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Introduction Evaluation Normal Histological Parameter Histochemistry and Immunohistochemistry Organelle Glossary
Introduction to Muscle Biopsy Head
University Neuromuscular Disease Center Staining Protocol
University Neuromuscular Disease Center
Mitochondrial myopathy link
Muscular Dystrophy Association (MDA)
result: A negative biopsy does not mean that the myopathy is not there. In
some myopathies, only certain muscle groups are affected. The absence of
evidence is not the evidence of absence..
1 piece for HE (formalin), histochemistry (fresh), EM (gluteraldehyde), and
biochemistry/genetics (fresh) respectively.
both longitudinal and cross sections should be cut. The best way to do it is to
process the whole piece of tissue (fixed in a clamp). After the tissue is
infiltrated with wax, cut the longitudinal and cross sections respectively
before embedding. Five levels should be obtained, especially for cases suspected
· Take biopsy from muscle with recent involvment, partial weakness, tender. In muscles with abnormal EMG, take biopsy from the contralateral side to avoid articacts produced by the electrodes.
take biopsy from the side where electromyography was done.
take biopsy from muscle that is not affected. You will see nothing.
take biopsy from muscle that is very weak. You will see only end stage changes.
Ocular muscles have very small motor units
and it may be difficult to tell if it is a neurogenic myopathy or myopathic
· Many metabolic and congenital myopathies,
particularly the milder form, have late juvenile or adult onset. The phenotype
in metabolic myopathies is particularly complex. Vladutiu
GD. Muscle Nerve 2000 Aug;23(8):1157-9.
variety of muscle diseases may produce identical clinical symptoms.
· A single
disease may produce several different patterns of injury.
a single pattern of injury may be observed in several diseases.
ocular muscles are frequently spared from many myopathies but they are affected in
mitochondrial myopathy, myotonic dystrophy, and congenital
major pieces of clinical information are critical for the pathologist:
Is the condition a long
standing or newly onset condition?
Is the condition
progrossive or relatively static?
Are there any associated
systemic conditions such as heart problem or autoimmune disease?
What is the serum CPK
helpful clinical information:
History of myoglobinemia. If yes, when.
Family history of
Sex and age.
Results of EMG studies.
Muscle groups being
groups being involved.
kinase level: The
pathologist can often use the creatine kinase level to rule out certainh
categories of myoapthies because different myopathies tend to generate a
different levels of elevation in CPK.
(e.g. Dystrophinopathies) 200-300 times of normal.
(e.g. Inflammatory myopathy) 20-30 times of normal.
(e.g. Neurogenic disorder) 2-5 times of normal.
Parameters to be evaluated:
Vasculitis (± fibrinoid
Endomysial and perimysial
Range of fiber caliber (very
important in pediatric cases)
Rounding of fiber contour
(better evaluated with frozen sections)
Variation of fiber diameter
(better evaluated with frozen sections)
Increase in number of
centrally located nuclei
Central capillary migration
HE frozen section
Modified Gomori Trichrome
Succinate dehydrogenase (SDH)
Cytochrome C oxidase (COX)
PAS with and without diastase
Oil red O
(size, density, and shape)
Normal Histologic Parameters Head
Shape and Diameter Muscle spindle Satellite cells Fiber typing Miscellaneous
Shape and Diameter: Head
Shape: Adult muscle fibers should look a little
polygonal. If they look round then some pathologic process is probably present. In contrast,
normal muscle fibers in children can look round.
diameter is an information particularly in infant and children.
The normal range of muscle diameter is around 40-100 micron. Because of
the shrinkage artifact introduced by paraffin embedding, it is more reliable to
assess fiber diameter and variation in fiber diameter in frozen sections.
Paraffin embedding will induce a 30% reduction in diameter. As high as up to 30%
reduction in size has been claimed but I suspect that this is an over
estimation. A 10-20% reduction is a better estimate.
muscles have larger mean fiber diameter (85-90 micron) for power generation.
muscles have a smaller mean fiber diameter (20 micron) for fine coordination.
general, fiber diameter is smaller in children and elderly and larger in active
fibers in man are usually larger (may be up to 10 mm)
than fibers in women.
infants and children, type 1 and 2 fibers are equal in size and have small
male, type 2 fibers are usually larger than type 1 fibers.
· In adult female, type 1 fibers are slightly larger than type 2 fibers.
Determination of muscle diameter: The diameter chosen is the shortest dimension
bisecting the muscle fiber in a plan through the center of the fibers. Diameter
of muscle fiber for children at different ages:
Data after Engel, A, 1998
spindles are mechanoreceptors arranged in parallel with the muscle fibers. They
are more often seen in biopsies of small muscles or in biopsies from children.
For unknown reasons, muscle spindles are rarely affected in many myopathies.
from 2 mm in infants to 7-8 mm in adult males. The total number of fibers varies
from 2 to 15.
Each spindle is innervated by one large afferent (sensory) myelinated fiber at
the equatorial zone. Axons from g
motor neurons and b motor
neurons provide efferent innervation and originate from the distal zone.
equatorial zone contains nuclei but no myofibrils and are unable to contract;
the distal parts contain striated fibers and are able to contract
The capsule: The
intrafusal fibers are contained with a fluid filled capsule that is composed of
multiple flattened lamellae. The structure is similar to the peirneurium and, in
fact, the capsule joins the perineurium of nerve supplying the spindle.
Three types of striated fibers are present. Each spindle contains up to 10 chain
fibers and 1-4 bag fibers. As a common feature, their nuclei are located in the
equatorial region of the spindle. The nuclei are concentrated in the equatorial
Nuclear bag 1-fiber:
smaller than bag-2 fiber.
Nuclear bag 2-fiber:
they are the largest and may contain up to 50 nuclei and measures up to 50 mm in diameter.
Nuclear chain fiber:
they are the thinnest and about 10-15 mm
also present. They lack basal lamina and can be readily recognized under
Tendon organ (Golgi organ): They are found in tendons lies at the transition
between the tendon and muscle. It is composed of collagen fibers of the tendon
intermingled with the nerve endings of a myelinated fiber. The fibers that
branches within the collagen bundles contain no myelin. When the muscle
contracts, the pressure on the nerve endings will generate nerve impulses.
· They are
small mononuclear cells that are normally found underneath the basal lamina of
muscle fibers and are considered as the reserve myogenic precursor cells. About
3.8% of the nuclei of extrafusal human adult fibers are satellite cells [Schmalbruch
H and Hellhammer U, 1976]. They are probably more numerous in newborn and
infants. They must be distinguished from other cells, such as plasma cells, that
may be occasionally found under the basal lamina in pathologic conditions.
· They can
be well demonstrated by electron microscopy, sometimes in semithin sections.
They can be easily demonstrated by immunostaining for N-CAM; they also express
cells are evenly spaced along the length of muscle fibers but they are increased
in number in the motor end plate and in intrafusal fibers.
satellite cells express Myo-D and myogenin [Rantanen
J et al., 1995].
Fiber Typing: Head
Although both oxidative enzymes and ATPase can be
used for fiber typing, ATPase is more reliable than oxidative enzyme
histochemistry. Atrophic change can induce an increase in oxdative enzyme
staining because the mitochondria and T-tubles may be more densely packed.
· Type 1 fiber (slow twitch): light staining in ATPase reaction, pH 9.4. They
are rich in mitochondria and triglyceride lipid. They contain less glycoen, less
ATPase, and enzymes that are associated with glycogen metabolism. Muscles that
are responsible for continual or postural functions, such as the calf and
gluteal muscles, contain more type 1 fibers. Variation also increase with depth
and the general trend is an increase of type 1 fibers in locations closer to the
bone. At the time of birth, some type 1 fibers have an unusually large diameter
and are formerly called “Wohfart type B” fibers.
· Type 2 (glycolytic) fiber (fast twitch): dark staining in ATPase reaction, pH 9.4. They are
rich in glycogen, myophosphorylase, phosphofructokinase and ATPase but contain
less mitochondria and triglyceride lipid. Type 2 fibers are further divided into
2A, 2B and 2C. ATPase reaction at pH 4.6, type 2c is the darkest, then 2b and
then 2a; type 1 is dark but not as dark as 2c.
differentiation into a checkerboard pattern of type 1 and 2 fibers is present
after about 28 weeks of gestation. Type 2C fibers seem to be the precursors of
type 1, 2A, and 2B fibers. Type 2C fibers are very rare in adults. At the time
of birth, about 10% of the fibers are type 2C fibers.
children, type 1 and type 2 fibers are in equal proportions. At about 1 years of
age, type 1 fibers comprise 60-65% of the fiber and 2A is still the predominant
type of type 2 fibers. In adult the ratio of type 1 to type 2 fibers can vary
but a typical muscle contains approximately twice as many type 2 (60-65%) than
type 1 fibers (35-40%).
Close to tendon:
in variation of muscle fiber diameter
in the amount of connective tissue
· Increased in the number of central nuclei
End plate: features include an array of nuclei with both
parallel and longitudinal arranagement in reference to the fiber. Small
capillaries is also a constant feature which may appear as a few red cells if
the end plate is seen en face.
Capillaries: can be visualized by PAS, Gordon and Sweet reticulin, immunostaining against factor VIII related antigens and also histochemistry using Ulex europaeus lectin.
Endomyseal connective tissue: they are best evaluated in frozen sections because
tissue shrinkage in paraffin sections will make the evaluation less reliable.
of nuclei: There should be about 4-8 peripherally and
subsarcolemmaly located nuclei in each fiber.
Capillaries are seen at the center of the fiber. This is a sign of chronic
Central nuclei: There should be about 3 central nuclei per 100
fibers. Too many central nuclei indicate a chronic condition.
Lipid: Increase of lipid can be detected by fat stain or
by EM. Mitochondria diseases often have increase in amount of lipid.
Click thumbnail to see abnormal accumulation of glycogen in McArdle's disease.
and Immunohistochemistry Head
ATPase Oxidative Enzymes Miscellaneous
ATPase at pH 9.4 (standard condition): type 1 with light staining and type 2 with dark
staining are distinguished. No intermediately staining fiber is seen.
ATPase at pH 4.6 (incomplete acid reversal): also known as the reverse ATPase staining. Type 1
with extremely dark staining, type 2A with virtually no staining, type 2B with
ATPase at pH 4.3 (complete acid reversal): type 1 with extremely dark staining, type 2A and
2B with no staining. Type 2C fibers have intermediate staining.
Oxidative enzymes (including NADH-TR, cytochrome C, succinic
dehydrogenase) are present in muscle These histochemical reactions essentially
distinguish fibers with low mitochondrial content (non-staining) to that with
high mitochondrial content (darkly stained). They are useful in fiber typing and
in investigating mitochondrial myopathy. Type 1 fiber is oxidative fiber
(intensely stained), type 2A is intermediately stained (and intermediately
oxidative) and type 2B is non-oxidative and lightly stained. In normal muscle,
oxidative enzyme stains will give a “lattice network” pattern. Mitochondria
in subsarcolemmal locations and around endplate will give a crescent shape
· NADH-tetrazolium reductase (NADH-TR):
this reaction detects a reductase that is present
in both mitochondria and sarcoplasmic reticulum, therefore, it is not a reliable
marker for mitochondria. SDH and COX are better markers of mitochondria. Tubular
aggregates accumulated in the sarcoplasmic reticulum can be stained by NADH-TR
but not succinic dehydrogenase. NADH-TR can also demonstrate the intermyofibril
network (T-tubules). A clear differentiation of fiber types is only seen in
healthy muscle and this is often disturbed in pathological muscle.
· Succinic dehydrogenase (SDH): stains for mitochondria. They give a staining
pattern similar to NADH-TR. However, succinic dehydrogenase is code by nuclear
DNA and is not usually deficient in mitochondrial myopathy. This is also a good
stain to demonstrate abnormal subsarcolemmal accumulation of mitochondria in
mitochondrial myopathies, see also ragged red fibers.
Cytochrome oxidase (COX):
stains the inner mitochondrial membrane. More prominent staining in type 1 than
type 2 fibers, reflecting the different number of mitochondria in these fibers.
Cytochrome oxidase is coded by mitochondrial DNA and is most frequently
deficient in mitochondrial myopathy.
Combined COX-SDH stain: This
is a very efficient screening test for mitchondrial diseases.
Combined NADH-TR-SDH stain: This combination allows differentiation between tubular aggregates
and mitochondrial accumulation.
Acid phosphatase: Lysosomal vacuoles, autophagic or due to lysosomal storage disease, are acid phosphatase-positive.
Acetylcholinerase: detection of end plates and muscle spindles.
Myoadenylate deaminase: Myoadenylate
deaminase is the muscle-specific isoform of adenylate deaminase.
Myoadenylate deaminase catalyses the deamination of AMP into IMP and
ammonia. IMP can subsequently be re-aminated to AMP. In cases of myoadenylate
deaminase deficiency, muscle biopsies are histologically normal but no
myoadenylate deaminase can be demonstrated by staining.
Non-specific esterase: they have increased staining in sites of lysosomal
and macrophage activity. They may also occur as small granules towards the edge
of fibers in muscle from aged individuals or as larger deposits at the sites of
is a good stain to identify macrophages.
and myotendinous junction are positive for esterase: The neuromuscular junctions
appears as small enzymatic reactive areas at the rim of the fibers.
staining is often excessively dark in denervated fibers but not in other
amount of esterase can also be seen in muscle fibers undergoing necrosis.
Type 2 fibers, particularly
type 2b, are dark and type 1 fiber are pale. Myophosphorylase is deficient in
can be used to detect glycogen storage. It can also be used to visualize the
basement membrane of the capillaries. Glycogen dissolve out easily in paraffin
section or cryostat section and give false negative results.
Modified Gomori’s Trichrome
stain contains Gomori’s trichrome (containing chromotrope 2R, fast
green, phosphotungstic acid, and glacial acetic acid) and hematoxylin. It
is used for frozen sections in muscle biopsy.
stain stains nuclei red-purple, normal muscle myofibrils green with
distinct A and I bands on longitudinal sections, intermyofibrillary
network red, and interstitial collagen green.
is useful in demonstrating the intermyofibril network, rimmed vacuoles in
inclusion body myopathies, and target fibers. These structures, however,
can also be demonstrated well with well-performed hematoxylin-eosin stain
on frozen muscle biopsy material.
useful in detecting ragged fibers in mitochondrial myopathy. The muscle
fibers are stained green and the ragged areas are stained red.
O or Sudan black: Stains for fat. Normal muscle fibers have evenly
distributed deposits of fine droplets. Type 1 fibers have more lipid than type 2
fibers but such separation is not reliable for fiber typing. Fat accumulation is
frequently seen in mitochondrial myopathy .
In normal muscle dystrophin
appears as a thin continuous sarcolemmal staining that is present in every
immunostaining or silver staining:
detection of nerve fibers or end aborization.
unit: is the longitudinal portions of a myofibril
between two Z-discs. Myofibrils in
adults is 0.5 to 1.2 mm in diameter, 10% wider in the A-band than the I-
I-band: is the light band (isotropic,
non-birefrigent). The I-band is composed of the light chains that are 5-6 nm in
diameter and roughly 1-1.25 mm in
length. The length of the I band is variable.
birefrigent) is the dark band and is composed of thick filaments that are 15-18
nm in diameter and a length of 1.6 mm.
The length of A-band is constant 1.6 mm.
means clear in german) is the region where the thick and thin filaments do not
overlap; the pseudo-H zone is the narrow clear area flanking the M line.
is at the center of the thick filaments and
contains fine interconnections between the thick filaments.
Regenerating muscle: Myofibril bundles are
irregular in shape in regenerating fibers.
comprise about 4% of volume in normal skeletal muscle cells. [Kiessling
KH et al., 1973].
· Distribution: Type
1 and 2A fibers have more mitochondria than type 2B fibers. They are usually
found in four locations: intermyofibrillar area adjacent to the Z-disc or
I-band, beneath the sarcolemma, at the poles of nuclei, and at motor end plates.
· Histochemistry: Distribution
of mitochondria can be well demonstrated by oxidative enzyme reactivity. The
intermyofibrillar mitochondria will give the characteristic “lattice
network” appearance with oxidative enzymes. Mitochondria at motor endplates
and subsarcolemmal regions will appear as crescents. Mitochondria are stained
red with Giomori’s modified trichrome. See “Oxidative enzymes” for
· Semithin sections: They
appear as tiny black dots around myofibrils or as delicate lattice network.
Subsarcolemal clusters of mitochondria large enough to appear as grayish
cresents are a normal finding, especially in the muscle of children. [Carpenter
and Karpati, 2nd edt., pp.162]
Electron microscopy: Intermyofibrillar
mitochondria should lie adjacent to the I-band/Z-disc, they are round on cross
section and elongated on longitudinal sections.
Mitochondrial myopathies: Features
of mitochondrial dysfunction include
accumulation of mitochondria- as
demonstrated by modified Gomori’s trichrome or SDH stain.
oxidase (COX) negative fibers with or
without subsarcolemmal accumulation of mitochondria.
Up regulation of antioxidants in
mitochondrial myopathies: Antioxidants including MnSOD, CuZnSOD and reduced glutathione are
up regulated in fibers with respiratory chain deficiency. The degree of MnSOD is
usually more obvious than CuZnSOD. Pattern of expression of reduced glutathione
(GSH) is similar to that of MnSOD. The degreee of antioxidant induction is
correlated with the degree of mitochondrial proliferation but not with clinical
phenotype, patients’ age, duration of disease, biochemical defects or
mitochondrial DNA abnormalities. [Filosto
et al., Acta Neuropathol 2002 103:215-220]
is synthesized in the cytoplasm and transported into the mitochondria.
Therefore, there is a pool of reduced glutathione in both the mitochondria and
(CAT): CAT is a peroxisomeal enzyme
that converts hydrogen peroxide into water and is found in the cytoplasm. With
the exception of heart muscle cells, hitochondria from most mammalian cells do
not contain CAT. The GSH system
is the only non-enzymatic system to reduce hydrogen peroxide. [Radi
R et al., J Biol Chem 1991 Nov 15;266(32):22028-34]
Differential diagnosis on intracytoplasmic inclusions
clear cytoplasmic vacuoles
due to improper freezing.
Real vacuoles: In
hypokalemic periodic paralysis (biopsy obtained at the time of attack).
spaces: They can be seen in non-lysosomal glycogen storage
disease and contain glycogen or filamentous polyglucosan. They may also be seen
in adult-onset centronuclear myopathy.
vacuoles (can be seen with light and electron microscopy).
Often found as an artifact in muscle that has has been placed in isotonic saline
for several minutes before fixation.
In stumps of muscle fibers that have undergone segmental necrosis. The best
example in human is dermatomyositis. Can also be seen in
dystrophy but they are far less common due to the extensive necrosis of fibers.
reticulum vacuoles: They
arise from the sarcoplasmic reticulum. They
can be seen in sarcotubular myopathy which is a rare congenital myopathy. Must
be distinguished from T-tube vacuoles.
vacuoles: They are acid phosphatase positive and can be
autophagic vacuoles or associated with a lysosomal storage disease. Differential
· Acid maltase deficiency:
can be seen in all variants.
· Batten’s disease:
Large vacuoles in rare cases of juvenile Batten’s disease.
Small vacuoles that can be demonstrated by autofluoresence or acid phosphatase
in most cases of Batten’s disease.
· X-linked vacuolar myopathy: Many of them are acid phosphatase positive and many
stain positively for calcium.
· Denervation atrophy: Acid phosphatase-positive vacuoles can be seen in the center of some muscle fibers.
Peroxisomal vacuoles: They
can be seen in Lafora’s disease as
multiple small clear vacuoles up to 8 mm
Glossary on Muscle Biopsy Head
Angulated fiber: Muscle fibers in neurogenic atrophy often appear angulated and small. In the initial stage of neurogenic atrophy, they may be found in a scattered pattern within other fibers. Angulated fibers, however, is not specific and can be seen in many other myopathies.
Centrally located nucleus: In normal conditions, there should be about 3 central nuclei per 100 fibers. An increase in centrally located nuclei is a common but non-specific pathologic features and is often seen in chrnic myopathy. A substantial increase of centrally located nuclei shoud raise the question of myotubular (centronuclear) myopathy.
capillary migration: This is
also a non-specific sign of chronic myopathy. They usually occur later than
the appearance of centrally located nuclei.
Centronuclear myopathy: a central or paracentral nucleus, which is
frequently separated from the surrounding sarcoplasm by a halo, is identified
within the majority of muscle fibers in transverse sections while there is an
apparent nearly total absence of peripheral, subsarcolemmal nuclei.
Cores are typically seen in central
core disease and multicore disease. They are not seen in
paraffin HE sections but best seen with Gomori’s modified trichrome. They are
amorphous round structures in muscles and characteristically lack oxidative
enzyme activity. The cores may be eccentric or central. Ultrastructurally, there
are two types of cores- structured (myofibrils are contracted and retain their
striated pattern and ATPase activity) and unstructured cores (severe disruption
of myofibrils and disruption of myofibrils and a lack of ATPase activity). All
cores show an absence of mitochondria although mitochondria may accumulate
in the core and non-core interface. Cores do not express desmin but the
surrounding non-core zones do. Target fibers and cores must be distinguished.
Target fibers for comparison
Histochemistry: They appear as round to irregular, 2-4 mm in
diameter, cytoplasmic bodies with a halo on HE frozen sections and as purple or
dark green bodies with a halo on Gomori’s trichrome. They have no enzymatic
activities and may be restricted to type 1 fibers.
microscopy: They appear as mesh works of thin filaments.
Delta lesion: This
term is applied to wedge shaped optically empty areas on cross sections that are
resulted from torn or over stretched segments in fibers with
hypercontraction. Particularly, this term implies necrosis of the fiber.
Disorganization of myofilament:
This includes Z-disck streaming, central cores and
nemaline bodies, cytoplasmic bodies, spheroid bodies and myofibrillar myopathy
with desmin accumulation.
Destructive myopathy: characterized by segmental necrosis of muscle
fibers followed by macrophage invasion and phagocytosis of muscle fiber debris.
Recurrent episodes of muscle cell necrosis and regeneration are accompanied by
fibrous scarring of the endomysium.
Fiber grouping: Abnormally small fibers may appear in groups in neurogenic atrophy. On ATPase reactions, these groups are composed of the same type of fiber. Fiber grouping is resulted from nerogenic degeneration and regeneration of motor units.
Heteroplasmic: Mixture of normal and abnormal genomes in the
Hyaline fiber: they have an abnormal increase in diameter
accompanied by a round configuration, a glassy and more deeply stained
appearance. The hyaline change reflects hypercontraction. They are most
frequently seen in Duchenne muscular dystrophy and, to a much lesser extent,
other muscular dystrophy.
fiber: they are fibers that has a lobulated cross section
on HE sections and are best demonstrated by NADH staining. Although they are
type 2 fibers, they may appear darker than usual because of the darker staining.
Lobualted fibers are seen in most myopathic cases and do not carry a special
Myositis: Myositis are characterized by inflammatory cell infiltration. The more common ones include dermatomyositis, polymyositis, and inclusion body myositis.
Nemaline bodies (nemaline
rods): Nemaline bodies have a
longitudinal striation with a periodicity of 8 nm; they have biochemical and
structural properties of Z-discs and
arise as a product of polymerization or proliferation of the lattice of the
Z-disc. Although nemaline bodies are the hallmark of nemaline body
they are also seen in fibers that have lost thick filaments, myopathy associated
with certain types of HIV virus, some cases of dermatomyositis and polymyositis.
Ragged-red fibers and pre-ragged-red fibers:
· Age: They are
not seen under the age of three.
The characteristic histochemical picture of a mitochondrial myopathy is ragged
red fibers, accumulation of lipid, and the absence of cytochrome oxidase
staining in a proportion of muscle fibers in the biopsy. Ragged red fibers
appear as blue muscle fibers with bright red irregular (moth eaten)
subsarcolemmal depositions with Gomori trichrome on frozen sections. The lipid
droplets appear as small vacuoles.
Ragged red fibers are more specifically demonstrated by succinate dehydrogenase;
these fibers are also known as ragged blue fibers (SDH stain is blue). Modified
SDH reaction stain Ragged red fibers and “ragged red equivalents”
(pre-ragged-red fibers that are not demonstrated by modified Gomori trichrome).
· Ragged-red equivalent:
The increase in mitochondria in these fibers can be demonstrated by SDH stain
but not enough to be demonstrated by Gomori’s modified trichrome.
· Differential diagnosis:
Ragged red fibers must be distinguished from nemaline bodies
which also stain red and tend to accumulate under the sarcolemma but have no
enzyme reactivity. Histochemical demonstration of oxidative enzymes may show
cresent shape reactivity in motor endplates where an increased number of
mitochondria is present, they must not be confused with Ragged red fibers.
Kearne-Sayre syndrome-oculocraniosomatic syndrome
Myoclonic epilepsy with ragged red fibers (MERRF)
Mitochondrial encephalomyopathy, lactic acidosis,
stroke-like episodes (MELAS)
Regenerating fibers: Takes
about three weeks to complete. Regenerating fibers express N-CAM, MyoD and
myogenin, and also embryonic and neonatal isoforms of myosin heavy chain. In
contrast to mature fibers, MCH class I histocompatibility complex is expressed
in regenerating fibers.
vacuoles: On cryostat sections, rimmed vacuoles are popcorn
like clear vacuoles with a densely blue rim in HE frozen sections. They are are
best demonstrated in MGT. The vacuoles are often assoctiated with cytoplasmic
and occasionally intranuclear eosinophilic inclusions. Rimmed vacuoles contain
amyloid ABeta, ApoE, and ubiquitin. On paraffin sections the granules are
dissolved so that only rather anonymous vacuoles remain. On semithin sections
there are round osmiophillic granules of various sizes that often occur in
clusters in subsarcolemmal locations. Differential
diagnoses include the followins:
body myositis (IBM):
Inclusion bodies are most frequently seen in IBM
but they are not pathogonomic and may also be seen in other conditions.
· Oculopharyngeal muscular dystrophy.
· Many cases of distal myopathy
(probably a type of familial inclusion body myopathy).
Chloroquine myopathy (with
acid phosphatase activity and are lysosomal).
X-linked vacuolar myopathy
(positive for calcium and acid phosphatase).
finding in other myopathies.
fiber: the ring is formed by a bundle of peripheral
myofibrils which are circumferentially oriented such that they encircle the
internal portion of the sarcoplasm which is normal in structure and orientation.
Rings are best seen with PTAH stains, semi-thin sections, or under phase
contrast or polarizing microscopes. Under the electron microscope the
pathologically oriented myofibrils are generally normal in structure except for
hypercontraction of the sarcomeres. Ring fibers are seen in myotonic
muscular dystrophy, limb girdle dystrophy and trauma.
coils myofibrils: This is
a non-specific change that can be seen in many situations incluing partial
denervation and limb-girdle dystrophy). Snake coils fibers are best demonstrated
in frozen sections and particularly well by Gomori’s trichrome and they
arrpear as a swirling pattern of the myofibrils.
Target fibers (as well as type grouping) are pathognomic of denervation but bone
fide target fibers are present in less than 25% of cases of neurogenic atrophy.
They are often strikingly abundant in subacute cases of Guillain-Barré
They difficult to be seen in paraffin sections but can be demonstrated nicely
with HE stain or mododified trichrome in frozen sections.
Histochemistry: Most target fibers are type 1
fibers. Target fiber has a distinct three-zone architecture that is best
demonstrated by NADH-TR histochemistry. Characteristically, there is a pale core
with no NADH-TR activity, rimmed by a darkly stained ring and surrounded by a
more normal staining peripheral zone. The core also lacks glycogen or
phosphorylase activity. Demonstration of target fibers with ATPase is possible
but unreliable. A fiber must have the three-zone architecture to be qualified as
target fiber. Some two-zone fibers may be seen and they are called targetoid
fibers. The distinction between target and targetoid fibers has no clinical
Cores for comparison
The cores in target fibers contain myofibrils with various forms of dissolution
and disorganization. Z-disc streaming is prominent.
Although targets fibers and cores are morphologically similar, they are
different and must be distinguished. Target
fibers must be distinguished from “motheaten change” that is frequently seen
in neurogenic atrophy.
aggregates: these are non-specific aggregates that can be seen
in myopathies. They are aggregates derived from the sarcoplasmic reticulum and
are, therefore, heavily stained by NADH-TR (present in both mitochondria and
sarcoplasmic tubules) and not by succinic dehydrogenase technique (present only
in mitochondria). A combination of NADH-TR and SDH allows differentiation
between tubular aggregates and mitochondrial accumulation.
loss: Selective Z-disc loss is
probably a result of necrosis. This can only be recognized at the early stage
and without contraction of the fibers. Once contraction is present, the lesion
will appear only as disoriented fibers.
streaming is a non-specific features that can be encountered in many
myopathies. They have also been described in normal control subjects. This is a result of
disorientation of Z-disc.It is a common and non-specific lesion.
Histochemistry: In Gomori’s trichome stain, they characteristically
appear as poorly defined patches of increased staining; histochemically, these
areas show considerable reduction in ATPase activity and often also as
negative areas on oxidative enzyme reactions.
they appear as flame-shaped structures under medium magnification. The number
of Z-discs being affected is highly variable. On longitudinal sections, they varies from focal
replacement of sarcomere by dense material that extends between Z-discs to
long and longitudinal stretches of dense materials in advanced lesions.
On higher magnification, they appear as collections of dark gray granular areas
within a fiber.
· Electron microscopy:
The streaming material is made up of electron dense granular substance. The
streaming, particularly in the smaller streams, starts and ends at the Z-disc.