JP3118625B2 - Blood cell separation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for separating blood cells from plasma (and / or serum) and blood cells from whole blood collected from a human body.

[0002]

BACKGROUND OF THE INVENTION Blood is composed of red blood cells, white blood cells, platelets and plasma (fibrinogen and serum). When blood is put into, for example, a test tube and left at room temperature for 5 to 10 hours, red blood cells, white blood cells, and platelets form a blood clot and settle at the lower part, and plasma (and / or serum) becomes the upper part and separates spontaneously. In order to artificially separate the plasma (and / or serum) and the blood cells, whole blood is conventionally put into a test tube, applied to a centrifuge, and subjected to separation using centrifugal force.

[0003]

The above-mentioned conventional separation method using a centrifuge not only requires a long time to separate plasma (and / or serum) and blood cells, but also uses a test tube containing blood. Each tube must be attached to a centrifuge, and the number of test tubes to be loaded into the centrifuge is limited. Whole blood collected from many people is separated into plasma (and / or serum) and blood cells. There were difficult problems to do.

[0004] The present invention has been made in view of the above points, and eliminates the above-mentioned problems. A plasma which can separate plasma (and / or serum) from blood cells with the lapse of a predetermined time only by adding and mixing a reagent. (And / or serum) and a blood cell separation method.

[0005]

In order to solve the above-mentioned problems, the present invention provides a method for preparing dextran, comprising:
It is characterized by adding and mixing vancomycin or a compound having a vancomycin structural formula, and separating plasma (and / or serum) and blood cells after a predetermined time.

In addition, dextran, vancomycin or a compound having the structural formula of vancomycin, and chloroquine are added to blood collected from a human body and mixed therewith, and plasma (and / or serum) and blood cells are separated after a predetermined time. It is characterized by.

Further, dextran, vancomycin or the compound having the structural formula of vancomycin, chloroquine, is dissolved in any one of physiological saline, water, and a buffer having the same properties as physiological saline, and then added and mixed. Features.

[0008] A predetermined amount of a solution obtained by mixing and dissolving dextran and vancomycin or a compound having the structural formula of vancomycin in a solvent in a predetermined amount is placed in a blood collection tube, and the solvent is volatilized. , Vancomycin or a compound having the structural formula of vancomycin is adhered to the inner wall of the blood collection tube, a predetermined amount of blood collected from the human body is put into the blood collection tube, and plasma (and / or serum) and blood cells are separated by a predetermined time. It is characterized by being separated.

A predetermined amount of a solution prepared by mixing and dissolving dextran, vancomycin or a compound having the structural formula of vancomycin, and chloroquine in a predetermined ratio in a solvent is introduced into a blood collection tube, and the solvent is volatilized to obtain a predetermined amount and a predetermined amount. Ratio of dextran, vancomycin or a compound having the structural formula of vancomycin, and chloroquine are adhered to the inner wall of the blood collection tube, and a predetermined amount of blood collected from a human body is put into the blood collection tube, and plasma (and / or Or serum) and blood cells.

[0010] The means for volatilizing the solvent is characterized by hot air drying or freeze drying.

[0011]

It is said that the aggregation of, for example, red blood cells in whole blood has the following five reasons. Reduces the zeta potential present on the surface of red blood cells. Change the specific potential constant of the cell suspension. Increase the cell surface area. Physically reduce the distance between cells. Changes the direction or distribution of the protein relative to the cell surface.

[0012] As described above, dextran and vancomycin or a compound having the structural formula of vancomycin are added to and mixed with whole blood collected from the human body. It is presumed that dextran acts to weaken the existing zeta potential, and that dextran forms a polymer bridge at different locations in the same cell, physically shortens the distance between cells and promotes aggregation. As a result, aggregation of blood cells in whole blood is promoted, and plasma (and / or serum) and blood cells are separated.

[0013] As described above, dextran and vancomycin or a compound having the structural formula of vancomycin are added to and mixed with whole blood collected from the human body to promote the aggregation of blood cells in whole blood and to increase plasma (and / or serum). And blood cells are separated, but about 20% of the sample does not coagulate, and the agglutination of the aggregated blood cells is weak, and the blood cells soar into the upper layer plasma (and / or serum) with a slight vibration. There is a problem. Thus, if chloroquine is added and mixed in addition to the above dextran and vancomycin or a compound having the vancomycin structural formula, all the specimens agglutinate rapidly in time, and the agglutinated blood cell portion is strengthened, so that the blood cell does not rise to the upper layer. . This is presumed to be due to the fact that chloroquine synergistically weakens the ion repulsion of blood cells and makes blood cell aggregation more robust.

Further, dextran, vancomycin or a compound having the structural formula of vancomycin is dissolved in a solvent, a predetermined amount of the solution is placed in a blood collection tube, and the solvent is evaporated to leave a mixture of dextran, vancomycin or a compound having the structural formula of vancomycin. When blood collected from the human body is put into the blood collection tube, dextran, vancomycin or a compound having the structural formula of vancomycin dissolves in the blood, and plasma (and / or serum) and blood cells are formed by the same action as described above. Separated. Furthermore, when a solution of chloroquine is added to cause residual adhesion, hemagglutination is accelerated and aggregation is strengthened.

[0015]

Embodiments of the present invention will be described below. The method of separating plasma (and / or serum) from blood cells is performed by adding the following first and second reagents to 1 milliliter (ml) of whole blood collected from a human body and placed in a test tube. Was.

The first reagent was mixed with 10 milligrams (mg) of vicomycin and dissolved in 1 ml of physiological saline, and 30 microliters (μl) was mixed and dissolved in 400 mg of the second reagent dextran with 1 ml of physiological saline. 25 μl of reagent,

To a test tube containing 1 ml of whole blood as described above, add 30 μl of the first reagent and 25 μl of the second reagent, invert the test tube about several times for contamination, and The test tube was set up and left at room temperature. As a result, plasma (and / or serum) and blood cells could be separated usually within 10 minutes. In addition, it takes 15 to 45 minutes for blood cells to completely separate from plasma (and / or serum).

A typical example in which blood cells are completely separated from plasma (and / or serum) with and without the use of the above reagent is shown below. The same whole blood sample is used here. When neither the first reagent nor the second reagent is added ...
········································································································ 50 minutes
·············· 51 minutes when first reagent + second reagent are added
… 28 minutes From this result, it can be seen that the first reagent obtained by mixing and dissolving vancomycin and physiological saline in whole blood and the second reagent obtained by mixing and dissolving dextran and physiological saline are added and mixed at an early stage. It can be seen that blood cells separate from plasma (and / or serum).

The following phenomena occur when examining how the separation of plasma (and / or serum) and blood cells becomes faster when the first reagent and the second reagent are added to and mixed with whole blood. It can be guessed that. It is said that the aggregation of blood cells in whole blood has the above five reasons.

Although the mechanism of hemagglutination is not clear, vancomycin does not act as a protein conjugate on the surface of blood cells, but is a polycation (multi-positive) compound. It is presumed to have the effect of weakening Also, dextran does not increase the zeta potential and does not affect the non-potential constant of the water-soluble suspension, because it is a non-electrode substance, but it probably forms polymer bridges at different places in the same cell, It is presumed that the above-mentioned action of physically shortening the distance between cells and promoting aggregation is exhibited. As a result, the action of vancomycin and dextran promotes the agglutination of blood cells in whole blood, resulting in rapid separation of plasma (and / or serum) and blood cells.

The mixing ratio of vancomycin and saline in the first reagent, the mixing ratio of dextran and saline in the second reagent, and the first
And the addition and mixing ratio of the second reagent is an example, and the present invention is not limited to this.
The features of the present invention are not lost.

The mixing ratio of vancomycin and physiological saline (vancomycin / physiological saline) in the first reagent is as follows:
Approximately 10 mg / ml is optimal. The optimal mixing ratio of dextran and saline in the second reagent (dextran / saline) is about 400 mg / ml. The optimal mixing ratio of the first reagent and the second reagent to 1 ml of whole blood is about 30 μl of the first reagent and about 25 μl of the second reagent.

In the above example, an example was shown in which vancomycin was used as the first reagent and dextran was used as the second reagent.
The first reagent, vancomycin, is not limited to this, and it has been experimentally confirmed by the present inventor that it is only necessary that vancomycin or a compound having the vancomycin structural formula be used. Further, vancomycin or a compound having vancomycin structural formula and dextran are dissolved in a solvent, respectively, to form a first reagent and a second reagent.
When vancomycin or the compound having vancomycin structural formula and dextran are easily dissolved in whole blood, it is not necessary to dissolve them in a solvent, and they may be directly added and mixed into whole blood.

By adding dextran and vancomycin or a compound having the structural formula of vancomycin to whole blood collected from a human body as described above, aggregation of blood cells in whole blood is promoted, and plasma (and / or serum) is added to whole blood. Blood cells are separated. However, about 20% of the sample does not agglutinate, and the agglutination of the agglutinated blood cells is weak. As shown in FIG. 1, the blood cells 2 that have condensed and settled in the lower part of the blood collection tube 1 are subjected to only a slight vibration. As shown in FIG. 2a, there is a problem in that the soot is soaked into the upper layer plasma (and / or serum) 3 and mixed therein.

Therefore, as a result of repeated experiments by the inventor of the present application,
When chloroquine is added and mixed in addition to the dextran and the compound having vancomycin or vancomycin structural formula, all the specimens are aggregated quickly in time, and the aggregated blood cell portion is strengthened to prevent rising to the upper layer. confirmed. Considering the reason, it is presumed that chloroquine synergistically weakens the ion repulsion of blood cells and makes hemagglutination more robust.

FIG. 2 shows the aggregation time of whole blood. In the figure, curve A is a case where physiological saline is added to whole blood and mixed (whole blood + saline), and curve B is a case where vancomycin or a compound having vancomycin structural formula and dextran are added to whole blood ( Whole blood + vancomycin or a compound having vancomycin structural formula + dextran),
Curve C shows the case where vancomycin or a compound having vancomycin structural formula is added to whole blood, dextran and chloroquine are added (whole blood + compound having vancomycin or vancomycin structural formula + dextran + chloroquine), respectively.

As shown in the figure, almost 100% of the hemagglutination occurs after 30 minutes in both the curves B and C, but the hemagglutination is more strongly agglutinated in the curve C (with chloroquine added). Here, in the curve B, the mixing ratio of vancomycin or vancomycin and physiological saline, and the mixing ratio of dextran and physiological saline are determined using the above-mentioned optimal first and second reagents, with respect to 1 ml of whole blood. About 30 μl of the first reagent and about 25 μl of the second reagent were added. In the curve C, about 50% of a reagent obtained by mixing and dissolving 8.26 milligrams (mg) of chloroquine with 1 milliliter (ml) of physiological saline was added. (Μl).

In the above embodiment, the following first and second reagents are added and mixed into blood collected from a human body. However, the method for separating blood cells of the present invention is not limited to this. . As shown in FIG. 3, vancomycin and dextran are mixed and dissolved in a predetermined ratio in a solvent such as water, physiological saline or a buffer solution of the same nature in the blood collection tube 1, and the solution is put into the blood collection tube and dried with a known hot air. The solvent is volatilized by freeze-drying, and a predetermined amount and a predetermined ratio of vancomycin or a mixture 4 of a compound having the structural formula of vancomycin and dextran are adhered to the inner wall of the blood collection tube 1, and a human body is inserted into the blood collection tube 1. By adding a predetermined amount of collected blood, plasma (and / or serum) and blood cells can be separated over a predetermined time.

When vancomycin or a compound having the structural formula of vancomycin or dextran is directly added to blood collected from the human body, it may not be dissolved in the blood. However, once dissolved in a solvent, the solvent is evaporated and the residual solvent is evaporated. Vancomycin and dextran have the property of easily dissolving in blood. Therefore, when the mixture of vancomycin and dextran is allowed to adhere to the inner wall of the blood collection tube 1 by evaporating the solvent as described above, the blood collection from the human body into the blood collection tube has vancomycin or vancomycin structural formula. The compound and dextran dissolve smoothly in the blood. As a result, plasma (and / or serum) and blood cells are separated after a predetermined time. It should be noted that chloroquine is added and mixed by the above-described method and remains adhered to the inner wall of the blood collection tube 1, whereby blood cells are aggregated strongly.

The ratio of the amount of the mixture 4 of vancomycin or a compound having vancomycin structural formula, dextran and chloroquine attached to the inner wall of the blood collection tube 1 and the ratio of the amount of blood to be put into the blood collection tube 1 becomes a predetermined value. (And / or serum) and blood cells are set so that they can be effectively separated. As a result, a predetermined amount and a predetermined ratio of vancomycin or a compound 4 having the structural formula of vancomycin, dextran and chloroquine adhere to the inner wall of the blood collection tube 1.

To explain an example of the hot air drying, water, physiological saline or a buffer having the same properties are used as a solvent, vancomycin or a compound having a structural formula of vancomycin, dextran and chloroquine are dissolved in the solvent, and this solution is used. After being put into the fixed-volume blood collection tube 1, the solvent is volatilized by hot air. As a result, a predetermined amount and a predetermined ratio of vancomycin or a mixture 4 of a compound having vancomycin structural formula, dextran and chloroquine adhere to the blood collection tube 1 and are enclosed.

One example of freeze-drying is to put a predetermined amount of vancomycin or a compound having the structural formula of vancomycin, dextran and chloroquine dissolved in water into a blood collection tube 1 and freeze it with liquid nitrogen or an ultra-freezer. When the blood collection tube is lightly capped and put in a freeze dryer, water is sublimated under a high vacuum, and the pressure is gradually reduced, whereby the internal pressure of the blood collection tube 1 is reduced and the cap is closed tightly.

[0033]

As described above, according to the present invention, the following excellent effects can be obtained. (1) A simple operation of adding and mixing vancomycin or a compound having vancomycin structural formula, dextran and a predetermined ratio to blood collected from a human body for a predetermined time without using a centrifuge as in the conventional method. With the passage of time, plasma (and / or serum) and blood cells can be separated. Furthermore, by adding and mixing chloroquine, separation of blood cells is fast and aggregation is strong.

(2) In addition, if the solvent is volatilized and a predetermined amount of vancomycin or a compound having vancomycin structural formula and dextran are adhered to the inner wall of the blood collection tube, the plasma ( And / or serum) can be separated from blood cells. Thus, by preparing in advance a number of blood collection tubes having dextran attached to such a vancomycin or a compound having the vancomycin structural formula, the plasma ( And / or serum) and blood cells are extremely easily separated. Further, by adding chloroquine and attaching the mixture of vancomycin or a compound having the structural formula of vancomycin, dextran and chloroquine to the inner wall of the blood collection tube, blood cells can be separated quickly and coagulation becomes strong.

[Brief description of the drawings]

FIG. 1 is a view showing a state of separation and aggregation of plasma (and / or serum) and blood cells in a blood collection tube.

FIG. 2 is a diagram showing a relationship between aggregation of whole blood and time.

FIG. 3 is a diagram showing a state in which vancomycin or a mixture of a compound having vancomycin structural formula and dextran is attached to the inner wall surface of a blood collection tube.

[Explanation of symbols]

 Reference Signs List 1 blood collection tube 2 blood cell 3 plasma (and / or serum) 4 mixture

──────────────────────────────────────────────────続 き Continued on the front page (56) References JP-A-7-124252 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 35/14 B01D 21/01 CA (STN ) MEDLINE (STN) EMBASE (STN)

Claims (6)

(57) [Claims] 1. Dextran and vancomycin or a compound having the structural formula of vancomycin are added to blood collected from a human body and mixed therewith.
Or serum) and blood cells. 2. A method in which dextran, vancomycin or a compound having a structural formula of vancomycin, and chloroquine are added to blood collected from a human body, and plasma (and / or serum) and blood cells are separated after a predetermined time. A method for separating blood cells, comprising: 3. The dextran, vancomycin or chloroquine, a compound having the structural formula of vancomycin, is dissolved in any one of physiological saline, water, and a buffer having the same properties as physiological saline, and then added and mixed. The method for separating blood cells according to claim 1 or 2, wherein the blood cells are separated. 4. A predetermined amount of a solution obtained by mixing and dissolving dextran and vancomycin or a compound having the structural formula of vancomycin in a solvent in a predetermined amount is placed in a blood collection tube, and the solvent is volatilized. And vancomycin or a compound having a vancomycin structural formula is adhered to the inner wall of the blood collection tube, and a predetermined amount of blood collected from a human body is put into the blood collection tube, and plasma (and / or serum) and blood cells are collected over a predetermined time. A method for separating blood cells, comprising separating blood cells. 5. A predetermined amount of a solution obtained by mixing and dissolving dextran, vancomycin or a compound having the structural formula of vancomycin, and chloroquine in a predetermined ratio in a solvent is introduced into a blood collection tube, and the solvent is volatilized. Ratio of dextran, vancomycin or a compound having the structural formula of vancomycin, and chloroquine are adhered to the inner wall of the blood collection tube, and a predetermined amount of blood collected from a human body is put into the blood collection tube, and plasma (and / or Or serum) and blood cells. 6. The method for separating blood cells according to claim 4, wherein the means for volatilizing the solvent is hot-air drying or freeze-drying.