Human Skeletal Muscle Biopsy Procedures Using the Modified Bergström Technique

Summary

The purpose of this video is to present the modified Bergström skeletal muscle biopsy technique on human subjects.

Abstract

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergström needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergström needle consists of an outer cannula with a small opening (‘window’) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90° and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

Introduction

The percutaneous or ‘semi-open’ biopsy technique is used to obtain skeletal muscle tissue specimens from human patients and research subjects. Duchenne (1806-1875) is credited as the first to construct a needle with a trocar to obtain skeletal muscle from living subjects through percutaneous biopsy¹. In the 1960’s, Bergström introduced a percutaneous biopsy needle similar to that described by Duchenne^2-4. Twenty years later, Evans et al.⁵ modified the technique by applying suction through the cutting trocar of the Bergström needle. This modification can enhance the yield of tissue 3- to 5-fold^6,7 and is employed in clinical and biomedical research settings. This technique has and will continue to foster the diagnosis of myopathies and our understanding of the structure and function of skeletal muscle.

The percutaneous muscle biopsy technique is straightforward. When done correctly, with strict adherence to aseptic technique, the associated risks are minimal. The muscle biopsy procedure is regarded as an investigational tool for research projects. Some academic institutions allow trained faculty researchers with a Ph.D., with direct physician oversight, to obtain muscle biopsies and others require a board-certified physician to perform the technique. The ASU biopsy team has successfully performed over 1,600 muscle biopsies during the past 13 years^8-15. The purpose of this video is to describe the modified Bergström needle percutaneous muscle biopsy technique⁵ to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects.

Protocol

The skeletal muscle procedure as described follows the guidelines of the Appalachian State University Institutional Review Board.

NOTE: The team consists of an operator (Ph.D. or M.D. trained in the biopsy technique), and at least one, but ideally, three or more technicians. The operator is responsible for conducting and overseeing all aspects of the skeletal muscle biopsy procedure. One technician (technician #1) is immediately responsible for assisting the operator with all aspects of muscle extraction. This includes being the “non-sterile hands” and applying suction with a sterile syringe. A second technician (technician #2) engages in conversation with subject and massages the subject’s opposite leg during procedure to minimize the subject's anxiety. The third technician (technician #3) is responsible for handling and processing the skeletal muscle sample. Downstream analytical measures dictate how many additional technicians are required for processing/preparing the skeletal muscle sample.

1. Subject Preparation

1. Gather, prepare, and organize the materials specified for the muscle biopsy procedure (Table 1). Decontaminate and sterilize the Bergström needle (outer cannula and inner trocar) and the plunger according to standard practice¹⁶.
2. Review the consent form with the subject. The skeletal muscle biopsy portion of the Consent to Participate form used at Appalachian State University is provided in online supplement 1.
   1. Talk to the subject about the general aspects of the muscle biopsy procedure with a focus on possible risks of the procedure, skin preparation, application of the anesthetic, typical sensations. Instruct the subject to carefully read the Consent to Participate form and sign it to confirm that the study design and procedures are understood.
3. Confirm that the subject is not allergic to “___caine”-type medications (e.g., lidocaine).
4. Instruct the subject to lie supine on a padded table with the thigh exposed. Place two disposable absorbent underpads with plastic backing under the subject’s leg. Position the leg in a relaxed manner with a small towel roll under the heel, such that the knee is fully extended (elevated ~1 cm), thus placing the vastus lateralis in a shortened position.
5. Have the operator instruct the subject to momentarily contract the exposed thigh muscle so that the biopsy site can be visualized. Note: The vastus lateralis biopsy site is just anterior to the fascia lata (illiotibial band), approximately one-third of the distance between the top of the patella and the greater trochanter⁷.
6. Have the operator mark just below (~0.5 cm) the incision site with a fine point permanent marker.
7. Have the operator determine the approximate skinfold thickness by pinching the skinfold.
8. Have the operator and technicians wash their hands with soap and warm water and don disposable gloves.
9. Have the operator remove the hair from a region ~15 cm x 15 cm around the biopsy site (via clippers). Note: clipping the hair from the biopsy site prevents hair from getting into the incision during the procedure and during closing, and hair removal allows for better contact between the tape closures and the skin (see 3.4).
10. Have the operator sterilize the area with swabs pre-soaked with a topical antiseptic, (such as povidone-iodine or chlorhexidine gluconate for subjects allergic to iodine/shellfish). Begin in the center and work in concentric circles towards the outer edge of the clipped area. At a minimum, repeat twice more with a new pre-soaked swab each time.

2. Biopsy Procedure

1. Have the operator remove disposable gloves, wash hands with soap and warm water, and don sterile surgical gloves using aseptic technique.
2. Have technician #1 present the sterile fenestrated drape to the operator. Have the operator, using aseptic technique, place the drape over the biopsy site to maintain a sterile field.
3. Have technician #1 present the operator a 5 ml syringe fitted with a 21 G needle while maintaining aseptic technique. Clean the top of the lidocaine vial with an alcohol swab.
   1. Have the operator immediately withdraw 5 ml of lidocaine. Remove the 21 G needle from the syringe and discard the needle into a sharps container.
   2. Have technician #1 present the operator with a 1-½ inch 25 G needle while maintaining aseptic technique. Have the operator place the 1-½ inch needle onto the syringe and evacuate the air bubbles from the syringe.
4. Have technician #1 spray ethyl chloride on the incision site (~0.5 cm above the indelible ink mark on the skin) until the skin appears to “blanch.”
   1. Have the operator insert the needle approximately horizontal to the skin into the dermis, aspirate the needle, and then infiltrate with ~100 µl of lidocaine to produce a “bleb” 2-4 mm in diameter.
      1. Have the operator aspirate the needle (slightly withdraw the plunger of the syringe) to confirm that the needle has not been placed in a blood vessel. If blood appears in the syringe, withdraw the needle, discard in a sharps container, and begin again at 2.3.
      2. Have the operator advance the needle into the subcutaneous tissue, aspirate the needle, and then infiltrate the tissue with ~1 ml of lidocaine to form a bleb. Once the bleb has subsided, insert the 1-½ inch needle on the lidocaine-loaded syringe vertically into the incision site, stopping superficial to the fascia. Note: The subject may feel a slight momentary stinging sensation upon the initial injection of lidocaine (similar to a bee sting).
   2. Ensure that the operator does not infiltrate the muscle with lidocoine because it is myotoxic^17-19.
   3. Have the operator aspirate the needle and then slowly inject the remaining 4 ml of lidocaine while withdrawing the needle from the thigh. Place sterile gauze over injection site and allow the subject to relax while the local anesthetic takes effect.
5. After 2-3 min, have technician #1 present the operator a scalpel while maintaining aseptic technique.
   1. Have the operator lightly probe the biopsy site with the tip of the scalpel to confirm that the area is anesthetized. If necessary, inject another 3-4 ml of lidocaine into the incision site, see 2.4.1.2.
6. Have the operator make a straight 1-cm incision through the skin and subcutaneous tissues (~2-3 mm above the ink mark) parallel to the femur.
   1. Have the operator insert the scalpel deeper to make an incision through the fascia into the muscle. Do this once in each direction. Note: The subject may feel a twinge or pressure with the deeper cut if the scalpel blade cuts into the muscle.
   2. Have technician #2 engage the subject in conversation and massage the opposite leg.
7. Have the operator place a liberal amount of sterile gauze over the incision and biopsy site and apply direct pressure to the incision to reduce bleeding.
   1. Have technician #1 connect the 3-way metal stopcock to the disposable 60 ml syringe and the 30 cm extension tubing (Figure 1).
   2. Have technician #1 connect one end of the tapered plastic tubing connecter into the free end of the extension tubing and firmly insert the other end of the tapered plastic tubing connecter into the large end of a 200 µl pipette tip with ~15-18 mm of the tip cut off.

Figure 1. The Bergström needle. (A) The Bergström needle (5 mm) is composed of the (a) outer cannula and (b) inner trocar, the associated components include (c) plunger, (d) 200 µl pipette tip with ~15-18 mm cut off, (e) tapered plastic connector, (f) 3-way metal stopcock, (g) 30 cm extension tubing, and (h) disposable 60 ml syringe. (B) The Bergström needle (5 mm) and associated components assembled. (C) The inner trocar of the Bergström needle (5 mm) withdrawn approximately 1 cm opens the window of the outer cannula.

9. After the bleeding has subsided, have technician #1 open the sterilized autoclave pouch and present the 5 mm biopsy needle and inner trocar (Figure 1) to the operator while maintaining aseptic technique.
   1. Have the operator assemble the needle (outer cannula and inner trocar), check alignment and sliding action.
10. Have the operator firmly hold the needle assembly (outer cannula and inner trocar) with both hands to prevent the inner trocar from rotating. Have technician #1 firmly insert the cut pipette tip into the top of the inner trocar portion of the biopsy needle with a slight downward twisting motion. Ensure that technician #1 does not touch the operator or the biopsy needle, only the pipette tip.
11. Have the operator introduce the biopsy needle into the tissue through the incision. Locate the incision in the fascia (an acquired “feel”) with the tip of the biopsy needle. Advance the needle just past the fascia and then angle the needle downward toward the floor as the needle is advanced into the muscle. Note: Subjects may experience some mild discomfort (e.g., “deep pressure,” or mild cramping sensation) as the incision through the fascia is located with the tip of the biopsy needle and the needle is advanced into the muscle.
    1. Have technician #2 instruct the subject to stay “relaxed” and not to contract their thigh muscles while the operator is advancing the needle into the muscle.
12. Once the biopsy needle is in position, have the operator signal technician #1 to open the disposable 60 ml syringe.
    1. Have technician #1 rapidly pull the disposable 60 ml syringe open to the 40-50 cc mark to create suction within the biopsy needle.
    2. Have the operator pull the inner trocar out approximately 1 cm to open the window of the outer cannula while maintaining the position of the outer trocar within the muscle. Have the operator rapidly close the inner trocar to cut (clip) and collect the muscle sample. Have the operator signal technician #1 to release suction.
    3. Have technician #1 open the stopcock to the room to release the suction.
    4. Have the operator rotate the biopsy needle 90° and repeat the process. If required, have the operator repeat the rotation procedure up to 3x (for a total of 4 clips).
13. Have the operator apply direct counter-pressure to the incision site with sterile gauze while removing the biopsy needle from the thigh, being careful not to catch fascia or skin. If significant resistance is felt, re-cut while applying a twisting motion to the inner trocar.
14. Upon removal of the biopsy needle from the thigh, have the operator pull the inner trocar back 1 cm to visually inspect the lumen of the outer cannula to estimate if an adequate amount of tissue was collected.
    1. Have the operator hand the biopsy needle to technician #1. To maintain aseptic technique ensure that technician #1 only touches the needle, not the operator. Have technician #1 hand the biopsy needle to technician #3.
15. Have technician #3 remove the tissue from the biopsy needle.
    1. Have technician #3 use the plunger (Figure 1) and a pair of fine tip forceps to ensure that all muscle tissue is removed from the inner trocar and outer cannula.
    2. Have technician #3 quickly weigh the sample to confirm that an adequate amount of muscle tissue was collected and place the samples on an ice-cold dissection block.
16. Have technician #3 carefully dissect visible connective tissue and fat from the muscle samples. Have technician #3 prepare the muscle samples for storage according to downstream analyses. For example, immediately place samples in a cryovial and snap freeze in liquid nitrogen, mount and freeze for histology, or place in a cryovial with RNase inhibitor, etc.
17. If necessary, repeat the procedure with a second sterilized biopsy needle while maintaining aseptic technique.

3. Closure

1. Have the operator apply direct pressure to biopsy site with sterile gauze and an ice pack for 10-15 min.
2. Once hemostasis is achieved, have the operator use alcohol prep pads to remove dried blood from the area around the incision.
3. Have technician #1 present a tube of topical surgical adhesive and applicator to the operator. Have the operator assemble the tube of surgical adhesive and applicator without touching the applicator tip or the top of the tube.
4. Have the operator pull the incision closed with one hand while applying a single layer of surgical adhesive over the top of the dry incision with the other hand. After the adhesive cures (~ 90 sec), apply tape closuresperpendicular to the incision. Alternatively, close the incision with two sterile 4-0 sutures.
5. Have the operator apply direct pressure by placing 3-4 2” x 2” non-sterile gauze pads on the incision site and secure with self-adhering adhesive wrap.
6. Have the operator provide the subject verbal and written instructions on proper wound care, normal and abnormal reactions, and activity guidelines for the following 1 to 4 days (see online supplement 2).

Results

The muscle biopsy procedure as described above allows the researcher to quickly and consistently collect skeletal muscle tissue samples. The typical yield in healthy, athletic subjects is 200 mg or more in a single pass with 3-4 clips. The procedure takes 15-20 min, most of which is spent in preparation for the incision. In exercise-based studies, muscle samples are often taken pre- and post-exercise, with one or two samples collected during recovery. With this design, the hair on the post-exercise (contralateral) thigh ...

Discussion

This video provides a step-by-step summary of the skeletal muscle biopsy procedure used at the ASU Human Performance Laboratory. This procedure, with small modifications, has been used to collect approximately 1,600 muscle biopsies during the past 13 years. The muscle biopsy samples have provided important data in sports nutrition based investigations, leading to important research discoveries.

There are many critical techniques to be aware of when performing the modified Bergström skelet...

Materials

Name	Company	Catalog Number	Comments
Bergström biopsy needle, 5 x 100 mm	Stille Surgical Inc.	119-29187-50	1 ea
Durasorb blue pad	Fisher	22-031-340	case of 300
Prep razor disposable	Moore Medical	89760	1 ea
Shave cream	Moore Medical	92869	1 can
Alcohol swab	Moore Medical	98721	box/200
Povidone-iodine swabsticks (3 pack)	Moore Medical	90691	pack of 50
Face masks	Fisher	19-039-690	case of 50
Sterile gloves; sz 6.5	Moore Medical	68128	box of 50 pairs
Sterile gloves; sz 7.0	Moore Medical	68129	box of 50 pairs
Sterile gloves; sz 8.5	Moore Medical	68132	box of 50 pairs
Fenestrated towel drape	Moore Medical	92712	case of 50
Lidocaine (1% HCl, w/o Epi)	Dealmed	427902	30 cc vial
Ethyl chloride spray	Dealmed	386020	3.5 fl. Oz. bottle
5 ml syringe w/ 21 G needle	Fisher	14-827-48	case of 100
25 G 5/8" needle	Fisher	14-826AA	case of 100
25 G 1 1/2" needle	Fisher	14-826-49	case of 100
Single-use scalpels w/ #11 blade	Fisher	0-8927-5B	box of 20
60 ml Syringe	Fisher	13-689-8	case of 40
~30 inch extension tubing	Fisher	14-169-7A	roll of 50 ft
Metal stopcock	Fisher	01-290-38	each
Polypropylene tubing connector w/ tapered ends, 3.2 to 5.5 mm	Fisher	15-315-28A	case of 100
Yellow pipette tips (1-200 µl)	Fisher	02-681-2	cut off ~15-18 mm of the tip
Topical surgical adhesive	Medline	MSC091076	case of 36 tubes
Topical surgical adhesive	Medline	MSC091076Z	box of 6 tubes
Butterfly stitches (adhesive strips) 1/2" x 4" (6 per pack)	Moore Medical	93050	box/300
Bandadge, Strip, 3/4 x 3 in.	Fisher	19-027-202	pack of 100
Adhesive bandage (5 m rolls)	Fisher	19-027-768	case of 36
1" paper surgical tape	Fisher	19-027-761	case of 12
Sutures (4-0 reverse cutting, FS2)	Moore Medical	82926	box of 36
4 x 4 Sterile gauze pads	Moore Medical	66949	pkg/50
2 x 2 Non-sterile gauze pads	Moore Medical	37336	pkg/200
3 x 4 Non-adherent pads	Moore Medical	74389	bx/100
Autoclave bags 3.5 x 9	Moore Medical	40390	box of 200
Autoclave bags 5.25 x 10	Moore Medical	40391	box of 200
Autoclave indicator tape	Moore Medical	31608	box of 250
Lab coats—small	Fisher	23-900-520A	10/pk
Lab coats—medium	Fisher	23-900-520B	10/pk
Lab coats—large	Fisher	23-900-520C	10/pk
Germicidal Disposable Cloth Wipes	Moore Medical	62879	Can/160