In vitro contracture testing (IVCT)

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  1. The minimum patient age for the muscle biopsy is 4 years but laboratories should not test children younger than 10 years of age without relevant control data. Laboratories may also set minimum body weight limits.
  2. The biopsy should be performed on the quadriceps muscle (either vastus medialis or vastus lateralis), using local (avoiding local anaesthetic infiltration of muscle tissue), regional or trigger-free general anaesthetic techniques.
  3. The muscle samples can be dissected in vivo or removed as a block for dissection in the laboratory within 15 min.
  4. The excised muscle should be placed immediately in precarboxygenated Krebs-Ringer solution with a composition of: 
    • NaCl 118.1 mmol L-1
    • KCl 3.4 mmol L-1
    • MgSO4 0.8 mmol L-1
    • KH2PO4 1.2 mmol L-1
    • Glucose 11.1 mmol L-1
    • NaHCO3 25.0 mmol L-1
    • CaCl2 2.5 mmol L-1
    • pH 7.4

    Freshly made or pharmaceutically stable Krebs-Ringer solution should be used. The ion concentration should be as stated with a maximal deviation of ± 10%, and its pH should be in the range 7.35-7.45 at 37°C.

  5. The muscle should be transported to the laboratory in Krebs-Ringer solution at ambient temperature. In the laboratory it should be kept at room temperature and carboxygenated.
  6. The time from biopsy to completion of the tests should not exceed 5 h.
  7. The tests should be performed at 37°C in a tissue bath perfused either intermittently or continuously with Krebs-Ringer solution and carboxygenated continuously. At least four tests should be performed, each one using a fresh specimen. These include two static caffeine tests (see 11 below) and two halothane tests. The halothane test could consist of either one static (see 12 below) and one dynamic test (see 13 below) or two static tests. Each laboratory should be consistent in the method employed. Separate tissue baths should be used for different agents.
  8. Muscle specimen dimensions. Muscle specimens suitable for in vitro investigation should measure 20-25 mm in length between ties with a thickness of 2-3 mm. For measurement of length, see 8 below. The weight of the specimens should be 100-200 mg. The specimens are blotted and weighed after the test, between sutures.
  9. Determination of specimen length and predrug force. The static tests (see 11 and 12 below) are performed at optimal length (l0) which is determined 5 min after suspension of the specimen in the tissue bath by slowly stretching the muscle to force of 2 mN (0.2 g). The length between sutures is measured (initial length). Leave the muscle for another 4 min at initial length, then commence electrical stimulation (see 10 below) and stretch the muscle slowly until optimal twitch results are obtained (usually corresponding to 2 -3 g  or to 120 - 150% of initial length). This new length is considered to be the optimal length (l0) and is recorded. The muscle is left at optimal length (l0) to stabilise for at least 15 min and until baseline force does not vary more than 2.0 mN (0.2 g) within a 10-min period. Drugs may then be added. The baseline force immediately before addition of drug is recorded as the predrug force.
  10. Electrical stimulation. To demonstrate viability, the muscle specimen should be electrically stimulated (field stimulation) with a 1-2 ms supramaximal stimulus at a frequency of 0.2 Hz. Following suspension of the muscle in the tissue bath and with the muscle at optimal length, current or voltage is slowly increased until twitch height does not increase any more (initial stimulus intensity). For the supramaximal stimulation, the current or voltage is increased to 120% of initial stimulus intensity.
  11. The static cumulative caffeine test and measurement of the caffeine threshold. The concentrations of caffeine (as free base, analytical grade) in the tissue bath should be increased stepwise as follows: 0.5; 1.0; 1.5; 2.0; 3.0; 4.0; and 32 mmol L-1. Each successive concentration of caffeine should be administered as soon as the maximum contracture plateau induced by the previous concentration of caffeine has been reached, or after exposure of the muscle to the caffeine concentration for 3 min if no contracture occurs. The muscle is not washed with fresh Krebs-Ringer solution between successive concentrations of caffeine. Caffeine should be added to the tissue bath either as a bolus by injection or, with low-volume (< 5 ml) baths, in the Krebs-Ringer perfusate. A rapid change of caffeine concentration must be achieved. The result of this test will be reported as the threshold concentration which is the lowest concentration of caffeine which produces a sustained increase of at least 2 mN (0.2 g) in baseline force from the lowest force reached. In addition, the maximum contracture achieved at 2 mmol L-1 caffeine should be reported. Please note that the lowest force is not necessarily the same as the predrug force.
  12. The static halothane test and measurement of static halothane threshold.The halothane threshold is obtained using the halothane concentrations 0.11; 0.22; 0.44 and an optional concentration of 0.66 mmol L-1 as equivalent to 0.5; 1.0; 2.0 and 3.0 Vol% respectively from a serviced and calibrated vaporizer. It is recommended that the halothane concentration in the gas phase should be measured close to the inlet port of the tissue bath and/or the tissue bath concentration should be measured regularly using gas chromatography (see below). The specimen should be exposed to each halothane concentration for at least 3 min or until maximum contracture is reached. The result of this test will be reported as the threshold concentration which is the lowest concentration of halothane which produces a contracture of at least 2 mN (0.2 g) measure as an increase in baseline force from the lowest force reached. The measurement of halothane should also be reported. For determination of halothane concentration see 14 below. The flow rate of gas should be set to maintain the correct halothane concentration in the tissue bath. The gas flow into the tissue bath should be controlled using a low-flow rotameter or similar device, situated close to the inlet port of the tissue bath. The time to reach equilibration of the halothane concentration in the bath should be determined in order to ensure that the muscle sample is exposed to the test drug for the required period. The equilibration time will depend on bath volume, gas flow rate, rate of perfusion and the dynamics of the tissue bath.
  13. The dynamic halothane test and measurement of dynamic halothane threshold. This test requires a motor to enable stretching and relaxation cycles of the muscle specimen at predefined constant rates. Initially, the muscle is stretched at a constant rate of 4 mm min -1 to achieve a force of approximately 30 mN (3 g) and held at this new length for 1 min. The stretching process is then reversed for 1.5 min. The movement of the transducer from the end of the 1-min rest period to the low force is measured accurately using a vernier scale. This measurement is then used to achieve all subsequent length/tension curves, i.e. the muscle is stretched and shortened 6 mm in each cycle. The muscle is allowed to rest for 3 min. The process is then repeated to obtain 3 control curves with 1 min rest at high force and 3 min rest at low force. At the end of the descent of the third control curve, the muscle is exposed to 0.11 mmol L-1 halothane (0.5 %) for 3 min and the stretch process is repeated. The procedure is repeated for 0.22 and 0.44 mmol L-1 halothane (1 and 2 %).The force is measured at the end of the 1-min rest after stretching and the dynamic halothane threshold is the lowest concentration increasing force 2 mN (0.2 g): the contracture at 0.44 mmol L-1 is also recorded.
  14. Laboratory diagnostic classification
    • MHShc: a caffeine threshold (as defined earlier) at a caffeine concentration of 2.0 mmol L-1 or less in at least one caffeine test, and a halothane threshold concentration at 0.44 mmol L-1 or less in at least one halothane test.
    • MHSh: a halothane threshold concentration at 0.44 mmol L-1 or less in at least one halothane test and a caffeine threshold at a caffeine concentration of 3 mmol L-1 or more in all caffeine tests
    • MHSc: a caffeine threshold at a caffeine concentration of 2.0 mmol L-1 or less and a halothane threshold concentration above 0.44 mmol L-1 in all halothane tests.
    • MHN: a caffeine threshold at a caffeine concentration of 3 mmol L-1 or more in all caffeine tests and a halothane threshold concentration above 0.44 mmol L-1 in all halothane test
  15. Quality control
    Viability in any specimen used should be demonstrated by twitches ³ 10 mN (1 g) at the beginning of a test, and/ or for the caffeine test a response to 32 mmol L-1 ³ 50 mN (5 g) at the end.  
    The concentrations of halothane and caffeine in the tissue bath should be checked at least every 6 months. The samples should be taken directly from the tissue bath under the same dynamic conditions as when testing. Samples for determination of halothane concentrations should be taken immediately after the gas flow has been stopped to avoid sampling from the gas phase. Halothane concentrations can be measured using GLC or HPLC and caffeine using UV spectroscopy.  
    Halothane 0.11 and 0.44 mmol L-1 and caffeine 0.5 and 2 mmol L-1 should be checked.  
    Accepted maximal deviation from the desired concentrations are ±10 %. Lambda halothane (air / Krebs-Ringer) is taken to be 0.72 at 37°C.  
    The vaporizer should be serviced and calibrated in accordance with the manufacturer’s recommendations.
  16. Control biopsies. Prospective MH units should test 30 control muscle samples according to this protocol before commencing their diagnostic programme. All MH units are asked to investigate further control samples when feasible. For control samples, the following groups of patients are considered suitable; healthy volunteers, patients having amputations for localized disease (not systemic of vascular disease), patients with varicose veins, brain-dead patients within the first 24 h, patients with fractures within the first 24 hours. Control biopsies should be conducted within the ethical framework of the local institutional review board or ethics committee.
  17. Optional tests. Tests with other drugs may be performed on an optional basis.  
    Results of optional tests are not used for diagnosis. However, to allow for comparison of results between centres it is recommended that optional tests are performed in a uniform way, agreed upon by the EMHG Board of Directors. At present, protocols exist for tests with ryanodine, sevoflurane and 4-chloro-m-cresol. These protocols may accessed through the EMHG homepage (www.emhg.org).
  18. Protocol review. The EMHG protocol for investigation of MH susceptibility by IVCT is reviewed annually.