Moderating Factors in Tissue Tonometry and Bioimpedance Spectroscopy Measures in the Lower Extremity of Healthy Young People in Australia and Myanmar

Abstract

Background: Expected values for tissue tonometry and bioimpedance spectroscopy (BIS) in the lower extremity of young people have not been established. These measures are commonly used to assess tissue changes in adult, breast cancer-related lymphedema of the arm. In tropical regions, identification of tissue changes in the lower limbs related to lymphatic filariasis is required; hence expected values and factors contributing to variation in tissue tonometry and BIS in two tropical populations were investigated. Methods and Results: A convenience sample of healthy volunteers aged 8–21 in Myanmar and Australia was recruited. Tissue compressibility at the calf and anterior and posterior thigh was measured using three tonometry devices and free fluid in each leg was assessed using BIS. Data were collected about possible modifiers: leg dominance, age, gender, body mass index (BMI), hydration, and menstrual cycle. Paired t-test and linear regression compared the objective measures with possible modifiers within each population. Statistical significance was set at p < 0.05 with a 95% confidence interval. In Myanmar, increases in free fluid, tissue compressibility, and limb circumference were associated with being older, female, underweight, or in the second half of the menstrual cycle. In young Australians, increases in tissue compressibility and limb circumference were associated with being older or in the second half of the menstrual cycle. Conclusion: When assessing tissue compressibility and free fluid in young people using tonometry and gender, BIS, limb dominance and BMI should be considered in a local context and attempts should be made to minimize the potential influences of hydration and the female menstrual cycle.

Introduction

The lymphatic system collects circulating proteins and fluid from the tissue spaces and returns them to the cardiovascular system. If lymphatic function is disrupted, a high protein edema accumulates around the distal lymphatics under the skin and is termed lymphedema.¹ Untreated lymphedema follows a predictable course of changes in the skin and underlying tissue. In the earliest stages, the edema is fluid and protein rich, the skin is soft to touch, and there is minimal or transient visible swelling. In the middle stages, the subcutaneous compartment visibly enlarges and slowly becomes filled with fat and fibrous tissue, which ultimately replaces the edematous tissue spaces, there is increased vascularization, and free fluid reduces. In later stages, the skin becomes thickened, the tissue feels hard on palpation, and there is little or no excess free fluid.² This spectrum of changes can be measured objectively using tissue tonometry to quantify changes in skin and subcutaneous tissue stiffness, bioimpedance spectroscopy (BIS) to detect increases in extracellular free fluid load, and a tape measure for changes in limb circumference.^3,4 These devices have been validated in research on breast cancer-related lymphedema (BCRL) of the arm, largely among postmenopausal women in affluent countries after unilateral intervention for breast cancer, where the contralateral “unaffected” limb is available as a within-subject control.⁵ However, lymphedema is a disease of many causes, affects all age groups and genders, and is influenced by environmental, familial and genetic factors.^6,7 When both legs are involved, there is no unaffected, control limb, and even when only one leg is involved, the “unaffected” leg may be in a state of covert or latent lymphedema.⁸ In either case, the contralateral leg is not a reliable control and there are little data available on the assessment of lymphedema of the lower limb.⁹

Tissue stiffness or compressibility can be measured using a hand-held device called a tonometer, where a small indenter depresses the skin and underlying tissue under a 200 g mass. The mechanical nature of this device requires that it is held steady in a vertical position, while two analogue dials are read. More recently, electromechanical devices employing fixed spring or force sensor mechanisms have been developed to overcome some of these limitations.¹⁰ Primarily used within airconditioned offices to assess within-subject tissue changes in BCRL of the arm, few moderating factors are routinely considered other than arm dominance to account for between-limb differences in underlying muscle mass. Arm dominance is also considered when using BIS in BCRL patients, and studies in children have shown that age, gender, and body mass index (BMI) also influence BIS measures.^11,12 It is likely that these similar factors will influence tissue compressibility, but no corresponding data are currently available. Ambient temperature, systemic hydration, hormonal changes during the menstrual cycle, underlying muscle mass, and tone in the measurement area due to limb use could also affect extracellular free fluid load, but few studies have addressed this.^13–15

Globally, 16 million people have lymphedema in one or both lower limbs due to lymphatic filariasis (LF), a parasitic disease closely associated with poverty in developing countries.¹⁶ Unlike latest trends in screening and early intervention for prevention of BCRL of the arm,^17,18 assessment of lower limb lymphedema in LF endemic regions depends on classification of visible and palpable tissue changes¹⁹ and few recommendations are made for early intervention.²⁰ Objective and inexpensive devices to quantify progression or treatment effectiveness in clinical settings and operational research on filarial lymphedema are needed.

In preparation for a study to investigate early lymphatic change in young people living in a filariasis-endemic area in Myanmar, limb circumference, tissue tonometry, and BIS data were collected on two sets of young people living in tropical settings. A Mechanical Tonometer was included to enable comparison with a previous study in young adults from Papua New Guinea (PNG).²¹ However, it is no longer commercially available and two newer, digital devices were also included. Reliability analysis on the devices found good to excellent intrarater reliability in measures of healthy young people in Australia and Myanmar, and although each device reports a different unit of measure, the coefficient of variation was low, indicating a good level of agreement at most measurement points.²² This study reports expected values and individual factors associated with variance in device measures in healthy young Myanmar and Australian people living in tropical regions. These findings may be of use to clinical lymphologists and researchers to identify and quantify tissue changes related to lymphedema. The population standards and range of variation in device measures will be especially useful for clinical trials or studies when the contralateral leg is not suitable for comparison.

Materials and Methods

This was a cross-sectional study on physical measures of the lower extremity in two tropical dwelling cohorts of healthy young people. All procedures were conducted in accordance with the Helsinki Declaration of 1975, as revised in 2008. The James Cook University Human Research Ethics Committee (approval numbers H5261 and H5497) and the Department of Health, Myanmar Ministry of Health, approved the study. Young people aged 18–21 years gave informed consent to participate; a parent or guardian of minors aged 8–17 years provided consent and was asked to remain with the child during all data collection. Verbal assent was reiterated before any measure was taken. Volunteers were excluded if they had any clinical sign of lymphedema or any injury to their lower limbs. Participants were asked “Which foot do you use to kick a ball?” to determine leg dominance. Time since the last drink was used as a proxy for hydration and young women were asked the number of days since their last menses. Data were collected between October 2014 and May 2015 and in Myanmar, only persons who tested negative for LF infection were included in this analysis.

In Myanmar, local research assistants under the supervision of the principal researcher (J.D.) interviewed the participants, measured height and weight, and completed all data collection sheets. In Australia, the participant, their guardian, or a peer filled in the data collection sheets. The principal researcher (J.D.) conducted all measurements.

Devices

Tissue compressibility measures were completed at the midpoint of the anterior thigh, posterior thigh, and calf, which were located using a tape measure and marked with a washable skin marker. The three hand-held devices were (1) the Digital Indurometer, (2) the Mechanical Tonometer (both Flinders Biomedical Engineering, Australia), and (3) the SkinFibroMeter (Delfin Technologies, Finland). The first two devices have a 7 cm diameter reference plate, which is placed flat to the skin with a 1 cm diameter indenter that extends beyond the reference plate. The Mechanical Tonometer uses a 200 g mass to drive the indenter into the skin and subcutaneous tissue, and the Digital Indurometer has an internal force sensor to detect when a similar load has been applied by the operator. In these devices, the displacement between the reference plate and the indenter is recorded in 0.01 units. Lower values represent more tissue stiffness and higher values indicate increased tissue compliance (more compressibility). The third device, the SkinFibroMeter, has a smaller reference plate and a fixed 1.25 mm indenter. The reference plate is touched evenly to the skin five times and the average resistance to 50 g is displayed digitally in Newtons. Using this device, lower values represent less resistance (more compressibility) and higher values indicate more resistance (increased tissue stiffness). All three tonometers were used in the Myanmar group, but only the two digital devices were used in the Australian group.

Whole-limb free-fluid in each leg was assessed using the SBF7 (Impedimed, Australia), a portable battery-powered BIS unit. Self-adhesive electrodes were applied according to the manufacturer's instructions for whole-limb analysis. A low-level multifrequency current is passed between the electrodes and impedance in the intracellular fluid (ICF) and extracellular fluid (ECF) compartments are recorded. Since homeostasis should maintain ICF levels within a tight range of normal, fluctuations in tissue fluid will most likely occur in the ECF (free fluid), therefore when assessing unilateral BCRL, ECF accumulation in the affected limb can be compared to the unaffected contralateral limb. However, for bilateral measures, a ratio of ICF resistance to ECF resistance (Ri:Re) allows for better comparison of ECF values in the same limb over time.²³ In our study on young healthy people, a lower Ri:Re indicated an increase in ECF load. Limb circumference measures were collected at the midpoints of the thigh and calf using a tape measure. More detailed data collection methods were previously published in a reliability study on the devices in the same populations.²²

Data analysis

Sample size was originally estimated for a longitudinal study of LF-infected and LF-uninfected young people in Myanmar. This study includes only the antigen-negative individuals in Myanmar and compares them to a similar size group of Australian participants. An online calculator was used to estimate a sample size of 32 subjects per group using an expected mean Digital Indurometer value of 2.5 (SD 0.7) at mid-calf²² with a 95% confidence interval and 80% power (http://biomath.info/power/ttestnoninf.htm).

Height and weight data were used to calculate BMI using the formula, kg/m². Growth charts for adolescents in the United States were used to identify underweight (in or below the 5th percentile) or overweight (in or above 85th percentile) participants. Charts were accessed online during June 2016 (www.cdc.gov/healthyweight/assessing/bmi/childrens_bmi/about_childrens_bmi.html). The effect of age was evaluated by dividing participants into younger (8–16 years) or older (17–21 years) age groups. In Australia, the average of three measures was recorded for all devices. In the Myanmar cohort, an average of either three or two measures was used, dependent on whether the participant had been included in a previous reliability study on the devices (three measures)²² or not (two measures). Between-country comparisons were made using independent sample t-tests for continuous variables and Chi-Square or Fisher's exact tests for categorical variables. Paired sample t-tests were used for between-leg comparisons (dominant vs. nondominant). Linear regression was used to determine how moderating factors were associated with variance in device scores. Due to the number of factors under investigation, the regression was repeated to produce a model with as few variables as possible by removing one insignificant factor at a time. Only factors which significantly predicted variance in scores have been reported. All analyses were completed using SPSS Version 23 (IBM Corp.).

Results

The study sites of Amarapura (near Mandalay, Myanmar, northern hemisphere) and Townsville (Queensland, Australia, southern hemisphere) are approximately equidistant from the equator and both locations have dry tropical climates. All the Australian volunteers and the uninfected participants in Myanmar were included in this analysis (n = 85). The mean cohort age was 15.18 years (SD 3.66, range 8–21) and there were more females: 57% in Myanmar and 60% in Australia. Mean height was 155.43 cm (SD 13.15, range 118.80–187.50 cm) and mean weight was 46.5 kg (SD 13.7, range 17.5–86.1 kg). Australian participants were significantly taller and heavier than the Myanmar cohort and did not differ markedly from U.S. anthropometric data. Table 1 shows the characteristics of the Myanmar and Australian cohorts by gender. A greater portion of Myanmar participants were in or below the 5th percentile for BMI, n = 16 (31.4%), compared to Australians, n = 3 (8.8%), but this was only statistically significant between the male cohorts. There were no overweight cases in either group and both cohorts were right-leg dominant, Myanmar 98% and Australia 94%. Fewer people in the Myanmar cohort had consumed a drink within the previous hour than their Australian counterparts (26.5% vs. 60.6%). There were no significant between-country differences in age groups or time since the last menstrual period (females).

Table 1.

Comparison of Participant Characteristics in Myanmar and Australian Cohorts

Characteristics	Gender	Myanmar (n = 51)	Australia (n = 34)	p
Aged 17–21 years, n (%)	Male	6 (27.3)	5 (35.7)	0.604
Aged 17–21 years, n (%)	Female	13 (44.8)	9 (45.0)	0.991
Height, cm, mean (SD)	Male	151.40 (13.97)	158.96 (20.97)	0.189
Height, cm, mean (SD)	Female^a	152.88 (9.13)	161.08 (9.03)	0.003
Weight, kg, mean (SD)	Male^a	40.77 (10.39)	51.92 (20.27)	0.037
Weight, kg, mean (SD)	Female^a	43.40 (9.67)	53.38 (12.94)	0.003
BMI, kg/m², mean (SD)	Male^a	17.46 (2.41)	19.62 (3.42)	0.033
BMI, kg/m², mean (SD)	Female^a	18.43 (2.83)	20.29 (3.31)	0.041
BMI ≤5th percentile,^b n (%)	Male^a	12 (54.5)	1 (7.1)	0.004
BMI ≤5th percentile,^b n (%)	Female	4 (13.8)	2 (10.0)	0.527
Last drink >1 hour before, n (%)	Male	13 (59.1)	5 (35.7)	0.194
Last drink >1 hour before, n (%)	Female^a	23 (79.3)	8 (40.0)	0.002
Last menses >14 days prior, n (%)	Female	10 (34.5)	8 (40.0)	0.672

Significant between-group differences.

CDC growth charts for children in United States www.cdc.gov/healthyweight/assessing/bmi/childrens_bmi/about_childrens_bmi.htm.

BMI, body mass index.

Devices, measuring points, and leg dominance

A consistent pattern of most compressible (softest) tissue over the anterior thighs and least compressible (stiffest) tissue at the calves was found with all three tonometers. Values for the posterior thighs fell between these and this pattern held true for age, gender, and country subgroups. There was significantly more free fluid in the dominant leg (7%, p = 0.034), but this pattern did not hold true for subgroups by country and gender. In both cohorts, being in the older age group was associated with larger limb circumference and being underweight was associated with smaller limb circumference.

In the Myanmar cohort, skin was less compressible (stiffer) over the dominant anterior thigh and thigh circumference was larger on the same side. On the nondominant leg, the skin was less compressible over the posterior thigh and calf, calf circumference was larger, and there was slightly more free fluid. In the Australian cohort, the circumference of the thigh and calf was both slightly larger on the dominant side with significantly more free fluid on that side (20.7%, p < 0.01), but no between-limb pattern of tissue compressibility. Mean device values and between-limb differences for Myanmar and Australian cohorts are provided in Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/lrb).

Mechanical tonometer

Used only in the Myanmar group, being in the older age group was associated with increased tissue compressibility at both anterior thighs. Higher compressibility scores were also associated with being female, or in the second half of the menstrual cycle at all posterior limb points. Increased tissue stiffness was associated with less recent hydration at all posterior limb points. Table 2 shows factors significantly associated with Mechanical Tonometer measures in the Myanmar group.

Table 2.

Factors Associated with Variation in Measures Using the Mechanical Tonometer (Myanmar Cohort)

Country/measure	Factor	Unstandardized coefficient (95% CI)	Direction	t
Anterior thigh
Dominant	17–21 years	0.921 (0.466, 1.376)	Softer	4.065^a
Nondominant	17–21 years	0.504 (0.056, 0.953)	Softer	2.264^b
Nondominant	Female	0.538 (0.101, 0.976)	Softer	2.474^b
Posterior thigh
Dominant	>1 hour since last drink	−0.891 (−1.494, −0.288)	Harder	−2.976^a
Dominant	>14 days since menses	0.883 (0.541, 1.226)	Softer	5.197^a
Nondominant	Female	0.998 (0.484, 1.511)	Softer	3.911^a
Nondominant	>1 hour since last drink	−0.895 (−1470, 0.319)	Harder	−3.129^a
Calf
Dominant	Female	1.290 (0.844, 1.736)	Softer	5.824^a
Dominant	>1 hour since last drink	−0.674 (−1.174, −0.174)	Harder	−2.715^a
Nondominant	Female	1.042 (0.629, 1.456)	Softer	5.077^a
Nondominant	>1 hour since last drink	−0.636 (−1.099, −0.172)	Harder	−2.762^a

p < 0.01.

p < 0.05.

Digital indurometer

In the Myanmar cohort, being in the older age group was associated with increased compressibility at the dominant anterior thigh. For all other measures, being female was associated with higher compressibility scores and having less recent hydration was associated with greater tissue stiffness. There was a significant association with being underweight and increased compressibility at the nondominant calf.

In the Australian cohort, increased compressibility was associated with being in the older age group at both posterior thighs and with being underweight at all posterior thigh and calf points. Significant factors associated with Digital Indurometer measures are given in Table 3.

Table 3.

Factors Significantly Associated with Variation in Compressibility Measures Using the Digital Indurometer

Country/measure	Factor	Unstandardized coefficient (95% CI)	Direction	t
Myanmar
Anterior thigh
Dominant	17–21 years	0.636 (0.276, 0.996)	Softer	3.548^a
Nondominant	Female	0.424 (0.039, 0.810)	Softer	2.216^b
Nondominant	>1 hour since last drink	−0.420 (0.852, 0.013)	Harder	−1.954^b
Posterior thigh
Dominant	Female	0.866 (0.419, 1.312)	Softer	3.900^a
Dominant	>1 hour since last drink	−0.668 (−1.169, −0.167)	Harder	−2.685^b
Nondominant	Female	0.962 (0.483, 1.441)	Softer	4.042^a
Nondominant	>1 hour since last drink	−0.777 (−1.314, −0.240)	Harder	−2.912^a
Calf
Dominant	Female	0.781 (0.448, 1.113)	Softer	4.725^a
Dominant	>1 hour since last drink	−0.419 (−0.792, −0.046)	Harder	−2.263^b
Nondominant	Female	0.898 (0.559, 1.237)	Softer	5.331^a
	>1 hour since last drink	−0.606 (−0.954, −0.257)	Harder	−3.499^a
	<5th percentile for BMI	0.526 (0.169, 0.882)	Softer	2.970^a
Australia
Posterior thigh
Dominant	17–21 years	0.788 (0.215, 1.360)	Softer	2.804^a
Dominant	<5th percentile for BMI	1.281 (0.287, 2.274)	Softer	2.628^b
Nondominant	17–21 years	1.126 (0.559, 1.692)	Softer	4.054^a
Nondominant	<5th percentile for BMI	1.220 (0.237, 2.202)	Softer	2.531^b
Calf
Dominant	<5th percentile for BMI	1.583 (0.635, 2.531)	Softer	3.400^a
Nondominant	<5th percentile for BMI	1.103 (0.137, 5.068)	Softer	2.326^b

p < 0.01.

p < 0.05.

SkinFibroMeter

In the Myanmar cohort, increased tissue compressibility at all measurement sites, was associated with being female or in the second half of the menstrual cycle, and with being in the older age group (17–21 years) only at the dominant anterior thigh. At both calves, increased tissue stiffness was associated with not having a drink in the previous hour.

In the Australian cohort, increased compressibility at the nondominant anterior and both posterior thighs was associated with being in the older age group. Being female was associated with more stiffness at the nondominant anterior thigh point, but the effect size was small (based on mean values found in Supplementary Table S1). Significant factors associated with SkinFibroMeter measures are given in Table 4.

Table 4.

Factors Significantly Associated with Variation in Compressibility Measures Using the SkinFibroMeter

Country/measure	Factor	Unstandardized coefficient (95% CI)	Direction	t
Myanmar
Anterior thigh
Dominant	17–21 years	−0.009 (−0.016, −0.002)	Softer	−2.607^a
Dominant	Female	−0.012 (−0.018, −0.005)	Softer	−3.455^b
Nondominant	>14 days since menses	−0.010 (9–0.014, −0.006)	Softer	−4.972^b
Posterior thigh
Dominant	Female	−0.026 (−0.036, −0.016)	Softer	−5.151^b
Nondominant	Female	−0.019 (−0.032, −0.007)	Softer	−3.109^b
Calf
Dominant	Female	−0.024 (−0.041, −0.006)	Softer	−2.746^b
Dominant	>1 hour since last drink	0.024 (0.004, 0.043)	Harder	2.432^a
Nondominant	Female	−0.024 (−0.041, −0.007)	Softer	−2.817^b
	>1 hour since last drink	0.019 (0.000, 0.038)	Harder	1.972^a
Australia
Anterior thigh
Nondominant	17–21 years	−0.011 (−0.020, −0.002)	Softer	−2.557^a
	Female	0.012 (0.003, 0.020)	Harder	2.642^a
Posterior thigh
Dominant	17–21 years	−0.020 (−0.036, −0.004)	Softer	−2.559^a
Nondominant	17–21 years	−0.021 (−0.036, −0.005)	Softer	−2.720^a

p < 0.05.

p < 0.01.

SBF7

In Myanmar, increased free fluid was associated with being in the older age group (dominant leg) or in the second half of the menstrual cycle (nondominant leg), and lower free fluid loads were associated with less recent hydration in both legs. There were no significant associations between any of the factors and BIS measures in the Australian group. Factors significantly associated with BIS measures are given in Table 5.

Table 5.

Factors Associated with Variation in Free Fluid Measures Using Bioimpedance Spectroscopy in the Myanmar Cohort

Limb	Factor	Unstandardized coefficient (95% CI)	Direction	t
Dominant leg	17–21 years	−0.342 (−0.575, −0.108)	More	−2.295^a
Dominant leg	>1 hour since last drink	0.415 (0.172, 0.658)	Less	3.445^a
Nondominant leg	>1 hour since last drink	0.329 (0.046, 0.612)	Less	2.347^b
Nondominant leg	>14 days since menses	−0.253 (−0.420, −0.087)	More	−3.065^a

p < 0.01.

p < 0.05.

Circumference

In addition to expected variances in limb circumference associated with age or being underweight, in the Myanmar group, being in the second half of the menstrual cycle was associated with a larger circumference at the midpoint of the dominant thigh, while a smaller circumference at the same point was associated with less recent hydration. In the Australian group, as well as the expected age- and underweight-related variances, being in the second half of the menstrual cycles was related to a larger circumference at both thigh and calf. Significant factors associated with limb circumference are given in Table 6.

Table 6.

Factors Significantly Associated with Variation in Measures of Limb Circumference

Country/measure	Factor	Unstandardized coefficient (95% CI)	Direction	t
Myanmar
Thigh
Dominant	17–21 years	5.199 (2.625, 7.773)	Larger	4.071^a
	<5th percentile for BMI	−5.244 (−7.869, −2.619)	Smaller	−4.026^a
	>1 hour since last drink	−2.779 (−5.318, −0.239)	Smaller	−2.205^b
	>14 days since menses	1.770 (0.827, 0.238)	Larger	−2.140^b
Nondominant	17–21 years	6.444 (4.190, 8.698)	Larger	5.749^a
Nondominant	<5th percentile for BMI	−6.470 (−8.819, −4.122)	Smaller	−5.540^a
Calf
Dominant	17–21 years	2.577 (1.348, 3.806)	Larger	4.217^a
Dominant	<5th percentile for BMI	−3.051 (−4.332, −1.771)	Smaller	−4.791^a
Nondominant	17–21 years	2.317 (1.113, 3.521)	Larger	3.869^a
Nondominant	<5th percentile for BMI	−2.936 (−4.191, −1.682)	Smaller	−4.706^a
Australia
Thigh
Dominant	17–21 years	6.746 (2.944, 10.547)	Larger	3.629^a
	<5th percentile for BMI	−11.562 (−17.374, −5.750)	Smaller	−4.069^a
	>14 days since menses	2.917 (0.723, 5.110)	Larger	2.720^b
Nondominant	17–21 years	6.800 (3.169, 10.430)	Larger	3.830^a
	<5th percentile for BMI	−11.689 (−17.240, −6.137)	Smaller	−4.306^a
	>14 days since menses	2.794 (0.699, 4.889)	Larger	2.728^b
Calf
Dominant	17–21 years	3.662 (1.458, 5.866)	Larger	3.398^a
	<5th percentile for BMI	−4.973 (−8.343, −1.604)	Smaller	−3.018^a
	>14 days since menses	1.509 (0.237, 2.780)	Larger	2.426^b
Nondominant	17–21 years	4.033 (1.819, 6.248)	Larger	3.726^a
	<5th percentile for BMI	−5.270 (−8.656, −1.885)	Smaller	−3.184^a
	>14 days since menses	1.369 (0.091, 2.646)	Larger	2.191^b

p < 0.01.

p < 0.05.

Discussion

Healthy, prepubescent children in all countries will undergo noticeable changes in body size and composition as they transition through adolescence and become young adults. In keeping with this expectation, limb circumference in both cohorts increased in an unremarkable association with age and decreased in those who were underweight.

There were significant directional changes associated with limb dominance in most measures and in the Myanmar cohort, this was consistent with expected muscle use during a kick. The kicking leg, labeled as dominant, had a slightly larger thigh circumference, while the leg supporting the weight of the body to propel it forward during the kick (nondominant leg) had a slightly larger calf circumference. Between-limb differences in tissue compressibility among Myanmar participants supported this pattern of muscle development (stiffer tissue over the front of the kicking leg and over the back of the supporting leg); however, there was no between-leg difference in free fluid loads. Taken together, between-leg measures suggest that neither lower limb is substantially more developed than the other in this cohort. In contrast, the young Australians appeared to be more homolateral with slightly larger thigh and calf circumferences and significantly more free fluid—all on the dominant side, but with no corresponding pattern of tissue compressibility. A similar study conducted in PNG on a comparable group of young people reported Mechanical Tonometer, circumference and BIS measures in an LF-endemic area. The same between-leg, “kicking” pattern, of tissue compressibility and circumference measures as found in the Myanmar cohort was reported among the young PNG participants (larger/stiffer dominant thigh, larger/stiffer non-dominant calf). The small difference in between-leg free fluid ratios was similar, but a different variable (ECF only) was used in the PNG analysis, so the results are not directly comparable.²¹ It is not possible from the data available to determine exactly why the between-leg patterns are not consistent in young people across all cohorts. Perhaps the young Myanmar and PNG people had spent more time kicking a ball than their Australian counterparts. The lack of any clear cross-cultural consistency in between-leg differences in any measure indicates that a universal assumption for leg dominance among young people cannot be made.

The units of mean compressibility measures from all devices are not directly equivalent, but when viewed together reveal expected tissue properties at each measurement site. Overlying the between-leg differences is a prevailing pattern that reflects normal fat distribution in the lower limbs and between-gender differences in muscle/fat ratios. The fatty layers over the anterior thighs were the most compressible (softest) while the mid-calf point, located over a dense tendomuscular junction with little overlying fat, was always the least compressible (stiffest). Universal patterns were seen less frequently in the regression models, but there were distinct trends; being older, female, or in the second half of the menstrual cycle was frequently associated with a larger, softer limb and more free fluid. Wherever such relationships were found, not having a drink within the previous hour was associated with increased tissue stiffness, and being underweight with increased compressibility.

Moderating factors were more frequently associated with variation in device measures in the Myanmar group than among the young Australians. In particular, there were multiple associations with hydration in Myanmar, but none in the Australian cohort, who were more recently hydrated. Significant associations with being underweight were only found when using the Digital Indurometer, but when considering BMI, adult values cannot be used for children,²⁴ and the World Health Organization (WHO) advises that normal BMI for adults in Asian countries can be 3 kg/m² less than Western standards.²⁵ In the absence of standardized growth data for young Myanmar people, using CDC growth charts may have misclassified some young Myanmar people of normal BMI (for Myanmar) to the underweight category; and given the small number of Australians who were classified as underweight (n = 3), reported variances associated with being underweight in either cohort should be viewed cautiously.

Mechanical Tonometry, which was used in both the Myanmar group and the PNG study, found softer tissues in the Myanmar group, while the Digital Indurometer, which was used in the Myanmar and Australian groups, found softer tissues in the Australian group. The direction of association between geographical locations—PNG, Myanmar, and Australia—and increasing tissue compressibility suggest that there may be a directional association between compressibility and economic development. If this is true, the cutoff figures proposed to identify subclinical change between LF-infected and LF-uninfected young people in the PNG study²¹ should not be generalized to other populations.

Among the LF-negative cases in the PNG group, between-leg differences in ECF values cases were consistent with the increased free fluid (lower Ri:Re values) found in the dominant leg in the Australian group, but not with the very small shift in the opposite direction among the young Myanmar participants. The lack of any homogeneous pattern of tissue compressibility or free fluid across all ethnic groups highlights the variation in expected values and difficulty in determining limb dominance in the lower extremities. Patterns of usual muscle activity should be taken into account when assigning criteria for leg dominance, and expected values in tissue compressibility and free fluid should be viewed in a local context.

When using these devices to track changes in the same subject over time, gender can be ignored, growth can be taken into account in the same way that changes in BMI are considered when assessing adults, and individual patterns of between-limb use should be defined. Of the other factors explored in this analysis, none had universal association with all measures, but most could be minimized with little effort. In the young Myanmar population, hydration contributed to variance in all devices, but this could be controlled by administering a standardized drink before measurements. In young women, the effect of regular hormonal fluctuations could be eliminated by timing repeated measures on young women to be taken at the same time of the monthly cycle.

Few studies have explored the effect of season or ambient temperature on lymphatic function. In a study on BCRL of the arm, Czerniec, and colleagues¹⁴ reported a slight increase in ECF associated with higher temperatures on the day before measurement (r = 0.27, p < 0.001), whereas a similar cohort of women without arm lymphedema had no seasonal variation in ECF. In this study, it was not possible to control for ambient temperatures, but the study sites were roughly equidistant from the equator in dry tropical zones with similar climates. The single operator could not be adequately blinded to any of the parameters measured, but the use of a scribe reduced the risk of bias. The sample was large enough to provide statistical significance for the between-leg differences and most of the regression analyses, but recruitment methods prevent the data from being truly representative of each population. Therefore, while it is not expected that these data will be directly transferrable to other settings, the mean values, standard deviations, and regression models provided may be useful in estimating expected values and cutoff points in future research on young populations at risk of lower limb lymphedema.

Conclusion

This study has demonstrated country-specific effects of age, gender, BMI, hydration- and menstrual cycle on tissue compressibility and free fluid loads in the lower limbs of young people. When using tissue tonometry and BIS, attempts to minimize moderating factors should be made such as administering a drink during the hour before measures and by collecting data on young women in the first half of the menstrual cycle. The definition of leg dominance and normal BMI should be determined in the context of local life.

Footnotes

Acknowledgments

Grateful thanks to the Myanmar Ministry of Health and Sports, the Vector Borne Disease Control Mandalay Regional Office, local administration center staff, and research assistants. A backup SBF7 Body Analyser and supply of electrodes was provided by Impedimed, Australia. The SkinFibroMeter was on loan from Delfin Technologies, Finland. The Physiotherapy Department, JCU Townsville, supplied the SBF7 Body Analyser, Digital Indurometer, and Mechanical Tonometer.

Author Disclosure Statement

This study was conducted as part of a longitudinal study on early detection of lymphatic dysfunction in Myanmar and undertaken as a part of the PhD research project of J.D. No competing financial interests exist.

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