Muscle biopsy essential diagnostic advice for pathologists

Abstract

Background
Muscle biopsies are important diagnostic procedures in neuromuscular practice. Recent advances in genetic analysis have profoundly modified Myopathology diagnosis.

Main body
The main goals of this review are: (1) to describe muscle biopsy techniques for non specialists; (2) to provide practical information for the team involved in the diagnosis of muscle diseases; (3) to report fundamental rules for muscle biopsy site choice and adequacy; (4) to highlight the importance of liquid nitrogen in diagnostic workup. Routine techniques include: (1) histochemical stains and reactions; (2) immunohistochemistry and immunofluorescence; (3) electron microscopy; (4) mitochondrial respiratory chain enzymatic studies; and (5) molecular studies. The diagnosis of muscle disease is a challenge, as it should integrate data from different techniques.

Conclusion
Formalin-fixed paraffin embedded muscle samples alone almost always lead to inconclusive or unspecific results. Liquid nitrogen frozen muscle sections are imperative for neuromuscular diagnosis. Muscle biopsy interpretation is possible in the context of detailed clinical, neurophysiological, and serum muscle enzymes data. Muscle imaging studies are strongly recommended in the diagnostic workup. Muscle biopsy is useful for the differential diagnosis of immune mediated myopathies, muscular dystrophies, congenital myopathies, and mitochondrial myopathies. Muscle biopsy may confirm the pathogenicity of new gene variants, guide cost-effective molecular studies, and provide phenotypic diagnosis in doubtful cases. For some patients with mitochondrial myopathies, a definite molecular diagnosis may be achieved only if performed in DNA extracted from muscle tissue due to organ specific mutation load.

Full text

R E V I E W Open Access
Muscle biopsy essential diagnostic advice
for pathologists
Ana Cotta
1*
, Elmano Carvalho
2
, Antonio Lopes da-Cunha-Júnior
3
, Jaquelin Valicek
2
, Monica M. Navarro
4
,
Sidney Baptista Junior
1
, Eni Braga da Silveira
5
, Maria Isabel Lima
5
, Bruno Arrivabene Cordeiro
5
,
Alexandre Faleiros Cauhi
6
, Miriam Melo Menezes
7
, Simone Vilela Nunes
7
, Antonio Pedro Vargas
7
,
Rafael Xavier Neto
7
and Julia Filardi Paim
1
Abstract
Background: Muscle biopsies are important diagnostic procedures in neuromuscular practice. Recent advances in
genetic analysis have profoundly modified Myopathology diagnosis.
Main body: The main goals of this review are: (1) to describe muscle biopsy techniques for non specialists; (2) to provide
practical information for the team involved in the diagnosis of muscle diseases; (3) to report fundamental rules for muscle
biopsy site choice and adequacy; (4) to highlight the importance of liquid nitrogen in diagnostic workup. Routine techniques
include: (1) histochemical stains and reactions; (2) immunohistochemistry and immunofluorescence; (3) electron microscopy;
(4) mitochondrial respiratory chain enzymatic studies; and (5) molecular studies. The diagnosis of muscle disease is a
challenge, as it should integrate data from different techniques.
Conclusion: Formalin-fixed paraffin embedded muscle samples alone almost always lead to inconclusive or unspecific
results. Liquid nitrogen frozen muscle sections are imperative for neuromuscular diagnosis. Muscle biopsy interpretation is
possible in the context of detailed clinical, neurophysiological, and serum muscle enzymes data. Muscle imaging studies are
strongly recommended in the diagnostic workup. Muscle biopsy is useful for the differential diagnosis of immune mediated
myopathies, muscular dystrophies, congenital myopathies, and mitochondrial myopathies. Muscle biopsy may confirm the
pathogenicity of new gene variants, guide cost-effective molecular studies, and provide phenotypic diagnosis in doubtful
cases. For some patients with mitochondrial myopathies, a definite molecular diagnosis may be achieved only if performed
in DNA extracted from muscle tissue due to organ specific mutation load.
Keywords: Muscle biopsy, Immunohistochemistry, Electron microscopy, Molecular diagnosis, Surgical pathology,
Mitochondrial respiratory chain, Inflammatory myopathy, Mitochondrial myopathy, Congenital myopathy, Muscular
dystrophy
Background
Muscle diseases are a subgroup from neuromuscular af-
fections in which the primary pathological process in-
volves the muscle.
This review provides text, tables and figures useful to
understand the indications of some ancillary techniques
performed for muscle biopsy interpretation in a
reference center. Figures 1,2,3,4,5and 6provide an
overall view of muscle biopsy procedures. The Tables
provide useful general myology information (Tables 1,2,
3,4), as well as relevant information for routine myology
practice (Tables 5,6,7,8), and a rationale for diagnostic
investigation (Table 9).
It is beyond the scope of this review to provide de-
tailed information to establish a Muscle Biopsy Refer-
ence Laboratory. Such information may be found on
specialized articles and myopathology books (Engel and
Franzini-Armstrong 2004; Karpati et al. 2010; Dubowitz
© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
* Correspondence: ana_cotta@yahoo.com.br
1
Department of Pathology, The SARAH Network of Rehabilitation Hospitals,
Av. Amazonas, 5953 Gameleira, Belo Horizonte, MG 30510-000, Brazil
Full list of author information is available at the end of the article
Surgical and Experimenta
l
Patholo
gy
Cotta et al. Surgical and Experimental Pathology (2021) 4:3
https://doi.org/10.1186/s42047-020-00085-w

et al. 2020; Dubowitz et al. 2013; Dubowitz and Sewry
2007; Dubowiz 1995; Dubowitz and Brooke 1973;
Loughlin 1993; Anderson 1985; Amato and Russell 2008;
Levy 1978; Dastgir et al. 2016), historical and contem-
porary overview of muscle biopsy procedures (Fardeau
2017; Malfatti and Romero 2017; Nix and Moore 2020),
didactic internet teaching videos (Table 1) with practical
steps for muscle biopsy freezing procedure in accord-
ance to the World Muscle Society standards and proto-
cols (Udd et al. 2019), and special recommendations for
patients with malignant hyperthermia (Table 1). It is also
beyond the scope of this work, the reproduction of the
39 pages monogenic nuclear genome neuromuscular dis-
orders Gene Table (Benarroch et al. 2019). An updated
gene table on nuclear genome neuromuscular disorders
is a reference for morphologic-molecular correlation,
which is published every year in the journal Neuromus-
cular Disorders and is freely accessible online (Table 1)
(Benarroch et al. 2019).
Some topics of this review were previously presented
at the Neuromuscular Diseases Scientific Department
Myopathy Course sponsored by the Brazilian Academy
of Neurology (ABN: Academia Brasileira de Neurologia).
Does the patient need a muscle biopsy?
Muscle biopsy is an invasive procedure that should be
performed after inconclusive noninvasive methods. Usu-
ally, muscle biopsies are useful for patients with: (1) ob-
jective muscle weakness, that is detectable through
physical examination, (2) increased muscle enzyme levels
(serum total creatine kinase and aldolase), (3) abnormal
muscle imaging, or (4) myopathic motor unit action po-
tentials on electromyogram (Amato and Russell 2008).
Muscle biopsy may not be performed in patients with
characteristic clinical and neurophysiological pattern, that
may be confirmed with molecular exams performed in
blood samples, such as dystrophinopathy (65 to 70% may be
diagnosed by molecular detection of deletions or duplica-
tions in the dystrophin gene), Steinert’s myotonic dystrophy,
facioscapulohumeral muscular dystrophy, and spinal muscu-
lar atrophy (Karpati et al. 2010;Cottaetal.2017).
In a previous publication from our group (Cotta et al.
2017), we studied the proportion of neuromuscular pa-
tients that reached a final diagnosis either by molecular
or by muscle biopsy studies. That 17 years retrospective
study, from 1999 to 2016, included 1603 patients that re-
ceived a final conclusive diagnosis at SARAH Network
neuromuscular outpatient clinic (Cotta et al. 2017). Mo-
lecular studies were responsible for 48.8% of the diagno-
sis, while muscle biopsies disclosed 51.2% of the
diagnosis in 16 neuromuscular diagnostic categories
(Cotta et al. 2017).
In the last years, our group has also performed muscle
biopsies in some patients previously submitted to mo-
lecular studies. For those patients, muscle biopsies have
been useful to confirm the pathogenicity of variants of
unknown significance (VUS). Some examples have been
observed in families with muscular dystrophies. They
presented inconclusive molecular studies but undoubtful
Fig. 1 Integrated approach, imaging, and types of muscle biopsy
exams. Patient evaluation should be performed by a multiprofessional
team with integration of: neurologic, surgical, laboratorial, radiological,
and therapy clinics for neuromuscular patient best care. The adequate
diagnostic approach includes the immediate separation of muscle
fragments for glutaraldehyde fixation for electron microscopy, liquid
nitrogen freeze in order to preserve histochemical reactions and
provide adequate samples for immunohistochemical, molecular, and
mitochondrial respiratory chain enzymatic studies. Evaluation of a
patient with central core congenital myopathy. aComputed
tomography demonstrated severe right vastus lateralis muscle fat
replacement (yellow arrow) with relative rectus femoris preservation
(blue arrow). band cRectus femoris muscle biopsy of the same
patient demonstrating round core structures (red arrows) devoid of
mitochondria. aComputed tomography of the pelvis, thighs and legs.
bSDH 100x, ctransmission electron microscopy 2.500x
Cotta et al. Surgical and Experimental Pathology (2021) 4:3 Page 2 of 20

Fig. 2 Muscle preparation for histochemical reactions. a. the fresh muscle sample is sectioned following the longitudinal direction of the fibers
and small fragments are immediately glutaraldehyde fixed for eventual electron microscopy studies, if necessary; cylindrical fragments are
separated to make liquid nitrogen frozen muscle blocks in order to preserve enzymatic functions. bOptimal cutting temperature (OCT) mounting
medium is applied on the surface of the previously identified cork. cThe muscle fragment is either cooled in isopentane (data not shown) or
involved in talcum powder in order to avoid freezing artifacts. dMuscle fragment over cork support. eLiquid nitrogen frozen muscle tissue. fThe
muscle fragment was frozen in liquid nitrogen and mounted in a cork and a pin. gThe muscle fragment was fixed inside the cryostat for the
performance of the frozen sections. iThe frozen section may be transferred either to a coverslip (photo) or to a glass slide (data not shown). j
Coverslip inside reaction dish. kFrozen sections in incubator. lthe incubated coverslips are extracted from the coverslip jar to mount the slides
for microscopic visualization
Cotta et al. Surgical and Experimental Pathology (2021) 4:3 Page 3 of 20

immunohistochemical deficiencies of specific proteins
(unpublished data).
How to choose the muscle site for biopsy?
In an ideal situation, muscle biopsy choice may be part
of an integrated multiprofessional approach (Fig. 1).
Muscle selection for biopsy may always consider muscle
strength. Whenever possible, the joint evaluation of
physical exam and imaging studies should be performed
to choose the most superficial muscles (Engel and Fran-
zini-Armstrong 2004; Amato and Russell 2008; Karpati
et al. 2010; Dubowitz et al. 2013).
Fig. 3 Normal control muscle histochemical reactions and stains. Muscle obtained from an amputation procedure for orthopedic deformity with
normal transverse (a) and longitudinal (b) architecture. Normal mitochondrial (c,f, and i), glycogen (d), and lipid (e) content. No inflammatory
activity is observed (kand l). Normal checkerboard with type 1 (number 1 in gand h) and type 2 (number 2 in gand h) fiber distribution. ldark
dots represent normal neuromuscular junctions (aHE 100x, bHE 400x, cModified Gomori trichrome 400x, dPAS 400x, eOil-red-O 400x, fSDH
400x, gATPase pH 9.4200x, hATPase pH 4.6200x, iCOX 200x, jNADH 400X, kAcid phosphatase 200x, lNonspecific sterase 200x)
Cotta et al. Surgical and Experimental Pathology (2021) 4:3 Page 4 of 20

Fig. 4 Morphological findings and histochemical reactions. aPerifascicular atrophy (arrow), ie, peripheral fibers with smaller diameter in patient
with dermatomyositis. bLymphocytic inflammatory infiltrate involving non-necrotic muscle fiber patient with sporadic inclusion body myositis. c
Rimmed vacuole (arrow) in patient with inclusion body myositis associated with HTLV-1 infection. dNecrosis (eosinophilic pale fibers, arrow),
phagocytosis (arrow head), and endomysial fibrosis in a patient with dystrophinopathy type Duchenne muscular dystrophy. eRagged red fiber
(arrow) in patient with mitochondriopathy type progressive external ophthalmoplegia. fGlycogen subsarcolemal accumulation in patient with
type V glycogenosis (McArdle disease). gNegative myophosphorylase reaction in McArdle disease (inset is normal control). hLipid accumulation
in patient with mitochondriopathy. inemaline rods (arrow) in patient with congenital nemaline myopathy, jCOX negative fibers (SDH reactive in
blue, arrow) in patient with mitochondriopathy subtype progressive external ophthalmoplegia. kClear central areas devoid of oxidative reaction
(core, arrow) in central core congenital myopathy. lLarge groups of type 2 atrophic (arrow) and large groups of type 1 hypertrophic fibers in
patient with neurogenic abnormality. (a. HE 100x, b. Gomori modified trichrome 400x, c. HE 400x, d. HE 200x, e. Gomori modified trichrome 400x,
f. PAS 400x, g. myophosphorylase 100x, h. Oil-red-O 400x, i. Gomori modified trichrome 200x, j. COX-SDH double reaction 100x, k. SDH 400x, l.
ATPase 9.4200x)
Cotta et al. Surgical and Experimental Pathology (2021) 4:3 Page 5 of 20

Fig. 5 Immunohistochemical evaluation. aInvasion (arrow) of non-necrotic muscle fiber by cytotoxic/ supressor CD8 positive T lymphocytes in a patient with
sporadic inclusion body myositis. bAbnormal sarcolemmal positivity for major histocompatibility complex class I (MHC-I) in patient with dermatomyositis (inset
shows normal control with blood vessel wall reaction). cIntrasarcoplasmic desmin reactive (arrow) material in patient with myofibrillar myopathy. dEctopic
intrasarcoplasmic dystrophin expression in patient with myofibrillar myopathy. eComplete immunohistochemical dystrophin deficiency in patient with
dystrophinopathy type Duchenne muscular dystrophy (inset shows normal control). fDecreased dystrophin reaction in patient with dystrophinopathy type
Becker muscular dystrophy. g.andhSerial sections showing abnormal utrophin reaction (g) in sarcoplasmic membranes of some fibers (asterisks) without signs
of regeneration, negative for neonatal myosin (h) in patient with dystrophinopathy type Becker (insets are normal controls). iand j. serial sections of negative
fibers in mosaic pattern in fibers without necrosis (with sarcolemma positive for spectrin in j) in a female symptomatic carrier of dystrophinopathy. k. normal
emerin immunophenotypic expression in nuclear membrane (arrow). l. normal sarcomeric (intrasarcoplasmic) telethonin expression (arrow). a. CD8 400x, b.
MHC-I 200x (inset normal control original magnification MHC-I 200x), c. desmin 200x, d. dystrophin carboxy-terminal DYS2 200x, e. dystrophin carboxy-terminal
DYS2 200x, f. dystrophin amino-terminal DYS3 200x, g.utrophin100x,h. neonatal myosin 100x, i. dystrophin amino-terminal DYS3 100x, j. spectrin 100x, k.
emerin 200x, l. telethonin 200x
Cotta et al. Surgical and Experimental Pathology (2021) 4:3 Page 6 of 20

In our service, after the implementation of routine
muscle imaging studies prior to muscle biopsy, the num-
ber of inadequate or end-stage biopsy specimens
dropped to less than 0.4% during the last 10 years (n=
804). All muscle imaging exams have been revised by
both radiologists and pathologists in advance in order to
determine the most suitable muscle biopsy site. For pa-
tients with advanced muscle fat replacement, an ultra-
sound guided muscle biopsy has been performed (Cotta
et al. 2014a,2014b).
There is consensus in the literature, that the muscle
submitted to biopsy may present intermediate force. The
Fig. 6 Ultrastructural evaluation. a. almost normal ultrastructural morphologic pattern. b. nemaline rods are rod shaped structures (arrows), c.
lattice structure similar to Z disks in nemaline bodies in a patient with nemaline congenital myopathy. d. abnormal mitochondrial accumulation
with paracrystalline inclusions (arrow) in patient with mitochondriopathy subtype progressive external ophthalmoplegia. e. tubulorreticular
inclusions (arrow) inside vessel wall endothelial cell cytoplasm in patient with dermatomyositis. f. 15 to 18 nm filamentous inclusion (arrow) in
patient with sporadic inclusion body myositis. g. area of myofibrillar disorganization (arrow) with few mitochondria (core) in central core
congenital myopathy. h. electrondense amorphous deposit (arrow) in patient with myofibrillar myopathy. i. elongated electrondense masses
(arrows) in patient with myofibrillar myopathy. (Transmission electron microscopy a. 2500x, b. 2500x, c. 150,000x, d. 25,000x, e. 15,000x, f. 20,000x,
g. 2500x, h. 30,000x, i. 5000x)
Cotta et al. Surgical and Experimental Pathology (2021) 4:3 Page 7 of 20

muscle submitted to biopsy may not be too weak or too
strong. Some authors recommend grade 3 or 4 muscle
strength as determined on neurological examination
using the MRC (Medical Research Council) scale (Pas-
noor and Dimachkie 2019) (Table 2) (Engel and Fran-
zini-Armstrong 2004), whereas others recommend grade
4 or 5 (Amato and Russell 2008).
Whenever available, muscle imaging exams may direct
the investigation to muscles with intermediate muscle
fat replacement. The muscle chosen for muscle biopsy
may be superficial and easily accessible to the surgeon,
and the most commonly biopsied are vastus lateralis and
biceps brachialis. In special situations, deltoid, triceps
brachialis, rectus femoris, soleus, tibialis anterior, gastro-
cnemius (Loughlin 1993) and short peroneus (Amato
and Russell 2008) may be chosen. In patients with suspi-
cion of distal myopathy, soleus, tibialis anterior, gastro-
cnemius, carpal radial extensor, and extensor digitorum
longus may be biopsied (Engel and Franzini-Armstrong
2004; Amato and Russell 2008).
Muscle fibers are classified in two great groups as type
1 or type 2. Type 1 fibers are predominantly aerobic
whereas type 2 are predominantly glycolytic. The per-
centage of each muscle type may vary for each specific
muscle (Table 3).
The distinction between fiber types is very useful for
the detection of: a) fiber type predominance, b) fiber
type disproportion, c) selective fiber type atrophy, and d)
fiber type grouping (Figs. 3and 4).
Fiber type predominance is detected when the mean
number of type 1 or type 2 fibers is 20% higher than the
expected mean for a given muscle (Table 3) (Loughlin
1993; Dastgir et al. 2016; Cotta and Paim 2016). For vas-
tus lateralis and biceps brachialis, the percentage of
muscle fiber types is usually of: one type 1 fiber for two
type 2 fibers. Type 1 fiber predominance is very com-
mon in congenital myopathies, but it also reflects long
standing myopathic processes, as in some types of mus-
cular dystrophies (Paim et al. 2013).
Fiber type disproportion reflects a morphometric
measure that quantifies how much type 1 fibers are
smaller than type 2 fibers (see section Morphometry
below). Congenital fiber type disproportion presents type
1 fiber predominance and almost all type 1 fibers are
smaller than type 2 fibers (Clarke 2011; Dubowitz et al.
2020).
Selective fiber type atrophy is usually considered when
isolated fibers of one type are smaller than the other
fiber type. Selective type 2 fiber atrophy although unspe-
cific, is very common in corticosteroid induced toxic
myopathy, and congenital myasthenic syndromes (Engel
and Franzini-Armstrong 2004; Scola et al. 2007; Dubow-
itz et al. 2013; Dubowitz et al. 2020).
Fiber type grouping is the morphological hallmark
or neurogenic disorders (Engel and Franzini-Arm-
strong 2004; Karpati et al. 2010; Dubowitz et al. 2020;
Dubowitz et al. 2013; Dubowitz and Sewry 2007;
Dubowiz 1995; Dubowitz and Brooke 1973;Loughlin
1993;Anderson1985; Amato and Russell 2008). It is
characterized by large groups of each fiber type, that
are formed during the process of reinnervation
(Fig. 4).
In special situations, it may be difficult to differenti-
ate which is the weaker muscle within a muscle
group. Detailed clinical exam with inspection and pal-
pation may provide useful data on muscle trophism.
Sometimes imaging studies may be very useful for
this evaluation, as observed in the example depicted
in Fig. 1. Physical examination disclosed a quadriceps
muscle with grade 4 strength (Table 2). A vastus
lateralis muscle biopsy could have provided an inad-
equate sample or end-stage muscle sample with ex-
tensive fibrous-fat replacement simulating a muscular
dystrophy. On the contrary, a rectus femoris muscle
biopsy provided an excellent sample that disclosed
type 1 fiber predominance and almost all fibers with
“central cores”, confirming the diagnosis of central
core congenital myopathy (Fig. 1).
Facial, cervical and hand intrinsic muscles are not usu-
ally biopsied due to functional and aesthetic reasons
(Engel and Franzini-Armstrong 2004).It is important to
perform the muscle biopsy on a contralateral muscle to
the side submitted to injections on neurophysiological
studies, in order to avoid needle artifacts such as inflam-
matory infiltrate (Engel and Franzini-Armstrong 2004;
Amato and Russell 2008). It is believed that the
Table 1 Useful free myology websites and information sources
http://anatpat.unicamp.br/eneuromusc.html
http://neuromuscular.wustl.edu/lab/mbiopsy.htm
https://www.jove.com/video/51586/
https://www.ncnp.go.jp/nin/guide/r1/video_e.html
http://www.scielo.br/pdf/rba/v63n1/en_v63n1a02.pdf
Malignant hyperthermia Hotline phone +55-11-55759873
http://www.musclegenetable.fr.
Table 2 Muscle strength grading according to MRC (Muscle
Research Council) (Pasnoor and Dimachkie 2019)
Grade Muscle movement on neurologic examination
1 Trace contraction of the muscle
2 Ability to move with gravity eliminated
3 Active movement against gravity
4 Ability to move the joint against combination of gravity and
some resistance
5 Normal power
Cotta et al. Surgical and Experimental Pathology (2021) 4:3 Page 8 of 20

inflammatory infiltrate may last for at least 4 or 6 weeks
after electromyogram (Engel and Franzini-Armstrong
2004).
In cases suspicious of congenital myasthenic syn-
dromes, ultrastructural evaluation of neuromuscular
junctions should be performed in part of the sample im-
mediately fixed in glutaraldehyde for electron micros-
copy studies. Exceptionally, a small formalin fixed
sample should be submitted to ultrastructural studies in
search of neuromuscular junctions.
For patients under investigation of malignant hyper-
thermia, it is recommended to search for a specialized
center equipped to perform the test (da Silva et al. 2013)
(Table 1).
What is necessary to do in advance to the muscle
biopsy procedure?
Before the performance of the muscle biopsy, it is advis-
able to get in touch with the laboratory that will receive
the patient or the muscle biopsy. It is mandatory to ask if
the laboratory is equipped to perform histochemical and
immunohistochemial studies in liquid nitrogen frozen
muscle. As fixation in formalin precludes the performance
of histochemical and various immunohistochemical tech-
niques, muscle biopsies should not be entirely formalin
fixed and paraffin embedded. In an ideal situation a small
sample is fixed in glutaraldehyde for eventual EM (elec-
tron microscopy) studies and the most substantial portion
is frozen in liquid nitrogen (Fig. 1). Nowadays, liquid ni-
trogen frozen specimens are the only way to preserve
histochemical and immunohistochemical muscle biopsy
properties for neuromuscular investigation (Engel and
Franzini-Armstrong 2004; Karpati et al. 2010;Dubowitzet
al. 2013; Dubowitz and Sewry 2007; Dubowiz 1995;
Dubowitz and Brooke 1973;Loughlin1993;Anderson
1985;AmatoandRussell2008;Levy1978; Dastgir et al.
2016;Fardeau2017; Malfatti and Romero 2017).
How to perform the muscle biopsy surgical
procedure and specimen handling?
The surgeon prepares the procedure without
cauterization of the muscle tissue, only the blood vessels
of the subcutaneous tissue should be cauterized. The
anesthetic should not be infiltrated inside the muscle
sample, in order to avoid artifacts that should preclude
adequate analysis. The skin is cleaned with antiseptic so-
lution. The skin and muscle fascia are infiltrated with
anesthetic, and the muscle is sectioned in the same dir-
ection of the fascicles of the muscle fibers in order to
perform transverse and longitudinal sections. After exci-
sion, the muscle is gently accommodated on a slightly
moist gauze. Immediately, one to four 0.1 × 0.1 cm
Table 3 Muscle fiber type percentage by muscle site (Loughlin 1993; Dastgir et al. 2016; Cotta and Paim 2016)
Muscle group Mean (%) Predominance (%)
Type 1 Type 2 Type 1 Type 2
Biceps brachii (surface) 42.3 57.7 > 62.3 > 77.7
Biceps brachii (deep) 50.5 49.5 > 70.5 > 69.5
Brachioradialis 39.8 60.2 > 59.8 > 80.2
Deltoid (superficial) 53.3 46.7 > 73.3 > 66.7
Deltoid (deep) 61.0 39.0 > 81.0 > 59.0
Extensor digitorum 47.3 52.7 > 67.3 > 72.7
Extensor digitorum brevis 45.3 54.7 > 65.3 > 74.7
Gastrocnemius (lateral head surface) 43.5 56.5 > 63.5 > 76.5
Gastrocnemius (lateral head deep) 50.3 49.7 > 70.3 > 69.7
Gastrocnemius (medial head) 50.8 49.2 > 70.8 > 69.2
Peroneus longus 62.5 37.5 > 82.5 > 57.5
Rectus femoris (lateral head surface) 29.5 70.5 > 49.5 > 90.5
Rectus femoris (lateral head deep) 42.0 58.0 > 62.0 > 78.0
Rectus femoris (medial head) 42.8 57.2 > 62.8 > 77.2
Tibialis anterior (surface) 73.4 26.6 > 93.4 > 46.6
Tibialis anterior (deep) 72.7 27.3 > 92.7 > 47.3
Triceps brachii (surface) 32.5 67.5 > 52.5 > 87.5
Triceps brachii (deep) 32.7 19.6 > 52.7 > 39.6
Vastus lateralis (surface) 37.8 67.3 > 57.8 > 87.3
Vastus lateralis (deep) 46.9 53.1 > 66.9 > 73.1
Cotta et al. Surgical and Experimental Pathology (2021) 4:3 Page 9 of 20

Table 4 Selected routine muscle stains and histochemical reactions (Dubowitz et al., 2020; Loughlin 1993; Udd et al. 2019)
1) Hematoxylin and eosin (HE)
General architecture, regeneration, necrosis, phagocytosis, perifascicular atrophy, nuclear internalization, rimmed vacuoles, and inflammation (Figs. 3
and 4).
2) Gomori modified trichrome:
Muscle fibers sarcoplasm is blue-green, nuclei are red, and collagen is light green (Figs. 3and 4). Sarcoplasmic reticulum, mitochondria, tubular aggre-
gates, cytoplasmic bodies and reducing bodies are dark red (Dubowitz et al., 2020; Loughlin 1993).
Congenital nemaline myopathy derives from the greek word “nema”that means “thread”(Dubowitz et al., 2020), that presents rod structures that
may be observed in red on the Gomori modified trichrome stain (Fig. 4). On electron microscopy, these structure are electrondense rods with a
lattice structure similar to the Z disks of the sarcomere (Fig. 6).
In mitochondrial myopathies, ragged red fibers, correspond to mitochondrial proliferation (Fig. 4).
3) Periodic acid Schiff (PAS) without and with diastase:
Glycogen, neutral mucopolysacarides, glycoproteins, mucoproteins, glycoproteins, glycolipids, some insaturated lipids and phopholipids (Figs. 3and
4) (Dubowitz et al., 2020; Loughlin 1993). The evaluation of PAS and PAS with diastase (glycogen is diastase labile) stains is useful to the diagnosis of
glycogenoses with intrasarcoplasmic glycogen accumulation (Fig. 4).
4) Oil-red-O or Sudan-black:
Neutral lipids stain red (Oil-red-O) (Figs. 3and 4) or black (Sudan black).
5) Succinate dehydrogenase (SDH):
Enzymatic activity of the nuclear encoded mitochondrial respiratory chain complex II that reacts selectively with mitochondria (Figs. 3and 4)
(Dubowitz et al., 2020; Loughlin 1993). It is very useful for the diagnosis of mitochondrial myopathies.
6) Cytochrome-c-oxidase (COX):
Enzymatic activity of the mitochondrial encoded respiratory chain complex IV that reacts selectively with mitochondria (Figs. 3and 4) (Dubowitz
et al., 2020; Loughlin 1993).
7) Combined Cytochrome-c-oxidase/ succinate dehydrogenase (double COX-SDH):
Histochemical reactions performed in sequence on the same frozen section that permits the visualization of COX negative fibers with retention of
SDH activity, useful for the diagnosis of mitochondrial myopathies (Fig. 4).
8) Nicotinamide adenine dinucleotide (NADH):
Oxidative enzyme on mitochondrial and endoplasmic reticulum, that is located in the sarcoplasm. It is very useful to demonstrate the muscle fiber
intermyofibrillar network (Figs. 3and 4) (Dubowitz et al., 2020; Loughlin 1993).
Congenital central core myopathy presents clear or empty areas on oxidative reactions COX, SDH, and NADH (Figs. 1and 4). The name central core
was given to this round structure that may sometimes occupy the central part of the muscle fibers and they may be visualized on transversal a
longitudinal section (Figs. 3and 4) (Dubowitz et al., 2020). However, these structure may present either central or peripheral disposition. On electron
microscopy, these structures correspond to areas of myofibrillar disorganization with absence of scarcity of mitochondria and sarcoplasmic reticulum,
that are responsible for the oxidative reactions (Figs. 1and 6) (Dubowitz et al., 2020).
9) Myosinic ATPase:
Differentiation of muscle fiber types. In normal muscle, muscle fibers types are alternately organized in a checkerboard pattern (Figs. 3and 4)
(Dubowitz et al., 2020; Loughlin 1993). The type of the fiber is determined by the motor neuron that innervates each fiber and varies in each muscle
(Table 3). This reaction if useful to demonstrate type grouping in neurogenic muscle abnormalities (Fig. 4). Type 1 fiber predominance is common in
congenital myopathies. In congenital fiber type disproportion, there is type 1 fiber predominance and atrophy.
10) Acid phosphatase:
Areas of increased lysosomal activity, necrotic fibers, and lipofuscin. It is very useful for the diagnosis of glycogenosis type II (Pompe disease) and
some vacuolar myopathies (Dubowitz et al., 2020; Loughlin 1993).
11) Myophosphorylase:
Enzyme present in the intermyofibrillar space aqueous sarcoplasm (Loughlin 1993). Its deficiency is useful for the diagnosis of glycogenosis type V
(McArdle disease) (Fig. 4) (Dubowitz et al., 2020).
12) Phosphofructokinase:
The reaction may be absent in patients with glycogenosis type VII (Tarui disease) (Dubowitz et al., 2020).
13) Myoadenylate deaminase:
The histochemical reaction that may be absent in some patients with exertional myalgia (Dubowitz et al., 2020).
14) Nonspecific esterase:
This stain highlights neuromuscular junctions, myotendinous junctions, phagocytosis, and small angulated denervated fibers (Dubowitz et al., 2020).
15) Alkaline phosphatase:
It highlights the normal blood vessels and it is increased in the perimysium in some inflammatory myopathies (Dubowitz et al., 2020).
16) Menadione-linked alpha-glycerophosphate:
This stain is very useful for the diagnosis of reducing body myopathy (Dubowitz et al., 2020).
17) Congo red:
This stain that permits the visualization of amyloid deposits (Dubowitz et al., 2020).
Cotta et al. Surgical and Experimental Pathology (2021) 4:3 Page 10 of 20

fragments are fixed in glutaraldehyde for electron mi-
croscopy, whenever possible. A muscle fragment is for-
malin fixed for transverse and longitudinal sections.
About 80 to 90% of the muscle sample is liquid nitrogen
frozen. Special recommendations for this procedure may
be found in the didactic videos following the World
Muscle Society instructions (Table 1) (Engel and Fran-
zini-Armstrong 2004; Karpati et al. 2010; Dubowitz et al.
2013; Dubowitz and Sewry 2007; Dubowiz 1995; Dubow-
itz and Brooke 1973; Loughlin 1993; Anderson 1985;
Amato and Russell 2008; Levy 1978; Dastgir et al. 2016;
Fardeau 2017; Malfatti and Romero 2017). It is
important to avoid freezing artifacts and most reference
laboratories use isopentane precooling for this purpose
(Table 1). An adaptation of these procedures using tal-
cum powder has been used with success in a reference
center for more than three decades (Fig. 2) with satisfac-
tory morphological (Figs. 3, 4, and 5) results (Werneck
and Silvado 1981;So1985). This procedure has also
been useful for complementary mitochondrial respira-
tory chain, Western blot, and Southern blot studies, per-
formed on archived remaining muscle tissue stored
either in liquid nitrogen containers or in −80 °C freezer
(Figs. 1and 2).
Table 5 Rimmed vacuoles in muscle biopsies: selected differential diagnosis
Gene Neuromuscular disorder
ANO5 Anoctaminopathy or limb girdle muscular dystrophy type R12, LGMD-R12 (in the old classification LGMD2L) (Hicks et al., 2011, Straub
et al., 2018)
CRYAB Alpha-beta-crystallinopathy distal myopathy or alpha-beta-crystallinopathy myofibrillar myopathy (Udd 2012)
DMD Dystrophinopathy type Becker muscular dystrophy (de Visser et al., 1990)
DNAJB6 Limb girdle muscular dystrophy type D1 (LGMD1D, in the old classification LGMD1D) (Hackman et al., 2011, Harms et al., 2012, Straub
et al., 2018)
DUX4 Facioscapulohumeral muscular dystrophy (FSHD) (Reilich et al., 2010)
DUX4/ SMCHD1 Facioscapulohumeral muscular dystrophy (FSHD) (Reilich et al., 2010)
EMD X-linked Emery-Dreifuss muscular dystrophy (Paradas et al., 2005)
FHL1 Reducing body myopathy (Waddell et al., 2011)
FKRP Fukutin-related proteinopathy or limb girdle muscular dystrophy type R9, LGMD-R9 (in the old classification LGMD2I) (Yamamoto
et al., 2008, Straub et al., 2018)
GNE Hereditary inclusion body myopathy (hIBM) or Nonaka distal myopathy or GNE myopathy (Nishino et al., 2015)
LAMA2 Merosinopathy or merosin deficient congenital muscular dystrophy (Rajakulendran et al., 2011)
LDB3/ ZASP Markesbery-Griggs distal myopathy (ZASP myofibrillar myopathy) (Udd 2012)
LMNA Laminopathy or Emery-Dreifuss muscular dystrophy (Fang et al., 1997)
MATR3 Vocal cord and pharyngeal weakness with distal myopathy (“VCPDM”) (Palmio et al., 2016)
MYHC-IIA Type 3 hereditary inclusion body myopathy (hIBM3)(Narayanaswami et al., 2014)
MYH2 Myosinopathy (subtype) (Oldfors 2007)
MYH7 Laing distal myopathy (myosinopathy subtype) (Lefter et al., 2015)
MYOT Myotilin distal myopathy or myotilin myofibrillar myopathy (Udd 2012)
MYOT Myotilinopathy (Pénisson-Besnier et al., 2006)
PABPN1 Oculopharyngeal muscular dystrophy (Engel and Franzini-Armstrong, 2004)
PNPLA2 PNPLA2 causes neutral lipid storage disease with myopathy and triglyceride deposit cardiomyovasculopathy (Kaneko and Aoki, 2014)
TCAP Telethoninopathy or limb girdle muscular dystrophy type R7, LGMD-R7 (in the old classification LGMD2G) (Moreira et al., 1997;
Negrão et al., 2010; Cotta et al., 2014a,b, Straub et al., 2018, Cotta et al., 2019)
TIA1 Welander distal myopathy (Udd 2012)
TNPO3 Transportinopathy or limb girdle muscular dystrophy type D2, LGMD-D2 (in the old classification LGMD1F) (Gamez et al., 2001, Straub
et al., 2018)
TTN Udd distal myopathy (titinopathy) (titin gene, allelic to Limb Girdle Muscular Dystrophy R10, LGMD-R10, in the old classification
LGMD2J) (Udd 2012, Straub et al., 2018)
VCP Hereditary inclusion body myopathy with Paget’s disease of bone and frontotemporal dementia “IBMPFD”(Narayanaswami et al.,
2014)
na Sporadic inclusion body myositis (sIBM) (Greenberg 2019)
na Dermatomyositis (Layzer et al., 2009)
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The talcum powder technique is based on the
principle of “powder coat ultrarapid freezing”, that pre-
vents the appearance of artifacts caused by ice crystals
(Moline and Glenner 1964; Werneck and Silvado 1981;
So 1985). In brief (Fig. 2): (1) prepare the intraoperative
laboratory room where the cryostat is located with: Petri
dish; tweezers; sharp blades; cryotubes; a glutaraldehyde
tube (Eppendorf size type); cork disks identified with the
number of the muscle biopsy and the initials of the pa-
tients written in a white adhesive surgical tape wrapped
around the cork disk; unscented dry talcum powder (Hy-
drous Magnesium Silicate); OCT (optimal cutting
temperature) mounting medium; glass slides; coverslips;
and HE (hematoxylin and eosin) staining kit; (2) after
the extraction of the muscle in the operating room, the
fragment is involved in a gauze slightly moisted in saline
for transport to the intraoperative laboratory room, lo-
cated inside the surgical center; (3) the muscle inside the
gauze is placed over a Petri dish; (4) the muscle is sepa-
rated from the gauze; (5) one to four 0.1 × 0.1 cm
muscle fragments are cut from the muscle sample and
immediately immersed in 2% glutaraldehyde; (6) the
fresh muscle tissue is oriented according to the muscle
fiber direction, if necessary, to make a transverse frozen
muscle block; (7) one extremity of the fragment is
chosen to serve as the base of the frozen block; (8) the
fresh muscle sample is covered in all sides, except the
base, by a generous amount of dry talcum powder; (9)
optimal cutting temperature (OCT) mounting medium
is applied on the surface of the previously identified
cork; (10) the mounting medium is used to connect the
base of the block to the previously identified cork disk;
(11) the cork disk with the muscle is immersed in liquid
nitrogen for 10 to 15 s; (12) all excess of talcum powder
should be mechanically removed with a precooled blade
before performing the cryostat sections; (13) the
remaining fresh muscle tissue without talcum is frozen
Table 6 Neuromuscular immunohistochemistry: selected
antibodies (Dubowitz et al., 2020; Dubowitz et al., 2013; Paradas
et al., 2005; Dastgir et al. 2016; Udd et al. 2019)
Reaction
Dystrophinopathies
Spectrin (sarcoplasmic membrane integrity marker) SM
Dystrophin Rod domain (DYS1) SM
Dystrophin carboxy-terminal (DYS2) SM
Dystrophin amino-terminal (DYS3) SM
Utrophin SM
Neonatal myosin S/ RF
Other muscular dystrophies
Spectrin (sarcoplasmic membrane integrity marker) SM
Alpha-sarcoglycan SM
Beta-sarcoglycan SM
Gamma-sarcoglycan SM
Delta-sarcoglycan SM
Dysferlin SM
Caveolin SM
Telethonin S
Emerin NM
Collagen VI EM
Laminin alpha 5 EM
Laminin beta 1 EM
Laminin gamma 1 (extracellular matrix integrity marker) EM
Merosin (alpha-2 laminin) (80 kDa, 300 kDa) EM
Integrin alpha-7 SM
Alpha-dystroglycan (VIA4, IIH6C4) EM
Beta-dystroglycan SM
nNOS (neuronal nitric oxide synthase) SM
Inflammatory myopathies
CD3 TL
CD4 TL
CD8 TL
CD20 BL
CD138 P
CD45 TL/ BL
CD68 Mac
CD31 C
MHC-I C
p62 A
C5b-9 C
Myofibrillar myopathies
Desmin S
Myotilin S
Beta-crystallin S
Table 6 Neuromuscular immunohistochemistry: selected
antibodies (Dubowitz et al., 2020; Dubowitz et al., 2013; Paradas
et al., 2005; Dastgir et al. 2016; Udd et al. 2019)(Continued)
Reaction
Sporadic inclusion body myositis
Ubiquitin S
Myosins
Rapid myosin S
Slow myosin S
Embryonic myosin S
Fetal myosin S
Reaction = immunohistochemistry reaction pattern. SM sarcoplasmic
membrane, Ssarcoplasm, EM extracellular matrix, NM nuclear membrane, L
lymphocyte, TL T lymphocyte, BL B lymphocyte, TL/BL T and B lymphocytes, C
capillary vessel wall, Pplasma cell (Dastgir et al., 2016), RF regeneration fiber,
Mac macrophage, Aaggregates
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in cryotubes for molecular or respiratory chain enzym-
atic studies whenever appropriate; (14) if there is
remaining fresh muscle, procedures “6”to “11”should
be performed for additional transverse or longitudinal
muscle blocks; (15) the transverse muscle block is cut at
−24 °C inside the cryostat with 8 to 10 μm thickness;
(16) a slide with a section correspondent to the first
transverse muscle block should be prepared and HE
stained, in order to give information about sample ad-
equacy to the surgeon, and to elaborate the intraopera-
tive report; (17) one section of each muscle block should
be cut and stained with HE; (18) a muscle sample is
fixed in buffered formaline; (19) all numbers and letters
written to identify the patient sample should be checked
again on the tubes and cork disks, carefully, before stor-
age either in liquid nitrogen containers or in −80 °C
freezer.
The talcum powder technique was chosen in our la-
boratory based on three negative characteristics from
isopentane. Isopenate is more expensive, it presents po-
tential explosion hazard, and it presents slower ultra-
rapid freezing properties. According to Moline and
Glenner (1964), powder coated liquid nitrogen achieved
−50 °C between 17 and 26 s, while isopentane cooled li-
quid nitrogen achieved the same temperature only after
34 to 74 s. The option to choose between the talcum
powder technique over the well known isopentane pre-
cooled technique should be careful. The talcum powder
technique should be used only if the technician is
instructed to mechanically remove all excess of talcum
powder using a precooled blade. Slide artifacts may be
created due to the abrasion of talcum against the cutting
blade if it is not appropriately removed.
Table 7 Immunohistochemistry and Western blot secondary antibody deficiency
Antibody Primary myopathy
Calpain Dysferlinopathy or LGMD-R2 dysferlin-related (Anderson et al., 2000)
Fukutin-related proteinopathy or LGMD-R9 FKRP-related (Yamamoto et al., 2008)
Titinopathy or LGMD-R10 titin-related (Pénisson-Besnier et al., 2010; Udd et al., 2005)
Anoctaminopathy or LGMD-R12 anoctamin-related (Penttilä et al., 2012)
Dysferlin Calpainopathy or LGMD-R1 calpain-related (Anderson et al., 2000, Groen et al., 2007)
Caveolinopathy (Müller et al., 2006, Matsuda et al., 2001)
Dystrophinopathy or Duchenne/ Becker muscular dystrophy (Izumi et al., 2015)
Sarcoglycanopathy or LGMD-R3-R4-R5-R6 or alpha, beta, gamma, delta sarcoglycan-related (Izumi et al., 2015)
Anoctaminopathy or LGMD-R12 anoctamin-related (Izumi et al., 2015)
GNE myopathy or Nonaka distal myopathy (Izumi et al., 2015)
Myotilinopathy (Izumi et al., 2015)
Caveolin Dysferlinopathy or LGMD-R2 dysferlin-related (Barresi 2011)
Calpain deficiency detectable by Western blot
Table 8 Morphometric muscle biopsy values (Bernier et al., 2002; Clarke 2011; Dubowitz et al., 2020)
Fiber size disproportion quotient
ðType 2D −Type 1DÞ
Type 2D Dubowitz criteria at least 25%
Clarke criteria at least 35 to 40%
Mean type 2 fiber diameter = Type 2D
Mean type 1 fiber diameter = Type 1D
Mitochondrial disorder diagnostic criteria
Below 16 yo More than 2% subsarcolemmal accumulation minor criteria
Below 30 yo Any ragged red fiber minor criteria
Between 30 and 50 yo 1–2% ragged red fibers minor criteria
Any age More than 2% ragged red fibers major criteria
Below 50 yo More than 2% COX negative fibers major criteria
Above 50 yo More than 5% COX negative fibers major criteria
Fiber size disproportion quotient: Mean type 2 fiber (Type 2 D) diameter less mean type 1 fiber (Type 1 D) diameter; the difference is divided by the mean type 2
fiber diameter (Type 2 D). Fiber size disproportion is observed in all Congenital Fiber Type Disproportion patients, but many other conditions may present fiber
size disproportion (nemaline myopathy, Steinert myotonic dystrophy, Spinal Muscular Atrophy) (Clarke 2011); yo: years old; subsarcolemal accumulations:
mitochondrial subsarcolemal accumulations on SDH reaction. Ragged-red fibers are usually quantified as ragged-red equivalent or ragged-blue fibers on SDH
reaction characterized by marked subsarcolemal and intrasarcoplasmic mitochondrial proliferation
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Intraoperative frozen section stained with
hematoxylin and eosin (HE)
Intraoperative frozen section is an excellent laboratorial
practice for rapid laboratory and neurologic clinic staff
communication. Immediately after the muscle biopsy
procedure, liquid nitrogen frozen sections are HE
stained. In few minutes, the valuable information about
sample adequacy is provided for the surgeon while the
patient is still inside the operative room. Therefore, the
patient is given the possibility to have another adequate
fragment extracted before the end of the surgery, if
necessary.
Transverse HE stain permits the evaluation of the gen-
eral architecture, muscle fibrous-fat replacement, fiber
size and shape, number and position of the nucleus,
sarcoplasmic basophilia (bluish stain) indicative of re-
generation, necrosis, and phagocytosis, perifascicular at-
rophy, nuclear internalization, and rimmed vacuoles
(Fig. 4) (Dubowitz et al. 2020; Loughlin 1993). Longitu-
dinal HE sections provide means to evaluate larger
muscle segments in order to identify focal inflammation
and nuclear internalization such as nuclear chains (e.g.,
congenital myotubular myopathy).
Which exams should be performed on the muscle
biopsy specimen?
Hematoxylin and eosin, histochemical stains and reac-
tions, morphometry, immunohistochemistry, immuno-
fluorescence, electron microscopy, mitochondrial
respiratory chain enzymatic studies, muscle tissue mo-
lecular studies (Western blot, Southern blot), and histo-
pathological study of paraffin embedded material
(Tables 4,5,6,7and 8) (Figs. 3,4,5and 6).
A fundamental part of morphologic muscle exam is
the evaluation of muscle enzymes that are viable only in
frozen unfixed sections as formalin fixation causes loss
of reactivity.
The most common stains and reactions usually per-
formed at a neuromuscular service are: hematoxylin and
eosin (HE), Gomori modified trichrome, PAS (Periodic
acid Schiff) with and without diastase, Oil-red-O or
Sudan-black, SDH (succinate dehydrogenase), COX (cyto-
chrome c oxidase), combined COX-SDH, NADH (nico-
tinamide adenine dinucleotide), myosinic ATPase, acid
phosphatase, nonspecific esterase, myophosphorylase,
phosphofructokinase, myoadenylate deaminase, alkaline
phosphatase, menadione-linked alpha-glycerophosphate,
and Congo red (Table 4)(Figs.3and 4).
Special microscopic findings: rimmed vacuoles
interpretation
Rimmed vacuoles (Fig. 4) are abnormal empty spaces or
cavities within the sarcoplasm, irregular or round, local-
ized in any part of the fiber, in which the rim of the
vacuole contains a basophilic granular material (HE) or
red granular material (modified Gomori trichrome)
(Engel and Franzini-Armstrong, 2004) (Fig. 4). They are
not infrequent in myology practice but their identifica-
tion should be a clue for the some specific diagnoses
(Table 5).
Sporadic inclusion body myositis is one of the most
common diagnoses in myopathology practice (Figs. 4,5
and 6). A clinically defined diagnosis of sporadic inclu-
sion body myositis is rendered when patients present,
after the age of 45 years, for more than 1 year, muscle
weakness that is worse on knee extension than hip
flexion, and worse on finger flexion than on shoulder ab-
duction, associated with serum creatine kinase increase
of less than 15 times the reference value (Rose and
ENMC IBM Working Group 2013). In this clinical set-
ting, a muscle biopsy with endomysial lymphocytic in-
flammatory infiltrate invading nonnecrotic muscle fibers
and rimmed vacuoles, that correspond to 15-18 nm
filamentous inclusions on electron microscopy, allows to
establish a definite diagnosis of sporadic inclusion body
myositis (Figs. 4,5and 6) (Rose and ENMC IBM Work-
ing Group 2013; Van De Vlekkert et al. 2015).
Nevertheless, in other cases, when the clinical presen-
tation is not characteristic of inclusion body myositis, it
is important to be cautious. Rimmed vacuoles should
not be considered sufficient for diagnosis of inclusion
body myositis, as they have been reported in various
muscle dystrophies, distal myopathies, and even in de-
nervated muscles (Table 5, Hicks et al. 2011, Straub et
al. 2018, Cotta et al. 2019, Udd 2012, de Visser et al.
1990, Hackman et al. 2011, Harms et al. 2012, Reilich et
al. 2010, Paradas et al. 2005, Waddell et al. 2011,
Table 9 Rationale for myopathy investigation
1. Complete anamnesis and neurological physical examination;
2. Family history with pedigree;
3. Exclude four common neuromuscular diagnosis that may be
diagnosed clinically and confirmed by molecular studies:
- Spinal muscular atrophy (SMA)
- Myotonic dystrophy type 1 (DM1, Steinert’s myotonic dystrophy)
- Facioscapulohumeral muscular dystrophy (FSHD)
- Dystrophinopathy type Duchenne muscular dystrophy
4. Electromyogram;
5. Muscle serum enzymes: creatine kinase, aldolase;
6. Muscle imaging: magnetic resonance imaging (MRI), ultrasound,
ocasionally computed tomography for patients that cannot be
submitted to MRI (e.g., pacemaker users, metal implants, etc.);
7. Brain imaging if suspected of encephalomyopathy.
8. According to the clinical suspicion: Muscle biopsy or Molecular
Studies such as Next Generation Sequencing (NGS) or targeted
molecular sequencing of the specific gene group related to muscle
biopsy findings.
Cotta et al. Surgical and Experimental Pathology (2021) 4:3 Page 14 of 20

Yamamoto et al. 2008, Nishino et al. 2015, Rajakulen-
dran et al. 2011, Fang et al. 1997, Palmio et al. 2016,
Narayanaswami et al. 2014, Oldfors 2007, Lefter et al.
2015, Pénisson-Besnier et al. 2006, Engel and Franzini-
Armstrong 2004, Kaneko and Aoki 2014, Moreira et al.
1997; Negrão et al. 2010; Cotta et al. 2014a,2014b,
Gamez et al. 2001, Greenberg 2019, Layzer et al. 2009).
Immunohistochemistry and immunofluorescence
Either immunohistochemistry or immunofluorescence in
frozen sections are diagnostic procedures that use anti-
bodies directed to specific muscle tissue proteins, that
may be absent, decreased or increased (Tables 6and 7)
(Fig. 5).
Immunohistochemistry is very useful for the diagnosis
of dystrophinopathies (in cases where molecular exams
are inconclusive), congenital muscular dystrophies, limb
girdle muscular dystrophies, X-linked, and inflammatory
myopathies (Table 5) (Dastgir et al. 2016;O’Connell et
al. 2004).
Patients with sporadic inclusion body myositis may
present invasion of nonnecrotic muscle fibers by T cell
cytotoxic/ suppressor CD8 positive lymphocytes (Fig. 5)
as well as abnormal expression of the major histocom-
patibility complex class I (MHC-I) on sarcoplasmic
membranes (Fig. 5).
Myofibrillar myopathies may present abnormal intra-
sarcoplasmic deposits reactive for desmine as well as ec-
topic intrasarcoplasmic dystrophin reactivity, in contrast
to normal sarcoplasmic membrane dystrophin expres-
sion (Fig. 5).
The different clinical presentations of dystrophinopa-
thy may present different patterns of dystrophin expres-
sion. Patients with Duchenne muscular dystrophy
usually present complete immunohistochemical dys-
trophin deficiency of three epitopes (Rod domain,
carboxy-terminal and amino-terminal) (Fig. 5). Patients
with less severe Becker muscular dystrophy clinical pres-
entation may present partial or decreased immunohisto-
chemical dystrophin expression (Fig. 5).
Some patients with Becker muscular dystrophinopathy
present only slight dystrophin decrease, associated with
abnormal sarcolemmal utrophin expression (Fig. 5). In
normal conditions, utrophin is expressed only in neuro-
muscular junctions, blood vessel walls, and regenerating
fibers (Karpati et al. 2010; Dubowitz et al. 2020). There-
fore it is important to analyze both utrophin and neo-
natal myosin to identify regenerating fibers (Fig. 5).
Patients with dystrophin deficiency present hyperexpres-
sion of utrophin, that is a dystrophin homologue codi-
fied on chromosome 6q24 (Karpati et al. 2010; Dubowitz
et al. 2020).
Immunohistochemistry for nNOS (neuronal nitric
oxide synthase) is also very useful for the diagnosis of
patients with Becker muscular dystrophy (Table 6)
(Dubowitz et al. 2020; Nix and Moore 2020). Some pa-
tients with Becker muscular dystrophy and in frame de-
letions of exons 45 to 51 may present deficient nNOS
with normal dystrophin antibodies to dystrophin Rod
domain (DYS1), Dystrophin carboxy-terminal (DYS2),
and Dystrophin amino-terminal (DYS3) (Dubowitz et al.
2020, Nix and Moore 2020). Therefore, for that group of
patients, nNOS deficiency may be the only clue to the
correct immunohistochemical diagnosis.
Female symptomatic carriers of dystrophinopathy
present a mosaic pattern of dystrophin deficiency, ie,
with groups of dystrophin positive and negative fibers
(Fig. 5), that in most cases are related to inactivation of
the mutated X chromosome in some fibers (Karpati et
al. 2010).
The performance of all immunohistochemical reac-
tions simultaneously with normal controls, cut in the
same slides, permits the comparison of the intensity of
the immunohistochemical reactions between patient and
control. The pattern of reaction varies in accordance to
the studied protein; it may be on the sarcoplasmic mem-
brane, blood vessels, nuclear membranes or contractile
proteins of the sarcoplasm (Fig. 5).
Immunohistochemical reactions may show primary or
secondary deficiencies. In primary deficiencies, the defi-
cient antibody reaction demonstrates concordance be-
tween the immunophenotypic and genotipic diagnosis.
The immunohistochemical deficiency of dysferlin is con-
sidered a primary deficiency when a patient presents a
pathogenic variant in the dysferlin gene. In this case, the
patient receives a definite diagnosis of phenotype and
genotype of Limb Girdle Muscular Dystrophy type R2 or
LGMD-R2 dysferlin-related (LGMD2B in the old classi-
fication) (Straub et al. 2018).
Secondary deficiencies (Table 7) reflect a discordance
between immunophenotypic and genotypic diagnosis.
Therefore, one must be cautious on immunohistochemi-
cal results interpretation, as secondary deficiencies may
occur (Table 7, Anderson et al. 2000; Yamamoto et al.
2008; Pénisson-Besnier et al. 2010; Udd et al. 2005; Pent-
tilä et al. 2012; Groen et al. 2007; Müller et al. 2006;
Matsuda et al. 2001; Izumi et al. 2015; Barresi 2011).
In order to choose the most appropriate immunohisto-
chemical panel, clinical data are or utmost importance.
It is also important to remember, that some muscle dys-
trophies that occur characteristically in adulthood, may
ocasionally, present early onset, such as dysferlinopathy
(Paradas et al. 2009), telethoninopathy (Ferreiro et al.
2011), and caveolinopathy (Madrid et al. 2005).
Morphometry in histochemical reactions
Morphometric analysis may be nowadays performed on
digital photographs with micrometric scales using free
Cotta et al. Surgical and Experimental Pathology (2021) 4:3 Page 15 of 20

softwares such as Image J (http://imagej.nih.gov/ij) and
LibreOffice (https://libreoffice.org) (Table 8).
Congenital fiber type disproportion presents type 1
fiber predominance and almost all type 1 fibers are
smaller than type 2 fibers. The exact percentage varies
according to the reference: at least 25% (Dubowitz et al.
2020) or at least 35 to 40% (Clarke 2011). This latter
more strict criteria aims to differentiate between fiber
type disproportion that may follow many different con-
genital myopathies from true cases of fiber type dispro-
portion congenital myopathy (Clarke 2011).
Morphometry is also useful for the evaluation of mito-
chondrial myopathies using SDH (succinate dehydrogen-
ase) and double COX-SDH (double cytochrome-c-
oxidase followed by succinate dehydrogenase) reactions.
The proportion of ragged red equivalent fibers or ragged
blue fibers and COX (citochrome c oxidase) negative fi-
bers are used as diagnostic morphological criteria (Table
8) (Bernier et al. 2002).
Electron microscopy
Transmission electron microscopy is used to evaluate
structural muscle components such as sarcolemma,
sarcomere, nucleus, mitochondria, and blood vessels.
Therefore, electron microscopy is useful for the diagno-
sis of congenital myopathy (specially recommended for
the diagnosis of core myopathies) (North et al. 2014),
myofibrillar myopathy, mitochondrial myopathy, and in-
flammatory myopathy. Among the inflammatory myop-
athies, electron microscopy is useful to confirm sporadic
inclusion body myositis (Fig. 6). Patients with dermato-
myositis may present tubulorreticular inclusions inside
the cytoplasm of endothelial cells of the capillary blood
vessels. These are considered early findings during dis-
ease evaluation, that may be found even before the ap-
pearance of the inflammatory infiltrate (Fig. 5) (Bronner
et al. 2008).
Muscle tissue molecular studies
Muscle biopsy tissue may be used to perform molecular
studies such as Southern blot and western blot.
Southern blot studies are very useful in molecular
pathology in order to identify mitochondrial DNA dele-
tions, as in progressive external ophthalmoplegia mito-
chondriopathy (Bohlega et al. 1996; Shapira and
DiMauro 2002). The identification of one single deletion
on the mitochondrial DNA, most frequently a 4977 base
pair deletion, “common deletion”, occurs frequently in
sporadic progressive external ophthalmoplegia (Shapira
and DiMauro 2002). On the other hand, patients with
progressive external ophthalmoplegia with autosomal in-
heritance may present multiple mitochondrial DNA
deletions.
Western blot may be used to quantify the decrease in
protein product in patients with muscular dystrophies.
The antibodies may include dystrophin, alpha-
dystroglycan, dysferlin, calpain, and telethonin, among
others.
In Becker muscular dystrophy, the milder form of dys-
trophinopathy compared to Duchenne muscular dys-
trophy, western blot may be helpful to detect only subtle
decrease in dystrophin bands.
Western blot studies are very useful for the diagnosis
of limb girdle muscular dystrophies. Calpain is an en-
zyme that presents rapid degradation, that precludes im-
munohistochemical detection (Groen et al. 2007).
Therefore, western blot is very useful for detection of
calpain deficiency in limb girdle muscular dystrophy R1
(LGMD-R1 calpain-related). Dysferlin deficiencies may
be detected either through western blot or through im-
munohistochemistry (with Polymer signal amplification).
Western blot may also be useful for the detection of
anoctamin-5 protein reduction in limb girdle muscular
dystrophy R12 (LGMD-R12 anoctamin-related) (Vihola
et al. 2018).
Southern blot studies received this name in honor to
the last name of the researcher that first detected spe-
cific DNA sequences in forensic medicine studies to de-
tected criminal DNA in corporeal fluids. He was able to
separate DNA fragments with restriction enzymes that
section the DNA and later identify the sequences with
labeled nucleic acid complementary probes (Nelson and
Cox 2008). Western blot studies are molecular studies
used to detect proteins using specific antibodies; this
name was an analogy to the Southern blot technique
that detects DNA fragments.
Mitochondrial respiratory chain enzymatic activity
studies
Mitochondrial respiratory chain enzymatic studies are
very useful to detect mitochondrial complex defects
(Barrientos 2002). Combined complex II and complex
III defects are indicative of Coenzyme Q10 deficiency
and patients may have benefit and clinical improvement
with Coenzyme Q10 treatment (Sobreira et al. 1997;
Cotta et al. 2020). Either patients with primary coen-
zyme Q10 deficiency, ie, with biochemical defects in Co-
enzyme Q10 biosynthesis, as well as patients with
secondary coenzyme Q10 deficiency may present clinical
improvement with coenzyme Q10 therapy (Trevisson et
al. 2011).
Histopathological study of formalin fixed paraffin
embedded muscle tissue
Formalin fixed muscle tissue alone is usually inadequate
to confirm a neuromuscular diagnosis. We strongly
agree with the World Muscle Society recommended
Cotta et al. Surgical and Experimental Pathology (2021) 4:3 Page 16 of 20

standards that liquid nitrogen frozen specimens are ne-
cessary for good muscle biopsy diagnosis (Udd et al.
2019).
In our experience, within a group of more than 1500
muscle biopsies evaluated during the last years, paraffin
embedded material was useful for diagnosis in less than
of the cases with concomitant liquid nitrogen frozen
confirmed diagnosis. Therefore, in this group of patients,
more than 99% of paraffin embedded muscle samples re-
sulted in nonspecific or inconclusive results, whereas the
liquid nitrogen frozen samples from the same patients
were diagnostic.
Special cases in which paraffin embedded material was
very useful included inflammatory infiltrate restricted to
the fascia and small amyloid deposits. Formalin fixed,
paraffin embedded (FFPE) tissue permits the diagnosis
of: neurogenic (fascicular) atrophy, the evaluation of the
degree of collagen (with a Gomori staining) and fat re-
placement, in both transverse and longitudinal sections.
The analyses of serial sections in the paraffin block is
useful for the diagnosis of vasculitis, and other inflam-
matory conditions, that may present focal or scattered
abnormalities in the tissue (Nix and Moore 2020).
Integrated molecular and morphologic myology
investigation
A rationale may be used for ordering either molecular
studies or muscle biopsy according to clinical diagnosis
(Table 9).
Future perspectives and advanced diagnostic
techniques
Mitochondrial diseases are peculiar. The study of mito-
chondrial myopathies is very challenging, even for the
most experienced myopathologists. Frequently, muscle
tissue is the only sample suitable for molecular mito-
chondrial diagnosis. Therefore molecular studies per-
formed in DNA extracted from blood samples may be
inconclusive. These characteristics are related to three
long known factors: heteroplasmy, the threshold effect,
and mitotic segregation (DiMauro and Schon 2003).
Normal subjects present homoplasmy, characterized
by identical normal mitochondriae in all cells. Patients
with mitochondriopathies present heteroplasmy, that is
and admixture of normal and mutant mitochondrial
DNA in different cells and tissues (DiMauro and Schon
2003). The threshold effect is the minimal number of
mutant mitochondrial DNA necessary to lead to oxida-
tive dysfunction in a given tissue (DiMauro and Schon
2003). Mitotic segregation is the random distribution of
mitochondriae at the time of cell division, resulting in
different proportions of mutant mitocondrial DNA in
different tissues (DiMauro and Schon 2003).
The omics era for muscle pathology has already begun
for patients suspected of mitochondrial myopathies and
encephalomyopathies (Rahman and Rahman 2018;
Hathazi et al. 2020). Omics technologies are promising
technologies that integrate various fields of molecular
analysis. They may be gradually incorporated to path-
ology routine practice during the next years or decades
(Karczewski and Snyder 2018; Rahman and Rahman
2018). Omics technologies include: genomics, tran-
scriptomics, proteomics, metabolomics, epigenomics,
and interactomics (Rahman and Rahman 2018).
Genomics includes the study of the nuclear and mito-
chondrial genome including: next generation sequencing
(NGS), whole exome sequencing (WES), and whole gen-
ome sequencing (WGS) (Rahman and Rahman 2018).
Transcriptomics is the study of RNA, the product of
DNA transcription. The RNA whole transcriptome is
analyzed using the technique of RNA sequencing (RNA-
seq) (Rahman and Rahman 2018). This technique allows
the identification of deep intronic variants affecting spli-
cing (Rahman and Rahman 2018). Sometimes, RNA se-
quencing may be helpful to understand the
consequences of DNA variants detected through WGS
(Rahman and Rahman 2018). This technique was crucial
for the discovery of the serum metabolic marker growth
differentiation factor-15 (GDF-15) (Kalko et al. 2014).
GDF-15 and fibroblast growth factor-21 (FGF-21) are
useful serum metabolic markers for the diagnostic inves-
tigation of mitochondrial disorders (Scholle et al. 2018;
Lehtonen et al. 2020; Hathazi et al. 2020).
Proteomics includes the quantitative study of proteins
using mass spectrometry. This technique allows the
study of the entire mitochondrial ribosome and the
post-translational modifications such as lysine acetyl-
ation, malonylation, and succinylation (Rahman and
Rahman 2018, Hathazi et al. 2020).
Metabolomics is a spectrometry-based technique. The
metabolome technique enables to profile thousands of
molecules. It allows the identification of products of oxi-
dative stress, redox imbalance, and energy deficiency
(Rahman and Rahman 2018).
Epigenomics includes the study of reversible alter-
ations in DNA expression that do not modify the DNA
sequence such as the downstream regulation of the Tri-
carboxylic acid cycle (Krebs cycle) induced by metabo-
lites and enzymes (Rahman and Rahman 2018).
Interactomics or integrated omics is the combined in-
terpretation of data from transcriptomics, proteomics,
and metabolomics. It may be useful to reveal the physio-
pathological pathways for mitochondrial cristae
organization, endoplasmic reticulum-mitochondrial
communication, and mitochondrial dynamics (mito-
chondrial motility within the cell) (Rahman and Rahman
2018, Hathazi et al. 2020).
Cotta et al. Surgical and Experimental Pathology (2021) 4:3 Page 17 of 20

Conclusions
In ideal situations, the choice of the muscle to be sub-
mitted to biopsy involves an integrated multiprofessional
approach to provide appropriate patient care. Liquid ni-
trogen specimens are necessary for effective muscle bi-
opsy diagnosis.
The choice of the special techniques and types of exams
should be individualized for each patient, according to the
clinical presentation, symptoms evolution, familial history,
physical and neurological exam, laboratorial, neurophysio-
logical, and imaging studies.
The interpretation of muscle abnormalities should be
performed together with clinical information in order to
avoid erroneous interpretation of morphological findings.
Muscle biopsy is useful for the differential diagnosis of
immune mediated myopathies, muscular dystrophies,
congenital myopathies, and mitochondrial myopathies.
With the advent of new molecular studies, muscle bi-
opsy has been necessary to confirm the pathogenicity of
new gene variants. In doubtful cases, muscle biopsy may
provide the morphologic phenotype necessary for diag-
nostic confirmation. Due to organ specific mutation load
in mitochondriopathies, the muscle tissue may be the
only source of representative DNA or RNA samples for
molecular diagnosis.
Abbreviations
A: Aggregates; ABN: Brazilian Academy of Neurology [Academia Brasileira de
Neurologia]; Alpha-dystroglycan (VIA4, IIH6C4): Alpha-dystroglycan antibody
clones VIA4 and IIH6C4; BL: B lymphocyte; C: Capillary vessel wall;
CD8: Cluster of differentiation 8 T lymphocyte; Complex II: Complex two of
the Mitochondrial Respiratory Chain; Complex III: Complex three of the
Mitochondrial Respiratory Chain; COX: Cytochrome c oxidase; COX-
SDH: Double cytochrome-c-oxidase followed by succinate dehydrogenase re-
action; DM1: Myotonic dystrophy type 1 (Steinert’s myotonic dystrophy);
DNA: Deoxyribonucleic acid; DYS1: Dystrophin - Rod domain;
DYS2: Dystrophin - carboxy-terminal; DYS3: Dystrophin - amino terminal;
EM: Extracellular matrix; HE: Hematoxylin and eosin; FGF-21: Fibroblast
growth factor-21; FSHD: Facioscapulohumeral muscular dystrophy; GDF-
15: Growth differentiation factor-15; hIBM: Hereditary inclusion body
myopathy; IBMPFD: Hereditary inclusion body myopathy with Paget’s disease
of bone and frontotemporal dementia; L: Lymphocyte; LGMD: Limb Girdle
Muscular Dystrophy; LGMD-R1 calpain-related: Limb Girdle Muscular
Dystrophy recessive type 1 related to the calpain gene; LGMD-R2 dysferlin-
related: Limb Girdle Muscular Dystrophy recessive type 2 related to the
dysferlin gene; LGMD-R3 alpha sarcoglycan-related: Limb Girdle Muscular
Dystrophy recessive type 3 related to the alpha-sarcoglycan gene; LGMD-R4
beta sarcoglycan-related: Limb Girdle Muscular Dystrophy recessive type 4
related to the beta-sarcoglycan gene; LGMD-R5 gamma sarcoglycan-
related: Limb Girdle Muscular Dystrophy recessive type 5 related to the
gamma-sarcoglycan gene; LGMD-R6 delta sarcoglycan-related: Limb Girdle
Muscular Dystrophy recessive type 6 related to the delta-sarcoglycan gene;
LGMD-R7 telethonin-related: Limb Girdle Muscular Dystrophy recessive type
7 related to the telethonin gene; LGMD-R9 FKRP-related: Limb Girdle
Muscular Dystrophy recessive type 9 related to the FKRP gene; LGMD-R10
titin related: Limb Girdle Muscular Dystrophy recessive type 10 related to the
titin gene; LGMD-R12 anoctamin-5 related: Limb Girdle Muscular Dystrophy
recessive type 12 related to anoctamin-5 gene; LGMD-D1 DNAJB6-
related: Limb Girdle Muscular Dystrophy dominant type 1 related to the
DNAJB6 gene; LGMD-D2 TNPO3-related: Limb Girdle Muscular Dystrophy
dominant type 2 related to the TNPO3 gene; Mac: Macrophage; MHC-I: Major
histocompatibility complex class I; MRI: Magnetic resonance imaging;
MRC: Medical Research Council; NADH: Nicotinamide adenine dinucleotide;
NGS: Next Generation Sequencing; NM: Nuclear membrane; PAS: Periodic
acid Schiff; P: Plasma cell; RF: Regeneration fiber; S: Sarcoplasm;
SDH: Succinate dehydrogenase; sIBM: Sporadic inclusion body myositis;
SM: Sarcoplasmic membrane; SMA: Spinal Muscular Atrophy; TL: T
lymphocyte; TL/BL: T and B lymphocytes; Type 1D: Mean type 1 fiber
diameter; Type 2D: Mean type 2 fiber diameter; VCPDM: Vocal cord and
pharyngeal weakness with distal myopathy; WES: Whole Exome Sequencing;
WGS: Whole Genome Sequencing
Acknowledgments
We would like to thank Cleides Campos de Oliveira and Simone Ferreira
Inacio for technical assistance. We thank Dr. Isabel Cristina Soares Brandão
for the critical review of the first draft of the manuscript.
Authors´ contributions
AC was responsible for the conception, design, organization, photographic
documentation, draft, and revision of the final version of the manuscript. EC
and JV contributed with neurophysiological data. ALdCJ contributed with
muscle imaging data. SBJ contributed with mitochondrial respiratory chain
studies. MMN, AFC, MMM, SVN, RXN, APV contributed with clinical data. EBS,
MIL, BAC contributed with electron microscopy information. AC and JFP
were responsible for muscle biopsy analyses. The author(s) read and
approved the final manuscript.
Funding
Not applicable.
Availability of data and materials
Not applicable.
Ethics approval and consent to participate
This manuscript has been approved by the Ethics and Research Comittee of
The SARAH Network of Rehabilitation Hospitals and “Plataforma Brasil”,
number 1.703.918. CAAE: 58086316.6.0000.0022.
Consent for publication
A consent for publication was not necessary as no individual data from
patients were used.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Pathology, The SARAH Network of Rehabilitation Hospitals,
Av. Amazonas, 5953 Gameleira, Belo Horizonte, MG 30510-000, Brazil.
2
Department of Neurophysiology, The SARAH Network of Rehabilitation
Hospitals, Belo Horizonte, MG, Brazil.
3
Department of Radiology, The SARAH
Network of Rehabilitation Hospitals, Belo Horizonte, MG, Brazil.
4
Department
of Pediatrics, The SARAH Network of Rehabilitation Hospitals, Belo Horizonte,
MG, Brazil.
5
Department of Electron Microscopy, The SARAH Network of
Rehabilitation Hospitals, Brasília, DF, Brazil.
6
Department of Surgery, The
SARAH Network of Rehabilitation Hospitals, Belo Horizonte, MG, Brazil.
7
Department of Neurology, The SARAH Network of Rehabilitation Hospitals,
Belo Horizonte, MG, Brazil.
Received: 29 October 2020 Accepted: 21 December 2020
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