The Procedure of Muscle Biopsy

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 ).