Biopsy procedure and sample handling
All research involving skeletal muscle biopsies must be approved by an institutional review board/ethics committee. The subject must be fully informed about the study and the potential risk and discomfort - this includes information about the potential establishment of muscle cell cultures and the long-term storage and use of the obtained biological material. For subjects undergoing skeletal muscle biopsies, we recommend careful history-taking and physical examination to exclude current infection and any disease which could interfere with the results of the study. Also avoid muscle that has recently suffered trauma. All medications should be discontinued at least one weak prior to biopsy. This is particularly important in subjects taking oral anticoagulants or other drugs interfering with haemostasis. Volunteers are usually admitted to the research centre after a standardised 12 h overnight fast and instructed to refrain from strenuous physical activity for a period of 48 h before the experiment.
1) Mark the biopsy site 10-15 cm above the patella on the vastus lateralis muscle and wash the skin with antiseptic solution (alcohol swaps).
2) Using a thin s.c. needle (0.6 x 30 mm), the skin and subcutaneous tissues down to the fascia are infiltrated with 4-10 ml of 2 % lidocaine, depending on the thickness of subcutis. It is important to avoid lidocaine in muscle. Try to locate the fascia by the tip of the needle. Otherwise, push the needle through the fascia, bend it against the fascia while slowly pulling it back, and sense the unbending of the needle as it passes back through the fascia.
3) Wait ~10 min before a skin incision is made. Prepare the biopsy table, as shown in Figure 14.2, and a sample handling table, which should include sterile gloves, IV needles for dissection of muscle samples, liquid nitrogen in a small container (200 ml), dry ice to pre-cool three vials with screw caps for the frozen muscle samples, small metal forceps for sample handling during freezing-procedure, Tissue-Tek (Sakura, Torrance, CA), glutaraldehyde and tubes with transport medium for the part of the muscle specimen that is used for isolation of satellite cells or mitochondria.
4) Using sterile gloves, disinfect the skin with chlorhexidine and drape the thigh, with the hole surrounding the biopsy site. Wash again and prick the skin with the tip of the
Figure 14.2 Biopsy Table The biopsy table, which is covered by 1) a utility drape (Klinidrape®45 x 75 cm; Mölnlycke Health Care, Göteborg, Sweden); contains 2) a small drape with adhesive aperture (Steri-Drape™ 40 x 40 cm, hole 6.3 cm in diameter; 3M Health Care, Borken, Germany); 3) a sterile scalpel (blade size 11); 4) two IV needles (1.2 x 40 mm); 5) one extra 20 ml syringe; 6) three low adherent adsorbent dressing (Melolin 10 x 10 cm; Smith & Nephew, Hull, England); 7) Steri-Strips™ (3M Health Care, Borken, Germany); 8) four gauze swabs, 8 ply (Multisorb® Cotton 5 x 5 cm; BSN medical GmbH & Co, Hamburg, Germany); 9) five cotton soaked in chlorhexidine placed in a sterile container 60 ml; and 10) a modified Bergström needle (4 mm) connected to a 20 ml syringe.
Figure 14.2 Biopsy Table The biopsy table, which is covered by 1) a utility drape (Klinidrape®45 x 75 cm; Mölnlycke Health Care, Göteborg, Sweden); contains 2) a small drape with adhesive aperture (Steri-Drape™ 40 x 40 cm, hole 6.3 cm in diameter; 3M Health Care, Borken, Germany); 3) a sterile scalpel (blade size 11); 4) two IV needles (1.2 x 40 mm); 5) one extra 20 ml syringe; 6) three low adherent adsorbent dressing (Melolin 10 x 10 cm; Smith & Nephew, Hull, England); 7) Steri-Strips™ (3M Health Care, Borken, Germany); 8) four gauze swabs, 8 ply (Multisorb® Cotton 5 x 5 cm; BSN medical GmbH & Co, Hamburg, Germany); 9) five cotton soaked in chlorhexidine placed in a sterile container 60 ml; and 10) a modified Bergström needle (4 mm) connected to a 20 ml syringe.
scalpel to ensure proper anaesthesia. Make a 3-5 mm skin incision with the scalpel, continue in one slow move through the subcutis, and penetrate the underlying fascia with the tip of the scalpel. Avoid making the incision in the fascia too large as this can make it difficult to achieve proper vacuum during suction.
5) Using sterile gauze swaps, apply firm pressure over the incision to halt any oozing of blood, which may otherwise coat the needle, contaminate the muscle specimen and cause freezing artifact in morphological studies and error in chemical analysis.
6) With the window closed, introduce the Bergstrom needle - attached to a 20 ml syringe, fitted to the top of the inner cutting cylinder - through the incisions in the skin and the fascia and insert it into the muscle so that the window of the needle is in the belly of the muscle, to a depth of approximately 2-5 cm below the level of the skin.
7) After applying suction to the needle using the syringe, rapidly pull the inner cutting cylinder back > 1 cm, allowing muscle to be drawn into the open window of the needle. Then quickly push the inner cylinder down again, cutting the muscle specimen. In this way the sample is guillotined. Several cuts can be made in quick succession by rotating the needle while in the muscle, but the procedure should not exceed 30 seconds. The window must be closed upon withdrawal of the needle from the muscle. A second sample can be taken through the same incision to provide additional material e.g. for establishment of cell cultures. In this way samples weighing 200-300 mg are easily obtained.
8) Quickly remove muscle tissue from the inner cutting cylinder of the needle and place the syringe on the shiny film side of a low adherent adsorbent (Melolin) dressing. The sample should be inspected by another person, and all visible blood, fat and connective tissue should be rapidly removed by rolling the tissue on the shiny surface of the adsorbent dressing using an IV needle.
9) Snap freeze pieces of ~50 mg muscle in liquid nitrogen within 30 sec of excision. For measurements of metabolites, protein expression, activity and phosphorylation, and gene expression, it is essential that the samples are frozen as quickly as possible.
10) Place small pieces of ~5 mg in a small folio vial and mount them in cryo embedding medium (Tissue-Tek), then rapidly freeze in isopentane, cooled to -160 °C in liquid nitrogen, for histochemistry/immunohistochemistry. Pre-fix small cubes of muscle (1mm3) in buffered (0.1 M cacodylate) 2-6 % glutaraldehyde for electron microscopy. Put the remaining muscle tissue into a tube containing medium for isolation of satellite cells (see later). If muscle is to be used for isolation of mitochondria, this part is done first (Table 14.1).
11) Upon withdrawal of the needle, apply firm pressure to the biopsy site, using sterile gauze swaps, for 5 min to assure haemostasis and prevent subsequent haematoma formation. Aseptic precautions should be maintained throughout the biopsy procedure. Oppose the edges of the small skin wound with one sterile tape (Steri-Strip), and cover the wound with a Melonin dressing and a plaster. The subject should wear a compression bandage for at least 3 h or until bedtime. Volunteers should be informed about potential complications and given the ability to get in touch with a doctor if they are concerned about anything.
12) It is important to keep the needle sharp. The cutting edge should be examined after each biopsy and sharpened if necessary (regularly).
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