When the pathologist is asked to evaluate a muscle biopsy, much of the interpretation is based on his or her previous experience and the recognition of similarities between the biopsy and muscle from known diseases. In some instances, such as an advanced dystrophy or a spinal muscular atrophy, the changes may be striking and unequivocal. In others, the changes may be more subtle and a systematic approach is required in the evaluation and interpretation. Once the pathology is defined, correlation with clinical features is essential and the pathology must be interpreted in the light of this. A muscle biopsy is only one piece of the jigsaw and has to be considered together with the family history, the clinical history and presentation and the results of any other investigations. Knowledge of serum creatine kinase (CK) levels, for example, is a useful indicator; no case of Duchenne muscular dystrophy has a normal CK, whereas levels are usually normal in congenital myopathies and neurogenic disorders. Knowledge of the distribution of muscle weakness, cardiac involvement, respiratory difficulties, abnormalities in brain and muscle magnetic resonance imaging (MRI), contractures and joint laxity are also valuable. Clinical spectra are widening with molecular advances and the increasing application of whole genome and exome sequencing, which has led to a wider appreciation of pathological features seen in muscle biopsies. There is frequently a lack of concordance between the severity of the pathological change and the degree of clinical disability, which places a constraint on the pathologist in giving a prognostication for a particular disease process. It is safer to define the type of pathological change and leave the assessment of clinical severity and prognosis to the clinician. Relating the pathology to the clinical picture is paramount to diagnosis. For example, an overtly dystrophic picture in a 6-month-old infant with hypotonia from birth may suggest the diagnosis of congenital muscular dystrophy, whereas a similar picture in a 14-year-old boy who was ambulant until the age of 10 would be characteristic of Duchenne muscular dystrophy. Similarly, an identical pathological picture is seen in a severely affected infant with Werdnig–Hoffmann disease (spinal muscular atrophy; SMA I) as in a milder case with later onset and better motor function and prognosis (SMA II).
There are considerable advantages if the clinician assessing the patient can also view and discuss the biopsy with the pathologist, and then provide a comprehensive diagnosis of the patient. This has always been our policy and gives continuity between the clinical diagnosis and other investigations. Regular multidisciplinary meetings between the clinicians, pathologists and other personnel involved in the diagnosis and care of a patient therefore have an important role.
During the preparation of the first edition of this book, a chapter was added, almost as an afterthought, which reflected many discussions on various aspects of interpretation of muscle pathology. It represented a synthesis of the approach to the interpretation of changes in a biopsy which we thought might be helpful, particularly to the novice in muscle pathology.
While retaining the same basic format as before, we have revised and extended the content of this chapter, updated each section, including the section on clinical features that may alert the pathologist to consider a possible diagnosis. Our aim is to summarize the main pathological features and to give a synopsis of their relevance. Subsequent chapters will give more detail on individual disorders. Part I attempts to place the biopsy into a normal or abnormal category and, if abnormal, to try and characterize the abnormality as myopathic/dystrophic or neurogenic, or some other pathological change. Part II attempts to provide a more specific diagnosis, based on comparison with the classical changes found in individual disorders. The sequence of features is expressed in the form of a flow sheet and is based primarily on the routine histological stains, haematoxylin and eosin (H&E), and Gomori trichrome, and the histochemical reactions for oxidative enzymes and adenosine triphosphatase (ATPase) and the additional techniques mentioned in Chapter 2 . Parts III and IV provide a similar approach to immunohistochemistry and electron microscopy, respectively. Part V lists some useful clinical features that are relevant to differential diagnosis and can alert the pathologist towards a possible diagnosis. It is not intended as a comprehensive or exclusive list of clinical features associated with particular disorders and should be read in conjunction with the chapters in Section B.
Part I
Histological Stains
With careful overall inspection of the stained sections it should be possible to answer the following:
Is the biopsy normal or abnormal?
Pay particular attention to:
Overall pattern of the muscle bundles
Shape and size of fibres
Position of nuclei
peripheral, number that are internal, central or multiple
Presence of focal changes in individual fibres
necrosis/overstained hypercontracted fibres
Distribution of connective and adipose tissue
Presence/absence of inflammatory cells
Is the abnormality gross and obvious, or minimal?
Is the abnormality diffuse or focal?
Histochemical Reactions
The following additional information should be obtained from the histochemical preparations.
Reduced Nicotinamide Adenine Dinucleotide-Tetrazolium Reductase (NADH-TR)
Differentiation into fibre types
Selective fibre type involvement
Architectural changes within the fibres:
intermyofibrillar network pattern
cores (areas devoid of stain)
moth-eaten fibres
coiled or whorled fibres
ring fibres
Aggregation of stain peripherally or internally
Excessive granularity or intensity of reaction product
Cytochrome c oxidase (COX); In addition to above
Absence or excess reaction product in some fibres
Size and distribution of mitochondria
Succinate Dehydrogenase (SDH)
Presence of reaction product in fibres devoid of COX
Excess reaction product
ATPase
Fibre typing
Size, shape, proportion and distribution of respective fibre types
Selective fibre type involvement
Periodic Acid–Schiff (PAS)
Distribution and amount of glycogen
Presence of ring or coil fibres
Absence of stain in some fibres (‘white’ fibres)
Presence of polyglucosan after digestion with α-amylase
Oil Red O (ORO) or Sudan Black
Distribution of lipid droplets
Excess lipid within fibres
Size of lipid droplets
Excess lipid outside the fibres
Artefact due to surface spreading of stain
Enzymatic Activity (of all Fibres)
Deficiency of:
Cytochrome c oxidase
Succinate dehydrogenase
Phosphorylase
Phosphofructokinase
Adenylate deaminase
Myosin ATPase
Enzyme activity:
Acid phosphatase associated with vacuoles
Acid phosphatase associated with macrophages
Acid phosphatase within fibres
Alkaline phosphatase in perimysium
Borderline Changes
One common difficulty is assessing the significance of pathological changes in borderline cases. Thus, one may attach different significance to the presence of one or two internal nuclei in occasional fibres, one or two internal nuclei in many fibres, many internal nuclei in occasional fibres and many internal nuclei in many fibres.
Tables 7.1–7.4 provide a systematic interpretation of individual changes in the course of analyzing a muscle biopsy.
| Feature | Implication |
|---|---|
| Fibre Size | |
| Changes in fibre size in relation to age | Biopsy is abnormal |
| Changes random and diffuse | Myopathy/dystrophy |
| Two distinct populations of small and large fibres | Characteristic of denervation or of type-specific atrophy or a congenital myopathy |
| Presence of small angulated fibres | Suggests denervation |
| Presence of small-group atrophy | Denervation |
| Presence of large-group atrophy | Denervation |
| Presence of perifascicular atrophy | Dermatomyositis |
| Presence of perifascicular necrosis | Suggests anti-synthetase syndrome |
| Nuclei | |
| Internal nuclei in more than: | |
| Mild non-specific abnormality in adults, may be significant in children |
| Suggests myopathy but is seen occasionally in chronic neuropathy |
| Suggests a chronic dystrophy |
| Think of myotonic dystrophies or myofibrillar myopathies |
| Centrally placed | Think of centronuclear myopathies, RYR1 -related core myopathy or congenital myotonic dystrophy |
| Nuclear clumps | Indicates fibre atrophy; common in denervation but also consider myotonic dystrophies and other chronic dystrophies |
| Fibres | |
| Presence of necrotic fibres | More common in dystrophies, some myopathies and inflammatory myopathies, rare in congenital myopathies, occasionally seen in denervation such as SMA III |
| Clusters of necrotic fibres | Dystrophies |
| Isolated necrotic fibres | Metabolic myopathy; disorders associated with rhabdomyolysis; immune-mediated necrotizing myopathy |
| Fibre splitting | Common in dystrophies, particularly limb-girdle forms |
| Basophilic fibres | If present to any degree, suggests a myopathy If in small groups, think of a dystrophy or MICU1- related myopathy |
| Phagocytosis | Feature of necrotic fibres, e.g. in a dystrophy, but may be seen in chronic neuropathies |
| Cellular reactions | |
| Presence of lymphocyte infiltration | Suggests a myopathy; think of inflammatory myopathies, facioscapulohumeral dystrophy or dysferlinopathy. Can occur in various other muscular dystrophies. (Distinguish normal neurovascular bundle and regeneration from inflammatory response) |
| Fibrotic Connective Tissue | |
| Confined to perimysium | Abnormal if abundant in an adult; often wide in neonates |
| Increased in endomysium | Abnormal |
| Mild | Non-specific but more common in myopathies; can occur in SMA III |
| Moderate | Suggests a dystrophy; can also occur in some congenital myopathies |
| Severe | Think of a dystrophy |
| Other Structures | |
| Are the vessels abnormal in size and number or have thickened basal lamina? | Number of capillaries low in neonatal muscle; vessel walls may be thickened in some inflammatory myopathies (e.g. ’pipestem’ capillaries) |
| Are the nerves abnormal? | Lack of Gomori trichrome staining may indicate a myelination problem; may be fibrotic; loss and size of axons assessed better in resin sections of sural nerve |
| Are muscle spindles abnormal? | Requires specialist knowledge |
| Presence of rods | Suggests nemaline myopathies; may also occur in core myopathies; presence at myotendinous junction is normal |
| Presence of vacuoles | Excessive in some glycogenoses; may have associated basophilia or red material with the Gomori trichrome stain in inclusion body myositis and various myopathies such as myofibrillar myopathies and distal myopathies; lined by sarcolemmal proteins in some vacuolar myopathies; invaginations of sarcolemma may appear as vacuoles. Congo red may be helpful |
| Feature | Implication |
|---|---|
| Presence of target fibres | Suggests reinnervation and thus, indirectly, denervation |
| Presence of targetoid fibres | A less-specific abnormality seen in both myopathies and denervation |
| Presence of central or peripheral cores | When numerous and large, suggests RYR1- related core myopathy; isolated cores may be difficult to distinguish from targetoid areas |
| Presence of minicores | Consider core myopathies; may occur in several clinical syndromes; assess longitudinal fibres if present |
| Large ‘wiped out’ areas devoid of stain | Think of myofibrillar myopathies |
| Uneven distribution of stain | May relate to uneven distribution of mitochondria and/or myofibrillar disruption; compare with staining for COX |
| Presence of moth-eaten and whorled fibres | Non-specific abnormality if a minor change; when numerous suggests a myopathy; when profuse consider a chronic dystrophy |
| Lobulated fibres | Non-specific but more common in limb-girdle 2A (calpainopathy); rare in children |
| Presence of dark-centred fibres | Indicates clusters of mitochondria; seen in centronuclear myopathies and various other disorders |
| Presence of tubular aggregates | Seen in a variety of disorders but when profuse consider ion channel disorders or myasthenic syndromes |
| Presence of small very dark angulated fibres | Suggests denervation but may occur in several myopathies; may contain fetal myosin |
| Feature | Implication |
|---|---|
| Fibre type pattern indistinct | Frequent in Duchenne dystrophy; also seen in RYR1- related core myopathy |
| Fibre type predominance | Type 1 fibre predominance common in most myopathies |
| Atrophy | Both types small suggests denervation |
| Type 1 atrophy/hypoplasia | Seen in myotonic dystrophy (DM1); common in congenital myopathies; may be smaller in laminopathies |
| Type 2 atrophy | Non-specific and may reflect disuse of the muscle; may be induced by corticosteroid therapy (often 2B atrophy); seen in some congenital myopathies; 2A atrophy common in DM2 and when MYH2A gene mutated |
| Largest fibres of one type | In motor neurone disease (amyotrophic lateral sclerosis) largest fibres are usually type 2; in chronic neuropathy type 1; in spinal muscular atrophy type 1 |
| Fibre type grouping | Pathognomonic of reinnervation and thus denervation. Must be distinguished from fibre type predominance |
| Ring fibres present in any of the stains (including periodic acid–Schiff) | When numerous, suggests a chronic myopathy such as limb-girdle dystrophy or myotonic dystrophy |
| Acid phosphatase present in vacuoles or visible as discrete focal areas present throughout a fibre | Acid maltase deficiency; marked in some vacuolar myopathies; focal perinuclear areas often relate to lipofuscin (normal) Necrotic fibres and macrophages within them Basophilic regenerating fibres |
| Absence of phosphorylase | Type V glycogenosis (McArdle disease); can also occur if glycogen synthesis is absent ( GYS1 ); fibres without glycogen negative |
Part II
In completing Part I, some idea will have been obtained as to whether an abnormal biopsy is due to a myopathic process or secondary to denervation. A few biopsies will not fit clearly into either category. Part II is concerned with placing the biopsy into a more specific diagnostic category. The biopsies will be considered under the headings: neurogenic, myopathic and others, and those additional changes that seem characteristic of a particular disease will be listed.
1 Neurogenic Biopsies
Spinal Muscular Atrophy
Severe infantile spinal muscular atrophy
(Werdnig–Hoffmann disease/SMA I)
Intermediate severity spinal muscular atrophy
(late infantile/juvenile/SMA II)
large-group atrophy
rounded atrophic fibres of both types
hypertrophied fibres mostly type 1 (ATPase)
Mild spinal muscular atrophy (Kugelberg–Welander disease/SMA III)
hypertrophic fibres often type 2
fibre type grouping
structural changes and internal nuclei in large fibres
mild ‘myopathic’ changes, including fibrosis
Motor Neurone Disease and Peripheral Neuropathies
Fibre hypertrophy (often type 2 in motor neurone disease)
Fibre type predominance (often type 2 in motor neurone disease)
Fibre type grouping
Small angulated fibres (dark with NADH-TR)
Target fibres
Nuclear clumps/bags
‘Myopathic’ changes, e.g. fibrosis, internal nuclei
2 Myopathic Biopsies
Duchenne Dystrophy
Wide variation in fibre size
Most fibres rounded
Necrotic fibres
Phagocytosis
Endomysial fibrosis
Basophilic fibres, often in clusters
Hypercontracted fibres
Internal nuclei increased but not a marked feature
Mild aggregation or loss of oxidative enzyme stains
Fibre typing indistinct
Split fibres
Whorled fibres
Becker Dystrophy
Increased variability in fibre size
Split fibres
Whorled fibres
Hypercontracted fibres
Endomysial fibrosis
Groups of small fibres
Necrosis and phagocytosis sometimes less than Duchenne
Basophilic fibres
Limb-Girdle Dystrophy
Good fibre type differentiation
Extensive fibre splitting
Excessive internal nuclei
Variable amount of fibrosis
Sometimes less basophilia and phagocytosis than Duchenne
Moth-eaten fibres common
Ring and whorled fibres
Lobulated fibres in adult cases
Facioscapulohumeral Dystrophy
Large fibres often type 2
Groups of small fibres
Endomysial fibrosis variable
Inflammatory response common
Small angulated fibres (may be the only abnormality)
Moth-eaten and whorled fibres
Changes often focal and inconspicuous
Occasionally extensive pathological change
Congenital Muscular Dystrophy
Forms with severe disability:
Variation in fibre size
Extensive endomysial fibrosis
Extensive adipose tissue
Necrosis, but may not be apparent
Type 1 predominance
Forms with mild disability:
Variation in fibre size, may be mild
Endomysial fibrosis and adipose tissue, may be mild
Necrosis may be present
Immunohistochemistry essential (see below)
Congenital Myopathies, e.g. Core Myopathies, Nemaline Myopathy, Centronuclear Myopathies, Cap Disease, Myosin Storage Myopathy
Distinctive pathological change implied in the name of the disease, e.g. nemaline rods
Selective type 1 fibre involvement
Type 1 fibre atrophy and/or fibre type disproportion
Type 1 fibre predominance or uniformity
Internal nuclei, may be central, e.g. in RYR1 core myopathy
Fibrosis may be present
Extensive adipose tissue may be present
Centronuclear Myopathies
X-linked myotubular myopathy:
Prominent single central nuclei in transverse section (may only be apparent in 10–20% of fibres owing to spacing of nuclei); dark central aggregation of stain and a pale peripheral halo with NADH-TR central ‘hole’ with ATPase; exclude congenital myotonic dystrophy;
‘Necklace’ fibres – loop of oxidative enzyme activity with associated nuclei
Autosomal centronuclear myopathies:
Radial strands with NADH-TR and PAS ( DNM2 )
‘Necklace’-like fibres without associated nuclei
Myotonic Dystrophies
Type 1 fibre atrophy (DM1); type 2 atrophy (DM2)
Excessive internal nuclei, central in congenital cases
Nuclear clumps (late in DM1, early in DM2)
Ring fibres
Sarcoplasmic masses
Moth-eaten fibres
Oculopharyngeal Dystrophy
Many rimmed vacuoles
Excessive internal nuclei
Inflammatory Myopathies
Hypertrophied fibres rare (except in IBM)
Perifascicular atrophy in dermatomyositis
Necrosis (perifascicular in anti-synthetase syndrome)
Phagocytosis
Variable degree of fibrosis
Moth-eaten whorled fibres
‘Ghost fibres’
Inflammatory cells (not universal)
Reduced number of capillaries (dermatomyositis)
Rimmed vacuoles in inclusion body myositis
Alkaline phosphatase in perimysium
Fibres devoid of COX in inclusion body myositis (more than related to age)
Immunohistochemistry essential (see below)
3 Other Biopsies
Myasthenic Disorders
Type 2 fibre atrophy, or type 1 atrophy/hypotrophy
Sometimes marked fibre hypertrophy
Sometimes small core-like areas
Tubular aggregates, particularly in cases with GFPT1 and DPAGT1 mutations
Aggregates of lymphocytes (myasthenia gravis)
Periodic Paralyses and Ion Channel Disorders
Tubular aggregates (particularly in cases with STIM1, ORAI1 or CASQ1 mutations)
Vacuoles
Mitochondrial Myopathies
‘Ragged-red’ or granular fibres
Fibres devoid of COX or SDH activity
Fibres with enhanced SDH or COX activity
Reduced COX staining in many fibres
Often minimal pathology, especially in children
Glycogenoses
Absence of phosphorylase (McArdle disease or GYS1 mutation only)
Absence of phosphofructokinase
Excess glycogen
Accumulation of polyglucosan material
Lipid Storage Myopathies
Excess intracellular lipid (often not seen in CPTII deficiency)
Part III
Immunohistochemistry
Assessment of a biopsy using immunohistochemical techniques is now essential. Several gene defects result in a change in the respective protein, particularly in the recessive muscular dystrophies. Secondary alterations can also be useful indicators. Many antibodies have been applied to muscle biopsies but Table 7.5 summarizes the localization in normal muscle of proteins relevant to diagnosis and the significance of their immunolabelling in neuromuscular disorders using a panel of commercially available antibodies.
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