The Reduction or Elimination of Tissue Autofluorescence in Formalin-Fixed, Paraffin-Embedded Renal Biopsy Images Using Spectral Imaging
JR Mansfield, CS Maki. Cambridge Research & Instrumentation, Woburn, MA
Background: For half a century, immunofluorescence (IF) on frozen sections has been the gold standard for immunochemical evaluation of renal biopsy specimens. Immunofluorescence (IF) labelling is not often used for formalin-fixed, paraffin-embedded (FFPE) specimens, the perception being that the inherent autofluorescence of such specimens makes high quality IF imaging difficult. This has placed two severe restrictions on investigators. First, it has limited fluorescence imaging to tissue cryosections and hence restricts analysis of clinical material. Second, as cell and tissue preservation is lower in cryosections than in FFPE sections, the quality of morphological findings is frequently compromised. However, advances in spectral imaging algorithms can provide a simple means of overcoming one of the major limiting factors on sensitivity and legibility of IF imaging of FFPE specimens, viz., tissue autofluorescence.
Design: A comparison between IF images taken of standard frozen-section renal biopsies and IF images taken of FFPE renal biopsy sections using a spectral imaging system was made for a variety of immunoglobulins and complements. Unlabeled FFPE sections and were used to develop spectral signatures of tissue autofluorescence, which were then used in conjunction with positive IF sections to develop the spectral signatures of the immunostain used (FITC).
Results: By utilizing correct spectral signatures of tissue autofluorescence and the immunostain (FITC), spectral imaging was shown to improve the contrast ratio in IF images of FFPE specimens by up to 100-fold, greatly increasing legibility and sensitivity. In addition, IF of FFPE sections had improved morphology compared to frozen sections.
Conclusions: The use of spectral imaging for acquiring IF images is an easy-to-implement methodology on any standard fluorescence microscope, and requires little to no extra time for imaging compared to regular imaging methods. By eliminating or greatly reducing the impact of tissue autofluorescence (by as much as two orders of magnitude) on image quality, spectral imaging can enable a shift from utilizing frozen sections for IF of renal biopsy specimens to utilizing more easily obtainable and preferable FFPE specimens.
Category: Kidney (does not include tumors)
Wednesday, March 24, 2010 1:00 PM
Poster Session VI # 254, Wednesday Afternoon