Introduction

Hepatic steatosis is a growing global concern that is often concurrent with iron overload, creating many morphological disturbances in the liver. In measuring hepatic steatosis, R2* correction is needed to improve the accuracy in quantification of fat fraction (FF).1 The current methods are single R2* correction (implies that water and fat share the same R2* and dual model R2* correction in which water and fat are independent. The use of both R2* correction models in the combined presence of iron and fat depends on the dephasing effects of iron deposits on water and fat protons, creating a dependence on the size and distribution of iron and fat particles.2 The size of in vivo hepatic fat droplets normally does not exceed 15-25 µm in diameter.3 However, current imaging phantoms that emulate hepatic steatosis often do not control the size of fat droplets and exceed the in vivo threshold.4 The primary goal of this study is to analyze the impact of fat droplet size on the response of R2* water and fat values in the presence of iron and fat and with respect to quantitative MRI.

Methods

Homogeneous fat-water-iron emulsion phantoms were produced at fat fractions of 0%, 10%, 20%, 30%, and 40% at iron concentrations of 0%, 0.0075%, 0.025%, and 0.06%. One set of phantoms was homogenized using a Benchmark Scientific D1000 homogenizer with a 10mm x 115mm saw tooth generator probe at approximately 15000 rpm, and the second set was mixed with a stir bar and magnetic hot plate at 1400 rpm. The fat source of these phantoms consisted of peanut oil emulsified with 43mM SDS. The iron nanoparticles (Bionized NanoFerrite, Micromod, Rostock, Germany) of concentration 29.2 mg/ml were held in suspension using 2% agar and were supplemented with 0.5 mM CuSO4 and 43 mM NaCl. A total of 40 phantoms were created (5 fat fractions x 4 iron concentrations x 2 sets). Fat droplet sizes were characterized with bright field light microscopy (Olympus BX-63) and quantified using the MorphoLibJ plugin on ImageJ.5 Magnetic resonance imaging was performed at 1.5T (MAGNETOM Avantofit, Siemens) using 2D GRE sequences with a monopolar gradient. Single R2* correction was performed with a mono-exponential model and NLSQ (Non-linear Least Squares) while dual R2* model was implemented with ARMA (Autoregressive Moving Average).6 Imaging parameters used included: TE1 = 1.2 ms, Echo Spacing= 1.44 ms, TR=200ms, echo train length=20, flip=35°, 104x128 matrix, and 5mm slices. MATLAB (R2020b) was used for ROI selection and R2*, fat fraction, and iron quantification.

Results & Discussion

The mean diameter size of the fat droplets was 14.31µm for the stir bar phantoms and 5.79µm for the homogenized phantoms as seen on Figure 1. Unlike prior literature findings in which mean fat droplet diameters were greater than 50µm, the mean fat droplet diameters of the traditional stir bar methods fell within the in vivo hepatocyte morphology range of less than 20µm, while the homogenized phantoms fell far below this value.4 The phantoms were processed through MATLAB to determine R2* values and FF by selecting ROI’s and finding the mean and standard deviation of each selection. As shown on Figure 2, increasing iron concentrations from 0% to 0.06% showed an increase in R2*. On Figure 4, the stir bar phantoms showed that higher FF’s have higher R2* values for all three R2* models, but the homogenized phantoms showed decreased R2* values for the mono exponential and ARMA models. This may be due to the reduced size of fat droplets in homogenized phantoms altering the distribution of iron particles. Figure 3 shows a comparison of the predicted FF against the estimated FF in both the NLSQ and ARMA models. At higher iron concentrations, both models failed to quantify FF accurately, however, the NLSQ model appeared to predict the FF slightly better than the ARMA model. This coincides with previous research in which NLSQ outperformed ARMA at high FF and high iron.6

Conclusion

Our study demonstrates that homogenization indeed decreases fat droplet size in MRI phantoms and decreases R2* (s-1), but this must be compared to in vivo R2* and FF data to determine if homogenization will provide a realistic tissue model of steatosis and iron overload in phantoms.

Acknowledgements

This project was funded by the National Institutes of Health (NIH) grant #1R21EB031298. The authors thank St. Jude Children’s Research Hospital for allowing the use of the MRI scanner.

References

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