The Evolving Role of Muscle Pathology
Muscle biopsy has been an important part of the assessment of patients with a neuromuscular disorder for many decades. The use of frozen sections and the application of histochemistry and electron microscopy have identified many pathological features that have defined and diagnosed a disorder. The molecular genetic revolution that began with the discovery of the gene responsible for Duchenne muscular dystrophy (DMD) has resulted in the identification of several hundreds of defective genes that cause a neuromuscular disorder. Muscle pathology has had a major role in directing molecular analysis, especially with increasing use of immunohistochemistry to visualize protein defects. In the past, molecular analysis was performed on a gene-by-gene basis but the rapidly advancing application of next-generation sequencing has led to the analysis of panels of genes, which is more time/cost-effective.
Coverage of genes, however, needs to be considered and some mutations – e.g. gross deletions, some duplications, re-arrangements and expansions – may escape detection by these sequencing panels. In addition, many variants may be identified, particularly in very large genes, and their pathological significance may be difficult to establish. The increasing application of whole exome/genome sequencing is not only identifying novel defective genes but also broadening the clinical phenotype associated with known genes and the muscle pathology associated with them. Muscle pathology also has an important role in establishing the pathogenicity of novel genes. In addition, epigenetic and gene silencing can be different in different tissues. In some mitochondrial diseases the guilty mitochondrial DNA mutation is only present in muscle and will not be identified without a muscle biopsy, which can then be analyzed by studies of protein expression, enzyme activities and morphological alterations in addition to gene analysis. In some clinical trials muscle pathology is being used as an important outcome measure, and an understanding of the advantages and limitations of techniques as well as the muscle pathology is necessary. Considering the increased demand for accurate genetic diagnosis, which is fundamental for prenatal diagnosis and patient management, all methods that can aid in reaching the correct diagnosis should be applied: thus, the role of muscle pathology is evolving but not redundant. The combination of whole genome/exome sequencing on DNA and cDNA from a muscle biopsy, in addition to all the other information that can be obtained by analyszing the affected tissue using advanced techniques such as laser capture, multiplex immunohistochemistry and methods to detect elements in tissues, will become increasingly important in the future. Modifying gene alterations, the influence of micro RNAs and the appearance of more than one pathogenic variant also have to be considered.
The Procedure of Muscle Biopsy
Muscle biopsy still has an important role as part of the diagnostic procedures in the assessment of a patient with a neuromuscular condition, despite the rapid molecular advances discussed previously. Accurate diagnosis and identification of a pathogenic molecular defect lead to better patient management and genetic counselling, and muscle pathology is contributing to the development of therapies and their application. Muscle biopsy is a relatively simple procedure; yet in the past it was frequently poorly done. The pathologist who receives a small fragment of an unnamed muscle, coiled into a disorientated ball after being dropped into formalin, is unlikely to get any meaningful information from it, no matter how careful the processing. With the upsurge of interest in neuromuscular disorders, clinicians and surgeons are now better informed on the handling of samples, which leads to useful information being obtained. The following are some guidelines worth following when planning a muscle biopsy.
Selection of the Patient
A full clinical assessment of the patient is essential. Diagnosis should always be based on a multidisciplinary approach with detailed clinical and family history, and clinical examination, in conjunction with any special investigations such as serum enzymes, biochemistry, muscle imaging and electromyography. A biopsy is an additional test of an underlying muscle and/or neural disorder. In general, the main indication for muscle biopsy is evidence of neuromuscular disease such as muscle weakness, muscle cramps or discomfort (especially on exercise) and muscle fatigue with activity. Pathological change may be found in some conditions in the absence of any apparent neuromuscular signs: for example, collagen vascular diseases. On the other hand, muscle pathology may be absent, minimal or non-specific in conditions such as some metabolic disorders, some myasthenic syndromes or ion channel disorders in which careful clinical and electrodiagnostic studies often provide the diagnosis.
In some conditions, such as spinal muscular atrophy, myotonic dystrophies and facioscapulohumeral muscular dystrophy (FSHD), molecular analysis is so reliable that it can provide direct confirmation of diagnosis without the need for a biopsy. In other conditions, there may be no specific muscle pathology, such as in disorders associated with mutations in the lamin A/C gene or limb-girdle muscular dystrophy caused by defects in the gene encoding anoctamin 5 ( ANO5 ), although there have been recent improvements in techniques (see Chapter 11, Chapter 6 ). Molecular analysis is then often the test of choice. Genotype and the results of DNA analysis, however, cannot always be related to phenotype and there are exceptions to every rule. This is well demonstrated in DMD, in which the molecular defect may not always correlate with the protein expression seen in the muscle ( ). More importantly, clinical severity cannot be judged by molecular analysis alone. In addition, with the increasing application of whole exome screening many changes in DNA are identified, but their significance in terms of protein expression and disease have to be interpreted. Pathology then has an important role. Muscle pathology complements modern techniques and is still an important component of patient assessment.
Selection of the Muscle
Selection of the muscle should be based on the distribution of the muscle weakness and the overall clinical assessment. In selecting the muscle for biopsy, it is important not to choose either a muscle which is so severely involved by the disease process that it will be largely replaced by fat or connective tissue (end-stage muscle) and show little recognizable trace of the underlying disease process or, alternatively, a muscle which is so little affected that it does not show sufficient change. Differential involvement of muscle occurs in several disorders, and ultrasound imaging is a simple, quick technique for assessing this that can be done in the outpatient clinic or at the bedside ( ). It can also help in the selection of the biopsy site. Magnetic resonance imaging (MRI) of muscle gives superior quality, and patterns associated with individual diseases are now recognized ( ).
In general, where the distribution of the weakness is proximal, a moderately affected proximal muscle is selected which is also reasonably accessible, such as the quadriceps (rectus femoris or vastus lateralis) in the leg or the biceps in the arm. In other circumstances, the deltoid or gastrocnemius is also a suitable muscle for biopsy. Where weakness is mainly distal, a more distal limb muscle may be selected, but even in these circumstances biopsy of a proximal muscle may reveal the underlying pathological process adequately.
In a chronic disease such as muscular dystrophy, a muscle with only moderate weakness may be the ideal site for biopsy. In an acute disease, on the other hand, because the process has not had time to progress to extensive destruction, a more severely involved muscle may be chosen. In addition, the biopsy technique (see next) may influence the choice of muscle. For example, with a needle technique the quadriceps is often considered relatively safe as the muscle is readily accessible and major nerves and blood vessels lie close to the femur and are unlikely to be damaged.
There are advantages in trying to limit the biopsies to certain muscles so as to be familiar with the normal pattern in that particular muscle. It is important to be aware of anatomical differences between muscles and be familiar with possible age-related changes. Thus, the distribution of fibre types and fibre sizes is well recognized in the biceps and the quadriceps but the pattern may be unfamiliar in such muscles as the intercostals, the abdominal muscles or the hand or foot muscles. Other muscles such as the paraspinal muscles frequently show abnormalities, and interpreting their significance is then difficult. In certain circumstances, for example when studying motor endplates, the muscle selected will be determined by the particular line of investigation. In this instance a motor-point sample is required, but in most institutes this is rarely performed and for diagnostic analysis of most muscle disorders it is not necessary. For any quantitative studies, adequate control determinations of the same muscle are essential. Sampling at the site of either electromyography or any form of injection should also be avoided as needling of any kind can produce changes in the muscle ( ). Similarly, sports injuries or other traumas, the use or disuse of the muscle, ageing, drugs and toxins and any possible effect of contractures should also be taken into account (see Chapter 19, Chapter 23 ).
For certain immunohistochemical studies, skin or buccal cells may be useful and for prenatal diagnosis chorionic villus samples can be used (see Ch. 6 ).
Technique of Biopsy
In the past we performed all our muscle biopsies in adults as well as infants under local anaesthesia and there was no justification for submitting patients who may already be at risk of respiratory deficit to general anaesthesia. In addition, there is a particular hazard of general anaesthesia and relaxant drugs in several conditions such as myotonic dystrophy, central core disease and malignant hyperthermia. Under local anaesthesia, the risks of muscle biopsy, as with other minor procedures, are negligible. Many hospitals, however, now insist that children are not aware of a procedure and general anaesthesia has to be used, with appropriate precautions, but this relies on available time on a theatre list. In our own unit we continue to do needle biopsies ourselves (see next) but these now have to be carried out under general anaesthesia. There is always merit in alerting a surgeon to the need for a muscle sample if a patient is undergoing some other surgical procedure. In these cases, particular note should be taken of the site sampled, as this may influence the pathology. For example, a biopsy taken near the tendon when the Achilles tendon is being lengthened may be very fibrous and difficult to interpret. Similarly, biopsies taken near any tendon or fascia may show features such as fibre splitting and internal nuclei (see Ch. 3 ).
For several decades in our centre in London we have used a needle biopsy technique for obtaining muscle samples, both for diagnostic and for research purposes, but an open technique is now sometimes performed by surgical colleagues and is often the preferred technique at other centres. Needle biopsy is a safe procedure, free of any complications, and the scar is often almost invisible with time. Open biopsies provide a larger sample, which may be useful for biochemical studies, or be required for clinical trials, but in most situations the same diagnostic conclusion can be reached in a needle sample. Developments in the sensitivity of biochemical and immunoblotting techniques have also reduced the need for large samples.
Although a needle for muscle biopsy was introduced more than 100 years ago by , the technique did not find wide application until relatively recent times. introduced a percutaneous needle with similar features to those of Duchenne’s, mainly for the study of normal muscle in relation to various physiological changes. Edwards and colleagues ( ) applied the Bergström needle for routine muscle biopsy mainly in adult patients, and for several decades we have found it to be suitable and satisfactory for neonates, infants and older children. We use mainly a 5-mm diameter needle and occasionally a smaller 4 mm one in newborn infants. Refinements to the prototype instrument have been made, but we have continued to use the original Bergström type. reviewed their experience in 1000 cases, and we reviewed 670, mainly childhood, needle biopsies ( ). Disposable needles based on the Bergstrӧm prototype are also now available (Laurane Medical) and have been successfully applied guided by ultrasound. Other types of needles (such as Trucut) do not produce adequate samples, with the exception of the conchotome, alligator-type forceps ( ). A recent publication has reviewed the use of the Bergstrӧm and conchotome types of needle ( ). The major advantages of the needle biopsy procedure over open biopsy are its simplicity, its speed and the fact that it can readily be done by physicians as a day case procedure, either under sedation or under general anaesthetic with an appropriate safe protocol. In addition, samples from multiple sites can be justified as the scar left is small. The choice may be dictated by patient’s age, anxiety level/cooperation and local hospital protocol.
In paediatric practice, needle biopsy can be performed under local anaesthesia with sedation, but it is important to ensure that a safe sedation protocol is in place, with adequate monitoring and resuscitation facilities to hand. In infants under 6 months, sedation is not normally used although chloral hydrate may be used (30–100 mg/kg). In children between 6 months and 10 years, we usually use chloral hydrate (80 mg/kg, maximum 1000 mg) if their weight is less than 15 kg and oral diazepam (0.2–0.4 mg/kg, maximum 10 mg) if their weight is above 15 kg. In our experience children heavier than 15 kg tend not to be well sedated with chloral hydrate, occasionally becoming hyperactive. If no sedation is achieved within 45 minutes, midazolam 0.1 mg/kg intranasally or orally (maximum 10 mg) can be given. The patient has to be connected to a saturation monitor and flumazenil (10 mg/kg) readily available in case the effect of midazolam has to be reversed (although we have never had a case in which this has been necessary). In older children and adults, the procedure can be performed without sedation.
Since 2008, following hospital protocol, we have performed needle muscle biopsies in children at our centre under general anaesthesia. We routinely ask screening questions for excessive bruising or bleeding to detect a possible coagulation disorder and for symptoms of sleep hypoventilation. Most of our biopsies are taken from the quadriceps (vastus lateralis) ( Figs. 1.1a–d, 1.2 ). When local anaesthesia is used, the skin is prepared in the usual way with antiseptic and draped. The skin and subcutaneous tissue down to the muscle sheath are infiltrated with 1% lidocaine (Xylocaine). It is important not to infiltrate the muscle, as this can cause artefacts. For general anaesthetic, our anaesthetic team uses propofol for total intravenous anaesthesia (TIVA), avoiding suxamethonium, halothane and the related inhaled anaesthetics to avoid malignant hyperthermia, and the rhabdomyolytic, hyperkalaemic, myoglobinuric crises which can occur in boys with Duchenne and Becker muscular dystrophy and some congenital myopathies. The procedure for obtaining the sample is similar, regardless of whether general or local anaesthetic is used. In most cases, we take the opportunity of obtaining a 4 mm skin punch biopsy to establish a fibroblast culture and sometimes for immunohistochemistry. A small incision is made with a scalpel blade, or through the skin biopsy site if this has been done, down into the muscle sheath, at approximately mid-thigh level, just lateral to the midline. In adults the degree of subcutaneous adipose tissue may influence the depth of the incision. Pressure is applied with a swab until any bleeding has completely stopped. The Bergström needle with the sliding cannula assembled and the window closed is then inserted into the muscle while the other hand steadies the thigh. The window is opened by sliding the cannula and the muscle gently squeezed so it goes into the window of the needle and ensures a reasonably sized specimen. After a quick to-and-fro movement of the cannula with the palm of the hand, the needle is withdrawn and the muscle sample removed. The to-and-fro movement should not be applied too often to avoid fragmentation of the sample. Sampling is rapid and takes only a few seconds. The sensation is of pressure within the muscle rather than pain. The needle can be reintroduced and multiple samples obtained through the same incision, if necessary, to produce an adequate quantity of muscle for biochemical studies. The quality of the sample, and whether it is adequate, should be assessed immediately under a dissecting microscope, and it is therefore advantageous to have a member of the laboratory staff close at hand, and not rely on samples being assessed sometime later in the laboratory. An average needle biopsy is approximately 3–4 mm in diameter and weighs about 20–50 mg. When muscle respiratory chain analysis or biochemical studies are likely to be required, an extra sample is taken via the same incision to aim for approximately 50 mg muscle. Samples for respiratory chain enzyme analysis should be frozen immediately or within 10–15 minutes.