Anesth Analg 2003;96:153-158
© 2003 International Anesthesia Research Society
ANESTHETIC PHARMACOLOGY
Extending the Skeletal Muscle Viability Period in the Malignant Hyperthermia Test
Saiid Bina, PhD,
Stephen Holman, MD, and
Sheila M. Muldoon, MD
Department of Anesthesiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland
Address correspondence and reprint requests to Saiid Bina, PhD, Department of Anesthesiology, USUHS, 4301 Jones Bridge Rd., Bethesda, MD 20814. Address e-mail to sbina{at}usuhs.mil
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Abstract
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The caffeine halothane contracture test (CHCT) is the only validated test for diagnosing malignant hyperthermia (MH) susceptibility (MHS) and phenotyping MHS families. Although most diagnostic laboratory tests can check intra- and interlaboratory consistency through the use of standard control samples, there has been no practical way to achieve this goal for the CHCT. The distances between diagnostic centers and time constraints of the CHCT protocol (5 h) prohibit centers from sharing tissue samples. In this study, we investigated varying storage conditions to extend the standard viability period of skeletal muscle to 24 h. Twenty MHS patients were tested according to the North America protocol. After standard CHCT, the surplus muscle samples were placed in one of the following four treatment groups. In Groups 1 and 2, muscles remained under tension and were stored in Krebs buffer (pH 7.4) at 23°C–25°C (clamped-warm) and 4°C (clamped-cold), respectively. In Groups 3 and 4, muscle strips were dissected, and the ends were tied with silk sutures, cut from the clamp, and placed in Krebs buffer at 23°C–25°C (free-warm) and 4°C (free-cold), respectively. The responses of the treatment groups to halothane (3%) and caffeine (0.5–32 mM) were tested at 22–26 h after excision. The clamped-warm storage group correctly diagnosed MHS in all patients.
IMPLICATIONS: Varying conditions for storage of muscle were investigated to extend the viability period of muscle in the malignant hyperthermia (MH) test from 5 to 24 h. Muscles stored for 24 h under tension at room temperature remained viable and correctly diagnosed MH susceptibility in all patients.
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Introduction
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Malignant hyperthermia (MH) is a genetic abnormality with autosomal dominant inheritance. MH episodes usually occur when MH susceptible (MHS) individuals are exposed to commonly used volatile anesthetics and depolarizing skeletal muscle relaxants. Symptoms including hypoxia, acidosis, hyperthermia, increased end-tidal CO2 production, lactic acidosis, and arterial oxygen desaturation result from excessive Ca2+ release from the sarcoplasmic reticulum in skeletal muscle. If the triggering drug is not stopped and therapy initiated immediately, the syndrome may lead to neurological, liver, and/or kidney damage and eventually death (1–3). The standard tests for the diagnosis of MHS and phenotyping MHS families are the North America (NA) caffeine halothane contracture test (CHCT) (4) and the European in vitro contracture test (IVCT) (5). Although most diagnostic laboratory tests can check intra- and interlaboratory consistency through the use of standard control samples, there has been no practical way to achieve this goal for the CHCT. The distances between diagnostic centers and time constraints for performance of the CHCT prohibit centers from sharing tissue samples.
Another major concern is the limited number of MH diagnostic centers in NA. In 1990, there were 18 MH diagnostic centers in NA (15 in the United States and 3 in Canada). Currently there are 11 centers in NA (8 in the United States) where the CHCT is performed. All the NA centers are under financial stress, and two may close in the near future. Current standards require that CHCT be completed within 5 h of muscle excision (4,5). Therefore, to ensure viability of the skeletal muscle samples, the biopsy must be conducted at one of the MH diagnostic centers. This logistic constraint contributes to patient costs and inconvenience. If the period of skeletal muscle specimen viability could be extended to 24 h, diagnostic centers could share samples. In addition, it may be possible to have the muscle biopsy performed at the patient’s local hospital and have the specimens delivered to the diagnostic center for CHCT. Previously, we evaluated muscles from normal and MH swine and found that muscle samples stored under tension at room temperature remained viable and accurately predicted MHS by CHCT at 24 h postexcision (6). The aim of this study was to evaluate whether these techniques are applicable in human vastus muscle specimens.
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Methods
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With institutional approved informed consent, 20 patients undergoing diagnostic CHCT for MHS participated in the study. The CHCT was performed by using vastus lateralis muscle in accordance with the NA MH protocol for CHCT (4) 2–4 h after muscle excision (standard CHCT) and after storage of surplus muscle for 22–26 h. Muscle samples were clamped under tension then excised and placed in room temperature Krebs bicarbonate buffer (pH 7.4) as previously described (4). A standard set of clamps was used to ensure that all muscles had the following approximate dimensions: 2–2.4 cm long, 1–1.5 cm wide, and 0.2 cm thick. In our MH diagnostic center, normal practice for CHCT is to use an experienced surgeon to excise muscle or one of the experienced researchers present to supervise the muscle biopsy.
After transfer of the muscle samples from local hospitals to the testing laboratory (1–2 h), muscles were placed in fresh Krebs bicarbonate buffer and aerated with carbogen (95% oxygen and 5% CO2) for 5 min. Muscle strips for standard CHCT were prepared by dissecting strips from the clamped muscle (2.1 ± 0.4 cm, 150 ± 35 mg). Both ends of each strip were tied with a silk suture, and the muscle ends were cut free from the biopsy clamp. One suture was secured to the lower end of a plastic frame that had two integrated platinum electrodes. The other suture was connected to a calibrated isometric force transducer (Model FTO3; Grass Medical Instruments, Quincy, MA). Muscle strips were immersed in organ chambers filled with 25 mL of Krebs bicarbonate buffer at 37°C and were aerated with carbogen, and isometric tension was recorded continuously (Model 3400; Gould Instrument Systems, Valley View, OH). A length-tension curve was performed on each specimen to determine the optimum resting tension. The muscle strips were allowed to stabilize for 15–30 min at the average resting tension of 2.2 ± 0.4 g (22 ± 4 mN). Muscle viability was assessed by evaluation of the twitch tension response to electrical stimulation delivered through platinum electrodes (duration 1 ms, frequency 0.2 Hz, voltage 12 V; Model S44; Grass Medical Instruments). The muscle responses to halothane (3%; 8 mM) and the cumulative addition of caffeine (0.5, 1, 2, 4, 8, and 32 mM) were performed within 2–4 h postexcision (standard CHCT, three or four strips for halothane and two or three strips for caffeine). After standard CHCT, the muscle strips were discarded.
After completion of the diagnostic standard CHCT, the surplus muscle tissues were allocated to one of the following four storage methods. In Group 1, muscle remained in the biopsy clamp under tension and was stored in Krebs bicarbonate buffer at 23°C–25°C (clamped-warm). In Group 2, it was treated as in Group 1 except for storage at 4°C (clamped-cold). In Group 3, muscle strips were dissected, and the ends were tied with silk sutures, cut from the clamp, and placed in Krebs buffer at 23°C–25°C (free-warm). Group 4 was treated as in Group 3 except for storage at 4°C (free-cold). The responses of the stored muscle to halothane or caffeine were tested between 22 and 26 h postexcision. The responses of treatment groups to halothane and caffeine were evaluated as described for the diagnostic CHCT protocol. In some cases, the surplus muscle specimens were not sufficient to examine all storage treatment groups. In those cases, the surplus specimens were allocated to the clamped storage treatment groups (Groups 1 and 2). This choice was made on the basis of data obtained on specimens from the first five patients and on previous swine data, which showed that free muscle specimens performed poorly (6).
Assessment of muscle specimen viability was based on the twitch tension response after electrical stimulation in accordance with the NA protocol (twitch tension  0.5 g). A positive halothane contracture test was defined as an increase in baseline tension of 0.7 g (7 mN) on exposure to 3% halothane. A positive caffeine contracture test was defined as an increase in baseline tension of 0.2 g (2 mN) in response to a total caffeine concentration of 2 mM. The increases in resting tensions were measured after each drug addition at the plateau of the contracture amplitude ( 1–5 min). An in-line infrared gas analyzer (Model 254; Datex, Helsinki, Finland), which was calibrated with standard calibration gas mixtures (Scott Medical Products, Plumsteadville, PA), monitored organ-bath halothane concentration. Halothane bath concentration was confirmed by a gas chromatographic procedure (7).
The patients were assigned the diagnosis obtained at the standard CHCT. Diagnostic criteria were as follows: a patient was considered to be MHS if at least one of the viable muscle strips demonstrated an abnormal contracture response after exposure to 3% halothane, 2 mM caffeine, or both. A patient was diagnosed MH negative (MHN) if no strip met positive criteria in response to 3% halothane or a 2 mM caffeine bath concentration.
Data are presented as mean ± SEM in grams. Analysis of variance (ANOVA) was used to analyze differences among the treatment groups. Significance within groups over time was determined by ANOVA for repeated measures. A P value of <0.05 was considered significant. A patient size of 9, with 29 (baseline) and 14 (clamped-warm) strips from MHS patients, had a 78% power to detect a difference in mean force of muscle contracture of 1.5 g, assuming a standard deviation difference of 1.3 g by using repeated-measures ANOVA.
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Results
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Two hundred twenty-nine muscle strips evaluated for standard CHCT and 22- to 26-h storage treatments; of these, 96 strips were from 9 MHS patients and 133 strips were from 11 MHN patients. In the standard CHCT, pretest twitch tensions in strips from MHS patients were not significantly different from twitch tensions in strips from MHN patients. Pretest twitch tensions in 106 of 111 muscle specimens were more than 0.5 g (5 mN) tension. Under all four storage conditions, the twitch tensions of the muscle bundles were significantly lower than standard CHCT twitch tensions (Table 1). The storage treatment that best preserved the twitch tension response at 24 h postexcision was the clamped-warm group (Group 1). Twitch tension in Group 1 decreased by 57% compared with standard CHCT (P < 0.05). Also in Group 1, a pretest twitch tension of 0.5 g (5 mN) was found in 33 of 48 strip specimens (Table 1). All strips in Group 1 had pretest twitch tensions of at least 0.1 g. However, in 19 of 42 muscle strips in Group 2 (clamped-cold), the pretest twitch response was totally absent. Also, in Groups 3 and 4 (free-warm and free-cold, respectively), the twitch response to 66% of strips was totally absent (Table 1).
The CHCT results from fresh and stored muscles are shown in Tables 2 and 3. All stored muscles had a decrease in the halothane (3%)-induced contractures, but halothane-induced contracture was best preserved in Group 1. Significantly larger decreases in the response to halothane were observed in Groups 2–4 (Table 2). Group I responses to caffeine (0.5–32 mM) were not significantly different from standard CHCT (Fig. 1). Thus, compared with the standard CHCT, strips in Group 1 had the least decrease in response to halothane and 0.5–32 mM caffeine. Group 2 mean responses to 2 mM caffeine (1.2 ± 0.5 g) in MHS subjects were more than in the standard CHCT. This may have been due to a large response in one of the strips. Significant decreases were found in the responses to 2 and 32 mM caffeine in Groups 3 and 4 and in Groups 2–4, respectively (Table 3).
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Table 2. Effect of Storage Treatment on the Halothane (3%)-Induced Contracture of Vastus Muscle Groups from Malignant Hyperthermia-Susceptible (MHS; n = 8) and Malignant Hyperthermia-Negative (MHN; n = 11) Patients
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Figure 1. Effect of clamped-warm storage treatment on caffeine-induced contractures. Baseline data were collected 2–4 h after muscle excision. The surplus muscle was stored for 22–26 h in the clamped-warm storage treatment, and the test was repeated. The patients were assigned the diagnosis obtained at baseline (5 MHS and 11 MHN). *Caffeine-induced contractures in strips from MHS patients at baseline and from the treatment group were not significantly different from each other. However, they were significantly different from the corresponding strips from MHN patients. MHS = malignant hyperthermia susceptible; MHN = malignant hyperthermia negative.
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Table 3. Effect of Storage Treatment on the Caffeine-Induced Contracture of Vastus Muscle Groups from Malignant Hyperthermia-Susceptible (MHS; n = 5) and Malignant Hyperthermia-Negative (MHN; n = 11) patients
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Discussion
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This study is the first report to show that human muscle bundles from MHS and MHN patients can accurately predict MHS by CHCT 24 hours postexcision of skeletal muscles. These findings are in agreement with previous findings in muscle bundles from normal and MHS swine (6) and may provide a practical means to establish a standard control for the CHCT. Until now, it has not been possible to share muscle samples between the diagnostic centers in NA because the distance between centers is too far to complete the test in the time limit set by the NA protocol.
Despite the overall decrease in the pretest twitch tension, clamped-warm was the only storage treatment group to correlate with the standard CHCT results. Thus, the CHCT results in the clamped-warm storage treatment group were 100% sensitive and specific (no false-positive or false-negative diagnosis resulted from the storage treatment). We found no correlation between twitch tension and halothane- or caffeine-induced contracture on muscle strips from normal or MHS patients, confirming previous reports (4,6). However, those muscle strips that did not twitch responded poorly to halothane and caffeine.
Despite standardization of the NA and European MH diagnostic protocols (4,5), significant intercenter variability of CHCT and IVCT results remains to be determined. The clamped-warm storage technique described in this study has the potential to be useful in investigating the center-to-center variability of test results. In addition, this technique may increase the potential for valid testing on patients who are far from test centers. Current standard NA CHCT protocols require testing to be completed within five hours of muscle excision and require the muscle specimens to be kept at room temperature until testing. This standard was arbitrarily set to minimize the effect of time-related muscle deterioration on CHCT results (false-positive or false-negative). However, there are only limited data to support or reject the five-hour limit. Britt et al. (8) reported no significant time effects on specimen performance over one hour. Gallant et al. (9) reported that increased postbiopsy time correlated significantly with enhanced sensitivity to halothane and that preparations using cut fibers were more sensitive to halothane than those with intact fibers. Later, Iaizzo and Lehmann-Horn (10) reported no significant difference in test results with IVCT between intact or cut fiber preparation.
The effect of transport time and diagnostic center on tissue viability has also been studied in multicenter investigations (6,11–13). Ording et al. (11) compared IVCT results obtained at two centers when the transport time of muscle specimens from one center to another was one to two hours and all tests were completed within five hours after excision of the muscle tissue. However, no comment could be made with regard to prolonging viability. Between centers, CHCT or IVCT variability could be mainly due to the surgical procedure, surgeon expertise, technical equipment, and laboratory technician expertise.
Regarding temperature, prior studies have addressed the temperature at which the test is conducted but have not addressed the optimal temperature of the muscle specimen during transport or before testing (14–16). Tonkin et al. (17) have described a method to transport human muscle specimens to a remote testing center for IVCT. They speculated that reducing muscle metabolism could extend viability. Because metabolism is temperature dependent, the free specimen was placed in a sealed container of ice-cold Krebs solution. On arrival at the diagnostic center, the specimen was dissected into strips at room temperature. The IVCTs were completed within four hours after the specimen was removed. Although the muscle specimen remained viable for IVCT, it was impossible to determine the effect of cooling on muscle specimen preservation without control data. Data from the current study showed that human vastus specimens stored at room temperature performed better than specimens stored at 4°C and that clamped specimens under tension performed better than free specimens. These results are comparable to a previous finding in muscles from normal and MHS swine (6).
Finally, how should viability be assessed? In this study, clamped muscle specimens stored at room temperature identified the correct phenotype for 86% of muscle strips tested at 22–26 hours postexcision. Identifying the correct phenotype is important in determining the sensitivity and specificity of a diagnostic test but cannot be used to determine viability, which must be determined before testing to eliminate false results due to nonviable specimens. The NA protocol states that good twitch viability should be demonstrated (0.5 g), and the European protocol requires a pretest twitch tension minimum of 1 g or a 32 mM caffeine-induced contraction of at least 5 g (50 mN). In addition, comparing absolute values of twitch tension may not be the best measure of viability, because we found no correlation between twitch tension and halothane or caffeine response on strips from MHS and MHN patients.
In conclusion, we showed that vastus skeletal muscle bundles from MHS and MHN patients can accurately predict MHS by CHCT at 22–26 hours postexcision when the muscle sample is clamped under tension and stored in Krebs buffer maintained at room temperature. Lack of tension and a cold storage temperature had negative effects on halothane- and caffeine-induced contractures. The current investigation was conducted in a single center with a relatively small number of patients. Confirmation of this method in a multicenter study may provide a comprehensive procedure to develop a standard control for a practical means to investigate center-to-center variability, a quality-assurance measure of CHCT/IVCT results, and remote testing for suspected MHS patients.
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Acknowledgments
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Supported by the Uniformed Services University of the Health Sciences, Bethesda, MD.
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References
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Accepted for publication October 1, 2002.
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Abstract
Introduction
