JPH11142401A - Cell-function measuring method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell-function measuring method by which gathered blood can be safely preserved until a measurement is performed in a cell-function measurement in which blood is used, and also a cell function can be measured precisely. SOLUTION: The subject measuring method of a cell function in blood is composed of a process in which gathered blood is preserved at a low temperature (e.g. over 0 deg.C and below 21 deg.C), a process in which the blood preserved at a low temperature is heated to a temperature near the body temperature (e.g. 21-45 deg.C) in advance of the measurement, a process in which a bioactive substance (e.g. tumor necrosis factor α (TNF α)) is produced by reacting blood cells in the blood with a stimulus (e.g. endotoxin), and a process in which the amount of the produced bioactive substance is measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for measuring cell functions in blood.

[0002]

2. Description of the Related Art Recent studies in immunology have shown that leukocytes such as granulocytes, monocytes, macrophages, and lymphocytes, which are blood cells, have various effects such as inflammatory reactions and immune reactions in blood and in various organs and organs. It is becoming clear that they play various roles in biological defense reactions. These cells play important roles in various disease states such as infectious diseases; inflammatory diseases such as hepatitis and nephritis; immunological and allergic diseases such as rheumatoid arthritis and asthma; and cancers. It is also known that the function of cells is suppressed or enhanced.

[0003] In addition, anti-inflammatory drugs,
Various drugs such as immunosuppressants, immunopotentiators, and anticancer drugs have been used, and it is also known that the function of these cells is suppressed or enhanced in such cases. Therefore, it is important to examine the functions of these cells in order to grasp the pathological state of various diseases, the effects or side effects of drugs, determine treatment guidelines, and determine the dose and timing of drugs.

Conventionally, in an examination room or an examination center of a hospital,
For the reasons described above, in order to measure such a cell function, a granulocyte phagocytosis function test, a granulocyte bactericidal ability (active oxygen producing ability) test, a lymphocyte blastogenesis test and the like have been performed. In recent years, surface antigen tests using a flow cytometry device and a fluorescently labeled monoclonal antibody against various immunocompetent cell surface antigens have been performed. However, the above test methods include cell isolation, cell culture,
Special techniques such as microscopic measurement were required, the measurement took time, and expensive equipment such as RI facilities and flow cytometry was required.

[0005] Recently, as a method improved from the conventional method,
Various methods for examining the cytokine production function from blood or leukocytes separated from blood have also been reported. For example, Japanese Unexamined Patent Publication No. Hei.
JP-A-3-285692 discloses that tumor necrosis factor α (TNFα) and interleukin-1 produced by reacting blood with lipopolysaccharide (LPS) or lectin.
Methods for measuring cytokines such as β (IL-1β) have been disclosed.

However, if blood is collected and stored at a low temperature in order to stably store the blood until measurement, when the blood is reacted with a stimulant, the amount of the physiologically active substance produced will cause the blood to be stored. However, there is a disadvantage that it is reduced as compared with the case of using without.

[0007]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for measuring cell functions using blood, in which the collected blood can be stably stored until the measurement is performed, and the cell functions can be accurately measured. It is to provide a method.

[0008]

A method for measuring cell function according to the present invention is a method for measuring cell function in blood, comprising the steps of storing collected blood at a low temperature, and prior to measuring the blood stored at a low temperature. Heating to a temperature close to body temperature, and then reacting the blood cells in the blood with a stimulant to produce a physiologically active substance, and measuring the amount of the produced physiologically active substance. It is characterized by.

Hereinafter, the method for measuring cell function of the present invention will be described in detail step by step. First, the blood used in the present invention is stored at a low temperature after blood collection and before cell function measurement. Low temperature refers to a temperature lower than body temperature,
It is preferably 0 ° C or more and less than 21 ° C, more preferably 0 to 1 ° C.
0 ° C. When the storage temperature is lower than 0 ° C., the cells are frozen, and the cells are liable to cause freezing damage. When the storage temperature is 21 ° C. or higher, a physiologically active substance is continuously produced from blood cells, so that there is a drawback that a difference occurs in the amount of the physiologically active substance immediately after blood collection and after storage.

[0010] As a low-temperature preservation method, blood is kept at 0 ° C or higher.
The method is not particularly limited as long as it can be kept at less than 1 ° C., and examples thereof include a method of storing in a refrigerator, a method of using a cooling agent, and a method of using a cooling box.

In the method of the present invention, the cryopreserved blood is then heated to a temperature close to body temperature prior to measurement. The temperature close to the body temperature is preferably 21 to 45 ° C, more preferably 25 to 40 ° C. The above temperature is 2
When the temperature is lower than 1 ° C., the production amount of the physiologically active substance from the blood cells becomes lower than that in the case of using blood immediately after blood collection, and the amount of production of the physiologically active substance is 45
When the temperature exceeds ℃, there is a disadvantage that the production amount of a physiologically active substance from blood cells decreases.

The time for heating to a temperature close to the body temperature is preferably 1 to 180 minutes, more preferably 2 to 180 minutes.
0-120 minutes. When the heating time is shorter than 1 minute, the production amount of the physiologically active substance from the blood cells is smaller than when using blood immediately after blood collection, and when it is longer than 180 minutes, the production amount of the physiologically active substance from the blood cells Is higher than when using blood immediately after blood collection.

The heating method is not particularly limited as long as the blood can be kept at 21 to 45 ° C., and examples thereof include a method using a thermostatic water bath and a method using a thermostatic bath.

In the method of the present invention, prior to the measurement, blood heated to a temperature close to body temperature is brought into contact with a stimulant, and the blood cells in the blood react with the stimulant to produce a physiologically active substance. Let it. The stimulant refers to a material that reacts with blood cells to produce a physiologically active substance.
Endotoxins consisting of various microorganisms such as dead bacteria and genus Corynebacterium, cell wall polysaccharides (lipopolysaccharides) derived from microorganisms; synthetic lipid A; pyran copolymer; phytohemagglutinin (PHA-L and PHA-P), concanavalin A, pork Lectins such as weed mitogen; poly I: poly C; purified protein-free tuberculin; OK
432 (Picibanil); PSK (Krestin); Lentinan; Zymozan; Interferon; Interleukin-1 (IL-1), Interleukin-6 (IL-
6), cytokines such as TNF; and the like.
In addition, materials obtained by immobilizing these materials on various natural or synthetic polymer materials by a known immobilization method may be used.

The stimulant is usually in the form of a powder or a liquid in which the stimulant is dissolved in a solvent such as water. Further, any known shape such as a fibrous shape, a hollow fiber shape, and a film shape may be used. The state of the stimulant in the reaction vessel may be solid, gel, or liquid. When the stimulant is water-soluble, the stimulant may be dissolved in an appropriate solvent and applied to or added to the inner wall surface of the reaction vessel, and then powdered.

Examples of the solvent include a buffer such as a phosphate buffer and Hanks buffer, MEM, RPMI-
Normal medium such as 1640 or water for injection (LPS-free water,
And Otsuka Pharmaceutical Co., Ltd.) and physiological saline.

The blood used in this reaction may be used after being diluted with the above-mentioned buffer solution or medium, if necessary, in addition to the collected whole blood.

The physiologically active substance referred to in the present invention is produced from blood cells when stimulated with the above-mentioned stimulants. For example, TNFα, IL-1β, IL-2, INF
L-4, IL-5, IL-6, IL-8, INF-γ,
Granulocyte Macrophage Colony Stimulating Factor
(GM-CSF), cytokines such as RANTES;
Prostaglandins such as PGE2, PG12; LTB
4, leukotrienes such as LTC4; various chemical mediators such as nitric oxide, active oxygen, histamine, platelet activating factor (PAF); and the like. Also, adhesion factors such as soluble ICAMI;
Soluble cytokine receptors such as receptors; eo
sinophile ceticic protei
n (ECP), major basic protein
n (MBP), eosinophile drive
d neurotoxin (EDN), eosinop
Granular proteins such as hill peroxidase (EPO) are also included, but are not limited thereto.

In collecting blood used in the method for measuring cell function of the present invention, it is necessary to use a blood anticoagulant to prevent blood from coagulating.

Examples of the blood anticoagulant include a heparin compound, a citrate compound, an oxalate compound, and the like. Heparin sodium is preferred because it does not inhibit the biological reaction of cells.

As a method for collecting blood used in the method for measuring cell function of the present invention, a blood collection device such as a vacuum blood collection tube or an injection syringe is used. Preferably, the blood collection device is a substance that induces the production of a physiologically active substance in blood cells non-specifically, for example, LPS (endotoxin),
It is preferable that β-1,3 glucan, antigen, and the like are not mixed.

In the vacuum blood collection tube, a plug is usually used to maintain the inside of the blood collection tube at a reduced pressure. As the material of the plug, for example, butyl rubber, chlorinated butyl rubber,
Thermoplastic elastomers and the like can be mentioned.

In the step of reacting blood cells in blood with a stimulant in the method of the present invention, it is preferable that a blood anticoagulant is present in the reaction vessel so that blood cells do not coagulate. Examples of the blood anticoagulant include those described above.For example, in the case of heparin sodium, when blood is contained in the reaction vessel, the concentration of sodium heparin in the whole liquid containing the blood decreases. Since blood coagulation is likely to occur, and if the blood pressure increases, cells may be unexpectedly activated or inactivated, the cells are preferably stored at 6 to 20 U / ml.

The material of the reaction container used in the step of reacting the blood cells in the blood with the stimulant may be, for example, plastic or glass. It is preferable that the material has a strength enough to withstand a centrifugal separation operation for measuring the concentration. As the plastic, any of a thermoplastic resin and a thermosetting resin can be used. As the thermoplastic resin, for example, polyethylene, polypropylene, polystyrene, polymethyl methacrylate, polyvinyl chloride, polyethylene terephthalate, styrene-acrylonitrile copolymer, styrene-maleic anhydride copolymer, styrene-acrylic acid copolymer, styrene -Methyl methacrylate copolymer, ethylene-propylene copolymer, ethylene-acrylic acid copolymer, ethylene-acrylic ester copolymer and the like. Examples of the thermosetting resin include an unsaturated polyester resin, an epoxy resin, and an epoxy-acrylate resin.

In the method of the present invention, usually, after heating blood stored at a low temperature, a reaction container containing a predetermined amount of a stimulant is added to the reaction container.
A method of adding and reacting is used. If the inside of the reaction container containing the predetermined amount of the stimulant is previously depressurized and sealed with a rubber stopper or the like, the inside of the reaction container and the container containing the blood to be measured are filled with a multiple injection needle or the like. By connecting, the blood can be more safely transferred into the reaction container.

Further, as the reaction vessel used in the method of the present invention, the vacuum blood collection tube used at the time of blood collection may be used as it is. As an example of this case, blood is collected in a blood collection tube in which a predetermined amount of a blood anticoagulant has been placed, and this blood is stored at a low temperature, and brought to a temperature close to the body temperature prior to the start of measurement. After heating, a method of adding a predetermined amount of a stimulant to the mixture and reacting the mixture is used.

The amount of blood used for measuring the cell function by the method of the present invention varies depending on the volume of the reaction vessel.
If a reaction vessel having a volume of 5 ml is used, 0.5
It may be about 2 ml.

The reaction temperature in the step of reacting blood cells in blood with a stimulant in the method of the present invention is a temperature at which the production of the physiologically active substance is carried out efficiently and does not cause excessive hemolysis. -42 ° C is preferred, and more preferably 30-40 ° C. The reaction time is preferably 1 to 48 hours, more preferably 2 to 24 hours, which is a reaction time at which the production of the physiologically active substance is efficiently performed and does not cause excessive hemolysis. In the above reaction,
It is preferable to shake the reaction container containing the blood, the stimulant and the blood anticoagulant.

Next, in the method of the present invention, the amount of the produced physiologically active substance is measured. As a method for measuring the amount of the physiologically active substance produced, for example, an enzyme immunization using an enzyme such as a monoclonal antibody or a polyclonal antibody against the physiologically active substance to be measured, peroxidase, alkaline phosphatase and a chromogenic substrate of each enzyme Measurement method: HPLC (High Performance Liquid Chromatography) method, but not limited thereto,
Known measurement methods for each physiologically active substance may be used.

[0030]

Now, the present invention will be described in further detail with reference to Examples and Comparative Examples of the present invention. The present invention is not limited to the following examples.

In the following Examples and Comparative Examples, endotoxin (LPS) was used as a stimulant, TNFα was used as a physiologically active substance produced and measured in blood cells, and a method for producing a reactor for measuring cell function was used. , TNF
The production reaction of α and the method of measuring the production amount were as follows.

(1) Method for Manufacturing Cell Function Measurement Reactor Eighteen LPS-free blood collection tubes (12.6φ × 75 mm, manufactured by Sekisui Chemical Co., Ltd.) made of polyethylene terephthalate were prepared.
LPS (manufactured by LBL) 200
0.05 ml of a solution of 0 EU (International Endotoxin Unit) / ml physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) was added to produce a cell function measurement reactor.

(2) Production reaction of TNF-α and method of measuring the amount of production A sample of LPS-free blood tubes made of polyethylene terephthalate (Sekisui Chemical Co., Ltd., 1
Heparin was sampled in 10 ml portions at 2.6 φ × 75 mm). 1 ml of the collected blood stored under the conditions of the following Examples and Comparative Examples was added to the above-described cell function measurement reactor, and the cells were cultured at 37 ° C. for 4 hours with shaking. The mixture was centrifuged under the conditions of minutes, and the supernatant plasma was collected. The amount of TNFα in the collected plasma was determined using an enzyme immunoassay kit (GenGen) using a monoclonal antibody.
(Zym). In addition, the detection limit concentration of this measurement method was 16 pg / ml. In addition, this production reaction was performed at a repetition number of 3 for each example.

Example 1 The collected blood was immediately stored at 4 ° C. for 24 hours.
After heating at 37 ° C. for 40 minutes, it was used for the above TNFα production reaction. Table 1 shows the average value, standard deviation, and coefficient of variation ((standard deviation / average value) × 100) of the TNFα concentration in the obtained plasma for each blood of volunteers A and B.

COMPARATIVE EXAMPLE 1 Immediately after blood collection without storing the collected blood,
It was used for α production reaction. For each blood of volunteers A and B, the mean, standard deviation and coefficient of variation ((standard deviation / mean) × 1) of the TNFα concentration in the obtained plasma
00) are shown in Table 1.

Comparative Example 2 The collected blood was immediately stored at 4 ° C. for 24 hours.
It was used for the above-mentioned TNFα production reaction in a state of cooling without heating. Table 1 shows the average value, standard deviation, and coefficient of variation ((standard deviation / average value) × 100) of the TNFα concentration in the obtained plasma for each blood of volunteers A and B.

[0037]

[Table 1]

Table 1 shows the following. 24 at 4 ° C
With the blood stored for a long time kept cold without heating,
When used for the production reaction of NFα (Comparative Example 2), TNFα was compared with the case where blood immediately after blood collection was used (Comparative Example 1).
Production was significantly reduced.

However, the blood stored at 4 ° C. for 24 hours
After heating at 37 ° C. for 40 minutes and using it for the TNFα production reaction (Example 1), the amount of TNFα production was almost the same as that when blood immediately after blood collection was used (Comparative Example 1).

As described above, when measuring the cell function of blood stored at low temperature after blood collection, before stimulating with a stimulant,
It was found that when a step of heating was added, the cell function could be measured with high accuracy as in the case immediately after blood collection.

[0041]

The structure of the method for measuring cell function of the present invention is as described above. When this method is used, in cell function measurement using blood, collected blood is stored at a low temperature until measurement is performed. Because it can be stored stably and it is not necessary to measure immediately after blood collection, sample processing efficiency can be increased, and blood stored at low temperature is heated to a temperature close to body temperature before measurement. The present invention provides a cell function measurement method capable of measuring cell functions with high accuracy despite being stored at a low temperature.

Claims (1)

[Claims] 1. A method for measuring cell function in blood, comprising: storing blood collected at a low temperature; heating the blood stored at a low temperature to a temperature close to body temperature prior to measurement;
A method for measuring a cell function, comprising the steps of: reacting blood cells in the blood with a stimulant to produce a physiologically active substance; and measuring the amount of the produced physiologically active substance.