JPS60115857A - Separation of serum and blood clot - Google Patents

Abstract

PURPOSE:To accelerate blood clotting and to improve sepn. of a blood-clot and serum and the yield of the serum by adding borate of specific bivalent or higher valency metallic ion to a blood and subjecting the blood to a centrifugal sepn. CONSTITUTION:Borate of vibalent or higher valency metallic ion selected from the group of Cu<2+>, Co<2+>, Fe<2+>, Ni<2+>, Mg<2+> and Ce<3+> as a blood clotting accelerator is added to a blood at <=1X10<-10>-10<-1>g for each 1ml the blood to clot the blood, then the blood is subjected to a centrifugal sepn. The clotting is completed within 30- 60min by using either glass or PE spitz for the blood vessel and the shrink characteristic of the blood-clot is good. The serum contg. no blood clot component is obtd. with a good yield by the centrifugal sepn. If said borate is added in the form of powder or concd. soln., there is the possibility of causing a deterioration in the properties of the protein component in the blood and if the soln. is too thin, the serum is diluted, thus hindering the inspection of the serum. It is therefore a good practice to disperse the borate deposited on a suitable carrier into 1.0wt% physiological salt soln. and adding such soln. to the blood.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for separating serum and blood ωF.
The present invention relates to a method for separating serum and blood clots from a whole blood sample of a subject by centrifugation.

Coupled with the remarkable progress in testing technology in recent years, blood tests have been made. jBlood tests such as biochemical tests, serum immunological tests, and hematology tests have become widely used and have greatly contributed to disease prevention and early diagnosis. Among these, serum tests are the main body of blood tests, and the serum required for this test is usually obtained by collecting blood into a blood test container, coagulating it, and then centrifuging it to produce blood clots with different specific gravities. That is, a gel-like mass containing fibrin and blood cells is separated, and the serum portion is placed in a bibet 1,
It is collected by sucking it up or by decantation.

However, blood generally takes some time to coagulate, making it difficult to conduct tests quickly. Even with glass blood test containers, which are said to have the shortest blood coagulation time, it takes 40 to 60 minutes for blood to coagulate after blood is injected.As for blood test containers made of synthetic resin, It takes 4 hours or more for blood to coagulate.

Furthermore, conventionally, when pure serum for use in testing is collected by phase-separating coagulated whole blood into serum and blood clots by centrifugation, the separability of serum is generally not good. Therefore, as mentioned above, even when the serum portion is pipetted or decanted, contamination with red blood cells is unavoidable even when careful care is taken, which may have a detrimental effect on clinical test results. There are also other problems, such as the need for centrifugation again.

The present invention has been made in order to solve the above-mentioned problems, and it is possible to significantly shorten the time required for blood coagulation and to improve the separation between serum and blood clot components. The purpose is to provide a separation method for

The present invention provides a method for separating blood into serum and blood clots by centrifugation, in which a metal salt of oxalic acid is used, the metal ion is divalent or higher, and the salt is present in the blood. This is a characteristic feature.

In the method of the present invention, blood is collected in a blood test container, and when the blood is coagulated, the oxalate is present in the blood as a blood coagulation promoter, but the blood test container is not particularly limited. , containers made of glass or resin that have been conventionally used may be used as appropriate.

The divalent or higher-valent metal ion in the oxalate used as a blood coagulation promoter in the method of the present invention is a metal ion selected from alkaline earth metals, transition metals, and rare earth metals, and has an ionic radius of 0. .50~1.
It is necessary to be within the range of 0.04 people. Preferably Cu
, Co, Fe.

Ni2-+, Mg2+, Ce6'', etc. are used. On the other hand, Mn has a small ionic radius and is not suitable for use.

Furthermore, since Ca2+, La''+ and the like have large ionic radii, they are not suitable for use in the present invention.

The present inventor already has carbonyl groups adjacent to each other in the molecule,
Furthermore, it was revealed that a group of cyclic organic compounds, which are three-dimensionally located on substantially the same plane, have a specific action as a blood coagulation factor, which is a protein (Japanese Patent Application No. 58-11). (No. 4659), when the metal ions forming oxalate are 2 fflli or more and the ionic radius is within the predetermined range as described above, oxalate ions can form via divalent metal ions. As a result of forming a chelate-like salt structure and inhibiting free rotation of the carbon-carbon bond, the molecule has adjacent carbonyl groups, which are sterically located on substantially the same plane. Although its mechanism of action is not clear, it appears to have a specific effect on blood coagulation factors in the same way as described above.

Therefore, when the metal ion is too small or too large, the carbon-
It probably does not act as a blood coagulant because it causes distortion in the carbon bonds, resulting in the inability of adjacent carbonyl groups to form substantially the same plane. In addition, when the metal ion is monovalent, the carbon-carbon bond in oxalate can rotate freely, and due to steric repulsion between carbonyl groups and electrostatic repulsion between two metal ions, To,
Since adjacent carbonyl groups are not on substantially the same steric plane, they probably do not have a specific effect on blood coagulation.

Incidentally, monovalent metal salts of oxalic acid, such as potassium oxalate and sodium oxalate, dissociate in the blood and form Ca2, which is an important blood coagulation factor and has a smaller tendency to ionize than K and Na. As a result, the coagulation mechanism of blood depleted of Ca2+ is inhibited, and therefore monovalent metal salts have been widely used as anticoagulants for blood.

In the present invention, in order to have the above-mentioned oxalate present in the blood as a blood coagulation promoter, it is added to the blood in the form of a powder as it is, or dissolved or dispersed in an appropriate solvent. However, if blood momentarily or partially comes into contact with high concentrations of these oxalates in a blood test container, protein components in the blood may be denatured, so use a carrier with a large specific surface area. It is preferable to carry oxalate on the oxalate and add this to the 1111 liquid in the blood test container. According to this method, oxalate is dispersed in the blood in a small uniform concentration, and unlike the case where a solution or dispersion of oxalate is diluted and added to the blood, the blood is diluted and tested. It does not cause any trouble.

As the carrier, various carriers can be used without particular limitation as long as they do not have a harmful effect on blood tests and have a large specific surface area, but for example,
Nonwoven fabrics, woven fabrics, resin beads, etc. can be suitably used. In order to support oxalate on such a carrier, for example, a solution or dispersion of the oxalate may be applied or immersed therein, followed by drying and adhering to the carrier. Further, particles supporting oxalate can also be obtained by mixing with an appropriate auxiliary agent such as gum arabic to form an aqueous dispersion, and then rapidly freeze-drying the resulting aqueous dispersion.

The amount of oxalate present in the blood as a blood coagulation promoter is at least 1 x '10-1 per ml of blood.
When the amount is less than 0g, the effect of promoting blood coagulation is poor. However, if it is present in too large a quantity, it may actually cause various problems in blood tests, so it is preferable to limit the amount to 10 g or less.

According to the method of separating blood serum and blood clots of the present invention, as described below, since oxalate as described above is present in the blood as a blood coagulation promoter, blood coagulation factors are rapidly removed. The time required for coagulation after blood is collected into a container is significantly shortened, and the separation of serum and blood clots is significantly facilitated, thus solving the problem of blood clot components being mixed in the separated and collected serum. will also be resolved. Further, according to the method of the present invention, blood clot components are sufficiently contracted, and as a result, the yield of serum is significantly increased.

The present invention will be explained below with reference to Examples, but the present invention is not limited to these Examples in any way.

Example 1 Mn salt, Cu salt, Ce salt, Fe salt, Ni salt of oxalic acid,
1.0 of each of Mg salt, Ce salt, Ca salt and La salt
A weight percent physiological saline dispersion was prepared.

5m of fresh blood per person in a commercially available 10m1 polyethylene spittoon
Next, add 50 μl of the above oxalate dispersion, stir gently, and leave at 20°C. Blood loses fluidity and serum begins to ooze out from part of the blood clot. The time was defined as the blood coagulation time, and immediately thereafter, centrifugation was performed at 3000 rpm for 5 minutes to observe the state of serum separation, and the supernatant serum was collected with a pipette 1, and the amount was defined as the serum yield.

The results are shown in the table.

Comparative Example 5 ml of fresh blood per person was injected into a commercially available 10 ml Spitz made of glass and polyethylene, and as in Example 7, the blood coagulation time and serum separation state were observed, and the serum yield was determined. The results are shown in the table.

Patent Applicant: Sekisui Chemical Co., Ltd. r1 Representative: Motoli Fujinuma Written Amendment to Procedures (Own Ship 1. Matters 11 Displayed in 1939)) Patent Application No. 225265 2, Title of Invention: Serum and Blood Clots Separation method 3, relationship with axle loader ('l) Patent applicant Postal code: 530 Address: 2-4-4 Nishitenma, Kita-ku, Osaka, Japan Patent Department Tokyo resident TE+, (03) 434-95524, Amendment Full text of the subject specification 5. Contents of amendment The full text of the specification attached to the IJI document will be amended as shown in the attached document.

6. Amended specification of list of attached documents (1 or more amended specification) Title of the invention Method for separating serum and blood clots 2 Feature f II: Claims +1+ Separating blood into serum and blood clots by centrifugation A method for separating serum and blood clots, the method comprising causing a metal salt of succinic acid to be present in the blood.

3. Detailed Description of the Invention] The present invention relates to a method for separating finished 1it serum and blood clots, and specifically relates to a method for separating blood t# and blood clots from a whole surface sample of a subject by centrifugation.

In recent years, with the remarkable progress in testing technology, blood tests such as serum chemistry tests, serum immunology tests, and hematology tests have become widespread, and have greatly contributed to disease prevention and early diagnosis. Among them, serum testing is the main body of blood testing.The serum required for this test is usually obtained by collecting blood in a blood test volume, coagulating it, and then centrifuging it to collect blood with different specific gravities. The mochi, that is, a gel-like mass containing a mixture of fibrin and blood cells, is separated, and the serum portion is collected by sucking it up with a pipette or by decantation.

However, it generally takes a considerable amount of time for blood to coagulate, making it difficult to perform tests quickly. Glass blood test containers, which are said to have the shortest blood coagulation time, Even after blood is added, it takes 40 to 60 minutes for it to coagulate, and synthetic resin blood test containers need to be left for 4 hours or more for blood to coagulate.

Furthermore, conventionally, in VC, in which pure serum for use in testing is collected by phase-separating the entire coagulated surface into serum and blood clots by centrifugation, the separability of serum is generally good. Therefore, as mentioned above, whether the serum portion is pipetted or decanted, contamination with red blood cells is unavoidable even with the utmost care, and there is no possibility that it will have a detrimental effect on the clinical test results. There are also problems such as the need for centrifugation again.

The present invention has been made in order to solve the above-mentioned NJJ problem, and it is possible to significantly shorten the time required for blood coagulation and to produce serum and blood clots that can effectively separate serum components and blood clot components. The purpose is to provide a method for separating

In the method of separating serum, blood clots, and VC from blood by centrifugation, a metal salt of succinic acid is used, and the metal ions constituting the metal salt are Cu2+ and Co2+.
, Fe2+ , N,2+ , , g2+ and ce”
This method is characterized in that succinate, which is a divalent or higher valent metal ion selected from co'm, is present in the blood.

In the method of R. Kinen 1, blood is collected in a blood test container, and when coagulating it, the above-mentioned succinate is present in the blood as a blood coagulation promoter, but the above-mentioned blood test container does not have any particular restrictions. Instead, containers made of glass or resin that have been conventionally used may be used as appropriate.

The metal ion constituting the succinate used as a blood coagulation promoter in the method of the present invention is Cu''.

(:o2+, Fe2+, HI2+, Δ(g 2
″- and Ce It is a divalent or higher metal ion selected from the 0 group.

Shukan 154 is a group of cyclic organic compounds that already have adjacent carbonyl groups in one molecule and that are sterically on substantially the same plane. It was revealed that it has a unique effect on coagulation factors (Japanese Patent Application No. 58-114659), but the metal ions that form succinate are the above-mentioned divalent or higher valent metal ions, and the succinate ion is When a chelate-like salt structure is formed through the metal ion and free rotation of the is-translated bond is inhibited, succinate has adjacent carbonyl groups in the molecule, and these are sterically Although the mechanism of action is not clear, it is thought that it has a specific effect on blood coagulation factors in the same way as above. It will be done.

Furthermore, when the metal ion is monovalent, the succinate il carbon-element bond can rotate freely, and the steric repulsion between the carbonyl groups and the electrostatic repulsion between the two metal ions can be avoided. Therefore, unless adjacent carbonyl groups are sterically on substantially the same plane, they do not have a specific i-function for blood coagulation.

Incidentally, monovalent metal salts of succinic acid, such as potassium succinate and sodium succinate, dissociate in the blood and are important blood coagulation factors. Since the monovalent metal salt forms a poorly soluble chelate compound with Ca2+, the coagulation mechanism of blood depleted of Ca2+ is inhibited, and therefore, the above-mentioned monovalent metal salts have been widely used as blood anticoagulants.

In the present invention, in order to have the above-mentioned succinate present in the blood as a blood coagulation promoter, it can be added to the blood in the form of a powder, or by dissolving or dispersing it in an appropriate solvent. However, if blood momentarily or partially comes into contact with high concentrations of these succinates in a blood test container, negative protein components in the blood may be denatured. It is preferable to carry succinate on a carrier having a large specific surface area and add this to the blood in a blood test container. By this method, succinate is dispersed in the blood at a uniformly small concentration, and unlike the case where a solution or dispersion of succinate is diluted and added to the blood, the blood is diluted. It will not interfere with the inspection.

As the carrier, various carriers can be used without particular limitation as long as they do not have a harmful effect on blood tests and have a large specific surface area, but for example,
Nonwoven fabrics, woven fabrics, resin beads, etc. can be suitably used. K, which supports succinate on such a carrier, may be applied, for example, by coating or immersing a solution or dispersion thereof, and then drying and adhering it to the carrier.

Further, particles carrying succinate can also be obtained by mixing with an appropriate auxiliary agent such as gum arabic to form an aqueous dispersion, and then rapidly freeze-drying the resulting aqueous dispersion.

The amount of succinate present in the blood as a blood coagulation promoter is at least 1X10 for blood 11R1Vc.
When g is less than IC1, the effect of promoting blood coagulation is poor. However, if too much M is present, it may cause various problems in blood tests, so it is preferable to limit the amount to 10 g or less.

According to the method of separating blood serum and blood clots of R. Kinen 1, as mentioned above, since the above-mentioned succinate is present in the blood as a blood coagulation promoter, blood coagulation factors are rapidly released. The time required for coagulation after blood is collected into a container is significantly shortened, and the separation of serum and blood clots is significantly facilitated, thus solving the problem of VC blood clot components being mixed in the separated and collected serum. will also be resolved. Furthermore, according to the method described in ``Saikan'', the blood clot components are sufficiently contracted, and as a result, the yield of serum is significantly increased.

The present invention will be described below with reference to VC examples, but the present invention is not limited to these examples in any way.

Example 1 A 10JIIi% physiological saline dispersion of Cu salt, Co salt, Fe salt, N1 salt, λ(g salt, Ce, and salt of succinic acid) was prepared.

Fresh blood cut into a commercially available 10me polyethylene spitz 5
Inject nIg and then add 50 μl of the earth succinate dispersion.
1 is added, stirred gently, and left at 20°C. The blood coagulation time is defined as the time when blood loses fluidity and serum begins to ooze out on the top of the clot. Immediately after this, the blood is heated at 3000 revolutions/min. Centrifuge for 5 minutes, observe the state of serum separation, and pipette the soil and surface liquid! f/: was taken, and the resistance was taken as the serum yield.

The results are shown in the table.

(The following is a blank space) Comparative Example 5 vre of freshly cut blood was injected into a commercially available 10U+ Subinotsu made of Megarasu and polyethylene, and the blood coagulation time and serum separation state were observed in the same manner as in the Examples, and the serum yield was determined. Show the results.

Special Ff [Applicant Shusui Chemical Industry Shiba Co., Ltd. Representative: Mototoshi Fujinuma

Claims (1)

[Claims] (1) In a method of separating blood into serum and blood clots by centrifugation, a metal salt of oxalic acid is used, the metal ion is 21a[i or more, and the ionic radius is 0.
A method for separating serum and blood clots, characterized by allowing 50 to 1.04 oxalate to be present in the blood.