Percutaneous Muscle Biopsy: History, Methods and Acceptability

Advances in histological, biochemical, physiological and molecular biological assays, as well as in microscopy and image analysis have allowed multiple analyses in muscle tissue (1-5). Muscle biopsy is invaluable in providing a definitive diagnosis of a wide range of myopathies (muscular dystrophies, glycogen storage diseases, inflammatory myopathies and congenital myopathies) and denervating disorders and gives important information on the course of the disease, informs treatment, disease stage as well as prognosis. Results of histopathological analyses should then be interpreted in context of the clinical history, examination and laboratory serum markers (1;6). Furthermore, access to muscle tissue provides the opportunity to assess morphological characteristics such as fibre composition, fibre cross sectional area and capillarisation (e.g. in ageing muscle) (7-9) as well as mRNA, protein abundance and muscle enzyme activity (10). Ultrastructural analyses to gauge response to intervention can be performed (11) as can studies of physiological characteristics of muscle such as single fibre contraction properties (2;8).

needle described by Duchenne, gained popularity through widespread use in diagnostic as well as research (study of normal muscle in relation to physiological change) purposes in both children and adults (3;4;16).Although continuously being refined (17;18), each iteration of the needle is true to its origins i.e. possess a sharp trocar, a cutting cannula that needs sharpening perhaps every 10 uses and a pushing rod to expel the tissue post biopsy (Figure 1).Muscle yields obtained from the needle biopsy have been reported to vary from 25-75 mg (2) 70 -140mg (4) and up to 143-293 mg after repeated sampling (19).
An alternative instrument to the Bergstrom biopsy needle is the Weil-Blakesley conchotome (Figure 2) (20)(21)(22).This instrument, designed like a forcep was described by Henriksson in 1979 (21) and consists of a sharp biting tip encompassing a hollow that can vary from 4-6 mm in width.In similar fashion to the Bergstrom needle, it is inserted through a skin incision 5-10mm in length but does not require a sharp trocar to aid muscle penetration.In addition, its design allows controlled tissue penetration and offers a degree of manoeuvrability (22).These features allow biopsy on a wider range of muscles and permits sampling of muscle groups where the pressure required for the needle procedure to penetrate muscle may not be advisable because of overlying neurovascular or underlying bony structures e.g. at the tibialis anterior (22;23).An additional benefit is that, unlike the Bergstrom needle, the conchotome biting tip does not need regular sharpening and has been reported to maintain sharpness for up to 4 years (17).Muscle yields from the conchotome technique in the earliest report by Henriksson (21) ranged from 26 to 296mg.In our practice, muscle weights ranged at a comparable 20-290mg (24).

Micro-biopsy
The 'semi-open' techniques described above requiring an incision may be considered too painful and the potential for scarring may be off putting for some.Furthermore, semi-open techniques may be impractical to study time course responses to intervention e.g.exercise or drug administration.As a consequence, minimally invasive techniques such as micro-biopsy have been explored (19;25;26).The device used for skeletal muscle micro-biopsy is a version of the popular spring loaded one-handed automated biopsy systems used to perform biopsies of the breast, prostate, kidney or liver.The assembly consists of a disposable core biopsy needle e.g.16G Magnum ® (Bard Ltd, UK) and an insertion cannula that will allow multiple biopsies to be performed via a single insertion site (Figure 3).The needle penetration depth can be pre-set by the operator after which release of a trigger unloads the spring and fires the needle into the muscle, excising a small piece of tissue.The validity of this method compared with the Bergstrom needle in histomorphometric analyses has been tested (19).
The semi-open muscle biopsy and micro-biopsy techniques are common, simple and easily learned procedures that have superseded open muscle biopsy in clinical as well as research practice (4;6;17).Any potential risks are reduced if performed correctly and with strict attention to asepsis.Muscles that can be subject to biopsy include the deltoid, biceps, triceps, tibialis anterior, gastrocnemius, soleus and the sacrospinal muscles (3;22;23;27;28).However, the most common site for biopsy both in clinical and research practice is the outermost portion of the vastus lateralis.This site, approximately two-thirds down a line from the anterior superior iliac spine to the patella is readily accessible and does not contain an overlying neurovascular bundle.Furthermore, extensive normative fibre histomorphometric data obtained from vastus lateralis biopsy exists in the literature that allows the recognition of normal and abnormal and facilitates comparison between studies (2;6).

Vastus lateralis muscle biopsy (Figure 4)
The biopsy technique using the Weil-Blakesley conchotome and the Bergstrom needle is described below.The technique for micro-biopsy is reviewed by Hayot et al (19); apart from the fact that a skin incision is not required, the procedure shares the same principle as for the conchotome and needle methods and is illustrated in Figure 5.
Equipment needed for percutaneous muscle biopsy 1. Liquid nitrogen and suitable cryovials 2. Isopentane cooled in liquid nitrogen 3. Cork disc 4. OTC mount 5. Fixative e.g.formalin, 3% Glutaralhedyde/4% formaldehyde 6. Dissecting microscope to orientate tissue sample so that sections with fibres in true cross-section can be obtained 7. Suitable blunt edged forceps for post procedure tissue handling In the clinical setting, muscle biopsies are conducted based on patient symptoms and the distribution of muscular weakness and generally do not require standardised conditions but do require the biopsy site to be free of previous muscle injury, contractures or prior instrumentation/therapy e.g.injection sites (1;6).In research or in quantitative studies, standard conditions should apply to all procedures.For example, studies may require participants to be fasted pre biopsy or the exclusion of diabetic subjects (24).
There is little guidance on the use of aspirin or other anti-platelet agents pre biopsy.Whereas warfarin and related anticoagulants would need to be stopped for at least one to two weeks prior to the procedure, aspirin was stopped 4-7 days prior to uneventful muscle biopsy in our research study (24).It is likely that the drug free duration would need to be longer for ADP receptor blockers e.g.clopidogrel.Obviously, risks of stopping such secondary prevention drugs must be considered case-by-case.
Participants are asked to lay supine, comfortably on a bed with the preferred thigh exposed from the groin crease.The operator should be positioned adjacent to the thigh however, a position adjacent to the contra-lateral thigh with the operator reaching over can also be assumed according to individual preference.The leg should remain straight and relaxed Fig. 2. The Weil-Blakelsley Cochotome with a 6mm bitting tip (From Patel HP et al [24])  Fig. 5. Micro-biopsy procedure with the Magnum ® biopsy system (Bard Ltd, UK) 19   throughout the procedure but the thigh can be tensed momentarily to accentuate the outline of the vastus lateralis to mark the biopsy site.Thereafter the skin surrounding the mark is shaved and cleaned with an alcohol swab.
The skin and overlying fascia is then infiltrated 5mls of 2% lidocaine local anaesthetic.The subcutaneous bleb is allowed to dissipate during which time the operator should dawn a sterile gown, gloves and create a sterile field on a suitable trolley.After opening the pack containing the Conchotome or biopsy needle, the skin should be sterilized with povidone iodine (Betadine) or a 2% Chlorhexidine gluconate/isopropyl alcohol solution according to local infection control protocols.A sterile drape with an adhesive aperture is then used to maintain a sterile field.With a size 11 scalpel, a 5-10mm incision is made on the skin and down to the fascia through which the closed biting tip of the conchotome is inserted.The conchotome is inserted at right angles to the long axis of the femur, facing away from the femur and to a depth averaging 2-5cm.The free hand of the operator can hold the thigh surrounding the biopsy site while the tip of the conchotome is opened and closed (scissor action) to interpose muscle tissue.The conchotome is then rotated through 90-180 to cut the muscle.Sampling takes a few seconds and can be repeated within the single site, where necessary, to obtain sufficient muscle tissue given the high probability of sampling adipose tissue initially (Figure 4 A-H).The biopsies should be placed on sterile saline-dampened gauze and then transferred into a universal container or petri dish and placed on ice for the remainder of the procedure.
Operator and skin preparation prior to using the using the needle biopsy or the microbiopsy gun is exactly the same as described for the conchotome (17;29).Constituents of the needle include include the trocar/needle, the cutting cannula and the clearing probe/rod (Figure 1A 1,2,3 respectively).The instrument should be assembled by inserting the cutting cannula into the trocar and checked to ensure alignment and sliding action.The width of a size 11 scalpel to make a skin incision and a track down to the fascia should suffice.The trocar is inserted through the incision, past the fascia where the operator should get a sensation of overcoming resistance; at this point the window of the trocar (Figure 1C) should be fully in the muscle.The cutting cannula is withdrawn a few centimetres and the needle angled to allow tissue to enter the instrument.Alternatively, an assistant can apply suction via a syringe applied to the cutting cannula (Figure 1B) to draw muscle into the cutting chamber before fully advancing the cannula to guillotine a section of muscle (18;30) (Figure 1D) The free hand of the operator can be used to steady the thigh.The yield of muscle can be increased by rotating the instrument by 90 and repeating the procedure (18).
Post procedure, direct pressure should be applied to the wound for 5-10 minutes prior to closure with steri-strips (Leukostrip, Smith and Nephew, UK).Sterile absorbent gauze is placed on the steri-strips and a clear adhesive film (Bioclusive™, Johnson and Johnson, UK) placed on the gauze.A two-layer compression bandage is then tied for up to 6 hours.The subject is asked to lay down for up to half an hour post procedure and is observed.The procedure, using the conchotome or the Bergstrom needle can take between 15 and 20 minutes; the majority of said time spent in preparation.

Post muscle biopsy patient/participant advice
Following the procedure it is common to experience some thigh stiffness that can be alleviated by gentle exercise e.g.walking.There are no real restrictions to mobility but in our practice we participants to avoid 1. vigorous activity for 72 hours (hill climbing, running, heavy lifting) and 2. Immersion in water for 48-72 hours (24).Participants can shower if the biopsy site was protected from water for example, wrapping polythene plastic/cling film around the thigh.Written instructions for post biopsy care, spare dressings and emergency contact details should be given to each participant or patient.To ensure full wound healing, that can take up to two weeks, the dressing should be changed after 4 days and steri-strips removed at day 7 post procedure.All those having the procedure should be warned about transient numbness around the biopsy site that may persist for up to two weeks as well as the remote possibility of wound infection that would necessitate a medical consult.

Acceptability and adverse events
When performed correctly and according to protocol, the semi-open and micro-biopsy techniques result only in minor discomfort for the subject that is qualitatively described as a ''pushing sensation" or a "deep pressure" (29) with very low pain scores (Table 1) (24).Post biopsy, there may be local soreness, stiffness or cramp that do not seem to limit normal activity including recreational sports e.g.golf, bowls (18;24) (Table 1).One participant in our research study reported running a Marathon two weeks after a conchotome vastus lateralis biopsy (24).
Where authors have reported complications, they have commonly been wound haematomas (4;20;22;26).For example, a subject had been taking aspirin at the time of the

VAS pain score (mm) Median (IQR)
During the procedure 7(1-34) research participants subject to the conchotome muscle biopsy.Pain scale was marked at 0mm -'no pain' and at 100mm -'pain as bad as it can be'.Typically pain scores were low during and post procedure.The majority of the participants resumed daily activity one-day post procedure (Adapted from Patel HP et al [24]) biopsy (20) or did not follow post biopsy and put an undue amount of strain onto the thigh shortly after the procedure (22).Pain only occurs if the fascia is caught in the needle or a nerve is sampled inadvertently and may persist for one to two weeks (6).There have been no reports in the literature of serious wound infections or disability post procedure.
Minimally invasive muscle biopsy techniques described above are both feasible and acceptable in clinical and research practice with good safety profiles.The choice of instrument depends on experience and preference, tissue requirement, location of muscle as well as clinical assessment of disease involvement.When performed correctly, all yield muscle tissue satisfactory for most histochemical and histological analyses.In addition, the semi-open techniques allow detailed morphological, biochemical and molecular biological analyses to be conducted on muscle tissue.

Fig. 1 .
Fig. 1. A. Components of the biopsy needle include 1. Trocar, 2. Cutting cannula, 3. Clearing probe/rod.B. Assembled for biopsy, with cutting cannula inserted within the trocar.A 20 ml syringe can be connected to the cutting cannula to increase the yield of tissue by suction.C. Cutting window visible.D. Window with cutting cannula fully depressed.

Fig. 3 .
Fig. 3. Spring loaded micro-biopsy system consisting of trigger housing, biopsy needle and the insertion cannula (not shown).(Illustration kindly provided by M Hayot, Service Central de Physiologie Clinique, Montpellier, France)

Table 1 .
Pain visual analogue scale (VAS) score and resumption of daily activity in 93