Validity of Methods Used for the Assessment of Body Components in Children

HORMONE RESEARCH

Horm Res 2006;66(suppl 1):5864 DOI: 10.1159/000096624

Published online: January 25, 2007

Validity of Methods Used for the Assessment of Body Components in Children: Pros and Cons of Modern versus Old Technology
Jonathan C.K. Wells
Childhood Nutrition Research Centre, Institute of Child Health, London, UK

Key Words Muscle mass Anthropometry Bioelectrical impedance Dual-energy X-ray absorptiometry Magnetic resonance imaging

Introduction

Abstract There is increasing interest in measuring muscle mass in paediatric research and clinical practice. The simplest approach is to measure lean mass and predict muscle mass as a proportion of it, but individuals and groups vary in this ratio. Calculation of arm muscle area from anthropometry has been shown to have poor accuracy in individuals; segmental impedance analysis, however, may perform better as a simple predictive method for limb composition, and merits further development. Biochemical techniques (e.g. urinary creatinine measurement) are cumbersome in children and their data are confounded by variability in maturation, diet and activity level. The best tools are radiographic imaging techniques that attempt direct measurement of muscle mass. The most widely available is dual-energy X-ray absorptiometry, which is capable of detecting the direction of differences or changes, but is less reliable regarding their magnitude because of error that varies with gender, size and fatness. Magnetic resonance imaging is currently the best technique available, as computed tomography has too high a radiation dose to justify its use in younger age groups. Regardless of which technique is used, reference data are required to aid interpretation of results, and are a current research priority.
Copyright 2006 S. Karger AG, Basel 2006 S. Karger AG, Basel 03010163/06/06670058$23.50/0 Accessible online at: www.karger.com/hre

Historically, the assessment of childrens body composition has focused on distinguishing the fat and lean components of body weight. As our understanding of growth has become more sophisticated, it has become important to develop methodologies capable of measuring more specific components. The process of growth comprises a number of different regulatory mechanisms, and specific treatments are now targeted at particular tissues. The aim of this article is to focus on the measurement of muscle mass. Muscle mass, present in the body in three forms (skeletal, smooth and cardiac muscle), represents the largest proportion of the fat-free mass, a term used synonymously in this article with lean mass. In adults, skeletal muscle represents about 30% of body weight in women and 40% in men [1]. In children, the contribution of muscle mass to weight varies with age, being lowest in infancy when body fat levels are greatest [2] (fig. 1). Skeletal muscle mass is primarily found in the limbs, particularly the legs, but is also present in the trunk and head [4]. As will be discussed in greater detail below, the most accurate methods for quantifying skeletal muscle mass are currently magnetic resonance imaging (MRI) and computed tomography (CT) scanning. Where relevant, other techniques are therefore evaluated in relation to these criterion methods. At the present time, the majority of research on measuring muscle mass remains foDr. Jonathan C.K. Wells Childhood Nutrition Research Centre Institute of Child Health, 30 Guilford Street London WC1N 1EH (UK) Tel. +44 207 905 2389, Fax +44 207 831 9903, E-Mail J.Wells@ich.ucl.ac.uk

Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com

cused on adults. This article incorporates adult studies to indicate relative merits between approaches, but draws wherever possible on equivalent work in children.
Measurement of Lean Mass

80 70 60 50 40 30 20 10 0 50

Weight (kg) Muscle mass (kg) % muscle mass

Quantification or estimation of lean mass represents the simplest approach to the estimation of muscle mass. Numerous techniques are now available for assessing lean mass in children. These include prediction methods (anthropometry and bioelectrical impedance analysis [BIA]) and more direct methods (isotope dilution, dualenergy X-ray absorptiometry [DXA], plethysmography, hydrodensitometry, potassium scanning and multicomponent models). These approaches vary in their accuracy, an issue discussed in more detail elsewhere [5] and below. Table 1 summarizes the main assumptions inherent in these techniques. Arguably the simplest method of all involves the prediction of body water from weight and height [6], from which lean mass and then muscle mass may in turn be estimated. However, accuracy of this equation is likely to be confounded by effects of disease such as oedema and wasting. At the other extreme, multicomponent models are currently regarded as the optimum source of chemical data on body composition, differentiating relatively accurately between fat, protein, water and mineral [7]. Multicomponent models can be applied with little practical difficulty in children aged 4 years and over, and are now also possible in neonates following the development of an infant whole-body plethysmograph [8]. For older infants and toddlers, fewer techniques are available due to the practical difficulties of making measurements. The most appropriate technique in this age category is isotope dilution, requiring only provision of a drink and the collection of saliva or urine samples. One benefit of isotope dilution is that it can be applied over the entire life course, from birth to old age. Of relevance to the estimation of muscle mass is the fact that variability in the proportion of lean mass that is muscle inevitably limits the value of this generic approach. Factors contributing to such variability include age, gender, ethnicity, hydration status, mineralization, physique, and the presence of disease. Thus, lean mass may be measured as a proxy for muscle mass, but it is likely that this approach will suffer from significant limitations in those patients in whom inter ventions on muscle mass are being administered. More direct measurements of muscle mass are therefore required.
Assessment of Body Components in Children

70

90

110

130

150

170

190

Height (cm)

Fig. 1. Relationship between height and weight or muscle mass using the reference data from Holliday [3]. The ratio of muscle to lean mass increases between early infancy and adulthood.

mass

Table 1. Alternative approaches to the measurement of lean

Method Prediction from weight and height Prediction from skin folds Prediction from BIA DXA Densitometry Potassium scanning Total body electrical conductivity MRI 4-component model

Assumed constancy Ratio of water to weight and height; hydration of lean mass Ratio of subcutaneous to total fat; density of lean mass Homogeneous distribution of water; hydration of lean mass X-ray attenuation of fat and lean; even tissue distribution Density of lean mass and fat mass Potassium content of lean tissue Electrical conductivity of lean tissue Electromagnetic properties of hydrogen nuclei Density of fat, protein and mineral

See [5] for further details. BIA = Bioelectrical impedance analysis; DXA = dual-energy X-ray absorptiometry; MRI = magnetic resonance imaging.

Horm Res 2006;66(suppl 1):5864

59

Table 2. Alternative approaches to the prediction of whole-body and regional skeletal muscle mass in adults

Method Anthropometry Whole body Anthropometry Impedance Urine Urine DXA DXA Potassium counting Neutron activation Regional DXA DXA

n 244 244 388 10 12 25 414 300 25 27 207

Predictor Weight, height Skin-fold thicknesses, body girths Height, impedance 3-methylhistidine (24-hour collection) Creatinine (24-hour collection) Appendicular lean tissue mass Limb lean tissue Body potassium content Total body nitrogen and potassium Thigh lean tissue Lower limb lean tissue

SEE (kg) 2.8 2.2 2.7 2.3 1.9 1.6 1.6 1.5 2.1 0.8 1.1

r2 0.86 0.91 0.86 0.77 0.85 0.90 0.96 0.96 0.90 0.79 0.88

Reference method MRI MRI MRI CT CT CT MRI MRI CT CT MRI

Reference [9] [9] [10] [11] [12] [12] [13] [14] [12] [15] [16]

Adapted and updated from [14]. Other studies could not be included due to lack of appropriate statistical data. CT = Computed tomography; DXA = dual-energy X-ray absorptiometry; MRI = magnetic resonance imaging; SEE = standard error of the estimate.

Prediction of Muscle Mass: Physical Methods

Various approaches to the prediction of whole body and regional skeletal muscle mass in adults are presented in table 2. Techniques such as anthropometry and BIA that estimate whole body composition are well known to suffer from a significant standard error. One approach to resolving this issue has been to measure regional rather than whole body composition. For example, arm anthropometry has been used for several decades to distinguish fat and lean masses. This method is based on the assumption that the arm can be considered as a cylinder of lean mass surrounded by a layer of fat tissue. By measuring mid-upper arm circumference (MUAC) and the thickness of the fat layer using caliper measurements of the triceps skin fold (TS), the cross-sectional area of lean tissue (AMA, containing muscle and bone) can be calculated according to the following formula [17]:
AMA = [MUAC (TS )]2/(4 )

Despite the widespread application of this technique in epidemiological and clinical research, little effort has been directed to evaluating its validity. A recent study in both healthy children and patients with cystic fibrosis compared rankings of fat and lean mass obtained by arm anthropometry with reference data obtained using DXA (limb body composition) and the 4-component model (whole body composition). In children, lean mass is closely associated with body size; hence, taller children tend to
60
Horm Res 2006;66(suppl 1):5864

have greater lean mass. Unless an adjustment is made for this correlation, agreement between techniques is artificially inflated. In healthy children and patients, after adjustment for body size, estimations of arm cross-sectional muscle area explained 1642% of arm lean mass and 2433% of whole body lean mass [18]. In adults, total muscle mass has also been predicted from measurements of skin folds and body girths [9]. The standard error of the estimate was around 10%, implying that this approach would again be more suitable for monitoring groups than individuals. In adults, muscle mass has been predicted directly from BIA measurements [10]. The standard error of the estimate was around 9%, of similar magnitude to anthropometric approaches. Part of the inaccuracy of whole body BIA derives from the inability of this technique to accommodate regional variability in body proportions. This issue is also relevant to the need for ethnic-specific BIA equations. Conventionally, BIA involves measurements between the hand and foot, and is intended to predict total body water. Accuracy of this approach is reduced by unequal distribution of bioelectrical resistance across anatomy resistance is greatest in thin narrow cylinders such as the forearm and lower leg, and lowest in broad cylinders such as the trunk; however, this distribution is the inverse of body weight distribution [19]. Thus, total body resistance is disproportionately accounted for by those anatomical regions that contribute least to variability in body weight. This
Wells

can be resolved by measuring impedance in more specific regions. In a study of pre-pubertal children, segmental impedance measurements were found to have high correlations (r2 values ranging from 0.69 to 0.88) with DXA measurements of limb lean mass [20]. This study addressed only agreement in ranking between techniques, and did not attempt to estimate actual limb lean mass; nor was adjustment for body size incorporated. Segmental BIA represents a relatively simple and convenient technique, useful for the collection of pilot data, or for assessing change in individuals in whom information about the direction of change is potentially more important than the magnitude. However, in the absence of reference data, it cannot provide absolute lean mass values and hence offers abstract output only. New 8-electrode BIA instrumentation addresses the same problem by making simultaneous measurements of impedance in the arms, legs and trunk. In a study of 40 adults and children ranging from 6 to 64 years in age, skeletal muscle mass estimated from BIA was strongly correlated with reference DXA measurements, with group mean values not differing significantly between techniques [21]. However, in this study, standard error of the estimate was not presented and the high correlation may derive in part from the wide range of ages, and hence body sizes, studied. Further work is required, particularly in children, to identify with greater confidence its accuracy in individuals. Potentially more sophisticated measurement techniques include nuclear methodologies, which address particular elements assumed to have a constant relationship with particular tissues. Total body potassium scanning, for example, assumes a constant potassium content of the body cell mass, whilst total body nitrogen can also be used as an index of whole body protein mass. Both techniques suffer from uncertainty as to the consistency of relationships between the element measured and the assumed tissue content, which is of particular relevance to the application of such techniques in children. In adults, prediction of muscle mass from whole body potassium scanning is justified by the high proportion (approximately 60%) of body potassium found in skeletal muscle [4]. In a study of 300 individuals over a wide age range, muscle mass was predicted with an error of around 4.5% [14]. Such an approach may be less successful in children, however, due to age-related variability in the potassium content of lean tissue. Potassium scanning estimates body cell mass and will provide inaccurate assessment of muscle mass in conditions in which the ratio
Assessment of Body Components in Children

of muscle to non-muscle cell mass is abnormal. In vivo neutron activation (IVNA) analysis is a technique that quantifies total body nitrogen and total body potassium, and calculates muscle mass by assuming specific proportions of each element in this tissue. Unlike potassium, the majority of body nitrogen is found in non-skeletal muscle mass; hence, simultaneous measurements potentially allow the ratio between the two compartments to be estimated. Although this approach compared favourably with anthropometry and BIA, Wang and colleagues reported better performance by DXA than by IVNA [12]. The low availability of IVNA also precludes its use except in a handful of specialized research centres.
Prediction of Muscle Mass: Biochemical Methods

An alternative approach to the prediction of total muscle mass is the measurement of muscle metabolites (table 2). The most widely used proxy is creatinine, of which approximately 98% of its precursor is found in skeletal muscle in the form of creatine phosphate [22]. Skeletal muscle mass can therefore be estimated from 24-hour urine collection, values being calculated by assuming a given muscle mass per gram of creatinine excreted. The validity of this approach is limited by several factors [4], including within-individual variability in the daily rate of creatinine excretion, the effect of dietary intake, and the protocol used for urine collection. Variability in relation to age, gender, maturation status and activity level is also predicted to confound the relationship between creatinine production and muscle mass, while clinical status may also exert effects. For these reasons, this method once again has significant limitations for application in the paediatric population [4]. An alternative metabolite measurable in urine is 3methylhistidine, an amino acid likewise located primarily in skeletal muscle [4]. Its concentration in muscle is relatively constant with age, but its accuracy as a marker of muscle mass may be confounded by the effect of nonskeletal muscle protein turnover. Both these metabolites may also be obtained from meat in the diet, which represents a further potential source of inaccuracy. In general, biochemical methods have limited accuracy and the protocol is over-complex for children in most circumstances [4].

Horm Res 2006;66(suppl 1):5864

61

3 2 1 0

**

***

***

***

Bias

1 2 3 4 5 6

Fig. 2. Inconsistency in bias between DXA and the criterion 4component model in groups of individuals differing according to size, gender, nutritional status and health status. Based on data from Williams et al. [24]. The number of asterisks indicates the strength of significance of the bias. GSD = Glycogen storage deficiency.

Direct Measurement of Muscle Mass

In contrast to all the methods described so far, radiographic approaches allow direct visualization and measurement of muscle mass (table 2). The most widely available radiographic technique is DXA. This technique exposes the individual to a low level of radiation, similar to a typical days background radiation on most instrumentation. Attenuation of the X-ray beam is influenced by tissue depth and composition, allowing the differentiation of tissues including fat, lean and bone. In any pixel, the instrument first differentiates bone and non-bone tissue, and then, in non-bone tissue, differentiates fat and lean. A range of different instrumentation is now available, along with a variety of different software packages. Initial assessments of validity were interpreted in terms of high accuracy and precision, and for some researchers the technique came to represent a new gold
62
Horm Res 2006;66(suppl 1):5864

standard for the measurement of body composition. However, more recent studies comparing DXA against improved reference methods have demonstrated significant limitations in relation to both random and systematic error [23, 24]. A recent study of children found that the accuracy of DXA for assessment of whole body lean mass varied in relation to age, size and body fatness [24] (fig. 2). From a theoretical perspective, error is predicted to be greatest in the trunk region due to the higher proportion of pixels containing bone. Thus, limb lean mass is predicted to be measured with greater accuracy, suggesting that DXA can be used to provide proxy data on limb muscle mass in children. In adults, studies likewise demonstrate the utility of DXA for estimating whole body and regional muscle mass as measured by MRI. Whole body muscle mass was estimated with a standard error equivalent to around 5% using Lunar instrumentation [13]. Further studies are required to establish its validity using other manufacturers instrumentation and in children. Several authors have also demonstrated the capacity of DXA to estimate regional muscle mass, although again these studies currently focus only on adults [15, 16, 25, 26]. Although these studies suggest an important role for DXA in the estimation of muscle mass, the inaccuracies described above caution its application in longitudinal studies. Given the relationship between DXA bias and tissue thickness or fatness [24], changes in body composition during an intervention are predicted to alter the accuracy of DXA between baseline and follow-up measurements. However, the technique can demonstrate the direction, if not the magnitude, of longitudinal changes in muscle mass, and may therefore play a valuable role in clinical management. The most accurate methods for muscle mass estimation comprise CT and MRI scanning. CT scanning measures X-ray attenuation in three dimensions, unlike DXA which uses only two dimensions. CT voxels therefore have greater accuracy than DXA pixels. The method provides high-quality images and clear differentiation of tissue types. However, muscle tissue varies in its attenuation of X-rays in relation to muscle location, and other factors such as nutritional status or health of the individual are also influential. MRI scanning evaluates the behaviour of hydrogen nuclei in a magnetic field when exposed to radiofrequency waves. The resulting images relate to the distribution of protons in water molecules, and discriminate different tissues according to their relative hydration. Both MRI and CT scanning have been evaluated with reference to cadaver studies. Mitsiopoulos and colleagues compared both techniques with a single cadaver leg and
Wells

Non-obese men

Non-obese women

Non-obese girls

Obese women

Non-obese boys

Obese children

Cystic fibrosis

GSD

arm, each sectioned and photographed at specific distances that were also imaged by the scanners [27]. The authors reported very high correlations between methods, but the standard error of the estimate was approximately 10% for both techniques. However, a proportion of the lack of agreement between techniques could be attributed to imprecision of the cadaver estimate, quantified as 8.5%. The authors concluded that both methods could be considered reference methods for skeletal muscle tissue. CT and MRI scanning are hence widely regarded as the most accurate methods for regional body composition analysis and have the greatest potential to measure muscle mass with accuracy. MRI measurements can be made without difficulty in neonates and young infants, and in children from around 6 years. Their main limitations comprise their high cost and limited availability. However, CT scanning exposes the individual to a high level of radiation that may be unacceptable in most paediatric patients.
Reference Data

Conclusions

As much of the variability in childrens muscle mass can be attributed to body size and gender, interpretation of all data, regardless of the measurement method, is difficult without reference data. This is particularly the case for longitudinal measurements where the individual grows in between time points. Thus, simpler techniques will maximize their utility when such data are available, and their derivation must be a research priority alongside the refinement of accurate methodologies.

Over the past decade, there have been significant methodological advances in the capacity to measure human body composition. Techniques that assess muscle mass indirectly either through regional anthropometry, whole body composition techniques, or biochemical methods have been replaced by imaging techniques that allow site-specific investigations and contribute improved accuracy in the differentiation of fat and lean tissue. The gold standard for muscle mass estimation remains cadaver dissection, and all in vivo measurement techniques suffer from limitations in their accuracy. However, studies are increasingly highlighting the capacity of new technology such as MRI and CT scanning to discern with acceptable accuracy the effect of diseases and treatments on human muscle mass. DXA appears to be the most accurate technique amongst those that are more widely available than CT or MRI; it is adequate for detecting differences between groups and for identifying the direction of changes within individuals over time, but does not discern the magnitude of such differences as accurately as CT or MRI. Simpler techniques such as anthropometry or BIA may be of value in detecting differences between groups, but have poor accuracy in individuals and, typically, variable accuracy across ethnic groups. At present, most studies remain focused on adults and there is a need for further research addressing both the measurement of muscle mass in younger age groups and appropriate reference data.

References
1 International Commission on Radiological Protection: Report of the task force on reference man. Oxford, Pergamon, 1975, pp 108 112. 2 Fomon SJ, Haschke F, Ziegler EE, Nelson SE: Body composition of reference children from birth to age 10 years. Am J Clin Nutr 1982; 35:11691175. 3 Holliday MA. Body composition and energy needs during growth; in Falkner F, Tanner JM: Human Growth: A Comprehensive Treatise, Vol 2. New York, Plenum Press, 1986, pp 101107. 4 Lukaski HC: Estimation of muscle mass; in Roche AF, Heymsfield SB, Lohman TG (eds): Human Body Composition. Champaign, IL, USA, Human Kinetics, 1996, pp 109128. 5 Wells JC, Fewtrell MS: Measuring body composition. Arch Dis Child 2006;91:612 617. 6 Wells JC, Fewtrell MS, Davies PS, Williams JE, Coward WA, Cole TJ: Prediction of total body water in infants and children. Arch Dis Child 2005;90:965971. 7 Wells JCK, Fuller NJ, Dewit O, Fewtrell MS, Elia M, Cole TJ: Four-component model of body composition in children: density and hydration of fat-free mass and comparison with simpler models. Am J Clin Nutr 1999; 69:904912. 8 Ma G, Yao M, Liu Y, Lin A, Zou H, Urlando A, Wong WW, Nommsen-Rivers L, Dewey KG: Validation of a new paediatric air-displacement plethysmograph for assessing body composition in infants. Am J Clin Nutr 2004;79:653660. 9 Lee RC, Wang Z, Heo M, Janssen I, Heymsfield SB: Total body skeletal muscle mass: development and cross-validation of anthropometric prediction models. Am J Clin Nutr 2000;72:796803. 10 Janssen I, Heymsfield SB, Baumgartner RN, Ross R: Estimation of skeletal muscle mass by bioelectrical impedance analysis. J Appl Physiol 2000;89:465471.

Assessment of Body Components in Children

Horm Res 2006;66(suppl 1):5864

63

11 Wang Z, Deurenberg P, Matthews DE, Heymsfield SB: Urinary 3-methylhistidine excretion: association with total body skeletal muscle mass by computerized axial tomography. J Parenter Enteral Nutr 1998; 22: 8286. 12 Wang ZM, Visser M, Ma R, Baumgartner RN, Kotler D, Gallagher D, Heymsfield SB: Skeletal muscle mass: evaluation of neutron activation and dual-energy X-ray absorptiometry methods. J Appl Physiol 1996; 80: 824831. 13 Kim J, Wang Z, Heymsfield SB, Baumgartner RN, Gallagher D: Total body skeletal muscle mass: estimation by a new dual-energy X-ray absorptiometry method. Am J Clin Nutr 2002;76:378383. 14 Wang Z, Zhu S, Wang J, Pierson RN, Heymsfield SB: Whole-body skeletal muscle mass: development and validation of total-body potassium prediction models. Am J Clin Nutr 2003;77:7682. 15 Wang W, Wang Z, Faith MS, Kotler D, Shih R, Heymsfield SB: Regional skeletal muscle measurement: evaluation of new dual-energy X-ray absorptiometry model. J Appl Physiol 1999;87:11631171. 16 Shih R, Wang Z, Heo M, Wang W, Heymsfield SB: Lower limb skeletal muscle mass: development of dual-energy X-ray absorptiometry prediction model. J Appl Physiol 2000;89:13801386.

17 Frisancho AR: Triceps skin fold and upper arm muscle size norms for assessment of nutrition status. Am J Clin Nutr 1974;27:1052 1058. 18 Chomtho S, Fewtrell MS, Jaffe A, Williams JE, Wells JCK: Evaluation of arm anthropometry for assessing pediatric body composition: evidence from healthy and sick children. Pediatr Res 2006;59:860865. 19 Fuller NJ, Fewtrell MS, Dewit O, Elia M, Wells JCK: Segmental bioelectrical impedance analysis in children aged 812 y: 1. The assessment of whole-body composition. Int J Obes 2002;26:684691. 20 Fuller NJ, Fewtrell MS, Dewit O, Elia M, Wells JCK: Segmental bioelectrical impedance analysis in children aged 812 y: 2. The assessment of regional body composition and muscle mass. Int J Obes 2002; 26: 692 700. 21 Pietrobelli A, Rubiano F, St-Onge M-P, Heymsfield SB: New bioimpedance analysis system: improved phenotyping with wholebody analysis. Eur J Clin Nutr 2004;58:1479 1484. 22 Borsook H, Dubnoff JW: The hydrolysis of phosphocreatine and the origin of urinary creatinine. J Biol Chem 1947; 168:493510. 23 Wong WW, Hergenroeder AC, Stuff JE, Butte NF, Smith EO, Ellis KJ: Evaluating body fat in girls and female adolescents: advantages and disadvantages of dual-energy X-ray absorptiometry. Am J Clin Nutr 2002; 76:384389.

24 Williams JE, Wells JCK, Wilson CM, Haroun D, Lucas A, Fewtrell MS: Evaluation of Lunar Prodigy dual-energy X-ray absorptiometry for assessing body composition in healthy individuals and patients by comparison with the criterion four-component model. Am J Clin Nutr 2006;83:10471054. 25 Fuller NJ, Hardingham CR, Graves M, Screaton N, Dixon AK, Ward LC, Elia M: Assessment of limb muscle and adipose tissue by dual-energy X-ray absorptiometry using magnetic resonance imaging for comparison. Int J Obes 1999;23:12951302. 26 Elia M, Fuller NJ, Hardingham CR, Graves M, Screaton N, Dixon AK, Ward LC: Modelling leg sections by bioelectrical impedance analysis, dual-energy X-ray absorptiometry, and anthropometry: assessing segmental muscle volume using magnetic resonance imaging as a reference. Ann NY Acad Sci 2000;904:298305. 27 Mitsiopoulos N, Baumgartner RN, Heymsfield SB, Lyons W, Gallagher D, Ross R: Cadaver validation of skeletal muscle measurements by magnetic resonance imaging and computerised tomography. J Appl Physiol 1998;85:115122.

64

Horm Res 2006;66(suppl 1):5864

Wells