A concerted protocol for the analysis of mineral deposits in biopsied tissue using infrared microanalysis

The mechanism(s) by which crystals are retained in the kidney resulting in stone disease remains unclear. Intratubular aggregation as well as crystal cell binding, or internalization and translocation, or alternatively nucleation and growth in the interstitial fluid are possible models. Our group is testing the hypothesis that calcium phosphate deposits in kidneys of patients with calcium renal stones arise in unique anatomical regions of the kidney. Furthermore, we believe that their formation is conditioned by specific stone forming pathophysiologies. To test this hypothesis, we performed intra-operative renal papillary biopsies during percutaneous nephrolithotomy of kidneys from 15 idiopathic calcium stone formers as well as kidney tissue from a patient who ingested ethylene glycol, and developed a new protocol to accurately identify the composition of the calcium deposits located in the renal tissue. We developed a new histological approach that incorporated a low-energy (low-E) reflective slide substrate that has similar characteristics to a common microscope slide and infrared absorption microspectroscopy. Infrared absorption microspectroscopy revealed the crystal deposits in the idiopathic calcium oxalate stone formers to be hydroxyapatite in composition with an occasional region of calcium carbonate, while calcium oxalate was the predominant mineral in the kidney of the patient who had ingested ethylene glycol. The results demonstrate that mixed sample types containing tissue and mineralized deposits are easily analyzed while mounted on a low-E slide using the attenuated total internal reflectance (ATR) method. Reflection/absorption (R/A) analysis allows one to quickly survey a tissue section and provides qualitative information about its components. Once interesting sites have been identified by R/A analysis, ATR analysis can then be used to collect the best data possible. ATR analysis provides spectra free from many of the artifacts associated with transmission and R/A analysis, and completes the full picture of the components contained in the crystal deposits and tissue. We present a method of analysis for mineralized materials embedded in kidney tissue that uses readily or easily obtainable materials and instrumentation. The sensitivity of this method allows tissue sections to remain unstained, alleviating the tedious and time-consuming constraints of earlier methods of visual analysis. The present method will save time and training, while simultaneously offering an unbiased analysis of mineralized components that is more accurate and conducive to patient treatments than previous methods.