Optical biopsy: a promising approach for real‐time liver steatosis grading

Optical biopsy is a diagnostic approach exploiting the photophysical phenomena arising from the interaction of ultraviolet-visible-near infrared radiations with matter to provide real-time information on the morpho-functional properties of a biological substrate, in the absence of sample removal. Among the photophysical phenomena, considerable attention is paid to autofluorescence or light-induced fluorescence, which is the fluorescence arising from a biological substrate upon excitation at a suitable wavelength, in the absence of exogenous markers, because of the presence of endogenous biomolecules acting as fluorophores. The endogenous fluorophores may be involved both in cell metabolic processes (for instance, pyridine coenzymes, flavins and lipofuscins) and in tissue histological organization (for instance, collagen and elastin). Because emission properties – such as amplitude and spectral shape – depend on the nature, amount, physicochemical state, intratissue distribution and microenvironment of fluorescing molecules, in close relationship with the morphological and metabolic conditions of the biological substrate, autofluorescence represents an intrinsic tissue diagnostic parameter (1, 2). The occurrence of either physiological changes or pathological conditions gives rise to alterations of the tissue morpho-functional state, resulting in changes of autofluorescence emission properties suitable for diagnostic purposes, provided that both endogenous fluorophores’ photophysical characteristics and tissue optical properties are defined. At least two major advantages emerge in the characterization of biological substrates through autofluorescence analysis: the widespread occurrence of endogenous fluorophores, allowing an extension of its application to various bio-medical fields, and the technological progress as to excitation sources, light delivery and detection system of fluorescence signals, allowing the high sensitivity of the fluorometric techniques to be fully exploited. In bulk tissues, autofluorescence analysis is widely considered for in vivo diagnostic purposes for both the detection of pathologies and the monitoring of organ functionality under normal, physiologically or purposely altered conditions (3). As to pathologies, optical biopsy diagnostic applications concern mainly the neoplastic growth [as reviewed by references (4, 5)]. The occurrence of a neoplasia alters the autofluorescence properties, acting on multiple factors, such as biochemical composition, histological organization and optical properties of the tissue. The optical properties, in particular, can affect the propagation of both excitation and emission light, thus influencing the amplitude and the spectral shape of the signal collected by the measuring probe, depending on the presence of non-fluorescent absorbers and scatterers within the tissue (6). As to the monitoring of organ functionality, works have been performed to assess the response to ischaemic conditions and the subsequent recovery ability. A device has been developed, for example, that combines the collection of the autofluorescence amplitude signal attributable to NAD(P)H as a marker of the mitochondrial redox state, with the monitoring of both microcirculatory blood flow and blood volume, to respond to the necessity of a real-time survey of organs’ vitality in clinics (7). The organ transplantation practice is a promising field of application (8, 9). In the case of the liver, to date, studies have mainly focused on the optimization of the preservation procedures and the prediction of organ viability, through a selective analysis of NAD(P)H and flavin autofluorescence as a probe of the energetic metabolism (10–13). The variety of additional endogenous fluorophores occurring in the liver depending on the multiple metabolic pathways carried out by the organ (such as fatty acids, lipopigments, vitamins and biliary salt derivatives) provides interesting perspectives for an extension of optical biopsy to the diagnosis of diseased conditions. A marked contribution is made by vitamin A to the liver overall emission because of both its relatively high quantum efficiency and its noticeable amount. When undesired – such as in the case of energetic metabolic studies based on NAD(P)H emission analysis – vitamin A contribution can be removed by a preliminary sample irradiation, exploiting its strong photolability. Among the altered morpho-functional states of liver, steatosis currently deserves particular attention because of its many implications for the outcome of liver transplantation. The efficacy of this therapeutic approach to end-stage hepatic insufficiency has led to a continuously increasing demand for organs. At present, steatosis occurs in about one-third of the livers becoming available for grafting. Organs with mildmoderate steatosis belong to the category of marginal livers, currently accepted despite the direct relationship between the presence of lipids and the enhancement of the risk of ischaemia reperfusion injuries, the cause of severe graft dysfunction (14). Currently, a precise diagnosis of steatosis requires a tissue histological examination, which provides an exact estimation of the lipid accumulation degree of the donor liver, but is time consuming and is generally available after several hours of cold ischaemia (15). Optical biopsy, in principle, can represent a good approach to overcome this limitation, through real-time detection and grading of steatosis in a donor liver. In fact, the accumulation of lipid droplets in hepatocytes’ cytoplasm is expected to modify the photophysical properties of liver tissue involving both its biochemical composition and its structural organization. The alteration of biochemical composition will affect the contribution of the endogenous fluorophores, influencing the autofluorescence spectral profile and signal amplitude; the alteration of the histological organization will affect the optical properties and therefore the light migration within tissue. In spite of this premise, only very recently optical biopsy has been considered as a suitable approach for the diagnosis of steatosis. In this view, the work by de Oliveira et al. (16) in this issue of Liver International is particularly appreciable. It refers mainly to the effect induced on the light migration in liver