Sample preparation method for measuring endotoxin bound to erythrocyte in blood

Abstract

To solve the problems that sepsis is a disease with a high mortality rate, that since sepsis caused by gram-negative bacteria has a particularly poor prognosis, early diagnosis and appropriate treatment are necessary, and although detection of endotoxin is an important method for quickly diagnosing sepsis, there are still challenges in detecting endotoxin in blood.SOLUTION: In the present invention, erythrocytes, which have not received much attention in a blood sample until now, are used as a target for measuring endotoxin, thereby establishing a method for measuring total endotoxin in blood, which contributes to diagnosis of gram-negative bacterial sepsis.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for preparing a sample for quantifying endotoxin in blood, which is one of the diagnostic methods for bacteremia caused by Gram-negative bacteria. [Background technology]

[0002] Once sepsis progresses, it is difficult to treat and can sometimes be fatal, even with advanced modern medical treatments. Currently, more than 300,000 sepsis patients are reported to be diagnosed annually in Japan, with more than 50,000 hospitalized patients dying from sepsis (Non-Patent Document 1). Sepsis is defined as life-threatening organ damage resulting from an inadequately controlled biological response to infection (Non-Patent Document 2). Therefore, it is important to recognize early signs of infection so that appropriate treatment can be administered to prevent the condition from worsening. Traditional methods for detecting infection include inflammatory cytokines and procalcitonin testing, and more recently, presepsin testing has been developed. At the same time, detecting the presence of bacteria in the blood has been considered particularly important. While culture methods have traditionally been used to directly test for the presence or absence of bacteria in the blood, they have the drawback of being time-consuming and not yielding as high a positive result as desired.

[0003] Endotoxin is a component of the outer membrane of gram-negative bacteria, and its chemical identity is lipopolysaccharide (LPS). Endotoxin is an important bacterial component that causes life-threatening conditions such as sepsis, septic shock, and multiple organ failure associated with gram-negative bacterial infections. Measuring the amount of endotoxin in the blood is believed to provide important information for the early diagnosis and treatment of gram-negative bacterial infections.

[0004] Currently, the most common method for measuring endotoxin is the Limulus test using horseshoe crab hemocyte extracts. Horseshoe crab hemocyte extracts contain a "factor C pathway" that specifically reacts with endotoxin. In the "factor C pathway," endotoxin binds tightly to factor C, activating it. Activated factor C activates factor B, which converts procoagulase into clotting enzyme. Clotting enzyme converts coagulogen into the clotting protein coagulin, resulting in gelation. In addition to the factor C pathway, horseshoe crab hemocyte extracts also contain a "factor G pathway" induced by β-D-glucan. β-D-glucan, a component of fungal cell walls, activates factor G. Similar to the activated factor B in the case of endotoxin, activated factor G converts procoagulase into clotting enzyme, which then converts coagulogen into the clotting protein coagulin. This results in gelation. Limulus tests are available in a variety of methods, including the gel inversion method (gelation method), the chromogenic synthetic substrate method (colorimetric method), and the turbidimetric time analysis method (turbidimetric method), which detects the turbidity change that occurs during coagulin formation. A bioluminescence method has also been developed. Recently, a method using recombinant factor C, which is the first to bind to endotoxin, has been developed (Non-Patent Documents 1 and 2). It has also become possible to use recombinant factor C, factor B, and procoagulant enzymes. Furthermore, methods that detect gelation electrochemically, using a quartz crystal oscillator, or mass change have become possible (Non-Patent Documents 1, 2, and 3). Currently, in Japan, a specific endotoxin assay has been developed, allowing accurate measurement of endotoxin in blood to diagnose sepsis caused by Gram-negative bacteria, and is covered by insurance. Furthermore, specific measurement of β-D-glucan has become possible, and is covered by insurance as a rapid diagnostic method for deep fungal infections.

[0005] Measuring endotoxin in blood using the Limulus test requires a pretreatment step to remove or inactivate interfering factors contained in plasma, etc. Factors contained in plasma, such as α2-plasmin inhibitor, antithrombin III, and α1-antitrypsin, are inhibitors of the Limulus test, while factors such as factor Xa, thrombin, and trypsin are stimulators of the Limulus test. These factors are inactivated by the plasma pretreatment step. Furthermore, endotoxin-degrading enzymes (acyloxyacyl hydrolases) derived from leukocyte granules and endotoxin-binding proteins (CAP-18 and BPI) are also present in plasma. It has been reported that these factors, which inhibit endotoxin measurement in the Limulus test, are increased in the plasma of patients with sepsis (Non-Patent Document 4).

[0006] The chromogenic synthetic substrate method, which was previously covered by health insurance, used the "perchloric acid method" as a pretreatment method for plasma. In this method, perchloric acid is added to plasma to denature and precipitate plasma proteins, and endotoxin in the centrifuged supernatant is measured. However, since most endotoxin is bound to plasma proteins, it was pointed out that endotoxin co-precipitates with the proteins, resulting in low measured values. Therefore, a "new perchloric acid method" was devised that solubilizes the precipitated proteins containing endotoxin, making it possible to measure protein-bound endotoxin as well (Non-Patent Documents 1 and 2).

[0007] The dilution and heating method is currently used as a pretreatment step in the turbidimetric method, which is covered by health insurance in Japan. The "dilution and heating method" involves diluting a blood-derived sample with water or a buffer solution, followed by heating to inactivate interfering factors. Unlike the conventional method using perchloric acid, this method does not require reagents; instead, it has the advantage of simply diluting the sample with water or other liquids and then heating it. Sample dilution is a procedure to prevent sample coagulation due to heating. Generally, when using serum or plasma, the sample is diluted 10-fold with water and heated at 70°C for 10 minutes. The pretreatment step for the turbidimetric method covered by health insurance in Japan involves diluting the sample with water containing 0.02% Triton X-100 (see Non-Patent Documents 1 and 2, and Patent Document 1).

[0008] Endotoxin in the blood forms a complex with LBP (LPS-binding protein) and then binds to the cell surface antigen CD14 on monocytes and granulocytes, which are white blood cells. Subsequently, it associates with MD-2 and TLR4 (a Toll-like receptor). As a result, endotoxin binding information is transmitted to the nucleus via intracellular signaling pathways, inducing the expression of inflammatory cytokine genes such as TNFα and IL-6, resulting in the production of these inflammatory cytokines (see Non-Patent Documents 1 and 2). In addition to specifically binding to TLR4, endotoxin also binds to CD11 / CD18, known adhesion molecules on the surface of white blood cells, or to scavenger receptors. Binding between hydrophobic moieties of endotoxin and hydrophobic moieties on the cell membrane has also been suggested. It has been reported that endotoxin bound to white blood cells is rapidly internalized into the cell (Non-Patent Document 5). Therefore, it is presumed that endotoxin in blood is not only present in plasma and serum, but also exists in a state bound to the membrane surface of leukocytes as endotoxin or incorporated into leukocytes. In other words, it is difficult to say that the amount of endotoxin in blood can be accurately quantified by measuring only the amount of endotoxin contained in plasma. If a certain amount of time has passed since infection before blood is collected, it is possible that the amount of endotoxin bound to the membrane surface of leukocytes or incorporated into leukocytes is predominant. In other words, endotoxin binding to the membrane surface of leukocytes induces cytokine production, resulting in pathological conditions such as septic shock. Therefore, the relationship with pathological conditions cannot be clarified without considering the amount of endotoxin bound to the membrane surface of leukocytes and endotoxin incorporated into leukocytes. Furthermore, it has been confirmed that endotoxin is present on the surface of gram-negative bacteria and can be measured as endotoxin even in bacteria (Non-Patent Document 2). In other words, endotoxin on the surface of gram-negative bacteria, like free endotoxin, will likely show a positive result in the Limulus test if it is recognized and bound to leukocytes or incorporated into cells, as long as the cells are destroyed. Therefore, when measuring the total endotoxin amount in a blood sample, not only endotoxin in the plasma but also endotoxin bound to leukocytes and endotoxin in the microorganisms themselves should be considered.

[0009] Previously, the present inventors have filed a patent application for a method for measuring endotoxin bound to or contained within white blood cells (including endotoxin present in the form of bacterial cells) (Patent Document 1). This method has attracted attention for the significance of measuring endotoxin bound to the membrane surface of white blood cells and endotoxin taken up into white blood cells. However, this method conversely excludes endotoxin contained in plasma, which was previously the target of measurement. The present inventors subsequently devised a method for simultaneously measuring endotoxin in white blood cells and plasma. In this method, plasma and white blood cells are collected separately and then mixed and measured, but this method requires centrifugation and is therefore a complicated procedure (Patent Document 2).

[0010] Red blood cells in blood undergo hemolysis during the pretreatment step of endotoxin quantification, and hemoglobin inhibits measurements using turbidimetric and colorimetric methods. Therefore, it is necessary to remove red blood cells as much as possible. Centrifuges are used to separate only red blood cells from blood, but this also sediments white blood cells, which are the target for endotoxin measurement. Therefore, the inventors devised a method for agglutinating and precipitating red blood cells, obtaining white blood cell-rich plasma for endotoxin measurement, and filed a patent application for this method. First, they devised a method using hydroxyethyl starch as a hemagglutinating agent (Non-Patent Document 6) (Patent Document 3), and then devised a method for obtaining white blood cell-rich plasma under ice-cooling using dextran, which also has hemagglutinating properties (Patent Document 4). The ice-cooling method is performed because endotoxin-inactivating factors in the blood of sepsis patients are thought to inactivate endotoxin in a temperature-dependent manner. In the following description, white blood cell-rich plasma is referred to as leukocyte-rich plasma (LRP). When an appropriate amount of hydroxyethyl starch is mixed with blood and left to stand at room temperature (Patent Document 3), or when dextran is mixed with blood and left to stand on ice (Patent Document 4), the red blood cells sediment due to their hemagglutinating properties, and LRP can be obtained in the supernatant in just about 15 to 30 minutes.

[0011] Measuring blood endotoxin levels is an important indicator for the early diagnosis and initiation of treatment of gram-negative bacterial infections. It is thought that in certain pathological conditions, endotoxins derived from bacteria normally present in the intestinal tract can enter the bloodstream even in smaller amounts than in sepsis due to increased permeability of the intestinal mucosa. However, the inventors have not clearly confirmed endotoxemia in any condition other than sepsis. Therefore, endotoxin measurement can be considered a diagnostic method limited to gram-negative bacterial infections such as sepsis. However, with conventional plasma-based assays, the rate of endotoxin positivity is at most 60–70% among patients suspected of gram-negative bacterial sepsis, and in some cases, it may be even lower. Therefore, the importance of this technique in clinical practice has gradually declined. Our technology for measuring endotoxin bound to leukocytes has been shown to correlate well with the clinical pathology of sepsis.

[0012] The inventors first devised a method for obtaining LRP using hydroxyethyl starch, but several problems were subsequently identified. Hydroxyethyl starch is poorly soluble in water, making it difficult to prepare a physiological saline solution. Furthermore, at the time, it was difficult to obtain a high-molecular-weight medical-grade hydroxyethyl starch solution with hemagglutinating properties. Therefore, a method was devised in which dextran, which also has red blood cell agglutinating properties, was used to prepare LRP instead of hydroxyethyl starch. High-molecular-weight dextran with red blood cell agglutinating properties is easily available, and preparing a physiological saline solution is much easier. Furthermore, it was found that the time required to obtain LRP was shorter than with hydroxyethyl starch.

[0013] When plasma is collected by centrifugation of anticoagulated blood, most red blood cells, white blood cells, and, depending on the centrifugal force, platelets are precipitated. In contrast, LRP collection is performed at 1 x g, and red blood cells that form clumps settle within 10 to 20 minutes, while white blood cells and platelets remain suspended. Bacteria, having a lower specific gravity than white blood cells, remain suspended in the LRP. Therefore, it is thought that the LRP contains all endotoxin detection targets other than endotoxin-containing blood cells.

[0014] Because plasma from septic patients exhibits greater endotoxin inactivation activity when heated to 37°C than plasma from healthy individuals, the inventors initially filed a patent application for a method for obtaining LRP using dextran under ice cooling (Patent Document 4). However, they subsequently found that LRP prepared at room temperature sometimes exhibited higher endotoxin levels than LRP prepared under ice cooling. This led to the invention of the 37°C heating method (LRP37 method). Specifically, the inventors devised a method in which blood is immediately warmed to 37°C after collection, and dextran is added to the blood, and the temperature is maintained until LRP is obtained, i.e., the LRP37 method (Non-Patent Document 7, Patent Document 5). The LRP37 method yielded higher endotoxin levels in many sepsis patient samples than the LRP0 method, in which blood is immediately warmed to 0°C after collection, and dextran is added to the blood, and the temperature is maintained until LRP is obtained. This indicates that the LRP37 method improves the positive rate of endotoxin in blood from sepsis patients.

[0015] As described above, the diagnostic efficiency of Gram-negative bacterial sepsis has been improved by measuring endotoxin bound to leukocytes as well as endotoxin in plasma using LRP (Non-Patent Documents 9, 10, 12). In this way, it was thought that by measuring endotoxin in leukocytes and plasma, it would be possible to measure all endotoxin in the blood, but the present invention relates to a method that also measures erythrocytes.

[0016] Although there are few reports that endotoxin acts on red blood cells, it has been shown that endotoxin causes morphological changes in human red blood cells (echinocytes) (Non-Patent Document 13), and that it actually binds to them has been demonstrated by quantifying hydroxy fatty acids, a component of endotoxin (Non-Patent Document 13). These reports suggest that red blood cell-bound endotoxin is the target of measurement. Ultimately, endotoxin in the body has the opportunity to randomly bind to red blood cells, white blood cells, platelets, and plasma proteins, including endotoxin-inactivating proteins, even though there are differences in the binding attraction between them. Therefore, we focused on red blood cells, which had previously been discarded, and added red blood cell-bound endotoxin to the measurement target, believing that this could improve the diagnostic efficiency of Gram-negative bacterial sepsis, leading to the invention of this patent.

[0017] However, a method for quantifying endotoxin (including red blood cells) targeting red blood cells, known as the "whole blood method," has been reported (Non-Patent Document 14, Patent Document 6). In this method, nitric acid or the like is added to whole blood, the resulting precipitate is removed by centrifugation, and the supernatant is subjected to endotoxin measurement. As in the discussion of the "perchloric acid method" and "new perchloric acid method" above, proteins are removed and the remaining endotoxin is quantified. Therefore, endotoxin presumably bound to membrane proteins in the destroyed red blood cells is thought to be precipitated and removed in the same way as plasma proteins, and as a result, red blood cell-bound endotoxin cannot be used for measurement.

[0018] Although not a method using the Limulus test, there is the endotoxin activity assay (EAA), a method for measuring endotoxin levels in whole blood using reactive oxygen species produced by leukocyte activation as an index (Non-Patent Document 15). This method uses a luminescence method to measure the amount of reactive oxygen species produced by leukocytes when C3b, which is produced when complement is activated by binding of anti-endotoxin antibodies to endotoxin in the blood, binds to the leukocyte membrane. However, it has been pointed out that this method simply measures leukocyte activation, as EAA values ​​increase when the leukocyte-activating factor interleukin-8 is added to blood (Non-Patent Document 16). Of course, it is believed that red blood cells are not involved at all. [Prior art documents] [Patent documents]

[0019] [Patent Document 1] Patent Publication No. 2004-1117127 Blood endotoxin measurement method (method for measuring endotoxin in white blood cells) [Patent Document 2] Patent No. 47611448 Blood endotoxin measurement method (method of separating plasma and white blood cells, then combining them and measuring) [Patent Document 3] Patent Publication 2013-124905: Method for preparing samples for measuring blood endotoxin (method for obtaining LRP using hydroxyethyl starch) [Patent Document 4] Patent Publication 2017-129429 Preparation method of LRP (preparation method using dextran under ice cooling) [Patent Document 5] Patent No. 6836700 Sample preparation method for measuring endotoxin in the blood of sepsis patients (LRP37 method) [Patent Document 6] JP 04-016765 Endotoxin measurement method (whole blood method) [Non-patent literature]

[0020] [Non-Patent Document 1] Imaeda T, et al, Trends in the incidence and outcome of sepsis using data from a Japanese nationwide medical claims database -the Japan Sepsis Alliance (JaSA) study group- Crit Care 25: 338, 2021 [Non-patent document 2] Singer M, et al. The third international consensus definitions for sepsis and septic shock (Sepsis-3) JAMA. 315: 801-810, 2016 [Non-patent document 3] Homepage (Inada Katsuya) Endotoxin Story http: / / www.asahi-net.or.jp / ~CP6K-IND / index.html (A website with comprehensive information on endotoxins, including a detailed description of Limulus ulcers) [Non-patent document 4] Endo, S. and Inada, K., Endotoxin and Pathogenesis. Health Publishing, 1995. (This book by the inventor is now out of print, but the contents are available on the website above.) [Non-patent document 5] Peng M, Electroochemical sensing strategies for the detection of endotoxin: a review. RSC Advances 3: 9606-9617, 2013 [Non-patent document 6] Olofsson P, et al, Endotoxin inactivation in plasma from septic patients: An in vitro study. World Journal of Surgery 10: 318-323, 1986 (the first paper to suggest that plasma should be cooled) [Non-Patent Document 7] Luchi M and Munford RS, Binding, internalization, and deacylation of bacterial lipopolysaccharide by human neutrophils. J Immunol 151: 959-969, 1993 (endotoxin is internalized within leukocytes) [Non-patent document 8] Graham JM, Isolation of human polymorphonuclear leukocytes (granulocytes) from a leukocyte-rich fraction. Scientific World Journal 2: 1393-1396. 2002 (Description of a method for isolating LRP using a hemagglutinating agent) [Non-Patent Document 9] Takahashi G, Inada K, Sato K, Inoue Y. A dextran-based warming method for preparing leukocyte-rich plasma and its clinical application for endotoxin assay. Biotechniques 68: 300-304, 2020(LRP37 method) [Non-Patent Document 10] Nonoguchi M, et.al, Development of new endotoxin measurement assay using bioluminescence method. J Iwate Med Assoc, 72:181-189, 2020 [Non-Patent Document 11] Inada K, et al, A silkworm larvae plasma test for detecting peptidoglycan in cerebrospinal fluid is useful for the diagnosis of bacterial meningitis. Microbiol Immunol 47:701-707, 2003

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

[0021] The problem we are trying to solve is how to obtain the red blood cell membrane (stroma) while avoiding the influence of hemoglobin in the red blood cells, and whether it is possible to measure the amount of endotoxin. [Means for solving the problem]

[0022] Therefore, we devised a method in which red blood cells are lysed in a hypotonic solution, and hemoglobin is transferred to the outside of the cells, so that the supernatant is removed by centrifugation, and the stroma is obtained as a sediment and used for endotoxin measurement. [Effects of the Invention]

[0023] The present invention makes it possible to detect endotoxin bound to erythrocytes in addition to leukocyte-bound endotoxin, and is expected to further improve the diagnostic efficiency of gram-negative bacterial sepsis. [Brief explanation of the drawings]

[0024] [Figure 1] Figure 1 shows two methods for extracting red blood cell stroma from blood. [Figure 2] Figure 2 shows the amount of endotoxin when the final sample was further washed. [Figure 3] Figure 3 shows the results of an investigation into factors affecting the Limulus test in the final sample. [Figure 4] Figure 4 shows the endotoxin levels in clinical samples. DETAILED DESCRIPTION OF THE INVENTION

[0025] Endotoxin measurements were performed using the clinical-grade Endotoxin Single Test Wako (Fujifilm Wako Pure Chemical Corporation) Limulus reagent and a Toxinometer MT-5500 (Fujifilm Wako Pure Chemical Corporation) using a turbidimetric method, i.e., evaluation of the increase in turbidity. All equipment, reagents, and water were endotoxin-free, meaning they contained only trace amounts of endotoxin that would not affect the endotoxin measurement results (the minimum detection sensitivity of the turbidimetric method is 0.05 pg / mL or less). Endotoxin-free pipette tips (Fujifilm Wako Pure Chemical Corporation Bioclean Tips) were used for the procedure. Commercially available test tubes (e.g., 15 mL Falcon graduated centrifuge tubes, polypropylene) that had been irradiated and confirmed to be endotoxin-free were used. [Example]

[0026] <Collection method 1 for specimens containing red blood cells> (Figure 1) After wetting the inner wall with heparin and discarding excess heparin (final heparin concentration: approximately 10 units), 0.5 mL of collected human blood is added to a 15 mL graduated plastic centrifuge tube (0101). Approximately 10 mL of cold water is added (0102) and the tube is centrifuged at 3,000 rpm (approximately 1,500 x g) for 10 minutes. Pink stroma precipitates (0103). The supernatant is carefully discarded, and cold water is added up to the 5 mL mark (0104). This results in a 10-fold dilution of the original blood volume. Endotoxin is measured using turbidimetry on this 200 μL tube. Due to this centrifugal washing and dilution process, it is believed that almost no plasma is present. This method is the simplest, although there is a possibility that leukocytes may be present and dissolved in the cold water, resulting in cell membrane fragments being precipitated along with the stroma during centrifugation. The obtained endotoxin value (pg / mL) was multiplied by 10 to obtain the final value.

[0027] <Collection method 2 for specimens containing red blood cells> (Figure 1) For example, 0.5 mL of heparinized human blood is added to a 15 mL graduated plastic tube (0101). The tube is centrifuged at 3,000 rpm (approximately 1,500 x g) for 10 minutes. The supernatant (plasma) (0105) and the white blood cell layer (0106), which is the upper cell layer (buffy coat), are carefully aspirated and discarded with a pipette. Approximately 10 mL of cold water is added to the red blood cell layer (0107) below (0108) and centrifuged at 3,000 rpm (approximately 1,500 x g) for 10 minutes. Stroma precipitates (0109), so the supernatant is carefully discarded and cold water is added up to the 5 mL mark (0110). This results in a 10-fold dilution of the original blood volume. This 200 μL is then measured for endotoxin using turbidimetry. Due to this centrifugal washing and dilution process, it is believed that almost no plasma remains. This is the sample to be measured. This method yields samples free of plasma and leukocyte contamination, but requires two centrifugation steps. The endotoxin level (pg / mL) obtained was multiplied by 10 to obtain the final value.

[0028] <Test to confirm whether endotoxin is bound to red blood cells; Addition experiment> The red blood cell layer (Figure 1(0107)) obtained from heparinized healthy blood was suspended in an equal volume of 2% human serum albumin in saline. 200 pg (10 μL) of Escherichia coli O111:B4 LPS (phenol-water extract, Sigma-Aldrich) was added to 1 mL of the red blood cell suspension and the mixture was shaken and warmed at 37°C for 20 minutes. Approximately 15 mL of cold water was added to the mixture to lyse the blood, and the mixture was centrifuged at 3,000 rpm for 10 minutes to obtain a pink stromal pellet. The supernatant was aspirated and discarded, and 1 mL of water was added to the pellet and thoroughly mixed using a vortex mixer. 100 μL of the diluted solution was diluted 10-fold with 0.002% Triton X-100, and 200 μL of the diluted solution was used to measure the endotoxin concentration by turbidimetry (one wash). Approximately 10 mL of cold water was added to the remaining 900 μL of stroma fluid, and the mixture was centrifuged at 3,000 rpm for 10 minutes. The supernatant was discarded, and 900 μL of water was added and thoroughly mixed using a vortex mixer. 100 μL of the mixture was diluted 10-fold with 0.002% Triton X-100, and 200 μL of the diluted mixture was used to measure endotoxin concentration by turbidimetry (washed twice). Approximately 10 mL of cold water was added to the remaining 800 μL of stroma fluid, and the mixture was centrifuged at 3,000 rpm for 10 minutes. The supernatant was discarded, and 800 μL of water was added and thoroughly mixed using a vortex mixer. 100 μL of the mixture was diluted 10-fold with 0.002% Triton X-100, and 200 μL of the diluted mixture was used to measure endotoxin concentration by turbidimetry (washed three times). <Result> As a result, as shown by ○-○ in Figure 2, endotoxin was sufficiently recovered from red blood cells even after washing three times. The endotoxin concentration in stromal fluid without added LPS was 0.5 pg / mL or less. The endotoxin concentration in water was 0.05 pg / mL or less. Figure 2 ●-● also shows the endotoxin levels of samples from patients with sepsis measured using Method 2, which were then centrifuged and washed with excess water.

[0029] <Consideration> If endotoxin had simply been present in the red blood cell suspension, most of the endotoxin level would have been lost by washing, but these results indicate that endotoxin is bound to the red blood cells.

[0030] <Factors remaining in the final sample that affect the Limulus test> Because the final sample (0104 and 0110 in Figure 1) contains almost no plasma, it is believed that the influencing factors in plasma are not present in the final sample. Furthermore, it is believed that any influencing factors that may be contained in white blood cells escape outside the cells when water is added, resulting in the destruction of the cells and therefore being almost absent in the final sample. Generally, when plasma is diluted 150-fold or more, the activity of the influencing factors becomes almost undetectable (Non-Patent Document 17). Therefore, to confirm this, we investigated whether it would be possible to omit the heating step, for example, at 70°C for 10 minutes, which inactivates the influencing factors, using the final processed sample. <Result> As shown in Figure 3, in the case of the final processed patient blood sample in which endotoxin was detected (solid line in the figure), even when heated at 70°C for 10 minutes, the value was slightly higher than when not heated (unheated), but there was no significant difference between the two groups (P>0.05, t-test).In addition, when a fixed amount of endotoxin (LPS derived from E. coli O111:B4) was added to the final sample using samples from healthy individuals, the recovery amounts of both groups were compared (dotted line in the figure), but the difference was not significant (P>0.05, t-test). <Consideration> Therefore, it was found that the final sample contained almost no factors affecting the Limulus test, and it was found that the final samples (0104 and 0110 in Figure 1) did not need to be heated to inactivate the factors, as was the case with plasma.

[0031] <Study on clinical samples> Using heparinized blood collected from patients with sepsis, the amount of endotoxin was measured by turbidimetry using Method 1 (a 0.02% Triton X-100 aqueous solution was used instead of water to make the total volume 5 mL). <Result> As a result, as shown in Figure 4, endotoxin was detected in one healthy subject (◯) at 0.5 pg / mL or less, and in five sepsis patients (●) (average of two measurements). <Consideration> It has been shown that the method for detecting endotoxin in red blood cell fractions devised by the inventors can be used as a method for measuring endotoxin in patients with sepsis.

[0032] The centrifugal acceleration of the centrifuge of approximately 1500 xg and the centrifugation time of 10 minutes are not particularly limited, and other conditions may be used as long as stroma can be obtained as a sediment.

[0033] The water used to lyse red blood cells in blood is preferably cold water cooled to around 4°C or below to avoid the action of endotoxin-inactivating factors contained in plasma, but there is no particular limit to this temperature. Furthermore, the amount of cold water used to lyse blood is approximately 20 times the volume of blood, but this amount is not limited.

[0034] In addition to water, ammonium chloride, amines, surfactants, etc. may also be used to lyse red blood cells as long as they do not affect the Limulus reaction.

[0035] An anticoagulant is added to the blood, and any common anticoagulant suitable for the Limulus test can be used. For example, heparin can be used 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.

[0036] To homogenize the stroma, it is desirable to thoroughly mix the stroma with a vortex mixer. In this case, homogenization procedures such as ultrasonic disruption, physical disruption, and freeze-thawing may also be used.

[0037] Limulus testing may be performed using commonly used methods such as gelation, colorimetric, turbidimetric, and endotoxin light scattering methods, or other methods. The horseshoe crab hemocyte extract used in this test is not particularly limited, as long as it is extracted from the hemocytes of horseshoe crabs of the genus Limulus, Tachypleus, or Carcinoscorpius, and can be used for standard endotoxin assays, such as those manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Furthermore, methods using recombinant factors rather than natural Limulus pathway factors may also be used. A recently developed bioluminescence method may also be used (Non-Patent Document 8). Furthermore, this method may also be applied to a blood endotoxin assay method that does not use a Limulus reagent, which is expected to be developed in the future (Non-Patent Document 1).

[0038] This method can be applied to the detection of endotoxins in biological samples containing red blood cells, such as umbilical cord blood, bone marrow cells, body fluids, cerebrospinal fluid, and urine, in addition to what is generally called blood. [Industrial Applicability]

[0039] Sepsis, once severe, is difficult to treat and can sometimes be fatal despite advances in modern medicine. Therefore, early detection of signs of infection and appropriate treatment before the condition worsens are crucial. Traditional methods include inflammatory cytokine and procalcitonin tests, and more recently, presepsin tests have been developed. The presence of gram-negative bacteria in the blood is thought to increase the risk of severe illness, as their endotoxin promotes abnormal cytokine production. Traditional culture methods for directly detecting the presence of bacteria in the blood have been around for a long time, but they have the drawback of being time-consuming and the positive rate is currently not as high as expected. Therefore, early detection of bacterial presence significantly contributes to treatment decisions. The blood endotoxin measurement method developed here can detect the presence of gram-negative bacteria within a maximum of two to three hours of blood collection, potentially providing important insights into appropriate antibiotic selection, rather than relying on empirical antibiotic treatment.

Claims

1. A method for preparing samples for endotoxin measurement using human red blood cells.

2. A method for preparing a sample for endotoxin measurement according to claim 1, in which approximately 20 volumes of cold water are added to one volume of blood collected using an anticoagulant to dissolve cells, the mixture is centrifuged at approximately 1500 x g for 10 minutes, and the resulting sediment is suspended in water.

3. A method for preparing a sample for endotoxin measurement according to claim 1, in which blood collected using an anticoagulant is centrifuged at approximately 1500 x g for 10 minutes to separate it into plasma and sediment, the plasma is removed by suction, and then approximately 20 volumes of cold water are added to one volume of sediment to dissolve the cells, and the resultant is centrifuged at approximately 1500 x g for 10 minutes, and the resulting sediment is suspended in water.

4. A method for preparing a sample for endotoxin measurement according to claim 1, in which blood collected using an anticoagulant is centrifuged in a centrifuge at approximately 1500 x g for 10 minutes to separate it into plasma and sediment, the plasma is removed by suction, and then the upper layer of the white blood cell layer out of the white blood cell layer and red blood cell layer that form the sediment is removed by suction, and approximately 20 times the volume of cold water per volume of red blood cell layer is added to lyse the cells, and the mixture is centrifuged in a centrifuge at approximately 1500 x g for 10 minutes, and the resulting sediment is suspended in water.

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