JP2008215901A - Qualitative and/or quantitative analyzing method of blood corpuscle and method of detecting blood deterioration - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel qualitative and/or quantitative analyzing method of a blood corpuscle. <P>SOLUTION: The qualitative and/or quantitative analyzing method of the blood corpuscle contains at least an electric field applying process for applying an electric field changeable in frequency to a sampled blood, a dielectric constant measuring process for measuring the dielectric constant of the sampled blood while changing the frequency of the electric field and a dielectric relaxing phenomenon analyzing process for analyzing a dielectric relaxing phenomenon from the measuring result obtained through the dielectric constant measuring process. Since the qualitative and/or quantitative analyzing method can be performed in a label free state without damaging a blood corpuscle cell or the like, it can be rapidly and simply performed and blood used in analysis can be used in blood transfusion or a blood preparation as it is. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for qualitative and / or quantitative analysis of blood cells. More specifically, the present invention relates to a method of analyzing blood cell morphology and / or density and the like by performing dielectric spectroscopy measurement on collected blood.

  Blood transports blood gases (oxygen, carbon dioxide), transports nutrients, transports and discharges metabolic waste, maintains acid-base balance in the body, regulates water metabolism, protects the body, regulates body temperature, transports bioactive substances It plays an important role in the maintenance of biological homeostasis, such as its metabolic regulation.

  Blood is roughly divided into blood cells and plasma, and the ratio is about 45:55. Among these, blood cells are roughly classified into red blood cells, white blood cells, and platelets. The cell components constituting the blood cell will be described below.

<Red blood cells>
Red blood cells occupy about 96% of blood cells and mainly carry blood gases (oxygen and carbon dioxide). The number of red blood cells is usually about 5 million for boys and about 4.5 million for girls in 1 mm ^{3 of} blood, but the number may be reduced due to iron deficiency anemia, aplastic anemia, hemolytic anemia, and the like.

  In addition, the red blood cells usually have a concave disk shape with a central portion of 7 to 8 μm in diameter, but the form may change depending on various diseases. For example, elliptical erythrocytes and sickle erythrocytes due to genetic causes, megaloblastic anemia, teardrop erythrocytes due to myelodysplastic syndrome, thin erythrocytes due to iron deficiency anemia, uremia-related urine Red blood cells, etc.). In addition, it is known that red blood cells have a ginger shape (uniform shape, scallop shape, mine shape) due to depletion of cell energy, and have a spherical shape as the number of storage days after blood collection elapses.

<White blood cells>
Leukocytes account for about 3% of blood cells, and are mainly involved in biological defense mechanisms. There are usually 4,000 to 10,000 leukocytes (average of about 7000) in 1 mm ³ of blood, but the number of leukocytes increases due to infection or allergy, or conversely normal leukocytes decrease due to leukemia. There is.

  Leukocytes are roughly classified into granulocytes, monocytes and lymphocytes, and granulocytes are further classified into neutrophils, eosinophils and basophils. The diameters are about 10 μm for neutrophils, about 13 to 18 μm for eosinophils, about 12 to 15 μm for basophils, about 12 to 20 μm for monocytes, and about 6 to 15 μm for lymphocytes. Abnormally shaped white blood cells (leukemia cells) may appear in large quantities.

<Platelets>
Platelets make up about 1% of blood cells and are involved in thrombus formation. There are usually 100,000 to 400,000 platelets in 1 mm ^{3 of} blood, but diseases in which platelets increase or decrease such as thrombocytopenia and thrombocytosis are also known.

  In addition, platelets have a disk shape with a diameter of 2 to 3 μm. However, it is known that platelets have a shape like gold saccharose when bleeding.

As a method for examining the morphology of blood cells, for example, Patent Document 1 discloses an apparatus and method for determining the shape of individual red blood cells by analyzing the asymmetry of a light scattering pattern generated by laser irradiation of cells. Yes. Patent Document 2 discloses a method for detecting an abnormal shape of red blood cells using a flow cytometer.
JP-T-2001-507122. JP-A-10-307135.

  As described above, since it is known that the amount and form of each component of blood cells change due to various diseases, etc., various diseases are diagnosed by examining the amount and form of each component of blood cells. be able to. Moreover, the preservation | save state (deterioration state) of blood can be analyzed by investigating the form of each component of the blood cell after blood collection.

  Therefore, the main object of the present invention is to provide a novel qualitative and / or quantitative analysis method for blood cells and to realize industrial use of the method.

  The inventors of the present application have conducted extensive research on qualitative and / or quantitative analysis methods for blood cells, and as a result, have found that qualitative and / or quantitative analysis of blood cells can be performed by analyzing dielectric relaxation phenomena of collected blood. It was.

First, in the present invention, a method for qualitative and / or quantitative analysis of blood cells, an electric field applying step of applying an electric field whose frequency can be changed to the collected blood, and a dielectric constant of the collected blood while changing the frequency of the electric field A blood cell qualitative and / or quantitative analysis method comprising at least a dielectric constant measurement step for measuring a dielectric constant and a dielectric relaxation phenomenon analysis step for analyzing a dielectric relaxation phenomenon from a measurement result obtained through the dielectric constant measurement step .
Blood containing blood cells exhibits different dielectric relaxation phenomena depending on the size, shape, cell membrane state, etc. of the blood cells, blood cell density, etc. Therefore, analyzing the dielectric relaxation phenomenon analyzes the blood cell morphology, density, etc. can do.
The dielectric relaxation phenomenon analysis can be performed, for example, by calculating a dielectric relaxation frequency, a dielectric relaxation time, and a dielectric relaxation strength from a measurement result obtained through the dielectric constant measurement step.
Moreover, the qualitative and / or quantitative analysis method according to the present invention can be used for analysis of all blood cells of red blood cells, white blood cells, and platelets.
Furthermore, the qualitative and / or quantitative analysis method according to the present invention can be applied to detection of deterioration of stored blood.

  Hereinafter, technical terms used in the present invention will be described.

  In the present invention, “blood” includes not only humans but also blood collected from animals. In addition, all blood including blood cells is included. For example, it includes not only blood containing all blood cells of red blood cells, white blood cells and platelets, but also blood containing any one or two blood cells.

  In the present invention, “dielectric relaxation” is a phenomenon in which electrification accumulates due to an applied voltage at the interface between a cytoplasm having high conductivity and a cell membrane having conductivity, and is known as typical interface polarization. For example, when the dielectric constant of blood containing blood cells as a main component is measured while changing the frequency, it indicates a phenomenon that shows a high dielectric constant on the low frequency side but drops to a low dielectric constant as it goes to the high frequency side.

  In the present invention, “dielectric relaxation frequency” refers to a frequency at which the dielectric constant changes most greatly in the course of the dielectric relaxation phenomenon.

  In the present invention, the “dielectric relaxation time” is a value characterizing the time scale of the dielectric relaxation phenomenon, and the measured data of the dielectric relaxation phenomenon is expressed by a theoretical or empirical relaxation function equation (for example, a “mathematical formula” described later). 1 ”etc.) is used to perform curve fitting, and is a quantity having a time dimension among the obtained parameters.

  In the present invention, “dielectric relaxation strength” means a value of a high dielectric constant observed at a frequency sufficiently lower than the relaxation frequency in a dielectric relaxation phenomenon, and a value of a low dielectric constant observed at a frequency sufficiently higher than the relaxation frequency. The difference between. Here, the sufficiently low (sufficiently high) frequency is desirably at least one digit lower (high). The accurate value of the dielectric relaxation strength is obtained by curve fitting the measured dielectric relaxation phenomenon data using a theoretical or empirical relaxation function formula (for example, “Formula 1” described later). Can be obtained.

  Since the qualitative and / or quantitative analysis method according to the present invention can perform qualitative and / or quantitative analysis of blood cells only by measuring the dielectric constant, it is less likely to damage blood cells and the like. There is no need to pre-label. Therefore, qualitative and / or quantitative analysis of blood cells can be performed quickly and easily. If the qualitative and / or quantitative analysis of blood cells according to the present invention is used, various diseases can be diagnosed and the blood deterioration state can be easily analyzed.

  DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, preferred embodiments for carrying out the invention will be described with reference to the drawings. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly.

<Qualitative and / or quantitative analysis method for blood cells>
FIG. 1 is a flow diagram of a qualitative and / or quantitative analysis method according to the present invention.

  In the qualitative and / or quantitative analysis method according to the present invention, first, an electric field applying step of applying an electric field to the collected blood is performed (see (I) in FIG. 1). A known method can be used to apply the electric field. For example, a method of using a pair of electrodes or using a coil can be employed freely. However, the electric field must be an electric field capable of changing the frequency. This is for analyzing a dielectric relaxation phenomenon described later.

Next, a dielectric constant measurement step is performed in which the dielectric constant of blood is measured while changing the frequency (see (II) in FIG. 1). The change range of the frequency can be appropriately set according to the blood cells (red blood cells, white blood cells, and platelets) to be qualitatively and / or quantitatively analyzed. For example, ¹⁰ 5 ^{to 10} 8 Hz in erythrocytes, the leukocytes ¹⁰ 5 ^{to 10} 8 Hz, the platelets it is preferable to sweep the range of ^{5 × 10 5 ~5 × 10 8} Hz.

  Since the qualitative and / or quantitative analysis method according to the present invention can perform qualitative and / or quantitative analysis of blood cells only by measuring the dielectric constant, it is less likely to damage blood cells and the like. Need not be pre-labeled. Therefore, qualitative and / or quantitative analysis of blood cells can be performed quickly and easily.

  Next, a dielectric relaxation phenomenon analysis step for analyzing the dielectric relaxation phenomenon is performed from the measurement result obtained through the dielectric constant measurement step (II) (see (III) in FIG. 1). The dielectric relaxation phenomenon analysis step (III) will be described in detail by taking the results of measuring the dielectric constant of red blood cells as an example.

  FIG. 2 is a diagram showing the results of measuring the dielectric constants of four types of red blood cells having different shapes while changing the frequency of the electric field (see Example 1 described later). These erythrocytes can be controlled by controlling the pH, normal form 1 (disc shape, see FIG. 3), sea urchin shape 2 (garage shape, scallop shape, mine shape, see FIG. 4), expanded shape 3 (see FIG. 5), spherical shape. 4 (see FIG. 6), all are set to substantially the same density.

  As shown in FIG. 2, each red blood cell shows a high dielectric constant on the low frequency side, whereas the dielectric constant decreases toward the high frequency side (hereinafter referred to as “dielectric relaxation phenomenon”). It can be confirmed that different phenomena are shown depending on the shape of each red blood cell.

  In this way, the dielectric relaxation phenomenon shows a phenomenon that varies depending on the size, shape, cell membrane state, etc. of the blood cell, the density of the blood cell, etc. Therefore, if the dielectric relaxation phenomenon of the blood cell is analyzed, the shape and density of the blood cell are analyzed. be able to.

  As an analysis method of the dielectric relaxation phenomenon, for example, there is a method of calculating a dielectric relaxation frequency (see (i) in FIG. 1). As shown in FIG. 2, it can be seen that the frequency at which the dielectric constant changes greatly, that is, the dielectric relaxation frequency, differs depending on the red blood cells of each shape. Therefore, the morphology and density of blood cells can be analyzed by calculating the dielectric relaxation frequency of blood cells from the measurement results obtained through the dielectric constant measurement step (II).

Further, the measurement result obtained through the dielectric constant measurement step (II) can be statistically curve-fitted (curve fitting) using a dielectric relaxation function or the like. Specifically, curve fitting is performed by using a real part, an imaginary part, or a real part and an imaginary part of a complex dielectric constant that varies depending on the frequency obtained by measurement. For example, the following formula 1 is used to determine a set of parameters (relaxation time, relaxation strength, relaxation time distribution, direct current conductivity, etc.) that minimize the residual sum of squares between the experimental value and the function value. Can do.

Where j is the imaginary unit, ω = 2πf is the angular frequency, Δε is the dielectric relaxation strength, τ is the dielectric relaxation time, ε _∞ is the high frequency limit of the dielectric constant, ε ₀ is the vacuum dielectric constant, and σ is the DC electrical conductivity , Α and β are parameters representing the distribution of dielectric relaxation time (hereinafter, α is referred to as “Cole-Cole parameter” and β is referred to as “Cole-Davidson parameter”).

  At this time, by performing analysis with β = 1 fixed (analysis by Cole-Cole equation), dielectric relaxation time τ, Cole-Cole parameter α, and dielectric relaxation strength Δε can be calculated ((ii) in FIG. 1). (See (iii)). Dielectric relaxation time τ, Cole-Cole parameter α, dielectric relaxation strength Δε, etc. also vary depending on the morphology and density of blood cells (see examples below), and from the measurement results obtained through the dielectric constant measurement step (II) By calculating the dielectric relaxation time τ, the Cole-Cole parameter α, and the dielectric relaxation strength Δε, the morphology and density of blood cells can be analyzed. The dielectric relaxation strength Δε value can be substituted by a change in the value of ε ′ at an arbitrary frequency from 10 kHz to 500 kHz without being analyzed by Equation 1.

  As described above, FIG. 2 used in the above description is explained by taking erythrocytes as an example. However, the qualitative and / or quantitative analysis method according to the present invention is not limited to qualitative and / or It can be used for quantitative analysis and is not limited to red blood cells.

  Moreover, the qualitative and / or quantitative analysis method according to the present invention may perform a step of pretreating the collected blood before the electric field application step I as necessary (see reference A in FIG. 1).

  For example, qualitative and / or quantitative analysis of leukocytes can be suitably performed by subjecting the collected whole blood to pretreatment with an erythrocyte hemolyzing agent or the like (see symbol A “collected blood pretreatment step” in FIG. 1). Moreover, qualitative and / or quantitative analysis of platelets can be suitably performed by performing pretreatment step A such as diluting the collected whole blood with an electrolyte solution.

<Blood deterioration detection method>
The qualitative and / or quantitative analysis method according to the present invention can be used for a blood deterioration detection method.

  For example, it is known that red blood cells are usually in the shape of a hollow disc in the center, but gradually change to a spherical shape as the number of storage days elapses after blood collection. Therefore, for example, if the relationship between the morphology of red blood cells and the dielectric relaxation phenomenon is examined in advance, the deterioration state of the stored whole blood is detected by measuring the dielectric relaxation phenomenon of the stored whole blood mainly composed of red blood cells. be able to.

  Preserved whole blood, particularly human whole blood, has an effective period of 21 days after blood collection at a storage temperature of 4 to 6 degrees. However, it is conceivable that the deterioration of blood progresses without waiting for the effective period, not only for the storage period but also for some other reason. In such a case, if the blood deterioration detection method according to the present invention is performed, it is possible to avoid the risk of erroneous use for blood transfusion and blood products.

  On the other hand, even though the effective period has passed, it is considered that there is blood that can be used sufficiently, but since the confirmation work is complicated, even blood that is originally effective is discarded. However, if the blood deterioration detection method according to the present invention is performed, it is possible to detect the deterioration state of individual blood rather than a uniform evaluation of the effective period. It can be used effectively.

  FIG. 7 is a drawing-substituting graph showing the relationship between the frequency and the dielectric constant of the rabbit-stored whole blood for each storage day (see Example 2 described later). As shown in FIG. 7, it can be confirmed that the dielectric relaxation phenomenon varies depending on the storage days. In particular, it can be confirmed that the first to ninth days show almost the same dielectric relaxation phenomenon, but the thirteenth day shows a completely different dielectric relaxation phenomenon. This is considered that blood deterioration progressed between the 9th day and the 13th day. Thus, if the dielectric relaxation phenomenon of blood is analyzed, blood deterioration can be detected.

  As a method of analyzing the dielectric relaxation phenomenon, there is a method of calculating a dielectric relaxation frequency, for example, as in the above-described qualitative and / or quantitative analysis method of blood cells. As shown in FIG. 7, it can be seen that the dielectric relaxation frequency differs for each storage day. Therefore, blood degradation can be detected by calculating the dielectric relaxation frequency of stored blood.

  In addition, as a method of analyzing the dielectric relaxation phenomenon, there is a method of calculating the dielectric relaxation time τ and the Cole-Cole parameter α, similarly to the above-described qualitative and / or quantitative analysis method of blood cells. FIG. 8 is a drawing substitute graph showing the relationship between the storage days and the dielectric relaxation time τ, and FIG. 9 is a drawing substitute graph showing the relationship between the storage days and the Cole-Cole parameter α (see Example 3 described later).

  As shown in FIG. 8, it can be seen that the dielectric relaxation time τ starts to increase from the 21st day. Also, as shown in FIG. 9, it can be seen that the Cole-Cole parameter α starts to decrease from the 21st day. That is, from the values of the dielectric relaxation time τ and the Cole-Cole parameter α, it is shown that the blood after the 21st day is further deteriorated. Therefore, blood deterioration can be detected by calculating the dielectric relaxation time τ and the Cole-Cole parameter α.

  Furthermore, as a method for analyzing the dielectric relaxation phenomenon, there is a method for calculating the dielectric relaxation strength Δε, similar to the above-described qualitative and / or quantitative analysis method for blood cells. FIG. 10 is a drawing-substituting graph showing the relationship between the storage days and the dielectric relaxation strength Δε (see Example 3 described later).

  As shown in FIG. 10, it can be seen that the dielectric relaxation strength Δε decreases with the number of storage days. The amount of decrease in the dielectric relaxation strength Δε has a substantially constant slope as shown by the straight line in FIG. Therefore, blood deterioration can be detected by calculating the dielectric relaxation strength Δε.

  In addition, by calculating the decrease rate of the dielectric relaxation strength Δε with respect to the storage days, the blood deterioration rate can be predicted. For example, when the rate of decrease of the dielectric relaxation strength Δε is larger than the rate of decrease in FIG. 10, it can be seen that the blood deterioration rate is high, and it can be predicted that there is a possibility that the blood is contaminated. As described above, by using the blood deterioration detection method according to the present invention, it is possible to avoid the risk of erroneously using the deteriorated blood in blood transfusions or blood products even within the legal storage period.

  Conversely, if the blood deterioration detection method according to the present invention is used, even blood that has passed the legal storage period can be detected without deterioration, until the blood is essentially deteriorated, It can be used effectively.

  Since the blood deterioration detection method according to the present invention can detect the blood deterioration state only by measuring the dielectric constant, it does not destroy cells such as blood cells in the blood and labels the blood cells in the blood in advance. Therefore, the blood deterioration state can be detected quickly and easily, and the blood used for blood deterioration detection can be used as it is for blood transfusions, blood products and the like. Therefore, even in a situation where the stored blood is insufficient, the stored blood that can be used safely can be identified without wasting the stored blood.

  As described above, the whole blood mainly composed of erythrocytes has been described as an example. However, the method for detecting blood deterioration according to the present invention includes concentrated erythrocytes (RC-MAPP), washed erythrocytes (WRC), leukocyte-removed erythrocytes. (LPRC), thawed erythrocyte concentrate (FTRC), synthetic blood (BET), etc. can also be used for detection of deterioration, and for other detection of deterioration such as concentrated platelet (PC), concentrated platelet HLA (PC-HLA), etc. Can also be used.

  Hereinafter, an example of a method for determining the availability of stored blood using the blood deterioration detection method according to the present invention will be described with reference to FIG. FIG. 11 is a flowchart showing an example of a method for determining the availability of stored blood using the blood detection method according to the present invention.

  In determining whether or not the stored blood can be used using the blood deterioration detection method according to the present invention, first, an electric field applying step (I) for applying an electric field to the stored blood subjected to the pretreatment step A is performed as necessary. Next, a dielectric constant measurement step (II) is performed in which the dielectric constant of the stored blood is measured while changing the frequency. Then, a dielectric relaxation phenomenon analysis step (III) for analyzing the dielectric relaxation phenomenon is performed based on the measurement result obtained through the dielectric constant measurement step (II).

  In the dielectric relaxation phenomenon analysis step (III), for example, during the legal validity period, the dielectric relaxation strength Δε is calculated, and the decrease rate with respect to the storage days is calculated, thereby predicting the deterioration rate of each stored blood. it can. For example, when the rate of decrease is extremely large, the stored blood is likely to be contaminated, and it is desirable to discard it even within the legal validity period.

  In order to further improve safety, it is desirable to calculate the dielectric relaxation frequency and dielectric relaxation time τ of stored blood during use. If the calculation result is out of the reference value, it can be predicted that the stored blood is deteriorating. Therefore, it is desirable to discard it even within the legally valid period. On the other hand, when the calculation result is within the standard, it can be used for blood transfusion, blood product and the like with peace of mind.

  When the legal validity period has passed, the stored blood has been uniformly discarded from the viewpoint of safety, but if the blood deterioration detection method according to the present invention is used, the stored blood is essentially deteriorated. It is possible to use effectively no stored blood.

  For example, in the same manner as described above, the dielectric relaxation frequency and dielectric relaxation time τ of stored blood that has passed the legal effective period are calculated. If the calculation result is out of the standard, it can be predicted that deterioration of the stored blood has progressed, so it must be discarded, but if the calculation result is within the standard, the legal expiration date has passed. It can be used safely. In this case, for example, it can be used for research purposes.

  In Example 1, it was examined whether the dielectric relaxation phenomenon differs depending on the shape of blood cells. Rabbit erythrocytes were used as an example of blood cells.

  First, the shape of red blood cells was changed to normal form 1 (see FIG. 3), sea urchin form 2 (see FIG. 4), expanded form 3 (see FIG. 5), and spherical form 4 (see FIG. 6) by pH control.

Next, an electric field was applied at a frequency of 10 ⁵ Hz at 25 ° C. and almost the same blood cell density (hematocrit value). The dielectric constant was measured while sweeping in the range of 10 ⁵ to 10 ⁸ Hz. The measurement results are shown in FIG.

  As shown in FIG. 2, each shape of red blood cells shows a high dielectric constant value on the low frequency side but drops to a low value on the high frequency side (dielectric relaxation phenomenon), but shows different dielectric relaxation phenomena. The data in FIG. 2 is obtained by correcting the contribution of electrode polarization by the method of Asami et al. (Reference: K. Asami, A. Irimajiri, T. Hanai, N. Shiraishi, K. Utsumi, Biochim. Biophys. Acta). 778, 559, 1984.).

From the measurement results of FIG. 2, each dielectric relaxation frequency, dielectric relaxation time τ, and dielectric relaxation strength Δε were calculated. Each calculation result is shown in Table 1.

  Further, the blood cell density (hematocrit value) of the spherical red blood cells 4 was changed, and the dielectric relaxation strength Δε was obtained in the same manner as described above. FIG. 12 shows the relationship between the dielectric relaxation strength Δε and the blood cell density (hematocrit value).

  In Example 1, it was found that different dielectric relaxation phenomena were exhibited depending on the form of blood cells (red blood cells) and the difference in blood cell density (hematocrit value). It was also found that the dielectric relaxation frequency, dielectric relaxation time τ, and dielectric relaxation strength Δε calculated from the dielectric relaxation phenomenon show different values depending on the blood cell morphology and blood cell density (hematocrit value). Therefore, it was found that by analyzing the dielectric relaxation phenomenon of blood cells, the morphology and blood cell density (hematocrit value) of blood cells can be analyzed.

  In Example 2, it was examined whether the qualitative and / or quantitative analysis method according to the present invention can be used for a blood deterioration detection method.

  First, rabbit-preserved blood (by Alsaver's solution) purchased from Kojin Bio Inc. (5-1-3 Chiyoda, Sakado City, Saitama Prefecture) was washed with a pH-controlled PBS buffer to prepare normal erythrocytes. Stored blood was obtained. The adjusted stored blood was stored at 5 ° C.

  Next, on the first day, the second day, the third day, the sixth day, the seventh day, the eighth day, the ninth day, and the thirteenth day, in the same manner as in Example 1, the dielectric of the adjusted stored blood The rate was measured. The measurement results are shown in FIG.

  As shown in FIG. 7, it can be confirmed that the first to ninth days show almost the same dielectric relaxation phenomenon, but the thirteenth day shows a completely different dielectric relaxation phenomenon.

  Moreover, the state of the prepared and preserved blood on the first day and the thirteenth day is shown in FIG. As shown in FIG. 13, when the adjusted and preserved blood on the 13th day was observed, discoloration was seen and it was completely deteriorated.

  In Example 2, it was found that when the deterioration of blood progresses as the storage days elapse, a different dielectric relaxation phenomenon is exhibited. It was also found that the dielectric relaxation frequency, dielectric relaxation time τ, and dielectric relaxation strength Δε calculated from the dielectric relaxation phenomenon show different values as the blood deteriorates as the storage days elapse. Therefore, it has been found that the qualitative and / or quantitative analysis method according to the present invention can be used for detection of blood deterioration.

  In Example 3, it was examined whether the qualitative and / or quantitative analysis method according to the present invention can be used for a blood deterioration detection method for unadjusted blood.

  First, the rabbit preserved blood purchased from Kojin Bio Inc. (according to Mr. Alsaber) was preserved at 5 ° C. without adjustment. Next, 1st day, 2nd day, 3rd day, 4th day, 7th day, 8th day, 9th day, 10th day, 14th day, 15th day, 16th day, 18th day On the 21st day, 22nd day, 23rd day, 24th day, 28th day, 29th day, 30th day, 31st day, 35th day, 36th day, the same method as in Example 1 The dielectric constant of the adjusted stored blood was measured. The measurement results are shown in FIG.

  As shown in FIG. 14, it can be confirmed that the dielectric relaxation phenomenon varies depending on the storage days.

From the dielectric relaxation phenomenon, the dielectric relaxation frequency, dielectric relaxation time τ, and dielectric relaxation strength Δε after each storage period were calculated. Table 2 shows the respective calculation results. FIG. 8 shows the relationship between the storage days and the dielectric relaxation time τ, FIG. 9 shows the relationship between the storage days and the Cole-Cole parameter α, and FIG. 10 shows the relationship between the storage days and the relaxation strength Δε.

  As shown in FIG. 8, it can be seen that the dielectric relaxation time τ starts to increase from the 21st day. Also, as shown in FIG. 9, it can be seen that the Cole-Cole parameter α starts to decrease from the 21st day. That is, from the values of the dielectric relaxation time τ and the Cole-Cole parameter α, it is shown that the blood after the 21st day is further deteriorated.

  In Example 3, it was found that the presence or absence of blood deterioration can be detected by analyzing the dielectric relaxation phenomenon even for unadjusted blood.

  Further, as shown in FIG. 10, it can be seen that the dielectric relaxation strength Δε decreases with the storage days. The amount of decrease in the dielectric relaxation strength Δε has a substantially constant slope as shown by the straight line in FIG.

  Therefore, in Example 3, it was found that the blood degradation rate can be predicted by calculating the dielectric relaxation strength Δε from the dielectric relaxation phenomenon and calculating the decrease rate with respect to the storage days.

  The qualitative and / or quantitative analysis method according to the present invention is a completely new method capable of performing qualitative and / or quantitative analysis of blood cells only by measuring the dielectric constant, and may damage blood cells and the like. And there is no need to pre-label blood cells. Therefore, qualitative and / or quantitative analysis of blood cells can be performed quickly and easily.

  The qualitative and / or quantitative analysis method for blood cells according to the present invention can be applied to novel diagnostic techniques for various diseases. Moreover, the qualitative and / or quantitative analysis method for blood cells according to the present invention can be easily used for a method for detecting deterioration of stored blood.

  By using the blood deterioration detection method according to the present invention, it is possible to individually detect the deterioration of stored blood within the effective period that could be used uniformly. The use of stored blood that has deteriorated due to the cause can be prevented. In addition, it is possible to individually detect the deterioration of stored blood after the expiration date for which the use was uniformly restricted, so that it is possible to detect the blood effectively without destroying the blood that is essentially effective. And can contribute to further development of medical field and research field.

It is a flow figure of the qualitative and / or quantitative analysis method concerning the present invention. In Example 1, it is a drawing substitute graph which shows the relationship between the dielectric constant and frequency of four types of red blood cells from which a shape differs. It is a drawing substitute photograph which shows normal form erythrocytes. FIG. 2 is a drawing-substituting photograph showing urchin-shaped (garlic, scalloped, mined) red blood cells. It is a drawing-substituting photograph showing expanded erythrocytes. It is a drawing substitute photograph which shows a spherical red blood cell. In Example 2, it is a drawing substitute graph which shows the relationship between the dielectric constant for every preservation | save days of a rabbit preservation | save whole blood (after adjustment), and a frequency. In Example 3, it is a drawing substitute graph which shows the relationship between storage days and dielectric relaxation time (tau). In Example 3, it is a drawing substitute graph which shows the relationship between storage days and Cole-Cole parameter (alpha). In Example 3, it is a drawing substitute graph which shows the relationship between storage days and dielectric relaxation intensity | strength (DELTA) epsilon. It is a flowchart which shows an example of the determination method of the availability of the preservation | save blood using the blood detection method which concerns on this invention. 6 is a drawing substitute graph showing the relationship between dielectric relaxation strength Δε and blood cell density (hematocrit value). In Example 2, it is a drawing substitute photograph which shows the mode of the preparation preservation | save blood of the 1st day and the 13th day. In Example 3, it is a drawing substitute graph which shows the dielectric constant and the relationship of a frequency for every preservation | save day of rabbit preservation | save whole blood (unadjusted).

Explanation of symbols

1 Normal shape red blood cell 2 Sea urchin shape (garage, scallop, mines) Red blood cell 3 Expanded red blood cell 4 Spherical red blood cell

Claims (8)

A method for qualitative and / or quantitative analysis of blood cells,
An electric field application step of applying an electric field whose frequency is variable to the collected blood;
A dielectric constant measurement step of measuring the dielectric constant of the collected blood while changing the frequency of the electric field;
Dielectric relaxation phenomenon analysis step of analyzing the dielectric relaxation phenomenon from the measurement result obtained through the dielectric constant measurement step,
A method for qualitative and / or quantitative analysis of blood cells containing at least   The qualitative and / or quantitative analysis method according to claim 1, wherein the dielectric relaxation phenomenon analysis step is a step of calculating a dielectric relaxation frequency.   The qualitative and / or quantitative analysis method according to claim 1, wherein the dielectric relaxation phenomenon analysis step is a step of calculating a dielectric relaxation time.   The qualitative and / or quantitative analysis method according to claim 1, wherein the dielectric relaxation phenomenon analysis step is a step of calculating a dielectric relaxation strength.   The method for qualitative and / or quantitative analysis of blood cells according to claim 1, wherein the blood cells are red blood cells.   The method for qualitative and / or quantitative analysis of blood cells according to claim 1, wherein the blood cells are leukocytes.   The method for qualitative and / or quantitative analysis of blood cells according to claim 1, wherein the blood cells are platelets.   A blood deterioration detection method using the analysis method according to claim 1.