature on energy turnover in dynamic exercise in a range of 34.2–38.3°C, ADP:O ratio has been shown to decrease at higher muscle temperatures (Brooks et al. 1971; Willis and Jackman 1994). During heavy exercise in the heat, non-specific proton leakage across the inner mitochondrial membrane is increased, resulting in a decrease in the efficiency of oxidative phosphorylation and increasing the resting metabolic rate (Willis and Jackman 1994). This will result in a reduction in GE. Willis and Jackman (1994) found a decrease of 10–20% in ADP:O ratio at muscle temperatures of 40°C and higher compared to 37°C. They found that this decrease resulted in a 400–800 ml min−1 increase in VO2. In the present study, a smaller but significant increase in VO2 of 124 ml min−1 was found. Mean Tmmax in heat was 38.7°C, lower than the 40°C as found in Willis and Jackman (1994), but higher than 34.2–38.3°C, the Tm range in which Ferguson et al. (2006) found no effect of increasing Tm on energy turnover in dynamic exercise. Since Tm was not measured in both climates, it cannot be confirmed if muscle temperature was significantly different. Tsk and Tre were significantly different but did not approach 40°C in either condition. It seems that, as suggested by Brooks (1971), the core of the body functions as a heat sink for the skeletal musculature helping to maintain Tm below the point where significant reductions in the ADP:O ratio occur.
Another potential cause for the decrease of GE in a hot ambient temperature was the larger vasodilatation of the skin to lose heat, as was shown by the significantly higher Tsk in H compared to N. The resulting decreased blood flow to exercising and respiratory muscles may be compensated by increasing cardiac output (Nielsen et al. 1990; Rowell et al. 1966), since a significant increase in heart rate was found in heat. The extra VO2 in the H condition is at least partially attributable to the extra myocardial VO2, since a higher cardiac output has to exist to continue supplying the muscles with the same blood flow, but have to send extra blood to the skin for cooling. Additionally, ventilation was increased, which may also lead to a reduction in GE. Assuming that the mechanical work per breath is 80–125 J (Milic-Amili et al. 2001), it can be estimated that the higher VE in this study can account for maximally 10% of the increase in VO2.
Lastly, it has to be noticed, that GE decreases if the proportion of energy expenditure that is used to maintain homeostasis is increased. Thus, the lower GE in H could have been solely due to an increase in resting metabolic rate, while net efficiency remained unchanged. This would be consistent with the hypothesis that muscle temperature in the hot conditions was not high enough to make mitochondrial leakage a likely explanation for the observed reduced GE. Unfortunately, resting metabolic rate has not been measured.
To determine the potential importance of the measured decrease in GE on time trial performance, a 20-km time trial, which was also studied by Tucker et al. (2004), was modeled by the use of the energy flow model (De Koning et al. 1999). A time trial of this distance can be seen as a mainly aerobic exercise bout. Tucker et al. (2004) found a difference in final time of 48 s between exercise in 35°C compared to 15°C. Using the energy flow model of De Koning et al. (1999), it was calculated that the measured difference in GE of 0.9% would lead to a difference in final time between H and N of 25.6 s. This explains about half of the 48 s found by Tucker et al. (2004). For the entire reduction in final time of 48 s, a decrease in GE of 2% would be necessary, which was not found. It can be concluded that about half of the decrease in time trial performance can be accounted for by the reduction in GE. It has to be noted that in this simulation, it is assumed that the difference in GE between H and N at higher intensities will not change. Although it has been shown that GE increases with exercise intensity, since the relative share of resting metabolism diminishes at higher sub-maximal intensities (Mosely and Jeukendrup 2001), this effect seems to be equal for both conditions and will at most have only a minor effect on the difference between conditions.
Conclusion
GE was lower in the heat. Tm was not high enough to make mitochondrial leakage a likely explanation for the observed reduced GE. Neither was the increased Tre. The extra VO2 in the H condition seems to be at least partially attributable to the extra myocardial VO2, since a higher cardiac output has to exist to continue supplying the muscles with the same blood flow, but have to send extra blood to the skin for cooling and thus impacted GE. Based on our findings under the current circumstances, it can be concluded that the temperature-induced change in GE could account for about half of the well-established performance decrements found during time trial exercise in the heat.


Value of tissue harmonic imaging (THI) and contrast harmonic imaging (CHI) in detection and characterisation of breast tumours

The purpose of this study was to investigate the extent to which tissue harmonic imaging (THI), speckle reduction imaging (SRI), spatial compounding (SC) and contrast can improve detection and differentiation of breast tumours. We examined 38 patients (14 benign, 24 malignant tumours) with different combinations of THI, SRI and SC. The effect on delineation, margin, tissue differentiation and posttumoral phenomena was evaluated with a three-point score. Additionally, 1oo not palpable tumours (diameters: 4–15 mm) were examined by contrast harmonic imaging (CHI) with power Doppler. After bolus injection (0.5 ml Optison), vascularisation and enhancement were observed for 20 min. The best combination for detection of margin, infiltration, echo pattern and posterior lesion boundary was the combination of SRI level 2 with SC low. THI was helpful for lesions OF more than 1 cm depth. In native Power Doppler, vessels were found in 54 of 100 lesions. Within 5 min after contrast medium (CM) injection, marginal and penetrating vessels increased in benign and malignant tumours and central vessels mostly in carcinomas (p<0.05). A diffuse CM accumulation was observed up to 20 min after injection in malignant tumours only (p<0.05). THI, SRI and SC improved delineation and tissue differentiation. Second-generation contrast agent allowed detection of tumour vascularisation with prolonged enhancement.
Introduction
As a technique complementary to mammography, high-resolution ultrasound (US) is well accepted for differentiating between cystic and noncystic putative tumour lesions [1–11]. New developments such as tissue harmonic imaging (THI) and contrast harmonic imaging (CHI) have enhanced its value, particularly for primary detecting tumours in dense inhomogeneous mammary gland tissue, identifying mammographically occult tumours and appraising abnormalities in tissue architecture [8, 12–22]. Lesions that are only a few millimetres in size become detectable, and tumours can be characterised more precisely. Ultimately, the exact localisation of small tumours is a prerequisite for ultrasound-guided needle biopsy, vacuum biopsy or preoperative wire placement [15, 23–25]. Harmonic imaging allows better visualisation of tissue architecture and facilitates recognition of parenchymal changes with tumour infiltration, making it easier to wire mark small lesions with THI [19, 26, 27].
For tumour characterisation, it is also essential to appraise vascularisation in a manner comparable to that achieved by dynamic contrast-medium-enhanced magnetic resonance imaging (MRI) [4, 12, 16, 17, 28–31]. Even if computed evaluation programmes with pixel analysis facilitate appraisal of tumour blood flow, native vascular US with power Doppler (PD) has proved to be relatively unreliable [4, 9, 13, 17, 18, 23, 24, 29]. Contrast medium enhancement already enables more distinct visualisation of intratumoral and peritumoral vessels. However, the brief diagnostic time window after bolus injection of US contrast medium (CM) is often a limiting factor. The combination of CHI with PD opens up promising vistas for appraisal of vascularisation. Preliminary investigations indicate that tumour detection, characterisation and US-guided intervention can be improved by using a CM of the second generation with modified CHI technique [18, 25, 32]. The aim of our study was to evaluate their potential benefits and limitations for detecting and differentiating breast tumours.
Materials and methods
Patients and US
A total of 138 patients (age: 25–79 years, median 54 years) were investigated in a multicentre trial with complementary mammary diagnostic procedures over a 36-month period (November 2002 to November 2005). All women gave informed written consent for their results to be used for programme research and evaluation. On the basis of US findings, each lesion was evaluated based on size, shape (ellipsoid, round, irregular), margins (smooth, macrolobulated, microlobulated, speculated, angulated, ill-defined), posterior sonographic artefacts (shadowing, through transmission, no posterior artefacts), echogenicity (hyperechogenic, isoechoic, hypoechoic), presence of calcifications and ductal extension. Investigations were supplemented by clinical examination and mammography in two planes. Ultrasound lesions were examined in three planes using PD and digital imaging with the GE Logiq 9 (GE Medical Systems, Milwaukee, WI, USA) US units. All investigations were carried out with a high-resolution multifrequency linear matrix array transducer (Linear Array M10L, 5–10 MHz) with the modalities of THI, spatial compounding (crossbeam), speckle reduction imaging (SRI) and contrast-medium-enhanced PD with CHI and additional three-dimensional (3D) imaging. Imaging documentation of digital raw data in single-image and short sequences was effected using a Picture and Communication System (PACS).
Study population
The patient population was divided into two groups. In group 1, we focused on the advantage of THI, SC and an adaptive algorithm for SRI. In group 2, we focused on CHI with PD.
Group 1
Thirty-eight patients (37–62 years) with 14 benign and 24 malignant tumours were examined in fundamental mode and with harmonic imaging with a high-frequency matrix-array transducer with the modalities of THI, SC and an adaptive algorithm for SRI. Prospectively, fundamental and tissue harmonic mode were used alone and in combination with all available levels of SC (cross beam: low, medium, high, maximum) and five levels of SRI. In patients without tumour lesions, criteria were differentiation of normal fibroglandular tissue from premammary and retromammary fat, including the Cooper’s ligaments and ducts. Criteria that were evaluated for differential diagnosis in patients with lesions were margin, infiltration of malignant tumours, echo pattern with tissue components, calcifications, posterior lesion boundary and posterior acoustic features. After optimising system settings, a three-level score was applied to enable comparison of the quality of scans with fundamental imaging (0=equal, −1=inferior, +1=superior).
Group 2
One hundred patients (25–76 years, median 53 years) comprised 29 probably benign [Breast Imaging Reporting and Data System (BI-RADS III)] and 71 malignant (BI-RADS IV–V) tumours confirmed by vacuum-assisted biopsy in 100/100 lesions and 78/100 cases additionally in the course of surgery. Tumour diameters varied between 4 and 15 mm (mean: 9 mm). Histological confirmation was obtained in all cases. All patients were comparatively investigated with fundamental B scan, THI and CHI with PD. After the B scan and evaluation of vascularisation in native PD, we used an echo signal amplifier of the second generation with low mechanical index (MI) technology. A bolus injection of Optison was administered (0.5 ml Optison diluted with 20 ml with NaCl solution and subsequent injection of another 20 ml NaCl). The bolus of CM was injected intravenously, and spreading of the contrast enhancement and washout in the tumours were followed for at least 20 min. A low MI (0.15–0.25) was chosen to avoid early destruction of the microbubbles.
CM enhancement was appraised for up to 20 min. Moreover, additional 3D evaluations of the cine sequences were also possible retrospectively in consequence of the archiving of dynamic digital images of up to 180 single images and 10- loop. The images archived in B scan and THI could also be processed retrospectively, with various phases of SRI, which enabled marginal contours of the tumours to be highlighted by homogenising tissue structures. The patients were given detailed information before each injection of CM, particularly with respect to a possible predisposition to allergic reactions. Written consent was obtained. Studies were performed only in patients with normal renal function. Approval had been obtained from the hospital’s ethics committee.
Histopathological correlation
Histopathological correlation was determined based on surgical and biopsy findings. Tumour size, grade, histological subtype and presence of invasion were documented. Pathological imaging correlation was performed in conjunction with both pathologist and breast imagers with regard to lesion location and size to ensure that the imaged lesions were evaluated histologically.
Breast biopsy
US-guided biopsies were performed as vacuum-assisted biopsies, mostly with an 11-gauge needle. Up to 12 representative tissue samples were taken. When there were malignant findings, US-guided wire marking was mostly undertaken before surgical excision. Afterwards, follow-up examinations were carried out at 6-month intervals.
Statistical analysis
We carried out statistical analyses:
Elementary statistics were computed.Fisher’s two-tailed exact test was applied for statistical assessment of powers for discriminating different vascularisation and comparison of benign tumours versus carcinomas after the use of Optison.
Results
Group1
THI has no effect on image quality in the near field. THI needs at least 1 cm of tissue penetration. The effect is best seen in the middle and far field, especially around the focus zone. Cutis and the first 10 mm of fat and Cooper’s ligaments are already seen in fundamental mode, with better visibility and delineation with SC (cross beam: level medium) and SRI. The reflex of the anterior mammary fascia appears thinner and brighter with SC (cross beam: level medium to high) than in fundamental imaging. Ducts in deeper areas are best visualised with a combination of SC and THI. The main disadvantage is a significant reduction of the frame rate caused by SC, especially when THI is possible in addition. The first impression from image analysis of all combinations of SC and SRI was that higher levels of SC could be avoided by using, in addition, an SRI level of 1 or 2, giving the same advantage over the fundamental image with less reduction of frame rate. The best compromise for screening examinations of breast tissue is the combination of SC (level low) and SRI (level 1 or 2) with THI in small breasts. Fat appears slightly more echogenic, which potentially helps to define small tumours. The main limitation of THI was the reduced penetration (16% of our patients). In patients with large breasts and abundant fibrous tissue, fundamental imaging should be preferred.
Lesion detection and differentiation
In four out of five cysts, THI clears artifactual echoes caused by reverberations, noise and speckle so that even small intracystic lesions could be depicted. Best visibility was achieved with a combination of THI, SC (level low) and SRI 2, giving clear cyst borders and the intracystic lesion. Using SC in two out of five cysts, the typical dorsal enhancement caused by sound transmission is less intense, as expected from the fundamental mode. Fibroadenomas were best detected with a combination of THI with SC (level medium) and SRI (2), showing better margin delineation, even in the edges. Better differentiation of tissue components and tumour infiltration could be achieved by the same combination (Fig. 1). Solid nodules generally appear more hypoechoic compared with surrounding breast tissue, which affords a better chance of detection [33].
Fig. 1Effect on delineation of tumour margin and tissue differentiation. On the left side is fundamental mode without spatial averaging and tissue harmonic imaging (THI). On the right side THI+cross beam level low+speckle reduction imaging (SRI) 2. a Improvement of delineation of tumour margin, especially if the tumour border is perpendicular to the ultrasound beam (fibroadenoma). b Improvement of tissue differentiation (haematoma)
Calcifications detected in mammography were seen in US in six out of ten patients. Visibility was increased, especially after using the combination of THI and SRI or SC and SRI (Fig. 2a,b). Results for malignant tumours are summarised in Table 1.
Fig. 2a, b Effect on visibility of calcifications and delineation of tumour margin a Fundamental B mode. b Tissue harmonic imaging (THI)+cross beam low+speckle reduction imaging (SRI) level 1. Well-differentiated invasive ductal carcinoma with microcalcification and infiltration better seen in cross beam+SRI 2. There is less attenuation retrotumoral due to spatial compounding (SC)Table 1Results of comparison of fundamental versus combination of tissue harmonic imaging (THI), spatial compounding (SC) (cross beam: level low) and speckle reduction imaging (SRI) level 1Criteria−10+1Margin11112Infiltration (existent in 19/24 malignant cases)1315Echo pattern/tissue components31011Calcifications123Posterior lesion boundary1716Posterior acoustic features996Score : −1=fundamental superior, 0=no difference, +1=combination superior
Group 2
Seventy-one cases confirmed by biopsy were classified as malignant (BI-RADS IV–V), and 29 cases were categorised as most likely benign (BI-RADS III). Histological confirmation by vacuum biopsy was also performed at patients’ expressed request and in cases of malignancy or borderline lesions also by surgery (82/100).
Histological appraisal
Benign lesions
Histological finding detected four cases with a scar or intraductal inflammatory origin or ductal lesions, two cases of papillomas, five cases of fibrocystic mastopathy and 18 cases of fibroadenomas.
Malignant lesions
Histological findings revealed 11 ductal carcinomas in situ (DCIS), two lobular carcinomas in situ (LCIS) and 58 cases of ductal invasive carcinomas. Sonomorphology defined in particular the extent of the tumour, marginal contours, change in tissue architecture, possible infiltration of surrounding tissue, intraductal spreading, altered echogenicity of lesions with possible dorsal sound enhancement or attenuation and unchanged compressibility. In PD, CM enhancement with harmonic imaging additionally enabled detection of peritumoral or intratumoral and irregular vessels penetrating the tumour as well as increased tumour vascularisation in intermittent dynamic appraisal. In particular, irregular penetrating vessels and prolonged enhancement for more than 5 min were found in only malignancies (65/71 cases: Fig. 3a–d, Fig. 4a–d and Fig. 5a–f). In six cases of DCIS with a maximum diameter of 5 mm and low grading (G I), increased vascularisation could not be demonstrated, even after CM enhancement. All were detected as small lesions using a combination of THI with SC. Five out of six showed tiny peritumoral vessels after contrast administration. Proliferating fibroadenomas and scars may show an increased peritumoral vascularisation but did not show prolonged enhancement in harmonic imaging with PD (Table 2).
Fig. 3a–d Well-differentiated invasive ductal carcinoma (75-year-old woman, non-palpable mass) a Irregular tumour of 12 mm with hidden distal border in fundamental B scan and tissue harmonic imaging (THI). b, c In combination with cross beam and speckle reduction imaging (SRI), better delineation of the pectoral (distal) aspect , in particular, showing infiltration of the pectoral fascia. d Good visibility of the biopsy needle in THI and THI with crossbeam in three-dimensional (3D) techniqueFig. 4a–d Same patient as Fig. 3 with invasive ductal carcinoma. a Unenhanced power Doppler (PD) shows few peripheral vessels. b–c After administration of 0.5 ml Option, increasing enhancement within the lesion with prolonged persistence of enhancement up to 6 min is seen. d Beginning washout after 8 minFig. 5a, b Ductal carcinoma in situ (DCIS) (35-year-old woman, no palpable mass). Cervical carcinoma, breast cancer in mother and sister. No clinical signs. Tumour proven by vacuum-assisted biopsy (DCIS) a Fundamental (B scan). b Tissue harmonic imaging (THI) showing a small, distinct, irregular mass of 8 mm. c–f Same patient as a, b. c Power Doppler (PD) before administration of contrast. d Pronounced visibility of the feeding vessel in the early phase. e Late and persistent enhancement of the tumour typical for malignant breast tumours. f Better delineation of the tumour even after 14 min in contrast harmonic imaging (CHI) modeTable 2Comparison of benign tumours versus carcinomas. Vascularisation after administration of CM [0.5 ml Optison] Benign tumours (n=29)Carcinomas (n=71)Power DopplerPower Doppler +CMPower DopplerPower Doppler + CMEarly phase (1–5 min)Marginal vessels11/29 (38%)24/29 (83%)43/71 (60%)67/71 (94%)Penetrating vessels4/29 (14%)12/29 (41%)28/71 (39%)65/71 (91%) (p<0.05)Central vessels0/29 (0%)6/29 (20%)21/71 (29%)48/71 (68%) (p<0.05)Late phase (5–20 min)Diffuse enhancement0/290/290/7165/71 (91%) (p<0.01)Marginal vessels are seen in both groups in a high percentage. Most carcinomas show diffuse enhancement through the late phase (statistical evaluation with the Fisher’s exact test, significant differences p<0.05)
The results of our own investigations indicate that the tumours can be identified better during biopsy in echo-inhomogeneous tissue using CHI in biopsies of smaller tumours under 10 mm in size. This enables false negative findings to be avoided in the histopathological investigations. After injection of CM, satellite foci and lymph nodes may also show increased vascularisation, which is manifested especially in harmonic imaging with PD.
Discussion
After clinical examination, breast US is the preferred method in symptomatic patients. In cases without symptoms, breast US is ascribed a higher sensitivity for detecting breast cancer in women with dense breast tissue, women under the age of 50 and high-risk women. Mammographically occult cancers can be detected by US in 10–40% of cases, depending on patients’ breast density and age. For women without symptoms, breast US should be mandatory and complementary to mammography in the case of breast density grade II [American College of Radiology (ACR)] or more [34]. US is now the more accurate imaging test in women 45 years or younger who present with breast symptoms and should be the initial imaging investigation [5, 7, 35]. In preoperative assessment of the local extent of breast cancer, US showed higher sensitivity for invasive cancer than for DCIS [8, 35, 41]. In nonfatty breasts, US and MRI were more sensitive than mammography for detecting invasive cancer, but both MRI and US involved the risk of overestimating tumour extent [4, 7–9, 12, 35]. Mammographic sensitivity decreased from 100% in fatty breasts to 45% in extremely dense breasts.
The respective diagnostic accuracy of the different breast imaging modalities, i.e. mammography (Mx), high-resolution breast US, and dynamic contrast-enhanced breast scanning (MRI) regarding early diagnosis of familial (hereditary) breast cancer were investigated. Breast cancer detection rates were: Mx: 42%, US 25% and MRI 83%, and positive predictive values were: Mx 29%, US 30% and MRI 43% [8].
THI in combination with digital encoding of the US beam is an increasingly common option for B-mode imaging. It reduces reverberations, sidelobe effects and speckle. Both image contrast and lateral resolution are improved in harmonic mode compared with conventional fundamental US [36]. First applications are reported for liver disease [37], lymph node enlargement in children [38] and patients with pancreatitis. Benefits of harmonic imaging are more apparent in obese patients, improving accuracy of diagnosis from 60% to 80% [39]. To determine the impact of THI on visualisation of focal breast lesions, Szopinski et al. [21] performed a prospective study on 219 female patients. THI improved the grey scale contrast between fatty tissue and breast lesions in 230 lesions (90.6%; p<0.001) compared with fundamental frequency images. Contrast improvement was greater in breasts with predominantly fatty or mixed (fatty/glandular) composition than in predominantly glandular breasts. Overall conspicuousness, lesion border definition, lesion content definition and acoustic shadow conspicuousness were improved or equal in the harmonic mode for all lesions.
After optimising the system settings, our results showed better delineation of tumour borders and infiltration with the combination of SC and SRI. Better differentiation of tissue structure was seen in the combination of THI and SRI; microcalcifications could be depicted best with a combination of harmonic mode, SRI and SC [19, 27, 40]. Retrotumoral tissue and the posterior lesion boundary of tumours are significantly better identified with high values of SC. One must be aware of the potential problem that the typical posterior acoustic enhancement is significantly reduced, with the result that a cyst can be misinterpreted as a solid tumour.
THI and CHI can be helpful for detecting tumours less than 10 mm in size in echo-inhomogeneous dense tissue, for characterising tumour morphology with dynamic enhancement studies and for performing a definitive core needle biopsy or vacuum-assisted biopsy in cases of tumour lesions less detectable in fundamental B scan or tumours located in the deep tissue near the intercostal muscles. Harmonic imaging in combination with SRI and cross beam can facilitate detection of intraductal tumours such as papillomas. With 3D imaging, the needle could be more easily placed in the middle of these small intraductal tumours during biopsies [15].
Various criteria of image morphology have proved useful in appraising whether a breast tumour is benign or malignant on the basis of US. Smooth marginal contour, dorsal US amplification, displacement margin, bilateral US extinction and absence of structural discontinuity in that order rather indicate a benign lesion. An echodense border, tumour outliers, jagged marginal contour, dorsal extinction and structural discontinuity point to malignant lesions. In consequence of the increasing improvements in tumour detection and morphological imaging, sensitivity <80% and specificities <95% can be achieved with US in tumour diagnostics [5, 7, 20, 21, 35]. An even higher diagnostic certainty is aspired to. This can already be attained with the combination of mammography and breast US, especially in dense and inhomogeneous glandular tissue [2, 7, 22, 35].
Tumour lesions of BI-RADS IV and V have to be clarified by biopsy, in particular, punch biopsy or vacuum biopsy. Consequently, US-guided biopsy is appropriate when it allows unequivocal lesion identification. US-guided interventions are required for further clarification in mammography occult findings and preoperative wire marking. In our own investigations, harmonic imaging was employed for definitive preoperative wire marking, to carry out a US-guided vacuum biopsy and also with CM enhancement for improved tumour detection.
Additional Doppler methods have been used to analyse breast tumour vascularity [42, 43]. Detection of penetrating or central vessels proved to be an accurate sign of malignancy [12, 13, 17, 22, 24, 25, 29, 31]. With the application of CM (Levovist), additional vessels were detected within the lesions, increasing diagnostic accuracy. Sensitivity of colour-coded US was improved from 64% up to 86% using the echo signal amplifier. Specificity was 86% without and 82% with echo signal amplifier. MRI was found to have a sensitivity of 100% and a specificity of up to 82% [12].
Our study with a modified technique of CM-enhanced PD and THI showed better visualisation of tumour vascularisation after bolus application of Optison. Persistent enhancement after 5 min was characteristic for malignant tumours, enabling tumour characterisation as has hitherto only been known from CM-enhanced MRI [4, 12, 16, 18, 24, 29–31, 44].
When using CM containing protein, special attention must be paid to an allergic predisposition and kidney function disorder. The use of second-generation CM necessitates a change in MI and imaging with harmonic imaging. However, the modified technique allows a differentiated diagnosis of tumour morphology and vascularisation [4, 12, 25, 29, 44, 45].
To improve the detection and assessment of intratumoral vessels in malignant breast tumours, techniques to enhance visualisation of vessels within focal lesions have been developed and automated. These techniques focus on the early perfusion phase following CM administration. Contrast-enhanced MRI allowed semiquantitative assessment of the kinetics of contrast media [4, 18, 31]. Artefacts caused by pulsation and patient movement should be avoided. More frequently, 3D postprocessing has become important in assessment of vasculature. CHI US is already used to distinguish scars from recurrent tumours as well as in the optimisation of US-guided biopsies [14, 15, 20, 32].
Conclusion
In conclusion, we feel confident that US using THI and CHI technologies is a valuable tool for evaluating focal breast lesions, in particular in mammography-dense breasts. If utilised extensively, it will be a cost-effective tool to facilitate detection and differentiation of malignant breast lesions in early, well-treatable stages and will thus help in reducing breast cancer mortality.

tissue harmonic imaging
contrast harmonic imaging
spatial compounding
breast ultrasound