Classification of red blood cell shapes in flow using outlier tolerant machine learning

The manual evaluation, classification and counting of biological objects demands for an enormous expenditure of time and subjective human input may be a source of error. Investigating the shape of red blood cells (RBCs) in microcapillary Poiseuille flow, we overcome this drawback by introducing a convolutional neural regression network for an automatic, outlier tolerant shape classification. From our experiments we expect two stable geometries: the so-called `slipper' and `croissant' shapes depending on the prevailing flow conditions and the cell-intrinsic parameters. Whereas croissants mostly occur at low shear rates, slippers evolve at higher flow velocities. With our method, we are able to find the transition point between both `phases' of stable shapes which is of high interest to ensuing theoretical studies and numerical simulations. Using statistically based thresholds, from our data, we obtain so-called phase diagrams which are compared to manual evaluations. Prospectively, our concept allows us to perform objective analyses of measurements for a variety of flow conditions and to receive comparable results. Moreover, the proposed procedure enables unbiased studies on the influence of drugs on flow properties of single RBCs and the resulting macroscopic change of the flow behavior of whole blood.Comment: 15 pages, published in PLoS Comput Biol, open acces

Rare Anemias: Are Their Names Just Smoke and Mirrors?

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Transitions in frailty state after kidney transplantation

Purpose: Frailty is the body’s failure to return to homeostasis after every day or acute stressful events, causing adverse outcomes. To study its dynamics in kidney transplant recipients (KTR), we determined whether the degree of frailty and its domains are affected by kidney transplantation (KT). Methods: Between 2015 and 2017, 176 KTR were included. Frailty scores were measured using the Groningen Frailty Indicator (GFI), assessed preoperatively and during follow-up. Transitions in frailty state and changes in the individual domains were determined. Results: Mean age (±SD) was 51.8 (± 14.1) years, and 63.1% of KTR were male. Thirty patients were considered frail (GFI ≥ 4) at baseline. After a mean follow-up of 22.8 ± 8.3 months, 34 non-frail patients (19.3%) became frail, 125 patients (71.0%) remained the same, and 17 frail patients (9.7%) became non-frail (GFI < 4). In the domain psychosocial functioning, 28.4% of the patients had an increase in GFI score after follow-up. Patients who scored a point in the domain cognition at baseline had a greater chance of becoming frail (OR 4.38, 95% CI 0.59–32.24). Conclusion: In conclusion, almost one-fifth of non-frail KTR transitioned to a frail state after their transplantation. These results could be used to predict the impact of KT on frailty course and help with implementing prehabilitation for patients at risk

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

PREhabilitation of CAndidates for REnal Transplantation (PreCareTx) study:protocol for a hybrid type I, mixed method, randomised controlled trial

INTRODUCTION: Kidney transplant candidates (KTCs) need to be in optimal physical and psychological condition prior to surgery. However, KTCs often experience compromised functional capacity which can be characterised as frailty. Prehabilitation, the enhancement of a person's functional capacity, may be an effective intervention to improve the health status of KTCs. The PREhabilitation of CAndidates for REnal Transplantation (PreCareTx) study aims to examine the effectiveness of a multimodal prehabilitation programme on the health status of KTCs, and to explore the potential of implementation of prehabilitation in daily clinical practice.METHODS AND ANALYSIS: This study uses a single centre, effectiveness-implementation hybrid type I study design, comprised of a randomised controlled trial and a mixed-methods study. Adult patients who are currently on the transplant waiting list or are waitlisted during the study period, at a university medical centre in The Netherlands, will be randomly assigned to either prehabilitation (n=64) or care as usual (n=64) groups. The prehabilitation group will undergo a 12-week home-based, tailored prehabilitation programme consisting of physical and/or nutritional and/or psychosocial interventions depending on the participant's deficits. This programme will be followed by a 12-week maintenance programme in order to enhance the incorporation of the interventions into daily life. The primary endpoint of this study is a change in frailty status as a proxy for health status. Secondary endpoints include changes in physical fitness, nutritional status, psychological well-being, quality of life and clinical outcomes. Tertiary endpoints include the safety, feasibility and acceptability of the prehabilitation programme, and the barriers and facilitators for further implementation.ETHICS AND DISSEMINATION: Medical ethical approval was granted by the Medical Ethics Committee Groningen, Netherlands (M22.421). Written informed consent will be obtained from all participants. The results will be disseminated at international conferences and in peer-reviewed journals.TRIAL REGISTRATION NUMBER: ClinicalTrials.gov, NCT05489432.</p

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

The Erythrocyte Sedimentation Rate and Its Relation to Cell Shape and Rigidity of Red Blood Cells from Chorea-Acanthocytosis Patients in an Off-Label Treatment with Dasatinib

Background: Chorea-acanthocytosis (ChAc) is a rare hereditary neurodegenerative disease with deformed red blood cells (RBCs), so-called acanthocytes, as a typical marker of the disease. Erythrocyte sedimentation rate (ESR) was recently proposed as a diagnostic biomarker. To date, there is no treatment option for affected patients, but promising therapy candidates, such as dasatinib, a Lyn-kinase inhibitor, have been identified. Methods: RBCs of two ChAc patients during and after dasatinib treatment were characterized by the ESR, clinical hematology parameters and the 3D shape classification in stasis based on an artificial neural network. Furthermore, mathematical modeling was performed to understand the contribution of cell morphology and cell rigidity to the ESR. Microfluidic measurements were used to compare the RBC rigidity between ChAc patients and healthy controls. Results: The mechano-morphological characterization of RBCs from two ChAc patients in an off-label treatment with dasatinib revealed differences in the ESR and the acanthocyte count during and after the treatment period, which could not directly be related to each other. Clinical hematology parameters were in the normal range. Mathematical modeling indicated that RBC rigidity is more important for delayed ESR than cell shape. Microfluidic experiments confirmed a higher rigidity in the normocytes of ChAc patients compared to healthy controls. Conclusions: The results increase our understanding of the role of acanthocytes and their associated properties in the ESR, but the data are too sparse to answer the question of whether the ESR is a suitable biomarker for treatment success, whereas a correlation between hematological and neuronal phenotype is still subject to verification

Singlet-Oxygen Generation by Peroxidases and Peroxygenases for Chemoenzymatic Synthesis

Singlet oxygen is a reactive oxygen species undesired in living cells but a rare and valuable reagent in chemical synthesis. We present a fluorescence spectroscopic analysis of the singlet-oxygen formation activity of commercial peroxidases and novel peroxygenases. Singlet-oxygen sensor green (SOSG) is used as fluorogenic singlet oxygen trap. Establishing a kinetic model for the reaction cascade to the fluorescent SOSG endoperoxide permits a kinetic analysis of enzymatic singlet-oxygen formation. All peroxidases and peroxygenases show singlet-oxygen formation. No singlet oxygen activity could be found for any catalase under investigation. Substrate inhibition is observed for all reactive enzymes. The commercial dye-decolorizing peroxidase industrially used for dairy bleaching shows the highest singlet-oxygen activity and the lowest inhibition. This enzyme was immobilized on a textile carrier and successfully applied for a chemical synthesis. Here, ascaridole was synthesized via enzymatically produced singlet oxygen. © 2020 Wiley-VCH Gmb