The Ability of a Cell Salvage System and Leukocyte Reduction Filter to Remove Hemangiosarcoma Cells from Dog Blood

Hemangiosarcoma is the neoplastic growth of primitive cells that resemble endothelial cells. Dogs with hemangiosarcoma often present on an emergency basis due to tumor rupture and intraabdominal bleeding. Treatment involves hemodynamic stabilization, surgery to remove the bleeding tumor and chemotherapy to address micrometastatic disease, which is presumed to be present at the time of diagnosis. Allogeneic blood transfusions are often required in the perioperative period to address blood loss. With recent advances in human medicine, the liberal use of allogeneic blood products in patients with cancer has been called into question. Intraoperative cell salvage offers an alternative method to restore blood volume in dogs with intraabdominal bleeding, but the concern for iatrogenic tumor cell dissemination exists. The first objective of this study was to determine if the administration of allogeneic blood products negatively affects time to disease progression in dogs undergoing surgery for hemangiosarcoma. The second objective of this study was to determine the ability of an intraoperative cell salvage system with a leukocyte reduction filter to remove hemangiosarcoma cells from dog blood ex vivo. Retrospective data were collected from two hospitals to include dogs undergoing surgery for hemangiosarcoma. Six variables were analyzed to determine predictors of time to disease progression. The administration of allogeneic blood products was found to be independently associated with shorter time to disease progression. For the second objective, cultured hemangiosarcoma cells were added to dog blood to represent the blood that may be encountered during surgery to address a bleeding tumor. The solution was processed through the intraoperative cell salvage machine followed by a leukocyte reduction filter. PCR, flow cytometry and cytologic examination were used to determine the presence of cultured hemangiosarcoma cells at different points in the intraoperative cell salvage system processing. The processing removed a majority hemangiosarcoma cells from dog blood

Optofluidique : études expérimentales, théoriques et de modélisation

This work focuses on the study of optical properties of fluids at the micrometer scale. To this end, we designed, implemented and studied different types of optofluidic micro- resonators in the Lab-on-Chip format. Our analysis is based on analytical and numerical modeling, as well as experimental measurements conducted on optical microcavities; we use one of them for refractometry applications on homogeneous fluids and on complex fluids, as well as for the localization of solid microparticles by optical trapping. We first focused on the study of a new form of Fabry-Perot micro-cavity based on curved mirrors between which a capillary tube is inserted for injecting a fluidic solution. Experimental results demonstrated the ability of this device to be used as a refractometer with a detection limit of 1.9 × 10-4 RIU for homogeneous liquids. Furthermore, for liquid containing solid particles, the ability to control the microparticles position either by optical trapping or optical binding effects has been successfully demonstrated. In a second step, an optical resonator is simply formed from a liquid droplet placed on top of a superhydrophobe surface. The resulting quasi-spherical shape supports resonant whispering gallery modes. It is shown that, up to millimeter size droplets, the proposed technique of free-space coupling of light is still able to access these modes with very low evanescent tail interaction, contrary to what was indicated in the literature so far. Such optofluidic droplet resonators are expected to find their applications for environmental air quality monitoring, as well as for incubator of living micro-organisms that can be monitored opticallyCe travail porte sur l'étude de propriétés optiques des fluides à échelle micrométrique. A cet effet, nous avons conçu, réalisé et étudié différents types de micro-résonateurs optofluidiques, sous forme de laboratoires sur puce. Notre analyse est fondée sur la modélisation analytique et numérique, ainsi que sur des mesures expérimentales menées sur des micro-cavités optiques; nous utilisons l'une d'entre elles pour des applications de réfractométrie de fluides homogènes et de fluides complexes ainsi que pour la localisation par piégeage optique de microparticules solides. Nous nous sommes d'abord concentrés sur l'étude d'une nouvelle forme de micro-cavité Fabry-Pérot basée sur des miroirs courbes entre lesquels est inséré un tube capillaire permettant la circulation d'une solution liquide. Les résultats expérimentaux ont démontré la capacité de ce dispositif à être utilisé comme réfractomètre avec un seuil de détection de 1,9 × 10-4 RIU pour des liquides homogènes. De plus, pour un liquide contenant des particules solides, la capacité de contrôler la position des microparticules, par des effets de piégeage optique ou de liaison optique, a été démontrée avec succès. Dans un second temps, un résonateur optique est formé simplement à partir d'une goutte de liquide disposée sur une surface super-hydrophobe. La forme quasi-sphérique résultante est propice à des modes de galerie. Il est démontré que, jusqu'à des tailles de gouttelettes millimétriques, la technique de couplage en espace libre est toujours en mesure d'accéder à ces modes à très faible queue évanescente d'interaction, contrairement à ce qu'indiquait jusqu'ici la littérature. De tels résonateurs optofluidiques à gouttelette devraient trouver leur application notamment comme capteur d'environnement de l'air ambiant ou encore comme incubateur de micro-organismes vivants pouvant être suivis par voie optiqu

On-Chip Fabry-Pérot Microcavity for Refractive Index Cytometry and Deformability Characterization of Single Cells

Une identification correcte et précise du phénotype et des fonctions cellulaires est fondamentale pour le diagnostic de plusieurs pathologies ainsi qu’à la compréhension de phénomènes biologiques tels que la croissance, les réponses immunitaires et l’évolution de maladies. Conséquemment, le développement de technologies de pointe offrant une mesure multiparamétrique à haut débit est capital. À cet égard, la cytométrie en flux est l’étalon de référence due à sa grande spécificité, sa grande sensibilité et ses débits élevés. Ces performances sont atteintes grâce à l’évaluation précise du taux d’émission de fluorophores, conjugués à des anticorps, ciblant certains traits cellulaires spécifiques. Néanmoins, sans ce précieux étiquetage, les propriétés physiques caractérisées par la cytométrie sont limitées à la taille et la granularité des cellules. Bien que la cytométrie en flux soit fondamentalement un détecteur optique, elle ne tire pas avantage de l’indice de réfraction, un paramètre reflétant la composition interne de la cellule. Dans la littérature, l’indice de réfraction cellulaire a été utilisé comme paramètre phénotypique discriminant pour la détection de nombreux cancers, d’infections, de la malaria ou encore de l’anémie. Également, les structures fluidiques de la cytométrie sont conçues afin d’empêcher une déformation cellulaire de se produire. Cependant, les preuves que la déformabilité est un indicateur de plusieurs pathologies et d’état de santé cellulaire sont manifestes. Pour ces raisons, l’étude de l’indice de réfraction et de la déformabilité cellulaire en tant que paramètres discriminants est une avenue prometteuse pour l’identification de phénotypes cellulaires. En conséquence, de nombreux biodétecteurs qui exploitent l’une ou l’autre de ces propriétés cellulaires ont émergé au cours des dernières années. D’une part, les dispositifs microfluidiques sont des candidats idéaux pour la caractérisation mécanique de cellules individuelles. En effet, la taille des structures microfluidiques permet un contrôle rigoureux de l’écoulement ainsi que de ses attributs. D’autre part, les dispositifs microphotoniques excellent dans la détection de faibles variations d’indice de réfraction, ce qui est critique pour un phénotypage cellulaire correcte. Par conséquent, l’intégration de composants microfluidiques et microphotoniques à l’intérieur d’un dispositif unique permet d’exploiter ces propriétés cellulaires d’intérêt. Néanmoins, les dispositifs capables d’atteindre une faible limite de détection de l’indice de réfraction tels que les détecteurs à champ évanescent souffrent de faibles profondeurs de pénétration. Ces dispositifs sont donc plus adéquats pour la détection de fluides ou de molécules. De manière opposée, les détecteurs interférométriques tels que les Fabry- Pérots sont sensibles aux éléments présents à l’intérieur de leurs cavités, lesquelles peuvent mesurer jusqu’à plusieurs dizaines de micromètres.----------Abstract Accurate identification of cellular phenotype and function is fundamental to the diagnostic of many pathologies as well as to the comprehension of biological phenomena such as growth, immune responses and diseases development. Consequently, development of state-of-theart technologies offering high-throughput and multiparametric single cell measurement is crucial. Therein, flow cytometry has become the gold standard due to its high specificity and sensitivity while reaching a high-throughput. Its marked performance is a result of its ability to precisely evaluate expression levels of antibody-fluorophore complexes targeting specific cellular features. However, without this precious fluorescence labelling, characterized physical properties are limited to the size and granularity. Despite flow cytometry fundamentally being an optical sensor, it does not take full advantage of the refractive index (RI), a valuable labelfree measurand which reflects the internal composition of a cell. Notably, the cellular RI has proven to be a discriminant phenotypic parameter for various cancer, infections, malaria and anemia. Moreover, flow cytometry is designed to prevent cellular deformation but there is growing evidence that deformability is an indicator of many pathologies, cell health and state. Therefore, cellular RI and deformability are promising avenues to discriminate and identify cellular phenotypes. Novel biosensors exploiting these cellular properties have emerged in the last few years. On one hand, microfluidic devices are ideal candidates to characterize single cells mechanical properties at large rates due to their small structures and controllable flow characteristics. On the other hand, microphotonic devices can detect very small RI variations, critical for an accurate cellular phenotyping. Hence, the integration of microfluidic and microphotonic components on a single device can harness these promising cellular physical properties. However, devices achieving very small RI limit of detection (LOD) such as evanescent field sensors suffer from very short penetration depths and thus are better suited for fluid or single molecule detection. In opposition, interference sensors such as Fabry-Pérots are sensitive to the medium inside their cavity, which can be several tens of micrometers in length, and thus are ideally suited for whole-cell measurement. Still, most of these volume sensors suffer from large LOD or require out-of-plane setups not appropriate for an integrated solution. Such a complex integration of high-throughput, sensitivity and large penetration depth on-chip is an ongoing challenge. Besides, simultaneous characterization of whole-cell RI and deformability has never been reported in the literature

Single cancer cell detection with optofluidic intracavity spectroscopy

2012 Summer.Includes bibliographical references.The detection of cancer cells is the basis for cancer diagnostics, cancer screening and cancer treatment monitoring. Non-destructive and non-chemical optical methods may help reduce the complexity and cost of related test, making them more available to the public. The label-free technique of optofluidic intracavity spectroscopy (OFIS) uses light transmitted through a cellular body in a microfluidic optical resonator to distinguish different types of cells by their spectral signatures. The OFIS chips are fabricated in the CSU semiconductor clean room and the fabrication process was reported by a previous Ph.D student, Hua Shao. She also did some initial exploration on combining dielectrophoresis (DEP) with the OFIS technique. Since then, some revisions to the fabrication technique have been made to improve the alignment, bonding and sealing of this microfluidic chip. In addition, new DEP electrode designs have been designed and fabricated to further improve the trapping performance of the traps and facilitate automated cell trapping and analysis. Viability tests were carried out to investigate the effect of heating (induced by DEP electrodes) on cells in chips built with borosilicate and sapphire substrates. These experiments used specially designed DEP electrodes that help more accurately control the DEP exposure time and strength. The survival rate of cells out of DEP enabled OFIS system is greatly affected by the substrate type and DEP exposure dose. The OFIS technique has differentiated red and white human blood cells, as well as canine lymphoma and lymphocytes based on their distinctive transmission spectra. Using OFIS chips fabricated with the modified process, OFIS spectra of settled cells from canine hemangiosarcoma (HSA) cell lines and monocytes in peripheral blood mononuclear cells (PBMCs) were collected and analyzed. To quantify the strength of transverse modes in their spectra, a single characteristic parameter was determined for each cell by forming a linear combination of the mean and standard deviation of the transmission spectra over one free spectral range excluding the residual longitudinal peaks of the bare Fabry-Pérot (F-P) cavities filled with cell suspending medium only. The difference in the characteristic parameters of HSA and monocyte samples was highly statistically significant with a p-value as low as 10-6. A receiver operating characteristic (ROC) curve constructed from t-distributions fit to the HSA and monocytes spectra indicates that the cell classification based on their characteristic parameters can achieve 95% sensitivity and 98% specificity simultaneously. Furthermore, some features observed in the spectra of HSA cells motivated a new optical model of the cell loaded F-P cavity. The OFIS spectra of individual cells from canine HSA and canine lymphoma cancer cell lines exhibit relatively uniformly spaced multiple transverse modes repeated in each free spectral range of a microfluidic F-P cavity while similar spectra of healthy canine monocytes and lymphocytes only have up to 2 or no transverse mode peaks. Modeling of the cells as thin lenses allows paraxial Gaussian beam resonator analysis that produces spectral features that quantitatively match the frequencies of transverse modes and qualitatively agree with the trends in maximum transmission of the modes when aperture losses are included. The extracted experimental focal lengths are significantly larger for cancerous cells than for noncancerous cells and can be used as a potential cell malignancy indicator. Furthermore, a thick lens model was developed, allowing manipulation of more parameters related to cell morphology and its location in the cavity. This model was used to interpret experimental results acquired from settled and suspended cells