Challenges in clinical analysis by infrared spectroscopy

En els últims anys, el desenvolupament de metodologies d’anàlisi clínic basades en espectroscòpia infraroja (IR) s’ha convertit en un camp molt actiu d’investigació. Millores en la instrumentació i en el processament de les dades han contribuït a que l’espectroscòpia IR s’utilitze per a obtindré informació de la composició de fluids i teixits biològics. Aquesta tesis consolida aquesta tècnica com una eina versàtil en el desenvolupament de metodologies per a extraure una gran quantitat d’informació en un ampli espectre de mostres mitjançant instrumentació senzilla, ràpida i econòmica. Tot i que la tècnica té problemes de sensibilitat i selectivitat, els resultats obtinguts en aquesta Tesi demostren que les tecnologies modernes poden assolir aquestes limitacions. Nous avanços en la quimiometría, la mesura o en la preparació de mostres han estat utilitzats per a millorar les característiques analítiques dels mètodes sense renunciar als beneficis generals de les metodologies basades en IR. Dels resultats obtinguts en aquesta tesi es pot concloure que: - L’anàlisi IR pot extraure informació de qualitat de les mostres clíniques sòlides, líquides o gasoses, incloent càlculs renals, teixits, l’alè, l'orina o el sèrum. - Quant a l'anàlisi de paràmetres clínics en mostres de sèrum, s'ha evidenciat que la naturalesa de les mostres integrades en el conjunt de calibratge és crítica, i es necessita una àmplia col•lecció de mostres amb diferents gammes de concentració dels anàlits i interferències per a proporcionar bones prediccions de mostres obtingudes de diferents orígens. - El principal coll de botella de les determinacions de paràmetres de rutina en sèrum és la sensibilitat. Els errors disminueixen en augmentar la concentració dels anàlits d'interès. Per als paràmetres proteics, triglicèrids i colesterol, les tècniques basades en IR estan preparades per a competir amb l'anàlisi pels mètodes de referència; en el cas de les lipoproteïnes, la glucosa, la immunoglobulina i la urea, la tècnica està limitada com a eina complementària de cribratge i per als anàlits amb concentració inferior a 10mg/dl és impossible realitzar qualsevol model predictiu amb l’adequada fiabilitat. - El problema endogen de la sensibilitat es por resoldre mitjançant la introducció de noves tècniques de pre-processament de mostres i mesures espectrals. Combinant una eficaç microextracció i la tècnica DF-ATR es va proposar una metodologia per a la determinació de lidocaïna en orina amb un límit de detecció al voltant d'1 ppm, i un límit de detecció de 32 ppb es va obtenir per a la quantificació d’isoprè en l'alè utilitzant una columna de preconcentració i una guia d’ones integrada (iHWG, per les seues sigles en anglès). -La metodologia DF-ATR proposada és una eina simple i eficaç per a millorar la selectivitat i la sensibilitat de les mostres clíniques. Aquesta estratègia millora la predicció dels paràmetres lipídics en sèrum i a més a més pot utilitzar-se per a establir un mètode ràpid per a l'elecció de les millors condicions de precipitació de proteïnes prèvies a l’anàlisi per cromatografia líquida i espectrometria de masses. - Altres tractaments quimiomètrics poden millorar la qualitat de la informació obtinguda a partir dels espectres IR dels sèrums. Els models locals basats en LW-PLS milloren els resultats obtinguts per PLS i els models de classificació bastats en LDA poden detectar la majoria de les mostres que es troben en el rang de normalitat dels paràmetres clínics. - Avui en dia hi ha una àmplia gamma d'algoritmes quimiomètrics (com PLS, LW-PLS, SBC, CLS o MCR-ALS) que es poden aplicar a determinacions multiparamètriques en mostres clíniques. Si es coneixen tots els components de la mostra, CLS proporciona prediccions simples i precises, i l’elecció entre PLS i SBC depèn de què és més fàcil, si mesurar un conjunt d’espectres i dades de referència representatiu o una matriu de soroll. MCR-ALS sembla ser el mètode adequat per a l’obtenció d’informació de les interferències. A més, la contribució dels gasos atmosfèrics a l’espectre es pot compensar fàcilment mitjançant un mètode de substracció directa. - Els potents algoritmes esmentats anteriorment poden conduir a resultats excessivament optimistes, especialment quan hi ha involucrat un procés de selecció de variables. L'ús d'un test de permutació pot ser utilitzat per a avaluar la significació estadística del model quan no es pot emprar un conjunt de validació externa. - Les mesures dels teixits de limfoma per transmissió i transflexió mostren imatges hiperespectrals comparables. A la regió de l'empremta digital no hi ha evidència de EFSW i les principals diferències estan connectades amb problemes de fixació de la mostra al suport . - Per als teixits de limfoma, existeixen diferències significatives entre els espectres de les mostres sensibles a la quimioteràpia i les quimioresistentes. Encara que els resultats són prometedors, es requereix un estudi més detallat i amb més mostres, per a establir models de diagnòstic fiables o connectar les diferències espectrals amb mecanismes biològics. Aquesta Tesi doctoral consolida l’espectroscòpia d'IR com una poderosa tècnica per a l'extracció d'informació d’una gran varietat de paràmetres clínics en una àmplia gamma de mostres. A dia d’avui, la instrumentació IR és assequible i compacta i la quantificació de compostos majoritaris es pot realitzar mitjançant el tractament quimiomètric de mesures directes o gairebé quasi-directes. D'aquesta manera, l’IR proporciona una alternativa econòmica i utilitzable en el PoC per a l’anàlisi clínica tradicional o per al desenvolupament de noves estratègies de diagnòstic com per exemple les basades en l'anàlisi no-invasiva de l’alè. Tot i que la tècnica té problemes de sensibilitat i selectivitat, els resultats obtinguts en aquesta Tesi demostren que les tecnologies modernes poden assolir aquestes limitacions. Nous avanços en la mesura (DF-ATR o iHWG) o en la preparació de mostres (microextracció) han estat utilitzats per a millorar les característiques analítiques dels mètodes sense renunciar als beneficis generals de les metodologies basades en IR. Entre d’altres, els pròxims reptes més importants són, d’una banda, la introducció de l'espectroscòpia IR a nivell comercial en la determinació de paràmetres clínics de rutina en sang i orina, que implicaria l'estalvi de grans quantitats de recursos i permetria realitzar anàlisis ràpides en farmàcies i ambulatoris. D’altra banda, l'ampliació de l’aplicació dels mètodes als estudis clínics. Les característiques d’aquests mètodes, que extrauen informació clínica de qualsevol mostra de forma econòmica i ràpida, els fan ideals per a la realització d’amplis estudis clínics, a un cost raonable i en un gran conjunt d’individus.In the last years, the development of IR-based methods in clinical analysis has become a very active field of research, driven by recent improvements in instrumentation and advanced data analysis strategies. In particular, IR spectroscopy has been applied to the clinical field in order to obtain information about the composition of biological fluids and tissues. This Thesis consolidates IR spectroscopy as a powerful technique for developing methodologies for extracting information about a large variety of clinical parameters in a wide range of samples. Although this technique suffers from selectivity and sensitivity, results obtained evidence that modern technologies concerning both, instrumentation and chemometrical treatment can be applied for correcting those drawbacks and improving the and improving the analytical features of the methods without renouncing to the benefits of the IR based methodologies. The main conclusions of the thesis are: i. IR analysis could extract high quality information of solid, liquid or gas samples, including renal stones, tissues, breath, sera or urine. ii. Regarding the analysis of clinical parameters on serum samples, it has been evidenced that the nature of the samples integrated on the calibration set is critical, and it is needed a wide collection of samples with different ranges of concentration of both, analyte and interferences for providing good predictions of samples from different origins. iii. The main bottleneck of the determination is the sensitivity. The errors obtained are related with the concentration of the target analyte. For protein parameters, triglycerides and cholesterol, IR based techniques are ready to compete with the reference analysis; in the case of lipoproteins, glucose, immunoglobulin and urea the technique is limited as a screening complementary tool and for analytes less concentrated than 10 mg dL-1 is impossible to perform any reliable prediction. iv. The endogenous sensitivity problem can be solved by introducing modern sample preprocessings and measurement techniques. A LOD around 1 ppm was measured for the determination of lidocaine in urine using ATR-FTIR measurements of DFs of organic extracts and a LOD of 32 ppb was calculated for the determination of isoprene in breath using a preconcentration column and an iHWG. v. ATR-FTIR measurements of DFs obtained from organic extracts is a straightforward approach for improving the selectivity and sensitivity of the clinical samples. This strategy improves the prediction of lipidic parameters in sera and this approach could be also used for establishing a cost-effective and fast back-up method for choosing the best protein precipitation conditions prior to liquid chromatography-mass spectrometry analysis. vi. Other chemometrical treatments are also useful to improve the quality of the information obtained from IR spectra of sera. Local models performed using LW-PLS could improve those obtained from PLS, and using LDA or PLS-DA could detect most of the samples which are located in the “healthy” range of clinical parameters. vii. Nowadays there is a wide range of chemometrical algorithms, such as PLS, LW-PLS, SBC, CLS or MCR-ALS which can be applied to the determination of analytes in clinical samples. If all sample components of the system are known, CLS provides a sufficiently accurate solution; the selection between PLS and SBC methods depends on whether it is easier to measure a calibration data set or a noise matrix; and MCR-ALS appears to be the most suitable method for detecting interferences within a sample. In addition, the contribution of atmospheric gases can be easily compensated using a straightforward method based on eluent subtraction algorithms for liquid chromatography-IR. viii. The aforementioned powerful algorithms could lead over-optimistic results as a result of the presence of chance correlations, especially when a variable selection process in involved. The use of a permutation test before the variable selection or including the variable selection process on the permutation can be used to provide statistical significance to the model when an external validation set is not available. ix. Transflection and transmission measurements of lymphoma tissues shows comparable IR hyperspectral images. In the fingerprint region there is no evidence of the EFSW, and the main differences are connected with fixation issues. x. There are significant differences between the spectra of chemosensitive and chemoresistant samples. Although results are promising, for obtaining a PLS-DA model for diagnosis purposes or connect the spectral differences with biological explanations a more detailed study with more samples is required

Combining Pharmacokinetics and Vibrational Spectroscopy: MCR-ALS Hard-and-Soft Modelling of Drug Uptake In Vitro Using Tailored Kinetic Constraints

Raman microspectroscopy is a label-free technique which is very suited for the investigation of pharmacokinetics of cellular uptake, mechanisms of interaction, and efficacies of drugs in vitro. However, the complexity of the spectra makes the identification of spectral patterns associated with the drug and subsequent cellular responses difficult. Indeed, multivariate methods that relate spectral features to the inoculation time do not normally take into account the kinetics involved, and important theoretical information which could assist in the elucidation of the relevant spectral signatures is excluded. Here, we propose the integration of kinetic equations in the modelling of drug uptake and subsequent cellular responses using Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) and tailored kinetic constraints, based on a system of ordinary differential equations. Advantages of and challenges to the methodology were evaluated using simulated Raman spectral data sets and real Raman spectra acquired from A549 and Calu-1 human lung cells inoculated with doxorubicin, in vitro. The results suggest a dependency of the outcome on the system of equations used, and the importance of the temporal resolution of the data set to enable the use of complex equations. Nevertheless, the use of tailored kinetic constraints during MCR-ALS allowed a more comprehensive modelling of the system, enabling the elucidation of not only the time-dependent concentration profiles and spectral features of the drug binding and cellular responses, but also an accurate computation of the kinetic constants

Fourier transform IR imaging of primary tumors predicts lymph node metastasis of bladder carcinoma

The process of metastasis is complex and often impossible to be recognized in conventional clinical diagnosis. Lymph node metastasis (LNM) of bladder carcinoma (BC) is often associated with muscle-invasive tumors. To prevent and recognize LNM, the standard treatment includes radical cystectomy with lymph node dissection and histological examination. Here, we propose infrared (IR) microscopy as a tool for the prediction of LNM. For this purpose, IR images of bladder biopsies from patients with diagnosed non-metastatic early (E BC) and advanced (A BC), as well as metastatic advanced (M BC) bladder cancer were first collected. Furthermore, this dataset was complemented with images of the secondary tumors from the lymph nodes (M LN) of the M BC patients. Unsupervised clustering was used to extract tissue structures from IR images to create a data set comprising 382 IR spectra of non-metastatic bladder tumors and 241 metastatic ones. Based on that, we next established discrimination models using PLS-DA with repeated random sampling double cross-validation, and permutation test to perform the classification. The accuracy of BC metastasis prediction from IR bladder biopsies was 83 % and 78 % for early and advanced BC, respectively, herein demonstrating a proof-of-concept IR detection of BC metastasis. The analysis of spectral profiles additionally showed molecular composition similarity between metastatic bladder and lymph node tumors. We also determined spectral biomarkers of LNM that are associated with sugar metabolism, remodeling of extracellular matrix, and morphological features of cancer cells. Our approach can improve clinical decision-making in urological oncology

Towards a Rapid Screening of Liver Grafts at the Operating Room Using Mid-Infrared Spectroscopy

The estimation of steatosis in a liver graft is mandatory prior to liver transplantation, as the risk of graft failure increases with the level of infiltrated fat. However, the assessment of liver steatosis before transplantation is typically based on a qualitative or semiquantitative characterization by visual inspection and palpation and histological analysis. Thus, there is an unmet need for transplantation surgeons to have access to a diagnostic tool enabling an in situ fast classification of grafts prior to extraction. In this study, we have assessed an attenuated total reflection−Fourier transform infrared (ATR−FTIR) spectroscopic method compatible with the requirements of an operation room for the evaluation of the lipid content in human livers. A set of human liver biopsies obtained from organs intended for transplantation were analyzed by expert pathologists, ATR−FTIR spectroscopy, lipid biochemical analysis, and UPLC−ESI(+/−)TOFMS for lipidomic profiling. Comparative analysis of multisource data showed strong correlations between ATR−FTIR, clinical, and lipidomic information. Results show that ATR−FTIR captures a global picture of the lipid composition of the liver, along with information for the quantification of the triradylglycerol content in liver biopsies. Although the methodology performance needs to be further validated, results support the applicability of ATR−FTIR for the in situ determination of the grade of liver steatosis at the operation room as a fast, quantitative method, as an alternative to the qualitative and subjective pathological examination

Label-free testing strategy to evaluate packed red blood cell quality before transfusion to leukemia patients

Abstract Patients worldwide require therapeutic transfusions of packed red blood cells (pRBCs), which is applied to the high-risk patients who need periodic transfusions due to leukemia, lymphoma, myeloma and other blood diseases or disorders. Contrary to the general hospital population where the transfusions are carried out mainly for healthy trauma patients, in case of high-risk patients the proper quality of pRBCs is crucial. This leads to an increased demand for efficient technology providing information on the pRBCs alterations deteriorating their quality. Here we present the design of an innovative, label-free, noninvasive, rapid Raman spectroscopy-based method for pRBCs quality evaluation, starting with the description of sample measurement and data analysis, through correlation of spectroscopic results with reference techniques' outcomes, and finishing with methodology verification and its application in clinical conditions. We have shown that Raman spectra collected from the pRBCs supernatant mixture with a proper chemometric analysis conducted for a minimum one ratio of integral intensities of the chosen Raman marker bands within the spectrum allow evaluation of the pRBC quality in a rapid, noninvasive, and free-label manner, without unsealing the pRBCs bag. Subsequently, spectroscopic data were compared with predefined reference values, either from pRBCs expiration or those defining the pRBCs quality, allowing to assess their utility for transfusion to patients with acute myeloid leukemia (AML) and lymphoblastic leukemia (ALL)

Metabolomic Diversity of Human Milk Cells over the Course of Lactation - A Preliminary Study

Human milk (HM) is a complex biofluid containing a wide cell variety including epithelial cells and leukocytes. However, the cellular compositions and their phenotypic properties over the course of lactation are poorly understood. The aim of this preliminary study was to characterize the cellular metabolome of HM over the course of lactation. Cells were isolated via centrifugation and the cellular fraction was characterized via cytomorphology and immunocytochemical staining. Cell metabolites were extracted and analyzed using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QqTOF-MS) in the positive and negative electrospray ionization modes. Immunocytochemical analysis revealed a high variability of the number of detected cells with relative median abundances of 98% of glandular epithelial cells, 1% of leukocytes, and 1% of keratinocytes. Significant correlations between the milk postnatal age with percentage of epithelial cells and leukocytes, and with total cell count were observed. Results from the Hierarchical Cluster Analysis of immunocytochemical profiles were very similar to those observed in the analysis of the metabolomic profiles. In addition, metabolic pathway analysis showed alterations in seven metabolic pathways correlating with postnatal age. This work paves the way for future investigations on changes in the metabolomic fraction of the cellular compartment of HM

Trends in biomedical analysis of red blood cells – Raman spectroscopy against other spectroscopic, microscopic and classical techniques

Application of modern and innovative spectroscopic and microscopic approaches to biomedical analysis opens new horizons and sheds new light on many unexplored scientific territories. In this review, we critically summarize up-to-date Raman-based methodologies for red blood cells (RBCs) analysis used in biology and medicine, and compare them with both classical, as well as other spectroscopic and microscopic approaches. The main emphasis is placed on the advantages, disadvantages and capabilities of each technique for detection of RBC deteriorations and RBC-related diseases. Although currently used classical techniques of medical analysts serve as a gold standard for clinicians in diagnosis of erythropathies, they provide insufficient insight into RBC alterations at the molecular level. In addition, there is a demand for non-destructive and label-free analytical techniques for rapid detection and diagnosis of erythropathies. Their recognition often requires multimodal methodology comprising application of methods including sophisticated spectroscopy-based techniques, where Raman-based approaches play an important role

Bypassing cellular EGF receptor dependence through epithelial-to-mesenchymal-like transitions

Over 90% of all cancers are carcinomas, malignancies derived from cells of epithelial origin. As carcinomas progress, these tumors may lose epithelial morphology and acquire mesenchymal characteristics which contribute to metastatic potential. An epithelial-to-mesenchymal transition (EMT) similar to the process critical for embryonic development is thought to be an important mechanism for promoting cancer invasion and metastasis. Epithelial-to-mesenchymal transitions have been induced in vitro by transient or unregulated activation of receptor tyrosine kinase signaling pathways, oncogene signaling and disruption of homotypic cell adhesion. These cellular models attempt to mimic the complexity of human carcinomas which respond to autocrine and paracrine signals from both the tumor and its microenvironment. Activation of the epidermal growth factor receptor (EGFR) has been implicated in the neoplastic transformation of solid tumors and overexpression of EGFR has been shown to correlate with poor survival. Notably, epithelial tumor cells have been shown to be significantly more sensitive to EGFR inhibitors than tumor cells which have undergone an EMT-like transition and acquired mesenchymal characteristics, including non-small cell lung (NSCLC), head and neck (HN), bladder, colorectal, pancreas and breast carcinomas. EGFR blockade has also been shown to inhibit cellular migration, suggesting a role for EGFR inhibitors in the control of metastasis. The interaction between EGFR and the multiple signaling nodes which regulate EMT suggest that the combination of an EGFR inhibitor and other molecular targeted agents may offer a novel approach to controlling metastasis

CMS physics technical design report : Addendum on high density QCD with heavy ions

Peer reviewe