In epidemiological studies: Findings from the hertfordshire sarcopenia study (HSS)

Abstract

Background

Sarcopenia, the loss of muscle mass and strength, is associated adversely with disability, morbidity and mortality. disability, morbidity and mortality. Epidemiological findings suggest influences operating across the life course may be important. course may be important. Our aim was to ascertain the feasibility and acceptability of obtaining muscle tissue from healthy older people in order to ultimately identify cellular mechanisms underlying life course influences on sarcopenia. influences on sarcopenia.

Methods

105 men with documented birth weight consented to detailed assessment of muscle mass and strength, and a biopsy of the vastus lateralis using the Weil-Blakesley conchotome. of muscle mass and strength, and a biopsy of the vastus lateralis using the Weil-Blakesley conchotome. Acceptability was ascertained by questionnaire and a 100mm pain Visual Analogue Scale (VAS). Acceptability was ascertained by questionnaire and a 100mm pain Visual Analogue Scale (VAS). 100mm indicated severe pain. indicated severe pain.

Results

Muscle biopsy was successfully carried out in 102 out of 105 participants, mean yield 107mg (range 20–290mg). There were no serious wound complications. Ninety-three participants completed feedback questionnaires. completed feedback questionnaires. The median pain VAS score during the procedure was 7mm (Interquartile range [IQR] 1–34), 4mm (IQR 0–16) one day after the procedure and 1mm (IQR 0–4) 7 days after the procedure. 60 (65%) participants were back to their normal levels of activity one day after the procedure. 85 (91%) found this procedure acceptable and would have the procedure again. 85 (91%) found this procedure acceptable and would have the procedure again.

Conclusion

Muscle biopsy using a Weil-Blakesley conchotome is both feasible and acceptable in community dwelling older men participating in epidemiological research. participating in epidemiological research. The excellent yield of biopsy tissue will allow morphological and molecular studies of muscle to be integrated into an epidemiological study facilitating investigation of the mechanisms underpinning sarcopenia that could potentially be altered by life course influences. the mechanisms underpinning sarcopenia that could potentially be altered by life course influences.

Introduction

Sarcopenia is the loss of muscle mass and strength with age (1). There is increasing recognition of the serious health consequences both for the individual in terms of disability, morbidity, impaired quality of life (2) and mortality (3, 4), and for the population in terms of significant healthcare costs (5). Given the expected rise in the number of older people in the next 20 years, it is clear that sarcopenia will pose a considerable social, health care and economic burden. Important determinants of muscle mass and strength including age, gender, size and physical activity and heritability have been well described (6, 7, 8, 9). However, there remains considerable unexplained variation in both muscle mass and strength of older men and women and interest in the potential for influences that operate earlier in the life course is growing. A number of epidemiological studies have now identified a relationship between small size at birth and lower adult muscle mass and strength, suggesting developmental influences are important (10). Research is now focussing on the mechanisms underlying these observations in both animal models and in human muscle tissue.

With advances in microscopy and image analysis as well as in histological, physiological and biochemical assays, multiple analyses can be achieved in muscle tissue (11, 12). Furthermore, access to muscle biopsies provides the opportunity to assess morphological characteristics (e.g. in ageing muscle) such as fibre composition, fibre cross sectional area and capillarisation (13, 14) as well as mRNA, protein abundance and muscle enzyme activity (13, 15). Ultra-structural analyses to gauge response to intervention can be performed (16) as can studies of physiological characteristics of muscle such as single fibre contraction properties (14).

Methodologies enabling the study of muscle in health and disease have relied on obtaining tissue through post mortem, open muscle biopsy (requiring general anaesthesia) and percutaneous semi-open techniques that require local anaesthesia. The semi-open muscle biopsy technique is a relatively common, simple and easily learned procedure which has superseded open muscle biopsy in clinical as well as research practice (17, 18). Muscles subject to biopsy in previous studies include the deltoid, biceps, triceps, tibialis anterior, gastrocnemius, soleus and the sacrospinal muscles (12, 19). 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 (17).

The first biopsy needle was introduced by Duchenne in 1868 and since then, numerous muscle biopsy needles have been described (20). However, the needle introduced by Bergstrom in 1962 (21) was the most popular and has gained widespread use in diagnostic as well as research purposes in both children and adults (12, 17, 18, 22). Muscle yields obtained from the needle biopsy have been reported to vary from 25-75 mg (11), 70 - 140mg (18) and up to 143-293 mg after repeated sampling (23).

An alternative to the biopsy needle is the Weil-Blakesley conchotome (Figure 1) (24, 25). This instrument is designed like a forcep and consists of a sharp biting tip encompassing a hollow that can vary from 4-6 mm in width. In similar fashion to the needle biopsy, 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 offers a degree of manoeuvrability (25). These features permit sampling of other muscle groups perhaps where the pressure required in the needle procedure to penetrate muscle may not be advisable because of underlying neurovascular or bony structures e.g. tibialis anterior. An additional benefit is that unlike the Bergstrom needle, the conchotome biting tip does not need regular sharpening (19).

Figure 1.

The Weil-Blakesley conchotome

It is well recognised that the muscle biopsy is extremely useful in the study of ageing muscle (11, 14, 26). However, the majority of human muscle biopsy studies to date have been carried out in clinical diagnostic or focused physiological research settings. Whether the muscle biopsy procedure is feasible and acceptable in an epidemiological study where larger numbers of participants are involved has not been previously described. Our objective for this study therefore was to ascertain the feasibility and acceptability of the muscle biopsy using the Weil-Blakesley conchotome in a group of community dwelling men from the well-established Hertfordshire Cohort Study (27) who were participating in the Hertfordshire Sarcopenia Study (HSS).

Methods

Study sample

Recruitment into the Hertfordshire Cohort Study has been previously described (27, 28). From 1931 to 1939, midwives collected detailed records including information on birth weight and weight at one year on 42,974 births in the county of Hertfordshire, UK. A total of 8650 men and women were subsequently traced through the National Health Service Central Registry (NHSCR) as still being alive and living in Hertfordshire in 1998. From these, 7106 were identified as registered with General Practitioners within Hertfordshire. Permission to contact a total of 6099 men and women was granted of whom 2997 agreed to both a home interview and clinic visit for the Hertfordshire Cohort Study. Participants from this cohort have been shown to be generally comparable to the nationally representative Health Survey for England (27). Out of the 2997 participants, 1086 men were identified as still residing in East and West areas of Hertfordshire. From these, a total of 375 men were identified as detailed below as potential participants for the Hertfordshire Sarcopenia Study (HSS), which was designed to investigate life course influences on muscle morphology, mass and strength in community dwelling older people.

Participants were recruited given the availability of their early life size data: birth weight and weight at one year old, and excluded on co-morbidity. For example they were excluded if they had a prior diagnosis of diabetes mellitus or myopathy - there is evidence of a change in fibre morphology as well as function in diabetes that may affect results obtained in this study (29, 30), as would changes in morphology that may accompany any myopathy. A further exclusion criterion was ischaemic heart disease in view of the future requirement to perform tests requiring physical exertion. In addition, participants did not have a biopsy if they were on anticoagulation drugs such as warfarin or medication such as steroids that could compromise wound healing.

One hundred and five participants consented to take part in the HSS. The study was run by a clinician in academic geriatric medicine and involved an overnight stay at the Wellcome Trust Clinical Research Facility (WTCRF) in Southampton. One hundred and two participants were eligible, and underwent percutaneous muscle biopsy with a Weil-Blakesley conchotome. The study received ethical approval from the Hertfordshire Research Ethics Committee (number 07/Q0204/68) and written informed consent was obtained from each participant.

Vastus lateralis muscle biopsy

All muscle biopsies were conducted while participants were fasted using the Weil-Blakesley conchotome with a 6mm biting tip (Gebrüder Zepf Medizintechnik, Dürbheim, Germany). Each participant was asked to lay supine on a bed and the preferred thigh was exposed from the groin crease down. The operator was positioned adjacent to the thigh however, a position adjacent to the contralateral thigh with the operator reaching over can also be assumed. The leg remained straight and relaxed throughout the procedure but the thigh was tensed momentarily to mark the biopsy site on the outer aspect of the vastus lateralis. Thereafter, the skin over the mark was shaved and cleaned with an alcohol swab.

The skin and overlying fascia was infiltrated with 5mls of 2% lidocaine (Lignocaine) local anaesthetic. The subcutaneous bleb was allowed to settle during which time, the operator created a sterile field and proceeded using an aseptic technique. The skin was sterilized with povidone iodine USP 10.0% (Videne™ alcoholic tincture) and a sterile drape with an aperture was used to maintain a sterile field. A size 11 scalpel was used to make a 5-10mm incision on the skin, and a track down to the fascia through which the closed biting tip of the conchotome could be inserted. The free hand of the operator held the thigh surrounding the biopsy site while the tip was opened and closed (scissor action) to interpose muscle tissue. The conchotome was rotated through 90° to cut the muscle. Sampling took a few seconds and the procedure was repeated within the single wound site, where necessary, to obtain sufficient muscle tissue given the high probability of sampling adipose tissue initially (Figure 2).

Figure 2.

The muscle biopsy procedure. A. The leg was exposed from the groin crease down to the ankle. The biopsy area over the vastus lateralis was shaved of hair, marked, infiltrated with local anaesthetic and cleaned with antiseptic B. The skin was isolated with a sterile drape that had a 10cm aperture. The skin and overlying fascia were then punctured with a size 11 scalpel. The conchotome was inserted into the track made by the scalpel and rotated through 90° to excise the muscle C&D. The 5-10mm wound was closed with steri-strips. Thereafter, a dry dressing and compression bandage was applied

The biopsies were placed on sterile saline-dampened gauze and then transferred into a universal container on ice for the remainder of the procedure. Direct pressure was applied to the wound for 5-10 minutes prior to closure with steri-strips (Leukostrip, Smith and Nephew, UK). Melolin gauze (Smith and Nephew, UK) was placed on the steri-strips and clear adhesive film (Bioclusive™, Johnson and Johnson, UK) placed on the Melolin. A two-layer compression bandage was applied to the thigh for 6 hours. There were no restrictions to mobility after the procedure but the participants were asked to avoid 1. vigorous activity for 72 hours (hill climbing, running, heavy lifting) and 2. Immersion in water for 48-72 hours. Written instructions for post biopsy care, spare dressings and emergency contact details were given to each participant. The participant was instructed to change the Melolin and Bioclusive™ dressings four days after the procedure. The procedure took between 15 and 20 minutes with the majority of said time spent ensuring a sterile field.

Acceptability and feasibility

Participants were asked to provide feedback on their experience of the study through an anonymous 12 point questionnaire that started on day after the clinic visit and was completed one week later. Acceptability and feasibility of the biopsy procedure were ascertained through questions within the feedback form (Table 1). Perception of pain intensity was obtained through a 100mm horizontal pain Visual Analogue Scale (31) with anchors stating ‘no pain’ (0mm) and ‘pain as bad as it can be (100mm)’ (31). Participants were asked to mark their perception of pain intensity during, one day post, and 7 days post procedure. The scores were marked in millimetres from the ‘no pain anchor’.

Table 1.

Muscle biopsy feedback questions

With respect to the muscle biopsy, was it	Better than expected? As expected? Worse than expected?
When did you resume your normal daily activities?	1 day after the biopsy2 days after the biopsy >2 days after the biopsy
Were there any complications with the wound in the days after having the procedure?
Would you be willing to have the muscle biopsy again for research purposes?

Statistical analyses

Subject characteristic variables were described with means and range. Pain scores were tabulated and expressed as median scores with an interquartile range (IQR) during, one-day post and 7 days post procedure. Questions with binary or ternary outcomes were expressed as frequencies and percentages. Data were entered into a Microsoft access database. All data was coded and analysed using STATA release 10 software (STATA Corp, Texas, 2007).

Results

Participant characteristics

Participant characteristics are shown in (Table 2). One hundred and five participants consented to take part in the study. The mean age of the participants was 72 (range 68 to 77 years), mean height and weight were 174 cm (158 to194) and 83 kg (58 to119 kg) respectively. The group mean BMI was 27 (19 to 38) kg/m². Mean birth weight was 3.5 kg (2.3-5.4).

Table 2.

Hertfordshire Sarcopenia Study (HSS) male participant characteristics

n=105	Mean (range)
Age (y)	72 (68-77)
Height (cm)	174 (158-194)
Weight (kg)	83 (58 -119)
BMI (kg/m2)	27 (19-38)
Biopsy mass (mg)	107 (20-290)∗
Birth weight (kg)	3.5 (2.3-5.4)

*Biopsy mass were available for 54 samples

Muscle biopsy

Muscle biopsies were successfully obtained in 102 out of 105 participants (three participants who did not have a biopsy were excluded because they were on treatment with drugs that could compromise wound healing [methotrexate n=1, long term prednisolone n=1], or predispose to haematoma formation [aspirin n=1]). Of the 102 samples, ~ 50% (n=54), were weighed upon retrieval to ascertain mean biopsy mass, which was 107mg (range 20 to 290mg) (Table 2).

Common feelings experienced during the biopsy were a pushing sensation, slight or moderate discomfort, and a very occasional transient sharp pain. After the biopsy there were often reports of thigh stiffness that was exacerbated by the pressure bandage. This was alleviated by mobilizing and by loosening the pressure bandage. There were no adverse events or further bleeding during the night after the bandage was removed.

Biopsy feedback

Descriptive analyses were performed on 93 complete feedback questionnaires. The majority of the participants did not have any adverse perceptions of the biopsy with a large proportion stating that the biopsy was better than expected (Table 3). The VAS pain scores showed low ratings during the procedure with equally small IQRs. The change in pain score from “during” to day 1 was from 7mm to 4 mm. All participants were mobile within half an hour of the procedure without any difficulty and 60 (65%) of the participants resumed their normal daily activity the next day (avoiding vigorous activity as detailed in the aftercare sheet). One participant reported running the London Marathon two weeks after having a biopsy. There were no serious wound complications such as sub-fascial haematomas or intramuscular infections, and there was full healing within a week leaving a small scar that faded over time. The one participant reporting a complication stated that there was a mildly tender lump beneath the scar for approximately 3 weeks after the procedure that did not limit any daily activity. Eighty-five (91%) of the participants were willing to have the biopsy again for research purposes. The 8 participants not willing to have the procedure again did not give a reason. Seven participants rated the procedure worse than expected but only 3 participants out of these were not willing to have the procedure again.

Table 3.

Biopsy feedback summary

n=93
Perceptions of the biopsy	n(%)
Better than expected	34 (37%)
As expected	52 (56%)
Worse than expected	7 (7%)
Pain VAS score (mm)	Median (IQR)
During	7 (1-34)
1 day post	4 (0-16)
7 days post	1 (0-4)
Resumption of daily activity	n(%)
1 day	60 (65%)
2 days	26 (28%)
> 2 days	7 (7%)
Wound complications n (%)	1(1%)
Willing to have the biopsy again n (%)	85 (91%)

Discussion

We have demonstrated that muscle biopsy using a Weil-Blakesley conchotome is both feasible and acceptable in community dwelling older men participating in an epidemiological study. One hundred and two muscle biopsies were successfully obtained and were processed according to standardized protocols used within our laboratory. The majority of the participants reported minimal pain after the procedure as evidenced by low VAS scores during, and in the days post procedure. Sixty five percent of participants were back to their normal daily activity within 24 hours and 85 (91%) of the participants were willing to have the procedure again, suggesting that the procedure was very well tolerated in this group of participants. There were no serious adverse events and the participants reported excellent rates of wound healing. This is consistent with previous reports describing a minimal degree of discomfort and risk of complications in older subjects (11, 18, 24).

The results from this study are consistent with other published work where the conchotome forcep had been used (19, 24, 25, 32). In a clinical study, Dorph et al. (32) investigated 122 patients, with median age of 56 (range 16-85) who had 149 biopsies for possible myositis and found that the biopsy yield varied between 23 and 123 mg. The sites used were vastus lateralis, tibialis anterior and deltoid, confirming the versatility of the conchotome forcep. In the study by Dorph et al. there were 4 complications, of which one was serious and involved a sub-fascial haematoma complicated by a DVT. The complication rate of just under 3%, compares with 1% in our study. In another clinical study by Dietrichson et al. (19) the biopsy mass range was 50-150 mg with the specimens giving good histological and histochemical diagnostic information. In this latter study, both the Bergstrom needle biopsy and conchotome were used. Patients, who experienced both, preferred the conchotome to the needle biopsy because it was less painful.

Limitations

There are limitations to this study that need to be acknowledged. The conchotome samples from the superficial surface rather than from deeper muscle layers that are possible by the needle biopsy. However, unless there is a difference in morphology such as fibre number or cross sectional area between superficial and deep layers (33), we do not anticipate this to be a problem in subsequent analyses. Nevertheless, despite this anatomical variation, so long as the method is consistent, as was the case in this study, direct comparisons between participants can be made. Furthermore, although a previous study suggested taking two biopsies at the same site and depth to characterise fibre type composition (34), we were limited to obtaining one bioptic sample for immunohistochemical analyses as the other samples were frozen and stored for later histological, molecular and biochemical analyses. Additional sampling could have been performed, however, we were of the opinion that this was not needed. We have shown that the biopsy technique is acceptable in healthy community dwelling older men but acknowledge that this may not necessarily apply to frail older men. Future muscle morphological studies that involve frail community dwelling older men, and women should ascertain feasibility and acceptability of muscle biopsies in these populations.

Strengths

The strengths of the HSS are that it is a large study of muscle biopsy feasibility with potential for investigations at the cellular and molecular level, in a group of participants from a well-characterised cohort, who have been shown to be generally comparable to the nationally representative Health Survey for England (27). In terms of the HSS sample being representative of older people with sarcopenia, previous studies by Melton et al. (35) and Baumgartner et al. (36) reported that the prevalence of sarcopenia in community dwelling older men and women increased with age; in the study by Melton et al. participants with sarcopenia were a mean age of 75.6 (comparable to 72.4 years in HSS), were likely to be male, and had associated difficulty in physical activities; in the study by Baumgartner et al. the prevalence of sarcopenia in men increased from 13% in those <70 years to >50% in those over 80 years.

Conclusion

Muscle biopsy using a Weil-Blakesley conchotome is both feasible and acceptable in community dwelling older men participating in epidemiological research. The excellent yield of biopsy tissue will allow morphological and molecular studies of muscle to be integrated into an epidemiological study facilitating investigation of mechanisms underlying sarcopenia, which may be influenced by life course events.

Acknowledgements: We thank all the men who took part in the Hertfordshire Sarcopenia Study and the Wellcome Trust Clinical Research Facility team. This work was supported by the Medical Research Council and the University of Southampton. Additional support was provided by a British Geriatrics Society SpR start-up grant.

Conflicts of interest declaration: The authors have no conflicts of interest.