Monitoring Graft Rejection Using Dual-Color Fluorescence Imaging of Formalin-Fixed Biopsies.
John E Tomaszewski, Tom Baradeti, James R Mansfield, Michael Feldman. University of Pennsylvania Health System, Pennsylvania; Cambridge Research & Instrumentation, Inc., Woburn, MA
Background: Rejection is the major cause of graft failure, and if the injury to the organ is severe, it may not recover; prompt diagnosis of acute rejection is therefore important, with the monitoring of capillary C4d deposition being a good indicator. For renal biopsies in particular, obtaining this diagnosis has often required taking two biopsies, one for frozen-section analysis, the other fixed in formalin. Currently, immunofluorescence (IF) on frozen sections is the standard for immunochemical evaluation of renal biopsy specimens. IF labeling is not often used for formalin-fixed, paraffin-embedded (FFPE) specimens because of their inherent autofluorescence makes high-quality IF imaging difficult. Advances in spectral imaging algorithms can provide a simple means of overcoming autofluorescence interference, enabling the use of FFPE sections for renal image assessment and eliminating the need for dual biopsies. Vessels from biopsies from transplants can be marked using one label (e.g. CD34) and C4d another, and automated morphology-based image analysis used to obtain an objective assessment of rejection.
Design: A series of matched formalin-fixed and frozen renal biopsy specimens were sectioned and stained for CD34 and C4d, and IF images of them acquired using a spectral imaging system. Multispectral images were then unmixed and analyzed using an automated morphologic image analysis software package to assess C4d staining levels in capillaries. An image-based objective measure of rejection status was then developed and compared to visual assessment.
Results: High-quality IF images from FFPE specimens were obtained using spectral imaging to remove autofluorescence, and automated morphologic analysis of the images was able to identify vessels and quantify the C4d intensities within those regions. Results from formalin-fixed specimens were comparable to those from frozen, and the image-based objective measure of rejection status gave good correlation to visual assessment.
Conclusions: Automated assessment of dual-labeled (CD34 and C4d) FFPE renal biopsy specimens can be achieved using spectral imaging and morphologic image analysis software, and gives a good correlation with results from frozen sections and against visual assessment. This methodology shows promise for becoming a routine method for clinical assessment of organ transplant biopsies and is amenable to studies of archival tissue.
Category: Kidney (does not include tumors)
Wednesday, March 2, 2011 1:00 PM
Poster Session VI # 278, Wednesday Afternoon