Calciphylaxis is a painful, debilitating, and premorbid condition, which presents as calcified vasculature and soft tissues. Traditional diagnosis of calciphylaxis lesions requires an invasive biopsy, which is destructive, time consuming, and often leads to exacerbation of the condition and infection. Furthermore, it is difficult to find small calcifications within a large wound bed. To address this need, a noninvasive diagnostic tool may help clinicians identify ectopic calcified mineral and determine the disease margin. We propose Raman spectroscopy as a rapid, point-of-care, noninvasive, and label-free technology to detect calciphylaxis mineral. Debrided calciphylactic tissue was collected from six patients and assessed by microcomputed tomography (micro-CT). Micro-CT confirmed extensive deposits in three specimens, which were subsequently examined with Raman spectroscopy. Raman spectra confirmed that deposits were consistent with carbonated apatite, consistent with the literature. Raman spectroscopy shows potential as a noninvasive technique to detect calciphylaxis in a clinical environment.
We report an overlooked source of artifacts for clinical specimens, where unexpected and normally negligible contaminants can skew the interpretation of results. During an ongoing study of bone fragments from diabetic osteomyelitis, strong Raman signatures were found, which did not correspond with normal bone mineral or matrix. In a bone biopsy from the calcaneus of a patient affected by diabetic osteomyelitis, Raman microspectroscopic analysis revealed regions with both abnormal mineral and degraded collagen in addition to normal bone. Additional bands indicated a pathological material. Stenotrophomonas maltophilia was identified in the wound culture by independent microbiologic examination. We initially assigned the unusual bands to xanthomonadin, a bacterial pigment from S. maltophilia. However, the same bands were also found more than a year later on a second specimen that had been noticeably contaminated with pathology marking dye. Drop deposition/Raman spectroscopy of commonly used pathology dyes revealed that a blue tissue-marking dye was responsible for the unusual bands in both specimens, even in the first specimen where there was no visible evidence of contamination.
Raman spectroscopy of bone has been used to characterize chemical changes occurring in diseases such as osteoporosis,
osteoarthritis and osteomyelitis. Metastasis of cancer into bone causes changes to bone quality that are similar to those
observed in osteoporosis, such as decreased bone strength, but with an accelerated timeframe. In particular, osteolytic
(bone degrading) lesions in bone metastasis have a marked effect on patient quality of life because of increased risk of
fractures, pain, and hypercalcemia. We use Raman spectroscopy to examine bone from two different mouse models of
osteolytic bone metastasis. Raman spectroscopy measures physicochemical information which cannot be obtained
through standard biochemical and histological measurements. This study was reviewed and approved by the University
of Michigan University Committee on the Care and Use of Animals. Two mouse models of prostate cancer bone
metastasis, RM1 (n=3) and PC3-luc (n=4) were examined. Tibiae were injected with RM1 or PC3-luc cancer cells, while
the contralateral tibiae received a placebo injection for use as controls. After 2 weeks of incubation, the mice were
sacrificed and the tibiae were examined by Raman microspectroscopy (λ=785 nm). Spectroscopic markers corresponding
to mineral stoichiometry, bone mineralization, and mineral crystallinity were compared in spectra from the cancerous
and control tibiae. X-ray imaging of the tibia confirmed extensive osteolysis in the RM1 mice, with tumor invasion into
adjoining soft tissue and moderate osteolysis in the PC3-luc mice. Raman spectroscopic markers indicate that osteolytic
lesions are less mineralized than normal bone tissue, with an altered mineral stoichiometry and crystallinity.
Raman spectroscopic measurement of bone composition has shown promise as a medical diagnostic by measuring the
molecular composition of the bone mineral and matrix. We previously demonstrated proof-of-principle transcutaneous
Raman spectroscopy bone measurements in human cadavers. In this paper, we discuss further optimization of the
instrumental configuration for efficient collection of bone signal using contact fiber-optic probe designs. To optimize
collection of Raman signal through overlaying soft tissue, novel geometrically-accurate tissue phantoms were prepared.
MRI and CT images of the human cadaveric specimens were used to create solid tissue phantoms with accurate
geometric dimensions. In these tissue phantoms, optical properties can be varied systematically. Raman spectra of the
prepared tissue phantoms were used to optimize the positions of the fibers in the fiber optic system, and the laser
illumination sequence in the measurements. Three fiber optic probes were developed and tested with both novel tissue
phantoms and human cadaveric specimens. The contact fiber optic probes were developed for arthroscopic
measurements of joints, for transcutaneous measurements of bone in situ, and for contact measurements of exposed
bone. By coupling the fiber optic probe to an imaging spectrograph, spectra were collected simultaneously at many
positions on the tissue. Furthermore, spectra were collected with several different excitation laser patterns to enhance the
effective spatial resolution of the measurements. Finally, a series of improvements were made in the data preprocessing
to improve the recovered spectral signal. Together, these modifications improve signal-to-noise and spatial resolution.
For many years, viscosity has been the primary method used by researchers in rheumatology to assess the physiochemical properties of synovial fluid in both normal and osteoarthritic patients. However, progress has been limited by the lack of methods that provide multiple layers of information, use small sample volumes, and are rapid. Raman spectroscopy was used to assess the biochemical composition of synovial fluid collected from 40 patients with clinical evidence of knee osteoarthritis (OA) at the time of elective surgical treatment. Severity of knee osteoarthritis was assessed by a radiologist using Kellgren/Lawrence (K/L) scores from knee joint x rays, while light microscopy and Raman spectroscopy were used to examine synovial fluid (SF) aspirates (2 to 10 µL), deposited on fused silica slides. We show that Raman bands used to describe protein secondary structure and content can be used to detect changes in synovial fluid from osteoarthritic patients. Several Raman band intensity ratios increased significantly in spectra collected from synovial fluid in patients with radiological evidence of moderate-to-severe osteoarthritis damage. These ratios can be used to provide a "yes/no" damage assessment. These studies provide evidence that Raman spectroscopy would be a suitable candidate in the evaluation of joint damage in knee osteoarthritis patients.
We describe the use of Raman spectroscopy to investigate synovial fluid drops deposited onto fused silica microscope slides. This spectral information can be used to identify chemical changes in synovial fluid associated with osteoarthritis (OA) damage to knee joints. The chemical composition of synovial fluid is predominately proteins (enzymes, cytokines, or collagen fragments), glycosaminoglycans, and a mixture of minor components such as inorganic phosphate crystals. During osteoarthritis, the chemical, viscoelastic and biological properties of synovial fluid are altered. A pilot study was conducted to determine if Raman spectra of synovial fluid correlated with radiological scoring of knee joint damage. After informed consent, synovial fluid was drawn and x-rays were collected from the knee joints of 40 patients. Raman spectra and microscope images were obtained from the dried synovial fluid drops using a Raman microprobe and indicate a coarse separation of synovial fluid components. Individual protein signatures could not be identified; Raman spectra were useful as a general marker of overall protein content and secondary structure. Band intensity ratios used to describe protein and glycosaminoglycan structure were used in synovial fluid spectra. Band intensity ratios of Raman spectra indicate that there is less ordered protein secondary structure in synovial fluid from the damage group. Combination of drop deposition with Raman spectroscopy is a powerful approach to examining synovial fluid for the purposes of assessing osteoarthritis damage.
Raman spectroscopy of bone is complicated by fluorescence background and spectral contributions from other tissues. Full utilization of Raman spectroscopy in bone studies requires rapid and accurate calibration and preprocessing methods. We have taken a step-wise approach to optimize and automate calibrations, preprocessing and background correction. Improvements to manual spike removal, white light correction, software image rotation and slit image curvature correction are described. Our approach is concisely described with a minimum of mathematical detail.
Human synovial fluid droplets were investigated using drop deposition in combination with Raman spectroscopy.
Following informed consent, synovial fluid was obtained from forty human patients with various severities of knee pain
and/or osteoarthritis at the time of knee arthroscopy or total joint replacement. Synovial fluid was aspirated from the
knee joint of each patient and stored at -80°C until examination by near-infrared Raman spectroscopy. Synovial fluid
aspirates from the knee joint of each patient were deposited onto a clean fused silica microscope slide and the droplet
dried under ambient laboratory conditions. Each droplet was illuminated by a line-focused or a ring-focused 785 nm
laser. As the droplet dries, biofluid components segregated based on solubility differences and a deposit that is spatially
heterogeneous was made. Spectra taken from the droplet edges and center were dominated by protein bands and showed
the presence of at least two protein moieties in the droplet. Band area and band height ratios (1410 cm-1/1450 cm-1)
showed the greatest change between specimens from patients with mild/early osteoarthritis compared to those with
severe/late stage osteoarthritis. The greatest differences were found in the center of the droplet, which contains more
soluble protein components than the edges.
We show that early indicators of osteoarthritis are observed in Raman spectroscopy by probing femur surfaces excised
from mouse models of early-onset osteoarthritis. Current clinical methods to examine arthritic joints include radiological
examination of the joint, but may not be capable of detecting subtle chemical changes in the bone tissue, which may
provide the earliest indications of osteoarthritis. Recent research has indicated that the subchondral bone may have a
more significant role in the onset of osteoarthritis than previously realized. We will report the effect of age and defective
type II collagen on Raman band area ratios used to describe bone structure and function. The carbonate-to-phosphate
ratio is used to assess carbonate substitution into the bone mineral and the mineral-to-matrix ratio is used to measure
bone mineralization. Mineral-to-matrix ratios indicate that subchondral bone becomes less mineralized as both the
wild-type and Del1 (+/-) transgenic mice age. Moreover, the mineral-to-matrix ratios show that the subchondral bone of
Del1 (+/-) transgenic mice is less mineralized than that of the wild-type mice. Carbonate-to-phosphate ratios from Del1
(+/-) transgenic mice follow the same longitudinal trend as wild-type mice. The ratio is slightly higher in the transgenic
mice, indicating more carbonate content in the bone mineral. Raman characterization of bone mineralization provides an
invaluable insight into the process of cartilage degeneration and the relationship with subchondral bone at the
ultrastructural level.
A novel application of surface-enhanced Raman spectroscopy (SERS) for in-vitro osteoarthritis (OA) biomarker detection is described. Hyaluronic acid (HA) is a potential OA biomarker and synovial fluid levels of HA have been correlated with progression of joint space narrowing. However, current immunoassay and chromatographic methods that identify HA in synovial fluid are cumbersome and often require sophisticated instrumentation. Raman spectroscopy may be an alternative to these analytical methods, providing rapid identification of HA using characteristic Raman bands. Yet, previous reports of un-enhanced Raman spectroscopy for hyaluronic acid are in aqueous solutions exceeding 1000X in-vivo concentrations because HA is a weakly scattering polysaccharide. Surface-enhanced Raman spectroscopy can improve detection limits by 100-1000 times and we present, to our best knowledge, the first surface-enhanced Raman spectra of hyaluronic acid. Moreover, the recent commercial availability of stable SERS gold substrates has enabled rapid SERS detection of this biomarker at concentrations diluted by more than an order of magnitude, compared to previous literature reports. Preliminary results of easily and rapidly observing hyaluronic acid at low concentrations in aqueous solutions supported further studies in synthetic models of biofluids, such as artificial synovial fluid, that contain HA at low concentrations. These complex fluids contain proteins that compete for the SERS-active sites on the substrate, and the resulting spectra are dominated by protein Raman bands. We apply a simple and validated protein precipitation protocol to artificial synovial fluid prior to deposition onto the SERS substrate. We find that HA is easily detected in these fluids after protein removal treatment.
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