Research Article: A guide for using NIH Image J for single slice cross-sectional area and composition analysis of the thigh from computed tomography

Date Published: February 7, 2019

Publisher: Public Library of Science

Author(s): Douglas E. Long, Alejandro G. Villasante Tezanos, James N. Wise, Philip A. Kern, Marcas M. Bamman, Charlotte A. Peterson, Richard A. Dennis, Sarah P. Psutka.


Reports using computed tomography (CT) to estimate thigh skeletal muscle cross-sectional area and mean muscle attenuation are often difficult to evaluate due to inconsistent methods of quantification and/or poorly described analysis methods. This CT tutorial provides step-by-step instructions in using free, NIH Image J software to quantify both muscle size and composition in the mid-thigh, which was validated against a robust commercially available software, SliceOmatic. CT scans of the mid-thigh were analyzed from 101 healthy individuals aged 65 and older. Mean cross-sectional area and mean attenuation values are presented across seven defined Hounsfield unit (HU) ranges along with the percent contribution of each region to the total mid-thigh area. Inter-software correlation coefficients ranged from R2 = 0.92–0.99 for all specific area comparisons measured using the Image J method compared to SliceOmatic. We recommend reporting individual HU ranges for all areas measured. Although HU range 0–100 includes the majority of skeletal muscle area, HU range -29 to 150 appears to be the most inclusive for quantifying total thigh muscle. Reporting all HU ranges is necessary to determine the relative contribution of each, as they may be differentially affected by age, obesity, disease, and exercise. This standardized operating procedure will facilitate consistency among investigators reporting computed tomography characteristics of the thigh on single slice images.

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Skeletal muscle accounts for approximately 40–45% of body mass and plays a vital role in health and disease through its influences on energy metabolism, glucose utilization, and physical function. Skeletal muscle is a remarkably plastic tissue continuously adapting to physiological and pathological conditions. The most potent modulators of the skeletal muscle phenotype are contraction and load, experienced during endurance and resistance exercise, whereas skeletal muscle loss is exacerbated by injury, illness, disuse, insufficient nutrient intake, and aging. This dramatic ability of skeletal muscle to adapt to various stimuli and its connection to morbidity and mortality make it the center of investigation in frailty, cachexia, sarcopenia, and metabolism-related research [1,2]. Thus, accurate measurements of muscle mass, size, and composition are critically important.

CT quantifies a variety of unique body composition characteristics in physiology research. However, the analysis of the produced images using complex or costly software systems may hinder the use of this technique. Two software packages are used regularly in the literature for the assessment of body composition by CT; SliceOmatic and NIH Image J, each having their own advantages and disadvantages that have been fully described elsewhere [12]. SliceOmatic comes with a very user-friendly interface and includes technical support, but contains costly start-up and yearly licensing fees. In addition, SliceOmatic allows the user to define HU ranges to delineate tissues such as fat, muscle, and bone. On the other hand, NIH Image J is freely available, but is limited to online developer resources making it difficult for a new user to effectively use the software. The NIH Image J website offers an online instruction manual for the various functions found within the software but none to our knowledge related to muscle size and composition determination. Typically, investigators using Image J must segment the region of interest by carefully tracing along the perimeter. Raw data (pixel counts) from the 16-bit user-traced region of interest is then exported into an excel program with automated area calculations for the determination of each tissue type based on pre-defined HU ranges using the histogram function (7–9). However, details of this process have not been fully described nor standardized. Here, we provide a step-by-step process for analyzing single-slice CT images of the mid-thigh using NIH Image J for the measurement of muscle and fat areas as well as mean attenuation in each soft tissue depot. This technique was chosen for three reasons: 1) to essentially eliminate inter-observer error; 2) to ensure correct and proper image calibrations within Image J rather than calculating it in an excel file; and 3) to threshold or segment HU ranges in an automated fashion within the program similar to SliceOmatic. Previously reported inter-observer coefficients of reliability between softwares are very high with values normally ranging from 0.98 to 1.00 for all comparisons between CSA measurements [12,13]. By utilizing the wand tool (as long as there is separation between the thighs), manual tracing differences between investigators are eliminated. Furthermore, Image J should automatically calibrate images to the proper scale based on the pixel width and pixel area; this eliminates the need to change pixel width in pre-set excel files as this could drastically affect calculated CSA data if it is not corrected for the field of view (FOV) of the obtained image. While we specifically measure the total mid-thigh region, this tutorial can be applied to individual muscle groups of the thigh, such as the vastus lateralis. More research is required to determine if this protocol will accurately quantify other anatomical regions of interest.

A limitation of this study is that CT scans were obtained from an older population that was generally healthy, predominately white, and had mostly normal or overweight BMIs. Generalizations to younger adults, or older adults with health conditions associated with obesity should be taken with caution as higher fat depots introduce measurement error. However, significant bias was not seen with abdominal visceral fat measures in abdominally obese individuals using either software package [12]. In addition, a general weakness of performing a single slice CT scan is that a detailed and precise standard operating procedure must be in place to position the scanner in order to get reliable, longitudinal values. To increase reliability, a scout scan should be performed so that anatomical landmarks of the femur can be used to determine the thigh midpoint.

The tissue density range used to define muscle and fat varies between studies and areas of intermediate density are often omitted. This CT tutorial provides valuable step-by-step instructions for using free, but technically challenging, software to quantify both muscle size and composition in the mid-thigh, which was validated against a robust commercially available software. Image J and SliceOmatic generate similar results for the measurement of muscle and fat of all densities at the mid-thigh. We show that by evaluating individual HU sub-ranges, as much as 7.5% of overall total thigh area could be unaccounted for by omitting areas of intermediate density such as VLDM and VHDM. Thus, we recommend all individual HU ranges be evaluated as interests dictate to be the most inclusive of skeletal muscle and to determine responses due to aging and diseases processes, nutritional intervention, or exercise training. This standardized operating procedure will facilitate consistency among investigators reporting computed tomography characteristics of the thigh on single slice images.




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