S. Agarwal1, B. Lloyd1, S. Nigwekar2, S. Loder1, K. Ranganathan1, P. Cederna1, S. Fagan2, J. Goverman2, M. Morris1, B. Levi1 1University Of Michigan,Surgery,Ann Arbor, MI, USA 2Massachusetts General Hospital,Boston, MA, USA
Introduction: Calciphylaxis is a painful and debilitating condition which creates large open wounds most frequently in patients with renal failure. Calciphylactic lesions are characterized by the precipitation of calcium deposits in the skin and soft tissues, resulting in vessel thrombosis and tissue necrosis. Diagnosis of calciphylaxis traditionally occurs via histologic evaluation of biopsy specimens. However, incisional biopsy of the affected site may result in further local inflammation leading to a cycle of further calcium deposition. We set out to develop a non-invasive diagnostic method which can identify calciphylaxis lesions and avoids creating local inflammatory trauma.
Methods: Two histology-confirmed human calciphylaxis biopsy specimens and normal surrounding tissue were examined using either a Raman microscope (interrogating a tissue area < 1 mm) or a hand-held Raman spectroscopy probe (interrogating a tissue volume < 1 mm3). Characteristic spectra for each specimen including the normal surrounding tissue and the known calciphylaxis regions were collected and compared to identify common peaks contributed by apatite. Spectra were pre-processed for removal of cosmic spikes and correction of spectrograph/detector alignment and grating-induced anamorphic magnification (curvature). Spectra were corrected for the fluorescence background by fitting background to a low order polynomial (Polynomial order = 5). Band heights and areas are measured. Concurrently nano-CT scans were performed to confirm the regions of calcification.
Results: Using nano-CT imaging, we demonstrate large areas of calcification including within the vasculature. Both calciphylaxis specimens exhibited a strong peak at 960 cm-1, consistent with Raman spectrum attributed to apatite and apatitic-like tissue components. Here, this strong peak is attributed to small calcium phosphate precipitates within the tissue. Normal tissue examined from these patients showed no Raman signature of calcium phosphate. Our results suggest that Raman spectroscopy can differentiate the calcium phosphate deposition of calciphylaxis from normal tissue.
Conclusion: Here we differentiate calciphylaxis and normal surrounding tissue based on the physical characteristics of the tissue using Raman spectroscopy. Although we have employed this technique in previously excised biopsy specimens, a hand-help, fiber-optic probe can be developed to analyze surface tissue in humans prior to biopsy. In the future, Raman spectroscopy may provide a rapid and non-invasive method for diagnosing calciphylaxis. By avoiding an incisional biopsy, we will be able to avoid exacerbating the cycle of inflammation which precipitates calcium phosphate deposition in these patients.
