Narahara Chari Dingari

Raman Spectroscopy Provides a Powerful Diagnostic Tool for Accurate Determination of Albumin Glycation

We present the first demonstration of glycated albumin detection and quantification using Raman spectroscopy without the addition of reagents. Glycated albumin is an important marker for monitoring the long-term glycemic history of diabetics, especially as its concentrations, in contrast to glycated hemoglobin levels, are unaffected by changes in erythrocyte life times. Clinically, glycated albumin concentrations show a strong correlation with the development of serious diabetes complications including nephropathy and retinopathy. In this article, we propose and evaluate the efficacy of Raman spectroscopy for determination of this important analyte. By utilizing the pre-concentration obtained through drop-coating deposition, we show that glycation of albumin leads to subtle, but consistent, changes in vibrational features, which with the help of multivariate classification techniques can be used to discriminate glycated albumin from the unglycated variant with 100% accuracy. Moreover, we demonstrate that the calibration model developed on the glycated albumin spectral dataset shows high predictive power, even at substantially lower concentrations than those typically encountered in clinical practice. In fact, the limit of detection for glycated albumin measurements is calculated to be approximately four times lower than its minimum physiological concentration. Importantly, in relation to the existing detection methods for glycated albumin, the proposed method is also completely reagent-free, requires barely any sample preparation and has the potential for simultaneous determination of glycated hemoglobin levels as well. Given these key advantages, we believe that the proposed approach can provide a uniquely powerful tool for quantification of glycation status of proteins in biopharmaceutical development as well as for glycemic marker determination in routine clinical diagnostics in the future.

Raman spectroscopy-based sensitive and specific detection of glycated hemoglobin.

In recent years, glycated hemoglobin (HbA1c) has been increasingly accepted as a functional metric of mean blood glucose in the treatment of diabetic patients. Importantly, HbA1c provides an alternate measure of total glycemic exposure due to the representation of blood glucose throughout the day, including post-prandially. In this article, we propose and demonstrate the potential of Raman spectroscopy as a novel analytical method for quantitative detection of HbA1c, without using external dyes or reagents. Using the drop coating deposition Raman (DCDR) technique, we observe that the nonenzymatic glycosylation (glycation) of the hemoglobin molecule results in subtle but discernible and highly reproducible changes in the acquired spectra, which enable the accurate determination of glycated and nonglycated hemoglobin using standard chemometric methods. The acquired Raman spectra display excellent reproducibility of spectral characteristics at different locations in the drop and show a linear dependence of the spectral intensity on the analyte concentration. Furthermore, in hemolysate models, the developed multivariate calibration models for HbA1c show a high degree of prediction accuracy and precision--with a limit of detection that is a factor of ~15 smaller than the lowest physiological concentrations encountered in clinical practice. The excellent accuracy and reproducibility achieved in this proof-of-concept study opens substantive avenues for characterization and quantification of the glycosylation status of (therapeutic) proteins, which are widely used for biopharmaceutical development. We also envision that the proposed approach can provide a powerful tool for high-throughput HbA1c sensing in multicomponent mixtures and potentially in hemolysate and whole blood lysate samples.

Incorporation of Support Vector Machines in the LIBS Toolbox for Sensitive and Robust Classification Amidst Unexpected Sample and System Variability

Despite the intrinsic elemental analysis capability and lack of sample preparation requirements, laser-induced breakdown spectroscopy (LIBS) has not been extensively used for real-world applications, e.g., quality assurance and process monitoring. Specifically, variability in sample, system, and experimental parameters in LIBS studies present a substantive hurdle for robust classification, even when standard multivariate chemometric techniques are used for analysis. Considering pharmaceutical sample investigation as an example, we propose the use of support vector machines (SVM) as a nonlinear classification method over conventional linear techniques such as soft independent modeling of class analogy (SIMCA) and partial least-squares discriminant analysis (PLS-DA) for discrimination based on LIBS measurements. Using over-the-counter pharmaceutical samples, we demonstrate that the application of SVM enables statistically significant improvements in prospective classification accuracy (sensitivity), because of its ability to address variability in LIBS sample ablation and plasma self-absorption behavior. Furthermore, our results reveal that SVM provides nearly 10% improvement in correct allocation rate and a concomitant reduction in misclassification rates of 75% (cf. PLS-DA) and 80% (cf. SIMCA)-when measurements from samples not included in the training set are incorporated in the test data-highlighting its robustness. While further studies on a wider matrix of sample types performed using different LIBS systems is needed to fully characterize the capability of SVM to provide superior predictions, we anticipate that the improved sensitivity and robustness observed here will facilitate application of the proposed LIBS-SVM toolbox for screening drugs and detecting counterfeit samples, as well as in related areas of forensic and biological sample analysis.

Laser-induced breakdown spectroscopy-based investigation and classification of pharmaceutical tablets using multivariate chemometric analysis

We report the effectiveness of laser-induced breakdown spectroscopy (LIBS) in probing the content of pharmaceutical tablets and also investigate its feasibility for routine classification. This method is particularly beneficial in applications where its exquisite chemical specificity and suitability for remote and on site characterization significantly improves the speed and accuracy of quality control and assurance process. Our experiments reveal that in addition to the presence of carbon, hydrogen, nitrogen and oxygen, which can be primarily attributed to the active pharmaceutical ingredients, specific inorganic atoms were also present in all the tablets. Initial attempts at classification by a ratiometric approach using oxygen (∼777 nm) to nitrogen (742.36 nm, 744.23 nm and 746.83 nm) compositional values yielded an optimal value at 746.83 nm with the least relative standard deviation but nevertheless failed to provide an acceptable classification. To overcome this bottleneck in the detection process, two chemometric algorithms, i.e. principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA), were implemented to exploit the multivariate nature of the LIBS data demonstrating that LIBS has the potential to differentiate and discriminate among pharmaceutical tablets. We report excellent prospective classification accuracy using supervised classification via the SIMCA algorithm, demonstrating its potential for future applications in process analytical technology, especially for fast on-line process control monitoring applications in the pharmaceutical industry.

Development of robust calibration models using support vector machines for spectroscopic monitoring of blood glucose.

Sample-to-sample variability has proven to be a major challenge in achieving calibration transfer in quantitative biological Raman spectroscopy. Multiple morphological and optical parameters, such as tissue absorption and scattering, physiological glucose dynamics and skin heterogeneity, vary significantly in a human population introducing nonanalyte specific features into the calibration model. In this paper, we show that fluctuations of such parameters in human subjects introduce curved (nonlinear) effects in the relationship between the concentrations of the analyte of interest and the mixture Raman spectra. To account for these curved effects, we propose the use of support vector machines (SVM) as a nonlinear regression method over conventional linear regression techniques such as partial least-squares (PLS). Using transcutaneous blood glucose detection as an example, we demonstrate that application of SVM enables a significant improvement (at least 30%) in cross-validation accuracy over PLS when measurements from multiple human volunteers are employed in the calibration set. Furthermore, using physical tissue models with randomized analyte concentrations and varying turbidities, we show that the fluctuations in turbidity alone causes curved effects which can only be adequately modeled using nonlinear regression techniques. The enhanced levels of accuracy obtained with the SVM based calibration models opens up avenues for prospective prediction in humans and thus for clinical translation of the technology.

Combined confocal Raman and quantitative phase microscopy system for biomedical diagnosis

We have developed a novel multimodal microscopy system that incorporates confocal Raman, confocal reflectance, and quantitative phase microscopy (QPM) into a single imaging entity. Confocal Raman microscopy provides detailed chemical information from the sample, while confocal reflectance and quantitative phase microscopy show detailed morphology. Combining these intrinsic contrast imaging modalities makes it possible to obtain quantitative morphological and chemical information without exogenous staining. For validation and characterization, we have used this multi-modal system to investigate healthy and diseased blood samples. We first show that the thickness of a healthy red blood cell (RBC) shows good correlation with its hemoglobin distribution. Further, in malaria infected RBCs, we successfully image the distribution of hemozoin (malaria pigment) inside the cell. Our observations lead us to propose morphological screening by QPM and subsequent chemical imaging by Raman for investigating blood disorders. This new approach allows monitoring cell development and cell-drug interactions with minimal perturbation of the biological system of interest.

Application of Raman Spectroscopy to Identify Microcalcifications and Underlying Breast Lesions at Stereotactic Core Needle Biopsy

Microcalcifications are a feature of diagnostic significance on a mammogram and a target for stereotactic breast needle biopsy. Here, we report development of a Raman spectroscopy technique to simultaneously identify microcalcification status and diagnose the underlying breast lesion, in real-time, during stereotactic core needle biopsy procedures. Raman spectra were obtained ex vivo from 146 tissue sites from fresh stereotactic breast needle biopsy tissue cores from 33 patients, including 50 normal tissue sites, 77 lesions with microcalcifications, and 19 lesions without microcalcifications, using a compact clinical system. The Raman spectra were modeled on the basis of the breast tissue components, and a support vector machine framework was used to develop a single-step diagnostic algorithm to distinguish normal tissue, fibrocystic change (FCC), fibroadenoma, and breast cancer, in the absence and presence of microcalcifications. This algorithm was subjected to leave-one-site-out cross-validation, yielding a positive predictive value, negative predictive value, sensitivity, and specificity of 100%, 95.6%, 62.5%, and 100% for diagnosis of breast cancer (with or without microcalcifications) and an overall accuracy of 82.2% for classification into specific categories of normal tissue, FCC, fibroadenoma, or breast cancer (with and without microcalcifications). Notably, the majority of breast cancers diagnosed are ductal carcinoma in situ (DCIS), the most common lesion associated with microcalcifications, which could not be diagnosed using previous Raman algorithm(s). Our study shows the potential of Raman spectroscopy to concomitantly detect microcalcifications and diagnose associated lesions, including DCIS, and thus provide real-time feedback to radiologists during such biopsy procedures, reducing nondiagnostic and false-negative biopsies.

Portable Optical Fiber Probe-Based Spectroscopic Scanner for Rapid Cancer Diagnosis: A New Tool for Intraoperative Margin Assessment

There continues to be a significant clinical need for rapid and reliable intraoperative margin assessment during cancer surgery. Here we describe a portable, quantitative, optical fiber probe-based, spectroscopic tissue scanner designed for intraoperative diagnostic imaging of surgical margins, which we tested in a proof of concept study in human tissue for breast cancer diagnosis. The tissue scanner combines both diffuse reflectance spectroscopy (DRS) and intrinsic fluorescence spectroscopy (IFS), and has hyperspectral imaging capability, acquiring full DRS and IFS spectra for each scanned image pixel. Modeling of the DRS and IFS spectra yields quantitative parameters that reflect the metabolic, biochemical and morphological state of tissue, which are translated into disease diagnosis. The tissue scanner has high spatial resolution (0.25 mm) over a wide field of view (10 cm×10 cm), and both high spectral resolution (2 nm) and high spectral contrast, readily distinguishing tissues with widely varying optical properties (bone, skeletal muscle, fat and connective tissue). Tissue-simulating phantom experiments confirm that the tissue scanner can quantitatively measure spectral parameters, such as hemoglobin concentration, in a physiologically relevant range with a high degree of accuracy (<5% error). Finally, studies using human breast tissues showed that the tissue scanner can detect small foci of breast cancer in a background of normal breast tissue. This tissue scanner is simpler in design, images a larger field of view at higher resolution and provides a more physically meaningful tissue diagnosis than other spectroscopic imaging systems currently reported in literatures. We believe this spectroscopic tissue scanner can provide real-time, comprehensive diagnostic imaging of surgical margins in excised tissues, overcoming the sampling limitation in current histopathology margin assessment. As such it is a significant step in the development of a platform technology for intraoperative management of cancer, a clinical problem that has been inadequately addressed to date.

Investigation of the specificity of Raman spectroscopy in non-invasive blood glucose measurements

Although several in vivo blood glucose measurement studies have been performed by different research groups using near-infrared (NIR) absorption and Raman spectroscopic techniques, prospective prediction has proven to be a challenging problem. An important issue in this case is the demonstration of causality of glucose concentration to the spectral information, especially as the intrinsic glucose signal is smaller compared with that of the other analytes in the blood–tissue matrix. Furthermore, time-dependent physiological processes make the relation between glucose concentration and spectral data more complex. In this article, chance correlations in Raman spectroscopy-based calibration model for glucose measurements are investigated for both in vitro (physical tissue models) and in vivo (animal model and human subject) cases. Different spurious glucose concentration profiles are assigned to the Raman spectra acquired from physical tissue models, where the glucose concentration is intentionally held constant. Analogous concentration profiles, in addition to the true concentration profile, are also assigned to the datasets acquired from an animal model during a glucose clamping study as well as a human subject during an oral glucose tolerance test. We demonstrate that the spurious concentration profile-based calibration models are unable to provide prospective predictions, in contrast to those based on actual concentration profiles, especially for the physical tissue models. We also show that chance correlations incorporated by the calibration models are significantly less in Raman as compared to NIR absorption spectroscopy, even for the in vivo studies. Finally, our results suggest that the incorporation of chance correlations for in vivo cases can be largely attributed to the uncontrolled physiological sources of variations. Such uncontrolled physiological variations could either be intrinsic to the subject or stem from changes in the measurement conditions.

Raman spectroscopy: a real-time tool for identifying microcalcifications during stereotactic breast core needle biopsies

Microcalcifications are an early mammographic sign of breast cancer and a target for stereotactic breast needle biopsy. We present here a Raman spectroscopic tool for detecting microcalcifications in breast tissue based on their chemical composition. We collected ex vivo Raman spectra from 159 tissue sites in fresh stereotactic breast needle biopsies from 33 patients, including 54 normal sites, 75 lesions with microcalcifications and 30 lesions without microcalcifications. Application of our Raman technique resulted in a positive predictive value of 97% for detecting microcalcifications. This study shows that Raman spectroscopy has the potential to detect microcalcifications during stereotactic breast core biopsies and provide real-time feedback to radiologists, thus reducing non-diagnostic and false negative biopsies.