JP2013124905A - Specimen preparation method for blood endotoxin measurement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve the problem in which, in measuring an endotoxin amount in leucocytes or blood plasma of infectious patients, a method for easily sampling a specimen is required to achieve promptness and deactivation of endotoxin should be prevented in preprocessing to measure a minute amount of endotoxin.SOLUTION: There is provided a specimen preparation method for blood endotoxin measurement, including adding a hydroxyethyl starch solution to a specimen, and then heating the specimen to prepare the specimen for endotoxin measurement. An endotoxin recovery rate is thereby improved. When a hydroxyethyl starch is added to the specimen as blood, leucocytes and blood plasma can be obtained easily by an erythrocyte flocculation effect of the hydroxyethyl starch without using a centrifugal separation operation, and a heating operation can serve also as a heating operation for removing limulus test interference factors in the blood plasma. Thereby, the specimen for endotoxin measurement can be obtained easily and activity of a little amount of endotoxin is not lost.

Description

The present invention is a sample used for measuring the endotoxin concentration in a gelation reaction by activation of an enzyme group generated when a blood cell extract of horseshoe crab reacts with endotoxin, which is generally called a Limulus test, and a method applied thereto. And a pretreatment liquid.

Endotoxin (endotoxin) is a component constituting the outer membrane of Gram-negative bacteria, and its chemical body is LPS (lipopolysaccharide: lipopolysaccharide). Endotoxin is generally considered to exist in the state of giant micelles.

Endotoxin is an important bacterial component that causes life-threatening pathologies such as sepsis due to Gram-negative infection, septic shock, or multiple organ failure. Measurement of endotoxin in blood contributes to elucidation of the relationship between endotoxin and various pathological conditions, early diagnosis and treatment of the pathological conditions.

Currently, the mainstream method for measuring endotoxin is the Limulus test.

There is a “factor C pathway” that specifically reacts with endotoxin in the blood cell extract of horseshoe crab. In the “factor C pathway”, endotoxin first binds tightly to factor C and activates factor C. Activated factor C activates factor B, and activated factor B turns the procoagulase into a coagulase. The clotting enzyme turns coagulogen into a coagulation protein, coagulin, resulting in gelation.

Further, in the blood cell extract of horseshoe crab, there is a “factor G pathway” induced by β-D-glucan in addition to the factor C pathway. β-D-glucan activates factor G, the activated factor G turns procoagulase into a coagulase as in endotoxin, and coagulase changes coagulogen into coagulin, a coagulation protein, Gelation occurs.

At present, measurement methods specific to endotoxin and β-D-glucan have been developed in Japan and are applied clinically as diagnostic methods for sepsis and deep mycosis caused by Gram-negative bacteria.

For the Limulus test, methods such as a gelation overturning method (gelation method), a chromogenic synthetic substrate method (colorimetric method), a turbidimetric time analysis method (turbidimetric method), etc., which are indicated by differences in determination or measurement methods are known . Recently, an endotoxin light scattering method, which is a gelation detection method using laser light, and a method using a genetically modified factor C to which LPS first binds have been developed (Non-patent Documents 1 and 2). .

In order to measure the endotoxin in blood by the Limulus test, a pretreatment step is necessary to remove or inactivate the Limulus test interfering factor contained in plasma or the like. Α2-plasmin inhibitor, antithrombin III, α1-antitrypsin and the like contained in plasma and the like are limulus test enhancing factors, and factor Xa, thrombin, trypsin and the like are suppressors of the limulus test.

As a pretreatment step, a PCA method, a New PCA method, an alkali treatment method and the like have been used so far, but a dilution heating method is used for the turbidimetric method.

The “dilution heating method” is a method in which water or a buffer solution is added to a blood-derived sample for dilution, and then the interference factor is inactivated by heating. When using serum or plasma, the dilution rate is 3 to 10 times, the temperature is in the range of 70 to 100 ° C., and the Limulus test interference factor is destroyed by heating for about 5 to 10 minutes. At this time, it may be diluted with water to which a surfactant such as Triton X-100 is added. The heating method is not limited as long as the component blood is sufficiently heated at the above temperature (see Non-Patent Documents 1 and 2 and Patent Document 1).

Endotoxin is thought to exist in the bloodstream after the death of Gram-negative rods, and plasma and serum separated from whole blood have been used as blood-derived samples that are conventionally used for the Limulus test.

Endotoxin in blood forms a complex with LBP (LPS binding protein: LPS binding protein) and then binds to cell surface antigen CD14 on monocytes and granulocytes contained in leukocytes. Subsequently, it associates with MD-2 and TLR4 (Toll-like receptor: one of Toll-like receptors). As a result, information on endotoxin binding is transmitted to the nucleus via an intracellular signal transduction pathway, and expression of inflammatory cytokine genes such as TNFα and IL-6 is induced, and these inflammatory cytokines are produced. (See Non-Patent Documents 1 and 2 above)

In addition to the specific binding described above, endotoxin binds to CD11 / CD18, which is known as an adhesion molecule on the leukocyte surface, or to a scavenger receptor or the like.

Therefore, endotoxin in the blood is bound to the membrane surface of leukocytes having endotoxin receptors in addition to being present on the surface of the cells or in the plasma or serum. It is thought that it exists even in the captured state. Therefore, it is difficult to say that the amount of endotoxin in blood is accurately determined only by measuring the amount of endotoxin contained in plasma. When a certain period of time elapses after the infection until blood is collected, the amount of endotoxin that is bound to the membrane surface of leukocytes or taken into the leukocytes may be dominant. In addition, pathological conditions such as symptoms such as septic shock are caused as a result of induction of cytokine production by binding endotoxin to the membrane surface of leukocytes as described above. Thus, unless the amount of endotoxin bound to the membrane surface of leukocytes or endotoxin taken into leukocytes is taken into account, the relationship with the disease state cannot be clarified. In addition, it can be said that endotoxin is inevitably present on the surface of leukocytes or inside cells even when gram-negative bacteria possessing endotoxin are recognized by leukocytes, bound and phagocytosed.

Therefore, the inventors have applied for a patent for a method for measuring endotoxin bound to leukocytes or contained therein (Patent Document 2). With this method, attention has been paid to the significance of measuring endotoxin bound to the membrane surface of leukocyte cells and endotoxin incorporated into leukocytes. However, this method, on the contrary, excludes endotoxin contained in plasma, which was a conventional measurement target.

The present inventors further devised a method for simultaneously measuring leukocyte and plasma endotoxins. In this method, plasma and white blood cells are collected by separate operations, and both are mixed and measured. However, there is a problem that a centrifugal separation operation is necessary (Patent Document 3).

Red blood cells are hemolyzed by a pretreatment step when quantifying endotoxin, and the sample removed as much as possible is used for measurement because it strongly interferes with the measurement system, but it is not easy to separate only red blood cells from white blood cells or plasma. In addition, white blood cells and plasma in the clinical field or each of them alone precludes rapid endotoxin quantification.

Appropriate amounts of ficoll, dextran, and hydroxyethyl starch are mixed with blood and allowed to stand at room temperature. When red blood cells are allowed to settle, plasma rich in white blood cells (polyleukocyte plasma) can be obtained in the supernatant. Conventionally used as a sampling method (Non-patent Document 3).

Ficoll, dextran, and hydroxyethyl starch affect the surface charge of red blood cells, promote recurring formation, and cause a difference in sedimentation rate from nucleated cells when left standing or centrifuged. Red blood cells are preferentially sedimented, and high leukocyte plasma can be obtained in the supernatant.

On the other hand, blood endotoxin levels in septic patients are often extremely small, from 1 pg / ml to 100 pg / ml. In order to more efficiently measure endotoxin in blood-derived samples, the steps of collection and pretreatment steps are required. Therefore, it is necessary to devise measures to prevent endotoxin from being inactivated.

Patent 2737514 JP 2004-117127 A Patent 4761448

Endo Shigetatsu, Inada Shinya, Endotoxin and Pathology. Helus Publishing, 1995. Homepage (Inada Katsuya) Endotoxin quantification method; Limulus test http://www.asahi-net.or.jp/~CP6K-IND/index.html Lecture formation education course: Blood rheology and physiological function, 2nd: Factors and analysis affecting blood viscosity, Shinji Maeda: Nihon Physiology Journal 2004, 66 (9), 287-297. Proc. Natl. Acad. Sci. USA, Vol. 92, pp 10119-10122 (1995), Indian J. et al. Med. Res. , Vol. 106, pp16-19 (1997) Current Status and Future Prospects of Hydroxyethy strach (HES) Formulation, Michiaki Sanin, Anesthesia 21 century, Vol. 11, No. 1-33, 2032-2046, 2009

There is a need for a technique for easily obtaining a sample for measuring endotoxin that contains white blood cells and plasma and does not contain red blood cells.

Furthermore, the amount of endotoxin in leukocytes and plasma is very small, and there is a problem that inactivation of endotoxin must be prevented in the pretreatment of the sample.

In the present invention, a hydroxyethyl starch solution is added to a sample for measuring the amount of endotoxin, and this is heated in the presence of hydroxyethyl starch to obtain a sample for measuring endotoxin.

White blood cells and plasma can be obtained as is well known by adding hydroxyethyl starch solution to blood and allowing to stand at room temperature, but when experimenting with white blood cells, the hydroxyethyl starch is removed by centrifugal washing. Usually, in the present invention, leukocytes coexisting with hydroxyethyl starch and plasma, that is, multi-leukocyte plasma, are diluted and heated to obtain a sample for endotoxin measurement. This operation can also serve as a “dilution heating method” which is a pretreatment step in measuring plasma endotoxin by the Limulus test.

By directly or appropriately diluting a sample containing a predetermined concentration of hydroxyethyl starch and then heat-treating it, an effect of improving the recovery rate of limulus activity possessed by endotoxin is brought about.

By adding hydroxyethyl starch to the blood, high leukocyte plasma can be obtained easily without centrifugation, and the subsequent pretreatment step, ie dilution and heating, is a method for removing interfering factors in the sample. An endotoxin recovery rate can also be improved, and a sample for measuring endotoxin can be obtained from blood easily and in an extremely short time.

When endotoxin (LPS) was heated in the presence of hydroxyethyl starch, the recovery rate in Limulus activity improved compared to heating in the absence of hydroxyethyl starch (LPS dissolved in physiological saline). LPS is added to the supernatant obtained by adding hydroxyethyl starch to the blood, diluted 10-fold with water, and heated at 70 ° C. for 10 minutes. Depending on the concentration of hydroxyethyl starch, Limulus Recovery in activity increased. LPS is added to healthy blood and heated at 37 ° C for up to 4 hours. Blood is collected at each time and the plasma or 6% hydroxyethyl starch is added to the supernatant. Showed Limulus activity. Endotoxin recovery up to 4 hours in high leukocyte plasma exceeded that in plasma.

The present invention relates to a method for pretreatment of a sample for measuring endotoxin, characterized in that hydroxyethyl starch is heated after being added to the sample, and the reaction of hydroxychiethyl starch is endotoxin-free and horseshoe crab blood cell extract and endotoxin It is an invention of a pretreatment solution for a sample for measuring endotoxin, which has a property that does not inhibit or promote the activity.

Hydroxyethyl starch has a high molecular weight (average 670 kD, medium molecular weight (average molecular weight 130-250 kD) and low molecular weight (average molecular weight 70 kD) used as a plasma substitute. If it exists, it will not ask | require especially about molecular weight.

An anticoagulant is added to the blood, but a general anticoagulant suitable for the Limulus test is used. For example, heparin may be added at a concentration that does not affect the Limulus test. In this case, the final concentration of heparin is preferably 10 to 100 units / mL.

The concentration of hydroxyethyl starch is preferably 0.01 to 6%, but is not particularly limited to this concentration. In general, a 6% hydroxyethyl starch aqueous solution and an equal amount of blood are mixed, mixed gently, and allowed to stand at room temperature. The concentration may be obtained by adding blood to the hydroxyethyl starch powder. Room temperature refers to about 10 ° C. to 25 ° C., but is not particular about this. The standing time is the time until erythrocytes settle, and is about 5 to 30 minutes because it is affected by the standing temperature. In either method, the layer above the sedimented erythrocyte layer is collected.

Dilution of multi-leukocyte plasma is an operation of diluting with water in advance to prevent coagulation due to heating of the sample in the next heating operation, similar to the dilution operation included in the plasma pretreatment step. 3 to 20 times is appropriate. However, if the endotoxin concentration does not decrease and does not fall below the measurement sensitivity by dilution, further dilution may be performed. The heating time is preferably 5 to 15 minutes, and the heating temperature is preferably about 60 ° C to 100 ° C. When the heat treatment also serves as a pretreatment step, the heat treatment is performed for the purpose of removing the interfering factor and improving the endotoxin recovery rate. In addition, when the interference factor of the Limulus test in the sample cannot be removed under the conditions of the heat treatment, heating may be newly performed for the purpose, or the interference factor may be removed by another method. When the destruction of leukocytes is not sufficiently performed by heating, another method for destroying leukocytes may be added.

If the sample is derived from blood, the leukocytes are destroyed by this water dilution and heating treatment, and the endotoxin inside the cells is exposed and measurement is possible. However, an operation to destroy the white blood cells may be added before or after heating. Absent. The methods include an ultrasonic destruction method, a physical disruption method, a freeze-thaw method, etc., but these methods are not limited as long as endotoxin is not destroyed and leukocytes can be sufficiently destroyed. I can't.

Any heating method may be used as long as the sample is sufficiently heated. For example, the container may be heated in a thermostat. The “constant temperature bath” may be a water bath or a heat block. Moreover, an air incubator may be used. The sample may be left to cool at room temperature after heating, or may be cooled by ice cooling or the like.

The “ultrasonic destruction method” is a method in which by applying ultrasonic waves to the sample, the cell membrane of white blood cells is destroyed by ultrasonic vibration, and the cell contents are eluted. The ultrasonic treatment may be performed using an apparatus such as an ultrasonic cell crusher (sonicator). The intensity of ultrasonic waves in this method can destroy the cell membrane of leukocytes, but may be within a range that does not destroy endotoxin, and may be appropriately adjusted depending on the amount of blood, the oscillation output of the ultrasonic waves, and the transmission time.

The “physical disruption method” is a method for destroying leukocytes by applying a physical external force to the sample. For the physical crushing treatment, an apparatus such as a homogenizer may be used. The strength of the external force in the method may be within a range that can destroy the cell membrane of leukocytes but does not destroy endotoxin.

The “freeze-thaw method” is a method for destroying leukocytes in blood by performing an operation of rapidly freezing the sample in liquid nitrogen or dry ice and then thawing it with warm water or the like. It is based on the principle of breaking the cell membrane by volume expansion due to crystallization of intracellular water by rapid freezing and subsequent crystal melting. The operation of freezing and thawing may be repeated a plurality of times until the white blood cells in the component blood are sufficiently destroyed.

The method of the Limulus test is not particularly limited as long as it is a generally performed method such as a gelation method, a color development method, a turbidimetric method, an endotoxin light scattering method, or the like. The horseshoe crab blood cell extract used in this case can be used for ordinary endotoxin measurement, for example, Wako Pure Chemical Industries, Seikagaku Biobusiness, Endosafe, Lonza, Limulus genus. There is no particular limitation as long as it is extracted from the blood cells of horseshoe crab belonging to the genus TachypleusT or the genus Carcinoscorpius.

The operation of heating a sample containing hydroxyethyl starch can also serve as inactivation of interference factors when the sample is derived from blood, but other methods such as PCA method, New PCA method, alkali can be used as inactivation of interference factors. There is no problem even if a treatment method is used.

This sample preparation method may also be used for the measurement of β-D-glucan using the Limulus test.

In addition to what is generally called blood, this method may be used for the purpose of removing red blood cells when detecting endotoxins such as umbilical cord blood, bone marrow cells, body fluids, spinal fluid, urine, etc. .

See FIG. It is assumed that the instruments, reagents, water, etc. used in the practice of the present invention are all endotoxin-free, or those containing an extremely small amount of endotoxin that does not affect the endotoxin measurement results. LPS (derived from Escherichia coli O111: B4, manufactured by Sigma) was suspended in phosphate buffered saline (PBS) at a concentration of mg / ml, and sufficiently suspended at the terminals of a vortex mixer and an ultrasonic generator. Using an endotoxin-free chip (Bioclean chip; manufactured by Wako Pure Chemical Industries, Ltd.), it was diluted to a predetermined concentration with PBS and used in the experiment. LPS having a final concentration of 20 pg / ml is contained in saline or hydroxyethyl starch (manufactured by Takara Bio Inc., dissolved in physiological saline) in a physiological saline solution having a concentration of 0.02% to 1.5%. After heating for 10 minutes, the Limulus activity after ice cooling was measured. Single test Wako (Wako Pure Chemical Industries, Ltd.) was used as the Limulus reagent, and the measurement was performed using Toxinometer MT-5500 (Wako Pure Chemical Industries). As a result, with respect to the limulus activity of LPS of 20 pg / ml, the recovery rate was the worst for physiological saline, but the recovery rate was improved depending on the concentration in the presence of hydroxyethyl starch. In addition, when LPS was added to hydroxyethyl starch at a concentration of 0.02% to 1.5% and limulus activity was examined without heating, the value was compared with the case where hydroxyethyl starch was not included. Almost unchanged, hydroxyethyl starch did not inhibit or promote the limulus activity of LPS.

See FIG. Equivalent saline solution containing 6% hydroxyethyl starch in the blood of healthy subjects (final concentration of hydroxyethyl starch 3%), 1/2 amount (2%), 1/3 amount (1.5%) 1/4 amount (1.2%), mixed and allowed to stand at room temperature. Red blood cells aggregated and precipitated in about 8 minutes, 11 minutes, 15 minutes, and 25 minutes, respectively. Could be collected. Endotoxin was measured by a Limulus test after adding LPS having a final concentration of 20 pg / ml to the polyleukocyte plasma. That is, 100 μl of this was added to 900 μl of water and diluted 10-fold (in this case, final concentrations of hydroxyethyl starch were 0.3%, 0.2%, 0.15%, 0.12%). This solution was heated in a heat block at 70 ° C. for 10 minutes for the purpose of exerting the effect of hydroxyethyl starch and for the purpose of inactivating the interference factor in plasma. Limulus activity was measured after ice cooling. As a result, when the activity of LPS without any treatment was taken as 100%, LPS dissolved and heated in physiological saline not containing hydroxyethyl starch was the worst (58.7%), and hydroxyethyl starch was added and collected. In the plasma containing leukocytes, the recovery rate improved depending on the concentration of hydroxyethyl starch (♦ in the figure). In the case of 1.2% hydroxyethyl starch to which LPS was not added, in the case of 3% hydroxyethyl starch, no Limulus activity was measured, indicating that there was no hydroxyethyl starch or endotoxin contamination during the experimental operation. (▲).

See FIG. LPS is added to human healthy blood collected by adding heparin at 20 pg / ml, heated to 37 ° C. for 4 hours, and after 0, 1, 2, 3, and 4 hours, a certain amount is taken. Limulus activity was examined. 1) Add an equal amount of blood to 6% hydroxyethyl starch and mix gently, then let stand at room temperature for 15 minutes. Collect the supernatant (rich leukocyte plasma), dilute 10 times with water and stir vigorously. Thereafter, the mixture was heated at 70 ° C. for 10 minutes and cooled with ice, and then the Limulus activity was examined (▲ in the figure). The value was multiplied by 10 to obtain the amount of endotoxin in the leukocyte plasma. 2) Blood is centrifuged at 1,500 rpm for 10 minutes with a table centrifuge (manufactured by Kubota Corporation), and the supernatant, ie plasma, is obtained, diluted 10-fold with water, heated at 70 ° C. for 10 minutes, and then ice-cooled. Limulus activity was examined. The value was multiplied by 10 to obtain the amount of endotoxin per ml of plasma. In both 1) and 2), the limulus activity of LPS immediately after addition of LPS (0) was expressed as 100%. As a result, the recovery rate of endotoxin in the leukocyte plasma collected by adding hydroxy starch was higher than the recovery rate of endotoxin in plasma.

INDUSTRIAL APPLICABILITY The present invention can provide a sample pretreatment method for measurement of a Limulus test that can be carried out by a simple operation without including an operation of centrifugation and has a good endotoxin recovery rate. In the case of measurement of mainly white blood cells, plasma, etc. derived from blood, it also serves to remove red blood cells that inhibit the measurement, and heat treatment can also serve to inactivate interference factors contained in plasma and destroy white blood cells. . The ability to easily remove red blood cells that have previously prevented the measurement of endotoxin in the blood and to conduct rapid measurement methods contributes to the diagnosis of septic and septic shock that requires urgency to establish a prompt treatment policy. be able to. This invention is considered to contribute greatly to clinical medicine.

Claims (8)

A method for preparing endotoxin measurement samples by the Limulus test, in which hydroxyethyl starch is added to the sample and heated. The method for preparing a sample for measuring endotoxin by the Limulus test according to claim 1, wherein the sample is blood. A method for preparing a sample for measuring endotoxin according to claim 1 or 2, comprising hydroxyethyl starch, leukocytes and plasma obtained by adding hydroxyethyl starch to a sample such as blood and allowing it to stand at room temperature to precipitate and remove erythrocytes. . The sample preparation method for measuring endotoxin according to any one of claims 1 to 3, wherein the final concentration of hydroxyethyl starch added to the sample is 0.01 to 10%, preferably 0.01 to 6%. The method for preparing a sample for measuring endotoxin according to any one of claims 1 to 4, wherein a preferable heating temperature of the sample containing hydroxyethyl starch is 70 ° C to 90 ° C, preferably 10 to 20 minutes. The method for preparing a sample for measuring endotoxin according to any one of claims 1 to 5, wherein the degree of dilution of the sample containing leukocytes and plasma is 3 to 20 times. The endotoxin according to any one of claims 1 to 6, wherein the operation of diluting and heating leukocytes containing hydroxyethyl starch and plasma can also serve as the diluting and heating operation of removing an interference factor of the Limulus test contained in these samples. How to make a sample for measurement. Hydroxyethyl starch is endotoxin free and is a pretreatment solution for preparing endotoxin measurement samples that has the property of not inhibiting or promoting the reaction between the blood cell components of horseshoe crab and endotoxin.

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