(19)
(11)EP 3 812 764 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
18.10.2023 Bulletin 2023/42

(21)Application number: 19819235.3

(22)Date of filing:  10.06.2019
(51)International Patent Classification (IPC): 
G01N 33/49(2006.01)
(52)Cooperative Patent Classification (CPC):
G01N 2015/0073; G01N 15/0618; G01N 33/4915
(86)International application number:
PCT/JP2019/022873
(87)International publication number:
WO 2019/240061 (19.12.2019 Gazette  2019/51)

(54)

METHOD FOR EVALUATING DEGREE OF AGING IN RED BLOOD CELLS

VERFAHREN ZUR BEWERTUNG DES ALTERUNGSGRADES IN ROTEN BLUTZELLEN

PROCÉDÉ D'ÉVALUATION DE DEGRÉ DE VIEILLISSEMENT DANS DES GLOBULES ROUGES


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 15.06.2018 JP 2018115021

(43)Date of publication of application:
28.04.2021 Bulletin 2021/17

(73)Proprietor: INSTITUTE OF RHEOLOGICAL FUNCTIONS OF FOOD
Kasuya-gun Fukuoka 8112501 (JP)

(72)Inventors:
  • FUJINO, Takehiko
    Fukuoka-shi, Fukuoka 813-0043 (JP)
  • MAWATARI, Shiro
    Fukuoka-shi, Fukuoka 813-0016 (JP)
  • KAWA, Tsunemichi
    Yokohama-shi, Kanagawa 222-0026 (JP)
  • KOYAMA, Tetsuji
    Yokohama-shi, Kanagawa 221-0866 (JP)
  • YAMASHITA, Toyoharu
    Fukuoka-shi, Fukuoka 812-0061 (JP)

(74)Representative: TBK 
Bavariaring 4-6
80336 München
80336 München (DE)


(56)References cited: : 
JP-A- H0 751 521
JP-A- 2005 241 378
JP-A- 2005 164 296
US-A1- 2009 285 892
  
  • Bosch F. H ET AL: "Determinants of red blood cell deformability in relation to cell age", European journal of haematology, 1 January 1994 (1994-01-01), pages 35-41, XP055812322, Oxford, UK DOI: 10.1111/j.1600-0609.1994.tb01282.x Retrieved from the Internet: URL:10.1111/j.1600-0609.1994.tb01282.x [retrieved on 2021-06-09]
  • NASH G.B. ET AL: "Red cell and ghost viscoelasticity. Effects of hemoglobin concentration and in vivo aging", BIOPHYSICAL JOURNAL, vol. 43, no. 1, 1 July 1983 (1983-07-01), pages 63-73, XP055812314, AMSTERDAM, NL ISSN: 0006-3495, DOI: 10.1016/S0006-3495(83)84324-0 Retrieved from the Internet: URL:https://pdf.sciencedirectassets.com/27 7708/1-s2.0-S0006349583X73590/1-s2.0-S0006 349583843240/main.pdf?X-Amz-Security-Token =IQoJb3JpZ2luX2VjENX//////////wEaCXVzLWVhc 3QtMSJHMEUCIFVw5bnPN4I14MnHYmVCLrkHz1Kav6c WpixEyBJeK1C+AiEAorPYjclKmPzM55NjZ4k7vD7J4 Dwb8NS/HD8s6kzik9sqgwQIjv//////////ARAEGgw wNTkwMDM1N>
  • SECHAN YOUN ET AL: "Cell Deformability Monitoring Chips Based on Orifice-Length-Dependent Digital Lysis Rates", MICRO ELECTRO MECHANICAL SYSTEMS, 2006. MEMS 2006 ISTANBUL. 19TH IEEE INTERNATIONAL CONFERENCE ON ISTANBUL, TURKEY 22-26 JAN. 2006, IEEE, PISCATAWAY, NJ, USA, 22 January 2006 (2006-01-22), pages 16-19, XP010914514, DOI: 10.1109/MEMSYS.2006.1627725 ISBN: 978-0-7803-9475-9
  • UESAKA, N.: "Deformability of red blood cells investigated using a new microporous filter (nickel mesh)", Journal of Japanese Society of Biorheology, vol. 5, no. 4, 25 December 1991 (1991-12-25), pages 23 (172)-32 (181), XP009524701, DOI: 10.11262/jpnbr1987.5.4_172
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

TECHNICAL FIELD



[0001] The present invention relates to a method for assessing aging of erythrocytes in blood, which is the cause of ghosting of capillary blood vessels.

BACKGROUND ART



[0002] By using microscopes for capillary blood vessel that are recently attracting press attention, it is possible to observe capillary blood vessels that cannot normally be seen, and it is possible to find ghosting of capillary blood vessels, such as disappearing, shortening or reducing in number of capillary blood vessels. This ghosting of capillary blood vessels can occur throughout the body, and there is a risk of inducing not only influence on skin state, which is visible, such as wrinkles or sagging of skin, but also severe diseases such as osteoporosis, dementia, and lifestyle-associated diseases (diabetes, hypercholesteremia, etc.)

[0003] What is important during the process in which the flow of capillary blood vessels worsen and ghosts, is erythrocytes. This is because there is no function of constriction or expansion in the capillary blood vessels which contain no smooth muscle, and the erythrocytes pass through capillary blood vessels (about 5 pm) that are narrower than its own diameter (about 8 pm) by changing their shape to circulate throughout the body. The number of erythrocytes per pL of blood is 4 to 5 millions, and the volume of erythrocytes reaches 40 to 50 % of the volume of blood. The lifetime of these erythrocytes is as short as about 120 days, and there exist erythrocytes at various stages ranging from juvenile erythrocytes to aged erythrocytes. Those associated with ghosting of capillary blood vessels the most are the aged erythrocytes of which deformability has decreased. The aged erythrocytes are clogged in the capillary blood vessels, and it becomes impossible to deliver substances necessary for life of the cells that form the capillary blood vessels ahead, and the ghosting is thus thought to progress.

[0004] Recently, methods or apparatuses for assessing such deformability of erythrocytes in blood are proposed. Apparatuses that have been developed in Japan include, for example, MCFAN HR 300 (Micro Channel Array Flow Analyzer, Japan, manufactured by MC health care). MCFAN is an apparatus of allowing collected blood to flow through something like a capillary blood vessel that has been artificially made with silicon, to observe the image. At first upon development, it has been admired as contributing a lot to the research of the deformability of erythrocytes. However, it has been then found that the credibility is low, and now it is only employed in a very limited number of hospitals.

[0005] Further, apparatuses that have been developed abroad include LORCA (Laser-assisted Optical Rotational Cell Analyzer, Netherlands, manufactured by Mechatronics) which deforms erythrocytes to an ellipse by imparting centrifugal stress by rotatable flow and assesses with the diffracted image of laser beam, and RheoScan-D (Korea, manufactured by RheoMeditech) which deforms erythrocytes to an ellipse by imparting shear stress with negative pressure, and assesses with an diffracted image of laser beam (non-patent document 1). RheoScan-D has characteristics that automatical measurement can be performed with whole blood, it is not necessary to clean after usage owing to disposable plastic microchips in the flow path, and the aggregation of erythrocytes can be also measured, etc.

[0006] However, the physiologic deformation of erythrocytes in vivo is a bended deformation, and attention should be paid to the fact that whether to measure the deformability by deforming the erythrocytes in an ellipse reflects the conditions of physiological microcirculation. Further, since the stress necessary for deformation in an ellipse is much larger than the stress necessary for bended deformation, there is a problem that particularly the measurement sensitivity is low with a low shear stress as compared to a method for measuring bended deformation.

[0007] According to non-patent document 2, red blood cell deformability is determined with an ektacytometer in fractions separated on the basis of differences in cell volume or density. Deformability is measured with ektacytometry (rpm-scan and osmo-scan). Three groups of red blood cells fractions are studied:
  1. 1. By counterflow centrifugation fractions of different cell age are obtainable which show a slight decrease in mean corpuscular hemoglobin concentration (MCHC) and an increase in surface-to-volume (S/V) ratio in fractions with older cells.
  2. 2. By Percoll fractionation fractions are obtainable which show a pronounced increase in MCHC but no change in S/V ratio.
  3. 3. By a combination of both fractionation techniques, fractions are obtainable which show an increased MCHC and an increase in S/V ratio.


[0008] Deformability in group 1, 2 and 3 show respectively no change, a moderate decrease and a pronounced decrease in fractions of older cells. A decline in deformability occurs during the aging process of the red blood cell. This decline in deformability in old red cells is greater than originally thought. This decline is the result of an increase in hemoglobin concentration and a second factor, probably a decrease in membrane elasticity.

[0009] According to non-patent document 3, to assess the influence of intracellular hemoglobin concentration on red cell viscoelasticity and to better understand changes related to in vivo aging, membrane shear elastic moduli (µ) and time constants for cell shape recovery (tc) are measured for age-fractionated human erythrocytes and derived ghosts. Time constants are also measured for osmotically shrunk cell fractions. Young and old cells have equal µ, but tc, is longer for older cells. When young cells are shrunk to equal the volume (and hence hemoglobin concentration and internal viscosity) of old cells, tc increases only slightly. Thus membrane viscosity (η = µ·tc) increases during aging, regardless of increased internal viscosity. However, further shrinkage of young cells, or slight shrinkage of old cells, causes a sharp increase in tc. Because this increased tc is not explainable by elevated internal viscosity, η increases, possibly due to a concentration-dependent hemoglobin-membrane interaction. Ghosts have a greater η than intact cells, with proportionally faster tc; their membrane viscosity is therefore similar to intact cells. However, the ratio of old/young membrane viscosity is less for ghosts than for intact cells, indicating that differences between young and old cell η may be partly explained by altered hemoglobin-membrane interaction during aging. In non-patent document 3, it is postulated that these changes in viscoelastic behavior influence in vivo survival of senescent cells.

[0010] Non-patent document 4 proposes to monitor cell deformability based on the lysis rate difference measured from the cells passing through a filter array having gradually increased orifice length. Compared to previous methods, the presented chips offer simple and inexpensive monitoring with high sensitivity. In the experimental study, normal and chemically treated erythrocytes are used to verify the performance of the presented chips. Using the fabricated chips, two different devices measure the maximum lysis rate difference between the normal and the treated erythrocytes at the second filter, respectively having 6.7µm and 4.3µm-long orifices. According to non-patent document 4, more than 40 times improvement in the ratio of the average signals for normal erythrocytes to that of chemically treated erythrocytes is achievable.

[0011] Patent document 1 discloses a method used for analyzing the membrane function of a living organism with a membrane structure. This method includes a process for immobilizing the living organism with a membrane structure in a measurement container under conditions that allow the living organism to maintain physiological functions, a process for bonding fluorescent marker molecules to the membrane structure of the living organism, and a process for measuring fluorescence intensity fluctuation of the marker molecules bonded to the membrane structure.

[0012] Patent document 2 discloses a blood diagnostic system, capable of conducting a plurality of blood diagnoses at the same time, by having to inject a micro amount of blood by providing a plurality of branch flow passages on a chip, and a plurality of scientific, biochemical and physical measuring mechanisms connected thereto. Information which is to some extent of expert knowledge is further acquired, based on the blood condition using a communication line, and prescription information is provided further for health care hereinafter.

[Prior Art Documents]


[Patent documents]



[0013] 

[Patent document 1] JP 2005 241378 A

[Patent document 2] JP 2005 164296 A


[Non-patent documents]



[0014] 

[Non-patent document 1] Patricia C. Sousa, Fernando T. Pinho, Man uel A. Alves and Monica S.N. Oliveira, A review of hemorheology: Measuring techniques and recent advances, Korea-Australia Rheolog y J, 28(1), 1-22 (2016)

[Non-patent document 2] Bosch F. H ET AL: "Determinants of red blood cell deformability in relation to cell age", European journal of haematology, 1 January 1994 (1994-01-01), pages 35-41, XP05581232 2, Oxford, UK, DOI: 10.1111/j.1600-0609.1994.tb01282.x

[Non-patent document 3] NASH G.B. ET AL: "Red cell and ghost viscoelasticity. Effects of hemoglobin concentration and in vivo aging", BIOPHYSICAL JOURNAL, vol. 43, no. 1, 1 July 1983 (1983 -07-01), pages 63-73, XP055812314, AMSTERDAM, NL, ISSN: 0006-3495, DOI: 10.1016/S0006-3495(83)84324-0

[Non-patent document 4] SECHAN YOUN ET AL: "Cell Deformability Monitoring Chips Based on Orifice-Length-Dependent Digital Lysis Rates", MICRO ELECTRO MECHANICAL SYSTEMS, 2006. MEMS 2006 ISTANBUL. 19TH IEEE INTERNATIONAL CONFERENCE ON ISTANBUL, pages 16-19, XP010914514, DOI: 10.1109/MEMSYS.2006.1627725


[Summary of the Invention]


[Object being solved by the Invention]



[0015] Conventional assessment methods assess aging of erythrocytes by using a deformability obtained with erythrocytes as a whole at various stages in the lifetime of about 120 days, and no apparatus that can obtain deformability of only aged erythrocytes, or erythrocytes excluding aged erythrocytes, or automated apparatus for this has been developed.

[0016] The ratio of aged erythrocytes, which is the cause of ghosting of capillary blood vessels, varies depending on each patient. If such assessment method or apparatus is developed, it would be possible to appropriately diagnose each patient for the development risks of complications such as diabetic retinopathy, nephropathy, etc., and it is estimated that clinical meaning is important.

[0017] The object of the present invention is to provide a method for assessing aging of erythrocytes that can assess aging of erythrocytes with higher accuracy and properly.

[Means to Solve the Object]



[0018] The present inventors focused on the rheology function such as flexibility or ability of flowing of erythrocytes in blood, which is very important for the prevention and treatment of lifestyle-associated diseases, and developed an apparatus for measuring deformability of erythrocytes using gravity nickel mesh filtration system, to provide erythrocyte deformability test.

[0019] By using this gravity nickel mesh filtration system (diameter of micropores of the filter: 3.2 pm), according to the results of an investigation by limiting to mild case of hyperlipidemia (total cholesterol level 260 mg/dl or less) for 139 subjects who had a medical check-up, the erythrocyte deformability showed a negative correlation between levels of neutral fat, and a positive correlation between HDL cholesterol levels (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 erythrocyte filterability : cross-sectional study of healthy subjects. Clin HemorheolMicrocirc 22 : 1-7, 2000.).

[0020] Further, for 101 patients having hypertension, an investigation using a filter (diameter of micropores: 4.94 pm) has revealed that the erythrocyte deformability showed a negative correlation between average blood pressure (K. Odashiro et al, Impaired deformability of circulating erythrocytes obtained from nondiabetic hypertensive patients: investigation by a nickel mesh filtration technique, Clin Hypertens. 21:17, eCollection (2015)).

[0021] The present inventors have further made keen studies to assess aging of erythrocytes in blood with higher accuracy and properly, and as a result, by separating erythrocytes plural times to obtain deformability of erythrocytes in each of separating step, assessed aging of erythrocytes by using these levels, they found out to be possible to assess aging of erythrocytes with higher accuracy and properly, and thus ghosting of capillary blood vessels. The present invention has been thus completed.

[0022] The object is achieved by a method having the features of claim 1. Further advantageous developments of the present invention are set out in the dependent claims.

[0023] Specifically, the present invention is as follows.
  1. [1] A method for assessing aging of erythrocytes by using at least two types of filters having a plurality of micropores, the method comprising an erythrocyte suspension-preparing step of preparing an erythrocyte suspension with a certain concentration of hematocrit from a blood sample;

    a first erythrocyte deformability calculating step of allowing the erythrocyte suspension to pass through a first filter, separating aged erythrocytes that do not pass through the first filter, and non-aged erythrocytes that pass through the first filter, and to calculate a deformability of erythrocytes contained in the erythrocyte suspension, which deformability is an index showing an ability to pass through the first filter;

    a second erythrocyte deformability calculating step of allowing the separated non-aged erythrocyte suspension to pass through a second filter having micropores of which diameter is smaller than the first filter, separating mild aged erythrocytes that do not pass through the second filter and juvenile erythrocytes that pass through the second filter, and to calculate a deformability of the non-aged erythrocytes contained in the non-aged erythrocyte suspension, which deformability is an index showing an ability to pass through the second filter; and

    an assessment step of assessing aging of erythrocytes based on a size of the deformability of erythrocytes calculated in the first erythrocyte deformability calculating step, and the deformability of non-aged erythrocytes calculated in the second erythrocyte deformability calculating step.

  2. [2] Preferably, a passing ratio of erythrocytes is further calculated in the first erythrocyte deformability calculating step and/or the second erythrocyte deformability calculating step, to use the passing ratio for the assessment in the assessment step.
  3. [3] Preferably, a diameter of micropores of the first filter is 5.50 to 8.00 pm.
  4. [4] Preferably, a diameter of micropores of the second filter is 3.00 to 6.00 pm.

[Effect of the present invention]



[0024] According to the method for assessing aging of erythrocytes of the present invention, aging of erythrocytes can be assessed with higher accuracy and properly. Therefore, the possibility of ghosting of capillary blood vessels can be accurately detected, and can be applied to diagnosis of various diseases.

[Brief Explanation of Drawings]



[0025] 

[Fig. 1]
It is a diagram showing the method for assessing aging of erythrocytes of one embodiment of the present invention.

[Fig. 2]
It is a brief figure explaining the apparatus used in the method for assessing aging of erythrocytes of one embodiment of the present invention.

[Fig. 3]
It is a figure showing "height-time curve" obtained by the measurement results of the apparatus shown in Fig. 2.

[Fig. 4]
It is a graph showing the deformability of erythrocytes passing through the filter when using the multistage method (Example 1) of the present invention.

[Fig. 5]
It is a graph showing the deformability of erythrocytes passing through the filter when using the conventional single stage method.

[Fig. 6]
It is a graph showing the number of erythrocytes (ratio) that have passed through the filter when using the multistage method (Example 1) of the present invention.

[Fig. 7]
It is a graph showing the number of erythrocytes (ratio) that have passed through the filter when using the conventional single stage method.


[Mode of practicing the Invention]



[0026] The assessing method for aging of erythrocytes of the present invention is a method for assessing aging of erythrocytes by using at least two types of filters having a plurality of uniform micropores, the method comprising

an erythrocyte suspension-preparing step of preparing an erythrocyte suspension from a blood sample;

a first erythrocyte deformability calculating step of allowing the erythrocyte suspension to pass through a first filter, separating aged erythrocytes that do not pass through the first filter, and non-aged erythrocytes that pass through the first filter, and to calculate a deformability of erythrocytes contained in the erythrocyte suspension;

a second erythrocyte deformability calculating step of allowing the separated non-aged erythrocyte suspension to pass through a second filter having micropores of which diameter is smaller than the first filter, separating mild aged erythrocytes that do not pass through the second filter and juvenile erythrocytes that pass through the second filter, and to calculate a deformability of the non-aged erythrocytes contained in the non-aged erythrocyte suspension; and

an assessment step of assessing aging of erythrocytes by using the deformability of erythrocytes calculated in the first erythrocyte deformability calculating step, and the deformability of non-aged erythrocytes calculated in the second erythrocyte deformability calculating step.



[0027] The method for assessing aging of erythrocytes of the present invention can assess aging of erythrocytes by using the apparatus for measuring deformability of erythrocytes described in "Toru Maruyama, Kazuhiko Okamoto, Quantitative analysis of deformability of erythrocytes by nickel mesh filtration system, Fukuoka Acta Medica, 95(6), 131-138(2004)".

[0028] In the method for assessing aging of erythrocytes of the present invention, in the first erythrocyte deformability calculating step and/or second erythrocyte deformability calculating step, it is preferred to calculate the passing ratio of erythrocytes, and to use this passing ratio in the assessment of the assessing step. Thereby, it is possible to make a more precise assessment of aging of erythrocytes.

[0029] Further, the method of the present invention can comprise further erythrocyte deformability calculating steps, such as a third erythrocyte deformability calculating step using a third filter, a fourth erythrocyte deformability calculating step using a fourth filter, etc., and to use the deformability or passing ratio of erythrocytes calculated in these steps for the assessment.

[0030] In the following, the method for assessing aging of erythrocytes of the present invention is explained in detail.

[0031] As shown in Fig. 1, the method for assessing aging of erythrocytes of one embodiment of the present invention comprises sequentially an erythrocyte suspension-preparing step (S1), a first erythrocyte deformability calculating step (S2), a second erythrocyte deformability calculating step (S3), and an assessment step (S4).

<Erythrocyte suspension-preparing step>



[0032] The erythrocyte suspension-preparing step (S1) is a step of preparing erythrocyte suspension from a blood sample, and for example is a step of preparing an erythrocyte suspension by washing a blood sample collected from a test subject. Specifically, for example, a treatment of washing the collected blood by centrifugation with a buffer is repeated plural times, and then is diluted with a buffer so that the hematocrit (HCT) has a certain concentration, to prepare an erythrocyte suspension.

< First erythrocyte deformability calculating step>



[0033] The first erythrocyte deformability calculating step (S2) is a step of passing the erythrocyte suspension prepared in the erythrocyte suspension-preparing step (S1) through a first filter, to separate aged erythrocytes that do not pass through the first filter and non-aged erythrocytes that pass through the first filter, and to calculate a deformability of erythrocytes contained in the erythrocyte suspension.

[0034] As for the first filter used in the present invention, to ensure high quantitativeness and reproducibility, uniform filters of which shape, number and distribution of micropores are similar are preferred. Examples include nickel mesh filter manufactured by combining a photoresist method and special plating method. The first filter is preferred to have a structure that hardly confer mechanical stimulation to leukocytes which are mixed during preparation of erythrocyte suspension.

[0035] The diameter of micropores of the first filter can be appropriately changed according to the situation of the test subjects, while generally, it is preferably 5.50 to 8.00 pm, more preferably 5.60 to 7.00 pm, and further preferably 5.70 to 6.50 pm.

[0036] In this step, the deformability of erythrocytes is calculated. The deformability is an index showing the ability of the erythrocytes (non-aged erythrocytes) contained in the erythrocyte suspension to pass through the first filter. Various levels calculated by so-called filtration method such as difference of pressure when the erythrocyte suspension passes through the micropores of the filter, the passing time that a certain amount of erythrocyte suspension passes through, the flow rate (Q) of erythrocytes, etc. can be used.

[0037] Specifically, the method for calculating the deformability of the present invention can be calculated for example by using an apparatus as shown in Fig. 2. As it is shown in Fig. 2, to the vertical glass tube 1, a nickel mesh filter 2 (for example, diameter of micropores: 6.0 pm) is mounted via a Tygon tube, and the erythrocyte suspension is filtered from a certain height (for example 15 cm). By continuously measuring the pressure at that time, the height (h in Fig. 2)- time curve is obtained (see Fig. 3). By comparing with the height-time curve of a buffer not containing erythrocytes similarly obtained, and comparing the time at the time point where it has been decreased to a certain height (for example 10 cm), the deformability is quantified.

[0038] In the present step, further, the passing ratio of erythrocytes, specifically the ratio of aged erythrocytes that do not pass through the first filter, and the non-aged erythrocytes that pass through the filter is preferably calculated. By using this passing ratio in the assessment step, a more precise assessment can be made. The calculation of the passing ratio can be obtained by using a well-known blood cell analyzer, etc., by measuring at least two of the total number of erythrocytes contained in the erythrocyte suspension, the number of aged erythrocytes, and the number of non-aged erythrocytes.

<Second erythrocyte deformability calculating step>



[0039] The second erythrocyte deformability calculating step (S3) is a step of allowing the non-aged erythrocyte suspension that has been separated in the above-mentioned first erythrocyte deformability calculating step (S2) to pass through a second filter having micropores of which diameter is smaller than the first filter, to separate the mild aged erythrocytes that do not pass through the second filter and the juvenile erythrocytes that pass through the filter, and to calculate a deformability of the non-aged erythrocytes contained in the non-aged erythrocyte suspension.

[0040] As the non-aged erythrocyte suspension used in this step, the suspension separated in the first erythrocyte deformability calculating step (S2) can be directly used, or can be diluted with a buffer so that the hematocrit (HCT) has a certain concentration, and used.

[0041] The treatment of the present step is basically similar to the treatment of the first erythrocyte deformability calculating step (S2), while the filter to be used is different. Specifically, in the present step, a second filter having micropores of which diameter is smaller than that of the micropores of the first filter is used. The diameter of micropores of the second filter can be appropriately changed according to the results of the first erythrocyte deformability calculating step (S2), etc., while generally, it is preferably 3.00 to 6.00 pm, more preferably 3.50 to 5.80 pm, further preferably 4.00 to 5.50 pm, and particularly preferably 4.50 to 5.50 pm. Further, it is preferable that the difference with the diameter of micropores of the first filter is 0.1 to 2.0 pm, more preferably 0.3 to 1.5 pm, and further preferably 0.5 to 1.0 pm.

<Assessment step>



[0042] The assessment step (S4) is a step of assessing the aging of erythrocytes using the deformability of erythrocytes calculated in the first erythrocyte deformability calculating step, and the deformability of non-aged erythrocytes calculated in the second erythrocyte deformability calculating step. In this step, additionally to the deformability of the erythrocytes and non-aged erythrocytes, it is preferred to use the passing ratio of the erythrocytes calculated in the first erythrocyte deformability calculating step and/or the second erythrocyte deformability calculating step. As such, a more precise assessment can be made.

[0043] Specifically, in this step, when the deformability is low, it is assessed that the erythrocytes are aged, and further by adding the assessment based on the passing ratio (the lower the passing ratio is, the more aged the erythrocytes are) at the same time or additionally, the aging of erythrocytes is assessed. As such, the possibility of ghosting of capillary blood vessels can be accurately detected, and can be applied to the diagnosis of the skin state such as wrinkles or sagging of skin, or diagnosis of osteoporosis, dementia, and lifestyle-associated diseases (diabetes, hypercholesteremia, etc.). Thus, early detection of diseases is possible.

[0044] Particularly, since the method for assessing aging of erythrocytes of the present invention assesses by using the deformability (and passing ratio) in at least two or more separation steps, an assessment of aging of erythrocytes that is more precise than a conventional method can be made. Further, by changing the combination of the size of micropores of the filters according to the situation of the test subjects (age, blood pressure, diseases suffering from, chronic disease, etc.), a more adequate assessment can be made.

[Example]



[0045] In the following, the present invention is explained in detail by referring to the Example. However, the present invention is not limited to the Example. The summary of the basic operations of the Example is shown in Fig. 1.

[Basic operations]


(Erythrocytes suspension-preparing step)



[0046] First, 30 cc of blood collected from a test subject is centrifuged at a rotation of 2500 rpm, for 10 minutes by using a centrifuge, and washed with a buffer. Then, by changing sequentially the rotation to 1950 rpm, 1700 rpm, 1550 rpm, centrifugation (10 minutes each) and washing with buffer are repeated to obtain washed erythrocytes. The obtained washed erythrocytes are diluted with a buffer, to prepare an erythrocyte suspension with a hematocrit (HCT) of 3%. The number of erythrocytes per µL of erythrocyte suspension is measured by using a blood cell analyzer.

(First erythrocyte deformability calculating step (stage 1))



[0047] A measurement apparatus mounted with a 6.00 µηι nickel mesh filter as shown in Fig. 2 is used. Erythrocyte suspension is put in a glass tube with a solution sending pump, to measure the deformability. Further, the number of erythrocytes per pL of non-aged erythrocytes suspension that have passed through the 6.00 µηι nickel mesh filter is measured by using a blood cell analyzer.

(Second erythrocyte deformability calculating step (stage 2))



[0048] The non-aged erythrocyte suspension is diluted with a buffer to prepare a non-aged erythrocyte suspension. After washing inside of the measurement apparatus with a buffer, the 6.00 µηι nickel mesh filter is changed to a 5.31 pm nickel mesh filter. Non-aged erythrocyte suspension is put in a glass tube with a solution sending pump, to measure the deformability. Further, the number of erythrocytes per pL of juvenile erythrocyte suspension that have passed through the 5.31 µηι nickel mesh filter is measured by using a blood cell analyzer.

(Assessment step)



[0049] By using each obtained deformability, each number of erythrocytes, the aging of blood is assessed.

[Example 1]



[0050] An example of using actual blood collected from human, and assessing aging of blood by following the above-mentioned basic operations is shown in the following.

[0051] By a method shown in the above-mentioned erythrocyte suspension-preparing step, an erythrocyte suspension (sample liquid) of HCT 3% was prepared from blood collected from human. The number of erythrocytes of the sample liquid at that time was 32 × 104/µl.

[0052] Further, for comparison, a comparative sample liquid added with 500 mM of a free radical producing substance AAPH (2,2'-azobis-2-methyl-propanimidamide,dihydrochloride) that decreases erythrocyte deformability was prepared (number of erythrocytes: 32 × 104/µl).

[0053] By using a 6.00 µηι nickel mesh filter, and employing the technique of the above-mentioned first erythrocyte deformability calculating step (stage 1), the deformability and the number of erythrocytes were measured. As it is shown in Fig. 4 (each graph on the left), the deformability of the sample liquid and that of the comparative sample liquid in stage 1 were 93% and 90%, respectively. Further, as shown in Fig. 6 (each graph on the left), the number of erythrocytes of the sample liquid that have passed through the filter in stage 1 was 26 × 104/µl (passing ratio: 81%), and the number of erythrocytes of the comparative sample that have passed through the filter was 26 × 104/µl (passing ratio: 81%).

[0054] The deformability of erythrocytes (%) was obtained as follows. The erythrocyte suspension (sample liquid or comparative sample liquid) was allowed to pass through the nickel mesh filter from a height of 15 cm, the pressure change during passing was continuously detected, to obtain height-time curve, and the erythrocyte deformability was assessed by using the height-time curve of the buffer not containing erythrocytes as control. The deformability at the time point where it has been decreased to 10 cm was quantified by comparing with the control.

[0055] The number of erythrocytes of the sample liquid and comparative sample liquid after the first erythrocyte deformability calculating step (stage 1) was diluted and adjusted to 9 × 104/µl. Further, by using a 5.31 pm nickel mesh filter, and employing the technique in the above-mentioned second erythrocyte deformability calculating step (stage 2), the deformability and the number of erythrocytes were measured.

[0056] As shown in Fig. 4 (each graph on the right), the deformability of the sample liquid and that of the comparative sample in stage 2 were 95% and 66%, respectively, resulting in a big difference of about 30%. Further, as shown in Fig. 6 (each graph on the right), the number of erythrocytes of the sample liquid that have passed through the filter in stage 2 was 8 × 104/µl (passing ratio: 89%), and the number of erythrocytes of the comparative sample that have passed through the filter was 7 × 104/µl (passing ratio: 78%). The difference here was also as large as about 11%.

[0057] On the other hand, as shown in Fig. 5, by a conventional method of a single stage using only a 5.31 pm nickel mesh filter (number of erythrocytes in the sample liquid: 34 × 104/µl), the deformability of the sample liquid and that of the comparative sample were 89% and 80%, respectively, of which difference was as small as less than 10%. Further, as shown in Fig. 7, the erythrocytes in the sample liquid that have passed through the filter was 25 × 104/µl (passing ratio: 74%), and the erythrocytes of the comparative sample that have passed through the filter was 26 × 104/µl (passing ratio: 76%), and there was almost no difference (the comparative sample liquid showed a larger value).

[0058] As it is stated in the above, according to the method of multistage of the present invention, the state of erythrocytes can be understood with excellent accuracy, allowing an accurate assessment, as well as a more sharp classification of right and wrong.

[Industrial Applicability]



[0059] The method of assessing aging of erythrocytes of the present invention can assess the aging of erythrocytes and is industrially useful.

[Explanation of codes]



[0060] 
  1. 1. glass tube
  2. 2. nickel mesh filter
  3. 3. constant temperature water tank



Claims

1. A method for assessing aging of erythrocytes by using at least two types of filters having a plurality of micropores, the method comprising

an erythrocyte suspension-preparing step of preparing an erythrocyte suspension from a blood sample; a first erythrocyte deformability calculating step of allowing the erythrocyte suspension to pass through a first filter, separating aged erythrocytes that do not pass through the first filter, and non-aged erythrocytes that pass through the first filter, and to calculate a deformability of erythrocytes contained in the erythrocyte suspension;

a second erythrocyte deformability calculating step of allowing the separated non-aged erythrocyte suspension to pass through a second filter having micropores which diameter is smaller than the diameter of micropores of the first filter, separating mild aged erythrocytes that do not pass through the second filter and juvenile erythrocytes that pass through the second filter, and to calculate a deformability of the non-aged erythrocytes contained in the non-aged erythrocyte suspension; and

an assessment step of assessing aging of erythrocytes based on the deformability of erythrocytes calculated in the first erythrocyte deformability calculating step, and the deformability of non-aged erythrocytes calculated in the second erythrocyte deformability calculating step.


 
2. The method for assessing aging of erythrocytes according to claim 1, wherein a passing ratio of erythrocytes is further calculated in the first erythrocyte deformability calculating step and/or the second erythrocyte deformability calculating step, to use the passing ratio for the assessment in the assessment step.
 
3. The method for assessing aging of erythrocytes according to claim 1 or 2, wherein a diameter of micropores of the first filter is 5.50 to 8.00 µm.
 
4. The method for assessing aging of erythrocytes according to any one of claims 1 to 3, wherein a diameter of micropores of the second filter is 3.00 to 6.00 µm.
 


Ansprüche

1. Verfahren zur Bewertung der Alterung von Erythrozyten unter Verwendung von mindestens zwei Arten von Filtern, die eine Vielzahl von Mikroporen haben, wobei das Verfahren Folgendes umfasst:

einen Erythrozytensuspensionsvorbereitungsschritt, in dem aus einer Blutprobe eine Erythrozytensuspension vorbereitet wird;

einen ersten Erythrozytenverformbarkeitsberechnungsschritt, in dem die Erythrozytensuspension durch einen ersten Filter gehen gelassen wird, gealterte Erythrozyten, die nicht durch den ersten Filter gehen, und nicht gealterte Erythrozyten, die durch den ersten Filter gehen, getrennt werden und um eine Verformbarkeit der Erythrozyten zu berechnen, die in der Erythrozytensuspension enthalten sind;

einen zweiten Erythrozytenverformbarkeitsberechnungsschritt, in dem die Suspension getrennter, nicht gealterter Erythrozyten durch einen zweiten Filter gehen gelassen wird, der Mikroporen hat, deren Durchmesser kleiner als der Durchmesser der Mikroporen des ersten Filters ist, leicht gealterte Erythrozyten, die nicht durch den zweiten Filter gehen, und juvenile Erythrozyten, die durch den zweiten Filter gehen, getrennt werden und um eine Verformbarkeit der nicht gealterten Erythrozyten zu berechnen, die in der Suspension nicht gealterter Erythrozyten enthalten sind; und

einen Bewertungsschritt, in dem beruhend auf der im ersten Erythrozytenverformbarkeitsberechnungsschritt berechneten Verformbarkeit der Erythrozyten und der im zweiten Erythrozytenverformbarkeitsberechnungsschritt berechneten Verformbarkeit der nicht gealterten Erythrozyten die Alterung der Erythrozyten bewertet wird.


 
2. Verfahren zur Bewertung der Alterung von Erythrozyten nach Anspruch 1, wobei in dem ersten Erythrozytenverformbarkeitsberechnungsschritt und/oder dem zweiten Erythrozytenverformbarkeitsberechnungsschritt außerdem ein Durchlassverhältnis der Erythrozyten berechnet wird, um das Durchlassverhältnis für die Bewertung im Bewertungsschritt zu verwenden.
 
3. Verfahren zur Bewertung der Alterung von Erythrozyten nach Anspruch 1 oder 2, wobei ein Durchmesser der Mikroporen des ersten Filters 5,50 bis 8,00 µm beträgt.
 
4. Verfahren zur Bewertung der Alterung von Erythrozyten nach einem der Ansprüche 1 bis 3, wobei ein Durchmesser der Mikroporen des zweiten Filters 3,00 bis 6,00 µm beträgt.
 


Revendications

1. Procédé d'évaluation du vieillissement d'érythrocytes utilisant au moins deux types de filtres ayant une pluralité de micropores, le procédé comprenant

une étape de préparation de suspension d'érythrocytes de préparation d'une suspension d'érythrocytes à partir d'un échantillon de sang ;

une première étape de calcul de déformabilité d'érythrocytes consistant à laisser la suspension d'érythrocytes traverser un premier filtre, séparer les érythrocytes vieillis qui ne traversent pas le premier filtre, et les érythrocytes non vieillis qui traversent le premier filtre, et calculer une déformabilité des érythrocytes contenus dans la suspension d'érythrocytes ;

une deuxième étape de calcul de déformabilité d'érythrocytes consistant à laisser la suspension d'érythrocytes non vieillis séparée traverser un deuxième filtre ayant des micropores dont le diamètre est inférieur au diamètre des micropores du premier filtre, séparer les érythrocytes légèrement vieillis qui ne traversent pas le deuxième filtre et les érythrocytes juvéniles qui traversent le deuxième filtre, et calculer une déformabilité des érythrocytes non vieillis contenus dans la suspension d'érythrocytes non vieillis ;

une étape d'évaluation d'évaluation des érythrocytes vieillis sur la base de la déformabilité des érythrocytes calculée dans la première étape de calcul de déformabilité d'érythrocytes, et la déformabilité des érythrocytes non vieillis calculée dans la deuxième étape de calcul de déformabilité d'érythrocytes.


 
2. Procédé d'évaluation du vieillissement d'érythrocytes selon la revendication 1, dans lequel un rapport de passage d'érythrocytes est en outre calculé dans la première étape de calcul de déformabilité d'érythrocytes et/ou la deuxième étape de calcul de déformabilité d'érythrocytes, pour utiliser le rapport de passage pour l'évaluation dans l'étape d'évaluation.
 
3. Procédé d'évaluation du vieillissement d'érythrocytes selon la revendication 1 ou 2, dans lequel un diamètre de micropores du premier filtre est de 5,50 à 8,00 µm.
 
4. Procédé d'évaluation du vieillissement d'érythrocytes selon l'une quelconque des revendications 1 à 3, dans lequel un diamètre de micropores du deuxième filtre est de 3,00 à 6,00 µm.
 




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Cited references

REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description




Non-patent literature cited in the description