Methods for evaluating the degree of aging of red blood cells

Abstract

The object of the present invention is to provide a method for evaluating the degree of aging of red blood cells, which can evaluate the degree of aging of red blood cells more accurately and appropriately. The solution is a method for evaluating the degree of aging of red blood cells, which comprises the following steps: a step of preparing a red blood cell suspension (S1), preparing a red blood cell suspension from a blood sample; a step of calculating the first red blood cell deformability (S2), passing the red blood cell suspension through a first filter, separating it into aged red blood cells that have not passed through the first filter and unaged red blood cells that have passed through the first filter, and calculating the deformability of the red blood cells; a step of calculating the second red blood cell deformability (S3), passing the separated unaged red blood cell suspension through a second filter having micropores smaller in diameter than the first filter, separating it into slightly aged red blood cells that have not passed through the second filter and immature red blood cells that have passed through the second filter, and calculating the deformability of the unaged red blood cells; and an evaluation step (S4), evaluating the degree of aging of red blood cells using the deformability of red blood cells and the deformability of unaged red blood cells.

Description

Technical Field

[0001] The present invention relates to a method for evaluating the degree of aging of red blood cells in blood that causes capillary ghosting. Background Art

[0002] In recent years, the use of capillaroscopy, which has also been introduced in the media, allows the observation of capillaries that are usually invisible, and the discovery of ghostly capillaries such as disappearance, shortening, and reduction in number of capillaries. Ghostly capillaries occur throughout the body, and therefore not only affect visible skin conditions such as wrinkles and sagging, but also have the risk of causing serious diseases such as osteoporosis, dementia, and lifestyle diseases (diabetes, hypercholesterolemia, etc.).

[0003] Red blood cells are important for the process of capillary flow deterioration and development into ghosting. This is because there is no smooth muscle in the capillaries, so there is no contraction and relaxation function. Red blood cells circulate throughout the body through capillaries (about 5 μm) thinner than their own diameter (about 8 μm) in a deformed state. The number of red blood cells per 1 μL of blood is 4 to 5 million, and the volume of red blood cells also accounts for 40% to 50% of the blood volume. The life span of these red blood cells is short, about 120 days, and there are red blood cells in various stages from young red blood cells to aged red blood cells. Among these stages, the most relevant to the ghosting of capillaries is the aged red blood cells with reduced deformability. It is believed that the aged red blood cells are blocked in the capillaries, and the substances necessary for cell survival cannot be delivered to the cells of the capillaries after the blockage is formed, causing the ghosting to develop.

[0004] In recent years, methods and devices for evaluating the deformability of red blood cells in blood have been proposed.

[0005] An example of a device developed in Japan is the MCFAN HR300 (Micro Channel Array Flow Analyzer, Japan, MC Healthcare, Inc.). MCFAN is a device that allows collected blood to flow through artificial capillary analogs made of silicon and observe images. In the early stages of development, it was praised as a great contribution to the study of red blood cell deformability, but it was later found to have low reliability and is now only used in a very limited number of hospitals.

[0006] In addition, as devices developed outside Japan, there are LORCA (Laser-assisted Optical Rotational Cell Analyzer, Mechatronics, Netherlands), which applies centrifugal stress through rotational flow to cause elliptical deformation of red blood cells and evaluates it with laser diffraction images, and RheoScan-D (RheoMeditech, South Korea), which applies shear stress through negative pressure to cause elliptical deformation of red blood cells and evaluates it with laser diffraction images (Non-Patent Document 1). RheoScan-D has the following features: it can automatically measure with whole blood, it does not need to be cleaned after use because a disposable plastic microchip is used in the flow path, and it can also measure the aggregation of red blood cells; etc.

[0007] However, the physiological deformation of red blood cells in the body is bending deformation, and one point that needs to be noted is whether the method of measuring the deformability of red blood cells by elliptical deformation can be considered to reflect the situation of physiological microcirculation. In addition, since the stress required for elliptical deformation is much greater than the stress required for bending deformation, there is a problem that the measurement sensitivity is low when a small shear stress is used, especially compared with the method of measuring bending deformation.

[0008] Prior art literature

[0009] Non-patent literature

[0010] Non-patent document 1: Patricia C. Sousa, Fernando T. Pinho, Manuel A. Alves and Monica S. N. Oliveira, A review of hemorheology: Measuring techniques and recent advances, Korea-Australia Rheology J, 28(1),1-22(2016) Summary of the invention

[0011] Problems to be solved by the invention

[0012] The conventional evaluation method is to evaluate the aging degree of red blood cells by using the deformability obtained from red blood cells at various stages of their life span of about 120 days as a whole, but a device capable of obtaining the deformability of aged red blood cells alone or the deformability of a portion after the aged red blood cells have been removed, or a device that automates the above-mentioned device, has not yet been developed.

[0013] The proportion of aged red blood cells that cause capillary ghosting is different for each patient. Therefore, it can be foreseen that if such an evaluation method and device can be developed, it will be possible to make appropriate diagnosis of the risk of progression of complications such as diabetic retinopathy and nephropathy in each patient, which will be of great clinical significance.

[0014] An object of the present invention is to provide a method for evaluating the degree of aging of erythrocytes which can more accurately and appropriately evaluate the degree of aging of erythrocytes.

[0015] Means for solving problems

[0016] The rheological properties of red blood cells such as flexibility and ease of flow are very important for the prevention and treatment of lifestyle diseases. With this in mind, the inventors of the present application developed a red blood cell deformability measuring device using a gravity-type nickel mesh filtration method, providing a red blood cell deformability test.

[0017] Using the gravity nickel mesh filtration method (the diameter of the pores of the filter is 3.2 μm), 139 subjects undergoing physical examinations were selected and only mild hyperlipidemia (total cholesterol value below 260 mg / dl) was studied. The results showed that red blood cell deformability was negatively correlated with neutral fat value and positively correlated with HDL cholesterol value (Ejima J, Ijichi T, Ohnishi Y, Maruyama T, Kaji Y, Kanaya S, Fujino T, Uyesaka N and Ohmura T: Relationship of high-density lipoprotein cholesterol and red blood cell filterability: cross-sectional study of healthy subjects. Clin Hemorheol Microcirc 22: 1-7, 2000).

[0018] In addition, a study was conducted on 101 patients with hypertension using a filter with a pore diameter of 4.94 μm, and it was found that erythrocyte deformability was negatively correlated with mean blood pressure (K. Odashiro et al., Impaired deformability of circulating erythrocytes obtained from nondiabetichypertensive patients: investigation by a nickel mesh filtration technique, Clin Hypertens. 21: 17, eCollection (2015)).

[0019] The inventors of the present application conducted further in-depth research in order to evaluate the degree of aging of red blood cells in the blood with higher accuracy and appropriateness. As a result, they found that by separating the red blood cells multiple times, calculating the deformability of the red blood cells in each separation step and using this to evaluate the degree of aging of the red blood cells, it is possible to evaluate the degree of aging of the red blood cells and, in turn, the ghosting of capillaries with higher accuracy and appropriateness, thereby completing the present invention.

[0020] In other words, the present invention is as follows.

[0021] [1] A method for evaluating the degree of aging of red blood cells, which is a method for evaluating the degree of aging of red blood cells using at least two filters having multiple micropores, characterized in that it comprises the following steps:

[0022] A red blood cell suspension preparation step, preparing a red blood cell suspension from a blood sample;

[0023] a first red blood cell deformability calculation step of passing the red blood cell suspension through a first filter to separate the red blood cells into aged red blood cells that have not passed through the first filter and unaged red blood cells that have passed through the first filter, and calculating the deformability of the red blood cells contained in the red blood cell suspension;

[0024] a second erythrocyte deformability calculation step, wherein the separated unaged erythrocyte suspension is passed through a second filter having pores smaller in diameter than the first filter, to separate the separated unaged erythrocytes into slightly aged erythrocytes that have not passed through the second filter and immature erythrocytes that have passed through the second filter, and the deformability of the unaged erythrocytes contained in the unaged erythrocyte suspension is calculated; and

[0025] The evaluation step evaluates the degree of aging of the erythrocytes using the deformability of the erythrocytes calculated in the first erythrocyte deformability calculation step and the deformability of the non-aged erythrocytes calculated in the second erythrocyte deformability calculation step.

[0026] [2] The method for evaluating the degree of aging of red blood cells as described in [1] above is characterized in that, in the first red blood cell deformability calculation step and / or the second red blood cell deformability calculation step, the passing ratio of red blood cells is further calculated and the passing ratio is used in the evaluation of the evaluation step.

[0027] [3] The method for evaluating the degree of aging of red blood cells as described in [1] or [2] above, wherein the diameter of the pores of the first filter is 5.50 to 8.00 μm.

[0028] [4] The method for evaluating the degree of erythrocyte aging according to any one of [1] to [3] above, wherein the diameter of the pores of the second filter is 3.00 to 6.00 μm.

[0029] Effects of the Invention

[0030] The method for evaluating the degree of aging of erythrocytes of the present invention can more accurately and appropriately evaluate the degree of aging of erythrocytes, and thus can accurately detect the possibility of ghosting of capillaries, and can be applied to the diagnosis of various diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] [ Figure 1 ] is a flow chart of a method for evaluating the degree of red blood cell aging according to one embodiment of the present invention.

[0032] [ Figure 2 ] is a schematic illustration of a device used in a method for evaluating the degree of red blood cell aging according to one embodiment of the present invention.

[0033] [ Figure 3 ] is to show that Figure 2 A graph of a "height-time curve" obtained as a result of measurement using the device shown.

[0034] [ Figure 4 ] is a graph showing the deformability of red blood cells when passing through a filter when using the multi-stage method of the present invention (Example 1).

[0035] [ Figure 5 ] is a graph showing the deformability of red blood cells when passing through a filter using a conventional single-stage method.

[0036] [ Figure 6 ] is a graph showing the number (ratio) of red blood cells that have passed through the filter when the multi-stage method of the present invention is used (Example 1).

[0037] [ Figure 7 ] is a graph showing the number (ratio) of red blood cells that passed through the filter when using the conventional single-stage method. DETAILED DESCRIPTION

[0038] The method for evaluating the degree of aging of red blood cells of the present invention is a method for evaluating the degree of aging of red blood cells using at least two filters having a plurality of uniform micropores, and is characterized in that it comprises the following steps: a step of preparing a red blood cell suspension, preparing a red blood cell suspension from a blood sample; a first red blood cell deformability calculation step, allowing the red blood cell suspension to pass through a first filter, separating it into aged red blood cells that have not passed through the first filter and unaged red blood cells that have passed through the first filter, and calculating the deformability of the red blood cells contained in the red blood cell suspension; a second red blood cell deformability calculation step, allowing the separated unaged red blood cell suspension to pass through a second filter having micropores smaller in diameter than the first filter, separating it into slightly aged red blood cells that have not passed through the second filter and immature red blood cells that have passed through the second filter, and calculating the deformability of the unaged red blood cells contained in the unaged red blood cell suspension; and an evaluation step, using the deformability of the red blood cells calculated in the first red blood cell deformability calculation step and the deformability of the unaged red blood cells calculated in the second red blood cell deformability calculation step, to evaluate the degree of aging of the red blood cells.

[0039] The method for evaluating the degree of erythrocyte aging of the present invention can be evaluated using the erythrocyte deformability measuring device described in "Maruyama Tetsu, Okamoto Kazuhiko, Quantitative analysis of erythrocyte deformability based on nickel mesh filtration method (Quantitative analysis of erythrocyte deformability by nickel mesh filtration method), Fukuoka Medical Journal, 95 (6), 131-138 (2004)".

[0040] In the method for evaluating the degree of aging of red blood cells of the present invention, it is preferred that the passing ratio of red blood cells is calculated in the first red blood cell deformability calculation step and / or the second red blood cell deformability calculation step, and the passing ratio is used in the evaluation in the evaluation step. Thus, the degree of aging of red blood cells can be evaluated more accurately.

[0041] In addition, the method of the present invention may further include a third red blood cell deformability calculation step using a third filter, a fourth red blood cell deformability calculation step using a fourth filter, and the like, and the deformability of red blood cells calculated in these steps may be evaluated by using a ratio.

[0042] Next, the method for evaluating the degree of aging of red blood cells of the present invention will be described in detail.

[0043] like Figure 1 As shown, a method for evaluating the degree of red blood cell aging according to one embodiment of the present invention sequentially comprises a red blood cell suspension preparation step (S1), a first red blood cell deformability calculation step (S2), a second red blood cell deformability calculation step (S3) and an evaluation step (S4).

[0044] <Red blood cell suspension preparation steps>

[0045] The red blood cell suspension preparation step (S1) is a step of preparing a red blood cell suspension from a blood sample, for example, a step of washing a blood sample collected from a subject to prepare a red blood cell suspension. Specifically, for example, the collected blood is first centrifuged and washed with a buffer solution repeatedly, and then diluted with a buffer solution so that the hematocrit (HCT) becomes a predetermined concentration to prepare a red blood cell suspension.

[0046] <1st red blood cell deformability calculation step>

[0047] The first red blood cell deformability calculation step (S2) is a step of passing the red blood cell suspension prepared in the red blood cell suspension preparation step (S1) through the first filter to separate it into aged red blood cells that have not passed through the first filter and unaged red blood cells that have passed through the first filter, and calculating the deformability of the red blood cells contained in the red blood cell suspension.

[0048] As the first filter used in the present invention, in order to ensure high quantitativeness and reproducibility, a filter having uniform shape, number and distribution of micropores is preferred, and for example, a nickel mesh filter manufactured by combining a photoresist method and a special plating method can be cited. It should be noted that the structure of the first filter is preferably a structure that is difficult to apply mechanical stimulation to white blood cells mixed in when preparing a red blood cell suspension.

[0049] The diameter of the pores of the first filter can be appropriately changed according to the condition of the subject, but is usually preferably 5.50 to 8.00 μm, more preferably 5.60 to 7.00 μm, and even more preferably 5.70 to 6.50 μm.

[0050] In this step, the deformability of the red blood cells is calculated. This deformability is an index showing the ability of the red blood cells (unaged red blood cells) contained in the red blood cell suspension to pass through the first filter, and various values ​​calculated by the so-called filtration method can be used, such as the pressure difference when the red blood cell suspension passes through the micropores of the filter, the passage time of a predetermined amount of the red blood cell suspension, and the flow rate (flow rate: Q) of the red blood cells.

[0051] As a method for calculating the deformation capacity of the present invention, specifically, for example, Figure 2 The calculation is performed using the device shown in FIG. Figure 2 As shown, a nickel mesh filter 2 (e.g., a pore diameter of 6.0 μm) is installed on a vertically erected glass tube 1 via a Tygon tube, and the red blood cell suspension is filtered from a predetermined height (e.g., 15 cm). By continuously measuring the pressure at this time, the height ( Figure 2 h)-time curve (see Figure 3). The height-time curve of the buffer solution without red blood cells obtained in the same manner is compared, and the time taken to descend to a predetermined height (eg, 10 cm) is compared to quantify the deformability.

[0052] In this step, it is preferred to further calculate the passing ratio of red blood cells, that is, the ratio of aged red blood cells that have not passed through the first filter to the non-aged red blood cells that have passed through the first filter. By using this passing ratio in the evaluation step, a more accurate evaluation can be performed. The passing ratio can be calculated by measuring at least two of the total number of red blood cells, the number of aged red blood cells, and the number of non-aged red blood cells contained in the red blood cell suspension using a conventionally known blood cell analyzer or the like.

[0053] <Second red blood cell deformability calculation step>

[0054] The second red blood cell deformability calculation step (S3) is a step of passing the unaged red blood cell suspension separated in the first red blood cell deformability calculation step (S2) through a second filter having pores with a smaller diameter than that of the first filter, separating the unaged red blood cells into slightly aged red blood cells that have not passed through the second filter and immature red blood cells that have passed through the second filter, and calculating the deformability of the unaged red blood cells contained in the unaged red blood cell suspension.

[0055] The unaged erythrocyte suspension used in this step may be the one separated in the first erythrocyte deformability calculation step (S2) as it is, or may be diluted with a buffer solution so that the hematocrit (HCT) becomes a predetermined concentration and used.

[0056] The processing of this step is basically the same as that of the first red blood cell deformability calculation step (S2), but the filter used is different. That is, in this step, a second filter having micropores with a diameter smaller than that of the first filter is used. The diameter of the micropores of the second filter can be appropriately changed according to the result of the first red blood cell deformability calculation step (S2), but usually, it is preferably 3.00 to 6.00 μm, more preferably 3.50 to 5.80 μm, further preferably 4.00 to 5.50 μm, and particularly preferably 4.50 to 5.50 μm. In addition, the difference in diameter from the micropores of the first filter is preferably 0.1 to 2.0 μm, more preferably 0.3 to 1.5 μm, and further preferably 0.5 to 1.0 μm.

[0057] <Evaluation Steps>

[0058] The evaluation step (S4) is a step of evaluating the aging degree of the red blood cells using the deformability of the red blood cells calculated in the first red blood cell deformability calculation step and the deformability of the non-aged red blood cells calculated in the second red blood cell deformability calculation step. In this step, it is preferred that the aging of the red blood cells is evaluated by using the passing ratio of the red blood cells calculated in the first red blood cell deformability calculation step and / or the second red blood cell deformability calculation step in addition to the deformability of the red blood cells and the non-aged red blood cells. This allows for a more accurate evaluation.

[0059] Specifically, in this step, when the deformability is low, the red blood cells are evaluated as aged, and the degree of aging of the red blood cells is evaluated by simultaneously or additionally considering the evaluation based on the passing ratio (the smaller the passing ratio, the older the red blood cells). As a result, the possibility of capillary ghosting can be accurately detected, and it can be applied to the diagnosis of skin conditions such as wrinkles and sagging, osteoporosis, dementia, lifestyle diseases (diabetes, hypercholesterolemia, etc.), etc., making it possible to detect diseases at an early stage.

[0060] In particular, the method for evaluating the degree of aging of red blood cells of the present invention utilizes the deformability (and the passing ratio) in at least two separation steps for evaluation, and thus can evaluate the degree of aging of red blood cells more accurately than before. In addition, by changing the combination of the size of the micropores of the filter according to the condition of the subject (age, blood pressure, diseases, chronic diseases, etc.), the evaluation can be performed more appropriately.

[0061] Example

[0062] The present invention is specifically described below by way of examples. It should be noted that the present invention is not limited to these examples. The basic operation of this example is briefly described as follows: Figure 1 shown.

[0063] [Basic Operation]

[0064] (Steps for preparing red blood cell suspension)

[0065] First, 30cc of blood collected from the subject was centrifuged at 2500rpm for 10 minutes using a centrifuge and washed with a buffer. Then, the speed was changed to 1950rpm, 1700rpm, and 1550rpm in sequence, and centrifugation (10 minutes each) and washing with a buffer were repeated to obtain washed red blood cells. The washed red blood cells obtained were diluted with a buffer to prepare a red blood cell suspension with a hematocrit (HCT) of 3%. The number of red blood cells per 1μL of red blood cell suspension was measured using a blood cell analyzer.

[0066] (1st step of calculating red blood cell deformability (stage 1))

[0067] Use as Figure 2 The measuring device shown is equipped with a 6.00 μm nickel mesh filter. The red blood cell suspension is fed into the glass tube by a liquid feeding pump to measure the deformability. In addition, the number of red blood cells per 1 μL of unaged red blood cells that have passed through the 6.00 μm nickel mesh filter is measured by a blood cell analyzer.

[0068] (Second red blood cell deformability calculation step (stage 2))

[0069] The unaged red blood cell suspension was diluted with a buffer to prepare an unaged red blood cell suspension. After the inside of the measuring device was washed with a buffer, the 6.00 μm nickel mesh filter was replaced with a 5.31 μm nickel mesh filter. The unaged red blood cell suspension was sent into a glass tube using a liquid delivery pump to measure the deformability. In addition, the number of red blood cells per 1 μL of the immature red blood cell suspension that passed through the 5.31 μm nickel mesh filter was measured using a blood cell analyzer.

[0070] (Evaluation Steps)

[0071] The degree of blood aging was evaluated using the obtained deformability and red blood cell count.

[0072] [Example 1]

[0073] The following is an example of evaluating the degree of blood aging using blood collected from real people according to the above basic operation.

[0074] By the method described in the above-mentioned erythrocyte suspension preparation step, an erythrocyte suspension (sample solution) with HCT 3% was prepared from blood collected from a human. The number of erythrocytes in the sample solution at this time was 32×10 4 Pieces / μl.

[0075] In addition, as a comparison, a comparative sample solution (red blood cell count 32×10 4 / μl).

[0076] Deformability and red blood cell count were determined by the method described in the first red blood cell deformability calculation step (stage 1) using a 6.00 μm nickel mesh filter. Figure 4 As shown in the left figures, the deformation capacity of the sample liquid in stage 1 is 93%, and the deformation capacity of the comparative sample is 90%. Figure 6 As shown in the left figures, the number of red blood cells in the sample solution that has passed through the filter in stage 1 is 26×10 4The number of red blood cells in the comparative sample that passed through the filter was 26×10 4 / μl (pass rate: 81%).

[0077] It should be noted that the deformability (%) of erythrocytes was determined as follows.

[0078] The red blood cell suspension (sample solution or comparison sample solution) was passed through a nickel mesh filter from a height of 15 cm, and the pressure change during the passage was continuously detected to obtain a height-time curve. The height-time curve of a buffer solution without red blood cells was used as a control to evaluate the deformability of red blood cells. The deformability at the time point when it dropped to 10 cm was compared with the control and quantified.

[0079] The red blood cell counts of the sample solution and the comparative sample solution after the first red blood cell deformability calculation step (stage 1) were adjusted to 9×10 4 In addition, the deformability and the number of erythrocytes were measured by the method in the second erythrocyte deformability calculation step (stage 2) using a 5.31 μm nickel mesh filter.

[0080] like Figure 4 As shown in the right figures, the deformation capacity of the sample liquid in stage 2 is 95%, and the deformation capacity of the comparison sample is 66%, showing a large difference of nearly 30%. Figure 6 As shown in the right figures, the number of red blood cells in the sample solution that passed through the filter in stage 2 was 8×10 4 The number of red blood cells in the comparative sample that passed through the filter was 7×10 4 pcs / μl (pass rate: 78%), and here too there was a large difference of about 11%.

[0081] On the other hand, Figure 5 As shown in the figure, in the conventional single-stage method using only a 5.31 μm nickel mesh filter (the number of red blood cells in the sample solution was 34×10 4 / μl), the deformation capacity of the sample liquid is 89%, and the deformation capacity of the comparison sample is 80%, and the difference is a small value of less than 10%. Figure 7 As shown in the figure, the number of red blood cells in the sample solution that passed through the filter was 25×10 4 The number of red blood cells in the comparative sample that passed through the filter was 26×10 4 pcs / μl (pass rate: 76%), and the difference is almost zero (in contrast, it is more in the case of the comparative sample solution).

[0082] As described above, the multi-stage method of the present invention can accurately grasp the state of red blood cells, enable accurate evaluation, and more finely classify the good and the bad.

[0083] Industrial Applicability

[0084] The method for evaluating the degree of aging of erythrocytes of the present invention can evaluate the degree of aging of erythrocytes and is therefore industrially useful.

[0085] Description of Reference Numerals

[0086] 1 Glass tube

[0087] 2 Nickel mesh filter

[0088] 3 thermostatic water tank

Claims

1. A method for evaluating the degree of aging of red blood cells, which is a method for evaluating the degree of aging of red blood cells using at least two filters having multiple micropores, characterized in that: The following steps are involved: a red blood cell suspension preparation step, preparing a red blood cell suspension having a predetermined hematocrit concentration from a blood sample; a first red blood cell deformability calculation step of passing the red blood cell suspension through a first filter having pores with a diameter of 5.50 to 8.00 μm to separate the red blood cells into aged red blood cells that have not passed through the first filter and unaged red blood cells that have passed through the first filter, and calculating a first deformability as an index indicating the ability of the red blood cells contained in the red blood cell suspension to pass through the first filter; a second erythrocyte deformability calculation step of passing the unaged erythrocyte suspension that has passed through the first filter through a second filter having pores with a diameter smaller than that of the first filter and ranging from 3.00 to 6.00 μm, separating the unaged erythrocyte suspension into slightly aged erythrocytes that have not passed through the second filter and immature erythrocytes that have passed through the second filter, and calculating a second deformability as an index indicating the ability of the unaged erythrocytes contained in the unaged erythrocyte suspension to pass through the second filter; and The evaluation step evaluates the degree of aging of the red blood cells based on the deformability of the red blood cells contained in the red blood cell suspension calculated in the first red blood cell deformability calculation step and the deformability of the non-aged red blood cells calculated in the second red blood cell deformability calculation step.

2. The method for evaluating the degree of aging of red blood cells according to claim 1, wherein: In the first erythrocyte deformability calculation step and / or the second erythrocyte deformability calculation step, a passage ratio of erythrocytes is further calculated, and the passage ratio is used in the evaluation in the evaluation step.

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