The influence of hypoxia in erythropoiesis and morphology of red blood cells in sickle cell disease and hereditary spherocytosis.

Die Lebensfahigkeit menschlicher Zellen h ¨ angt in hohem Maße vom Sauerstoff ab, der von den roten ¨ Blutkorperchen transportiert und zur Verf ¨ ugung gestellt wird. In dieser Arbeit wird untersucht, wie ¨ Sauerstoff die Physiologie und Pathophysiologie jener roten Blutkorperchen beeinflusst. Die Arbeit ¨ ist in zwei wesentliche Teile gegliedert. Zunachst werden Ergebnisse einer gr ¨ oßeren Studie mit Probanden vorgestellt, die in großer H ¨ ohe ¨ (3450 m) durchgefuhrt wurde. In dieser H ¨ ohe sorgt der reduzierte Luftdruck f ¨ ur eine Abnahme ¨ der Sauerstoffsattigung im Blut und f ¨ uhrt so zu einer erh ¨ ohten Produktion an roten Blutk ¨ orperchen ¨ (Erythropoese). Nach der Ruckkehr aus großer H ¨ ohe f ¨ allt die Zahl an roten Blutk ¨ orperchen nach ¨ kurzer Zeit wieder auf das vorherige Niveau Es ist bislang nicht verstanden, ob die neugebildeten roten Blutzellen dazu abgebaut werden. Der hyptothetische Mechanismus der Neozytolyse (engl. Neocytolisis) geht davon aus, dass ausgerechnet die neu gebildeten, roten Blutzellen als erste wieder abgebaut werden, wenn die Ruckkehr auf Meeresniveau erfolgt. Die wissenschaftliche Fragestellung, ¨ die im Rahmen dieser Studie beantwortet wurde, ist, ob und in welchem Maß Neocytolyse nach einem 3-wochigen Aufenthalt in großer H ¨ ohe stattfindet. Dazu wurden zun ¨ achst Untersuchungen an aller ¨ Probanden mittels in-vitro Zellkulturen durchgefuhrt, um sowohl die Erythropoese als auch (neu ent- ¨ standende) Retikulozyten zu charakterisieren. Die Ergebnisse zeigen eine beschleunigte Reifung der Vorlauferzellen in Zellkulturen bei niedrigerem Sauerstoffgehalt (3%) verglichen mit normaler Atmo- ¨ sphare (20%) und eine unerwartet verbesserte ¨ Uberlebensrate der Retikulozyten. Dies stimmt mit dem ¨ Ergebnis der Studie uberein, dass nach der R ¨ uckkehr von der gew ¨ ahlten H ¨ ohe keine Neozytolyse, d.h. ¨ kein bevorzugter Abbau von neu gebildeten roten Blutzellen, auftrat, was die Hypothese eines selektiven, vorzeitigen Abbaus von unreifen roten Blutzellen widerlegt. Weiterhin wurde in der Zellkultur unter verringerter Sauerstoffgabe eine erhohte Zahl bikonkaver Zellen beobachtet, was eine typische ¨ Gestalt fur einen fortgeschrittenen Reifegrad ist. Diese Beobachtung k ¨ onnte ein Anhaltspunkt daf ¨ ur¨ sein, dass der Reifeprozess von roten Blutzellen durch die Reduzierung des atmospharischen Sauer- ¨ stoffgehaltes begunstigt werden kann. Im Rahmen dieser Arbeit wurde zus ¨ atzlich ein Protokoll zur ¨ Isolation der Retikulozyten vom Vollblut der Probanden entwickelt, um die pure Zell-RNA jeweils vor und in großer Hohe zu sequenzieren. Jedoch muss die Ausbeute an RNA weiter optimiert werden, ¨ um einen detaillierten Vergleich der Gen-Expressions-Niveaus anstellen zu konnen. ¨ Der zweite Teil der Arbeit konzentriert sich auf die Untersuchung der Morphologie der roten Blutkor- ¨ perchen bei zwei Arten von Anamie. Aufgrund der hohen Empfindlichkeit der roten Blutk ¨ orperchen ¨ gegenuber Formvariationen, mussten die Proben vor jeder experimentellen Manipulation fixiert wer- ¨ den. Dazu musste ein angemessenes Verfahren entwicklelt werden, das im Rahmen dieser Arbeit vorgestellt wird. Zunachst wird auf die Sichelzellenan ¨ amie eingegangen, bei der es unter Desoxy- ¨ genierung des Hamoglobins zur starken Verformung der roten Blutk ¨ orperchen (Sichelzellen) kommt. ¨ Dies beintrachtigt sowohl deren Funktion als auch Lebensdauer. Eine quantitative Analyse von Zell- ¨ Projektionsbildern aus konfokalen Mikroskopieaufnahmen wurde mit Hilfe eines maßgeschneiderten Computerprogramms im Rahmen einer klinischen Pilotstudie der Phase II zur Therapie der Sichelzellanamie durchgef ¨ uhrt. Es konnte gezeigt werden, dass diese Methode in Kombination mit anderen ¨ experimentellen Verfahren ein wirkungsvolles Instrument zur Beurteilung des Zellhydratationszustands von Sichelzellenpatienten ist. Daher kann diese Technik zur Beurteilung der Wirksamkeit von Sichelzellen-Therapien oder zur Beurteilung des Zustands der roten Blutkorperchen eines Pa- ¨ tienten verwendet werden. Da die roten Blutkorperchen bei verschiedenen Arten von An ¨ amie auch ¨ verschiedene Formvariationen aufweisen, wurde eine weitere Blutkrankheit, die hereditare Sph ¨ arozy- ¨ tose, untersucht. In diesem Fall lag der Schwerpunkt auf der automatisierten Formerkennung der roten Blutkorperchen, die in der Regel manuell durchgef ¨ uhrt wird und daher einer Bewertungsinkonsistenz ¨ unterliegt. Die Untersuchung nutzt die 3D-Rekonstruktion der Zellen aus konfokalen Mikroskopieaufnahmen und die anschließende Formerkennung mittels kunstlicher neuronaler Netze. Die Beurteilung ¨ dieses Systems zeigte sowohl eine sehr gute Erkennungsrate, hohe Prazision, eine schnelle Prozesszeit, ¨ als auch ein objektives Ergebnis verglichen mit der manuellen Klassifikation. Verglichen mit der Analyse von 2D Mikroskopieaufnahmen von Blutabstrichen, konnten durch die Auswertung korrespondierender 3D Aufnahmen außerdem andere Formspektren abgeleitet werden. Dies legt die Empfehlung nahe, die manuelle Klassifizierung von Zellformen (Stand der Technik) im Kontext von hereditarer Sph ¨ arozytose zu ¨ uberdenken.Human cell viability highly depends on oxygen, which is carried and provided by red blood cells. This thesis aimed to investigate how oxygen influences physiology and pathophysiology of red blood cells and is divided in two main parts. The first one presents results that are part of a larger study performed at high altitude (3450 m). Here, the reduced air pressure causes a decrease in blood oxygenation, which is balanced by an increase in red blood cells production (erythropoiesis). Upon return from high altitude, the amount of red blood cells is restored to the original levels within a few days, which is in contrast with the average red blood cell lifespan of 120 days. The reasons leading to such red blood cells premature clearance are not well understood. A hypothetical mechanism previously proposed is defined as neocytolisis, i.e. the selective clearance of the red blood cells formed at high altitude upon return to sea level. The scientific question of the study was therefore to assess if and how neocytolisis occurs after a 3-week stay at high altitude. The investigations performed in this thesis involved in vitro culture of erythroid precursors of the donors participating in the study to characterize both erythropoiesis and the obtained immature red blood cells, namely reticulocytes. Results highlighted an accelerated maturation of erythroid precursors in cultures performed at lower oxygen (3%) compared to atmospheric oxygen (20%) ones and an unexpected improved cell survival of the obtained reticulocytes. This was in accordance with the finding that after the stay at the chosen altitude no neocytolisis occurred, denying the hypothesis of a higher fragility of cells formed at low oxygen causing their selective premature clearance upon return from high altitude. Moreover, cultures performed at low oxygen resulted in the formation of more biconcave cells, the typical shape of mature red blood cells. This suggests that reducing oxygen levels in cultures may contribute to advance their maturation in vitro. In addition to cell cultures, another objective was to perform RNA sequencing of isolated reticulocytes from whole blood of the donors to compare pre- and high altitude conditions. A protocol for the isolation of a pure fraction of reticulocytes and their RNA was developed. However, total RNA yield needs to be increased to perform an accurate comparison of gene expression levels. The second part of the thesis focused on studying red blood cell morphology in two types of anemia. Because of the high sensitivity of red blood cells to shape variation, samples were always fixed before any experimental manipulation. A thorough study describing how to perform red blood cell fixation is presented. The first blood disease of study was sickle cell anemia. In this pathology, deoxygenation of hemoglobin causes the deformation of red blood cells to the shape of a sickle that impairs their functions and lifespan. The quantitative analysis of cell projections from confocal images by means of a customized computer program was employed within a pilot phase II clinical trial for the therapy of sickle cell disease. The obtained results combined with other experimental evaluations showed that red blood cell shape analysis of sickle cell disease patients is a simple but powerful tool to evaluate cell hydration state. Therefore, this technique may be used for the assessment of the efficacy of sickle cell disease therapies or to evaluate the state of red blood cells of a patient. Since red blood cells display shape variations in different types of anemia, a second blood disease was investigated, namely hereditary spherocytosis. In this case, the focus was the automation of red blood cells shape recognition, which is usually performed manually and therefore subjected to evaluation inconsistency. The investigation made use of 3D cell reconstructions from confocal images and automated shape recognition by means of artificial neural networks. System benchmarks showed a good recognition performance, high accuracy, fast processing time as well unbiased results compared to the manual classification. Moreover, the application of 3D imaging in contrast to the traditional 2D-microscopy typically employed in blood smear analysis revealed a different red blood cell shapes spectrum. These results therefore suggest to revise the state-of-the art manual shape classification applied in hereditary spherocytosis

Rare Anemias: Are Their Names Just Smoke and Mirrors?

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Effect of Cell Age and Membrane Rigidity on Red Blood Cell Shape in Capillary Flow

Blood flow in the microcirculatory system is crucially affected by intrinsic red blood cell (RBC) properties, such as their deformability. In the smallest vessels of this network, RBCs adapt their shapes to the flow conditions. Although it is known that the age of RBCs modifies their physical properties, such as increased cytosol viscosity and altered viscoelastic membrane properties, the evolution of their shape-adapting abilities during senescence remains unclear. In this study, we investigated the effect of RBC properties on the microcapillary in vitro flow behavior and their characteristic shapes in microfluidic channels. For this, we fractioned RBCs from healthy donors according to their age. Moreover, the membranes of fresh RBCs were chemically rigidified using diamide to study the effect of isolated graded-membrane rigidity. Our results show that a fraction of stable, asymmetric, off-centered slipper-like cells at high velocities decreases with increasing age or diamide concentration. However, while old cells form an enhanced number of stable symmetric croissants at the channel centerline, this shape class is suppressed for purely rigidified cells with diamide. Our study provides further knowledge about the distinct effects of age-related changes of intrinsic cell properties on the single-cell flow behavior of RBCs in confined flows due to inter-cellular age-related cell heterogeneity

The Evolution of Erythrocytes Becoming Red in Respect to Fluorescence

Very young red blood cells, namely reticulocytes, can be quite easily recognized and labeled by cluster of differentiation antibodies (CD71, transferrin receptor) or by staining remnant RNA with thiazol orange. In contrast, age specific erythrocyte labeling is more difficult in later periods of their life time. While erythrocytes contain band 4.1 protein, a molecular clock, so far it has not been possible to read this clock on individual cells. One concept to track erythrocytes during their life time is to mark them when they are young, either directly in vivo or ex vivo followed by a transfusion. Several methods like biotinylation, use of isotopes or fluorescent labeling have proved to be useful experimental approaches but also have several inherent disadvantages. Genetic engineering of mice provides additional options to express fluorescent proteins in erythrocytes. To allow co-staining with popular green fluorescent dyes like Fluo-4 or other fluorescein-based dyes, we bred a mouse line expressing a tandem red fluorescent protein (tdRFP). Within this Brief Research Report, we provide the initial characterisation of this mouse line and show application examples ranging from transfusion experiments and intravital microscopy to multicolour flow cytometry and confocal imaging. We provide a versatile new tool for erythrocyte research and discuss a range of experimental opportunities to study membrane processes and other aspects of erythrocyte development and aging with help of these animals

Cross-talk between red blood cells and plasma influences blood flow and omics phenotypes in severe COVID-19

Coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and can affect multiple organs, among which is the circulatory system. Inflammation and mortality risk markers were previously detected in COVID-19 plasma and red blood cells (RBCs) metabolic and proteomic profiles. Additionally, biophysical properties, such as deformability, were found to be changed during the infection. Based on such data, we aim to better characterize RBC functions in COVID-19. We evaluate the flow properties of RBCs in severe COVID-19 patients admitted to the intensive care unit by using microfluidic techniques and automated methods, including artificial neural networks, for an unbiased RBC analysis. We find strong flow and RBC shape impairment in COVID-19 samples and demonstrate that such changes are reversible upon suspension of COVID-19 RBCs in healthy plasma. Vice versa, healthy RBCs resemble COVID-19 RBCs when suspended in COVID-19 plasma. Proteomics and metabolomics analyses allow us to detect the effect of plasma exchanges on both plasma and RBCs and demonstrate a new role of RBCs in maintaining plasma equilibria at the expense of their flow properties. Our findings provide a framework for further investigations of clinical relevance for therapies against COVID-19 and possibly other infectious diseases

Glutaraldehyde – A Subtle Tool in the Investigation of Healthy and Pathologic Red Blood Cells

Glutaraldehyde is a well-known substance used in biomedical research to fix cells. Since hemolytic anemias are often associated with red blood cell shape changes deviating from the biconcave disk shape, conservation of these shapes for imaging in general and 3D-imaging in particular, like confocal microscopy, scanning electron microscopy or scanning probe microscopy is a common desire. Along with the fixation comes an increase in the stiffness of the cells. In the context of red blood cells this increased rigidity is often used to mimic malaria infected red blood cells because they are also stiffer than healthy red blood cells. However, the use of glutaraldehyde is associated with numerous pitfalls: (i) while the increase in rigidity by an application of increasing concentrations of glutaraldehyde is an analog process, the fixation is a rather digital event (all or none); (ii) addition of glutaraldehyde massively changes osmolality in a concentration dependent manner and hence cell shapes can be distorted; (iii) glutaraldehyde batches differ in their properties especially in the ratio of monomers and polymers; (iv) handling pitfalls, like inducing shear artifacts of red blood cell shapes or cell density changes that needs to be considered, e.g., when working with cells in flow; (v) staining glutaraldehyde treated red blood cells need different approaches compared to living cells, for instance, because glutaraldehyde itself induces a strong fluorescence. Within this paper we provide documentation about the subtle use of glutaraldehyde on healthy and pathologic red blood cells and how to deal with or circumvent pitfalls

Acanthocyte Sedimentation Rate as a Diagnostic Biomarker for Neuroacanthocytosis Syndromes: Experimental Evidence and Physical Justification

(1) Background: Chorea-acanthocytosis and McLeod syndrome are the core diseases among the group of rare neurodegenerative disorders called neuroacanthocytosis syndromes (NASs). NAS patients have a variable number of irregularly spiky erythrocytes, so-called acanthocytes. Their detection is a crucial but error-prone parameter in the diagnosis of NASs, often leading to misdiagnoses. (2) Methods: We measured the standard Westergren erythrocyte sedimentation rate (ESR) of various blood samples from NAS patients and healthy controls. Furthermore, we manipulated the ESR by swapping the erythrocytes and plasma of different individuals, as well as replacing plasma with dextran. These measurements were complemented by clinical laboratory data and single-cell adhesion force measurements. Additionally, we followed theoretical modeling approaches. (3) Results: We show that the acanthocyte sedimentation rate (ASR) with a two-hour read-out is significantly prolonged in chorea-acanthocytosis and McLeod syndrome without overlap compared to the ESR of the controls. Mechanistically, through modern colloidal physics, we show that acanthocyte aggregation and plasma fibrinogen levels slow down the sedimentation. Moreover, the inverse of ASR correlates with the number of acanthocytes (R 2 = 0.61, p = 0.004). (4) Conclusions: The ASR/ESR is a clear, robust and easily obtainable diagnostic marker. Independently of NASs, we also regard this study as a hallmark of the physical view of erythrocyte sedimentation by describing anticoagulated blood in stasis as a percolating gel, allowing the application of colloidal physics theory

Red blood cell lingering modulates hematocrit distribution in the microcirculation

The distribution of red blood cells (RBCs) in the microcirculation determines the oxygen delivery and solute transport to tissues. This process relies on the partitioning of RBCs at successive bifurcations throughout the microvascular network, and it has been known since the last century that RBCs partition disproportionately to the fractional blood flow rate, therefore leading to heterogeneity of the hematocrit (i.e., volume fraction of RBCs in blood) in microvessels. Usually, downstream of a microvascular bifurcation, the vessel branch with a higher fraction of blood flow receives an even higher fraction of RBC flux. However, both temporal and time-average deviations from this phase-separation law have been observed in recent studies. Here, we quantify how the microscopic behavior of RBC lingering (i.e., RBCs temporarily residing near the bifurcation apex with diminished velocity) influences their partitioning, through combined in vivo experiments and in silico simulations. We developed an approach to quantify the cell lingering at highly confined capillary-level bifurcations and demonstrate that it correlates with deviations of the phase-separation process from established empirical predictions by Pries et al. Furthermore, we shed light on how the bifurcation geometry and cell membrane rigidity can affect the lingering behavior of RBCs; e.g., rigid cells tend to linger less than softer ones. Taken together, RBC lingering is an important mechanism that should be considered when studying how abnormal RBC rigidity in diseases such as malaria and sickle-cell disease could hinder the microcirculatory blood flow or how the vascular networks are altered under pathological conditions (e.g., thrombosis, tumors, aneurysm)

Erysense, a Lab-on-a-Chip-Based Point-of-Care Device to Evaluate Red Blood Cell Flow Properties With Multiple Clinical Applications

In many medical disciplines, red blood cells are discovered to be biomarkers since they “experience” various conditions in basically all organs of the body. Classical examples are diabetes and hypercholesterolemia. However, recently the red blood cell distribution width (RDW), is often referred to, as an unspecific parameter/marker (e.g., for cardiac events or in oncological studies). The measurement of RDW requires venous blood samples to perform the complete blood cell count (CBC). Here, we introduce Erysense, a lab-on-a-chip-based point-of-care device, to evaluate red blood cell flow properties. The capillary chip technology in combination with algorithms based on artificial neural networks allows the detection of very subtle changes in the red blood cell morphology. This flow-based method closely resembles in vivo conditions and blood sample volumes in the sub-microliter range are sufficient. We provide clinical examples for potential applications of Erysense as a diagnostic tool [here: neuroacanthocytosis syndromes (NAS)] and as cellular quality control for red blood cells [here: hemodiafiltration (HDF) and erythrocyte concentrate (EC) storage]. Due to the wide range of the applicable flow velocities (0.1–10 mm/s) different mechanical properties of the red blood cells can be addressed with Erysense providing the opportunity for differential diagnosis/judgments. Due to these versatile properties, we anticipate the value of Erysense for further diagnostic, prognostic, and theragnostic applications including but not limited to diabetes, iron deficiency, COVID-19, rheumatism, various red blood cell disorders and anemia, as well as inflammation-based diseases including sepsis