The Application of Raman Spectroscopic Analysis for the Evaluation of Hepatic Steatosis

Abstract

Background: Livers with significant steatosis are considered extended criteria organs for transplantation because of increased rate of non-function and reduced survival in recipients. The evaluation of steatosis, and decision to use an organ, is most often made by the retrieving surgeon, but is accurate only 30-70% of the time. Raman spectroscopy is an imaging technique with the potential to allow for accurate real-time evaluation of steatosis at the time of retrieval. Methods: Male C57BL/6 mice were fed either a control or a methionine and choline deficient (MCD) diet to induce steatosis. At 2, 3 and 4 weeks, mice were sacrificed and livers harvested. Livers samples were analyzed by: 1) Oil Red O staining and scored by a histopathologist; 2) Biochemical determination of triglyceride and cholesterol content; 3) Raman spectroscopic analysis using a 515 nm-laser confocal microscope, or 785 nm-laser Raman probe; 4) intensity of triglyceride-specific Raman spectra were correlated with the degree of steatosis as determined by 1) and 2). Results: Pathology for all control mice revealed no steatosis, whereas MCD-fed mice were all scored as positive. Triglyceride content for MCD-fed mice was significantly elevated at all time-points, compared with controls, and correlated with the length of time the mice were fed an MCD diet. Upon Raman spectroscopic analysis, control livers did not show any consistent shift in the Raman spectra from 1000cm-1 to 4000cm-1, whereas MCD-fed mouse livers demonstrated shifts at 1146 cm-1, 1313 cm-1, 1460 cm-1, 1664cm-1 and 2896 cm-1, consistent with that of adipocytes and triglyceride. Conclusion: Livers with significant steatosis, as demonstrated by pathology and biochemical analysis, can be distinguished from non-fatty livers by a Raman spectral signature consistent with that of triglycerides and adipocytes. Raman spectra from mouse livers can be obtained immediately after the organ is retrieved, or while still inside the animal with the use of a portable, hand-held Raman spectrometer, which we hope to employ in future in vivo experiments. Further work will optimise the conditions for quantitative analysis of liver fat content which might be applied in the operating room. This is a promising technique for real-time evaluation organ quality at the time of retrieval.