Intraoperative spectroscopic evaluation of sentinel lymph nodes in breast cancer surgery.

BACKGROUND AND OBJECTIVES: Sentinel lymph node (SLN) biopsy is a standard procedure for patients with breast cancer and normal axilla on imaging. Positive SLNs on histological examination can lead to a subsequent surgery for axillary lymph node clearance (ALNC). Here we report a non-destructive technique based on autofluorescence (AF) imaging and Raman spectroscopy for intra-operative assessment of SLNs excised in breast cancer surgery. METHODS: A microscope integrating AF imaging and Raman spectroscopy modules was built to allow scanning of lymph node biopsy samples. During AF-Raman measurements, AF imaging determined optimal sampling locations for Raman spectroscopy measurements. After optimisation of the AF image analysis and training of classification models based on data from 85 samples, the AF-Raman technique was tested on an independent set of 81 lymph nodes comprising 58 fixed and 23 fresh specimens. The sensitivity and specificity of AF-Raman were calculated using post-operative histology as a standard of reference. RESULTS: The independent test set contained 66 negative lymph nodes and 15 positive lymph nodes according to the reference standard, collected from 78 patients. For this set of specimens, the area under the receiver operating characteristic (ROC) curve for the AF-Raman technique was 0.93 [0.83-0.98]. AF-Raman was then operated in a regime that maximised detection specificity, producing a 94% detection accuracy: 80% sensitivity and 97% specificity. The main confounders for SLN metastasis were areas rich in histiocytes clusters, for which only few Raman spectra had been included in the training dataset. DISCUSSION: This preliminary study indicates that with further development and extension of the training dataset by inclusion of additional Raman spectra of histiocytes clusters and capsule, the AF-Raman may become a promising technique for intra-operative assessment of SLNs. Intra-operative detection of positive biopsies could avoid second surgery for axillary clearance.

Intra-operative assessment of sentinel lymph nodes for breast cancer surgery: An update.

Lymph node (LN) involvement is the strongest prognostic factor in operable breast cancer (BC). Therefore, accurate assessment of LN status is essential for management of BC patients. The introduction of sentinel LN approach reduced the need for extensive axillary surgery to achieve accurate staging. However, positive sentinel LN as determined on postoperative histological examination often leads to a second axillary operation to ensure an accurate staging and that positive non-sentinel LNs are removed. Although preoperative assessment of LN has improved significantly, its accuracy remains insufficient to avoid further axillary surgery and is not sufficient to predict the status of the LN. Therefore, intraoperative evaluation of the sentinel LN to determine the need for completing lymph node dissection in case of metastasis can provide an important approach to guide BC management decision making. This article reviews the techniques available and under development for intraoperative detection of sentinel LN metastasis in BC surgery. The key features of each technique are described in detail, emphasising the benefits offered by label-free optical techniques: minimal sample preparation, high spatial resolution, and immediate on-site implementation. Optical techniques have the potential to provide a cost-effective and accurate intraoperative platform for the assessment of SLN within the operating theatre.

Probing Nanoparticle-Cell Interaction Using Micro-Raman Spectroscopy: Silver and Gold Nanoparticle-Induced Stress Effects on Optically Trapped Live Red Blood Cells.

Advancements in the field of nanotechnology have resulted in the emergence of a large variety of engineered nanomaterials for innumerable applications. Despite the ubiquitous use of nanomaterials in daily life, concerns regarding the potential toxicity and safety of these materials have also been raised. There is a high demand for assessing the unwanted effects of both gold and silver nanoparticles, which is increasingly being used in biomedical applications. This paper deals with the study of stress due to silver and gold nanoparticles of varying size on red blood cells (RBCs) using Raman tweezers spectroscopy. RBCs were incubated with nanoparticles of size in the 10-100 nm range with the same concentrations, and micro-Raman spectra were recorded by optically trapping the nanoparticle-treated live RBCs. Spectral modifications implicating hemoglobin deoxygenation were observed in all nanoparticle-treated RBCs. One of the probable reason for the deoxygenation trend can be the adhesion of nanoparticles onto the cell surface causing imbalance in cell functioning. Moreover, the higher spectral variations observed for silver nanoparticles indicate that oxidative stress is involved in cell damage. These mechanisms lead to the modification in the hemoglobin structure because of changes in the pH of cytoplasm, which can be detected using Raman spectroscopy.

Micro-Raman Spectroscopy Analysis of Optically Trapped Erythrocytes in Jaundice.

Derangements in bilirubin metabolism and/or dysfunctions in the hepato-biliary system lead to the unhealthy buildup of bilirubin in blood, resulting in jaundice. During the course of this disorder, circulating red cells are invariably subjected to toxic effects of serum bilirubin and an array of inflammatory compounds. This study aimed to investigate the vibrational spectroscopy of live red cells in jaundice using micro-Raman spectroscopy combined with optical-trap. Red cells from blood samples of healthy volunteers and patients with jaundice were optically immobilized and micro-Raman probed using a 785 nm diode laser. Raman signatures from red cells in jaundice exhibited significant variations from the normal and the spectral-markers were obtained from multivariate analytical methods. This research gives insightful views on how different pathologies can act as "stress-milieus" for red cells in circulation, possibly impeding their normal functions and also exasperating anemia. Raman spectroscopy, an emerging bio-analytical technique, is sensitive in detecting molecular-conformations in situ, at cellular-levels and in real-time. This study could pave way in understanding fundamental red cell behavior in different diseases by analyzing Raman markers.

Effect of infrared light on live blood cells: Role of ß-carotene.

We have utilized Raman tweezers to measure and assign micro-Raman spectra of optically trapped, live red blood cells (RBCs), white blood cells (WBCs) and platelets. Various types of WBCs- both granulocytes, lymphocytes, and their different types have been studied. The Raman bands are assigned to different biomolecules of blood cells. The Raman spectra thus obtained has been enabled detection of ß-carotene in these blood cells, the spectral features of which act as a signature that facilitates experimental probing of the effect of 785nm laser light on different blood cells as a function of incident laser power in the mW range. The spectral changes that we obtain upon laser irradiation indicate that, both haemoglobin as well as the cell membrane sustains damage. In case of lymphocytes and platelets the peaks corresponding to ß-carotene showed drastic changes. Thorough analysis of the spectral changes indicates possibility of free radical induced damage of ß-carotene in lymphocytes and platelets. Among different blood cells, RBCs have a power threshold of only 10mW. The power threshold for other types of blood cells is somewhat higher, but always below about 30mW. These values are likely to serve as useful guides for Raman tweezers based experiments on live cells.

Probing differentiation in cancer cell lines by single-cell micro-Raman spectroscopy.

Single-cell micro-Raman spectroscopy has been applied to explore cell differentiation in single, live, and malignant cells from two tumor cell lines. The spectra of differentiated cells exhibit substantial enhancement primarily in the intensities of protein peaks with concomitant decrease in intensities of O−P−O asymmetric stretching peaks in DNA/RNA. Principal component analyses show that the spectral score of differentiated cells tends to asymptotically approach that of spectra obtained from normal neural stem cells/progenitors. This lends credence to the notion that the observed spectral changes are specific to differentiation, since upon differentiation, malignant cells become less malignant and tend toward benignity.

A micro-Raman study of live, single red blood cells (RBCs) treated with AgNO3 nanoparticles.

Silver nanoparticles (Ag NPs) are known to exhibit broad antimicrobial activity. However, such activity continues to raise concerns in the context of the interaction of such NPs with biomolecules. In a physiological environment NPs interact with individual biological cells either by penetrating through the cell membrane or by adhering to the membrane. We have explored the interaction of Ag NPs with single optically-trapped, live erythrocytes (red blood cells, RBCs) using Raman Tweezers spectroscopy. Our experiments reveal that Ag NPs induce modifications within an RBC that appear to be irreversible. In particular we are able to identify that the heme conformation in an RBC transforms from the usual R-state (oxy-state) to the T-state (deoxy-state). We rationalize our observations by proposing a model for the nanoparticle cytotoxicity pathway when the NP size is larger than the membrane pore size. We propose that the interaction of Ag NPs with the cell surface induces damage brought about by alteration of intracellular pH caused by the blockage of the cell membrane transport.