Muscle Biopsy

Muscle biopsy is used to:

• Establish the diagnosis in an inflammatory
myopathy before treatment
• Establish the diagnosis in weakness of unknown
cause
• Establish the diagnosis in hereditary myopathies
and dystrophies
• Identify a treatable disorder
• Evaluate carrier status in female relative of a boy
(X-linked)

with Duchenne muscular dystrophy >baby have normal muscle movement

↳ but cannot walk
I not treatable
as
grown
Diseases of muscle
• Myopathies – intrinsic abnormality of muscle
➢Inflammatory myopathies
➢Metabolic myopathies
➢Periodic paralysis (Chanelopathies)
➢Myopathies associated with systemic disease

• Muscular dystrophies – inherited intrinsic defect

• Neurogenic disorders – disease of innervating

neurons
 Selection of muscle sample
• Chronic disease: Muscle with moderate, but not
severe, weakness
• Acute disease: Muscle with severe or moderate
weakness
• Best specific muscles: Deltoid; Biceps; Quadriceps
• Sample site information must be included in
request form
Selection of muscle sample
• Normally avoid
➢Clinically uninvolved muscle
➢End-stage muscle
➢Site of recent trauma
Biopsy Types
large samplesize
⑨
• Open biopsy
L

I more invasives
inflammation and degeneration of
skeletal muscle
↑
➢For disorders with patchy pathology, e.g. polymyositis

small samplesize
I

• Needle biopsy less invasives

➢Minimize trauma
➢May miss patchy or epimysia pathology
➢Difficult to handle
Main changes seen in
muscle biopsies
• Necrosis
• Atrophy Idecrease in sizes

• Hypertrophy (increase in size)

• Regeneration
Muscle Biopsy Handling
do (artifect may introduced)
not directly soak muscle in saline

• Received fresh, wrapped in a saline-

moistened gauze pad
• Avoid drying artefact
• Specimen should not be immersed in saline
Muscle Biopsy Handling
for opensection
I
• Bench rest for 5 min. after removal before
dissection
• Reduce muscle contraction
• Needle bx : to exam under stereo-microscope to
ensure cross-sectional orientation during
embedding
Muscle Biopsy Handling
• 4 types of processing
*
• Frozen section for histochemistry
• Paraffin for histology
• EM studies
• Biochemical analysis

• Pathological interpretation of neuromuscular

disorders is based essentially on
histochemistry and EM
EM studies
• Transmission Electron Microscopy
• muscle is fixed in a properly accession number
labeled bottle of 3% glutaraldehyde in 0.1M sodium
cacodylate-HCl buffer pH 7.4 at 4°C
• Ultrastructural analysis
• Good visualization of muscle endomysial capillaries,
inclusions, mitochondria……
Paraffin for histology
• Useful for inflammation & morphology of
inflammatory cells
• But poor muscle fiber morphology
Frozen Sections
fat is difficult to be removed
X
• Trim off subcutaneous fat and connective tissues

• Ideal size (cross section 0.5x0.5x1cm)

• Immediate snap freeze to avoid ice crystal artefact

↑
• Large ice crystals may form when cooling is slow

• For muscle fiber morphology, enzyme

histochemical studies & IHC
Frozen Sections
• FS are cut at a thickness of 6-8 µm for special
& enzyme histochemical staining
• The sections for histochemical staining set
consecutive

should be serial sections

1

• Store frozen sections at – 20°C

• Remaining frozen blocks are kept in pre-
chilled, well labeled ampoules and stored in -
70°C freezer
Freezing agents
Pre-cooled isopentane
(by liquid nitrogen)

1 -
1300)
Isopentane
• Intermediate cooling liquid
①
• Does not itself penetrate tissue
②
• Nor alter histochemical reactions
• Can be pre-cooled to the temp of liq N2

• Inexpensive, reusable, available commercially

• Vaporize at RT if not kept sealed, potentially
explosive
• Safe in capped bottle and kept in fridge
Liquid nitrogen
Block Freezing
• Specimen is rolled in
freezing
Lensure evenly ofspecimens

talcum powder until

I
evenly coated remove excess
talcum powder
Block Freezing
• A small piece of
aluminium foil is
folded to provide a
groove onto which a
small amount of OCT
is applied
Block Freezing
• The muscle is placed into
the OCT with the muscle
fibres running in parallel
with the groove
• Correct orientated
transverse section is
essential for making
diagnosis
Block Freezing
• The aluminium foil is held
in a pair of forceps and
plunged into the freezing
agent

L liquid nitrogen)
pre-cooled isopentence
Block Freezing
• When large bubbles stopped forming, the
aluminium foil, together with the frozen tissue, is
transferred into the cryostat at -20°C
I artifact
change in temperature induced
• Excessive OCT is trimmed away using a precooled
razor blade to provide a flat base for embedding
• The tissue is embedded for cryosectioning with pre-
cooled forceps
↑ pre-cooled all equipment
PrestoCHILL
I freeze the block from frozen section
 ↑ crossectionthing artefact)

lateral section (Cross-section

is preferred
⑨
2

I S S
[
freezing artefact cross-section
1
 Freezing artefacts
Causes Remedy
Direct freezing of samples 1. use of freezing bath
in liquid N2 produce bubbles 2. wrap sample in Al foil prior
↓
insulator
that prevent rapid freezing to freezing in liquid N2
powder
talcum
X
3. coat sample in talc prior to
freezing in liquid N2
 Freezing artefacts
Causes Remedy
Frozen sample partial thaw
1. while placing into 1. precool container in
specimen container cryostat
2. while attaching to cryostat 2. ensure specimen
& transfer thermostat
using
chucks are stored in
block should be cooled the cryostat
↓
3. while removing from chuck 3. ensure this is carried
out rapidly
4. while transferring to low temp storage 4. use a cooled flask for
transporting
 Characteristics of holes/vacuoles in
muscle sections
Freezing crystal artefact
Irregular shapes
Various sizes
Often confined to one area
particularly edge of section
No storage material demonstrated
Metabolic abnormalities
Round ‘punched out’
vacuoles
All small & same size
Throughout area of
section but often random
distribution
Glycogen or lipid
demonstrated
 Skeletal Muscle Biopsy
• Traditional staining is sufficient to show
– inflammatory responses
– variation in fibre size
• Enzyme histochemistry
– Differentiate between various muscle fibre
types
 Muscle Fibre Typing
• principally two muscle fiber types : based
upon the oxidative and glycolytic activity of
the muscle
 Muscle Fibre Typing
• Type 1 muscle fibers
– high in oxidative activity
– low in glycolytic activity
– red grossly because of a high content of myoglobin and
mitochondrial cytochromes
– contract slowly but are capable of repeated or continuous
contraction.
– "ONE SLOW RED OX"
& high oxidative
lonoglycolytic
• Type 2 muscle fibers
– low in oxidative activity
– high in glycolytic activity
– white grossly
– capable of rapid contraction, but cannot maintain repeated
contraction indefinitely
– "TWO FAST WHITE SUGAR"
I

high gly
 XMuscle Fibre Typing
• Type 1
– Red, high content of myoglobin, cytochrome, plentiful
mitochondria, energy from oxidative phosphorylation
• Type 2A
– Intermediate between red & white fibres
• Type 2B
– White, low content of myoglobin, cytochrome,
mitochondria, energy from anaerobic glycolysis
• Type 2C
– Rare, present in developing muscle and in pathological
situation
 Routine Panel of Demonstration nucleus in

peripheral
Non-enzyme stains
I

• HEEPESA
IX differentiate
ABRE
in acid alcohol)
differentiate
Eosin CaCk
+
+

(xacetic acid)
LillieMayer ↳ dissolvenuclearstain

➢ general architect and morphology

➢ Correlate histological & histochemical features
(one-step trichrome)

• Gomori’s trichrome ↳ counterstain nucleibynx @ Scottloptional) trichrome

➢ Connective tissues, abnormal structures (red ragged
fibres, nemaline bodies)
[abnormal substance
• PAS -> do not place section in
↳
H20 for too long
glycogen:watersoluble
->
immediately
fixed in
chloroform

➢ screen for glycogen storage disease & hemaline bodies

 Routine Panel of Demonstration type I muscle fibers

N
Non-enzyme stains V

• ORO/SBB
➢any excess accumulation of lipid
2 type Imusclefibers
• Congo red / Crystal violet
↑ bi-refringence I metachromasia

➢amyloid
 Routine Panel of Demonstration
Enzyme histochemical stains
• ATPase
➢ Fiber typing

• NADH Diaphorase (reduced nicotinamide adenine

dinucleotide)
➢ Mitochondria activity
➢ sarcoplasmic structural detail

• SDH (succinate dehydrogenase)

➢ Mitochondria activity, oxidative enzyme activity
 Routine Panel of Demonstration
Enzyme histochemical stains
↑ if substance is soluble in H20 aqueous mount
• COX (Cytochromeoxidase if substance is not soluble DP X
->

type I muscle
stained deeply
➢ mitochondria type I stained pale
↳ PEO(disease):VSDA stain but XCOXstam
• Acid phosphatase
➢ Lysosomal activityin degenerating fibers
• Phosphorylase
➢ Glycolytic activity
➢ Deficient in striated muscle cells but present in smooth muscle
cells of blood vessels in McArdle’s disease
• Acid phosphatase/esterase
➢ Lysosomal activity in degenerating fibers
 Adenosine triphosphatase
• adenosine triphosphatase (ATPase) is the
most reliable way to distinguish between
muscle fiber types
• ATPase removes the terminal phosphate
group from ATP
• by varying pH during the staining
procedure, different muscle fiber types may
be differentially stained
 Adenosine triphosphatase
Dam, mountw/DPX

A-P~P~P + H2O A-P~P + H3PO4 + E

~P: energy rich phosphate bond
X from muscle
fiber
from buffer section
precipitate
W on

ATPase Ca++ /
ATP PO4 cobaltchloride Ca3(PO4)2
X
Ca3(PO4)2 + CoCl2 CaCl2 + Co3(PO4)2
X ammoniaquite
& blackprecipitate
Co3(PO4)2 + (NH4)2S CoS
-
Metallic coupling method
toxic perform in fame hoods
to remove (NH4S
flush in tap water thoroughly
cut consecutive sections
↳ stained in diff. pH
(compare diff.pH, distinguish
muscle types)

ATPase pH4.3 ATPase pH4.6 ATPase pH9.4

 NADH Diaphorase
• “Diaphorase” is a term given to flavoprotein
enzymes that have the property of transferring
hydrogen from reduced nicotinamide adenine
dinucleotide (NADH) to various dyes
• The hydrogen transfer reduces the dye
 NADH Diaphorase
alcohol soluble mount
Haosoluble but
slightly aqueous

⑱not
->

• Usually, tetrazolium compounds e.g., nitro blue

tetrazolium (NBT), function as the hydrogen
V

acceptor when diaphorases are being demonstrated

histochemically
• With the addition of hydrogen, the tetrazolium is
reduced to purple blue water-insoluble formazan
pigment marking the site of enzyme activity
 NADH-tetrazolium reductase
• A flavin enzyme catalyse the oxidation of NADH
• Takes up H reversibly and transfer to the acceptor,
a tetrazolium salt (nitroblue T)
Nicotinamide adenine
dinucleotide (reduced form)

NADH NAD + H

tetrazole H2 tetrazole
colored
 Succinic dehydrogenase
I detect mitochondrial proliferation

• SDH is a soluble iron flavoprotein that

catalyzes the reversible oxidation of
succinic acid to fumaric acid
• Demonstration of the enzyme activity is
achieved by incubation of fresh frozen
sections with a succinate substrate in the
presence of a tetrazolium compound
↑
:
-
SBB
 Histochemical Reactions in Human
Muscle
Muscle fibre Type 1 2A 2B 2C
Routine ATPase pH9.4 1+ 3+ 3+ 3+
ATPase pH4.6 3+ – 3+ 3+
ATPase pH4.3 3+ – – +/2+
NADH 3+ 2+ 1+ 2+
SDH 3+ 2+ 1+ 2+
PAS +/2+ 3+ 2+ 2+
Phosphorlase +/– 2+ 3+ 3+
COX 3+ 2+ 1+ 1+
SBB 3+ 2+/3+ 1+ +/–
 Histological &
Histochemical Assessment
• Overall structure
• Cellular reactions
• Structural abnormalities
• Deficiency of enzymes
• Accumulation of products
• EM findings
Interpretation of Muscle Disease
• Muscle fibers are only one component of muscle
• Interfascicular and endomysial fibrous tissue,
lipid, arterioles and capillaries, intramuscular
nerve bundles and nerve endings, and sensory
receptors, including free, unmyelinated C fibre
endings, pacinian corpuscles and muscle
spindles, are of major importance in muscle
contraction
• All may be affected in different ways by disease
END