JP2009229340A - Method and device for creating information on severity of systemic inflammatory response syndrome - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and device for easily crerating information indicating whether a patient having undergone an operation using an artificial heart-lung machine, especially a patient carried to an ICU (intensive care unit) may be suffering from SIRS (systemic inflammatory response syndrome). <P>SOLUTION: The method includes a step of acquiring information regarding the concentration of interleukin-1 acceptor antagonist (IL-1RA) contained in the blood extracted from the patient having undergone the operation using the artificial heart-lung machine, and a step of generating the information regarding the severity of systemic inflammatory response syndrome based on the information on the concentration of IL-1RA acquired in the above process. Preferably, the blood is sampled in 15 to 100 minutes after detachment of the artificial heart-lung machine, and the patient may be dosed with a steroid agent. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method and apparatus for generating information regarding the severity of a systemic inflammatory response syndrome that develops after surgery using an oxygenator.

Excessive biological reactions caused by various invasions such as surgery, trauma, burns, and sepsis have been covered by a new concept called systemic inflammatory response syndrome (SIRS). A concept announced by ACCP / SCCM in 1992. Abnormality in two or more of the following four items (body temperature, heart rate, respiratory rate, peripheral blood leukocyte count) reflecting systemic inflammatory response to clinical invasion A state with is defined as SIRS.
(I) Body temperature> 38 ° C. or <36 ° C. (ii) Heart rate> 90 / min (iii) Respiration rate 20 / min or Pa (CO ₂ ) <32 Torr (iv) White blood cell count 12000 / mm ³ ultra or less 4000 / mm ^3, or immature granulocytes than 10%

  When severe SIRS occurs, organ dysfunction, circulatory insufficiency (lactic acidosis, oliguria, acute consciousness disorder, etc.) or decreased blood pressure (systolic blood pressure <90 mmHg or blood pressure decreased by 40 mmHg or more than normal systolic blood pressure) are likely to occur. Become. Furthermore, multiple organ dysfunction (MODS), which is a syndrome in which multiple vital organs or system dysfunctions occur at the same time, or sepsis that spreads throughout the body due to the invading disease causing the pathogen to enter the blood, will die. It will be every case. Since the progression to sepsis and multi-organ failure (MODS) proceeds relatively shortly after entering the intensive care unit (ICU), patients with SIRS are frequently monitored by the ICU and rapidly. It is required to respond quickly to changes in symptoms. On the other hand, if there are no complications, there is SIRS that can be removed from SIRS within 3 days after the operation, and can be transient. Thus, SIRS includes severe SIRS that may cause complications and death, and SIRS that does not. Therefore, it is considered reasonable to generate information about the severity of the patient's SIRS and to distinguish between critical and non-critical patients in ICU monitoring.

  Surgery is one of the major invasions of living bodies, and the incidence of SIRS is high. According to “Critical Care SIRS / Shock / MODS”, pp. 63-64 (non-patent document 1), the incidence of SIRS after digestive surgery is 100% for esophagectomy and hepatectomy, and 78% for colectomy. And high rate. In surgery using a heart-lung machine (CBP), activation by contact of blood with an artificial surface by CBP circuit or intracardiac suction, non-physiological blood flow such as non-pulsatile flow, ischemia reperfusion Significantly exacerbates the systemic inflammatory response (Non-Patent Document 2: Anesthesia for cardiac surgery, 3rd edition, Tokyo, Medical Science International, 2004) Effect on immunity, 1. Systemic inflammatory reaction).

  Thus, SIRS occurs at a high rate due to surgery, but digestive surgery and circulatory surgery cannot be taken in the same way. For example, in the case of gastrointestinal surgery, the incidence of SIRS is high in relation to high infection at the surgical site, and there is a high possibility of reaching severe SIRS. Therefore, a high degree of monitoring is required for all patients. On the other hand, in the case of cardiac surgery using an artificial cardiopulmonary apparatus, it is possible to administer an anti-inflammatory agent such as a steroid during surgery for infection. Therefore, steroid administration can reduce the onset of SIRS due to infection and reduce SIRS symptoms. Therefore, high level monitoring is not required for all patients after surgery.

  In other words, in surgery using a heart-lung machine, if information on the severity of a patient's SIRS can be generated to distinguish between severe SIRS and non-SIRS patients in advance, high efficiency such as ICU monitoring systems and medication schedules can be achieved. And rationalization can be achieved.

  In SIRS, as a local or systemic reaction, inflammatory cytokines centered on TNF-α, IL-1, IL-6, IL-8, etc .; anti-inflammatory such as IL-4, IL-1RA, IL-10, etc. It is known that sex cytokines are elevated.

  Therefore, there is a concept of distinguishing severe SIRS from other SIRS using markers such as cytokines. For example, in Japanese Translation of PCT International Publication No. 2005-521882 (Patent Document 1), CD64, CD11, HLA detected using flow cytometry or the like is used as a method for monitoring SIRS patients at risk of developing sepsis that may occur in the near future. It has been proposed to measure cell surface markers such as DR, markers such as IL-6 and IL-10 that can be measured using an immunosorbent assay, and identify early sepsis using a classification tree. In the examples, patients who developed SIRS after being accommodated in a surgical intensive care unit of a hospital were examined, and blood samples were collected every day for a maximum of 14 days or until the patient developed sepsis from the date of study. , Monitoring markers and identifying patients with SIRS who are prone to progress to sepsis. However, Patent Document 1 does not present a method that can distinguish patients who are likely to die of sepsis or death at an early stage.

  In addition, Special Table 2006-219473 (Patent Document 2) removes leukocytes including neutrophils from the blood of a subject for the purpose of suppressing infection at a surgical site that occurs at a high rate in digestive surgery. Regarding the method, there is the following description regarding cytokines produced by surgery. That is, in Example 6 and paragraphs 0083 to 0090, in patients undergoing colorectal cancer surgery, the white blood cell count increased during the surgery, and a significant increase in the white blood cell count occurred within 2 hours after the induction of anesthesia. IL-6 reaches its maximum at 4 hours after induction of anesthesia, IL-1RA shows a significantly higher value at 4 hours after induction of anesthesia, and the highest value at 2 hours after operation. It is explained that it was found to reach

  Furthermore, Non-Patent Document 1 discloses that in esophageal cancer surgery, the serum IL-6 value reaches a maximum value at 6 hours after the operation, and then gradually decreases, and the serum IL-8 value is the highest. It is disclosed that the value is observed on the first disease day slightly later than IL-6. And, it is stated that monitoring of cytokines such as IL-6 and IL-8 under invasion is useful for predicting the occurrence of organ damage. Moreover, since sTNF-RI and IL-1RA, which are inhibitors of TNF and IL-1, both show the highest value during the operation and show an extremely early response to the surgical invasion, these are postoperative organ damages. It is described that it becomes an index of onset monitoring (FIGS. 2-3 and 2-4 on page 13 and 14). However, the relationship between SIRS symptoms (severe SIRS and non-SIRS) has not been clarified.

  Furthermore, Non-Patent Document 1 shows that IL-6 and IL-8, which are inflammatory cytokines, are significantly increased from 60 minutes after release of aortic block in open-heart surgery compared to before introduction and 60 minutes after aortic block. And IL-10, which is an anti-inflammatory cytokine, IL-1RA, which is an antagonist, soluble TNF (sTNF) -αRI, and sTNF-αRII, have been disclosed that production increases from 60 minutes after the release of aortic blockade. . However, it has not been shown whether it is possible to distinguish severe SIRS from other SIRS by judging how to use any kind of cytokine.

  Thus, although it is known that SIRS is related to the production of various inflammatory cytokines and anti-inflammatory cytokines, the production behavior of cytokines for surgery cannot be said to be the same depending on the operative method. The current situation is that a cytokine useful for distinguishing non-SIRS cannot be narrowed down to any cytokine.

Special table 2005-521882 Special table 2006-219473 "Critical Care SIRS / Shock / MODS" School of Medicine, 2001 Anesthesia for cardiac surgery, 3rd edition, Tokyo, Medical Science International, 2004

  The present invention has been made in view of such circumstances, and an object thereof is a method and apparatus for generating information on the severity of systemic inflammatory response syndrome in a patient who has performed an operation using an oxygenator. Is to provide. In particular, it is an object of the present invention to provide a method and an apparatus capable of easily generating information indicating whether a patient carried to an ICU has severe SIRS or not.

  That is, the method for generating information on the severity of the systemic inflammatory response syndrome of the present invention is an interleukin 1 receptor antagonist (IL-1RA) contained in blood collected from a patient after surgery using an oxygenator. Obtaining information relating to the concentration of the patient, and generating information relating to the severity of the systemic inflammatory response syndrome of the patient based on the information relating to the concentration of IL-1RA obtained in the obtaining step.

  The blood collection is preferably performed 15 to 100 minutes after leaving the heart-lung machine, and the patient may be administered a steroid during surgery.

  The information generating step is preferably a step of generating information indicating whether or not the patient has a severe systemic inflammatory reaction syndrome based on information on the concentration of IL-1RA. Further, the information regarding the concentration of IL-1RA obtained in the acquisition step is a value regarding the concentration of IL-1RA, and the information generation step includes a value regarding the concentration of IL-1RA and a predetermined threshold value. In comparison, when the value relating to the concentration of IL-1RA is higher than the threshold, it is more preferable to generate information indicating that the patient has a severe systemic inflammatory response syndrome.

  The apparatus for generating information on the severity of systemic inflammatory response syndrome according to the present invention provides information on the concentration of interleukin 1 receptor antagonist (IL-1RA) contained in blood collected from a patient after a cardiopulmonary apparatus operation. An information acquisition unit for acquiring; an information generation unit for generating information on the severity of a systemic inflammatory reaction syndrome of a patient based on information on the concentration of IL-1RA obtained by the information acquisition unit; and the information generation unit An output unit for outputting the generated information is included.

  It is preferable that the information generating unit generates information indicating that the patient has a severe systemic inflammatory reaction syndrome based on information on the IL-1RA concentration.

  The method for generating information on the severity of systemic inflammatory response syndrome according to the present invention is obtained by collecting blood of a patient after surgery using an oxygenator and obtaining information based on the IL-1RA concentration. Can be distinguished whether the patient is a severe SIRS patient.

  The method of generating information regarding the severity of systemic inflammatory response syndrome in a patient of the present invention is an interleukin 1 receptor antagonist (IL-1RA) contained in blood collected from a patient after surgery using a cardiopulmonary apparatus. Obtaining information on the concentration of the patient, and generating information on the severity of the systemic inflammatory response syndrome of the patient based on the information on the concentration of IL-1RA obtained in the obtaining step.

The patient targeted by the method of the present invention is a patient who has undergone surgery using a heart-lung machine. A heart-lung machine is a device used for extracorporeal circulation designed to safely block cardiac blood flow. The pump part plays the role of the heart that pumps blood, and oxygen is added to the blood to eliminate carbon dioxide. It is a device provided with an artificial lung part that functions as a lung.
Surgery using a heart-lung machine specifically includes coronary artery bypass surgery, thoracic aortic aneurysm surgery, aortic valve replacement surgery, artificial valve replacement surgery, venthal surgery, mitral valve surgery, mitral valve replacement surgery, intraventricular Surgery involving the heart, such as perforation of the membrane.

  The patient includes a patient who received steroid administration during surgery. As described above, in surgery using a heart-lung machine, it is possible to administer an anti-inflammatory agent such as steroid to the patient during surgery. For this reason, in surgery using an oxygenator, there are many cases where severe SIRS caused by infection can be suppressed. Therefore, generating information on the severity of SIRS of a patient who has undergone surgery using a heart-lung machine is important in improving the efficiency and streamlining of the ICU monitoring system and medication schedule. That is, if a patient who has a high risk of complications such as sepsis and multiple organ failure can be distinguished from a patient who is not so in an ICU (intensive care unit) after surgery, the monitoring efficiency of the patient in the ICU can be increased.

  The collection of blood is preferably performed after surgery, that is, after suturing the thoracotomy and before entering the ICU. Specifically, although it depends on the time required for movement in the hospital facility from the operating room to the ICU, the time required for X-ray imaging, etc., it is usually 15 minutes to 100 minutes after the cardiopulmonary bypass, preferably Perform between 20 minutes and 90 minutes. This is because the blood concentration of IL-1RA appears to be particularly high during this degree. It is possible to rationally plan the medication and monitoring schedule in the ICU by distinguishing whether it is severe SIRS or not in the situation immediately after entering the ICU.

  The information regarding the concentration of IL-1RA in the present invention is information indicating the IL-1RA concentration directly or indirectly. More specifically, it is information obtained by measuring the IL-1RA concentration described later. For example, the information obtained by directly measuring the IL-1RA concentration includes the IL-1RA concentration. Further, information indirectly indicating the IL-1RA concentration includes absorbance, reflection intensity, fluorescence intensity, and the like.

  The sample for obtaining information on the concentration of IL-1RA contained in the collected blood may be the collected blood itself, or a plasma component excluding red blood cells and peripheral blood mononuclear cells by centrifugation. It may be used. A plasma component is preferred. In the operation using the heart-lung machine, the concentration of IL-1RA begins to rise after the heart-lung machine is released, reaches a peak in about 20 minutes after the heart-lung machine, and then decreases. Therefore, the concentration of IL-1RA is excellent as a marker for generating information regarding the severity of SIRS of a patient in a test performed during ICU movement after surgery. In this regard, IL-6, IL-8, etc. are cytokines associated with SIRS symptoms, but these cytokine concentrations in samples collected after surgery and during ICU transfer differed between severe SIRS and other SIRS. Is not as pronounced as IL-1RA.

  The method for measuring the concentration of IL-1RA can be performed by a known method. For example, measurement methods such as a radioimmunoassay method (RIA method), an enzyme immunization method (EIA method), or a fluorescence immunization method (FIA method) can be mentioned. Of these, the EIA method is preferable, and the ELISA method using an antibody immobilized on a solid phase is particularly preferable among the EIA methods.

  Information about the severity of the patient's systemic inflammatory response syndrome is generated based on the measured concentration of IL-1RA. Here, as information regarding the severity of the systemic inflammatory reaction syndrome, for example, information indicating whether or not it is severe can be mentioned. In addition, information indicating the severity of SIRS in stages is also included. Information that shows the severity of SIRS in stages includes information using severe, moderate, and mild.

  The information regarding the severity of the systemic inflammatory response syndrome of the patient of the present invention is preferably information indicating whether or not the patient has a severe systemic inflammatory response syndrome. Here, the generation of information indicating whether or not the patient has a severe systemic inflammatory response syndrome is performed based on the measured concentration of IL-1RA in the blood of the patient.

  Information indicating whether or not the patient has severe systemic inflammatory response syndrome can be generated by, for example, comparing the measured concentration of IL-1RA in the blood of the patient with a predetermined threshold. That is, when the measured concentration value of IL-1RA is higher than a predetermined threshold value, information indicating a severe systemic inflammatory response syndrome can be generated. On the contrary, when it is lower than the predetermined threshold value, information indicating that it is not a severe systemic inflammatory reaction syndrome can be generated.

  In addition, information indicating the severity of SIRS in a stepwise manner can be generated by providing a predetermined threshold. That is, when the measured IL-1RA concentration value falls within the threshold range, information indicating that the SIRS is moderate is generated. And when larger than the upper limit of the width | variety of a threshold value, the information which shows that it is severe SIRS is produced | generated. On the contrary, when it is smaller than the lower limit of the threshold width, information indicating that the SIRS is mild is generated. Furthermore, it is possible to generate more detailed information by setting a plurality of widths to a predetermined threshold.

  In addition, the information which shows whether it is a serious systemic inflammatory reaction syndrome is produced | generated by comparing with the value of the density | concentration of IL-1RA measured with the blood used as control other than the method using the above-mentioned threshold value. You can also. That is, if the measured value of IL-1RA concentration in the patient's blood is higher than a certain level than the measured value of IL-1RA concentration in the control blood, the information indicates severe SIRS. Can be generated. On the other hand, when it is low, information indicating that the SIRS is not severe can be generated. Examples of blood used as a control include blood of healthy persons and preoperative patients.

In the present invention, severe SIRS refers to SIRS that may lead to sepsis or multiple organ failure. More specifically, it refers to the severity of SIRS of a patient corresponding to three or more items among the following items (i) to (iv).
(I) Body temperature above 38 ° C. or below 36 ° C. (ii) Heart rate above 90 / min (iii) Respiration rate above 20 / min or Pa (CO ₂ ) below 32 Torr (iv) White blood cell count 12000 / mm ³ greater or less than 4000 / mm ^3, or immature granulocytes than 10%

  That is, if the method for generating information on the severity of SIRS of a patient according to the present invention is used, severe SIRS patients can be distinguished without examining the above four items. Usually, as a patient's symptom, it is determined as SIRS when two items among items (i) to (iv) correspond, but severe SIRS to be determined in the present invention corresponds to three or more items. Is. Usually, the two items (i) and (iv) are first applicable, and the item (ii) or (iii) appears in the more severe case.

  The predetermined threshold used to generate the information can be derived from IL-1RA concentration data for a number of patients who have undergone surgery using a heart-lung machine. More specifically, it is possible to derive from IL-1RA concentration data 15 to 100 minutes after cardiopulmonary withdrawal for patients with severe SIRS and those who are not. These may vary depending on the accumulation and accuracy of data. For example, a threshold value of IL-1RA concentration in distinguishing severe SIRS patients from other patients is 5 ng / ml or more, preferably 10 ng / ml. The above is mentioned. By setting the predetermined threshold value to 5 ng / ml or more, it is possible to distinguish between the case where three or more items among the SIRS determination items (i) to (iv) are applicable and the case of two items or less. Thus, the method of generating information regarding the severity of SIRS of a patient of the present invention makes it possible to distinguish between patients with severe SIRS and those who are not.

[Information generator]
The information generation apparatus regarding the severity of SIRS of the present invention is information for acquiring information regarding the concentration of interleukin 1 receptor antagonist (IL-1RA) contained in blood collected from a patient after surgery using a heart-lung machine. An acquisition unit; an information generation unit that generates information on the severity of a systemic inflammatory response syndrome of a patient based on information on the concentration of IL-1RA obtained by the information acquisition unit; and the information generation unit An output unit for outputting information is included.

An embodiment of the information generation apparatus of the present invention will be described based on the hardware configuration example of FIG.
The information generating apparatus according to the present embodiment is mainly composed of measuring apparatus 150 and computer 100. The computer 100 mainly includes an information generation unit 110, a display 120, and an input device 130. The information generation unit 110 mainly includes a CPU 110a, a ROM 110b, a RAM 110c, a hard disk 110d, a reading device 110e, an input / output interface 110f, a communication interface 110g, and an image output interface 110h, and the CPU 110a and the ROM 110b. The RAM 110c, the hard disk 110d, the reading device 110e, the input / output interface 110f, the image output interface 110h, and the communication interface 110g are connected by a bus 110i so that data communication is possible.
The output unit corresponds to the display 120.

  The measuring device 150 is provided with a measuring device for acquiring measurement data relating to the IL-1RA concentration of a blood sample collected from a patient. The measurement apparatus differs depending on the method for measuring the IL-1RA concentration. For example, when measuring by ELISA, a commercially available absorption microplate reader can be used as the measurement apparatus 150.

  The measurement data regarding the IL-1RA concentration acquired by the measurement device 150 is sent as a digital signal to the information generation unit 110 through the communication interface 110g. The measurement data varies depending on the measurement method, but includes data that can calculate the IL-1RA concentration, such as absorbance, reflection intensity, and fluorescence intensity. For example, when the IL-1RA concentration is measured by an absorption microplate reader, a digital signal related to absorbance is sent to the information generation unit 110 through the communication interface 110g. Note that the communication interface 110g also has a function for transmitting a command from the CPU 110a for controlling the operation of the measuring apparatus 150.

  Note that IL-1RA concentration measurement data may be input using an input device 130 such as a keyboard or a mouse, and sent to the information generation unit 110 via the input / output interface 110f. That is, when the user measures the IL-1RA concentration using an external measuring device, the measurement data can be taken into the information generation unit 110 from the input device 130.

  The ROM 110b is configured by a mask ROM, PROM, EPROM, EEPROM, or the like, and records a program necessary for starting the computer 100.

  The RAM 110c is configured by SRAM, DRAM, or the like. The RAM 110c is used for reading computer programs recorded in the ROM 110b and the hard disk 110d. Further, when these computer programs are executed, they are used as a work area of the CPU 110a. Measurement data (or a digital signal corresponding to this) input from the input / output interface 110f may be temporarily stored.

  The hard disk 110d includes an arithmetic processing program for calculating the IL-1RA concentration from the measurement data, information on the IL-1RA concentration and the severity of SIRS, and an operating system (for example, Windows (registered trademark) manufactured and sold by Microsoft Corporation, USA). ) And other computer programs to be executed by the CPU 110a, such as an operating system that provides a graphical user interface environment) and application programs. An application program 140a described later is also installed in the hard disk 110d.

  The reading device 110e is configured by a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, or the like, and can read a computer program or data recorded on the portable recording medium 140. In addition, the portable recording medium 140 may store an application program 140a for causing the computer 100 and the measurement device 150 to function as the information generation device according to the present invention. The computer 100 can read the application program 140a according to the present invention from the portable recording medium 140 and install the application program 140a in the hard disk 110d.

  The application program 140a is not only provided by the portable recording medium 140, but also from an external device that is communicably connected to the computer 100a via an electric communication line (whether wired or wireless). It is also possible to provide through. For example, the application program 140a is stored in a hard disk of a server computer on the Internet. The computer 100 can access the server computer, download the computer program, and install it on the hard disk 110d. is there.

  The image output interface 110h is connected to a display 120 constituted by an LCD, a CRT, or the like, and outputs a video signal corresponding to the arithmetic processing result given from the CPU 110a to the display 120. The display 120 displays the processing result in the information generation unit 110, that is, information on the severity of SIRS of the subject patient on the screen according to the input video signal. Data that uniquely corresponds to the concentration of IL-1RA or the concentration of IL-1RA (absorbance, reflection intensity, fluorescence intensity, etc.) may be displayed together.

  The CPU 110a can execute a computer program stored in the memory and a computer program loaded in the RAM 110c. The information generation unit 110 of the computer 100 functions when the CPU 110a executes an application program 140a described later. Specifically, an arithmetic processing program for calculating the IL-1RA concentration from the measurement data stored in the hard disk 110d is executed, and the measurement data regarding the IL-1RA concentration input from the measurement device 150 to the information generation unit 110 Based on the above, the IL-1RA concentration or the IL-1RA concentration in the blood of the patient from whom the blood was collected is uniquely calculated. Based on the IL-1RA concentration and SIRS severity information stored in the hard disk 110d and the IL-1RA concentration or corresponding data, the CPU 110a generates information on the patient's SIRS severity. The For example, when the information on the IL-1RA concentration and the severity of SIRS is a predetermined threshold of the concentration of IL-1RA for distinguishing whether or not it is severe SIRS, the IL in the patient's blood is more than the threshold. When the -1RA concentration is high, information indicating severe SIRS can be generated. The generated information regarding the severity of the SIRS of the patient is displayed on the display 120 which is an output unit.

[Method for preparing sample for cytokine measurement]
(1) Preparation of a sample for cytokine measurement from collected blood After adding an equal amount of physiological saline to blood collected by a vacuum blood collection tube and diluting it twice, on Ficoll (specific gravity solution for lymphocyte isolation) Layered and centrifuged to separate erythrocytes, peripheral blood mononuclear cells and plasma components. After centrifugation, plasma components were collected and used as cytokine measurement samples (plasma samples).
Unless otherwise specified, the sample could be used for measurement without dilution. However, if the sample measurement data exceeded the calibration curve range, the sample was diluted at an appropriate magnification (10 times or 50 times). ) And again used for measurement.

[Method for measuring cytokine concentration]
(1) Kit used for measurement Each cytokine was measured using an ELISA kit shown in Table 1.

(2) Measurement of each cytokine concentration of the cytokine measurement sample In parallel with the measurement sample (plasma sample), the dilution series of the calibrator adjusted for the calibrator attached to each measurement kit is also processed according to the kit protocol and calibrated. While creating a line, the cytokine concentration is calculated from the measured value of the plasma sample.
A calibration curve for each cytokine is prepared by plotting the calibrator concentration on the X-axis and the absorbance on the Y-axis, and using the least square method.

(2-1) Measurement of IL-1RA A calibrator is diluted according to Table 2, and a calibrator dilution series is adjusted.

  100 μl of the diluted solution is added to each well containing 50 μl of each concentration calibrator or prepared plasma sample, and incubated at room temperature for 2 hours. After completion of the reaction, the wells are washed three times with Wash buffer, 50 μl of biotinylated anti-IL-1RA antibody and 100 μl of HRP (House radish peroxidase) -labeled streptavidin are added to each well, and incubated at room temperature for 30 minutes. After completion of the reaction, the plate is washed 3 times with Wash buffer, 100 μl of substrate is added, and incubated at room temperature for 20 minutes. After 20 minutes, 50 μl of the reaction stop solution is added, and the absorbance at 450 nm is measured using a microplate reader. Based on the calibration curve prepared using the dilution series of the calibrator, the IL-1RA concentration is calculated from the absorbance of the plasma sample.

各濃度のキャリブレータあるいは調製した血漿サンプルを５０μｌずつ各ｗｅｌｌに加え、その後５０μｌのビオチン化抗ＩＬ−１β抗体を添加し、室温で２時間インキュベートする。反応終了後、Ｗａｓｈバッファーにより３回洗浄し、各ｗｅｌｌに１００μｌのＨＲＰ標識ストレプトアビジンを添加し、室温で３０分間インキュベートする。反応終了後、Ｗａｓｈバッファーにより３回洗浄し、基質を１００μｌ添加し、３０分間室温でインキュベートする。３０分後、反応停止溶液を５０μｌ加え、マイクロプレートリーダーを用いて４５０ｎｍの吸光度を測定する。キャリブレータの希釈系列を用いて作成した検量線に基づいて、血漿サンプルの吸光度からＩＬ−１β濃度を算出する。 (2-2) Measurement of IL-1β A calibrator is diluted according to Table 3, and a calibrator dilution series is adjusted.

50 μl of each concentration of calibrator or prepared plasma sample is added to each well, and then 50 μl of biotinylated anti-IL-1β antibody is added and incubated at room temperature for 2 hours. After completion of the reaction, the wells are washed 3 times with Wash buffer, 100 μl of HRP-labeled streptavidin is added to each well, and incubated at room temperature for 30 minutes. After completion of the reaction, the plate is washed 3 times with Wash buffer, 100 μl of substrate is added, and incubated at room temperature for 30 minutes. After 30 minutes, 50 μl of the reaction stop solution is added, and the absorbance at 450 nm is measured using a microplate reader. Based on the calibration curve created using the dilution series of the calibrator, the IL-1β concentration is calculated from the absorbance of the plasma sample.

５０μｌのビオチン化抗ＩＬ−６抗体を各ｗｅｌｌに加えた後、キャリブレーターあるいは調製した血漿サンプルを５０μｌずつを添加し、さらに５０μｌの希釈溶液を加えて、室温で２時間インキュベートする。反応終了後、Ｗａｓｈバッファーにより３回洗浄し、各ｗｅｌｌに１００μｌのＨＲＰ標識ストレプトアビジンを添加し、室温で３０分間インキュベートする。反応終了後、Ｗａｓｈバッファーにより３回洗浄し、基質を１００μｌ添加し、３０分間室温でインキュベートする。３０分後、反応停止溶液を１００μｌ加え、マイクロプレートリーダーを用いて４５０ｎｍの吸光度（リファレンス：５５０ｎｍ）を測定する。測定された４５０ｎｍの吸光度から５５０ｎｍの吸光度を引いた値を測定値とし、キャリブレータの希釈系列を用いて作成した検量線に基づいて、血漿サンプルの吸光度からＩＬ−６濃度を算出する。 (2-3) Measurement of IL-6 A calibrator is diluted according to Table 4, and a calibrator dilution series is adjusted.

After 50 μl of biotinylated anti-IL-6 antibody is added to each well, 50 μl of calibrator or prepared plasma sample is added, 50 μl of diluted solution is added, and incubated at room temperature for 2 hours. After completion of the reaction, the wells are washed 3 times with Wash buffer, 100 μl of HRP-labeled streptavidin is added to each well, and incubated at room temperature for 30 minutes. After completion of the reaction, the plate is washed 3 times with Wash buffer, 100 μl of substrate is added, and incubated at room temperature for 30 minutes. After 30 minutes, 100 μl of the reaction stop solution is added, and the absorbance at 450 nm (reference: 550 nm) is measured using a microplate reader. The value obtained by subtracting the absorbance at 550 nm from the measured absorbance at 450 nm is used as a measurement value, and the IL-6 concentration is calculated from the absorbance of the plasma sample based on a calibration curve created using a dilution series of a calibrator.

キャリブレーターあるいは調製した血漿サンプルを５０μｌずつ各ｗｅｌｌに加えた後、５０μｌの希釈溶液を加えて、室温で１時間インキュベートする。反応終了後、Ｗａｓｈバッファーにより３回洗浄し、各ｗｅｌｌに５０μｌのビオチン化抗ＩＬ−８抗体を添加し、室温で１時間インキュベートする。反応終了後、Ｗａｓｈバッファーにより３回洗浄し、１００μｌのＨＲＰ標識ストレプトアビジンを添加し、室温で３０分間インキュベートする。反応終了後、Ｗａｓｈバッファーにより３回洗浄し、基質を１００μｌ添加し、３０分間室温でインキュベートする。３０分後、反応停止溶液を１００μｌ加え、マイクロプレートリーダーを用いて４５０ｎｍの吸光度（リファレンス：５５０ｎｍ）を測定する。測定された４５０ｎｍの吸光度から５５０ｎｍの吸光度を引いた値を測定値とし、キャリブレータの希釈系列を用いて作成した検量線に基づいて、血漿サンプルの吸光度からＩＬ−８濃度を算出する。 (2-4) Measurement of IL-8 A calibrator is diluted according to Table 5, and a calibrator dilution series is adjusted.

Add 50 μl of calibrator or prepared plasma sample to each well, add 50 μl of diluted solution, and incubate at room temperature for 1 hour. After completion of the reaction, the well is washed 3 times with Wash buffer, 50 μl of biotinylated anti-IL-8 antibody is added to each well, and incubated at room temperature for 1 hour. After completion of the reaction, the plate is washed 3 times with Wash buffer, 100 μl of HRP-labeled streptavidin is added, and incubated at room temperature for 30 minutes. After completion of the reaction, the plate is washed 3 times with Wash buffer, 100 μl of substrate is added, and incubated at room temperature for 30 minutes. After 30 minutes, 100 μl of the reaction stop solution is added, and the absorbance at 450 nm (reference: 550 nm) is measured using a microplate reader. The value obtained by subtracting the absorbance at 550 nm from the measured absorbance at 450 nm is used as a measurement value, and the IL-8 concentration is calculated from the absorbance of the plasma sample based on a calibration curve prepared using a dilution series of a calibrator.

キャリブレーターあるいは調製した血漿サンプルを５０μｌずつ各ｗｅｌｌに加えた後、５０μｌのビオチン化抗ＩＬ−１０抗体を添加し、さらに５０μｌの希釈溶液を加えて、室温で２時間インキュベートする。反応終了後、Ｗａｓｈバッファーにより３回洗浄し、１００μｌのＨＲＰ標識ストレプトアビジンを添加し、室温で３０分間インキュベートする。反応終了後、Ｗａｓｈバッファーにより３回洗浄し、基質を１００μｌ添加し、３０分間室温でインキュベートする。３０分後、反応停止溶液を１００μｌ加え、マイクロプレートリーダーを用いて４５０ｎｍの吸光度（リファレンス：５５０ｎｍ）を測定する。測定された４５０ｎｍの吸光度から５５０ｎｍの吸光度を引いた値を測定値とし、キャリブレータの希釈系列を用いて作成した検量線に基づいて、血漿サンプルの吸光度からＩＬ−１０濃度を算出する。 (2-5) Measurement of IL-10 A calibrator is diluted according to Table 6, and a calibrator dilution series is adjusted.

After 50 μl of calibrator or prepared plasma sample is added to each well, 50 μl of biotinylated anti-IL-10 antibody is added, 50 μl of diluted solution is added, and incubated at room temperature for 2 hours. After completion of the reaction, the plate is washed 3 times with Wash buffer, 100 μl of HRP-labeled streptavidin is added, and incubated at room temperature for 30 minutes. After completion of the reaction, the plate is washed 3 times with Wash buffer, 100 μl of substrate is added, and incubated at room temperature for 30 minutes. After 30 minutes, 100 μl of the reaction stop solution is added, and the absorbance at 450 nm (reference: 550 nm) is measured using a microplate reader. The value obtained by subtracting the absorbance at 550 nm from the measured absorbance at 450 nm is used as a measurement value, and the IL-10 concentration is calculated from the absorbance of the plasma sample based on a calibration curve created using a dilution series of a calibrator.

５０μｌの希釈溶液を各ｗｅｌｌに加えた後、キャリブレーターあるいは血漿サンプルを５０μｌずつ添加し、室温で１時間インキュベートする。反応終了後、Ｗａｓｈバッファーにより３回洗浄し、１００μｌのビオチン化抗ＴＮＦ−α抗体を添加し、室温で１時間インキュベートする。反応終了後、Ｗａｓｈバッファーにより３回洗浄し、１００μｌのＨＲＰ標識ストレプトアビジンを添加し、室温で３０分間インキュベートする。反応終了後、Ｗａｓｈバッファーにより３回洗浄し、基質を１００μｌ添加し、３０分間室温でインキュベートする。３０分後、反応停止溶液を１００μｌ加え、マイクロプレートリーダーを用いて４５０ｎｍの吸光度（リファレンス：５５０ｎｍ）を測定する。測定された４５０ｎｍの吸光度から５５０ｎｍの吸光度を引いた値を測定値とし、キャリブレータの希釈系列を用いて作成した検量線に基づいて、血漿サンプルの吸光度からＴＮＦ−α濃度を算出する。 (2-6) Measurement of TNF-α A calibrator is diluted according to Table 7 to adjust a calibrator dilution series.

After adding 50 μl of diluted solution to each well, add 50 μl of calibrator or plasma sample and incubate at room temperature for 1 hour. After completion of the reaction, the plate is washed 3 times with Wash buffer, 100 μl of biotinylated anti-TNF-α antibody is added, and incubated at room temperature for 1 hour. After completion of the reaction, the plate is washed 3 times with Wash buffer, 100 μl of HRP-labeled streptavidin is added, and incubated at room temperature for 30 minutes. After completion of the reaction, the plate is washed 3 times with Wash buffer, 100 μl of substrate is added, and incubated at room temperature for 30 minutes. After 30 minutes, 100 μl of the reaction stop solution is added, and the absorbance at 450 nm (reference: 550 nm) is measured using a microplate reader. The value obtained by subtracting the absorbance at 550 nm from the measured absorbance at 450 nm is used as a measurement value, and the TNF-α concentration is calculated from the absorbance of the plasma sample based on a calibration curve created using a dilution series of a calibrator.

血漿サンプルを希釈溶液で２．５倍希釈したサンプル溶液あるいはキャリブレーターを１００μｌずつ各ｗｅｌｌに添加し、室温で１時間インキュベートする。反応終了後、Ｗａｓｈバッファーにより４回洗浄し、１００μｌのＣｏｎｊｕｇａｔｅ ｓｏｌｕｔｉｏｎを添加し、室温で１時間インキュベートする。反応終了後、Ｗａｓｈ ｂｕｆｆｅｒにより４回洗浄し、基質を１００μｌ添加し、３０分間室温でインキュベートする。３０分後、反応停止溶液を１００μｌ加え、マイクロプレートリーダーを用いて４５０ｎｍの吸光度（リファレンス：６２０ｎｍ）を測定する。測定された４５０ｎｍの吸光度から６２０ｎｍの吸光度を引いた値を測定値とし、キャリブレータの希釈系列を用いて作成した検量線に基づいて、血漿サンプルの吸光度からＩＬ−１８濃度を算出する。 (2-7) IL-18 measurement The calibrator is diluted according to Table 8 to adjust the calibrator dilution series.

100 μl of a sample solution or calibrator obtained by diluting a plasma sample 2.5 times with a diluted solution is added to each well and incubated at room temperature for 1 hour. After the reaction is complete, wash 4 times with Wash buffer, add 100 μl of Conjugate solution, and incubate at room temperature for 1 hour. After completion of the reaction, the plate is washed 4 times with a wash buffer, 100 μl of substrate is added, and incubated at room temperature for 30 minutes. After 30 minutes, 100 μl of the reaction stop solution is added, and the absorbance at 450 nm (reference: 620 nm) is measured using a microplate reader. The value obtained by subtracting the absorbance at 620 nm from the measured absorbance at 450 nm is used as a measurement value, and the IL-18 concentration is calculated from the absorbance of the plasma sample based on a calibration curve created using a dilution series of a calibrator.

[Methods of patients providing measurement samples and SIRS applicable items]
For 15 patients who were able to collect blood before, during, and after surgery after obtaining informed consent, the surgical procedure (both using a cardiopulmonary device), the types of applicable items among the conventional SIRS criteria, and Table 9 shows the number of items. All 15 patients received steroids during surgery. The contents of the corresponding items in Table 9 are as follows.
Body temperature: body temperature above 38 ° C. or below 36 ° C. Heart rate: heart rate above 90 / min Respiration rate: respiratory rate above 20 / min or Pa (CO ₂ ) below 32 Torr White blood cell count: white blood cell count 12000 / mm ³ ultra or less than 4000 / mm ^3, or immature granulocytes than 10%

As can be seen from Table 9, SIRS onset was 13 cases (87.7%) out of 15 cases using this heart-lung machine, among which 3 or more of SIRS judgment items are applicable. Only 3 cases (20%) had severe SIRIS.
Note that the determination of SIRS determination items and the sampling for determination are performed during ICU movement.

[Sampling of measurement samples and measurement of each cytokine concentration-primary screening of appropriate markers]
Patient No. who underwent surgery (using a heart-lung machine) shown in Table 9. About 1-15, the informed consent was received and the blood was extract | collected on the day after ICU admission before surgery, immediately after heart-lung removal, after completion | finish of an operation (after completion | finish of suture of a thorax), and ICU admission. The time from the end of the operation (after the suture of the thoracotomy) to the entrance to the ICU varies depending on the patient, and the time from the time of the heart-lung removal to the sampling timing in each patient varies. Between 100 minutes and 100 minutes. The next day after entering the ICU is 15 to 20 hours after leaving the heart-lung machine.

A sample for measurement is prepared from the blood of each patient according to the above preparation method, and IL-1RA, IL-1β, IL-6, IL-8, IL-10, IL-18, TNF are prepared based on the above measurement method. The concentration of -α was calculated.
Patient No. which corresponds to 3 items among SIRS judgment items (corresponds to severe SIRS). No. 8 and Patient No. which did not correspond to SIRS determination at all (not SIRS). The changes in the concentration of each cytokine in 11 patients are shown in FIGS. 2 and 3, respectively.
From a comparison between FIG. 2 and FIG. 3, no change in the concentration of IL-18 with the progress of surgery was observed. Regarding TNF-α and IL-1β, changes in the concentration with the progress of surgery were observed, but the concentration itself is very low, and thus it is considered unsuitable as a SIRS marker.
IL-6, IL-8, IL-10, and IL-1RA are considered to be usable as markers because their concentration changes with the progress of surgery and the cytokine concentration is relatively high.

[Sampling of measurement samples and correlation between cytokine levels-secondary screening of appropriate markers]
Patient No. shown in Table 9 For 1 to 15, IL-6, IL-8, IL-10, IL-1RA concentrations in blood collected immediately after cardiopulmonary withdrawal, ICU movement, and the day after ICU were examined for the relationship with the number of SIRS criteria . The results are shown in FIGS. In each figure, (a) Immediately after cardiopulmonary withdrawal, (b) During ICU movement, (c) Measurement results on the next day of ICU. Further, the vertical axis represents the concentration of each cytokine, and the horizontal axis represents the number of corresponding items of SIRS determination items (see Table 10). However, “−1” indicates the concentration before surgery, and “−2” indicates the concentration of healthy volunteers.

In the case of IL-6, immediately after cardiopulmonary withdrawal (FIG. 4 (a)), the corresponding item is 2 items or less (when it is not mild SIRS or SIRS) and 3 items or more (severe SIRS), Although there was a difference in concentration, no significant difference was observed when ICU was moved (FIG. 4 (b)).
In the case of IL-8 (FIG. 5) and IL-10 (FIG. 6), when the corresponding item is 2 items or less immediately after the cardiopulmonary apparatus is released, either immediately after the cardiopulmonary apparatus is removed or when the ICU is moved (mild SIRS) Alternatively, there was no difference in cytokine concentration between the case of not SIRS and the case of 3 or more items (severe SIRS).
In the case of IL-1RA (Fig. 7), immediately after cardiopulmonary withdrawal and ICU movement, the corresponding item is 2 items or less (if it is not mild SIRS or SIRS) and 3 items or more (severe SIRS) There was a significant difference in the concentration of cytokines. In particular, when the concentration difference at the time of ICU movement (Fig. 7 (b)) is large and the threshold is set in the range of 5-10 ng / ml, the corresponding item is 2 items or less (if it is not mild SIRS or SIRS) ) And 3 or more items (severe SIRS) can be clearly distinguished.

  The method and apparatus for generating information regarding the severity of SIRS of a patient according to the present invention relates to the severity of SIRS of a patient based on samples collected from the end of surgery using the cardiopulmonary apparatus until entering the ICU. Information can be generated. Thus, using the method and apparatus of the present invention, it is possible to distinguish between severe SIRS and non-critical SIRS. Therefore, it is possible to monitor a patient who is likely to have sepsis or multiple organ failure within a few days after entering the ICU separately from a patient who is not at risk. As a result, efficiency and rationalization of the hospital ICU monitoring system and medication schedule can be achieved.

It is a block diagram which shows the structure of the determination apparatus of this invention. Patient No. It is a graph which shows the measurement result of cytokine concentration of 8. Patient No. It is a graph which shows the measurement result of 11 cytokine concentration. Patient No. It is a graph which shows the state of a change of the IL-6 density | concentration of 1-15. Patient No. It is a graph which shows the state of a change of the IL-8 density | concentration of 1-15. Patient No. It is a graph which shows the state of a change of the IL-10 density | concentration of 1-15. Patient No. It is a graph which shows the state of a change of the IL-1RA density | concentration of 1-15.

Claims (7)

Obtaining information on the concentration of interleukin 1 receptor antagonist (IL-1RA) contained in blood collected from a patient after surgery using an oxygenator; and IL-1RA obtained in the obtaining step A method for generating information on the severity of a patient's systemic inflammatory response symptom military comprising the step of generating information on the severity of the patient's systemic inflammatory response syndrome based on the information on the concentration. The method according to claim 1, wherein the blood is collected 15 to 100 minutes after the cardiopulmonary apparatus is detached. The method according to claim 1 or 2, wherein the patient is administered a steroid during surgery. The method according to any one of claims 1 to 3, wherein the information generating step generates information indicating whether or not the patient has a severe systemic inflammatory response syndrome based on information on the concentration of IL-1RA. . The information relating to the IL-1RA concentration obtained in the obtaining step is a value relating to the IL-1RA concentration, and the information generating step compares the value relating to the IL-1RA concentration with a predetermined threshold value. 5. The method of claim 4, wherein if the value for the concentration of IL-1RA is higher than the threshold, information is generated indicating that the patient has severe systemic inflammatory response syndrome. An information acquisition unit that acquires information on the concentration of an interleukin 1 receptor antagonist (IL-1RA) contained in blood collected from a patient after surgery using a heart-lung machine;
An information generation unit that generates information on the severity of a systemic inflammatory reaction syndrome of a patient based on information on the concentration of IL-1RA obtained by the information acquisition unit; and outputs the information generated by the information generation unit The apparatus which produces | generates the information regarding the severity of the systemic inflammatory reaction syndrome of a patient containing the output part to perform. The apparatus according to claim 6, wherein the information generation unit generates information indicating that the patient has a severe systemic inflammatory reaction syndrome based on information on the IL-1RA concentration.

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