Method and device for determining red blood cells deformability

A method for in vitro determination of variation of deformability of red blood cells of a tested individual, the method comprising the steps of:

• a) determining a first amount of red blood cells having a tank-treading motion in a population of red blood cells, said population of red blood cells being obtained from a sample of said tested individual, by performing the following steps:
• i. subjecting the population of red blood cells to a flow within a circulation member having a boundary wall, the flow having a flow gradient and the boundary wall being perpendicular to the direction of the flow gradient, the flow having a direction and further having a wall shear rate from 1 s−1 to 50 s−1 and a viscosity from 10−3 Pa·s to 10−1 Pa·s,
• ii. recording in greyscale images, using a visualisation member connected to a recording member, motions and trajectories of at least a part of the red blood cell population in an observation field of the visualisation member and obtaining at least one video being a sequence of successive images of movement of the recorded red blood cells across the observation field, wherein the observation field is arranged perpendicularly to the direction of the flow gradient with a focus at a distance h where the flow has constant shear rate, wherein the images of the sequence comprise projected shapes of the red blood cells in a plane perpendicular to the direction of the flow gradient and parallel to the boundary wall of the circulation member, wherein the projected shapes of each red blood cell present a minor and a major axis,
• iii. individually tracking in all the images of the sequence of successive images the projected shapes relating to at least a part of the recorded red blood cells, obtaining a motion sequence of individual images for each red blood cell and determining for each projected shape of a particular red blood cell a grey level intensity profile from the greyscale images,
• iv. analysing evolution of the projected shapes of each tracked red blood cell in its motion sequence and classifying a motion type of the said each tracked red blood cell as follows:
• (1) a red blood cell is classified as having a tank-treading motion on conditions that a ratio of the minor to major axis of its projected shape is greater than 0.45 in all images of its individual motion sequence and that the grey level intensity profile in at least 90% of its projected shapes has two extrema separated by at least 1 μm,
• (2) a red blood cell is classified as having another motion on condition that the ratio of the minor to major axis of its projected shape is less than 0.45 in at least two images in the sequence,
• v. determining the first amount as a ratio of the number of red blood cells classified as having a tank-treading motion to a sum of the number of red blood cells classified as having a tank-treading motion and the number of red blood cells classified as having another motion,
• wherein steps ii. to v. are implemented by a control module and performed automatically,
• b) calculating an absolute value of a difference between
• (1) said first amount of red blood cells having a tank-treading motion in a population of red blood cells, and
• (2) a second amount being a calculated mean of the determined amount of red blood cells having a tank-treading motion obtained in reference populations of red blood cells, said calculated mean of the determined amount being obtained along with a value of a standard deviation of the red blood cells having a tank-treading motion obtained in the reference populations of red blood cells,
• wherein said reference populations are obtained either from at least two independent samples of said tested individual, or from at least one sample of at least two individuals, said at least two individuals being different from said tested individual,
• c) determining said absolute value is greater than twice the value of said standard deviation
• d) concluding the red blood cells of said population present a significant variation of their deformability compared to the red blood cells of said reference populations.