DS0413 - Technologies pour la santé

Cellular Ultrasonic imaging Modality to assess red Blood cell Aggregation in vivo – CUMBA

Cellular Ultrasonic imaging Modality to assess red Blood cell Aggregation in vivo

Evaluate continuously the red blood cell aggregation to provide a method for early diagnosis of inflammation

Context

Red blood cells (RBCs) in normal blood flowing through human vessels constitute reversible aggregates. Aggregates usually form rouleaux or complex three-dimensional structures. The aggregation phenomenon is normal and occurs in the circulation of many mammalian species. However, hyperaggregation is a well-known hemorheologic disorder that leads to microvascular dysfunctions and venous thrombosis. It is mainly correlated to the increase of inflammatory plasmatic macromolecules. RBC hyperaggregation is a global marker of inflammation during illness or surgery, and is found in a variety of chronic states (obesity, diabetes, atherosclerosis) and sepsis. <br />In the context of organ transplantation, monitoring RBC aggregation could be a relevant marker of inflammation and tissue perfusion to verify the quality of the organ to transplant. Indeed, most of organs for transplantation are procured from brain-dead donors. However, organs from brain dead-donors exhibit increased inflammatory and immunological responses that degrade their integrity and thus reduce the number of transplantable organs. In order to provide a method for early diagnosis of inflammation, we propose to continuously evaluate RBC aggregation in potential donors by using a Cellular Ultrasound Imaging Modality. We propose to use it to assess real-time variations of RBC aggregation as a surrogate marker of inflammation before the organ retrieval. The medical outcome of this study would be beneficial in online, continuous monitoring of the brain-death patient’s condition in intensive care units to detect any alteration in RBC aggregation level and possible association with inflammatory conditions of the organs to transfer.

The main goal of CUMBA project is to develop a cellular ultrasonic imaging modality to assess the RBC aggregation under in vivo and in situ flow conditions. From the spectral analysis of the ultrasonic backscattered signals from blood, the cellular ultrasound imaging tool we propose will allow extracting structural quantitative ultrasound parameters describing the level of aggregation: the aggregate mean radius, compactness, shape and orientation. This imaging tool will allow quantifying the hyperaggregation in specific arteries or veins of the donor. Before assessing the performance of the new cellular imaging method for pathophysiological human studies, calibration and assessment of accuracy are required. To reach this goal, an original experimental cone-plane flow device will be developed to combine optical and ultrasound measurements on hemoglobin free RBCs or transparent particles mimicking of RBCs. Hence we will evaluate quantitatively the performance of the ultrasonic technique using the optical detection of cell boundaries as the gold standard measure of the aggregate structure. The tests of reliability on the proposed experimental set-up will imply the production of specific RBC-mimicking phantoms. A microfluidic device will be thus developed to produce monodisperse transparent particles in the 10µm diameter range with controllable mechanical and aggregation properties that will mimic RBCs. Furthermore, a new theoretical development of the cellular ultrasound imaging technique will be proposed to incorporate the aggregate anisotropy in order to provide an optimal model for the inversion of experimental data, and to differentiate between normal and pathological aggregates. Finally, a preclinical study will be conducted to evaluate the local inflammation on a brain-death porcine model using our cellular ultrasonic imaging tool.

The main results are as follows:
- The effective medium theory was developed to take into account the coherent and incoherent components of the scattering by aggregates of RBCs in order to improve the modeling at high frequency range. A theoretical development of the cellular ultrasound imaging technique was proposed to incorporate the aggregate anisotropy.
- The effective medium theory combined with the polydisperse structure function was proposed for estimating the distribution of aggregate sizes. We propose two successive experiments: a first experiment on blood sheared in a Couette flow device coupled with an ultrasonic probe, and a second experiment, on the same blood sample, sheared in a plate rheometer coupled to a light microscope. Strong correlation (R2=0.95) was obtained between the aggregate size distributions estimated with ultrasound and optics.
- An experimental cone-plane flow device was developed to combine optical and ultrasound measurements on transparent suspensions of PMMA microspheres.
- A microfluidic device was developed to produce monodisperse microbeads of calcium alginate of diameter 20 microns. The microbeads generation is reproducible thanks to the controlled flow of the solutions and the optical microscopy observation allows to follow the decrease in microbead sizes.

The works in progress are as follows:
- The microbead production with controllable mechanical and aggregation properties that will mimic RBCs.
- The simultaneous measurements in US and optics on hemoglobin free RBCs to evaluate quantitatively the performance of the ultrasonic technique.
- The experimental validation of the anisotropic US scattering modeling.

Paper in refereed journals
[1] De Monchy R., Destrempes F., Saha R. K., Cloutier G. & Franceschini E., Coherent and incoherent ultrasound backscatter from cell aggregates, J. Acoust. Soc. Amer. 140(3) 2173-2184, 2016

Conference proceedings
[2] de Monchy R., Chayer B., Cloutier G. & Franceschini E., Effective Medium Theory combined with a polydisperse Structure Factor Model for characterizing red blood cell aggregation, Proceedings of the 2016 IEEE International Ultrasonics Symposium, doi:10.1109/ULTSYM.2016.7728606, Tours, 18th-21th September, 2016
[3] de Monchy R., Destrempes F., Saha R. K., Cloutier G. & Franceschini E., Coherent and incoherent ultrasound backscatter from cells in aggregated conformation, Proceedings of the 2016 IEEE International Ultrasonics Symposium, doi:10.1109/ULTSYM.2016.7728605, Tours, 18th-21th September, 2016
[4] de Monchy R., Chayer B., Cloutier G. & Franceschini E., Experimental assessment of quantitative ultrasound technique for estimating aggregated red blood cell structures, 10th International Conference on Ultrasonic Biomedical Microscanning, Bonaire, 1-4 May, 2016

CONTEXT. Red blood cells (RBCs) in normal blood flowing through human vessels constitute reversible aggregates. Aggregates usually form rouleaux or complex three-dimensional structures. The aggregation phenomenon is normal and occurs in the circulation of many mammalian species. However, hyperaggregation is a well-known hemorheologic disorder that leads to microvascular dysfunctions and venous thrombosis. It is mainly correlated to the increase of inflammatory plasmatic macromolecules. RBC hyperaggregation is a global marker of inflammation during illness or surgery, and is found in a variety of chronic states (obesity, diabetes, atherosclerosis) and sepsis.
In the context of organ transplantation, monitoring RBC aggregation could be a relevant marker of inflammation and tissue perfusion to verify the quality of the organ to transplant. Indeed, most of organs for transplantation are procured from brain-dead donors. However, organs from brain dead-donors exhibit increased inflammatory and immunological responses that degrade their integrity and thus reduce the number of transplantable organs. In order to provide a method for early diagnosis of inflammation, we propose to continuously evaluate RBC aggregation in potential donors by using a Cellular Ultrasound Imaging Modality. We propose to use it to assess real-time variations of RBC aggregation as a surrogate marker of inflammation before the organ retrieval. The medical outcome of this study would be beneficial in online, continuous monitoring of the brain-death patient’s condition in intensive care units to detect any alteration in RBC aggregation level and possible association with inflammatory conditions of the organs to transfer.

OBJECTIVES. The main goal of CUMBA project is to develop a cellular ultrasonic imaging modality to assess the RBC aggregation under in vivo and in situ flow conditions. From the spectral analysis of the ultrasonic backscattered signals from blood, the cellular ultrasound imaging tool we propose will allow extracting structural quantitative ultrasound parameters describing the level of aggregation: the aggregate mean radius, compactness, shape and orientation. This imaging tool will allow quantifying the hyperaggregation in specific arteries or veins of the donor. Before assessing the performance of the new cellular imaging method for pathophysiological human studies, calibration and assessment of accuracy are required. To reach this goal, an original experimental cone-plane flow device will be developed to combine optical and ultrasound measurements on hemoglobin free RBCs or transparent particles mimicking of RBCs. Hence we will evaluate quantitatively the performance of the ultrasonic technique using the optical detection of cell boundaries as the gold standard measure of the aggregate structure. The tests of reliability on the proposed experimental set-up will imply the production of specific RBC-mimicking phantoms. A microfluidic device will be thus developed to produce monodisperse transparent particles in the 10µm diameter range with controllable mechanical and aggregation properties that will mimic RBCs. Furthermore, a new theoretical development of the cellular ultrasound imaging technique will be proposed to incorporate the aggregate anisotropy in order to provide an optimal model for the inversion of experimental data, and to differentiate between normal and pathological aggregates. Finally, a preclinical study will be conducted to evaluate the local inflammation on a brain-death porcine model using our cellular ultrasonic imaging tool.

PARTNERSHIP. This project brings together four research laboratories with complementary competences: the LMA with an expertise in quantitative ultrasound techniques for tissue characterization, the IRPHE in bio-fluid mechanics and in vascular biomechanics, the CINaM in micro-particle generation and the LBUM in ultrasound imaging and in preclinical studies (international partner with own funds).

Project coordination

Emilie Franceschini (Centre National de la Recherche Scientifique Délégation Provence et Corse, Laboratoire de Mécanique et d'Acoustique)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CNRS DR12 _CINaM Centre National de la Recherche Scientifique délégation Provence et Corse _ Centre Interdisciplinaire de Nanoscience de Marseille
LBUM University of Montreal Hospital Research Center (CRCHUM), Laboratory of Biorheology and Medical Ultrasonics
CNRS DR12_IRPHE Centre National de la Recherche Scientifique délégation Provence et Corse_Institut de Recherche sur les Phénomènes Hors Equilibre
CNRS DR12_LMA Centre National de la Recherche Scientifique Délégation Provence et Corse, Laboratoire de Mécanique et d'Acoustique

Help of the ANR 566,384 euros
Beginning and duration of the scientific project: January 2016 - 48 Months

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