(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 (R2=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.

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.

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.

The investigation of cell shapes mostly relies on the manual classification of 2D images, causing a subjective and time consuming evaluation based on a portion of the cell surface. We present a dual-stage neural network architecture for analyzing fine shape details from confocal microscopy recordings in 3D. The system, tested on red blood cells, uses training data from both healthy donors and patients with a congenital blood disease, namely hereditary spherocytosis. Characteristic shape features are revealed from the spherical harmonics spectrum of each cell and are automatically processed to create a reproducible and unbiased shape recognition and classification. The results show the relation between the particular genetic mutation causing the disease and the shape profile. With the obtained 3D phenotypes, we suggest our method for diagnostics and theragnostics of blood diseases. Besides the application employed in this study, our algorithms can be easily adapted for the 3D shape phenotyping of other cell types and extend their use to other applications, such as industrial automated 3D quality control.

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.