How to Read a Biopsy





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.



TABLE 7.1

H&E, Gomori Trichrome



















































































































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:






    • 3% of the fibres


Mild non-specific abnormality in adults, may be significant in children






    • 10% of the fibres


Suggests myopathy but is seen occasionally in chronic neuropathy






    • 30% of the fibres


Suggests a chronic dystrophy






    • 60% of the fibres


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


TABLE 7.2

NADH-TR (Reduced Nicotinamide Adenine Dinucleotide-Tetrazolium Reductase) Reaction








































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


TABLE 7.3

ATPase (Adenosine Triphosphatase) Reaction, pH 9.4































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


TABLE 7.4

Other Stains









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.


Feb 23, 2021 | Posted by in MUSCULOSKELETAL MEDICINE | Comments Off on How to Read a Biopsy
Premium Wordpress Themes by UFO Themes