Elsevier

Experimental Eye Research

Volume 90, Issue 2, February 2010, Pages 330-336
Experimental Eye Research

Transscleral visible/near-infrared spectroscopy for quantitative assessment of melanin in a uveal melanoma phantom of ex vivo porcine eyes

https://doi.org/10.1016/j.exer.2009.11.010Get rights and content

Abstract

Optical spectroscopy has been used as a supplement to conventional techniques for analyzing and diagnosing cancer in many human organs. Because ocular tumors may be characterized by their different melanin content, we investigated the feasibility of using transscleral visible/near-infrared spectroscopy (Vis/NIRS) to estimate the quantity of melanin in a novel uveal melanoma phantom of ex vivo porcine eyes. The phantoms were made by injecting a freshly prepared suspension of 15% (wt/vol) gelatin, 10 mg/ml titanium dioxide (TiO2), and natural melanin, isolated from the ink sac of cuttlefish (Sepia officinalis), into the suprachoroidal space of 30 enucleated porcine eyes. The melanin concentrations used were 1 mg/ml, 2 mg/ml, and 3 mg/ml, with 10 eyes in each group. After gelation, the size and location of the phantoms were documented by B-scan ultrasonography and transillumination. Vis/NIRS recordings, covering the wavelength region from 550 to 1000 nm, were performed with two optical fibers separated by 6 mm to deliver and collect the light through the sclera. During all measurements, the exact pressure exerted by the fiber probe on the scleral surface was monitored by placing the eye on an electronic scale. Transscleral Vis/NIRS was performed across the phantom inclusion, as well as on the opposite (normal) side of each eye. A total of three consecutive measurements were carried out alternately on each side of the globe. The spectral data were analyzed using partial least squares regression. In the melanin concentration groups of 1 mg/ml (n = 10), 2 mg/ml (n = 10), and 3 mg/ml (n = 10), the largest basal phantom diameters (mean ± SD) were 14.9 ± 1.6 mm, 14.6 ± 1.5 mm, and 14.3 ± 1.0 mm, respectively (p > 0.05). The largest phantom thicknesses (mean ± SD) were 4.0 ± 0.5 mm, 4.4 ± 0.7 mm, and 4.5 ± 0.5 mm, respectively (p > 0.05). Statistical regression modeling of the Vis/NIRS data revealed that it was possible to correctly classify the phantoms according to their melanin concentrations in 84.4% of cases. The correct classification rate for phantoms with the lowest (1 mg/ml) and highest (3 mg/ml) melanin concentrations was 99.2%. The study demonstrates that transscleral Vis/NIRS is a feasible and accurate method for predicting the content of melanin in choroidal lesions.

Introduction

Ultrasonography plays an important role in the characterization of intraocular tumors, including uveal melanomas, which typically demonstrate medium to low internal reflectivity and characteristic features like acoustic hollowness and choroidal excavation (Sobottka et al., 1998). Computed tomography and magnetic resonance imaging (MRI) have proved to be accurate in determining the location and size of intraocular tumors and evaluating possible extraocular tumor extension, but lesions with a thickness of less than 2–3 mm are generally poorly visualized by these imaging techniques (Peyman and Mafee, 1987). Owing to the paramagnetic properties of melanin, uveal melanomas often appear hyperintense on T1-weighted and hypointense on T2-weighted MRI (Mafee et al., 1986). However, the predictive value of MRI is limited by overlap of signal intensities between uveal melanomas and intraocular hemorrhages, metastases and other solid tumors (Ferris et al., 1993, Lemke et al., 2001). In general, current imaging modalities provide precise information on the topography of intraocular lesions, but there is still a need to further refine the analysis of tumor tissue and morphology by ancillary diagnostic methods.

Visible/near-infrared spectroscopy (Vis/NIRS) is an optical non-invasive or minimally invasive method that offers qualitative and quantitative analysis of tissues (Richards-Kortum and Sevick-Muraca, 1996). It relies on measuring the absorption imprint by different tissue chromophores, e.g., oxy- and deoxyhemoglobin, melanin, carotenes, lipids, and water. The near-infrared part of the spectrum (700–1000 nm) has a good penetration depth in tissue. However, in this wavelength range, scattering dominates heavily over absorption. The scattering originates from refractive-index mismatches within the cellular or extracellular tissue structures, whereas absorption is caused by electronic transitions within the chromophore molecules. The interpretation of the resulting absorption and scattering spectra is often combined with models of light propagation and statistical regression models to predict the composition of unknown tissue samples. Based on spectral differences associated with parameters like edema, fibrosis, vascularization, and oxygenation, Vis/NIRS has been successfully used to detect and characterize cancer and precancerous lesions in the breast (Tromberg et al., 2005), prostate (Svensson et al., 2007), lung (Bard et al., 2006), female reproductive organs (Hornung et al., 1999), and gastrointestinal tract (Kondepati et al., 2007). Because optical spectroscopy provides quantitative information about specific chromophores such as melanin and hemoglobin, the method has also been comprehensively investigated for the evaluation of melanocytic skin tumors (McIntosh et al., 2001, Murphy et al., 2005). The spectroscopic identification of melanin is difficult because it has no distinct absorption bands. The light absorption increases linearly in the wavelength range between 720 and 620 nm and then exponentially toward shorter wavelengths. According to Kollias and Baqer, 1985, Kollias and Baqer, 1987, the gradient of this absorption spectrum is a sensitive indicator for the total melanin content in the skin. Similarly, Dwyer et al. (1998) found that the absorbance difference between 400 nm and 420 nm makes it possible to estimate the density of cutaneous melanin. Moreover, it has been shown that laser-induced fluorescence of melanin could serve as a sensitive approach for early diagnosis of melanomas (Schneider et al., 2005, Eichhorn et al., 2009).

Transpupillary reflectance spectroscopy has been widely applied to study pathophysiological aspects of pigmentation and vascularization in the ocular fundus (Hammer and Schweitzer, 2002, Hardarson et al., 2006, van de Kraats et al., 2008). For the purpose of analyzing choroidal tumors, the usefulness of this approach is limited by media opacities, the retinal pigment epithelium (RPE), and possible retinal hemorrhages or metaplasia that may obscure the real nature of the lesions. The relative homogeneous structure of the sclera, the location of various choroidal lesions immediately underneath it, and the fact that melanin is a spectroscopically quantifiable marker suggest that optical spectroscopy via a transscleral approach could be of value in the differentiation of choroidal tumors. Herein, we report the feasibility and accuracy of using transscleral Vis/NIRS to estimate the content of natural melanin in a novel uveal melanoma phantom of ex vivo porcine eyes.

Section snippets

Porcine eyes

Thirty eyes from domestic pigs (Norwegian Landrace) of both sexes, each with a live weight of about 75 kg and an age of 6–7 months, were obtained from a local abattoir. The enucleations were carried out within 12 h post-mortem, and the eyes were stored at 4 °C in a moist chamber until preparation. All experiments were performed within 3–5 days after the death of the animals. The study adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Preparation of injection suspensions

A freshly prepared

Uveal melanoma phantoms

The injection procedure was successfully performed in all eyes. Ultrasonography showed that the suspension was localized within a limited area of the suprachoroidal space without any penetration through the retina or into the vitreous cavity. In all cases, the phantom presented as a solitary dome-shaped tumor with medium internal reflectivity (Fig. 2A). In the melanin concentration groups of 1 mg/ml (n = 10), 2 mg/ml (n = 10), and 3 mg/ml (n = 10), the largest basal phantom diameters

Discussion

Within the field of ocular oncology, the technical complexity and potential risks associated with intraocular biopsies have led to a demand for ancillary diagnostic tests. The present study demonstrates that transscleral Vis/NIRS is a possible diagnostic method for predicting the content of melanin in choroidal tumors. To the best of our knowledge, the feasibility and utility of using this method to evaluate choroidal lesions have not been previously reported. The reasons for deviating from a

Acknowledgments

The study was supported by grants from the Western Norway Regional Health Authority and a Linnaeus grant for the Lund Laser Centre.

References (40)

  • J.D. Ferris et al.

    Quantification of melanin and iron content in uveal malignant melanomas and correlation with magnetic resonance image

    Br. J. Ophthalmol.

    (1993)
  • M. Hammer et al.

    Optical properties of ocular fundus tissues – an in vitro study using the double-integrating-sphere technique and inverse Monte Carlo simulation

    Phys. Med. Biol.

    (1995)
  • M. Hammer et al.

    Quantitative reflection spectroscopy at the human ocular fundus

    Phys. Med. Biol.

    (2002)
  • S.H. Hardarson et al.

    Automatic retinal oximetry

    Invest. Ophthalmol. Vis. Sci.

    (2006)
  • M.J. Hogan et al.

    Ultrastructure of malignant melanomas of the choroid

    Invest. Ophthalmol.

    (1962)
  • R. Hornung et al.

    Quantitative near-infrared spectroscopy of cervical dysplasia in vivo

    Hum. Reprod.

    (1999)
  • D.N. Hu et al.

    Uveal melanocytes, ocular pigment epithelium, and Müller cells in culture: in vitro toxicology

    Int. J. Toxicol.

    (2002)
  • V.R. Kondepati et al.

    CH-overtone regions as diagnostic markers for near-infrared spectroscopic diagnosis of primary cancers in human pancreas and colorectal tissue

    Anal. Bioanal. Chem.

    (2007)
  • J. van de Kraats et al.

    Lutein and zeaxanthin measured separately in living human retina with fundus reflectometry

    Invest. Ophthalmol. Vis. Sci.

    (2008)
  • J. Krohn et al.

    Corrosion casts of the suprachoroidal space and uveoscleral drainage routes in the pig eye

    Acta Ophthalmol. Scand.

    (1997)
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