Biopsy a clinically weak muscle (MRC grade 4/5).

Biopsy is taken from the belly of the muscle, and it is important to avoid the region of the tendon.

Each specimen should be about 1-2 cm in length and 0.5 cm in width.  

The specimen should be wrapped in slightly moist gauze  and placed in separate labeled sterile containers until they reach the laboratory.  It is important not to place the specimens in a container of saline else this will lead to artifact.  Nor should the entire specimen be placed in fixative else the important histochemistry stains, protein/enzyme analysis, and mutation analysis cannot be performed.  

Obtain at least two separate specimens as muscle disorders can be multifocal (e.g., inflammatory myopathies).  

Freeze the specimen in isopentane cooled in liquid nitrogen.  The frozen tissue is then sectioned and stained for routine histochemistry. 

A separate piece of muscle is fixed in formalin or Bouin's fluid for paraffin sections. 

Another piece of muscle is taken for ultrastructural examination by EM.  This small piece of muscle tissue is secured on a clamp or stretched out by suturing the muscle over a tongue blade, in order to prevent hypercontraction artifact. The tissue is fixed in glutaraldehyde for plastic (resin) embedding for EM.

Teaching Video

muscle biopsy (univ of MI)

MUSCLE BIOPSY TISSUE  SUBMISSION PROTOCOL

The muscle biopsy should be taken from the belly of a moderately affected muscle (avoid EMG and injection sites).  

Ideally, the muscle biopsy is 2.5 – 3.0 cm length by 1.0 cm width for an adult or 2.0 cm length by 0.5 cm width for a child.  

Divide the muscle into three portions: 

a. 1.5 cm fresh (ideal) tissue portion on a saline-moistened Tefla gauze received on wet ice at refrigerator temperature. 

b. 1.0 cm in 10% neutral buffered formalin (for Histology). 

c.  0.5 cm in glutaraldehyde (for Electron Microscopy). 

The fresh tissue, once received, will be frozen by MLabs.  This specimen is most important for diagnosis as it is used for histochemical stains. If the muscle biopsy is from a child, omit the formalin fixed specimen and provide two fresh (or frozen) specimens along with the glutaraldehyde fixed specimen.  Please complete the MLabs Muscle/Renal/Nerve Biopsy Requisition that is provided in the Muscle Biopsy collection kit and send the completed requisition back to MLabs along with the specimens.   Specimen Handling Fresh tissue specimen is preferred provided that the specimen can arrive at MLabs within 4 hours of collection, Monday through Friday 7:00 am – 5:00 pm. Otherwise, the tissue must be frozen (see frozen instructions). FRESH TISSUE The first piece of muscle, 1.5 cm length by 1.0 cm width will be used for histochemical stains. Do not tease or pinch with forceps or let the tissue dry out. Wrap the tissue in a saline-moistened Telfa pad and place in a screw cap container. Do not immerse the tissue in saline. Container should be kept cold (wet ice) in transit; do not freeze. If the specimen cannot arrive at MLabs within 4 hours of collection, it must be frozen in the following manner: FROZEN TISSUE If liquid nitrogen is available: a. b. Isopentane method (PREFERRED): Add 50-100 mL of isopentane (2-methybutane) to a Nalgene or metal beaker. Suspend the beaker in a bath of liquid nitrogen and wait until the isopentane freezes to a white, chalky substance. Remove the beaker from the liquid nitrogen bath. Carefully drop or set the muscle on the frozen isopentane while keeping the muscle fibers straight and running in the same direction. Freeze the muscle for about 10 – 15 seconds. Place the frozen muscle in a Styroform container filled with 7 lbs of dry ice or in a -70ºC freezer. If isopentane is not available, roll the muscle gently in talc or glove powder, wrap it loosely in a piece of foil (do not flatten the muscle!), and immerse it into liquid nitrogen for 20 – 30 seconds. Place the frozen muscle in a Styrofoam container filled with 7 lbs of dry ice or in a -70ºC freezer until ready to ship. Avoid any thawing of specimen! If it thaws and re-freezes, the specimen may not be salvageable. Page 1 MLAB-OPS-xxxx-xxx1 2019-09-15MUSCLE BIOPSY TISSUE  SUBMISSION PROTOCOL If liquid nitrogen is not available: roll the muscle gently in talc or glove powder, wrap it loosely in a piece of foil (do not flatten the muscle!) and place in a Styrofoam container filled with 7 lbs of dry ice. Avoid any thawing of specimen! FORMALIN FIXED TISSUE The second piece of muscle, approximately 1.0 cm length by 1.0 cm width will be used for paraffin sections. Stretch the muscle longitudinally on a piece of tongue blade using sutures tied at either end, then place specimen side down in a container with 10% neutral buffered formalin (NBF). If the muscle biopsy is from a CHILD, omit the formalin fixed specimen; instead provide two fresh (or frozen specimens) along with the glutaraldehyde fixed specimen. Specimen should be sent room temperature, do not freeze.   GLUTARADLEHYDE FIXED TISSUE For the third piece of muscle tissue, 0.5 cm for electron microscopy, use a metal clamp or small piece of a sterile wooden tongue blade (size of a toothpick) to suture to a 3 mm thin portion of muscle in situ. Place this sample with the clamp or tongue blade attached in a vial containing 3% glutaraldehyde / 3% formaldehyde. Specimen should be sent at room temperature, do not freeze.  Required Information and Shipping 1. 2. 3. 4. 5. Label the containers with the patient’s name and a 2nd patient identifier such as a date of birth or a medical record number. Complete the Renal/Muscle/Nerve Biopsy Patient Clinical Requisition and the MLabs Muscle/Nerve/Renal Requisition to accompany the specimens. The formalin and glutaraldehyde fixed specimens must be shipped in a room temperature container, separate from cold or frozen material. Care must be taken that these specimens do not freeze. Do not place these in the Styrofoam container with wet or dry ice. Once the formalin and glutaraldehyde specimens have been placed in their liquid containers and the tops secured, place inside the ziploc specimen biohazard bag with the absorbent sheet and seal. Wrap the bag with bubble wrap and place inside transport box. Please be careful to not contaminate the outside of the transport bags, paperwork or box when handling. Transport at room temperature. If shipping by UPS, place closed box inside Express Pak and seal according to instructions on the Express Pak. UPS, call 800.742.5877 to schedule pick up. Specimens should be sent Monday – Thursday (preferred); if sending on Friday is necessary, clearly indicate Saturday delivery required. Questions Call 800.862.7284 or you may visit mlabs.umich.edu 

HFH:  

GENERAL MORPHOLOGIC FEATURES 

Myofiber size: variable 

Angulated fibers: present 

Fiber groups: absent 

Perifascicular atrophy: absent 

Myofiber types (ATPase pH4.3, 4.6 and 9.4): Equal populations of both fiber 

types; normal sized and atrophic fibers of both types 

Blood vessels: normal 

Amyloid (Congo red): absent 

MYOPATHIC FEATURES 

Necrotic fibers (Gomori trichrome, nonspecific esterase, CD68): present 

De-/Regenerating fibers: present 

Internal nuclei: present 

Endomysial fibrosis (Masson Trichrome): mild 

Fatty metaplasia: present 

INFLAMMATION (CD3, CD20) 

Perivascular: absent 

Endomysial: absent 

Vasculitis: absent 

CYTOARCHITECTURAL FEATURES 

Vacuoles (Gomori trichrome): absent 

Target/Targetoid fibers (NADH-TR): absent 

Small dark angulated fibers (NADH-TR): present 

Lobulated Fibers (NADH-TR): absent 

Cores (NADH-TR): absent 

Minicores (NADH-TR): absent 

Subsarcolemmal deposits: absent 

Ragged Red Fibers (Gomori trichrome): absent 

Nemaline Rods (Gomori trichrome): absent 

Tubular Aggregates (Gomori trichrome): absent 

COX-negative fibers (COX-SDH): within normal limits 

Subsarcolemmal nuclear clusters: present 

IMMUNOHISTOCHEMISTRY 

MHC Class I (B1): Increased sarcolemmal staining in necrotic fibers 

CD3 (A2): virtually absent 

CD20 (A2): virtually absent 

CD163 (A2): macrophages in endomysium and myophagocytosis 

GROSS DESCRIPTION 

A through C.  Muscle, quadriceps, left, biopsy:  

CONTAINER LABEL: Left quadriceps - fresh muscle biopsy for protocol. 

RECEIVED: Unfixed on a saline soaked Telfa pad. 

PIECES: 1.  SIZE: 1.7 x 1.4 x 1 cm.  PROCEDURE: (A) Sections are taken for 

routine processing and paraffin-embedding; (B and C) samples are snap frozen 

for histochemistry and placed in glutaraldehyde for possible electron 

microscopy/Epon embedding; one piece is retained for frozen storage. 

CASSETTES/PARTS: A1: Paraffin-embedded block; B1: Snap frozen block; C1-6: 

Epon-embedded blocks, NS. 

lmb/11/17/2023 

Candi Bennett 

MICROSCOPIC DESCRIPTION 

STAINS 

Paraffin sections (tissue block A1): Hematoxylin and eosin, Masson trichrome, 

Congo red, CD163 (scavenger receptor cysteine-rich [SRCR] 1), CD3 (T cell 

marker), CD20 (L26, B cell marker) 

Cryosections (tissue block B1): Hematoxylin and eosin, Gomori trichrome, 

alkaline phosphatase, nonspecific esterase, NADH-tetrazolium reductase, 

succinate dehydrogenase, cytochrome oxidase-succinate dehydrogenase, ATPase 

(pH4.3, 4.6 and 9.4), MHC Class I 

Plastic-embedded sections (tissue blocks C1-6): Paragon 

TISSUE QUALITY: Within normal limits 

GENERAL MORPHOLOGIC FEATURES 

Myofiber size: Mild variation 

Angulated fibers: Rare 

Fiber groups: Absent 

Perifascicular atrophy: Absent 

Myofiber types (ATPase pH4.3, 4.6 and 9.4): Equal populations of both fiber 

types; normal sized and atrophic fibers of both types 

Blood vessels: Within normal limits 

Endomysium: Mild increase in collagen 

Amyloid (Congo red): Absent, including sarcoplasmic compartment 

MYOPATHIC FEATURES 

Necrotic fibers (Gomori trichrome, nonspecific esterase, CD163): Present 

(myophagocytosis) 

De-/Regenerating fibers: Present 

Internal nuclei: Present 

Endomysial fibrosis (Masson trichrome): Mild 

Fatty metaplasia: Mild 

INFLAMMATION (CD3, CD20) 

Perivascular: Sparse 

Endomysial: Sparse 

Vasculitis: None identified 

CYTOARCHITECTURAL FEATURES 

Red-rimmed vacuoles (Gomori trichrome): Absent 

Vacuoles (H&E): Present (not a specific finding) 

Target/Targetoid fibers (NADH-TR): Absent 

Small dark angulated fibers (NADH-TR): Very rare 

Lobulated Fibers (NADH-TR): Absent 

Cores (NADH-TR): Absent 

Minicores (NADH-TR): Absent 

Subsarcolemmal deposits: Absent 

Ragged Red Fibers (Gomori trichrome): Absent 

Nemaline Rods (Gomori trichrome): Absent 

Tubular Aggregates (Gomori trichrome): Absent 

COX-negative fibers (COX-SDH): NOT increased 

Subsarcolemmal nuclear clusters: Absent 

Other: Not applicable 

IMMUNOHISTOCHEMISTRY 

MHC Class I (B1): Increased sarcolemmal labeling (patchy), labeling of 

inflammatory cells and perimysium 

CD3 (A1): Scattered peri- and endomysial T cells predominantly in a 

perivascular distribution 

CD20 (A1): NO significant B cell infiltrate is seen in this stain 

CD163 (A1): highlights macrophages as part of the perivascular infiltrate and 

foci of myophagocytosis 

PARAGON-STAINED SECTIONS 

NO further pathologic features are recognized in plastic-embedded and 

Paragon-stained sections 

Appropriate controls are reviewed and found satisfactory 

GROSS DESCRIPTION 

A.  Muscle for Light Microscopy, bicep, right:  

CONTAINER LABEL: Right bicep muscle 

FIXATIVE: fresh on ice 

GENERAL: 

Received is a 0.7 x 0.5 x 0.3 cm segment of muscle submitted in a clamp. All 

segments are sectioned into several segments and entirely submitted. 

SECTIONS: A portion in formalin for light microscopy (A1), a portion frozen 

for histochemistry (B1), a portion in glutaraldehyde for plastic embedding (C) 

and 3 pieces (5 mg) for frozen storage. 

B.  Muscle for Histochemistry:  

C.  Muscle for Electron Microscopy:   

mcs/5/21/2024 

Janela-Michelle Mojica. 

MICROSCOPIC DESCRIPTION 

STAINS 

Paraffin sections (tissue block A1): Hematoxylin and eosin, Masson trichrome, 

Congo red, CD163 (scavenger receptor cysteine-rich [SRCR] 1), CD3 (T cell 

marker), CD20 (L26, B cell marker), HLA-B 

Cryosections (tissue block B1): Hematoxylin and eosin, Gomori trichrome, 

alkaline phosphatase, nonspecific esterase, NADH-tetrazolium reductase, 

succinate dehydrogenase, cytochrome oxidase-succinate dehydrogenase, ATPase 

(pH4.3, 4.6 and 9.4) 

Plastic-embedded sections (tissue blocks C1-3): Paragon 

TISSUE QUALITY: within normal limits 

GENERAL MORPHOLOGIC FEATURES 

Myofiber size: variable 

Angulated fibers: rare 

Fiber groups: absent     

Perifascicular atrophy: vague areas 

Myofiber types (ATPase pH4.3, 4.6 and 9.4): Equal populations of both fiber 

types; normal sized and atrophic fibers of both types with more type 2 

atrophic fibers 

Blood vessels: focally decreased 

Amyloid (Congo red): absent 

MYOPATHIC FEATURES 

Necrotic fibers (Gomori trichrome, nonspecific esterase, CD163): few 

De-/Regenerating fibers: few 

Internal nuclei: absent     

Endomysial fibrosis: absent 

Fatty metaplasia: absent 

INFLAMMATION (CD3, CD20) 

Perivascular: focal 

Endomysial: mild     

Vasculitis: absent 

CYTOARCHITECTURAL FEATURES 

Vacuoles (Gomori trichrome): absent 

Target/Targetoid fibers (NADH-TR): absent 

Small dark angulated fibers (NADH-TR): absent 

Lobulated Fibers (NADH-TR): absent 

Cores (NADH-TR): absent 

Minicores (NADH-TR): absent 

Subsarcolemmal deposits: absent 

Ragged Red Fibers (Gomori trichrome): absent 

Nemaline Rods (Gomori trichrome): absent 

Tubular Aggregates (Gomori trichrome): absent 

COX-negative fibers (COX-SDH): within normal limits 

Subsarcolemmal nuclear clusters: absent 

IMMUNOHISTOCHEMISTRY 

HLA-B (A1): Increased sarcolemmal staining 

CD3 (A1): few endomysial and perivascular T cells 

CD20 (A1): virtually absent B cells 

CD163 (A1): few endomysial and perivascular macrophages 

Appropriate controls are reviewed and found satisfactory. 

Univ of MI

Microscopic Description: 

Formalin-fixed, paraffin-embedded (FFPE) H&E: Hematoxylin- and 

eosin-stained sections of the paraffin-embedded tissue show minimal 

variation in muscle fiber diameters, no degenerating/regenerating muscle 

fibers, and no endomysial fibrosis. There is no endomysial inflammatory 

infiltrate. The epimysial and perimysial vessels show no perivascular 

inflammation and no vasculitis. Central nuclei are not increased. 

FFPE Slow Myosin (S-MYOS) and Fast Myosin (F-MYOS): Immunohistochemistry 

for slow myosin shows the muscle fiber type distribution to be 

approximately 50% slow-myosin-expressing, without obvious type 1 groups. 

Immunohistochemistry for fast myosin shows the muscle fiber type 

distribution to be approximately 50% fast-myosin-expressing, without 

obvious type 2 groups. 

Frozen H&E: Hematoxylin- and eosin-stained sections of the frozen section 

material show similar findings to the paraffin-embedded tissue with no 

degenerating/regenerating muscle fibers, no endomysial fibrosis, no 

inflammation, and no increase in central nuclei. The muscle fiber diameters 

range from 40 - 60 microns. 

Frozen Trichrome: Trichrome staining shows no endomysial fibrosis, no 

ragged red fibers, no rimmed vacuoles, and no nemaline rods. 

Frozen NADH: NADH staining shows no moth-eaten fibers, no fibers with 

scalloping of the myofibrillary matrix, and no fibers with subsarcolemmal 

crescents. 

Frozen SDH: SDH staining shows no fibers with subsarcolemmal mitochondrial 

aggregates. 

Frozen COX/SDH: COX/SDH staining shows no COX-negative fibers. 

Frozen PAS: PAS stain shows no definite accumulation of PAS-positive 

material. 

Frozen Oil Red O: There is no significant increase in lipid in muscle 

fibers. 

Frozen Fast and Slow Myosin Dual Stain, Frozen ATPase pH 4.6 and Frozen 

ATPase pH 9.4: Fiber typing stains show the muscle fiber type distribution 

to be approximately 40% type 1 fibers and 60% type 2 fibers, with a single 

rare type 1 group, scattered type 2 groups, and no grouped atrophy. 

Frozen Congo Red: There is no increase in congophilic material (also 

evaluated by polarized light). 

Metabolic panel: 

Frozen phosphofructokinase (pH 7.0 and 8.6): Shows retained staining, 

comparable to normal controls. 

Frozen myoadenylate deaminase:  Shows retained staining, comparable to 

normal controls. 

Frozen acid phosphatase: No increased acid phosphatase activity in muscle 

fibers is seen. 

Frozen myophosphorylase: Shows retained staining, comparable to normal 

controls. 

Frozen PASD: There in no increase in PAS-positive, diastase-resistant 

material.