Changes in Blood Cell Deformability in Chorea-Acanthocytosis and Effects of Treatment With Dasatinib or Lithium

Misshaped red blood cells (RBCs), characterized by thorn-like protrusions known as acanthocytes, are a key diagnostic feature in Chorea-Acanthocytosis (ChAc), a rare neurodegenerative disorder. The altered RBC morphology likely influences their biomechanical properties which are crucial for the cells to pass the microvasculature. Here, we investigated blood cell deformability of five ChAc patients compared to healthy controls during up to 1-year individual off-label treatment with the tyrosine kinase inhibitor dasatinib or several weeks with lithium. Measurements with two microfluidic techniques allowed us to assess RBC deformability under different shear stresses. Furthermore, we characterized leukocyte stiffness at high shear stresses. The results showed that blood cell deformability–including both RBCs and leukocytes - in general was altered in ChAc patients compared to healthy donors. Therefore, this study shows for the first time an impairment of leukocyte properties in ChAc. During treatment with dasatinib or lithium, we observed alterations in RBC deformability and a stiffness increase for leukocytes. The hematological phenotype of ChAc patients hinted at a reorganization of the cytoskeleton in blood cells which partly explains the altered mechanical properties observed here. These findings highlight the need for a systematic assessment of the contribution of impaired blood cell mechanics to the clinical manifestation of ChAc.


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Supplementary Information

RT-FDC gating strategies
Lymphocytes and myelocytes are distinguished by size like shown in figure S5A. In RT-FDC leucocyte measurements, red blood cells are not used in the analysis and filtered out in a first step by only allowing cells with an aspect ratio (x-size/y-size) smaller than 2. For ChAc patients, a lot of RBCs were found in the deformation-size region described for leucocytes. To exclude these events from the analysis, additionally all measurements were gated for brightness values within the contour, fitting for lymphocytes or myelocytes respectively depicted for one exemplary measurement in figure S5B (for a detailed explanation of the gating strategy, see Töpfner et al., 2018 (1)). Additionally, the contours are checked for convexity and an event is only considered in the WBC analysis if the area of the convex hull of the contour deviates by a maximum of 5% from the area of the raw contour. For RBC measurements a deviation by 10% was allowed and events were filtered in a size range of 20-50 µm 2 .

Shape Analysis of in-vitro Dasatinib treated RBCs
Blood samples were obtained via venipuncture and collecting the blood in vacutainers containing the anticoagulant heparin. An amount of approximately 100 µl was suspended in 1ml phosphate buffered saline (PBS) and centrifuged at 1500 g for 5 min. After this centrifugation, the supernatant was discarded and the residual pellet of RBCs is resuspended in PBS. This process is repeated three times in total, leaving a dense pellet of RBCs devoid of blood plasma and other cellular constituents. To obtain the final blood solution used in the microfluidic experiments, a volume of 10 µl of these RBCs was filled to 1 ml total volume with a solution of PBS and 1 mg/ml bovine serum albumin (BSA), yielding a hematocrit of 1%.
For the Dasatinib measurements, a stock solution of 0.5 mg Dasatinib per 1 ml dimethyl sulfoxide (DMSO), yielding a concentration of ca. 1 µmol/l, was created. From this stock solution, an additional amount of 10 µl was then added to the formerly described final blood solution. In addition, the RBCs were incubated in 10 µl of the previously described stock solution, filled to 1ml total volume with PBS, at room temperature for at least 3 h prior the actual measurement.
By applying a set of discrete pressure drops in a regime up to 700 mbar to a connected Eppendorf tube containing the final blood solution, we advected this solution through microfluidic channels with a cross-section of (w x h) 12 µm x 10 µm, and a length of ca. 4 cm.
The flowing RBCs were recorded with a brightfield microscope equipped with an oil-immersion objective (Nikon Plan Apo 60x) at a distance 10 mm away from the channel entrance to neglect transient cell shapes (cf. references (2,3) for more information). With the aid of a custom-tailored particle tracking algorithm, we extract cropped images of single flowing RBCs together with their individual velocities and centroid positions.
Cells flowing due to a distinct pressure drop are grouped and from this set the mean velocity was obtained. Analogously, cells associated to one distinct pressure drop were manually categorized into a binary scheme of healthy shapes (hs) and acanthocytes (ac).
The results are shown in main text figure 2F. It can be seen that the number of acanthocytes decreases with the flow velocity. This is because the typical morphologic acanthocyte features can vanish with cell deformation. There was no significant difference between the control and Dasatinib-treated sample.

RBC treatment with glutaraldehyde and diamide
To see how the deformation of RBCs changes when artificially altering the cells' material properties, we incubated them with different concentrations of glutaraldehyde (0.0001-0.005 v%; Sigma-Aldrich, Cat# G6257) or diamide (0.1-10 mM, Sigma-Aldrich, Cat# D3648) for 20 min in the measurement buffer before measuring. While glutaraldehyde crosslinks proteins throughout the whole cell, diamide specifically targets the spectrin network at the RBC cytoskeleton. The results for the measured projected areas and deformations are given in figure S3.
For glutaraldehyde, cells decrease in size for concentrations up to 0.001 v% with remaining or slightly increasing deformation values. At the concentration of 0.0025 v% most cells are fixed in shape, which leads to two populations for the measured size (dependent on the direction of the cell when the image is taken) and also for the deformation, while deformation values clearly drop at these concentrations. This highlights that a stiffening of RBCs not necessarily leads to a decrease of the deformation value in RT-FDC and even the opposite can be observed.