Ex Vivo Fluorescence Confocal Microscopy in Specimens of the Liver: A Proof-of-Concept Study

Abstract

Ex vivo Fluorescence Confocal Microscopy (FCM) is a technique providing high-resolution images of native tissues. The method is increasingly used in surgical settings in areas of dermatology and urology. Only a few publications exist about examinations of tumors and non-neoplastic lesions of the liver. We report on the application of FCM in biopsies, surgical specimens and autopsy material (33 patients, 39 specimens) of the liver and compare the results to conventional histology. Our preliminary examinations indicated a perfect suitability for tumor diagnosis (ĸ = 1.00) and moderate/good suitability for the assessment of inflammation (ĸ = 0.4-0.6) with regard to their severity and localization. Macro-vesicular steatosis was reliably detected, micro-vesicular steatosis tended to be underestimated. Cholestasis and eosinophilic granules in granulocytes were not represented in the scans. The tissue was preserved as native material and maintained its quality for downstream histological, immunohistological and molecular examinations. In summary, FCM is a material sparing method that provides rapid feedback to the clinician about the presence of tumor, the degree of inflammation and structural changes. This can lead to faster therapeutic decisions in the management of liver tumors, treatment of hepatitis or in liver transplant medicine.

Keywords: digital pathology; fluorescence confocal microscopy; hepatitis; liver biopsies; liver transplantation; liver tumors.

Figure 1

Processing of the native material for FCM. (A): The native material was transferred into ethanol, AO and saline solution and finally positioned on a slide specially equipped with magnets. The tissue was spread evenly on the microscope slide using an additional foam pad (not shown). This process to prepare the native material for confocal microscopy was simple and took approximately 3–4 min. Depending on the size of the material examined, the scan took between 2–5 min. The resulting digital image was viewed approximately 10 min later. (B,C): Matching visualization of the tissue in FCM (B) and conventional histology (C) is seen. The tumor infiltrate detected in the biopsy is easily recognizable in both images.

Figure 2

Representation of malignant primary tumors of the liver in FCM scans. (A,B): Hepatocellular carcinoma (grade 2) in FCM (A) and conventional histology ((B), HE staining). Infiltrative growth and trabecular architecture were easily recognizable in FCM scans. Note the missing representation of Mallory bodies (arrows) in the FCM scans in comparison to the HE-slide. (C,D): Intrahepatic cholangiocellular carcinoma of the liver in FCM (C) and conventional histology ((D), HE staining). Infiltrating groups of neoplastic glands were reliably recognizable in FCM. The dashed line marks the invasion front between tumor formations (left) and pre-existing liver parenchyma (right).

Figure 3

Representation of benign primary tumors and masses of the liver in FCM scans. (A,B): Focal nodular hyperplasia in FCM (A) and conventional histology with special staining ((B), Masson’s trichome). Note the radial scar and fibrous septa are well represented in FCM. (C,D): A case of hemangioma in FCM (C) and conventional histology ((D), HE staining). Good representation of vessels and a fibrous matrix in the FCM scans. Note the lining of unsuspicious endothelial cells in FCM.

Figure 4

Representation of metastatic neoplasms of the liver in FCM scans. (A,B): Metastasis of colorectal carcinoma in FCM (A) and conventional histology ((B), HE staining). Typical glandular patterns of the primary tumor were preserved in this case and clearly recognizable in the FCM scans. Characteristic expression of Cytokeratin 20 (inlay) was preserved in the material. (C,D): Liver biopsy with focal manifestations of poorly differentiated neuroendocrine carcinoma in FCM (C) and conventional histology, ((D), HE staining) showing no characteristic features. Immunohistology showed preserved expression of neuroendocrine markers (Chromogranin A, inlay). (E,F): Metastasis of dedifferentiated pancreatic carcinoma in FCM (E) and conventional histology ((F), HE staining) presenting sarcomatoid morphology. Immunohistology (Vimentin, inlay) was not helpful in this case. The final diagnosis was established with knowledge of the pancreatoduodenectomy specimen.

Figure 5

Representation of inflammatory changes in FCM scans. (A–C): Liver biopsy of a case with methotrexate induced liver injury in FCM (A) and conventional histology ((B), HE staining) with special stains ((C), Masson’s trichrome). Portal and interface inflammation are well represented in FCM. Stellate cell hyperplasia (arrows) can be recognized in FCM comparable to conventional histology. Fatty vacuoles (asterisks) are more difficult to recognize in FCM. Fibrotic fibers are not represented in FCM and HE stain, the real amount of fibrosis is only recognized in Masson’s trichrome staining. (D,E): Liver biopsy of a case with drug induced liver injury in FCM (D) and conventional histology ((E), HE staining). Portal and periportal inflammatory infiltrates are shown in a comparable way. Note the missing representation of eosinophilic granules in the FCM scans (arrows).

Figure 6

Limited representation of cytoplasmatic changes in FCM scans. (A,B): Neonatal giant cell hepatitis in FCM (A) and conventional histology ((B), HE staining). FCM scans show a marked lobular inflammatory infiltrate and trabeculae of multi-nucleated hepatocytes. Note the discrepancy in the presentation of cytoplasmic bile accumulation. In FCM, cytoplasmatic accumulation of bile (arrows) does not give a signal and is only represented as empty cytoplasmic vacuoles. (C,D): A Case with alcoholic steatohepatitis in FCM (C) and conventional histology ((D), HE staining). Macrovesicular steatosis is very well represented in FCM scans of native liver tissue. Ballooning degeneration (asterisks) and Mallory bodies (arrows), that are clearly visible in the HE-slides, are very subtle in FCM scans and were missed by the blind assessment. There is also no correlate for canalicular bile stasis visible in the HE-slide.

Figure 7

Manifestations of Echinococcus multilocularis in FCM scans and conventional histology. A/B: Echinococcus multilocularis in FCM (A) and conventional histology ((B), HE staining). One of multiple daughter cysts are shown in the pictures with characteristic layers. The outer layer contains granulation tissue with marked inflammatory infiltrates and foreign body reaction. Characteristic laminated acellular material of the middle layer is represented also in FCM scans (inlay).