Assessing Graft Rejection by Automated C4d and CD34 Quantitation and Co-Localization
John E Tomaszewski, Timothy Baradet, Clifford C Hoyt, James R Mansfield, Michael Feldman. University of Pennsylvania Health System, Pennsylvania, PA; Caliper Life Sciences, Hopkinton, 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 reliable early indicator of humeral rejection. For renal biopsies in particular, testing requires taking two biopsies, one for frozen-section analysis with immunofluorescence (IF) and the other for formalin fixation and visual assessment with histochemical stains. IF labeling is not often used for formalin-fixed, paraffin-embedded (FFPE) renal specimens because of its inherent autofluorescence, which makes IF imaging and marker quantitation difficult. Spectral imaging can overcome autofluorescence interference, enabling the use of FFPE sections for renal imaging. Vessels in graft sections can fluorescently labeled for CD34 and C4d and then automated morphology-based image analysis and quantitation can be used to obtain an objective assessment of rejection.
Design: A cohort 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. These were analyzed using an automated morphologic image analysis software package to assess C4d staining levels in capillaries. An objective measure of rejection status was then developed using a trainable pattern-recognition-based image analysis tool. This automatically identifies capillaries and measures C4d in capillary walls and immediately surrounding the capillaries. Automated measures were compared to visual assessments.
Results: Autofluorescence-free IF images from FFPE specimens were obtained using spectral imaging. Automated morphologic analysis of the images identified vessels and quantified the C4d intensities within those regions. Results from FFPE specimens were comparable to those from frozen, and the image-based objective measure of rejection status gave good correlation to visual assessment.
Conclusions: Automated quantitation 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 21, 2012 1:00 PM
Poster Session VI # 298, Wednesday Afternoon