Muscle biopsy

Dr Neha Mahajan

Normal Muscle

Organization of Skeletal Muscle Including Connective Tissue (CT) Compartments

EPIMYSIUM•Loose CT

•Blood vessels

PERIMYSIUM

•Septa

•Nerve branches

•Muscle spindles

•Fat

•Blood vessels

ENDOMYSIUM

•Muscle fibers

•Capillaries

•Small nerve fibers

Perimysial

connective tissue

Endomysial

connective tissue

Normal H&E-stained frozen cross-section of skeletal muscle

Note uniform sizes, polygonal shapes, and eccentric nuclei.

Normal muscle Adult Normal Muscle child

Ultrastructure of a Sarcomere

Extends from Z-band to Z-band.

Note arrangement of thick and thin filaments.

Z ZM

H band

Actin

MyosinI bandI band

A band

A band includes overlap of actin & myosin.

Indications of Muscle biopsy

General reasons:Weakness of uncertain cause-generalised, proximal ,floppy infant syndromeMuscle pain ,cramps, stiffnessPersistently elevated muscle enzymes(CK)Specific reasons:Hereditary muscle disease in other family membersCarrier detectionSystemic connective tissue disease & vasculitisCertain metabolic diseases such as storage diseaseSuspicion of steroid myopathy in treated myositisExclude drug induced myopathyConflicting clinical ,EMG or lab findingsConfirm/reinforce clinical diagnosis

Contraindications

1.Electrolyte disturbance2.Most endocrine 3.Malignant hyperthermia4.Periodic paralysis5.Poor nutrition6.Prior Trauma

Site of biopsy

• Muscle with moderate disease, NOT severe

• Muscle belly, not from tendon

• Proximal myopathies/generalised systemic disease- Vastus Lateralis

• Other sites-Biceps,gatronemius

• Avoid Deltoid,muscle that are site of EMG,injections/trauma

• Imaging used to select pathological muscle site in difficult cases.

Bergston NeedleTechnique

1.Needle Biopsy

2.Open Biopsy- indicated for disorders with patchy pathology

Processing

Transportation:Muscle may be saved in saline moistened guage for several hrsKeep the specimen coolDo NOT immerse in saline ,fixative or other liquid

Fresh Fixed

Glutaraldehyde

RESIN section/EM( 1mm x 0.5 cm)

Formalin

PARAFFIN(0.5x0.5cm)

Snap freeze

HISTOCHEMISTRY(0.5x0.5cm)

Sample size: 0.5 cm diameter & 1 1cm length

Biopsy is processed:

1.Paraffin embedding2.Histochemistry3.For electron microscopy4.For molecular biology

Stains1. H&E- gen architecture & morphology2.Masson`s trichome-Collagen,fibrosis3.Mod Gomori`s Trichome- red ragged fibres,nemaline

bodies,nuclei,myelinated fibres4.PAS-glycogen5.Oil red O-neutral lipid6.Acid phosphatase-lysosomal enzymes,necrotic fibres7.Crystal voilet, Congo Red-amyloid8.ATPase PH 9.4 Type 1 fibres pale

Type 2 fibres dark9. ATPase PH 4.6 Type 1 fibres dark

Type 2 fibres pale10. NADH- Sarcoplasmic structural details11.SDH- oxidative enzyme activity12.Cytochrome C Oxidase- mitochondrial enzyme activity13.NSE-lysosomal &macrophage activity

Type I fibers are light

Type II fibers are dark

Normal ATPase pH 9.4

Rapid gomori`s trichomestainRed ragged fibers,nemalinebodies,myelinated fibers

Nuclei :red purpleNormal muscle myofibrils:BlueGreenIntermyofibril muscle membrane: RedInterstitial collagen: Green

NADH-TR stainSarcoplasmic structural

details

SDH stainFor mitochondrial activity

Cytochrome c oxidase•Mitochondrial activity•Type 1 fibers are darkerthan Type2

OIL RED OAccumulation of lipid

Esterase stainLysosomal & phagocytic activity

Macrophages are ingesting the remnants of a degenerating fiber. This is a non-specific myopathic finding.

Neuromuscular junction

Observations in routine paraffin sections

H & EUsed to evaluate gen architecture of muscle and variation in morphology of individual fibres

•Variation in fascicular architecture•Variation in fiber size•Necrosis and degeneration of muscle fibres•Nuclear characteristics•Type & distribution of inflammatory infiltrate•Interstitial changes

Architecture of muscle fascicles -scanner view

PATCHY Inflammatory myopathies

FOCAL Neurogenic DIFFUSE Dystrophy

Nuclear changes

Normal Internalisation of nuclei

Internalisation of nuclei

• Myotendinous insertion

• Centronuclear myopathy

• Myotonic dystrophy

• Fiber regeneration

• Fiber atrophy

• Chronic neuropathies

Ring fibres

•Limb girdle dystrophy•Myotonic dystrophy

Hyaline Fibres

Duchene muscular dystrophy

Whorled fibres

•Limb girdle muscular dystrophy•Chronic neuropathies

Pathologic features Disease

Small groups of necrotic fibres

DMD

Perifascicular necrosis Dermatomyositis

Random fibre necrosis PM,IBM

Infarcts with large areas of necrosis

PAN

Extensive,diffuse Rhabdomyolysis

Fibre necrosis seen in biopsy specimen

Fiber necrosis Perifascicular necrosis

INCLUSIONS

Nuclear inclusions:Oculopharyngeal dystrophy

Sarcoplasmic Inclusions:Myofibrillar myopathyInclusion body myositis

Inflammation

Pathologic feature Disease

Perivascular, angiocentric DM, connective tissue disease ,FSHD

Endomysial, aroundfibres

PM, IBM,viral

Nodular Rheumatoid arthritis, granuloma

Polymorphs with eosinophils

PAN, drug reactions, trichinosis, eosinophilicfascitis

Inflammatory myopathy

Atrophy

Type 1 fiber atophy Type 2 fiber atophy

•Myotonic dystrophy•Nemaline myopathy•Centronuclear myopathy•Congenital fibre type disproportion

Corticosteroid therapyMyasthenia GravisDisuse AtrophyAcute denervationParaneoplastic myopathy

Pattern of atrophy:Grouped atrophy Chronic neurogenic disordersPanfascicular ISMA(Infantile spinal muscular atrophy)Perifascicular DM(Dermatomyositis)

Normal Atrophy

Hypertrophy

Type 1 fiber hypertrophy

Type 2 fiber hypertrophy

Type 1&2 fiber hypertrophy

ISMANormal Atheletes

SprintersCongenital type disproportion

Limb girdle dystrophy,IBM,myotonia congenita,acromegaly

Normal Hypertrophy

Fiber shape

Normal muscle-polygonalAngular-neurogenic Rounded-myopathic

Fibre splittingLimb girdle dystrophyIBM

Cores and targets

Oxidative enzymes are ideal to assess depleted enzyme activityCores : Neurogenic atrophy, central core diseaseTarget fibres: Chronic neuropathies

CORE TARGET

Central cores Target fibres

Mottled fibresFSHD

Nemaline rodsDetected by RTC(Rapid gomori trichome)Seen in Nemaline myopathy

Mitochondrial Abnormalities

Ragged red fibres(RTC stain)

Sarcoplasmic vacuoles seen in biopsy specimen

Pathologic feature Disease

In centre arranged in size gradient

Freezing artifact

In scattered fibres ,small round osmiophilic ,oil red O positive

Lipid storage disorders,Mitochondrial myopathies

Often subsarcolemmalPAS +

Glycogen storage

Rimmed, ubiquitin + IBM ,Distal myopathy, OPMD

Freezing artifact Rimmed Vacuole

Vacuoles in glycogen storage disease

Parasite ( Trichenella Spiralis)

Granulomatous Myositisin a Patient with Sarciodosis

Granulomas tend not to cause significant

damage to adjacent myofibers.

Giant cell

Pyomyositis Gram Positive Cocci

History & examination

History:• Four major pieces of clinical information are critical for the pathologist:• Is the condition a long standing or newly onset condition?• Is the condition progressive or relatively static?• Are there any associated systemic conditions such as heart problem or

autoimmune disease?• What is the serum CPK level?• Other helpful clinical information:• History of myoglobinemia. • Family history of neuromuscular disease.• Sex and age.• Results of EMG studies.• Muscle groups being affected.• Presence of contracture.• Muscle groups being involved.• Site of biopsy.• Therapy and medication at the time of biopsy.

LAB Investigations:

Creatine kinase level: To rule out certain categories of myopathies because

different myopathies tend to generate a different levels of elevation in CPK.

•High: (e.g. Dystrophinopathies) 200-300 times of normal.

•Intermediate: (e.g. Inflammatory myopathy) 20-30 times of normal.

•Low: (e.g. Neurogenic disorder) 2-5 times of normal.

INHERITEDMuscular dystrophies DMD,BMDFSHDLGDDistal myopathyOPMDMyotonic muscular dystrophyCongenital myopathyCentral core diseaseMulticore diseaseCentronuclearCong fibre type disproportionMyofibtillar myopathyMetabolic diseasesGlycogen storage diseasesMitochondrial diseasesMyoadenylate deficiencyChannelopathies

MyopathiesACQUIREDInflammatory-PM,DM,IBM,granulomatousInfectionsTrichinosis, cyticercosis,toxoplasma,HIV,Coxackie A&BToxicAlcohol, Vit E, OPP, snake venomsDrugsSteroids,statins, b blocker, zidovudin, amiodarone,Chloroquine, chlofibrate,vincristine,cyclosporine,opiatesEndocrine & metabolicHypo/HyperthyroidismAcromegalyCushing`s syndromeConn`s diseaseOsteomalaciaHypercalcemiaHypokalemiaNEOPLASTICBenign-Rhabdomyoma Malignant-RMS

Muscular dystrophies

•Usually not congenital•Onset in childhood ,young adults•Hereditary diseases ,often with a family history•Weakness frequently severe ,variable distribution•Proximal in DMD,BMD,LGD•Facial in fascioscapulohumeral dystrophy•Distal in distal myopathy MD•Progressive course•Fiber destruction pathological, damage is random ;not all fibers are affected

Duchenne muscular dystrophy• Inheritance- X- linked recessive disorder

• Defective gene- Dystrophin

• Onset- usually b/n 3-5yrs age

• C/F – progressive weakness of the girdle muscles

- difficulty running , jumping, hopping, unable to get up from the floor (Gower’s maneuver)

-enlargement of muscles of lower leg a/w weakness-Pseudohypertrophy

- contractures( hip, knee, elbow, wrist) with chest deformities →severe pulmonary infections → death at age 16-18yrs

Others : cardiomyopathy , mental retardationLab. – Serum CK : elevated 20-100x normal

- EMG : myopathic features

- Muscle biopsy- Fiber necrosis & regenerationVariation in fiber size,internalisation of nuclei

proliferation of endomysial connective tissue.Deficiency of dystrophin seen on western blot analysis & immunohistochemical staining.

- DNA analysis : mutation of gene that encodes dystrophin

Becker muscular dystrophy• Inheritance – X- linked recessive disorder

• Defective gene – dystrophin

• Onset- experience difficulty b/n 5- 15yrs of age

• C/F – proximal muscles especially of lower extremities are prominently involved.

- hypertrophy of muscles , particularly the calves, is an early & prominent finding.

- cardiomyopathy may occur , MR is less common

• Lab. – CK : elevated

- EMG : myopathic

- muscle biopsy : similar to DMD

: reduced amount or abnormality of dystrophin( Dx)

- DNA analysis : deletions or duplications( Dx)

• Treatment – supportive

• Survival : survive in to the 4th to 5th decade

Duchene muscular dystrophyWestern Blot

DMDEnd stages-extensive replacementby adipose tissue andfibrosis

NORMAL DMD

Myotonic dystrophy

• Inheritance : AD

• Defective gene: two types with distinct molecular genetic defects

-DM1 : expansion CTG repeat

- DM2 ( proximal myotonic myopathy – PROMM ): CCTG repeat

• C/F – myotonia : usually appears by age 5 yrs

- Hatchet- faced appearance: temporalis , masseter , facial muscle atrophy & weakness

- frontal baldness in men

- foot drop : ankle dorsiflexor weakness

- weakness of wrist extensors , finger extensors, & intrinsic hand muscles

- early involvement of neck muscle flexors, sternocleidomastoids

- dysarthritic speech, nasal voice, swallowing problems due to palatal , pharyngeal, and tongue involvement

- respiratory insufficiency : diaphragm & intercostal muscle involvement

- cardiac disturbances : conduction block with sudden death

: CHF from cor pulmonale 2ry to respiratory failure

Cont,d

• - other system manifestations : intellectual impairment, hypersomnia, cataract, gonadal atrophy, insulin resistance, reduced esophageal & colonic motility

• Lab. – Dx ; usually based on clinical findings

- CK : N or mildly elevated

- EMG : evidence of myotonia

Biopsy : variation in fiber size,

increase in internal nuclei(chains)

ring fibers, atrophy which

selectively involves type – 1 fibers in 50%

Myotonic dystrophy

Oculopharyngeal dystrophy

• Inheritance: AD with complete penetrance

• Defective gene: expansion, poly-A-RNA binding protein

• Onset – usually late onset ( 4th – 5th decade )

• C/F – progressive external ophthalmoplegia ( slowly progressive ptosis, limitation of eye movements with sparing of pupillary rxns.

- dysphagia : can be life threatening

: may result in repeated episodes of aspiration

- mild weakness of the neck and extremities

• Lab. – EMG: myopathic features

- CK : 2-3x N

- BIOPSY : distinct features – presence of tubular filaments in muscle

cell nuclei, mild dystrophic changes with

nuclear internalisation ,fiber atrophy,

interstital fibrosis, rimmed vacuoles in type 1 fibres

Fascioscapulohumeral /FSH/ muscular dystrophy

• Inheritance: AD

• Onset : childhood or young adulthood

• Defective gene: deletion, distal 4q

• C/F- facial weakness: initial manifestation

- weakness of shoulder girdle muscles : weak arm elevation

: scapular winging

- weak wrist extension > wrist flexion

- foot drop : weakness of anterior compartment muscles of the legs

- weakness of the pelvic girdle muscles : 20%

- other organ ( rarely) : labile HTN, nerve deafness

• Lab. – CK : N or elevated

- EMG: myopathic pattern

- BIOPSY: Atrophic muscle fibres in clusters/groups in absence of necrosis,

Moth eaten fibres and perivascular inflammatory infiltrate

Distal myopathies• Notable for their preferential distal distribution of muscle weakness in contrast to

most muscle conditions associated with proximal weakness

• Four types : mode of inheritance, age of onset, pattern of weakness

1. Welander DM : AD

2. Tibial MD : AD

- late onset, usually after age 40; start in the hands

3. Nonanka DM: AR

4. Miyoshi myopathy : AR

- early onset in late teens or twenties; start in the lower limbs

• Lab. – CK : only slightly elevated except in Miyoshi myopathy-

EMG : myopathic

Biopsy : non- specific dystrophic changes

selective type1 fiber atrophy in welander DM

Inflammatory and immunemediate myopathies

1.Dermatomyositis2.Polymyositis3.Inclusion body myositis

Dermatomyositis

• Inflammatory myopathy– Prevalence: 1:100,000 in

general population

– Female to male prevalence of 2:1

– peak incidence ages 40-50

– Immune complex deposition in the vessels considered to be part of a complement-mediated vasculopathy

Hematoxylin and eosin stain (20x) of a muscle biopsy from a patient with dermatomyositisshowing perivascular and perimysial inflammation, as well as perifascicular necrosis.

Diagnostic Criteria

• Bohan and Peter Criteria:– Symmetric proximal muscle weakness

• most common symptom

– typical rash

– elevated serum muscle enzymes

– myopathic changes on EMG

– characteristic muscle biopsy abnormalities and absence of histopathologic signs of other myopathies

Grotton’ s Sign: An erythematous, scaly eruption over the extensor surfaces of the metacarpophalangeal joints and digits

Heliotrope rash:A reddish-purple eruption on the upper eyelidaccompanied by swelling of the eyelidMost specific rash in DM

Dermatomyositis

Perifascicular atrophy

Degeneration

Inflammatory cells in the perimysiumsurrounding a blood vessel

Inflammatory cells tend to be B-cells.

PolymyositisSymmetrical proximal muscle involvement similar to DMLack of cutaneous involvement,endomyseal inflammatory involvement

Inclusion body myositisInvolvement of distal muscles,esp extensors of knee and flexors of wrist,ASYMMETRICAL

PM DM IBM

Age at onset >18yrs Adulthood, childhood >50yrs

sex M=F F>M M>F

Weakness proximal proximal Proximal, early distal

involvement

Familial association No No Yes, in some cases

/familial inflammatory

myopathies /

Response to treatment good better poor

CTDs yes yes Yes, in up to 20%

malignacy No yes, in up to 15% of

cases

No

Rash Absent Present Absent

Biopsy “primary” inflammation

with the CD8/MHC-I

complex & vacuoles

Perifascicular,

perymysial, or

privascular infiltrates,

perifascicular atrophy

Primary inflammation with

CD8/MHC-I complex;

vacuolated fibers with

b-amyloid deposits ,

cytochrome oxygenase-

negative fibers ; signs of

chronic myopathy

POLYMYOSITIS, usually endo-myseal

INCLUSION BODY MYOSITIS, “rimmed” vacuoles

Congenital myopathy• Rare disorders distinguished from muscular dystrophies by the presence of specific

histochemical & structural abnormalities in muscle fibers.

• Onset : infancy or childhood

• C/F - progressive muscle weakness ( proximal> distal, legs> arms) & limpness, hypotonia & delayed milestones

- skeletal deformities (kyphoscoliosis, club foot, hip dislocation)

• Lab. - CK: usually N or slightly elevated

- EMG : myopathic/ mostly/; positive sharp waves, myotonic discharges

- Biopsy : features specific to each type

Congenital myopathies

1.Central core disease

2.Multicore disease

3.Nemaline(Rod) myopathy

4.Centronuclear myopathy

5.Congenital fibre type disproportion

6.Myofibrillar myopathy

Central core disease•AD Ryanodine receptor(RYR1)Gene 19q13.1•Early onset hypotonia, floppy infant,associated skeletal abnormalities,malignant hyperthermia•Biopsy- cytoplasmic cores in type 1Fibres(NADH-TR stain)

Multicore disease

•Cong non progressive myopathy(gen weakness,hypotonia)

•Biopsy –type 1 fiber predominance& minute core like structures in majority of fibres

Nemaline (Rod)myopathy•AR/AD•Childhood weakness•Ebbing of strength is more in facial & proximal limb muscle•Facial dysmorphism•Aggregates of subsarcolemmal spindle shaped particles(nemaline rods) occuringpredominantlyIn type 1 fiber best seen RTC stain

Myotubular/Centronuclearmyopathy

•AD/AR/XL•MTM 1 gene(Xq27-28) XL•Clinical findings-severe congenital hypotoniaFloppy infants, poor prognosis, extraocular

palsies & facial asthenia occur

simultaneously with involvement of

appendicular muscle•BIOPSY- Abundance of centrally located nuclei involving majority of muscle fibres(mostly in type1 fibres)

Congenital fibre type disproportion

•Atrophy of type 1 fibres, hypertrophy of type 2 fibres•Detectable at birth, paucity of motor activity & diminished tone•Muscle deterioration tends to continue throughout first decade& then ceases or undergo reversal•Skeletal deformities-hip dislocation ,kyphoscoliosis ,joint contractures

Myofibrillar myopathies

•Heterogenous group of disorders(protein surplus myopathies)•Accumulation of intermediate filaments including desmin,actin,myosin,ab crystalline & myomotilin within fibres•Adult onset•Distal weakness,dysphagia & cardiac involvement

Desmin myopathy (RTC)

General questions to keep in mind during reporting muscle biopsy

• Are the muscle fibres abnormal??

• Is the pathologic process-neurogenic/myopathic??

• What is the distribution of pathology??

• Are there any diagnostic features??

Are the muscle fibers abnormal??

Size-small or large

Shape-rounded or angular

Type-grouping, fiber predominance

Internal architecture-disordered/lost, vacuoles,Internal nuclei, inclusions

Storage/acumulated material-glycogen, lipid, mitochondria

Is the pathologic process

NEUROGENIC/MYOGENIC

• Shape of muscle fibers

ROUND Myopathic

ANGULAR Neurogenic

• Distribution of atophic fibers

Grouped-Denervation ,dystrophinopathies

Scattered-Acute neuropathy or myopathy

ACUTE OR CHRONIC

Acute:

Myopathy-Muscle fiber regeneration &denevation

Neuropathy-Small angular muscle fibers

Chronic:

Myopathy-increased endomysial connective tissue, muscle fiber hypertrophy

Neuropathy-fiber type grouping ,pyknotic nuclear clumps

• What is the distribution of the pathology??UNIFORM-Dystrophy, fiber type predominance

REGIONAL

Patchy fascicular changes-inflammatory myopathies,focal denervation

Group of muscle fibers

Neuropathy -Progressive denervation with reinnervation

Myopathy- Myopathic grouping, perifascicular atrophy

SCATTERED muscle fibers: Acute myopathy, Acute neuropathy

• Are there diagnostic feaures??INFLAMMATION

SITE

STORAGE MATERIAL

PATHOLOGY other than muscle fibers

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Stephen S sternburg, Donald A.A,Daryl. Carter,Stacey.E, ObermanH.A. Diagnostic Surgicl Pathology.3rd ed. Newyork: LippincortWilliams &Wilkins;1999.p.1701-1784.

C. Sundaram and Megha S.Uppin,Approach to the interpretation of muscle biopsy ,Nizam institute of medical sciences, Hyderabad.

Ivan Damjanov,Anderson`s Pathology.10th

ed.Kansas:Elsevier;2009.Vol2.p2653-2692.

WellerR.O.Systemic Pathology,Nervous system,muscle & eyes.3rd

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