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Biomedical Imaging and Instrumentation

Biomedical Imaging and Instrumentation - Poster Session B

Poster G6 - Examining Feasibility of Diffuse Reflectance Spectroscopy for Analysis of Formalin Fixed Paraffin Embedded Cancer Samples

Thursday, October 24, 2024

2:45 PM - 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Micah Dick

Student
University of Arkansas
Wichita, Kansas, United States

Primary Investigator(s)

Narasimhan Rajaram, PhD

Associate Professor of Biomedical Engineering
University of Arkansas
Fayetteville, Arkansas, United States

Introduction: Diffuse reflectance spectroscopy (DRS) is a low-cost and non-invasive method that uses a low-power, non-ionizing, visible to near-infrared light source and optical fibers to collect diffusely reflected light from samples. The non-destructive nature of the light interaction provides information regarding absorption from hemoglobin, oxygen saturation, and other absorbers as well as scattering from cells, organelles, and collagen. This information has been used for various applications, such as cancer diagnosis, monitoring response to therapy, and delineating tumor surgical margins. Formalin Fixed Paraffin Embedded (FFPE) samples are a commonly used method to prepare and store patient biopsies for oncology and cancer research. They are easy to obtain, affordable, and stable. Due to their popularity within cancer research, we propose to investigate the feasibility of DRS to obtain spectra from FFPE samples.

Materials and

Methods: The DRS system consists of a halogen lamp as its light source and a USB spectrometer for capturing spectra. The multimodal handheld probe features a central 300 μm core filtered fiber, housed in an SMA connector for light excitation. Light is gathered through six 300 μm fibers, positioned in a circular arc 2.5 mm away from the core fiber. The probe includes a single input connection for interfacing with the halogen lamp source. Depending on the size of the FFPE blocks, about 5-20 spectra were obtained from each tumor block using a 500 ms integration time. We acquired spectra from a total of 29 tumor blocks. The location of the spectrum was then annotated on a photo of each block. Background subtraction was performed in MATLAB and the data was then exported to Microsoft Excel where specific absorbance and scattering ratios were calculated. These ratios were based on reflectance values at specific wavelengths and were 584/545 for total hemoglobin, 539/545 for oxygen saturation, and 490/600 for scattering.

Results, Conclusions, and Discussions: By gathering multiple spectra per sample, DRS could identify the properties of individual areas within each sample which could be compared to the entire sample. The hemoglobin ratio was effective in determining which samples contained blood, which was demonstrated by a large range between the ratios of the samples. By plotting the spectra and noting those with high hemoglobin absorbance, Figure 1 shows the difference between the samples with high hemoglobin absorbance and the other samples using a total hemoglobin ratio. It became evident that the paraffin wax that the tumors were embedded in influenced the spectra, so it was important to understand when it was affected or if it was possible to remove it through processing. Additionally, small samples did not seem overly impacted by the paraffin wax, though the probe was occasionally larger than the sample itself.

Historically, FFPE samples have been challenging to image using optical techniques due to the contributions of the formalin crosslinks to autofluorescence from the sample. Our data suggests that DRS is a feasible method to analyze samples embedded in paraffin wax. We will present detailed spectral maps from each tumor block demonstrating DRS spectra and ratios that resemble those obtained from tissue.

Acknowledgements (Optional): Funding provided by National Science Foundation Grant: EWFD 2243953.