Do formalin fixation and freeze‐thaw affect near‐infrared Raman spectroscopy of cartilaginous tissue? A preliminary ex vivo analysis of native human articular cartilage

Near-infrared (NIR) Raman microprobe spectroscopy has been applied to the non-invasive characterization of the biochemical structure of extracellular matrix in articular cartilage, a step forward along the path of in vivo diagnostic application of chondropathy. In most studies handling ex vivo cartilage specimens, formalin fixation or freeze-thaw treatments have been applied in order to stabilize tissue and cell constituents prior to spectroscopic measurements. However, these pre-processing manipulations might significantly affect certain target bands of the cartilage spectra, thus introducing biases in the characterizations, and potentially leading to data misinterpretation. In this study, we evaluated how formalin fixing and freeze-thaw processes affect Raman spectra from human femur cartilage. Healthy cartilage specimens were fixed/stored either in a 10% neutral buffered formalin solution or in a deep freezer set at −80 °C. The results of this study show that formalin fixation significantly affects the NIR Raman spectra of cartilage specimens due to concurrent formalin absorption and water dehydration within both collagen and glycosaminoglycan macromolecules. Water dehydration was also confirmed in the amide I structure in the frozen-thawed specimen, but to a much lesser extent. Furthermore, soaking the tissues in phosphate-buffered saline solution minimized the storage-induced Raman artifacts, but its immersion had limited effectiveness in formalin-fixed specimens, predominantly due to an overlap of signals from the formalin liquid (i.e. emitting at 1046 and 1492 cm−1). Therefore, to provide a highly accurate biochemical evaluation of extracellular matrix using NIR Raman spectroscopy, freeze-thaw processes are more suitable for ex vivo samples of human cartilage than formalin fixation. Copyright © 2015 John Wiley & Sons, Ltd.