Statistical Dynamics of Flowing Red Blood Cells by Morphological Image Processing

Blood is a dense suspension of soft non-Brownian cells of unique importance. Physiological blood flow involves complex interactions of blood cells with each other and with the environment due to the combined effects of varying cell concentration, cell morphology, cell rheology, and confinement. We analyze these interactions using computational morphological image analysis and machine learning algorithms to quantify the non-equilibrium fluctuations of cellular velocities in a minimal, quasi-two-dimensional microfluidic setting that enables high-resolution spatio-temporal measurements of blood cell flow. In particular, we measure the effective hydrodynamic diffusivity of blood cells and analyze its relationship to macroscopic properties such as bulk flow velocity and density. We also use the effective suspension temperature to distinguish the flow of normal red blood cells and pathological sickled red blood cells and suggest that this temperature may help to characterize the propensity for stasis in Virchow\u27s Triad of blood clotting and thrombosis

Numerical Model for the Determination of Erythrocyte Mechanical Properties and Wall Shear Stress in vivo From Intravital Microscopy.

The mechanical properties and deformability of Red Blood Cells (RBCs) are important determinants of blood rheology and microvascular hemodynamics. The objective of this study is to quantify the mechanical properties and wall shear stress experienced by the RBC membrane during capillary plug flow in vivo utilizing high speed video recording from intravital microscopy, biomechanical modeling, and computational methods. Capillaries were imaged in the rat cremaster muscle pre- and post-RBC transfusion of stored RBCs for 2-weeks. RBC membrane contours were extracted utilizing image processing and parametrized. RBC parameterizations were used to determine updated deformation gradient and Lagrangian Green strain tensors for each point along the parametrization and for each frame during plug flow. The updated Lagrangian Green strain and Displacement Gradient tensors were numerically fit to the Navier-Lame equations along the parameterized boundary to determined Lame's constants. Mechanical properties and wall shear stress were determined before and transfusion, were grouped in three populations of erythrocytes: native cells (NC) or circulating cells before transfusion, and two distinct population of cells after transfusion with stored cells (SC1 and SC2). The distinction, between the heterogeneous populations of cells present after the transfusion, SC1 and SC2, was obtained through principle component analysis (PCA) of the mechanical properties along the membrane. Cells with the first two principle components within 3 standard deviations of the mean, were labeled as SC1, and those with the first two principle components greater than 3 standard deviations from the mean were labeled as SC2. The calculated shear modulus average was 1.1±0.2, 0.90±0.15, and 12 ± 8 MPa for NC, SC1, and SC2, respectively. The calculated young's modulus average was 3.3±0.6, 2.6±0.4, and 32±20 MPa for NC, SC1, and SC2, respectively. o our knowledge, the methods presented here are the first estimation of the erythrocyte mechanical properties and shear stress in vivo during capillary plug flow. In summary, the methods introduced in this study may provide a new avenue of investigation of erythrocyte mechanics in the context of hematologic conditions that adversely affect erythrocyte mechanical properties

The effect of red blood cell deformability on microscale blood flows

The non-Newtonian nature of blood arises from the presence of suspended formed elements which are the red blood cells (RBCs), white blood cells (WBCs) and platelets. Red blood cells or erythrocytes are the predominant constituent elements of blood, hence their role on haemodynamics is of great importance. Their remarkable deformability enables their flow in microvessels and is vital to oxygen delivery to tissue. Different diseases, such as malaria, sickle cell anaemia, diabetes etc. affect the mechanical properties and mainly the deformability of RBCs leading to pathological conditions and disorders in the microcirculation. However, the exact role of RBC deformability in microvascular flows has not been established hitherto. In this study, the role of red blood cell deformability on microscale haemodynamics was examined by perfusing artificially hardened RBCs in straight and bifurcating microchannels mimicking the microvasculature. RBC microchannel flows were resolved using brightfield micro-PIV methods. Advanced image processing routines were implemented in MATLAB to simultaneously determine the velocity and haematocrit distributions for a range of flow rates and feed haematocrit conditions. At low feed haematocrits (5%) hardened RBCs were found to be more dispersed in the straight microchannel flows compared to healthy RBCs, consistent with reports of decreased migration of hardened cells. At high haematocrits (25%) hardened RBCs produced less blunted velocity profiles compared to healthy RBCs, implying a reduction in the shear thinning behaviour of the suspensions. However, the haematocrit profiles appeared to also be sharper indicating some complex interactions between hardened cells. These findings were supported by cell tracking experiments which produced similar cell distributions for fluorescent hardened RBCs in a hardened RBC suspension, in contrast to observed margination of the same cells when suspended in healthy RBCs suspensions. Experiments with higher aspect microchannels confirmed the same trends, indicating that the latter were not due to confinement. The extent of RBC aggregation – indicated by the bluntness of the velocity and haematocrit profiles as well as the standard deviation of the image intensity – was found to be decreased in flows of hardened RBCs, compared to healthy ones in the whole range of the measured flow rates. RBC flows showed a higher level of heterogeneity in the bifurcating microchannels with both haematocrit and velocity profiles downstream of the T-junction bifurcation, exhibiting skewness the extent of which depended on the flow ratio between branches and RBC properties. RBC aggregation appeared to affect the non-uniformity of the haematocrit and velocity distributions downstream the bifurcation to a larger extent than RBC hardening which showed smaller variations compared to healthy non-aggregated RBC suspensions. Finally, the parent branch flow rate affected the redistribution of RBCs downstream of the bifurcation producing less skewed distributions with increasing flow rate. The thesis elucidated the physics of RBCs flows with impaired deformability providing thus the fundamental knowledge that is required for the development of medical diagnostic tools able to capture and assess the severity of diseases associated with impaired RBC deformability

Flow Morphometry of Red Blood Cell Storage Quality Based on Neural Networks

Red blood cell transfusion is routinely performed to improve tissue oxygenation in patients with decreased hemoglobin levels and oxygen-carrying capacity. Generally, blood banks process and store packed red blood cells as RCCs. During storage, RBCs undergo progressive biochemical and morphological changes which are collectively described as storage lesion. According to regulatory guidelines, the quality of RCCs is assessed by quantifying hemolysis before transfusion. However, the hemolysis level only gives an indication of the already lysed erythrocytes; it does not indicate the degree of deterioration of aged cells, which are known to compromise the post-transfusion survival. Morphological analysis, a method that has the potential to provide a simple and practical diagnosis, is suitable for indicating the degradation of RBCs and thus has considerable power to predict actual post-transfusion survival. Microfluidic systems with suspended RBCs can enable fully automated morphological diagnosis based on image analysis with large cell statistics and high sample throughput. The previous version of the flow morphometry system, which was based on a binary decision tree was able to show in a first attempt that spherocytes are a suitable candidate for such a morphological storage lesion marker. However, due to the low classification resolution (three morphology classes), possible shear-induced morphology changes of the measurement system could not be evaluated. In this study, the image classification of the flow morphometry system was substantially enhanced by using a convolutional neural network to strongly improve the resolution and accuracy of the morphology classification. The resulting CNN-based classification achieved a high overall accuracy of 92% with RBCs being classified into nine morphology classes. Through this improved classification resolution, it was possible to assess degradation-induced morphologies at high resolution simultaneously with shear-induced morphologies in RCCs. The overall goal was to provide a robust and strong marker for storage lesion that reflects post-transfusion survival of RBCs. Therefore, it was necessary to analyze the extent to which the shear in the microfluidic system affected the morphological transients between RBC classes. Indeed, it could be shown that shear-induced morphology changes appear dependent on the position of the focal plane height in the flow chamber. The proportion of stomatocytes is increased near the surfaces of the laminar flow chamber. This temporary shear-induced morphology transformation can occur only in flexible erythrocytes with intact membrane properties. Therefore, these cells should be considered a subset of healthy erythrocytes that can reversibly alter from stomatocyte to discocyte morphology. The nine RBC morphology classes of the improved classification resolution were further analyzed to determine whether they exhibit a particular pattern based on their relative proportions during storage that could be used as a storage lesion marker. All individual RBC classes, except for the spherical morphologies, undergo reversible transitions among themselves that are related to the SDE sequence and result in a low signal-to-noise ratio. The proportions of the irreversible spherical morphologies, spheroechinocytes and spherocytes, were defined as the lesion index. This lesion index showed a strong correlation to hemolysis levels. In fact, the correlation between the hemolysis level and the lesion index was so good that it persisted at an individual RCC level. A preliminary lesion index threshold of 11.1% could be established, which is equivalent to a hemolysis threshold of 0.8% established in regulatory guidelines, to assess whether an RCC is of appropriate quality for transfusion. However, the lesion index, besides predicting the hemolysis level, can also be used to generate more information about post-transfusion survival, since it consists exclusively of the RBC morphologies that are removed by the body in a very short time after transfusion in the recipient. Finally, we translated the newly established lesion index and standard biochemical parameters into a quality assessment of RCC shipped and transported repeatedly on air rescue missions to assess an eventual deterioration of the RBCs. We showed that the quality of RCCs was not inferior to control samples after repeated air rescue missions during storage. German regulations allow RCCs to be stored for 42 days in a temperature range of +2°C to +6°C. Compliance with this regulation can be secured during air rescue missions by means of suitable logistics based on a rotation system. By using efficient cooling devices, the logistics and maintenance of the thermal conditions are both safe and feasible. A well-defined rotation system for the use of RCCs during routine air rescue missions offers a resource-saving option and enables the provision of RCCs in compliance with German transfusion guidelines. This innovative concept enables life-saving prehospital transfusions directly at the incident scene. CNN-based flow morphometry and the calculated lesion index allow a reliable assessment of RCC quality. The method also decreases the demand for complex laboratory procedures. Therefore, it is highly advisable to include the lesion index as an additional marker for storage lesion in routine clinical practice. Unlike hemolysis, the lesion index may serve as a good indicator of post-transfusion survival. Thus, both measurements together could provide increased safety and efficacy of stored RCCs

Asymmetric-detection time-stretch optical microscopy (ATOM) for ultrafast high-contrast cellular imaging in flow

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Deformability-induced effects of red blood cells in flow

To ensure a proper health state in the human body, a steady transport of blood is necessary. As the main cellular constituent in the blood suspension, red blood cells (RBCs) are governing the physical properties of the entire blood flow. Remarkably, these RBCs can adapt their shape to the prevailing surrounding flow conditions, ultimately allowing them to pass through narrow capillaries smaller than their equilibrium diameter. However, several diseases such as diabetes mellitus or malaria are linked to an alteration of the deformability. In this work, we investigate the shapes of RBCs in microcapillary flow in vitro, culminating in a shape phase diagram of two distinct, hydrodynamically induced shapes, the croissant and the slipper. Due to the simplicity of the RBC structure, the obtained phase diagram leads to further insights into the complex interaction between deformable objects in general, such as vesicles, and the surrounding fluid. Furthermore, the phase diagram is highly correlated to the deformability of the RBCs and represents thus a cornerstone of a potential diagnostic tool to detect pathological blood parameters. To further promote this idea, we train a convolutional neural network (CNN) to classify the distinct RBC shapes. The benchmark of the CNN is validated by manual classification of the cellular shapes and yields very good performance. In the second part, we investigate an effect that is associated with the deformability of RBCs, the lingering phenomenon. Lingering events may occur at bifurcation apices and are characterized by a straddling of RBCs at an apex, which have been shown in silico to cause a piling up of subsequent RBCs. Here, we provide insight into the dynamics of such lingering events in vivo, which we consequently relate to the partitioning of RBCs at bifurcating vessels in the microvasculature. Specifically, the lingering of RBCs causes an increased intercellular distance to RBCs further downstream, and thus, a reduced hematocrit.Um die biologischen Funktionen im menschlichen Körper aufrechtzuerhalten ist eine stetige Versorgung mit Blut notwendig. Rote Blutzellen bilden den Hauptanteil aller zellulären Komponenten im Blut und beeinflussen somit maßgeblich dessen Fließeigenschaften. Eine bemerkenswerte Eigenschaft dieser roten Blutzellen ist ihre Deformierbarkeit, die es ihnen ermöglicht, ihre Form den vorherrschenden Strömungsbedingungen anzupassen und sogar durch Kapillaren zu strömen, deren Durchmesser kleiner ist als der Gleichgewichtsdurchmesser einer roten Blutzelle. Zahlreiche Erkrankungen wie beispielsweise Diabetes mellitus oder Malaria sind jedoch mit einer Veränderung dieser Deformierbarkeit verbunden. In der vorliegenden Arbeit untersuchen wir die hydrodynamisch induzierten Formen der roten Blutzellen in mikrokapillarer Strömung in vitro systematisch für verschiedene Fließgeschwindigkeiten. Aus diesen Daten erzeugen wir ein Phasendiagramm zweier charakteristischer auftretender Formen: dem Croissant und dem Slipper. Aufgrund der Einfachheit der Struktur der roten Blutzellen führt das erhaltene Phasendiagramm zu weiteren Erkenntnissen über die komplexe Interaktion zwischen deformierbaren Objekten im Allgemeinen, wie z.B. Vesikeln, und des sie umgebenden Fluids. Darüber hinaus ist das Phasendiagramm korreliert mit der Deformierbarkeit der Erythrozyten und stellt somit einen Eckpfeiler eines potentiellen Diagnosewerkzeugs zur Erkennung pathologischer Blutparameter dar. Um diese Idee weiter voranzutreiben, trainieren wir ein künstliches neuronales Netz, um die auftretenden Formen der Erythrozyten zu klassifizieren. Die Ausgabe dieses künstlichen neuronalen Netzes wird durch manuelle Klassifizierung der Zellformen validiert und weist eine sehr hohe Übereinstimmung mit dieser manuellen Klassifikation auf. Im zweiten Teil der Arbeit untersuchen wir einen Effekt, der sich direkt aus der Deformierbarkeit der roten Blutzellen ergibt, das Lingering-Phänomen. Diese Lingering-Ereignisse können an Bifurkationsscheiteln zweier benachbarter Kapillaren auftreten und sind durch ein längeres Verweilen von Erythrozyten an einem Scheitelpunkt gekennzeichnet. In Simulationen hat sich gezeigt, dass diese Dynamik eine Anhäufung von nachfolgenden roten Blutzellen verursacht. Wir analysieren die Dynamik solcher Verweilereignisse in vivo, die wir folglich mit der Aufteilung von Erythrozyten an sich gabelnden Gefäßen in der Mikrovaskulatur in Verbindung bringen. Insbesondere verursacht das Verweilen von Erythrozyten einen erhöhten interzellulären Abstand zu weiter stromabwärts liegenden Erythrozyten und damit einen reduzierten Hämatokrit

Quantifying Embolism: Label-Free Volumetric Mapping of Thrombus Structure and Kinesis in a Microfluidic System with Optical Holography

Embolization of thrombotic material may lead to acute events such as ischemia and myocardial infarction. The embolus is the physical detachment from a primary thrombus that has developed under fluid shear rates. The physical characteristics (surface area coverage, volume, mass, and packing density) of a thrombus influence the overall flow dynamics of an occluding blood vessel. Here, the effectiveness of holographic quantitative phase microscopy (QPM) in identifying multiple morphological parameters of a thrombus (volume, surface area, and height) formed over collagen‐coated microfluidic channels by exerting a range of shear rates with anticoagulated platelet‐rich plasma (PRP) and whole blood is demonstrated. QPM enables the recording of entire thrombus volumes in real‐time using PRP and observed both growth and contraction trends of thrombi, without need for biochemical labeling. The process of emboli detachment in a microfluidic channel under pathophysiological shear rates (7500 and 12 500 s−1) is quantified. Rapid and direct quantification of an embolizing thrombus can enable the study of events during undesirable vessel occlusion and lead to targeting and early diagnosis of acute coronary and venous events.The authors received funding from the National Health and Medical Research Council of Australia and the Australian Research Council