METHOD FOR EVALUATING FUNCTION OF A LIVER FOR TRANSPLANTATION, PROGRAM FOR EVALUATING FUNCTION OF A LIVER FOR TRANSPLANTATION, AND APPARATUS FOR EVALUATING FUNCTION OF A LIVER FOR TRANSPLANTATION

Abstract

This functional assessment method for an organ for transplantation includes performing, in a perfusion solution, perfusion on an organ for transplantation removed from a living body, the method comprising: a step for preparing data indicative of temporal change in the contents of multiple marker substances caused by perfusion; a step for collecting a first tissue sample from the organ for transplantation at a first timing during perfusion; a step for collecting a second tissue sample from the organ for transplantation at a second timing subsequent to the first timing during perfusion; a step for measuring the contents of the respective marker substances in the first tissue sample; a step for measuring the contents of the respective marker substances in the second tissue sample; a step for determining changes in the contents of the respective marker substances at the second timing as compared with the first timing; and a step for determining an index for functional assessment of the organ for transplantation using the determined content changes and content data.

Description

[Technical field]

[0001] The present invention relates to a method for evaluating the function of an organ for transplantation, a program for evaluating the function of an organ for transplantation, and an apparatus for evaluating the function of an organ for transplantation. [Background technology]

[0002] As a technique for resolving the chronic shortage of organs in transplantation medicine, JP 2018-154617 A (Patent Document 1) discloses a method for preserving organs for transplantation for a long period of time while maintaining their functions. In Patent Document 1, by perfusing an organ after cardiac arrest with a perfusion solution containing an oxygen carrier and a blood coagulation inhibitor, it is possible to suppress tissue damage caused by warm ischemia and restore the organ to a state close to that before cardiac arrest. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2018-154617 A Summary of the Invention [Problem to be solved by the invention]

[0004] In conventional organ transplants, the decision as to whether an organ is in a state suitable for transplantation is based solely on the experience (tacit knowledge) of the transplant surgeon and rapid histopathological diagnosis. If an organ that is not actually functional is transplanted despite being deemed suitable for transplantation based on the transplant surgeon's tacit knowledge, there is a risk of graft failure (Primary Non-Function: PNF). In particular, after transplantation of the heart, lungs, and liver, which are known as vital organs, there is a risk of the patient's death if re-transplantation is not performed promptly.

[0005] As described above, in the conventional method, since there is no objective criterion for judging the potential function or the degree of irreversible damage of an organ, it is not possible to accurately judge the suitability of the organ before transplantation, and the judgment is left to experienced transplant surgeons. Therefore, even in the case of organs extracted after cardiac arrest whose function has been restored by the technology described in the above Patent Document 1, it may be ultimately judged as untransplantable in consideration of PNF, and there is a concern that the solution to the shortage of donors will be halted. Even if the organ is transplanted, there is always a risk of losing the patient due to graft loss caused by PNF.

[0006] The present invention has been made to solve these problems, and its object is to provide an index for objectively evaluating the suitability of a transplant organ for transplantation. [Means for solving the problem]

[0007] The method for evaluating the function of an organ for transplantation according to the first aspect of the present invention is , irrigation preparing data indicative of a time-dependent change in content of a plurality of marker substances due to the flow; Reproduction of organs for transplant Obtaining a first tissue sample from the organ at a first time during perfusion; Re The method includes the steps of: obtaining a second tissue sample from the organ for transplantation at a second timing after the first timing of perfusion; measuring the contents of multiple marker substances in the first tissue sample; measuring the contents of multiple marker substances in the second tissue sample; determining a change in content at the second timing relative to the first timing for each of the multiple marker substances; and determining an index related to functional evaluation of the organ for transplantation using the determined change in content and the data. Effect of the Invention

[0008] According to the present invention, it is possible to provide an index for objectively evaluating the suitability of a transplant organ for transplantation. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a diagram for explaining the processing procedure from liver removal to reperfusion, and the timing of obtaining tissue samples. [Diagram 2] FIG. 2 is a diagram showing a schematic configuration example of a perfusion circuit. [Diagram 3] FIG. 2 is a schematic diagram illustrating the configuration of a mass spectrometer and an evaluation device. [Figure 4] FIG. 13 is a diagram for explaining the evaluation criteria in the first evaluation. [Diagram 5] FIG. 13 is a diagram for explaining the evaluation criteria in the second evaluation. [Figure 6] FIG. 13 is a diagram for explaining the evaluation criteria in the second evaluation. [Figure 7] 1 is a flowchart for explaining a method for evaluating liver graft function according to the present embodiment. [Figure 8] 8 is a flowchart for explaining the procedure of a first evaluation process shown in step S100 of FIG. 7. [Figure 9] 8 is a flowchart for explaining a first modification of the first evaluation process shown in step S100 of FIG. 7. [Figure 10] 8 is a flowchart for explaining a second modification of the first evaluation process shown in step S100 of FIG. 7. [Figure 11] 8 is a flowchart illustrating the procedure of the second evaluation process shown in step S200 of FIG. 7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In the following, the same or corresponding parts in the drawings are denoted by the same reference characters, and their description will not be repeated in principle.

[0011] [Overview of methods for evaluating the function of organs for transplantation] In summary, the method for evaluating the function of an organ for transplantation in this embodiment involves mass spectrometry of the organ for transplantation to obtain quantitative data on pre-specified marker substances, and evaluating the obtained quantitative data in accordance with pre-set evaluation criteria to determine whether the organ for transplantation is in a state where it can be transplanted.

[0012] In the following, first, the evaluation criteria used in the function evaluation method according to the present embodiment will be described, followed by the function evaluation method using the evaluation criteria.

[0013] In the present specification, rats are used as mammals that serve as organ donors. The organ for transplantation is the liver. However, the mammals can be appropriately selected depending on the recipients to whom the organs are to be transplanted, and in addition to rats, for example, humans, pigs, cows, monkeys, dogs, cats, etc. can be applied. This is because metabolomics analysis is used to obtain quantitative data on the marker substances. Among omics analyses, metabolomics analysis targets low molecular weight metabolites, but there are no species differences in low molecular weight metabolites, and some metabolic pathways, such as glycolysis, are common to almost all organisms. Therefore, it is already known that the results of animal experiments, such as rats, can be easily extrapolated to other species, such as humans (see, for example, Aono, R., et al. A pentose bisphosphate pathway for nucleoside degradation in Archaea. Nature Chemical Biology, 11(5), 355-360 (2015) and Yoshida et al., "Diagnosis of gastrointestinal cancer by metabolome analysis - Search for pancreatic cancer biomarkers by metabolomics -", ​​Journal of the Japanese Society of Clinical Laboratory Medicine, 63(4):450-456). In addition to the liver, organs that can be used include, for example, the heart, kidneys, lungs, pancreas, stomach, small intestine, large intestine, testes, ovaries, and eyes.

[0014] (1) Liver function evaluation criteria In order to create a standard for evaluating the function of livers for transplantation (hereinafter also referred to as liver grafts), the inventors of the present application perfused three types of livers with different liver function states under common conditions and measured the amount of metabolites contained in each liver using mass spectrometry. In the measurement, in order to know how the amount of metabolites changes during the process from liver removal to reperfusion, tissue samples were taken at multiple times and mass spectrometry was performed.

[0015] FIG. 1 is a diagram for explaining the processing procedure from liver removal to reperfusion and the timing of obtaining tissue samples.

[0016] (1-1) Collection of tissue samples The samples used were livers excised from Wistar rats. In the example of Figure 1, sample A is a normal liver excised from a living Wistar rat.

[0017] Sample B is a liver after cardiac arrest (DCD: Donor after circulatory death). Sample B is a liver excised from a warm ischemic model created by allowing a Wistar rat to stand for 10 minutes after cardiac arrest.

[0018] Sample C is a liver from a cardiac arrest (DCD). Sample C is a liver extracted from a warm ischemic model rat that was created by leaving a Wistar rat for 30 minutes after cardiac arrest. Five pieces each of Sample A, Sample B, and Sample C were prepared.

[0019] In the extraction of the liver, a perfusate inflow cannula was inserted into the portal vein of the liver, a perfusate outflow cannula was inserted into the vein of the liver, and a bile outflow cannula was inserted into the bile duct. With these cannulae fixed to the liver, the liver was extracted from the Wistar rat and placed in a storage container.

[0020] Here, the time from cardiac arrest to liver removal is different between sample B and sample C. This time is the time of "warm ischemia" when blood flow to the liver stops. In a warm ischemic liver, cellular swelling disorder and waste accumulation occur due to depletion of ATP (adenosine triphosphate). Therefore, when transplantation is completed and blood flow to the liver is resumed, a large amount of active oxygen is generated due to the rapid metabolism of waste by the oxygenated perfusate, which may result in liver damage. Therefore, in conventional liver transplants, if the warm ischemic state continues for several minutes, it is judged that the liver is not transplantable. This judgment of transplantability is based solely on the experience of the transplant surgeon and histopathological findings, and usually, a liver that has been in a warm ischemic state for 10 minutes is judged to be transplantable. On the other hand, a liver that has been in a warm ischemic state for 30 minutes is judged to be not transplantable.

[0021] In the following description, the post-cardiac arrest liver in which the warm ischemic state continued for 10 minutes is also referred to as "mild DCD". The post-cardiac arrest liver in which the warm ischemic state continued for 30 minutes is also referred to as "severe DCD". In this embodiment, sample A (normal liver) and sample B (mild DCD) correspond to livers with normal function. On the other hand, sample C (severe DCD) corresponds to livers with poor function.

[0022] Next, for each of samples A to C, washing (flushing) of the blood vessels in the liver was performed. Physiological saline was used for washing. Physiological saline was allowed to flow in through the perfusion fluid inflow cannula, and was allowed to flow out through the perfusion fluid outflow cannula. The outflowing physiological saline was not circulated to the perfusion fluid inflow cannula, but was collected.

[0023] Next, after washing, the liver was poured with UW solution (University of Wisconsin solution) to replace the blood with UW solution, and then the liver was immersed in UW solution and cold-preserved for four hours. This cold preservation was performed based on the assumption that a liver graft extracted from a donor would be transported to a recipient in an actual medical setting. The cold preservation time was set at four hours, estimating the maximum time required for transportation. This four-hour period was taken into consideration when using a liver graft from a remote location.

[0024] After the cold storage, the liver was rewarmed to bring its temperature back up to the body temperature of the Wistar rat (approximately 37°C) for 10 minutes.

[0025] Next, the liver was reperfused while maintaining the liver temperature at 37° C. Krebs buffer solution was used as the perfusion fluid. FIG. 2 is a diagram showing a schematic configuration example of a perfusion circuit. Referring to FIG. 2, the perfusion circuit has a storage container 10, perfusion fluid reservoirs 50, 60, a perfusion fluid inflow path 20, a perfusion fluid outflow path 30, a gas exchange mechanism 70, and a pump 22.

[0026] A liver 1, which is a sample, is accommodated in the storage container 10. The perfusion fluid reservoirs 50, 60 are containers in which perfusion fluid is stored. A temperature mechanism (not shown) is connected to the perfusion fluid reservoirs 50, 60. The temperature mechanism is configured to maintain the perfusion fluid stored in the perfusion fluid reservoirs 50, 60 at a constant temperature (approximately 37° C.).

[0027] The perfusion fluid inflow path 20 is connected to a perfusion fluid inflow cannula 12 provided in the storage container 10. A pump 22 is inserted into the perfusion fluid inflow path 20. The perfusion fluid outflow path 30 is connected to a perfusion fluid outflow cannula 14 provided in the container 10.

[0028] The perfusion fluid inflow path 20 uses a pump 22 to supply perfusion fluid from a perfusion fluid reservoir 50 to the liver 1 via a perfusion fluid inflow cannula 12. At this time, the pump 22 is controlled by a control unit (not shown) so that the pressure of the perfusion fluid is 3 to 8 mmHg.

[0029] The perfusion fluid flowing out from the liver 1 is collected in the perfusion fluid reservoir 60 via the perfusion fluid outflow cannula 14 and the perfusion fluid outflow path 30. The gas exchange mechanism 70 is configured to return the perfusion fluid to the perfusion fluid reservoir 50 after dissolving the gas by supplying a gas such as oxygen to the perfusion fluid stored in the perfusion fluid reservoir 60.

[0030] 2 shows an example of the configuration of a circulation type perfusion circuit in which the perfusion fluid is circulated, but the perfusion fluid after use may be collected without being circulated. Bile output from the bile duct of the liver 1 during reperfusion is collected in a container 80 via a bile outflow cannula 40.

[0031] During the execution of the series of steps described above, a tissue sample S was collected for each specimen by cutting a piece of tissue from the liver 1. Figure 1 shows timings T1 to T4 at which the tissue samples were collected.

[0032] Timing T1 is set to a timing after washing of the liver and before cold preservation, and timing T2 is set to a timing immediately after cold preservation, i.e., timings T1 and T2 are set before and after cold preservation, respectively.

[0033] The timing T3 is set to the early reperfusion period. The early reperfusion period is a period in which liver function is recovering. In the example of FIG. 1, the timing T3 is set to 15 minutes after the start of reperfusion.

[0034] Timing T4 is set in the late reperfusion period. The late reperfusion period is a period in which recovery of liver function reaches its peak. In the example of FIG. 1, timing T4 is set to 60 minutes after the start of reperfusion. That is, timings T3 and T4 are set during reperfusion when the liver function states are different from each other.

[0035] (1-2) Mass spectrometry of tissue samples Next, the tissue samples of specimens A to C collected at the above-mentioned times T1 to T4 were screened using mass spectrometry. Liquid chromatography / mass spectrometry (LC / MS) was used for the mass spectrometry.

[0036] Specifically, 500 μL of the internal standard solution was added to a tissue sample (approximately 50 mg), and the mixture was homogenized using a BioMasher (Nippi Corporation). Next, 250 μL of water was added to the homogenate and stirred, and then 500 μL of chloroform was added and stirred. After centrifugation (15,000 rpm, 4°C, 5 minutes), 500 μL of the upper layer was extracted. Furthermore, the extract was dried using a centrifugal evaporator, and the residue was redissolved in 50 μL of water to prepare a measurement sample.

[0037] LC / MS analysis was performed using an LCMS-8060 (Shimadzu Corporation). Multiple reaction monitoring (MRM) and scan analysis were used as the measurement modes for LC / MS analysis.

[0038] Fig. 3 is a schematic configuration diagram of mass spectrometer 100 and evaluation device 120. Referring to Fig. 3, mass spectrometer 100 has a liquid chromatography / mass spectrometry (LC / MS) unit 102, a data processing unit 104, and a control unit 106.

[0039] The LC / MS section 102 has a liquid chromatograph equipped with a column (not shown) and a mass spectrometry section. The sample supplied to the column is separated into individual sample components as it passes through the column, and is then sequentially guided to the mass spectrometry section in a vacuum state for mass spectrometry. This allows different spectra to be obtained depending on the retention time.

[0040] The mass spectrometry section includes, for example, an ionization chamber, an ion trap, and a TOFMS (time-of-flight mass spectrometer). The ionization chamber ionizes the sample components separated by the liquid chromatograph using an ionization method such as ESI (electrospray ionization). The ionization method for the sample components is not limited to ESI, and various methods such as APCI (atmospheric pressure chemical ionization) can be used.

[0041] The ion trap is, for example, a three-dimensional quadrupole type, and can trap the ions obtained in the ionization chamber and selectively leave some of the trapped ions in the ion trap and dissociate them by CID (collision-induced dissociation). The dissociated ions are supplied from the ion trap to the TOFMS.

[0042] In TOFMS, ions accelerated by an electric field formed in a flight space are separated in time according to their mass-to-charge ratio while flying through the flight space, and are detected sequentially by an ion detector. The detection signal by the ion detector is input to a data processing unit 104 and converted into digital data, after which various data processing operations such as the creation of a mass spectrum, a mass chromatogram, and a total chromatogram are performed.

[0043] The control unit 106 controls the operation of the LC / MS unit 102. The functions of the data processing unit 104 and the control unit 106 can be realized by a personal computer equipped with predetermined control and processing software.

[0044] In this embodiment, a configuration in which a liquid chromatograph mass spectrometer is used as the mass spectrometer 100 has been described, but it is also possible to use a mass spectrometer that uses a PESI ion source based on a probe electrospray ionization (PESI) method (a PESI ionization mass spectrometer).

[0045] The control unit 106 transfers the data created by the data processing unit 104 to the evaluation device 120. The evaluation device 120 is communicatively connected to the mass spectrometer 100. The communication between the mass spectrometer 100 and the evaluation device 120 may be realized by wireless communication or by wired communication.

[0046] The evaluation device 120 has, as main components, a CPU (Central Processing Unit) 122 which is a calculation unit, a storage unit 124, a display unit 126, and an input unit 128. The evaluation device 120 may be, for example, a personal computer.

[0047] The CPU 122 reads out and executes a program stored in the storage unit 124 to control the operation of each unit of the evaluation device 120. Specifically, the CPU 122 executes the program to realize a functional evaluation of a liver for transplantation, which will be described later. Note that, although the example of Fig. 3 illustrates a configuration in which there is a single CPU 122, the evaluation device 120 may have a configuration in which there are multiple CPUs.

[0048] The storage unit 124 is realized by a non-volatile memory such as a random access memory (RAM), a read only memory (ROM), or a flash memory. The storage unit 124 stores a program executed by the CPU 122, data used by the CPU 122, or the like. The program may be stored in a non-transitory computer-readable medium.

[0049] A display unit 126 and an input unit 128 are connected to the CPU 122. The display unit 126 is composed of a liquid crystal panel capable of displaying images, etc. The input unit 128 accepts user operation input to the evaluation device 120. The input unit 128 is typically composed of a touch panel, a keyboard, a mouse, etc.

[0050] The evaluation device 120 acquires mass spectrum data of tissue samples at times T1 to T4 for each of the specimens A to C, and then performs quantitative analysis of metabolites contained in the specimens based on the acquired mass spectrum data. The quantitative data acquired at this time is used to set evaluation criteria used for functional evaluation of the liver graft.

[0051] (1-3) Setting evaluation criteria The liver function evaluation method according to this embodiment is composed of two stages of evaluation. The first evaluation is performed using quantitative data acquired before reperfusion, and evaluates the quality of the function of the liver graft. The second evaluation is performed using quantitative data acquired during reperfusion, and evaluates whether or not the liver graft whose function has been maintained or restored by reperfusion can be transplanted. Note that the first evaluation is a preliminary evaluation for the final second evaluation, and therefore may be omitted.

[0052] (i) Evaluation criteria for the first evaluation In the first evaluation, a marker substance for evaluating liver function and a threshold value for determining whether liver function is good or bad are used as evaluation criteria. Therefore, based on the quantitative data of the above-mentioned three types of samples A to C, the marker substance was identified and a threshold value was set for each marker substance.

[0053] Specifically, based on the quantitative data of samples A to C collected at times T1 and T2, candidate marker substances were identified from the viewpoint that liver grafts with normal function showed high values ​​and liver grafts with poor function showed low values.

[0054] In the quantitative data at timing T1, before cold storage, two metabolites were identified as potential markers, showing high values ​​in sample A (normal liver) and sample B (liver after mild cardiac arrest) and low values ​​in sample C (liver after severe cardiac arrest). These two metabolites are uridine and adenylosuccinic acid.

[0055] On the other hand, in the quantitative data at timing T2 after cold storage, one metabolite was identified as a candidate marker substance, which showed high values ​​in sample A (normal liver) and sample B (liver after mild cardiac arrest) and low values ​​in sample C (liver after severe cardiac arrest). This one metabolite was uridine.

[0056] FIG. 4 shows the content of the marker substance in the quantitative data at timing T1 and the content of the marker substance in the quantitative data at timing T2. FIG. 4(A) is a graph showing the content of uridine at timing T1 for each of samples A to C, and FIG. 4(B) is a graph showing the content of adenylosuccinic acid at timing T1 for each of samples A to C. The vertical axis of each graph corresponds to the value obtained by dividing the peak area ratio of the mass spectrum by the protein amount (peak area ratio of the mass spectrum / protein amount). The peak area ratio of the mass spectrum is the ratio of the peak area of ​​the target component to the peak area of ​​the internal standard. The protein amount is the protein amount of each tissue sample measured by quantitative analysis.

[0057] According to the graph in Figure 4(A), at time T1, the uridine content decreases in the following order: normal liver, liver after mild cardiac arrest, and liver after severe cardiac arrest. Therefore, a threshold U1 is set between the normal liver and liver after mild cardiac arrest and liver after severe cardiac arrest to determine whether the liver is functioning properly. Specifically, the average value of the average value of the uridine content of the normal liver and liver after mild cardiac arrest and the average value of the uridine content of the liver after severe cardiac arrest is set as the threshold U1.

[0058] As shown in the graph of FIG. 4(B), at time T1, the content of adenylosuccinic acid decreases in the following order: normal liver, liver after mild cardiac arrest, and liver after severe cardiac arrest. Therefore, a threshold value A1 for determining the quality of liver function was set between the normal liver and liver after mild cardiac arrest and the liver after severe cardiac arrest. Specifically, the average value of the average value of the adenylosuccinic acid content of the normal liver and liver after mild cardiac arrest and the average value of the adenylosuccinic acid content of the liver after severe cardiac arrest was set as the threshold value A1.

[0059] On the other hand, as shown in the graph of FIG. 4(C), at time T2, the uridine content decreases in the order of normal liver, liver after mild cardiac arrest, and liver after severe cardiac arrest. Therefore, a threshold U2 for determining the quality of liver function was set between the normal liver and liver after mild cardiac arrest and the liver after severe cardiac arrest. Specifically, the average value of the average value of the uridine content of the normal liver and liver after mild cardiac arrest and the average value of the uridine content of the liver after severe cardiac arrest was set as the threshold U2.

[0060] (ii) Evaluation criteria for the second evaluation In the second evaluation, the evaluation criteria are a marker substance for evaluating the maintenance and recovery of liver function by reperfusion and a threshold value for judging the feasibility of transplantation. Based on the quantitative data of the above-mentioned three types of samples A to C, the marker substances are identified and a threshold value is set for each marker substance.

[0061] Specifically, based on the quantitative data of samples A to C collected at times T3 and T4, candidate marker substances were identified from the viewpoint of changes in content during reperfusion. Ten metabolites were identified as candidate marker substances whose content changed between times T3 and T4. These ten metabolites were sedoheptulose 7-phosphate, adenosine triphosphate, glucose-6-phosphate, succinyl coenzyme A, dimethylglycine, choline, 2-aminobutyric acid, uric acid, pyruvic acid, and inosine.

[0062] Fig. 5 shows the content of the marker substance in the quantitative data at timing T3 and the content of the marker substance in the quantitative data at timing T4. Fig. 5(A) to Fig. 5(J) are graphs showing the content of the marker substance in each of samples A to C at timings T3 and T4.

[0063] In each figure, A (T3) is the content of the marker substance in sample A (normal liver) at time T3, and A (T4) is the content of the marker substance in sample A (normal liver) at time T4. B (T3) is the content of the marker substance in sample B (liver after mild cardiac arrest) at time T3, and B (T4) is the content of the marker substance in sample B (liver after mild cardiac arrest) at time T4. C (T3) is the content of the marker substance in sample C (liver after severe cardiac arrest) at time T3, and C (T4) is the content of the marker substance in sample C (liver after severe cardiac arrest) at time T4. The vertical axis of each graph corresponds to the value obtained by dividing the peak area ratio of the mass spectrum by the amount of protein (peak area ratio of the mass spectrum / amount of protein).

[0064] As shown in Figure 5(A), the content of sedoheptulose 7-phosphate increased in normal liver and liver after mild cardiac arrest, and decreased in liver after severe cardiac arrest. As shown in Figure 5(E), Figure 5(F), Figure 5(G), Figure 5(H), Figure 5(I), and Figure 5(J), the contents of dimethylglycine, choline, 2-aminobutyric acid, uric acid, pyruvic acid, and inosine also showed a similar tendency to that of sedoheptulose 7-phosphate.

[0065] In contrast, as shown in Figure 5(B), the content of adenosine triphosphate decreased in normal liver, increased in liver after mild cardiac arrest, and decreased in liver after severe cardiac arrest. As shown in Figures 5(C) and 5(D), the contents of glucose-6-phosphate and succinyl CoA increased in normal liver, liver after mild cardiac arrest, and liver after severe cardiac arrest.

[0066] Figure 6 shows the tendency of the content change from timing T3 to timing T4 for the 10 marker substances shown in Figure 5. Figure 6 is a table showing the tendency of the content change in a total of five samples for each of samples A to C, expressed as a score. In this table, for each marker substance, when the content increases from timing T3 to timing T4, the score is set to "1", and when the content decreases, the score is set to "0".

[0067] The increase / decrease in the content may be determined based on the magnitude of the content between timings T3 and T4. In this case, if the content at timing T4 is greater than the content at timing T3, the score is set to "1," and if the content at timing T4 is smaller than the content at timing T3, the score is set to "0."

[0068] Alternatively, the increase / decrease in the content may be determined based on the rate of change in the content between timings T3 and T4. In this case, the ratio of the content at timing T4 to the content at timing T3 is calculated. When the contents at timings T3 and T4 are equal, the ratio = 1. If the content at timing T4 is greater than the content at timing T3, the ratio is > 1, and if it is less than the content at timing T3, the ratio is < 1.

[0069] A first threshold value greater than 1 and a second threshold value less than 1 are preset for the ratio, and the calculated ratio is compared with the first and second threshold values. When the calculated ratio is greater than the first threshold value, the content is determined to have increased, and a score of "1" is assigned. When the calculated ratio is less than the second threshold value, the content is determined to have decreased, and a score of "0" is assigned.

[0070] 6, for example, in the case of sedoheptulose 7-phosphate, in sample A (normal liver), of the five samples A1 to A5, two samples A1 and A3 showed a score of "1," and the remaining three samples A2, A4, and A5 showed a score of "0." In contrast, in sample B (liver after mild cardiac arrest), all five samples B1 to B5 showed a score of "1," and in sample C (liver after severe cardiac arrest), all five samples C1 to C5 showed a score of "0."

[0071] In the case of glucose-6-phosphate, dimethylglycine, choline, 2-aminobutyric acid and uric acid, all five samples in sample A (normal liver) and sample B (liver after mild cardiac arrest) showed a score of "1", while in sample C (liver after severe cardiac arrest), all five samples C1 to C5 showed a score of "0".

[0072] Once the scores were calculated for each marker substance, the sum of the 10 scores was calculated for each sample, i.e. the highest total score was "10" and the lowest was "0".

[0073] As shown in Figure 6, the total scores of sample A (normal liver) and sample B (liver after mild cardiac arrest) are distributed to values ​​close to the maximum value of "10." In contrast, the total score of sample C (liver after severe cardiac arrest) is distributed to values ​​close to the minimum value of "0."

[0074] According to the table in Fig. 6, for 9 of the 10 marker substances, the tendency of change in the content of the marker substance from timing T3 to timing T4 is consistent between sample A (normal liver) and sample B (liver after mild cardiac arrest). As a result, the total scores of the normal liver and the liver after mild cardiac arrest are similar.

[0075] In contrast, for nine of the ten marker substances, sample A (normal liver) and sample C (liver after severe cardiac arrest) show different trends in the amount of the marker substance that changes from time T3 to time T4. As a result, the total scores of the normal liver and the liver after severe cardiac arrest are not close to each other, but are far apart. In the table of FIG. 6, the total score of the liver after severe cardiac arrest is significantly lower than that of the normal liver.

[0076] Based on the table in Figure 6, the inventors set a threshold value for the total score to distinguish between normal livers and livers after mild cardiac arrest and livers after severe cardiac arrest, and used this threshold value as a standard value for determining whether a liver graft has recovered function through reperfusion or not.

[0077] Specifically, while the total scores for normal liver and liver after mild cardiac arrest range from "6" to "10," the total score for liver after severe cardiac arrest is "0" or "1," so the reference value was set at "2."

[0078] According to this, the total score can be used to determine whether the liver graft after reperfusion is in a state suitable for transplantation. Specifically, if the total score of the liver graft is higher than the reference value "2", the liver graft is determined to be suitable for transplantation. In this case, it can be determined that the tendency of the change in the content of the marker substance from time T3 to time T4 of the liver graft is consistent with that of a normal liver. Therefore, it can be determined that the function of the liver graft has been restored to a level suitable for transplantation by reperfusion.

[0079] On the other hand, if the total score of the liver graft is equal to or less than the threshold value "2", the liver graft is judged to be untransplantable. In this case, it can be judged that the tendency of the change in the content of the marker substance from time T3 to time T4 of the liver graft is different from that of the normal liver. Therefore, it can be judged that the function of the liver graft has not been restored to a transplantable level even by reperfusion.

[0080] In the second evaluation, the total score of the scores of the 10 marker substances is used to determine whether transplantation is possible, which makes it possible to absorb the variation in the scores of each marker substance. In other words, even in normal livers, there is variation in the tendency of increase / decrease in the content among multiple samples, so if the tendency of the change in the content of each marker substance is determined to be consistent with that of normal livers, it may be judged incorrectly. By using the total score of all marker substances to comprehensively judge whether the tendency of the change in the content of the entire marker substances is consistent, such an erroneous judgment can be prevented.

[0081] (2) Liver function evaluation method Next, a method for evaluating the function of a liver graft using the above-mentioned evaluation criteria will be described.

[0082] Fig. 7 is a flowchart for explaining the method for evaluating the function of a liver graft according to the present embodiment. Fig. 7 shows a flowchart for explaining the procedure from removal to reperfusion of the liver graft (left diagram of Fig. 7), a flowchart for explaining the procedure in a mass spectrometer (center diagram of Fig. 7), and a flowchart for explaining the procedure in an evaluation device (right diagram of Fig. 7).

[0083] 7, in step S10, a liver graft is extracted from a donor. In step S20, a flushing process is performed to wash blood vessels in the extracted liver graft. For example, physiological saline can be used for the washing. The washed liver graft is immersed in UW solution.

[0084] In step S30, a tissue sample is taken from the liver graft. The taking of the tissue sample in step S30 corresponds to the timing T1 in FIG.

[0085] In step S40, a cold preservation process for the liver graft is performed. The time for cold preservation is not particularly limited, and can be set appropriately by the user in consideration of the transportation time of the liver graft from the donor to the recipient. In addition, the preservation solution is not limited to the UW solution, and can be selected appropriately by the user.

[0086] Once the cold preservation process (step S40) is completed, a tissue sample is taken from the liver graft in step S50. The taking of the tissue sample in step S50 corresponds to the timing T2 in FIG.

[0087] In step S60, a rewarming process of the liver graft is performed to return the temperature of the liver graft to about 37°C.

[0088] In step S70, reperfusion of the liver graft is performed while maintaining the temperature of the liver graft at about 37° C. The reperfusion can be performed, for example, using the perfusion circuit shown in Fig. 2. However, the perfusion solution is not limited to Krebs buffer solution and can be selected appropriately by the user.

[0089] When reperfusion is started, a tissue sample is collected from the liver graft at a predetermined timing in the early stage of reperfusion (e.g., 15 minutes after the start of reperfusion) in step S80. Collection of the tissue sample in step S80 corresponds to timing T3 in FIG. 1. Furthermore, a tissue sample is collected from the liver graft at a predetermined timing in the final stage of reperfusion (e.g., 30 minutes after the start of reperfusion) in step S90. Collection of the tissue sample in step S90 corresponds to timing T4 in FIG. 1.

[0090] In step S31, mass spectrometry is performed on the tissue sample collected in step S30 in mass spectrometer 100. Mass spectrum data acquired by mass spectrometry is transferred to evaluation device 120.

[0091] Thereafter, in steps S51, S81, and S91, mass spectrometry is performed on the tissue samples collected in steps S50, S80, and S90, respectively, in mass spectrometer 100. Mass spectrum data acquired in each mass spectrometry process is transferred to evaluation device 120.

[0092] In the evaluation device 120, based on the mass spectrum data transferred from the mass spectrometer 100, an evaluation is made as to whether or not the liver graft is in a state suitable for transplantation.

[0093] Specifically, first, in step S32, evaluation device 120 acquires mass spectrum data of a tissue sample collected at timing T1 before cold storage (S40), and then in step S52, acquires mass spectrum data of a tissue sample collected at timing T2 after cold storage (S40). In step S100, evaluation device 120 executes a first evaluation process using these two mass spectrum data.

[0094] Fig. 8 is a flowchart for explaining the procedure of the first evaluation process shown in step S100 in Fig. 7. The flowchart in Fig. 8 can be executed by the CPU 122 of the evaluation device 120.

[0095] 8, in step S101, CPU 122 detects the content M1(T1) of uridine, which is a marker substance, from the mass spectrum data at timing T1. Then, in step S102, CPU 122 detects the content M1(T2) of uridine from the mass spectrum data at timing T2. In steps S101 and S102, CPU 122 detects the content M1 of uridine by dividing the peak area ratio of the mass spectrum by the amount of protein.

[0096] Next, in step S103, the CPU 122 compares the uridine content M1(T1) with a threshold value U1. The threshold value U1 is the uridine content M1(T1) in the normal liver at the timing T1 in FIG. Light This was set based on the uridine content in liver from mild cardiac arrest and severe cardiac arrest.

[0097] When the uridine content M1 (T1) is equal to or less than the threshold value U1 (NO in S103), the CPU 122 determines in step S106 that the liver graft is malfunctioning.

[0098] On the other hand, when the content M1 (T1) is greater than the threshold U1 (S10 3If the answer is YES at step S104, the CPU 122 subsequently compares the uridine content M1(T2) with a threshold value U2. The threshold value U2 is the uridine content M1(T2) of the normal liver at the timing T2 in FIG. Light This was set based on the uridine content in liver from mild cardiac arrest and severe cardiac arrest.

[0099] If the uridine content M1(T2) is equal to or less than the threshold U2 (NO in S104), the CPU 122 determines in step S106 that the liver graft is malfunctioning. On the other hand, if the uridine content M1(T2) is greater than the threshold U2 (YES in S104), the CPU 122 determines in step S105 that the liver graft is functioning normally.

[0100] In the flowchart shown in Fig. 8, the function of the liver graft is determined based on the uridine content at timings T1 and T2, but as shown in Fig. 9, the function of the liver graft can also be determined based on the uridine and adenylosuccinic acid content at timing T1. Alternatively, as shown in Fig. 10, the function of the liver graft can also be determined based on the uridine content at timing T2.

[0101] FIG. 9 is a flowchart for explaining a first modification of the first evaluation process shown in step S100 of FIG.

[0102] 9, the CPU 122 detects the content M1(T1) of uridine, which is a marker substance, from the mass spectrum data at the timing T1 in step S101. 7 Thus, the content M2(T1) of adenylosuccinic acid is detected from the mass spectrum data at the timing T1.

[0103] Next, the CPU 122 compares the uridine content M1(T1) with the threshold U1 in step S103. If the uridine content M1(T1) is equal to or less than the threshold U1 (NO in S103), the CPU 122 determines in step S106 that the liver graft is malfunctioning.

[0104] On the other hand, when the content M1 (T1) is greater than the threshold U1 (S10 3 If the answer is YES at step S10, the CPU 122 proceeds to step S10. 8 The content of adenylosuccinic acid M2(T1) is compared with the threshold value A1. The threshold value A1 is the content of adenylosuccinic acid M2(T1) in normal liver at the timing T1 in FIG. 4(B), Light This was set based on the adenylosuccinic acid content in liver from mild cardiac arrest and severe cardiac arrest.

[0105] When the content of adenylosuccinic acid M2(T1) is equal to or less than the threshold value A1 (S10 8 On the other hand, if the content M2(T1) of adenylosuccinic acid is greater than the threshold A1 (S10), the CPU 122 determines in step S106 that the liver graft is malfunctioning. 8 If the answer is YES at step S104, the CPU 122 determines in step S105 that the liver graft is functioning normally.

[0106] FIG. 10 is a flowchart for explaining a second modification of the first evaluation process shown in step S100 of FIG.

[0107] 10, in step S102, CPU 122 detects the content M1(T2) of uridine, which is a marker substance, from the mass spectrum data at timing T2.

[0108] Next, the CPU 122 compares the uridine content M1(T2) with a threshold value U2 in step S104. If the uridine content M1(T2) is equal to or less than the threshold value U2 (NO in S104), the CPU 122 determines in step S106 that the liver graft is malfunctioning. On the other hand, if the content M1(T2) is greater than the threshold value U2 (YES in S104), the CPU 122 determines in step S105 that the liver graft is functioning normally.

[0109] 7, in step S82, evaluation device 120 acquires mass spectral data of a tissue sample collected at timing T3 of the early reperfusion stage, and then in step S92, acquires mass spectral data of a tissue sample collected at timing T4 of the late reperfusion stage. In step S200, evaluation device 120 executes a second evaluation process using these two mass spectral data.

[0110] Fig. 11 is a flowchart for explaining the procedure of the second evaluation process shown in step S200 in Fig. 7. The flowchart in Fig. 11 can be executed by the CPU 122 of the evaluation device 120.

[0111] 11, in step S201, the CPU 122 selects a target marker substance Mi (i is an integer between 1 and 10) from among the 10 marker substances. Next, in step S202, the CPU 122 calculates the content of a metabolite that is the target marker substance Mi from the mass spectrum data at timing T3. X In step S203, the CPU 122 detects the content of the target marker substance Mi from the mass spectrum data at the timing T4. X i(T4) is detected. Step S20 2 ,S20 3 Then, the CPU 122 calculates the content of the target marker substance by dividing the peak area ratio of the mass spectrum by the protein amount. X Detect i.

[0112] Next, in step S204, the CPU 122 calculates the content from the timing T3 to the timing T4. X In step S204, for example, the CPU 122 calculates the trend of change in i. X i(T3) and X Based on the magnitude of i(T4), the content X The change trend of i (increase / decrease) is determined. In this case, the CPU 122 X i(T3)< X If i(T4), then the content X It is determined that i has increased, X i(T3)> X If i(T4), then the content X It is determined that i has decreased.

[0113] Alternatively, the CPU 122 may X i(T3) and X Rate of change of i(T4) (= X i(T4) / X i(T3)) based on the content X The CPU 122 can determine an increase / decrease in the content X Content relative to i(T3) X Calculate the ratio of i(T4). X i(T3) = X When i(T4), the ratio is 1. On the other hand, X i(T4)> X At the time of i(T3), the ratio is > 1, X i(T4)< X When i(T3), the ratio is <1.

[0114] The CPU 122 compares the calculated rate of change with a first threshold value (first threshold value>1), and when the rate of change is greater than the first threshold value, X In addition, the CPU 122 compares the rate of change with a second threshold value (second threshold value<1), and when the rate of change is smaller than the second threshold value, determines that the content X It is determined that i has decreased.

[0115] Next, the CPU 122 proceeds to step S205, and X It is determined whether the trend of change in i coincides with the trend of change in the content of the marker substance Mi in a normal liver. The trend of change in the content of the marker substance Mi in a normal liver is based on the content of the marker substance in the normal liver (sample A) at timings T3 and T4 shown in Figure 5. The CPU 122 can calculate the trend of change in the content of the marker substance Mi in a normal liver using a method similar to the method described in step S204.

[0116] In step S205, if the tendency of change in the content of the marker substance Mi in the liver graft coincides with the tendency of change in the content of the marker substance Mi in the normal liver (YES in S205), the CPU 122 sets the score for the marker substance Mi to "1" in step S206. On the other hand, if the tendency of change in the content of the marker substance Mi in the liver graft does not coincide with the tendency of change in the content of the marker substance Mi in the normal liver (NO in S205), the CPU 122 sets the score for the marker substance Mi to "0" in step S207.

[0117] In addition, if the score when the tendency of the change in the content of the marker substance Mi in the liver graft coincides closely with the tendency of the change in the content of the marker substance Mi in the normal liver is different from the score when the degree of coincidence is small, the score value is not limited to "1" or "0". The absolute value of the change may be calculated, and when the degree of coincidence is large, the score may be set to "1", when the degree of coincidence is small, the score may be set to "0", and in between, the score may be set to "0.5".

[0118] Next, the CPU 122 determines whether or not all of the 10 marker substances have been selected as the target marker substances in step S208. If all of the marker substances have not been selected (NO in S208), the CPU 122 returns the process to step S201, selects the next marker substance M(i+1) as the target marker substance, and executes step S2 The process from 02 to S207 is executed again.

[0119] On the other hand, if all of the 10 marker substances have been selected as the target marker substances (YES in S208), the CPU 122 proceeds to step S209 and calculates a total score by adding up the scores of all the marker substances.

[0120] In step S210, the CPU 122 compares the total score calculated in step S209 with a reference value (=2). This reference value is set based on the total scores of the normal liver, the liver after mild cardiac arrest, and the liver after severe cardiac arrest shown in FIG.

[0121] If the total score is higher than the reference value (YES in S210), the CPU 122 determines in step S211 that the liver graft is in a transplantable state. On the other hand, if the total score is equal to or lower than the reference value (NO in S210), the CPU 122 determines in step S212 that the liver graft is in an untransplantable state.

[0122] In other words, the score for each marker substance or the total score serves as an index for evaluating the function of the liver graft.

[0123] 7, in step S300, the CPU 122 displays the evaluation results in the first evaluation process (S100) and the second evaluation process (S200) on the display unit 126. Based on the evaluation results displayed on the display unit 126, a user such as a transplant surgeon can determine whether or not to transplant the liver graft into a recipient.

[0124] As described above, the method for evaluating the function of an organ for transplantation according to this embodiment performs mass analysis of the organ for transplantation to obtain quantitative data on a pre-specified marker substance, and evaluates the obtained quantitative data according to a pre-set evaluation criterion to determine whether the organ for transplantation is in a state where it can be transplanted. This makes it possible to objectively determine whether an organ for transplantation can be transplanted, which has been conventionally determined based on the experience of transplant surgeons and histopathological findings. In addition, since the feasibility of an organ for transplantation can be determined before the organ is transplanted, the number of organs available for transplantation can be increased.

[0125] Furthermore, according to the method for evaluating the function of an organ for transplantation in this embodiment, it is possible to increase the possibility that not only brain-dead livers but also post-cardiac arrest livers whose function has been restored by reperfusion can be used for transplantation, thereby contributing to resolving the shortage of donor organs.

[0126] [Other embodiments] In the above embodiment, the degree of agreement between the tendency of change in the content of the marker substance Mi in the liver graft and the tendency of change in the content of the marker substance Mi in the normal liver is scored, and the sum of the scores of each marker substance is calculated to evaluate the function of the organ for transplantation. In another embodiment, the function of the organ for transplantation may be evaluated by regression analysis without scoring. For example, a regression equation may be created by using the amount of change in the content of the marker substance Mi as an explanatory variable and the normality of the function of the organ for transplantation as a target variable.

[0127] [Aspects] It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0128] (Item 1) A method for evaluating the function of an organ for transplantation according to one embodiment of the present invention comprises the steps of: Vessel A functional evaluation method, comprising the steps of: preparing data showing changes over time in contents of a plurality of marker substances due to perfusion; Reproduction of organs for transplant Obtaining a first tissue sample from the organ at a first time during perfusion; Re The method includes the steps of: obtaining a second tissue sample from the organ for transplantation at a second timing after the first timing of perfusion; measuring the contents of multiple marker substances in the first tissue sample; measuring the contents of multiple marker substances in the second tissue sample; determining a change in content at the second timing relative to the first timing for each of the multiple marker substances; and determining an index related to functional evaluation of the organ for transplantation using the determined change in content and the data.

[0129] According to the function evaluation method described in paragraph 1, Re By indexing the changes in the content of each of the multiple marker substances during perfusion, it is possible to determine whether or not an organ can be transplanted based on this index. This makes it possible to objectively determine whether an organ can be transplanted before transplantation. In addition, it also increases the possibility that organs after cardiac arrest that have recovered function through reperfusion can be used for transplantation, which can contribute to resolving the shortage of donor organs.

[0130] (Item 2) In the method for evaluating the function of an organ for transplantation according to item 1, the step of determining the change includes a step of determining the trend of change in the content. The step of determining an index includes a step of scoring the trend of change in the content. The step of scoring includes a step of making the score higher when the trend of change in the content in the organ for transplantation matches the trend of change in the content in an organ with normal function than when they do not match.

[0131] According to the function evaluation method described in paragraph 2, Re By scoring the tendency of change in the content of each of multiple marker substances during perfusion based on the tendency of change in the content of each of multiple marker substances in a functionally normal organ, the suitability of transplantation can be objectively determined based on this score.

[0132] (Item 3) In the method for evaluating the function of an organ for transplantation described in item 1 or 2, each of the steps of measuring the content of multiple marker substances in a first tissue sample and the step of measuring the content of multiple marker substances in a second tissue sample includes a step of measuring the content of the multiple marker substances using mass spectrometry.

[0133] According to the function evaluation method described in item 3, by performing mass spectrometry on the first and second tissue samples, Re The change in the content of each of a plurality of marker substances during perfusion can be indexed.

[0134] (Item 4) In the method for evaluating the function of an organ for transplantation described in items 1 to 3, the step of determining the change further includes a step of determining an increase in the content when the rate of change of the content at the second timing relative to the content at the first timing is greater than a first threshold value greater than 1, and determining a decrease in the content when the rate of change is smaller than a second threshold value less than 1.

[0135] According to the function evaluation method described in item 4, Re The changes in the content of each of the multiple marker substances during perfusion can be appropriately scored regardless of the variations in tissue samples, allowing the feasibility of transplantation to be determined with high accuracy.

[0136] (Item 5) In the method for evaluating the function of an organ for transplantation according to items 1 to 4, the organ for transplantation is a liver, and the plurality of marker substances include at least two of sedoheptulose 7-phosphate, adenosine triphosphate, glucose-6-phosphate, succinyl coenzyme A, dimethylglycine, choline, 2-aminobutyric acid, uric acid, pyruvic acid, and inosine.

[0137] According to the function evaluation method described in item 5, Re By indexing the changes in the content of each of the multiple marker substances during perfusion, it is possible to determine whether or not a liver graft can be transplanted based on this index. This makes it possible to objectively determine whether or not a liver graft is in a state where it can be transplanted before transplantation. In addition, it also increases the possibility that a post-cardiac arrest liver whose function has been restored by reperfusion can be used for transplantation, which can contribute to solving the shortage of donor organs.

[0138] (Item 6) The method for evaluating the function of an organ for transplantation according to items 1 to 5 includes, after removal from a donor, Re A step of cold preserving the organ for transplantation until perfusion, a step of taking a third tissue sample from the organ for transplantation at a third timing before the start of the cold preservation, and a step of taking a third tissue sample from the organ for transplantation at a third timing after the end of the cold preservation and Re The method further includes the steps of obtaining a fourth tissue sample from the organ for transplantation at a fourth timing before the start of perfusion, measuring the content of a first marker substance in the third tissue sample, measuring the content of the first marker substance in the fourth tissue sample, and determining whether the function of the organ for transplantation is normal based on the content of the first marker substance at the third timing and the fourth timing.

[0139] According to the function evaluation method described in item 6, Re The function of organs for transplantation before perfusion can be assessed.

[0140] (7) The method for evaluating the function of an organ for transplantation according to any one of the first to fifth aspects includes, after removal from a donor, Re A step of cold preserving the organ for transplantation before perfusion, and after completion of cold preservation and Re The method further includes the steps of obtaining a fourth tissue sample from the organ for transplantation at a fourth timing before the start of perfusion, measuring the content of a first marker substance in the fourth tissue sample, and determining whether the function of the organ for transplantation is normal based on the content of the first marker substance at the fourth timing.

[0141] According to the function evaluation method described in item 7, Re The function of the organ for transplantation before perfusion can be determined.

[0142] (Item 8) The method for evaluating the function of an organ for transplantation according to items 1 to 5 includes, after removal from a donor, Re The method further includes the steps of cold preserving the organ for transplantation until perfusion, taking a third tissue sample from the organ for transplantation at a third timing before the start of cold preservation, measuring the contents of the first and second marker substances in the third tissue sample, and determining whether the function of the organ for transplantation is normal based on the contents of the first and second marker substances at the third timing.

[0143] According to the function evaluation method described in item 8, Re The function of organs for transplantation before perfusion can be assessed.

[0144] (Item 9) In the method for evaluating the function of an organ for transplantation according to item 6 or 7, the organ for transplantation is a liver, and the first marker substance is uridine.

[0145] According to the function evaluation method described in item 9, Re The function of the liver graft before perfusion can be determined.

[0146] (Item 10) In the method for evaluating the function of an organ for transplantation according to item 8, the organ for transplantation is a liver. The first marker substance is uridine, and the second marker substance is adenylosuccinic acid.

[0147] According to the function evaluation method described in paragraph 10, Re The function of the liver graft before perfusion can be determined.

[0148] (Item 11) The program for evaluating the function of an organ for transplantation according to item 11 is a program for evaluating the function of an organ for transplantation extracted from a living body by using a computer having a calculation unit. Evaluate the functionality of The program includes steps of preparing data showing changes in the contents of a plurality of marker substances over time due to perfusion; Reproduction of organs for transplant Obtaining a first tissue sample from the organ at a first time during perfusion; Re The calculation unit executes the steps of: collecting a second tissue sample from the organ for transplantation at a second timing after the first timing of perfusion; measuring the contents of multiple marker substances in the first tissue sample; measuring the contents of multiple marker substances in the second tissue sample; determining the change in content at the second timing relative to the first timing for each of the multiple marker substances; and determining an index related to functional evaluation of the organ for transplantation using the determined change in content and the data.

[0149] According to the program described in paragraph 11, ReBy indexing the changes in the content of each of the multiple marker substances during perfusion, it is possible to determine whether or not an organ can be transplanted based on this index. This makes it possible to objectively determine whether an organ can be transplanted before transplantation. In addition, it also increases the possibility that organs after cardiac arrest that have recovered function through reperfusion can be used for transplantation, which can contribute to resolving the shortage of donor organs.

[0150] (Item 12) The apparatus for evaluating the function of an organ for transplantation according to item 12 includes a calculation unit Including The function evaluation device prepares data showing changes over time in the contents of a plurality of marker substances due to perfusion. Reproduction of organs for transplant The contents of a plurality of marker substances are measured in a first tissue sample taken from the transplant organ at a first time point during perfusion. The calculation unit is The contents of the plurality of marker substances are measured in a second tissue sample taken from the transplant organ at a second time point during perfusion that is later than the first time point. The calculation unit is For each of a plurality of marker substances, a change in content at a second timing relative to a first timing is obtained, and an index relating to functional evaluation of an organ for transplantation is obtained using the obtained change in content and the data.

[0151] According to the function evaluation device described in paragraph 12, Re By indexing the changes in the content of each of the multiple marker substances during perfusion, it is possible to determine whether or not an organ can be transplanted based on this index. This makes it possible to objectively determine whether an organ can be transplanted before transplantation. In addition, it also increases the possibility that organs after cardiac arrest that have recovered function through reperfusion can be used for transplantation, which can contribute to resolving the shortage of donor organs.

[0152] The embodiments disclosed herein should be considered to be illustrative and not restrictive in all respects. The scope of the present invention is defined by the claims, not the above description, and is intended to include all modifications within the meaning and scope of the claims. [Explanation of symbols]

[0153] 1 liver, 10 storage container, 12 perfusate inflow cannula, 14 perfusate outflow cannula, 20 perfusate inflow path, 22 pump, 30 perfusate outflow path, 40 bile outflow cannula, 50, 60 perfusate reservoir, 70 gas exchange mechanism, 80 container, 100 mass spectrometer, 102 LC / MS unit, 104 data processing unit, 106 control unit, 120 evaluation device, 122 CPU, 124 memory unit, 126 display unit, 128 input unit.

Claims

1. A method for evaluating the function of a liver for transplantation excised from a living body, comprising the steps of: preparing reference data showing time-dependent changes in content of a plurality of metabolites contained in a sample taken from a perfused liver; The step of preparing the reference data includes: obtaining first quantitative data of metabolites contained in samples taken at a first timing during reperfusion for each of the normally functioning liver and the poorly functioning liver; obtaining second quantitative data of metabolites contained in samples taken at a second time point after the first time point of reperfusion for each of the normally functioning liver and the poorly functioning liver; Identifying the plurality of metabolites based on the first and second quantitative data, provided that the plurality of metabolites includes metabolites whose content changes between the first timing and the second timing and whose content changes in the normal functioning liver and the poorly functioning liver have different tendencies; generating, as the reference data, data indicating a time change in the content of the plurality of metabolites from the first time point to the second time point based on the first and second quantitative data for each of the normally functioning liver and the impaired functioning liver; taking a first sample from the liver for transplant at the first time point during reperfusion of the liver for transplant; taking a second sample from the liver for transplant at the second time point during reperfusion of the liver for transplant; measuring the content of the plurality of metabolites in the first sample; measuring the content of the plurality of metabolites in the second sample; determining a change in content at the second time point relative to the first time point for each of the plurality of metabolites; and determining an index relating to functional evaluation of the liver for transplantation using the determined change in the content of the plurality of metabolites and the reference data; The step of determining the change includes a step of determining a trend of change in the content of each of the plurality of metabolites; The step of determining the index includes a step of scoring the tendency of change in the content of each of the plurality of metabolites using the reference data; A method for evaluating the function of a liver for transplantation, the scoring step including a step of making the score larger when the trend of change in the content in the liver for transplantation matches the trend of change in the content in a liver having normal function in the reference data than when they do not match.

2. each of the steps of acquiring the first quantitative data and acquiring the second quantitative data includes measuring the abundance of the metabolite using mass spectrometry; The method for evaluating the function of a liver for transplantation as described in claim 1, wherein each of the steps of measuring the content of the plurality of metabolites in the first sample and the step of measuring the content of the plurality of metabolites in the second sample includes a step of measuring the content of the plurality of metabolites using mass spectrometry.

3. 3. The method for evaluating the function of a liver for transplantation according to claim 1 or 2, wherein the step of determining the change further includes a step of determining that the content has increased when a rate of change of the content at the second timing relative to the content at the first timing is greater than a first threshold value greater than 1, and determining that the content has decreased when the rate of change is smaller than a second threshold value less than 1.

4. 4. The method for evaluating function of a liver for transplantation according to claim 1, wherein the plurality of metabolites include at least two of sedoheptulose 7-phosphate, adenosine triphosphate, glucose-6-phosphate, succinyl coenzyme A, dimethylglycine, choline, 2-aminobutyric acid, uric acid, pyruvic acid, and inosine.

5. each of the normally functioning and the poorly functioning liver is cold preserved prior to reperfusion; The step of preparing the reference data includes: obtaining third quantitative data of metabolites contained in samples taken at a third time point before the start of the cold preservation for each of the normally functioning liver and the poorly functioning liver; obtaining fourth quantitative data of metabolites contained in samples taken at a fourth time point after the end of the cold preservation and before the start of the reperfusion for each of the normally functioning liver and the poorly functioning liver; and identifying a first metabolite based on the third and fourth quantitative data, provided that the content at the third and fourth timings is high in the liver having normal function and is low in the liver having poor function; The liver for transplantation is cold-preserved after removal from the donor and before the reperfusion; collecting a third sample from the liver for transplant at the third timing of the liver for transplant; Collecting a fourth sample from the liver for transplant at the fourth timing of the liver for transplant; measuring the content of the first metabolite by mass spectrometry of the third sample; measuring the content of the first metabolite by mass spectrometry of the fourth sample; and determining whether or not the function of the liver for transplantation is normal based on the content of the first metabolite at the third time point and the fourth time point; A method for evaluating the function of a liver for transplantation described in any one of claims 1 to 4, wherein the determining step includes a step of determining that the liver for transplantation is normal when the content of the first metabolite is greater than a threshold value at each of the third timing and the fourth timing.

6. each of the normally functioning and the poorly functioning liver is cold preserved prior to reperfusion; The step of preparing the reference data includes: obtaining fourth quantitative data of metabolites contained in samples taken at a fourth time point after the end of the cold preservation and before the start of the reperfusion for each of the normally functioning liver and the poorly functioning liver; and identifying a first metabolite based on the fourth quantitative data, provided that the content at the fourth timing is a high value in the liver having normal function and a low value in the liver having poor function; The liver for transplantation is cold-preserved after removal from the donor and before the reperfusion; Collecting a fourth sample from the liver for transplant at the fourth timing of the liver for transplant; measuring the content of the first metabolite by mass spectrometry of the fourth sample; and determining whether or not the function of the liver for transplantation is normal based on the content of the first metabolite at the fourth timing. A method for evaluating the function of a liver for transplantation described in any one of claims 1 to 4, wherein the determining step includes a step of determining that the liver for transplantation is normal if the content of the first metabolite is greater than a threshold value at the fourth timing.

7. each of the normally functioning and the poorly functioning liver is cold preserved prior to reperfusion; The step of preparing the reference data includes: obtaining third quantitative data of metabolites contained in samples taken at a third time point before the start of the cold preservation for each of the normally functioning liver and the poorly functioning liver; and identifying the first and second metabolites based on the third quantitative data, provided that the contents at the third timing indicate a high value in the liver having normal function and a low value in the liver having poor function; The liver for transplantation is cold-preserved after removal from the donor and before the reperfusion; collecting a third sample from the liver for transplant at the third timing of the liver for transplant; measuring the content of the first and second metabolites by mass spectrometry of the third sample; and determining whether or not the function of the liver for transplantation is normal based on the contents of the first and second metabolites at the third timing.

5. A method for evaluating the function of a liver for transplantation according to any one of claims 1 to 4, wherein the determining step includes a step of determining that the liver for transplantation is normal if, at the third timing, the content of the first metabolite is greater than a first threshold value and the content of the second metabolite is greater than a second threshold value.

8. 7. The method for evaluating the function of a liver for transplantation according to claim 5, wherein the first metabolite is uridine.

9. the first metabolite is uridine; The method for evaluating function of a liver for transplantation according to claim 7 , wherein the second metabolite is adenylosuccinic acid.

10. A computer-readable storage medium having recorded thereon a program for evaluating the function of a liver for transplantation extracted from a living body, using a computer having a calculation unit and a storage unit, The program is storing reference data in the storage unit, the reference data indicating time-dependent changes in the content of a plurality of metabolites contained in a sample taken from the perfused liver; Obtaining measurement data of the contents of the multiple metabolites in a first sample collected from the liver for transplant at a first timing during reperfusion of the liver for transplant; Obtaining measurement data of the contents of the multiple metabolites in a second sample collected from the liver for transplantation at a second timing after the first timing during reperfusion of the liver for transplantation; determining a change in content at the second time point relative to the first time point for each of the plurality of metabolites; and calculating an index relating to functional evaluation of the liver for transplantation using the calculated change in the content of the plurality of metabolites and the reference data. The step of storing the reference data in the storage unit includes: obtaining first quantitative data of metabolites contained in samples taken at the first timing during reperfusion for each of the normally functioning liver and the poorly functioning liver; obtaining second quantitative data of metabolites contained in samples taken at the second timing during the reperfusion for each of the normally functioning liver and the poorly functioning liver; Identifying the plurality of metabolites based on the first and second quantitative data, provided that the plurality of metabolites includes metabolites whose content changes between the first timing and the second timing and whose content changes in the normal functioning liver and the poorly functioning liver have different tendencies; generating, as the reference data, data indicating a time change in the content of the plurality of metabolites from the first time point to the second time point based on the first and second quantitative data for each of the normally functioning liver and the impaired functioning liver; The step of determining the change includes a step of determining a trend of change in the content of each of the plurality of metabolites; The step of determining the index includes a step of scoring the tendency of change in the content of each of the plurality of metabolites; A program for evaluating the function of a liver for transplantation, wherein the scoring step includes a step of making the score larger when the trend of change in the content in the liver for transplantation matches the trend of change in the content in a liver with normal function in the reference data than when they do not match.

11. An apparatus for evaluating the function of a liver for transplantation extracted from a living body, the apparatus comprising: The calculation unit is obtaining first quantitative data of metabolites contained in samples taken at a first timing during reperfusion for each of the normally functioning liver and the poorly functioning liver; obtaining second quantitative data of metabolites contained in samples taken at a second time point after the first time point of reperfusion for each of the normally functioning liver and the poorly functioning liver; Identifying a plurality of metabolites based on the first and second quantitative data, provided that the plurality of metabolites includes metabolites whose content changes between the first timing and the second timing and whose content changes in the normal functioning liver and the poorly functioning liver have different tendencies; generating, as reference data, data indicating a time change in the content of the plurality of metabolites from the first time point to the second time point based on the first and second quantitative data for each of the normally functioning liver and the impaired functioning liver; storing the generated reference data in the storage unit; The calculation unit is Obtaining measurement data of the contents of the plurality of metabolites in a first sample collected from the liver for transplant at the first timing during reperfusion of the liver for transplant; obtaining measurement data of the contents of the plurality of metabolites in a second sample collected from the liver for transplant at the second timing during reperfusion of the liver for transplant; For each of the plurality of metabolites, a tendency of change in content at the second time point relative to the first time point is obtained; and and determining an index for functional evaluation of the liver for transplantation by scoring the tendency of change in the content of each of the plurality of metabolites using the reference data, The calculation unit is an apparatus for evaluating the function of a liver for transplantation, which gives a higher score when the trend of change in the content in the liver for transplantation matches the trend of change in the content in a liver having normal function in the reference data, than when they do not match.

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