Clinical Validation of a Raman Spectroscope to Determine Hepatic Fat Content

Overview

Fatty liver disease including liver steatosis (fat deposition) is a major health problem worldwide. It is considered pathological when fat accumulation exceeds 5% of the liver weight. Transplantation represents the ultimate treatment for end stage liver disease. However, the discrepancy between the demand for donor organs and their availability presents one of the greatest hurdles of liver transplantation. Therefore, extended criteria organs have to be considered to overcome this shortcoming. Hepatic steatosis is one of the most important criteria defining extended-criteria donor liver. In liver transplantation, 'severe' fat deposition in donor liver is a major cause of graft failure while 'mild' deposition is considered transplantable and 'moderate' deposition represents the gray zone and remains difficult to assess. Surgeons usually perform a hepatic steatosis assessment during liver donor surgery. However, this method is very subjective and difficult especially when inexperienced surgeons or trainees are concerned. Furthermore, it has recently been documented that the assessment of steatosis is challenging even in the hands of experienced surgeons. Theoretically, a better way to assess steatosis before organ procurement would be a non-invasive imaging technique. However, some of these techniques appear to lack the sensitivity to accurately quantify hepatic fat content while others are complex, expensive and inconvenient to use in the setting of organ procurement. Therefore, liver biopsy remains the reference for grading hepatic steatosis. In daily practice the assessment and quantification of steatosis by a pathologist during organ procurement is more complex related to the lack of availability of dedicated hepatopathologists outside of office hours and in smaller community hospitals. A simple and short technique is therefore required to assess liver steatosis before retrieval. We have recently demonstrated that Raman spectroscopy could provide an accurate, rapid and real-time assessment of hepatic fat content and correlated highly with the gold standard (i.e. histopathological assessment of liver sections) in an animal model of liver steatosis. The purpose of this study is to validate the use of Raman spectroscopy for quantitative assessment of hepatic steatosis. In the hands of the surgeons this device can provide an immediate, robust tool to assess the suitability of donor livers at the site of retrieval prior to liver transplantation.

Currently there are three main methods reported to assess liver steatosis before deceased donor liver retrieval: Noninvasive imaging techniques, evaluation of a liver biopsy sample by an experienced pathologist and assessment by transplant surgeon. Imaging techniques that are currently in use include ultrasonography (USG) , computerized tomography (CT) and magnetic resonance imaging (MRI). Although it has an acceptable level of sensitivity, USG does not provide reproducible quantitative information. It can be used in most centers but it is operator dependent. Likewise, the diagnostic performance of non-enhanced CT scan for the accurate quantification of steatosis is unreliable. MRI is highly sensitive and specific in diagnosing hepatic steatosis and it has been reported that fat quantification using a 3.0-T MRI can provide sufficient sensitivity to detect and quantify steatosis in live liver donor candidates. However, this complex technology limits its use in routine organ procurement. The microscopic evaluation of a liver biopsy sample by an expert pathologist is the gold standard for steatosis assessment. The presence and type of steatosis can be determined by microscopic evaluation. The grade of steatosis can be determined according to the international standards. Despite being gold standard, biopsies are recorded for only 23% of transplanted livers in United States. Since a pathologist is not always available at the time of procurement, many centers rely mainly on the assessment of steatosis and organ quality by the procurement surgeon (i.e. transplant surgeon). During this assessment the donor liver is evaluated according to parenchymal texture criteria such as degree of yellowness (normal, mild, moderate, severe) and degree of firmness (soft, normal; mild, moderate, severe). However, studies which investigated how accurately a transplant surgeon could predict hepatic steatosis concluded that these assessments could be significantly misleading even in experienced hands. Since all these three methods have their limitations, there is a need for a convenient and simple technique to better assess hepatic fat content prior to transplantation. Raman spectroscopy is an inelastic light scattering technique that is sensitive to molecular vibrations, the symmetry and frequencies of which are unique to the type of atoms and their spatial arrangement. Sensitivity to these properties is the basis of its ability to provide a spectral fingerprint of molecules in the illuminated region. It has been used to detect amino acids, nucleotide bases, fatty acids, saccharides, primary metabolites and other constituents that form the protein, carbohydrates, fats and DNA/RNA of biological tissues. It is an attractive technology for bioanalysis because no sample preparation is needed. Raman spectroscopy is currently under investigation for use in clinical settings and it is carried out mainly for diagnostic purposes. It is currently approved for use in Canada for skin cancer diagnosis. There are a variety of different Raman spectroscopy systems. We have recently designed a high throughput Raman spectroscopy system which is smaller than the others, portable and robust. It enables in situ scans of tissue to be undertaken with very little experience required of the operator. This system uses 'near infrared (NIR) excitation wavelength to improve the penetration depth of the laser while reducing the possibility of tissue damage. It gives information in the form of Raman spectra, analysis of which gives a Principal Component (PC) score. This specially designed system could accurately quantify steatosis in mice and rat livers. The PC scores exhibited a significant correlation with histopathological examination results. Since steatosis in human liver tissue exhibit changes similar to the left liver lobe of mice and rats, we concluded that this technique could be applied to the field of transplantation. Clinical validation and subsequent adoption of this technique can provide transplant specialists with a valuable tool to obtain real time information on the severity of hepatic steatosis to assess the likelihood of a successful outcome and reduce inadvertent organ discard rates. OBJECTIVE The aim of this study is to validate Raman spectroscopy (by means of our specially designed high throughput fiber optic Raman system) in a clinical setting in assessment of hepatic fat content. The in vivo Raman spectra (PC scores) will be compared to the findings of a pre-operative imaging method (MRI-fat quantification) and the current gold standard, liver biopsy (i.e. histopathological assessment of the liver specimen). SPECIFIC RESEARCH QUESTIONS 1\. Can Raman spectroscopy utilizing a portable fiber optic system rapidly assess the fat content of the liver in situ in clinical setting? 2. Do Raman spectra (principal component scores) correlate well with the findings of MRI-fat quantification and histopathological assessment of the liver biopsy specimen? STUDY DESIGN: This study is conceived as a single-centre, prospective validation study. METHODS Partial liver resection is the surgical removal of a portion of the liver. Most partial liver resections are performed for the treatment of hepatic neoplasms. Patients who will undergo an open partial liver resection surgery in our institution will be reviewed with the circle of care team and approached for consent if deemed suitable for this study. A Nova Scotia Health Authority Research Ethics Board-approved informed consent form will be utilized in the consent discussion (run by the clinical fellow or the research associate- both of whom are investigators participating in this project) and patients will be provided time to read the document in full before being asked for a decision on participation. Patients who give their consent for the study will be assigned to the study group. Assignment process will continue until 25 patients are included. Study participants will undergo a pre-operative MRI for fat quantification. They will be asked to fast for at least 8 hours prior to MRI. Fat quantification technique (proton density fat fraction technique) will be performed using the IDEAL pulse sequence, which has been Health Canada approved for assessment of hepatic fat fraction, on a 3.0-T MR unit (GE 750; GE Healthcare). An attending fellowship trained abdominal radiologist who is experienced in MR image interpretation will measure the hepatic fat fraction (FF) on FF maps, which will be generated automatically. The automatically calculated hepatic FF will be displayed as a percentage on a full scale of 0-100. During the liver resection procedure a region of the specimen that is to be removed but not the actual lesion will be illuminated by the Raman spectroscope fiber-optic probe. The Raman spectra provided and recorded in real-time (within a few seconds) will subsequently be analyzed (principal component analysis). This analysis will provide a PC score. Following the application of Raman spectroscopy the operative procedure will continue as planned. Histochemical stains, H\&E (hematoxylin-eosin) and trichrome, are routinely used for the interpretation of liver specimens in our anatomic pathology laboratory. As a special stain Oil Red O will need to be used to demonstrate the presence and extent of the fat (i.e. fat quantification) in liver tissue. Since it can only be performed on frozen sections a frozen section will be obtained from the removed liver specimen section to allow for Oil Red O staining- specific for diagnosing and quantifying fat droplets. An attending pathologist who is blinded to the MRI findings will evaluate these sections and determine the grade of steatosis as per the international standards: M0: no macrovesicular steatosis M1: mild focal macrovesicular steatosis (\<30% hepatocytes are involved) M2: moderate, zonal macrovesicular steatosis (\>30 and \<60% hepatocytes involved) M3: severe, panlobular macrovesicular steatosis (\>60% hepatocytes involved) Correlation analysis will be performed between hepatic FF percentages, PC scores and steatosis grades for each study participant. Study participants will be followed after the surgery according to our standard post-liver resection follow-up protocol without any deviations from the standard of care. STATISTICAL ANALYSIS For statistical analysis, quantitative data will be expressed as means and standard deviations and qualitative data will be expressed as numbers. Linear regression will be performed between macrovesicular steatosis grade and hepatic FF percentages and PC scores. Receiver operating characteristics (ROC) curves will be generated for the PC scores and hepatic FF percentages to identify their ability predict the grade of macrovesicular steatosis. All statistical analysis will be performed with commercially available statistical software (XLSTAT 2014). Personal health information will not be used for the conduct of this project. Code numbers will be assigned for each participant. A database which will be accessible only to the investigators will be stored in a secured computer (requiring password entry) in transplant office (Queen Elizabeth II Health Sciences Centre, Victoria General Site, 4th floor, Dickson Building, Room 4074). This database will include the code numbers of the participants and the data elements such as hepatic FF percentage, PC score and steatosis grade.