Awareness of a patient’s malignant hyperthermia predis position based on diagnostic testing is essential to preventing serious malignant hyperthermia events
The in vitro contracture test after surgical muscle biopsy is a well-established and comprehensive diagnostic test
Genetic testing can be used to confirm malignant hyper thermia susceptibility but not to exclude it
In vivo ultrasound (shear-wave) elastography detected a temporary increase in local tissue stiffness soon after intramuscular injection of halothane and caffeine in malignant hyperthermia–susceptible pigs
Quantitative shear-wave elastography allowed earlier detection of reactions to halothane and caffeine than did measurement of local lactate concentrations in microdialysis fluids, although the responses were analogous
Halothane and caffeine induce excessive sarcoplasmic calcium liberation and skeletal muscle contracture in patients susceptible to malignant hyperthermia (MH) and are utilized for diagnosis in the in vitro contracture test. Intramuscular injection previously caused a marked local lactate increase in MH-susceptible but not in MH-nonsusceptible individuals in vivo. Using shear-wave elastography, this study evaluated localized changes in muscle stiffness after intramuscular injection of halothane and caffeine.
Microdialysis probes were placed into the gracilis muscle of 16 pigs (9 MH-susceptible and 7 MH-nonsusceptible). After local injection of either halothane or caffeine in different concentrations, changes of tissue elasticity surrounding the probe were examined by quantitative shear-wave elastography. Local lactate concentrations were analyzed spectrophotometrically.
Ultrasound elastography detected a temporary increase in local muscle rigidity in MH-susceptible but not in MH-nonsusceptible pigs after 2.5 and 5 vol% halothane and after 10, 40, and 80 mM caffeine, whereas there were no differences in the control groups (median [interquartile range] for maximum effect after 5 vol% halothane: MH-susceptible: 97 [31 to 148] vs. MH-nonsusceptible: 5 [−6 to 18] kPa; P = 0.0006; maximum effect after 80 mM caffeine: 112 [64 to 174] vs. −3 [−6 to 35] kPa; P = 0.0002). These effects were seen rapidly within 5 min. Local lactate concentrations were higher in MH-susceptible versus nonsusceptible pigs after 1 and 2.5 vol% halothane and 10, 40, and 80 mM caffeine (2.5 vol% halothane: MH-susceptible: 2.8 [1.9 to 4.4] vs. MH-nonsusceptible: 0.6 [0.6 to 0.7] mmol/l; P < 0.0001; 80 mM caffeine: 5.2 [4.1 to 6.3] vs. 1.6 [1.2 to 2.4] mmol/l; P < 0.0001). After 10 vol% halothane, rigidity and lactate levels were increased in both MH-susceptible and MH-nonsusceptible animals.
This pilot study revealed shear-wave elastography as a suitable technique for real-time detection of altered tissue elasticity in response to pharmacologic stimulation. By considering the variability of these results, further test protocol optimization is required before elastography could serve as a minimally invasive MH diagnostic test.
MALIGNANT hyperthermia (MH) is a genetically determined disorder leading to skeletal muscle hypermetabolism in response to volatile anesthetics and succinylcholine.1 The incidence of apparent MH episodes during anesthesia is estimated between 1 in 10,000 and 1 in 220,000, but genetic prevalence is predicted to be as high as 1 in 2,000.2 For the prevention of serious MH events, diagnostic testing and awareness of a patient’s MH predisposition are absolutely essential. Because most MH-susceptible individuals do not have muscular symptoms in everyday life, patients with undiagnosed MH susceptibility are at risk of developing this life-threatening anesthetic complication.3
The latest diagnostic guidelines issued by the European Malignant Hyperthermia Group (Herlev, Denmark) contain two possible diagnostic approaches: the in vitro contracture test after surgical muscle biopsy as the most comprehensive and well established but comparatively invasive procedure and the molecular genetic detection of a known causative mutation located on the gene of the ryanodine receptor 1 or the dihydropyridine receptor.4,5 Because of the heterogeneity of MH, genetic testing can be safely applied only to confirm but not to exclude MH susceptibility.
In a search for a less invasive alternative for the in vitro contracture test, a diagnostic test based on intramuscular injection of small amounts of triggering agents and subsequent monitoring of local lactate concentrations by microdialysis was proposed.6 This test showed promising results in MH-susceptible pigs7 as well as in a pilot study with probands and patients.8,9 Local lactate levels were significantly increased in MH-susceptible compared with MH-nonsusceptible individuals after halothane or caffeine application. However, because of the time-consuming and elaborate test setup, the need for sufficient immobilization of a patient’s leg for more than 60 min, and the requirement of a laboratory facility for further processing of the microdialysis probes, this test does not seem to be ideal for bedside application in daily routine.
Recent developments in ultrasound techniques led to the question of whether the localized intramuscular reactions could be visualized by using ultrasound imaging. Shear-wave elastography is an ultrasound technique that enables quantification of elastic tissue properties. Tissue stiffness is calculated as Young’s modulus in kPa and displayed in real time as a color-coded image.10–12 Ultrasound elastography is well established in the diagnostics of liver disease and breast, thyroid, and prostate lesions.13 In the anesthetic field, it has been proposed for the prevention of intraneural injections during ultrasound-guided peripheral nerve blocks.14,15 Musculoskeletal applications include examination of muscle and tendon disease and evaluation of effects due to treatment or exercise.16–18 Different studies have proven shear-wave elastography to be sufficient for quantification of muscular contraction19,20 and for detection of pathologic muscle stiffness.21,22
This is a porcine pilot study evaluating the opportunities of shear-wave elastography for differentiation between MH-susceptible and MH-nonsusceptible individuals. We hypothesized that the intramuscular injection of halothane and caffeine in pigs with and without predisposition to MH causes not only localized metabolic muscular reactions but also regional changes in muscle stiffness, which could be detected by shear-wave elastography.
Materials and Methods
With approval of the responsible animal care committee (Government of Unterfranken, Wuerzburg, Germany, No. 39/13), an experimental study in 16 pigs was conducted. The animals were obtained from a local farmer (Farm Lippert, Germany), were derived from several long-standing colonies, and were related to one another to varying degrees up to first degree. According to previous DNA analysis (GeneControl GmbH, Germany), nine pigs were homozygous for the Arg615Cys mutation on ryanodine receptor 1, which causes a phenotype similar to human MH in pigs (MH-susceptible), whereas seven were homozygous negative for the mutation (MH-nonsusceptible).23 The pigs were investigated in random order according to availability from the animal breeder.
After insertion of a venous cannula into an ear vein, the pigs received general anesthesia with thiopental (10 mg/kg) and fentanyl (0.01 mg/kg). An orotracheal tube with a 9-mm internal diameter (Teleflex Medical GmbH, Germany) was inserted, and the pigs were mechanically ventilated with an inspiratory oxygen fraction between 0.4 and 0.5. Ventilator settings were adjusted to maintain the end-tidal carbon dioxide (ETCO2) between 32 and 39 mmHg (respiratory rate 10 to 16 breaths/min, tidal volume 8 to 12 ml/kg, positive end-expiratory pressure 5 mmHg). Anesthesia was maintained with thiopental (10 mg · kg−1 · h−1) and fentanyl (0.01 to 0.02 mg · kg−1 · h−1). Standard monitoring (electrocardiography, oxygen saturation measured by pulse oximetry [Spo2], rectal temperature), as well as an arterial line in the tibial artery for invasive blood pressure monitoring and intermittent arterial blood gas analysis, were established. After completion of the experiments, the pigs were euthanized in deep anesthesia by thiopental bolus application (0.1 g/kg).
Custom-made microdialysis probes with attached microtubing for injection of testing agents (MAB-7; Microbiotech Sweden) were inserted into the gracilis muscle after skin incision and surgical exposure of the muscle. Ten probes were used per animal, five on each leg. A distance of more than 10 mm between two adjacent probes was ensured to avoid interference. Adequate positioning of the probes was confirmed by B-mode ultrasonography. Microdialysis probes were perfused with Ringer’s solution (1 µl/min) for at least 30 min to allow equilibration. After collection of baseline values, testing agents were injected at each probe. 300 µl halothane (Sigma-Aldrich Chemie, Germany) dissolved in soybean oil (Lipofundin 20%; B. Braun, Germany) in concentrations of 0, 1, 2.5, 5, and 10 vol% and 300 µl caffeine solution in concentrations of 0, 1, 10, 40, and 80 mM (Merck, Germany) were used as established in previous studies.7 Samples were collected in 15-min intervals.
Sonographic evaluation was performed by quantitative shear-wave elastography (Aixplorer; Supersonic Imagine, France). A linear ultrasound probe (4 to 15 MHz; SuperLinear SL 15-4, Supersonic Imagine, France) was used to visualize the microdialysis probe and the surrounding muscle tissue (B-mode) and to determine tissue elasticity (shear-wave elastography). The specific ultrasound transducer simultaneously induces shear waves. As these shear waves propagate through the tissue, their velocity, which changes with different elastic tissue properties, is measured by ultrafast ultrasound. The transducer was positioned in line with the direction of muscle fibers because previous studies have confirmed this position as suitable for the detection of muscular contractions by shear-wave elastography.24 A measuring field of 3 mm in diameter was targeted in the surrounding of the microdialysis probe aiming for the area of highest muscle stiffness. To exclude artifacts caused by the probe, the probe itself was excluded from the measuring field. Tissue elasticity was measured quantitatively as Young’s modulus in kPa before and at time intervals of 1, 5, 15, 30, 45, and 60 min after application of the testing agents. Sufficient blinding of the examiner was not feasible because pigs with and without susceptibility to MH are easily distinguishable based on anatomical features. For standardization, all shear-wave elastography examinations were performed by the same operator.
Lactate concentrations in the dialysate were measured spectrophotometrically before and 15, 30, 45, and 60 min after injection of testing agents. In detail, the dialysate samples were incubated with lactate reagent (Trinity Biotech, Ireland), and after enzymatic conversion, a chromogene dye correlating directly to lactate concentration was measured by spectrophotometry (Agilent 8453; Agilent Technologies, Australia) at 540 nm.
Because this was a pilot study, it was not possible to estimate effect sizes without any preexisting data. Therefore, an a priori power calculation was not conducted. A necessary sample size of a minimum of seven animals per group was determined based on rational expectations and on experience with previous microdialysis studies. Statistical evaluation was performed by using GraphPad Prism 6.0 for Mac OS X (GraphPad Software, USA). Parametric distribution of the data was confirmed by using the D’Agostino-Pearson normality test. To better allow evaluation of skew at different time points, data are presented as median and interquartile range. Two-way ANOVA was calculated with the factors MH diagnosis and time separately for each examined concentration level and followed by post hoc Sidak’s test for multiple comparisons. P < 0.05 was considered statistically significant.
There were no relevant differences in biometric data between MH-susceptible and MH-nonsusceptible pigs (age: MH-susceptible 69 [68 to 71] days vs. MH-nonsusceptible 69 [67 to 70] days; weight: MH-susceptible 30 [21 to 32] kg vs. MH-nonsusceptible 25 [23 to 28] kg). Vital signs (heart rate, Spo2, mean arterial pressure, and body temperature), as well as metabolic parameters (pH, Paco2, potassium, and lactate), were comparable between the MH-susceptible and MH-nonsusceptible groups before and after the experiment. Within the groups of MH-susceptible and MH-nonsusceptible animals, the application of the testing agents had no effect on vital and systemic metabolic parameters (tables 1 and 2).
A significant increase in local muscle stiffness compared with baseline was detected by shear-wave elastography in MH-susceptible but not in MH-nonsusceptible animals 1 min after application of 2.5 vol% halothane (MH-susceptible: 64 [39 to 148] kPa vs. MH-nonsusceptible: 2 [−45 to 9] kPa; P < 0.0001) and at 1 and 5 min after application of 5 vol% halothane (1 min: MH-susceptible: 88 [44 to 160] kPa vs. MH-nonsusceptible: 4 [−7 to 37] kPa; P = 0.0009; 5 min: 97 [31 to 148] kPa vs. 5 [−6 to 18] kPa; P = 0.0006). The most distinct differences were present within 5 min after injection (figs. 1 and 2). After the maximum concentration of 10 vol% halothane was injected, increasing stiffness was detected in MH-susceptible and in MH-nonsusceptible muscle alike (5 min: MH-susceptible: 53 [30 to 106] kPa vs. MH-nonsusceptible: 41 [36 to 104] kPa; P > 0.999; fig. 3). Controls did not show any alteration in elasticity after injection of soybean oil without halothane in either MH-susceptible or MH-nonsusceptible animals.
Local lactate concentrations significantly increased in MH-susceptible versus MH-nonsusceptible muscle after injection of 1 and 2.5 vol% halothane (1 vol% halothane: MH-susceptible: 2.3 [1.9 to 2.8] mmol/l vs. MH-nonsusceptible: 0.7 [0.5 to 0.8] mmol/l; P < 0.0001; 2.5 vol% halothane: 2.8 [1.9 to 4.4] mmol/l vs. 0.6 [0.6 to 0.7] mmol/l; P < 0.0001), and there was a pronounced lactate increase in both groups after injection of 5 and 10 vol% halothane. The peak lactate levels were reached 30 min after halothane injection (fig. 4). Two-way ANOVA revealed significant interaction between MH diagnosis and time for elastography results at 2.5 and 5 vol% halothane and for lactate concentration at 1 and 2.5 vol%.
Again, with a maximum effect within 5 min after injection, a significant increase in local muscle rigidity compared with baseline was detected in MH-susceptible but not in MH-nonsusceptible pigs 1 min after injection of 10 mM caffeine (MH-susceptible: 58 [38 to 98] kPa vs. MH-nonsusceptible: 18 [4 to 40] kPa; P = 0.0064) and 1 and 5 min after injection of 40 mM caffeine (1 min: MH-susceptible: 102 [87 to 192] kPa vs. MH-nonsusceptible: 14 [7 to 22] kPa; P < 0.0001; 5 min: MH-susceptible: 86 [23 to 114] kPa vs. MH-nonsusceptible: 4 [–4 to 12] kPa; P = 0.0075). After injection of 80 mM caffeine, significant differences in muscle rigidity were present between 1 and 15 min after injection with the maximum difference after only 1 min (MH-susceptible: 112 [64 to 174] kPa vs. MH-nonsusceptible: −3 [−6 to 35] kPa; P = 0.0002; figs. 5, 6, and 7). There were no differences between MH-susceptible and MH-nonsusceptible animals in the control group and after injection of 1 mM caffeine.
Significantly higher lactate concentrations in MH-susceptible compared with MH-nonsusceptible muscle were noticed after injection of 10, 40, and 80 mM caffeine (maximum at 80 mM caffeine: MH-susceptible: 5.2 [4.1 to 6.3] mmol/l vs. MH-nonsusceptible: 1.6 [1.2 to 2.4] mmol/l; P < 0.0001), whereas control and 1 mM caffeine showed no relevant differences. Similar to halothane, lactate levels peaked 30 min after injection of caffeine (fig. 8). The interaction between MH diagnosis and time was significant for elastography results at 40 and 80 mM caffeine and for lactate levels at 10, 40, and 80 mM.
Excluded Data and Missing Values
Because of insufficient image quality, the following elastography measurements had to be excluded from evaluation: 0 vol% halothane: pigs 10 and 11; 1 vol% halothane: pigs 5 and 10; 0 mM caffeine: pigs 1 and 10; 1 mM caffeine: pigs 8 and 16; and 40 mM caffeine: pig 14. Single values were missing due to inaccurate labeling of the images for pig 16 at 5 min after application of 5 vol% halothane, pig 2 at 45 min after 0 mM caffeine, and pigs 10 and 13 at 5 min after 40 mM caffeine. Due to dysfunction of the microdialysis probes, no lactate concentrations were recorded for pig 17 after 1 mM caffeine, for pigs 3 and 16 after 10 mM caffeine, and for pig 13 after 40 mM caffeine.
In this study, we applied ultrasound elastography to reveal a localized skeletal muscular reaction to pharmacologic stimulation in real time. Shear-wave elastography indicated a temporary increase in local tissue stiffness rapidly after application of halothane and caffeine analogous to the intramuscular metabolic reaction detected by monitoring of local lactate concentrations.
In accordance with previous studies, injection of small amounts of halothane and caffeine induced a reaction in skeletal muscle, resulting in a local lactate increase with significant differences between MH-susceptible and MH-nonsusceptible animals. Interestingly, the results of ultrasound imaging matched these findings. Similar to both techniques and in line with previous findings,7 a muscular reaction was provoked in a dose-dependent manner also in MH-nonsusceptible muscle at the highest halothane concentration. This was not surprising because halothane is known to activate ryanodine receptor 1–mediated calcium release and cause contractions in the in vitro contracture test dose-dependently also in muscle of MH-nonsusceptible individuals.25,26 As hypothesized, shear-wave elastography allowed considerably earlier detection of intramuscular reactions compared with microdialysis. The maximum effects were recognized by elastography within 5 min after injection and lasted up to 15 min. This result was illuminating because both onset and decay of the reaction were visualized by elastography in real time. In contrast, maximum changes in local lactate concentration occurred between 15 and 30 min after trigger injection. Detection of the metabolic reaction by microdialysis was delayed by the process of lactate liberation and diffusion from the skeletal muscle cells to interstitial tissue and the microdialysis probe and obviously by the dead space of the microdialysis probes, which was responsible for an almost 15-min delay at the applied flow rate of 1 µl/min. Additional time was necessary for subsequent spectrophotometric processing of the microdialysis samples. In summary, at least 2 h were needed from trigger application until a result of the microdialysis measurements was achieved.
Previous studies already excluded systemic effects of the injection and indicated that the impact of the applied amounts of test substances is limited to an area of less than 10 mm around the injection site.7 The presented results are in accordance with these findings. Local injection of the testing agents was safe and did not trigger any MH events. There was no influence on hemodynamic or systemic metabolic parameters. Interference between different probes has been prevented by ensuring a sufficient distance between the probes.
The distribution of the injected agents into the muscle tissue has been visualized by B-mode ultrasonography. It slightly varied between different microdialysis probes due to the individual position and the specific tissue structure and texture of the area around the probe. By reviewing the elastography images, the area where elasticity changes happen is obviously related to the microdialysis probe at the site of injection. However, the size of this area and the exact location in relation to the probe show considerable variations that could be based on individual differences in distribution of the injected agents. Therefore, it was impossible to specifically define a region to measure elastographic changes because this might have completely missed the reaction in some cases. The integrated Aixplorer software allows quantitative measuring of a circular field (Q-Box) of variable diameter within the borders of the elastography area. Choosing a measuring field consisting of a large portion of muscle with the microdialysis probe in the center was no option because the hardness of the microdialysis probe itself would have been accountable for the highest elasticity values in that area. Instead, we decided to use a relatively small measuring field of 3 mm in diameter and screened the muscle area surrounding the probe for the region of maximum stiffness. Ideally, an area of muscle where no probe or catheter could interfere with shear-wave elastography would be desirable for the test, but withdrawal of the injection catheter after application would raise doubts about the reliable identification of the injection site for repeated shear-wave elastography measurements. The use of smaller and softer catheters for injection could minimize this issue in future studies, when simultaneous microdialysis will not be necessary anymore.
One reason for variation within the MH-susceptible and MH-nonsusceptible groups was technical artifacts that were noticed especially when dialysis probes were placed in comparably slim layers of the gracilis muscle or superficially with only minor distance between the region of interest and the ultrasound transducer. Our study protocol included previous surgical exposure of the investigated muscle in order to limit the possible influence of skin and subcutaneous tissue. In retrospect, this would not have been necessary. Especially in slim portions of the muscle, it further reduced the distance between the transducer and the investigated region and may have facilitated artifacts. As specified above, in some cases inconsistent shear-wave elastography results at baseline resulted in exclusion of the particular probe from further investigation. There are general concerns about the use of shear-wave elastography in very superficial structures.27 Based on our findings, we believe that a previously proposed minimum distance between the ultrasound probe and assessed tissue of 3 mm is not sufficient in our setup.28
In order to limit further influencing factors on the measurement results, we have decided in favor of one single operator performing all shear-wave elastography examinations. Therefore, interexaminer reliability of the procedure has not been evaluated and needs to be tested with future studies.
The presented investigation was intended as a feasibility study. Further efforts are needed to evaluate this new approach as a potential diagnostic test procedure. Some adjustments of the procedure, such as the definition of a minimum distance of presumably 10 mm between the transducer and investigated muscle region and the usage of catheters causing fewer artifacts on shear-wave elastography, have already been discussed. So far, hardly any data about shear-wave elastography in pigs are available,29 and it remains unclear if skeletal muscle elasticity values are comparable between species. However, we expect no benefit from further optimization of this test in the pig model but aim to evaluate the principle at the bedside in patients with and without predisposition to MH. A larger trial is needed, sufficiently powered to define diagnostic cutoff values. Before being considered for use in clinical routine, diagnostic sensitivity and specificity need to fit with the in vitro contracture test, which is still the accepted standard of MH diagnostics.4
After shear-wave elastography has been frequently investigated and has proven a suitable technique for assessment of elastic properties in different tissues, in this pilot study it has been utilized to observe a skeletal muscle response to pharmacologic stimulation. Our data revealed a novel approach for real-time monitoring of an intramuscular reaction after local injection of halothane and caffeine. Further studies are needed to evaluate this technique for potential use as a rapid and minimally invasive diagnostic test for MH susceptibility at the bedside.
The authors thank Judith Skirde, chief technician, Center for Malignant Hyperthermia, University of Wuerzburg, Wuerzburg, Germany, for essential advice and technical assistance with this study.
Support was provided solely from institutional and/or departmental sources.
The authors declare no competing interests.