Malignant Hyperthermia Diagnostic Centre

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle that presents as a hypermetabolic response to potent volatile anaesthetic gases such as halothane, sevoflurane, desflurane, isoflurane and the depolarizing muscle relaxant as succinylcholine. The syndrome is inherited in an autosomal dominant pattern. Uncontrolled rise of myoplasmic calcium, which activates biochemical process related to muscle activation leads to the pathophysiologic changes. This condition is potentially dangerous: it causes the uncontrolled increase of muscle tissue’s oxidative metabolism which exceeds the organism’s capacity to provide oxygen and to dispose of carbon dioxide, leading ultimately to a cardiovascular collapse and to the patient’s death. The disease is completely asymptomatic, making its identification extremely difficult.

In its clinical expression the MH is a rare affection. The global incidence is estimated around 1:15.000 anaesthesia in pediatric population and 1:50.000 in adults with a small predisposition for male and for paediatric people, while not making recognisable predicting factors (a subject already exposed to trigger factors, in which the syndrome isn’t disappeared, can’t to be considered without risk to a further exposure). The mortality stands actually around the 7% cases all of the world.

The MH is a channelopathy caused usually by gene mutation which encodes for the ryanodine receptor (Ryr1), a protein found in the sarcoplasmic reticulum of skeletal muscle’s fibrocellular, which controls the opening and the closing of a channel through which calcium is freed in fibrocellular cytoplasm. The calcium is the ionic element which average the mechanism of contraction/relaxation muscle, therefore as a result of the administration of those anaesthetics, a calcium flux not-settled means a pathological muscle contractures, followed by an increase of the metabolic activity, excessive consumption of energy and then of oxygen. Due to the considerable demand, the production of energy becomes insufficient and consequently increased the production of the heat and the body temperature until to 42-45°C, the alteration of cell membrane’s integrity, followed by Rabdomiolisi (the breaking of cell membrane) and the augmentation of enzymes of muscle necrosis in the blood (cpk,ldh,sgot,aldolasi).

The MH is a genetic pathology with autosomal dominant transmission for which a parent, carrying the genetics, can transmit the disease to 50% of children.
Studies of molecular genetics have shown that the MH’s primary defect resides in the calcium channel of skeletal muscle commonly known as Ryanodina receipt (RYR1).
The gene which codes this protein, in tetrameric form constitutes the calcium channel, is located on the Chromosome19. Currently the studies of molecular genetics have allowed to identify 34 causative mutations which codes for the type1 ryanodine receptor (RYR1), localised to chromosome19q13.1.
The genetics analysis in MHS patients has been expanded also to the mutation research both in CACNA1S gene and CACNL2A and SCN4A gene localised to other chomosomes involved themselves in the Malignant Hypertermia, but object of study yet. At the Centre of Biotechnology was conducted the study of RYR1 gene in 14 families tested positive at the IVCT (In Vitro Contracture Test): the study has made it possible to identify 10 known mutations and 10 new mutations. The aim of the genetics study is to develop a non-invasive method which is able to substitute the IVCT and then the surgical operation for the IM diagnosis. The genetics study until now has made it possible to ask genetics diagnosis in only the 40% of susceptible patients at the MH diagnostic test.

The triggers drugs in the susceptible individuals provoke a prolonged opening of calcium channels with an abnormal increase of calcium ion’s concentration in the fibrocellular muscle’s cytoplasm. The non-regulated flux of calcium causes a pathological muscle contractures and increases the muscle’s metabolic activity.
The muscles, which are active in these conditions, consume energy and so an excessive amounts of oxygen, with subsequent emission of heat, water, carbon dioxide and lactates.
The release of large quantities of heat by the muscles subjected to excessive activities provokes the increase of the patient’s temperature more quickly than the natural system of the thermoregulation can keep under control. In a few minutes may occur an increase of temperature until to 42-45°C.
The energy production becomes insufficient and disappears the cell membrane’s integrity, proteins as Creatine Kinasi (CPK), LactateDehydrogenase (LDH), S-Glutammic-Oxaloacetic-Transaminase (SGOT), Aldolase and Myoglobin release into the blood (Rhabdomyolysis). The absence of oxygen can cause brain damage; the increase of carbon dioxide into the blood stimulates a quick and rapid breathing.
The breakdown of skeletal muscle is associated with excretion of myoglobin in the urine where it can cause an acute renal failure. The potassium increase in the blood provokes tachycardia and tachyarrhythmia until the cardiac arrest, if action is not taken promptly.

The “gold standard” for diagnosis of MH is currently an in vitro contracture test, which is based on contracture of muscle fibers in the presence of halothane or caffeine, according to the criteria by the Protocol of the MH European Group (EMHG:European Malignant Hyperthermia Group – www.emhg.org). The Test is carried out on muscle fibers of vastus medialis or vastus lateralis of quadriceps muscle, removed through muscle biopsy in locoregional anaesthesia.
During the test the muscle fibers are stretched at 2000mg and subsequently stimulated to a frequency of 0.2 Hz. It is necessary to stabilize the muscle , during the stimulation, of at least 20 minutes or however of the appropriate time to show a stable baseline, so that it does not suffer the variations more than 2mN in order to register the real variations of the answer to drugs.
Finally the fibers is stimulated with an increasing solution of Halothane and Caffeine.
Two contraction tests both at Caffeine and Halothane are carried out, as provided by the European MH Group’s protocol.
The positive test and so the diagnosis of MH susceptibility is determined in order to the increase of the muscle tension, measured by the isometric transducer and registered.
The answers at the IVCT test are classified as follows:
MHS (Malignant Hyperthermia Susceptibility): when the muscle answers to the test with an increase of the muscle tension equal or greater of 0,2g (2mN), as a result of the administration of Halothane to the percentage concentration up to 2% and of Caffeine to the concentration of 2mM.
MHE (Malignant Hyperthermia Equivocal): this diagnosis class si divided in MHEh if the test is only positive to the Halothane and MHEc if the test is only positive to the Caffeine.
MHN (Malignant Hyperthermia Non-Susceptible): there isn’t increases of the muscle tension equal to or greater than 0,2g (2mN) to the useful concentrations.
The IVCT of European protocol presents 99% of Sensibility and 94% of Specifity

Diagnostic laboratory

The Centre for the Malignant Hyperthermia study of A.O.R.N. “A.Cardarelli” of Naples starts its activity in this area in 1989 and until the 31/12/2015 have been reported, especially by anaesthetists of several health services , both public and private, predominantly of the Central-South Italy, 2163 patients, 1835 of them subjected to outpatient visit and 820 of them subjected to IVCT test: the 35% of them was found susceptible to MH.

In the 2015 were carried out 42 muscle biopsies, subdivided as follows:

• MHN    30        (Malignant Hyperthermia Negative: Negative patients)
• MHS       7         (Malignant Hyperthermia Susceptible: Positive patients)
• MHEh    5         (Malignant Hyperthermia Equivocal Halotane: Positive patients)
• MHEc    0         (Malignant Hyperthermia Equivocal Caffeine)

The “diagnostics iter” includes:

1. Patient reporting for the followings reasons: Family Clinical Case, Fatal familial case, elevated CPK, rhabdomyolysis, myophaties.
2. Outpatient visit in which is practised an accurate anamnesis, both personal and familiar, clinical examination, checks of other tests with a subsequent differential diagnosis with other diseases.
3. The visit can be reserve at the CUPA through a free telephone number 800 019 774 at the following timetables: 12,00 – 12,30 / 14,00 – 18,30. The demanding must be endorsed by the hospital’s ticket office, located at the Palermo Bulding on the groundfloor. The demanding must contain the following doctor’s note: “It is required a visit for suspicious susceptibility at the Malignant Hyperthermia”.
4. Hospitalization in Day-surgery to perform the muscle biopsy ( The biopsy is carried out in DS at the plastic surgery department).
5. Laboratory for the diagnostics test, localised at the Centre of Biotechnologies , where the muscle is subjected to the IVC Halothane – Caffeine Test.
6. Patient followed up until the removing stitches

In the Centre has been achieved information material (brochure, informative articles, posters for the operating room) to promote a continuous updating, both for the healthcare professionals and for the patients.

In the Centre is present a bank of blood and muscle collecting of the patients subjected to the IVCT test.

In 2015 have been frozen 42 samples of blood and muscle tissue.