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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 ¯ophage 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
References Vinay Kumar,Abul.Kabbas,Nelson Fausto. Robbins & Cotran
Pathological Basis of Disease.8th ed. Chicago,Illinois:Elsevier.2010 .p. 905-969
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
ed/vol4.Chuechill Livingston;1990.p.580-665.