Interpretation of Muscle biopsy with special reference to Muscular Dystrophy and Myopathies

Dr. Adrija Pathak

The Weil-Blakelsley Cochotome with a 6mm bitting tip

Collection & Preparation of Biopsy Specimen

Clinical informationAppropriate muscle-

representative of ds -ds process is active and

evolvingBefore excision muscle

maintained in isometric state by introducing it into clamp

Normal Muscle

Normal muscle (transverse section). The fibers are typically polygonal, and the sarcolemmal nuclei are located peripherally.

Skeletal mucle is composed of elongated, multinucleate ,unbranched contractile cell described as mucle fibre

Characteristic cross-striations seen on LM d/t arrangement of contractile protein

• Individual muscle fibers are surrounded by endomysium and are grouped in fascicles which are surrounded by a small amount of connective tissue known as perimysium. • Epimysium, in turn, is the connective tissue which surrounds multiple muscle fascicles• In normal muscle, the endomysium is so inconspicuous that individual muscle fibers appear to abut one another.

Normal muscle. In the alkaline adenosine triphosphatase (ATPase) reaction, type 1 fibers are light, and type 2 fibers are dark because of their high content of ATPase for usein the glycolytic pathway.(ATPase, pH 9.4, counterstained with eosin).

‘Reverse’ ATPase ph 4.3 showsthe normal distribution of dark type 1 fibres, pale type 2A fibres and alsointermediate type 2B fibres.

ATPase at ph 9.4 shows a normal ‘checkerboard’ or ‘mosaic’ distribution of fibre types 1 and 2. Type 2 fibres stain darkly.

Frozen section stained for the oxidative enzyme NADH-tetrazolium reductase shows darkly stained type 1 fibres.

High power of NADH-TR stained frozen section shows positive staining of both the sarcoplasmic reticulum and mitochondria, the latter more numerous in type 1 fibres.

Stain for succinic dehydrogenase is paler and has a particulate appearance due to selective staining of mitochondria.

Staining for cytochrome oxidase (COX) shows a similar distribution toSDH staining (more prominent in Type 1 fibres) but in this stain the end product is golden brown.

Frozen section stained for phosphorylase. Type 2 fibres are stained darkly but this reaction is not used routinely to demonstrate fibre type differentiation. Complete absence of staining is typical of McArdle’s disease (Type V Glycogenosis).

A modified PAS stain to demonstrate glycogen. Type 2 fibres which are dependent on intrinsic glycogen stain darkly.

Verhoeff Van-Gieson (VVG) stain of frozen tissue to show fibrous tissue, elastin and myelinated nerve fibres. The fine black dots representmitochondria (hence the darker staining of type 1 fibres) and the intermyofibrillary network.

Oil Red-O in frozen section demonstrates normal distribution of fine lipid droplets within muscle fibres, more prominent in type 1 fibres (arrow).

The modified Gomori trichrome stain identifies mitochondria as small reddots within the muscle fibre, most numerous in type 1 fibres and at the fibre periphery, in the subsarcolemmal zone (arrow). This biopsy containsa normal number of mitochondria in usual distribution.

Working Classification of Muscular Diseases

Neurogenic

Neuromuscular Disorder

Primary Myopathic Changes

Inflammatory

Dystrophy

Congenital

Metabolic

EndocrinopathiesToxic-Drug Induced

DuchenneBeckerFSHDLimb-GirdleOPMDDistal MyopathyMyototic

Central CoreMulticoreNemalineCentronuclearFibre type DisproportionMyofibrillar

PMDMIBMSarcoidosisViral

GlycogenosisLipid StorageMitochondrialMalig HyperpyrexiaMyoglobinuria

Pathological Reactions of Muscle

Nuclear internalization. Many fibers contain one or more internal, often pyknotic nuclei.

Nuclear Internalization

Ring Fibres

Hyaline Fibres

Circumferential orientation of the peripheral myofibrils produces a striated ring that encircles a transversely sectioned fiber in the center of the field (periodic acid-Schiff stain).

The fiber in the center of the photograph is rounded, ellarged and darkly stained, opaque sarcoplasm.

- Myotonic dystrophy- Limb-Girdle dystrophy

-Early stage of necrosis- Duchenne muscular dystrophy

Fiber Necrosis

The necrotic process in the fiber at the centre of this longitudinal section is recognized by a loss of cross striations and early phagocytosis.

Fiber Regeneration

Sarcolemmal nuclei are large and vesicular, and they contain prominent nucleoli. The sarcoplasm is basophilic.

Inclusions

Nuclear inclusion- Oculopharyngeal dystrophy- EM- Inclusion Body Myositis- LM

Oculopharyngeal muscular dystrophy. High-power electron micrograph showing intranuclear inclusion composed of 8.5-nm tubulofilamentous material.

Inclusion body myositis. An intranuclear inclusion is shown at the center of the picture. The inclusion is eosinophilic and smudged; it is located within a sarcolemmal nucleus.

Sarcoplasmic inclusion- Myofibrilllar myopathy

Inflammation

Inflammatory myopathy. Sheets of lymphocytes expand the endomysial spaces and surround the fibers.

Frozen section in inflammatory myopathy illustrating perivascular andendomysial inflammation (black arrow), individual necrotic fibres (yellow arrow) and fibres undergoing phagocytosis (blue arrow).

Multiple discrete granulomas are seen in this case of sarcoidosis.

•Fibrosis and Fatty infiltration

•Atrophy and HypertrophyMost common form of atrophy-denervation

Type 1 fibre hypertrophy- specific for infantile spinal muscular atrophy (ISMA). Also seen in athletes undergoing endurance training

Type 2 fibre hypertrophy- sprinters& congenital fibre type disproportion

Hypertrophy involving both fibres- limb-girdle dytrophy, IBM, myotonia congenita & acromegaly

ATPase ph 9.4 shows diffuse selective atrophy of type 2 fibres. This was a common finding in biopsies from patients attending the Rheumatology clinic.

Type 2 atrophy in a patient with malignancy and cachexia (immunostain for fast myosin).

dermatomyositis

denervation

chronicdenervation with reinnervation

Fibre type predominance is present when Type 1 fibres constitute more than 55% of the total fibre population or when more than 80% of fibres are Type 2.

A predominance of Type 1 fibres is seen in Charcot-Marie Tooth disease and Type 2 fibres are predominant in Motor Neuron Disease.

Fibre type deficiency is confirmed when less than 10% of fibres constitute either group. A deficiency of Type 2 fibres may be seen in limbgirdle dystrophy

Chronic neurogenic atrophy. Grouping of many small angular fibers is evident.

Neurogenic atrophy. Many atrophic fibers are angular (adenosine triphosphatase, pH 9.4).

Infantile spinal muscular atrophy. Most of the fibers in the fascicle are atrophic and rounded.

Fiber Shape

Mottled Fibers

Fiber Splitting

Several hypertrophic fibers are seen. The fiber at the bottom and center is divided into two smaller subunits (frozen section, rapid Gomori trichrome).

The sarcoplasm appears moth eaten as a result of the presence of patchy areas of poor staining due to lack of mitochondria& destruction of myofilament (NADH-TR).

- Limb-Girdle dystrophy- Denervation- IBM

-FSHD-Limb-Girdle-Denervation

Cores & Targets

The focal areas of reduced enzyme activity are single, and they are centrally positioned within many fibers (NADH-TR).

Neurogenic atrophy. In target fibers, an inner, unstained zone is surrounded by a rim of increased enzyme activity (NADH-TR).

Cores- in variety of diseases, most prevalent in neurogenic atrophy

Target- pathognomic for neurogenic atrophy

Nemaline Rods

Collections of dark, rod-shaped structures are evident in many of the fibers (frozen section, rapid Gomori trichrome).

Ultrastructurally, rods are osmiophilic and

elongated or rectangular, resembling Z-bands.-Nemaline myopathy-Muscular dystrophy-PM

Mitochondrial Abnormalities

Ragged red fiber. Collections of mitochondria appear as red-stained, irregular, subsarcolemmal areas within the involved fiber (frozen section, rapid Gomori trichrome).

Ragged red fiber is seen with abnormally large mitochondria, several of which contain paracrystalline inclusions.

Vacuolar changes

Lipid storage myopathy. Numerous osmiophilic, lipid-containing vacuoles are evident in the sarcoplasm of the fiber at the center (resin section, toluidine blue).

A rimmed vacuole contains abundant red, granular material (frozen section, rapid Gomori trichrome).

Artifact & Pitfall

Freezing artifact. Extensive vacuolar change is caused by improper freezing. Many of the vacuoles have linear, noncircular geometric shapes.

Contraction artifact. Darker contraction bands and disrupted lucent zones are seen in several longitudinally oriented fibers (periodic acid-Schiff stain).

Frozen section has partially lifted off the slide. Tissue twists create artifact seen as fiber curling with striped and ring structures in the fibers (ATPase, pH 9.4, counterstained with eosin).

Tendinous insertion. In this location, the muscle fibers normally vary in size, and they are often surrounded by fibrous tissue (Gomori trichrome).

Muscle specimen submitted in saline. Fluid between fibers mimics edema. Several fibers are damaged and disrupted and appear blown out.

During the biopsy procedure, the muscle has been roughly handled, leading to a pseudovasculitis in the perimysium. Neutrophils are marginating in the vessel lumina and beginning to traverse the vessel walls.

Non-specific features- thyroid ds, statin therapyPrior trauma during EMG, i/m inj – necrosis, regeneration, inflamm, endomysial fibrosisNot all muscle look alike. Ex. Paraspinal muscle- internal nuclei, grps of fasicles seperated by abundant conn ts resembling fibrosisCrush artifact- fibres appear atrophicFatty infiltration seen in obese

Neurogenic AtrophyLMN ds- poliomyelitis, amylotrophic lateral

sclerosis,spinal muscular atrophy & peripheral neuropathy

Bx- early denervation- random atrophy of both fibre

mainly type 2- Angulated- Small and later large groups of atrophied fibre- Target fiber- Denervation & renervation loss of

checkerboard pattern- Motor unit territory enlarges newly recruited

fibre converted to single type fibre type grouping

H&E frozen section showing large group atrophy

Small group atrophy seen in H&E stained frozen section

The small angulated fibres stain darkly in NADH-TR reaction.

Reinnervation is evident in fibre type grouping

A group of target fibres in NADH-TR reaction. A clear central zone is surrounded by a densely stained intermediate zone

Chronic denervation with reinnervation. Type grouping replaces the normal checkerboard staining pattern (adenosine triphosphatase, pH 9.4).

Duchenne Muscular DystrophyMost common dystrophyMost severeX-linked recessive- affects boysNeurologically intact at birthFirst sign when child attempts to

walk/standWeakness begins in pelvic girdle muscle

then extent to shoulder girdle sparing face muscle and swallowing

Psedohypertrophy of calves and buttock- fatty infiltration and reactive fibrosis

Elevated serum creatine kinase- first decade of life

Early death d/t cardiomyopathyMultiple exonic deletion DMD gene

on chr Xp21 encoding dystrophinBx- fiber necrosis and

regeneration - hyaline fibersImmunostain for membrane

associated dystrophin- absence of immunostaining diagnostic of disease

Absent staining for Dystrophin in the majority of fibres in a case ofDuchenne dystrophy

Normal immunostaining pattern for dystrophin. The sarcolemmal regions of the fibers are outlined

Becker Muscular DystrophyLess severeRate of progression is slowContains dystrophin but of

abnormal size/structureBx-variation in fibre size - nuclear internalization - necrosis, phagocytosis,

regeneration - endomysial connective ts

proliferation

Facioscapulohumeral DystrophyMild myopathyInvolves face, shoulder & upper

extrimitiesOnset in 2nd-3rd decadeBx- atrophic muscle clustered

together - moth eaten fibers - perivascular lymphocytes - absence of fiber necrosis/

regeneration

Limb-Girdle DystrophyCollection of myopathiesInv of proximal axial musclesOnset in young adult2B- Dysferlinopathy- most

commonBx- nuclear internalization - variability of fiber diameter - fiber splitting

Oculopharyngeal Muscular DystrophyLate onset- middle lifeBenign outcomeHeralded by ptosis

ophathalmopegia & dysphagiaBx-mild dystrophic change

(nuclear internalization, atrophy & interstitial fibrosis)

EM- nuclear inclusion

Myotonic Muscular Dystrophy3rd-4th decadeBegins with weakness of facial

muscle and acral muscle of extremities

C/F- ptosis, expressionless visage, dysphagia

Myotonia –inability of muscle to relax once contracted

A/W- cataracts, testicular atrophy, DM, CMP, mild dementia

AD- incresed CTG trinucleotide repeat of gene at chr 19

Bx- Early stage- pyknotic int nuclei -selective atrophy of type 1 fiber -ring fibre Chronic- fiber destruction, regeneration & fibrosis

A group of ‘ring’ fibres. This abnormality may be a feature in chronic myopathies e.g. myotonic dystrophy

Central Core/ Multicore Disease

Lack of muscular vitality noted in infancy

Mutation in RYR1 gene-ryanodine receptor protein that is a portion Ca release channel of sarcoplasmic reticulum l/t Malignant hyperthermia

Bx- cores type 1 fibre

predominant

Multicore disease. Numerous small globular-shaped cores are seen in the fibers. Cores appear unstained with oxidative enzyme reactions (nicotinamide adenine dinucleotide, reduced).

Nemaline (Rod) MyopathyFemaleFacial dysmorphism-

face elongated, jaw prognathic, high vaulted palate

Histochemical rxn- selective atrophy of oxidative fiber

This is an example of nemaline myopathy seen in a biopsy ofparavertebral muscle taken during spinal surgery for kyphoscoliosis in a young girl.Frozen section H&E shows numerous rods in most fibres with many grouped in peripheral clusters.

Modified trichrome stain highlights rod bodies

Centronuclear Myopathy/ Myotubular Myopathy

Age of onset not uniform: infancy-7th decade

Extaocular palsies & facial asthenia with inv of appendicular muscles

Bx- central/paracentral nucleus in most muscle fibre resembling those indeginious to fetal myotube stage of development

• Nuclei exceed the normal size and have vesicular chromatin• Sarcoplasm surrouding central nucleus is disrupted ultrastructurally and appear clear or vacuolated in frozen section• few if any peripheral nuclei

Congenital Fiber-Type Disproportion

Atrophy of type 1 fibers and hypertrophy of type 2 fibers

Paucity of motor activity & decresed muscle tone at birth

Deterioration throughout 1st decade then cease/reversal

Skeletal deformities-hip dislocation, kyphoscoliosis & joint contracture

Congenital fiber:type disproportion with hypertrophy of some type 2 fibers and atrophy of type 1 fibers (nicotinamide adenine dinucleotide, reduced)

Myofibrillar MyopathyProtein surplus myopathyAccumulation of intermediate

filaments- desmin, actin, myosin, æßcrystalline, myotilin

Sarcoplasmic inclusionsAdult onset, slowly progressiveDistal weakness, dysphagia &

cardiac involvement

Sarcoplasmic inclusion- Myofibrilllar myopathy

Desmin myopathy. Two fibers contain slightly basophilic smudged regions within the sarcoplasm, which represent collections of myofibrillar material (frozen section, rapid Gomori trichrome).

Cytoplasmic body. Circumscribed inclusion with three dense, red central foci surrounded by green filamentous material (paraffin, Gomori trichrome stain).

Hyaline body has distinct margins and a subsarcolemmal location. The finely red granular appearance of the mitochondria in the normal sarcoplasm is absent from the more dense, homogeneous look of the hyaline body (frozen section, rapid Gomori trichrome).

Polymyositis and DermatomyositisCommon myopathies of adultMore prevalent in women, 20-40yrsAbrupt onset, rapidly progressiveRemission & exacerbationsProximal muscle weaknessDM- violaceous rash on eyelid, face

and extensor surface of digitsDM- a/w ca lung, colon, breast

↑ ESR, creatine kinaseAb in serum- anti-Jo-1, anti-PM-1EMG- small, brief and polyphasic motor

activityMHC class1 antigen expressed

sarcolemmal surface when examined by immunoperoxidase

Bx- fiber necrosis & inflammatory rxn - long standing ds- atrophy &

endoperimysial fibrosis - DM- perifascicular atrophy is hallmark -perivascular lymphocytic infiltrate

Dermatomyositis. The fibers at the edge of the fascicle at the top are atrophic.

Endomysial inflammation in H&E paraffin section in a case of polymyositis

Inclusion Body MyositisWithering of acral muscle esp extensor

compartment of armMen, 50-70 yrsDoes not respond to steroidsFrozen section necessary for diagnosisBx- small, angular & grouped fiber - inflammation - fiber hypertrophy & splitting - variable necrosis/regeneration - MHC class 1 expression - rimmed vacuoles, inclusions, ragged red

fiber

Many fibres contain ‘rimmed vacuoles’ in this biopsy from a patient with Inclusion Body Myositis (IBM). Myopathic features such as increased variability in muscle fibre diameters and increased central nuclei are present and neurogenic features may also be identified in such biopsies. Congo Red staining may reveal deposits of beta amyloid within the fibres.

‘Rimmed vacuoles’ contain basophilic granular inclusions and have a basophilic rim. Electron microscopy will show membranous whorls (‘myeloid bodies’) within the vacuoles.

Carbohydrate Storage Ds1 ACID MALTASE DEFICIENCY Infants Prog weakness, hypotonia, macroglossia, cardiomyopathy,

organomegaly PAS positive, diastase labile vacuoles of varying sizes EM: membrane bound glycogen filled vacuoles Biochemical analyses of tissue is necessary for diagnosis2 MCARDLE’S DISEASE AR, in childhood/adolscence Muscle weakness, pain & stiffness exacerbated by excercise Many crescentric PAS positive vacuoles in sub sarcolemmal position. Histochemical reactions showing absence of phosphorylase activity.3 PFK DEFICIENCY AR, in childhood Muscular pain & stiffness induced by exercise. Hemolytic anemia in few pts In frozen, PAS positive crescents adj to sarcolemma PFK can be demons histochemically unreliable. Confirmation by biochemical analysis.

Lipid Storage Ds1 CARNITINE DEFICIENCY Infancy to middle age. Systemic / skeletal ms. Ac encephalopathy, heart failure, Diffuse vacuolization of ms fibres. Fat stains/ EM: dysmorphic, enlarged

mitochondria with paracrystalline inclusions

2 CARNITINE PALMITOYLTRANSFERASE DEFICIENCY

Weakness, myalgias ppt by exercise or fasting Lipid vacuoles may be normal or increased Detected by biochemical reactions

Mitochondrial MyopathyPrimary or secondary to lipid

storage ds/ hypothyroidsm1 Kearns-Sayre syndrome- ptosis, ext

ophthalmoplegia, pigmentary retinal degeneration, heart block, cerebellar ataxia & short stature

2 Myopathy, encephalopathy, lactic acidosis, strokes syndrome (MELAS)

3 Myoclonus Epilepsy with ragged red fiber syndrome (MERRF)

Increased staining of mitochondria may be evident in H&E frozen section in mitochondrial myopathy. Note basophilic stippling in several fibres, particularly in sub-sarcolemmal zones.

Prominent subsarcolemmal clumping of abnormal mitochondria.

increased red staining in subsarcolemmal zonesdue to aggregates of abnormal mitochondria

Increased mitochondrial staining associated with vacuolation at periphery of muscle fibre.

References Sternberg’s diagnostic surgical

pathology, 5th editionDubowitz V, Sewry CA, Muscle

Biopsy: a Practical approach, 3rd edition, Saunders, 2006

Robbins & Cotran pathological basis of disease, 8th edn

Theory and practice of Histological techniques: John D Bancroft, 6th edn

Wheater’s funtional histology, 5th edn