Malignant hyperthermia (MH) is a pharmacogenetic disease triggered by inhalational anaesthetics and depolarizing muscle relaxants. It is a potentially life-threatening syndrome, caused by an alteration of the myoplasmic calcium (Ca2+) homeostasis . The clinical features of MH are generalized muscle spasms, hypermetabolism with heat production and hypoxaemia, rhabdomyolysis with resulting potassium elevation and acid-base disturbance, cardiac dysrhythmia and renal failure . MH is autosomal dominantly transmitted in human beings, and for about 50% of the families there is a linkage to the gene that codes for the skeletal muscle ryanodine receptor (RYR1) [3-5]. The RYR1 is the calcium release channel of the sarcoplasmic reticulum and point mutations in this gene were found to co-segregate with MH susceptibility [5-10].
Despite advances in genetic diagnosis, the 'gold standard' for MH diagnosis is the in vitro muscle-contracture test (IVCT) that is done on freshly biopsied skeletal muscle bundles according to the protocols of the European and North American Malignant Hyperthermia Groups (EMHG, NAMHG). Muscle bundles are tested for abnormal sensitivity to halo-thane and caffeine .
Due to the high affinity of the RYR1 for the plant alkaloid ryanodine, an optional muscle-contracture test using ryanodine was proposed to improve the specificity in MH diagnosis [12-15]. This optional test is used in some MH investigation units [15-20]; however, some variability of the results obtained at various MH investigation units makes it impossible to use one specific value that unequivocally distinguishes MH susceptible (MHS) from MH negative (MHN) subjects [17-19]. Having performed the ryanodine in vitro muscle-contracture test (RCT) under standardized conditions for many years, we retro-spectively analysed the results for MHN and MHS subjects in order to investigate, whether we could define cut-off values separating MHN from MHS subjects. Additionally, in order to clarify whether or not there are differences between individual in vitro effects of ryanodine, halothane and caffeine, we compared the results of RCT with muscle contractures and threshold values for halothane and caffeine, which were obtained using IVCT.
In a retrospective study, we evaluated the protocols of 113 patients who underwent both IVCT and RCT performed between 1994 and 2001 in our MH investigation unit. The patients or at least one member of their family had a previous history of a clinical MH episode. Before performing the investigations a complete personal and family history was taken from all patients, and written, informed consent was obtained. The true MH status, MHS or MHN, was defined using the results of IVCT with halothane and caffeine. Patients classified as MH equivocal were excluded from the study.
Muscle biopsies were taken from quadriceps muscles (vastus medialis or lateralis) under regional anaesthesia (3-in-1 nerve block) with mepivacaine 1% 30-40 mL. The muscle specimens were immediately placed in pre-oxygenated Krebs-Ringer solution and brought to the laboratory.
In vitro muscle-contracture tests
All tests were performed within 6 h after biopsy. For the halothane and caffeine tests: the muscle bundles were suspended in a 3 mL test bath, perfused with Krebs-Ringer solution and bubbled with carbogen (oxygen 95%, carbon dioxide 5%). RCT was performed in a 20 mL test bath. Temperatures in the test baths were kept between 36.5°C and 37.5°C. Only viable muscle strips with a twitch response to supra-maximal electrical stimulation (single twitch, 100 V, square wave 2 ms and frequency 0.2 Hz) of > 10 mN were included in this study. After stretching the muscle bundles to 150 ± 10% of their initial length and equilibrating until a stable baseline tension was obtained, the study agent was applied to the bath.
For all subjects, IVCT was performed on four muscle bundles according to the protocol of the EMHG (two static halothane tests and two static caffeine tests)  and RCT was performed on one surplus muscle bundle. Administration of halothane at concentrations of 0.11, 0.22, 0.44 and 0.66 mmol or caffeine at concentrations of 0.5, 1.0, 1.5, 2.0, 3.0, 4.0 and 32 mmol was started. Baseline tensions were recorded before drug administration as well as the maximum muscle contractures at each of the drug concentrations. Halothane was administered using a carboxygen flow at a rate of 200 mL min−1 (Fluotec® 3; Cyprane, Keighley, UK). Immediately before the IVCT, a solution of caffeine dissolved in a Krebs-Ringer solution at the indicated concentrations was prepared.
For each test the following parameters were recorded: weight and length of the muscle bundle, maximum preload and twitch height before the drug application. MH status was evaluated according to the thresholds indicated in the EMHG protocol: MHS equals a muscle contracture ≥2 mN at a halothane threshold concentration of ≤0.44 mmol and a caffeine threshold concentration ≤2.0 mmol; MHN equals a muscle contracture of ≥2 mN at a halothane threshold concentration >0.44 mmol and a caffeine thresh-old concentration >3.0 mmol [11,21].
The RCT was performed according to the EMHG guidelines for optional IVCT . The test was performed at optimal muscle bundle length with a stimulation of a 1 ms supra-maximal stimulus at a frequency of 0.2 Hz. Baseline tension did not vary more than 2 mN within the 10 min period before the addition of ryanodine. Immediately before the ryanodine challenge, the perfusion of the bath was stopped. Ryanodine was applied to the test bath with a single bolus technique using a stock solution with a ryanodine concentration of 100 μmol in distilled water in order to reach a final bath concentration of 1 μmol. High-purity ryanodine (C25H35NO9) was used (CALBIOCHEM®; La Jolla, CA, USA).
The parameters analysed (Fig. 1) were as follows: time from the addition of ryanodine (1 μmol) until muscle tension exceeds pre-drug tension value equals to onset time (Otp); time from the addition of ryanodine to the development of a 10 mN muscle contracture above pre-drug tension is 10tp; time interval between Otp and 10tp is dtp; maximum muscle contracture of the muscle bundle is Tmax. Results were evaluated by segregating for gender and age (over and under 40 yr). Eight cut-off values with an interval of 2.5 min were defined for Otp and for each value the sensitivity, specificity and Youden index  were calculated on the basis of the MH diagnosis by IVCT. These cut-off values were used to calculate the receiver operating characteristic (ROC) curve .
Comparisons between subjects and specimen characteristics and different end-points for RCT were performed using the U-rank sum test for non-parametric variables. Correction for ties was used. The correlations between parameters of the RCT and the IVCT were examined by logistic regression analysis and Spearman's rank correlation coefficient . For all these tests, StatView® (ADEPT, Hertfordshire, UK) was used. P < 0.05 was considered significant.
The study included 113 subjects. Seventy-seven subjects were MHS and 36 subjects were MHN. The MHS subjects were from 33 unrelated families, three of these families each had more than five MHS members. For patient characteristics data and pre-test characteristics see Table 1.
Otp was significantly shorter for MHS than for MHN individuals (median 4.8 min and 20.1 min, respectively, P < 0.0001, Table 2 and Fig. 2). A total of 72 (93.5%) MHS subjects had an Otp < 15 min, whereas 27 (75%) MHN subjects had an Otp > 15 min. There was no significant effect of age or gender on Otp. Subjects <40 yr had a median Otp of 4.2 min vs. 4.8 min in subjects >40 yr (n.s.). Males had a median Otp of 4.2 min, while females had 5.4 min (n.s.).
The results for 10tp were comparable to those for Otp. Subjects diagnosed as MHS had shorter 10tp than MHN subjects (median 12.0 min vs. 29.6 min, respectively, P < 0.0001). No influence of gender or age was found. As expected, dtp proved to be shorter for MHS subjects (P < 0.0004). Subjects diagnosed as MHS also developed significantly greater maximal muscle contractures (see Table 2).
The best cut-off value calculated by the Youden index was 10 min with sensitivity of 0.78 and specificity of 0.94 (Table 3). Shorter cut-off values separated groups better, but sensitivity decreased. The area under the ROC curve for the RCT was 0.93.
For MHS subjects there was a correlation between Otp in RCT and muscle contractures developed in IVCT (Fig. 3). Patients with short Otp developed higher muscle contractures at halothane 0.44 mmol and caffeine 2 mmol, although this correlation was weak (r = 0.40 and 0.36, respectively, P < 0.0004 and P < 0.0015, respectively). A stronger correlation existed between Otp and the threshold concentrations of halothane and caffeine, with ρ = 0.47 and 0.52, respectively (P < 0.0001) (Fig. 4). MHS specimens showing a high susceptibility towards halothane and caffeine also showed a high susceptibility to ryanodine (short Otp). This correlation was also found between 10tp and the threshold concentration of halothane and caffeine (ρ = 0.51 and 0.58, respectively, P < 0.0001).
MHS subjects had significantly shorter Otp in RCT compared with MHN subjects (median 4.8 vs. 20.1 min, respectively) independent of age or gender. MHS subjects also had shorter 10tp than MHN subjects (median 12.0 vs. 29.6 min, respectively), and dtp also was shorter for MHS subjects. Muscle bundles from MHS subjects also developed a significantly greater maximum muscle contracture in RCT than did those from MHN subjects. Thus, results of the RCT significantly differed between MHS and MHN subjects.
The optimal cut-off value for Otp for distinguishing MHS from MHN subjects was 10 min. This value has been suggested as optimal in dog experiments  and similar values have been suggested for human muscle [17,19]. This time interval accurately reflects individuals who are truly MHS with almost no false-positive test results (two of 62 (3.2%) according to the MH diagnosis by IVCT), with a high specificity (94%). However, not all MHS subjects were identified within this interval, as its sensitivity was only 78%. To improve sensitivity to >94%, the interval had to be increased to 15 min. This resulted in an overlap of about 11% (9/81) false-positive test results and a decrease in specificity; thus, in contrast to other investigations [18,19], we were not able to define end-points for the RCT that separated MHS from MHN subjects without having this overlap.
Hopkins and colleagues  attempted to identify common cut-off values for the RCT from results obtained in 11 different MH investigation units of the EMHG. They found a high degree of variability in RCT results between the units suggesting it inappropriate to define common values until the reasons for this discrepancy are clarified. Although there were some methodological differences in the RCTs, they did not correlate with the differences found in RCT results. It is possible that inherent population differences with a varying mutation prevalence accounts for this . Different mutations also show different muscle contractures and threshold values for halothane and caffeine in IVCT [27,28], indicating that the genetic background influences test results. For the IVCT, Ording and colleagues  found identical diagnostic results between two centres in only 56% of the tested patients. These differences were almost exclusively seen in cases with contractures of <5 mN and abnormal results in only one or two muscle bundles. With increasing contractures (≥5 mN) and contractures in at least 75% of the probes, this discrepancy vanished. Nevertheless, common threshold concentrations of halothane and caffeine are used successfully by all centres, reflecting the robust nature of the current diagnostic criteria of the IVCT. Whether general cut-off values for the RCT will prove practical requires further evaluation.
The negative predictive value (NPV) of the IVCT using halothane and caffeine, that is, the calculated conditional probability of a true-negative subject, given a negative result, is high because of the IVCT high sensitivity of about 99% [21,22]. This high NPV is required clinically for the safety of patients. The positive predictive value (PPV) of the IVCT is the calculated conditional probability of a true-positive subject, that is, MHS, given a positive result. It is a function of the pre-test probability (PTP) and the specificity. The PTP is defined as the probability, or clinical suspicion, that a person has disease before testing. A high likelihood of MH (high PTP) reduces false-positive results and improves PPV. In contrast, with a low PTP, a negative test result would effectively rule out MH, since the NPV approaches 1.0 . In this case, a positive test result is more likely to be a false-positive result than a true-positive result. To date, there is no safe way to distinguish between true-positive and false-positive results. Therefore clinicians should always act as if the subject's result is true positive, because exposing a susceptible patient to triggering anaesthetics may be deleterious.
For the IVCT, 95% confidence intervals for specificity range between 71-85% for the NAMHG and 89-96% for the EMHG [21,22]. Our RCT results did not improve specificity of MH diagnosis without losing sensitivity. We speculate that a specific pharmacogenetic disposition of an MHS subject may lead to similar IVCT results using different agents, that is, it is unlikely that there is a drug which can significantly improve the specificity of both halothane and caffeine. Diagnostic limitation of the RCT reflected by an overlap of 3.2-11% in our hands may be explained by at least two reasons: firstly, inappro-priate pre-test classification caused by known limitations of IVCT (99% sensitivity, 94% specificity) . Secondly, limitation of discrimination using RCT as supported by the findings of Hopkins  and our results.
MHS subjects had a positive correlation between the threshold values for halothane and caffeine obtained in IVCT and the Otp in RCT. High susceptibility towards halothane and caffeine correlated with high susceptibility towards ryanodine, indicated by a short Otp and vice versa. This is a key finding in our study because it may reflect the individual pharmacogenetic variability of MH. Other authors have also described these correlations . The genotype as well as a variety of environmental causes may influence the phenotype presentation provoked by the use of different agents for IVCT. However, due to the limited number of investigated subjects in this study we cannot rule out that the RCT might be a useful tool for further investigation of pharmacogenetic mechanisms of MH.
In conclusion, the 'gold standard' in MH diagnosis is the IVCT using halothane and caffeine. The optional RCT using a bolus application technique with a ryanodine test bath concentration of 1 μmol distinguished MHS and MHN patients, but a small overlap between the two groups was found. Age and gender had no influence on test results. Cut-off values of <10 min increased the specificity but showed reduced sensitivity and vice versa. A correlation between halothane and caffeine susceptibility and ryanodine susceptibility was found for most MHS subjects. This correlation may reflect the pharmacogenetic variability of MH.
The authors thank Mrs Joan Etlinger for excellent secretarial assistance.
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