| Abstract|| |
Histopathological analysis of muscle biopsy is a prerequisite in the evaluation of neuromuscular disorders, particularly inflammatory myopathies, metabolic myopathies, congenital myopathies, muscular dystrophies and differentiating myopathies and neurogenic disorders with overlapping clinically features. It not only provides useful information that helps in the diagnosis but also treatment and management. Fundamental skills and basic knowledge regarding handling, processing and analyzing a muscle biopsy are required in any specialized or a general pathology lab supporting neuromuscular clinical services. Care during transport of the muscle biopsy, sample receipt in the laboratory and grossing is very important. Standard operating procedure should be followed for the preanalytical steps (freezing and cryomicrotomy), routine and special staining (enzyme and non enzymatic) and immunohistochemistry. A well organized neuromuscular laboratory with good quality management system is necessary for the practice of myopathology. This article gives an overview of establishing such a laboratory.
Keywords: Cryomicrotomy, enzyme histochemistry, muscle biopsy, myopathology, snap-freezing
|How to cite this article:|
Nandeesh BN, Narayanappa G, Yasha T C. Basic requirements to establish a neuromuscular laboratory. Indian J Pathol Microbiol 2022;65, Suppl S1:233-40
|How to cite this URL:|
Nandeesh BN, Narayanappa G, Yasha T C. Basic requirements to establish a neuromuscular laboratory. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 May 24];65, Suppl S1:233-40. Available from: https://www.ijpmonline.org/text.asp?2022/65/5/233/345059
| Introduction|| |
The diagnosis of neuromuscular disorders requires a thorough clinical examination with electrophysiological evaluation, histopathological study and genetic testing along with radiology. Histopathological study acts as an excellent supplementary testing tool in providing useful diagnostic evidence that guides in management of the patient. Muscle biopsy is indicated in suspected cases of inflammatory myopathies, metabolic myopathies, congenital myopathies, muscular dystrophies, spinal muscular atrophies, vasculitis, and cases with overlapping clinical features. Fundamental skills and basic knowledge about handling, processing and systematic interpretation of muscle biopsy are prerequisites for a pathologist serving in a neuromuscular laboratory.
Inspite of the advances in high throughput molecular genetic testing, muscle biopsy and the histolopathological evaluation is the gold standard when all other ancillary testings including genetics gives doubtful results.
Fixation compromises the biochemical properties of the tissue and only limited stains such as hematoxylin and eosin, Masson's trichrome, phosphotungstic acid hematoxylin can be performed, which is inadequate in providing essential information. Hence fresh muscle subjected to enzyme histochemistry and additionally, immunohistochemistry, western blot techniques and mitochondrial respiratory chain complex assays are required. DNA and RNA isolated from the fresh muscle tissue is essential for genetic analysis and further research. For this, a well organized neuromuscular laboratory with a good quality control system is necessary. This article gives an overview of establishing such a laboratory.
Basic neuromuscular lab: A 20 × 30 ft well lit/ventilated room with AC, working benches, storage cupboards, sink with pedal-operated tap, and provision to house a cryostat, 11/25 liters liquid nitrogen dewar flask, 32°C incubator, -20°C, -80°C freezers and 4°C laboratory refrigerator (one each to begin with). Additional space of 15 × 20 sq ft and 30 × 40 sq ft is required to perform muscle/nerve/skin biopsy in the vicinity of the basic lab
Preanalysis and preanalytical requirements
Muscle biopsy is invaluable in the evaluation of neuromuscular diseases. It is recommended to perform the muscle biopsy in close proximity (adjoining) to the neuromuscular lab. The staff should take utmost care in handling the tissue right from the time of biopsy to its transportation and receipt in the lab followed by snap freezing, cryotomy, staining, storage of remaining tissue and preparation of slides for further analysis. For electron microscopic (EM) studies (available in house or at a referral center), a small fragment of the tissue must be fixed in 2.5% buffered glutaraldehyde for immediate or future studies.
Muscle biopsy procedure and its requirement
Muscle biopsy can be obtained either by an open biopsy procedure or needle technique. The former is most commonly used and recommended as it provides a larger tissue sample than the needle biopsy (uses a modified Bergstrom needle). The biopsy should always accompany a request form with pertinent clinical information. If the lab is situated at a different location, due diligence should be given for proper packing of the specimen and shipping it promptly. Peripheral nerve and skin biopsies may also be performed along with the muscle biopsy in suspected cases of vasculitis, metabolic or syndromic disorders. Unlike muscle, nerve and skin biopsies should be fixed in appropriate fixative depending on the test requested. However, skin punch biopsy done in cases with metabolic myopathies for fibroblast cultures should be placed in culture media soon after biopsy.
The biopsy should be from the muscle belly. It is to be noted that a region close to myotendinous area must be avoided and should be at least 2 cm away. Muscles with any previous injury, local injection and sites of electromyography needle insertion should be avoided., The requirements for performing the biopsy are as per minor surgical procedure which includes general antiseptic preparations, scalpel with the blade, curved artery forceps, local anesthesia, needle and suture material, bandage/dressing material, Personal protective equipment (PPE) and general nursing measures.
Choice of muscle/biopsy site
A moderately involved muscle [MRC grade 3 or 4] should be chosen in chronic diseases. In contrast, in cases with the acute presentation, a severely affected muscle may be chosen. Recently, magnetic resonance imaging (MRI) and ultrasound guided selection of affected muscle are being recommended.,
A superficial and easily accessible muscle is considered for biopsy. The most common muscles for biopsy include vastus lateralis and biceps brachialis. However, deltoid, triceps brachialis, rectus femoris, soleus, and peroneus brevis muscles may be biopsied, depending on the clinical indication. In a suspected case of distal myopathy, distal limb muscles that include soleus, tibialis anterior, gastrocnemius, carpal radial extensor, and extensor digitorum longus may be considered for biopsy.,,
It is noteworthy that the muscle tissue should not be subjected to any cauterization and anesthesia. Only overlying blood vessels may be cauterized and skin and fascia may be infiltrated with the anesthesia. Local antiseptic and sterility precautions should be taken during the biopsy procedure. The muscle tissue should be removed with minimal or no trauma in the same direction as the fascicles of the muscle fibers. The muscle must not be clamped, damaged (e.g., tied to tongue blades or squeezed with forceps), or cauterized during the procedure. If the tissue removed appears less and additional testing is expected, extra tissue from the same region may be removed. The ideal size of the muscle tissue would be a longitudinal piece measuring 2 to 3.0 cm in length and 2.0 cm in diameter. However, obtaining a larger size may be difficult in infants and children, and a strip of 0.5 cm in diameter and 2 cm in length would be satisfactory.
The lab must be informed prior to the sample being sent for adequate preparation to receive the biopsied tissue. There is no transport medium that preserves the muscle's physical, chemical, and physiological properties. Hence, one must plan for a rapid transfer (ideal transit time of 20 minutes) to the neuromuscular laboratory.
Immediately after the muscle biopsy procedure, the fresh muscle tissue must be placed on a moistened gauze (moistened by sprinkling a few drops of saline). Care should be taken not to soak the gauze with excess saline and not to immerse the tissue in saline, as this produces saline artefacts causing difficulty in interpreting the tissue and carrying out specialized tests required for diagnosis. Arrangement has to be made to transport and deliver the muscle tissue to the neuromuscular laboratory without delay in order to avoid loss of integrity and denaturation of enzymes/proteins.
The tissue wrapped in a saline-moistened gauze pad should be placed inside an airtight/screw cap container/zip lock bag referred to as the specimen container. This represents the primary packing. The primary package should accompany details of the patient and the sample. Biopsies from separate sites should be labelled and placed in separate specimen containers, however can be transported in one common shipping/transportation container. An additional bubble wrap may be used. NOTE: When a triple (muscle, nerve and skin) or double (muscle and nerve OR muscle and skin biopsy) biopsy is done, nerve and skin should be fixed in appropriate fixative (10% buffered formalin or 2.5% gluteraldahyde) and placed in a separate container and not in the same container/package as the fresh muscle. This is because the vapors emanating from the fixative fixes the fresh muscle.
The next step is the secondary packing where the primary container with the tissue is placed in a sealed (water tight), leak proof plastic box/wide mouthed flask/thermocol container with adequate quantities of dry ice/ice cold packs to maintain cold chain (4–8°C) during transit and has to be sealed securely. It is to note that the specimen should not come in direct contact with ice at any time. Furthermore, the secondary pack must be placed in an outer most pack, which is the shipping/transportation container that should be sufficiently strong (to protect the contents from physical damage) and heat resistant. The outer box must to be sealed well and the address of the receiving lab be written legibly along with the sender's name and address in clear bold print. Also, the package should have arrows to indicate UP and DOWN side of the package and carry biohazard label for category B. The duly filled in requisition form should accompany the specimen and should include patient details (Name, preferably in upper case, age, gender, hospital/ward, the muscle biopsied (site, laterality), time and date of biopsy, name and contact details of the referring doctor, relevant clinical information and tests including electrophysiological (NCS and EMG), imaging, family history and any other information required for correlation. Fresh muscle should be delivered well before the closing time to carry out preanalytical procedures (freezing, sectioning, and staining particularly enzyme histochemistry). [Figure 1] shows the sequence of steps involved in transportation of the muscle tissue.
|Figure 1: showing the sequence of steps involved in transportation of the muscle biopsy. (a, b)- muscle tissue placed over a gauze piece sprinkled/moistened with a few drops of saline, (c) – Primary packing using a airtight/ziplock cover, (d, e) – secondary packing filled with ice and holds the primary package|
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If the sample is fixed either in 10% buffered formalin or 2.5% buffered glutaraldehyde, it can be transported at room temperature without requirement of the cold chain. Muscle tissue fixed in formalin can be used only for routine histological stains, while tissue fixed in glutaraldehyde is used for electron microscopy (EM). Tissues fixed in formalin and Formalin-fixed, paraffin-embedded (FFPE) block is not suitable for EM study.
Sample receipt in the laboratory
Requirements: Accession register, if the barcode is used – barcode reader, barcode generator, stickers and stationery. A photographic record of the sample at receipt also can be considered if the resources permit.
After receipt of the sample, the details on the request form and details on the specimen container have to be cross checked and verified. The entry of the details on the package and the inner containers at receipt have to be recorded in the laboratory accession document/register (either in digital or physical form) including the time, date, and name of the person receiving and the person handing over. Most importantly, the status of the container and the tissue at the time of the receipt have to be recorded. A unique laboratory identification number has to be assigned to the specimen and the specimen to be labelled and shifted to the laboratory workstation that handles the muscle tissue.
The laboratory technician receiving and handling the specimen should be aware that formalin fixation impedes the various enzyme histochemical and immunohistochemical techniques. Hence, any kind of exposure of the muscle biopsies to either liquid or gaseous form of formalin will compromise further steps. It is recommended that the package is handled in a formalin and alcohol free zone.
Preanalytical steps in the laboratory
Requirements: Accession register, if the barcode is used – barcode reader, barcode generator, stickers/labels and stationery. A photographic record of the sample at receipt can also be considered if the resources permit.
After receipt of the sample, the details on the request form and details on the specimen container have to be cross checked and verified. The entry of the details on the package and the inner containers at receipt have to be recorded in the laboratory accession document/register (either digital or physical form) including the time, date, and name of the person receiving and the person handing over. Most importantly, the status of the container and the tissue at the time of the receipt have to be recorded. A unique laboratory identification number has to be assigned to the specimen and the specimen has to be labelled and shifted to the laboratory workstation that handles the muscle biopsy.
The laboratory technician receiving and handling the specimen should be aware that formalin fixation impedes the various enzyme histochemical and immunohistochemical techniques. Hence, any kind of exposure of the muscle biopsies to either liquid or gaseous form of formalin will compromise the further steps. It is recommended that the package should be handled in a formalin and alcohol free zone.
Grossing and the preanalytical steps in the laboratory
The pathologist/trained technician has to examine and document the size, gross features, intactness and the status (discolored, softened) of the specimen along with the presence of any aponeurosis myotendinous bands, blood vessel and if the muscle is foul-smelling due to autolysis.
The muscle tissue is generally divided into three bits: The largest and the main portion (70 – 80%) of the muscle biopsy tissue is snap frozen in liquid nitrogen. This procedure preserves the muscle's microanatomy, enzymes, and antigens intact, which is crucial for further analysis. Hence, care should be taken in handling, freezing, and other steps.,,
Of the remaining two small bits, one small portion of the muscle tissue is fixed in 10% buffered formalin for paraffin embedding. The second small bit is fixed in buffered glutaraldehyde (2.5%) for eventual EM studies. It is essential to preserve, freeze, and store a fragment of the fresh tissue in cryovials at -80°C, preferably liquid nitrogen container for further investigations. Alternatively, an additional small portion can be preserved during the biopsy procedure itself for mitochondrial assays and western and southern blot investigations.
Freezing and cryomicrotomy
Cryostat – with or without digital/automation, high profile cryostat blades, anti-roll, clean glass slides, cover slips, long tweezers, brush to clean the surface of anti-roll, aluminum foil, and slide labelers.
Note: The equipment has to be under annual/comprehensive maintenance and calibrated periodically. It is also recommended to have a backup instrument.
- Wide moth metal beakers with a long slender handle of 100 ml capacity
- Liquid nitrogen containers of at least 5 to 25 litres capacity with a regular supply of liquid nitrogen.
- -20°C and 80°C freezers for the storage of enzyme substrate, slides with frozen sections and frozen tissue
- 4°C laboratory refrigerator for storage of buffers and incubating medium and chemicals.
The main, well-preserved portion of the muscle is snap-frozen using isopentane precooled in liquid nitrogen for cryosectioning. Slow cooling results in freezing artifacts due to generation of intracellular micro-ice crystals that aggregate and damage the cells causing vacuolation in the myofibers compromising the histological interpretation. The following are the steps -
- The muscle tissue should be correctly oriented to ensure transverse sections are obtained and placed on a flat piece of cork for further steps. A specimen with a diameter of 4 mm and a length of 8 to 10 mm is considered optimum as thicker/larger tissue results in freeze artefacts. To facilitate this and to trim the tissue, a razor blade split into two (with one half/cutting edge being available) may be used. For better visualization and orientation, a magnifying glass may be used. The trimmed fragment is placed on a small flat cork or a plastic piece and secured using OCT mounting medium around the specimen.
- Around 50-100 ml of isopentane is taken in a small cylindrical container/metal beaker with a handle or a glass beaker with a string tied to the neck and immersed in liquid nitrogen (-160°C) taking care that the liquid nitrogen does not enter the isopentane. We recommend a metal beaker with a wide mouth and long narrow handle (small bucket) for immersion. Once the isopentane is adequately frozen (frozen chalky white phase and liquid phase), the beaker is taken out and the specimen is immersed with the help of long tweezers into the liquid phase of isopentane for about 20-30 seconds. Too short or too long duration of cooling results in ice crystal formation and cracking of the tissue, respectively.
- The tissue is removed and without delay placed on a metal chuck provided in the cryostat. The temperature of the cryostat should be maintained at -20oC - 22oC and the knife/blade is placed before transferring the tissue for sectioning. Ensure the knife is sufficiently cooled before sectioning. Eight to ten micron (μm) thick transverse sections is collected on plain slides by a simple touch of the slide surface over the tissue section. The temperature difference between the slide and the tissue sections enables easy adherence to the slide. Utmost care should be taken to prevent folds and wrinkles in the sections. This is achieved by gently placing the anti-roll plate (provided in the cryostat) parallel to the knife/blade for sections to pass between the two. At least two sections are collected per slide. The slides with frozen sections are transferred into a slide box and stored at -80oC freezer until further use. Before collecting the sections, the slides are numbered using a diamond marker with the patient's unique histopath ID and in the sequence of collection. The cryosection thus collected is utilized for Enzyme histochemistry (EHC), Immunohistochemistry (IHC) and other specialized stains. The first section is stained with hematoxylin and eosin (HE) to check the orientation and also serves an internal quality check. An adequate number of sections including a few extra have to be collected for routine and special stains, enzyme histochemistry, immunohistochemistry, and in the event any of the stains needs to be repeated. Prior to storage, the labeling on all the slides and the tissue samples has to be checked and verified. After the staining process, the unstained extra sections and the portion of the tissue remaining after sectioning can be stored in deep freezers (-80oC) and liquid nitrogen respectively for future use. [Figure 2] shows the sequence of events involved in freezing, precryotomy, and cryotomy.
|Figure 2: showing the sequence of events involved in freezing, precryotomy and Cryotomy: optimal orientation (a), Isopentane in a convenient container (b), freezing of the isopentane by introducing into the liquid nitrogen container (c), snap- freezing of the muscle tissue by dipping in the liquid phase of isopentane (d), transferring the snap frozen muscle tissue to the crysostat (e), Cryosectioning (f)|
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It is advisable to use precooled aluminum foil to wrap the slides with the sections or a slide box for storage. However, the tissue has to be stored in special cryotubes. If additional sections are required for analysis, then the stored flash-frozen tissue can be sectioned again by transferring it to the cryostat and acclimatize in the cryostat for about 15- 20 min.
Enzyme histochemical staining
Substrates, buffers, glycerin jelly and coverslip
pH meter, weighing balance (good precision level), and measuring range 20 mg to 200 g
Glassware, staining rack, moist chamber, filter paper and gloves.
32°C incubator to carry out enzyme histochemical staining.
Surgical trays with stands to lay slides flat for enzyme staining
Glassware (beakers, conical flask, measuring cylinders, pipettes, etc)
Manual IHC/Automated IHC – but needs standardization, validation, titration, and verification. Primary, secondary antibody and the IHC related consumables including buffer blocking solutions, charged slides, pipette with tips, mounting media and coverslips.
Prior to staining, the tissue sections should be brought to room temperature for about one minute. Care should be taken not to allow the sections to dry as it results in cracks. The sections are then subjected to a battery of stains including routine hematoxylin and eosin (H and E/HE). [Table 1] shows the routine and special stains that are essential. [Table 2] shows additional stains that can be performed based on the clinical indication/histopathological assessment.
|Table 1: List of essential stains (routine and special) and its usefulness|
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|Table 2: Additional stain that may be required based on the case indication|
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The study of longitudinal sections provides added advantages, especially the presence of segmental necrosis, focal inflammation and nuclear internalization such as nuclear chains (e.g., centronuclear/myotubular myopathy) as larger muscle segments/zones can be assessed on longitudinal sections.
The following are the routine and special stains that are to be performed:,
Based on the clinical suspicion and the histological findings, additional stains are performed that include:,,
Depending on the clinical features and histological findings, immunohistochemical markers (antibodies directed against specific skeletal muscle proteins) should be available in the laboratory. The result may indicate an absent, decreased, or increased protein contents/expression. The important immunohistochemical (IHC)/immunoblot markers include the list shown in [Table 3].,,,,,,,
The histopathological study of a muscle biopsy tissue provides most information when it is received fresh (with the guidelines mentioned in the main text) allowing to perform a battery of tests as described above. [Figure 3] and [Figure 4] indicate the algorithm of the workflow depending on the facilities available.
|Figure 3: Algorithm – 1 shows the work flow for a basic laboratory with minimal facilities and no cryostat|
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|Figure 4: Algorithm 2 shows the work flow for a neuromuscular laboratory with more facilities|
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In conclusion, the present article attempts to introduce the basic requirements regarding establishing a muscle laboratory. Muscle biopsy, though an invasive procedure, remains the gold standard for the diagnosis of neuromuscular disorders, especially when genetics study is not contributory. Any laboratory or a laboratory professional catering to a neuromuscular clinical service should be aware of the preanalytical requirements (transport, freezing), staining, the analytical methodology that includes interpretation of stains, including enzyme histochemistry, immunohistochemistry, and clinical correlation. Basic knowledge and skill of handling, staining and histological analysis will enormously assist in providing good quality neuropathology services that can support the treating clinician and facilitate further workup, patient management, treatment, and follow-up.
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Conflicts of interest
There are no conflicts of interest.
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Bevinahalli N Nandeesh
Additional Professor, Department of Neuropathology, National Institute of Mental Health and Neurosciences, NIMHANS - 560 034, Bangalore, Karnataka
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]