Method for analyzing neonatal specimens by using specimens non-invasively isolated from newborns

Abstract

The present invention relates to a method for analyzing neonatal specimens by using specimens non-invasively isolated from newborns. Through the present invention, it is expected that neonatal specimens can be non-invasively, quickly, and accurately analyzed, and information necessary for the diagnosis and prediction of neonatal or maternal diseases can be easily provided therefrom.

Description

A method for analyzing neonatal samples using noninvasively isolated samples from newborns

[0001] This invention was made possible with the support of the Ministry of Health and Welfare, under the research identification number 1465039092 and sub-project number HR22C1363020023. The research management organization for this project is the Korea Health Industry Development Institute, and the research project name is "Research-Oriented Hospital Promotion R&D." This research was conducted by Samsung Medical Center from January 1, 2023 to December 31, 2023, under the research project "Discovering Candidate Substances for Rare and Intractable Disease Treatments and Developing In Vitro Gene Therapy."

[0002] This invention was made possible by the support of the Ministry of Science and ICT (MSIT) under the grant number 1711181340 and the sub-grant number 2022R1F1A1063243. The research management organization for the said project is the National Research Foundation of Korea, and the research project name is "Individual Basic Research." This research was conducted by Samsung Medical Center in Seoul from March 1, 2023 to February 29, 2024, under the research project titled "Discovery of biomarkers predicting death, chronic lung disease, and necrotizing enterocolitis in extremely premature infants: Analysis of extracellular vesicles in gastric aspirates of premature infants."

[0003] The present invention relates to a method for analyzing a newborn sample using a sample noninvasively separated from the newborn.

[0004] Knowing what environment a newborn was exposed to at birth, what their current health status is, what diseases they may have, and predicting their future prognosis can provide important directions for future treatment and care of newborns, and can serve as important data for treatment and drug administration.

[0005] Amniotic fluid contains many diagnostically significant components that can represent the newborn's birth environment, but it must be collected invasively and has the problem of causing side effects such as fetal damage, uterine wall damage, and placental damage.

[0006] Additionally, in the case of intrauterine infection, it takes a long time of about a week to get the analysis results, which can lead to the problem of missing the timing and direction of treatment.

[0007] However, gastric aspirate, which can be collected noninvasively from newborns immediately after birth, has a high viscosity or is coagulated due to meconium and gastric substances, making accurate analysis through liquid biopsy difficult.

[0008] Accordingly, there is an urgent need to develop an analytical method that can rapidly and accurately analyze specimens that can be collected non-invasively from newborns, such as gastric aspirates.

[0009] Accordingly, the inventor of the present invention developed a method for analyzing a newborn sample using a sample noninvasively separated from the newborn, thereby completing the present invention.

[0010] The present invention provides a method for analyzing a newborn's sample, comprising: a collection step of non-invasively collecting a sample isolated from a newborn; a homogenization step of homogenizing the sample by mixing the sample with a sample homogenization composition; and an analysis step of analyzing the homogenized sample.

[0011] Another object of the present invention is to provide a method for providing information necessary for diagnosing and predicting a disease in a newborn or mother, comprising a collection step of non-invasively collecting a sample isolated from a newborn; a homogenization step of mixing the sample with a sample homogenization composition to homogenize the sample; and an analysis step of analyzing the homogenized sample.

[0012] Another object of the present invention is to provide a method for diagnosing and predicting a disease in a newborn or mother, comprising a collection step of non-invasively collecting a sample isolated from a newborn; a homogenization step of homogenizing the sample by mixing the sample with a sample homogenization composition; and an analysis step of analyzing the homogenized sample.

[0013] The present invention relates to a method for analyzing a newborn sample using a sample noninvasively separated from the newborn.

[0014] Hereinafter, the present invention will be described in more detail.

[0015] One aspect of the present invention relates to a method for analyzing a sample from a newborn, comprising: a collection step of non-invasively collecting a sample isolated from a newborn; a homogenization step of homogenizing the sample by mixing the sample with a sample homogenization composition; and an analysis step of analyzing the homogenized sample.

[0016] In the present invention, the non-invasively separated sample may be at least one selected from the group consisting of gastric aspirate, saliva, amniotic fluid, and lung aspirate, and may be, for example, gastric aspirate, but is not limited thereto.

[0017] In the present invention, the specimen homogenization composition may include at least one selected from the group consisting of PBS and saline solution.

[0018] In the present invention, the homogenization step may be mixing the sample and the sample homogenization composition in a volume ratio of 1:0.01 to 10, 1:0.01 to 9, 1:0.01 to 8, 1:0.01 to 7, 1:0.01 to 6, 1:0.01 to 5, 1:0.01 to 4, 1:0.1 to 10, 1:0.1 to 9, 1:0.1 to 8, 1:0.1 to 7, 1:0.1 to 6, 1:0.1 to 5, 1:0.1 to 4, 1:1 to 10, 1:1 to 9, 1:1 to 8, 1:1 to 7, 1:1 to 6, 1:1 to 5, or 1:1 to 4, for example, 1:3. there is.

[0019] In the present invention, the homogenization step may additionally include an extrusion step of sucking a mixture of a sample and a sample homogenization composition through a suction device and then discharging it.

[0020] In the present invention, the suction device may be a syringe or a dropper.

[0021] In the present invention, the extrusion step may be a step of inhaling a mixture of a sample and a sample homogenization composition through a syringe or a dropper at least once and then discharging it, and for example, it may be a step of inhaling 1 to 30 times, 1 to 20 times, 5 to 30 times, or 5 to 20 times and then discharging it, but is not limited thereto.

[0022] In the present invention, the needle of the syringe may have a diameter of 21 to 34G, 21 to 30G, or 21 to 26G, for example, 22G, but is not limited thereto.

[0023] In the present invention, the extrusion step may be performed at a temperature of, but is not limited to, 2 to 8°C, 2 to 7°C, or 2 to 6°C. When the extrusion step is performed within the above temperature range, the long-term storage of the specimen becomes easier, and when testing is to be performed immediately without storage, the extrusion step can be performed at room temperature.

[0024] In the present invention, the analysis step may be analyzing one or more selected from the group consisting of microbiome, protein, genome, and exosome in a homogenized sample, but is not limited thereto.

[0025] In the present invention, the analysis step may be performed by one or more analysis methods selected from the group consisting of Western blot, ELISA analysis, Next Generation Sequencing (NGS) technique, lateral flow analysis, immunochromatography, and protein mass spectrometry, but is not limited thereto.

[0026] Another aspect of the present invention relates to a method for providing information necessary for diagnosing and predicting a disease in a newborn or mother, comprising: a collection step of non-invasively collecting a sample isolated from a newborn; a homogenization step of homogenizing the sample by mixing the sample with a sample homogenization composition; and an analysis step of analyzing the homogenized sample.

[0027] In the present invention, the disease may be at least one selected from the group consisting of intrauterine infection, necrotizing enterocolitis, neonatal pulmonary hypertension, neonatal respiratory distress syndrome, and chorioamnionitis, but is not limited thereto.

[0028] In the present invention, the non-invasively separated specimen may be at least one selected from the group consisting of gastric aspirate, saliva gastric aspirate, saliva, amniotic fluid, and lung aspirate, and may be, for example, gastric aspirate, but is not limited thereto.

[0029] In the present invention, the specimen homogenization composition may include at least one selected from the group consisting of PBS and saline solution.

[0030] In the present invention, the homogenization step may be mixing the sample and the sample homogenization composition in a volume ratio of 1:0.01 to 10, 1:0.01 to 9, 1:0.01 to 8, 1:0.01 to 7, 1:0.01 to 6, 1:0.01 to 5, 1:0.01 to 4, 1:0.1 to 10, 1:0.1 to 9, 1:0.1 to 8, 1:0.1 to 7, 1:0.1 to 6, 1:0.1 to 5, 1:0.1 to 4, 1:1 to 10, 1:1 to 9, 1:1 to 8, 1:1 to 7, 1:1 to 6, 1:1 to 5, or 1:1 to 4, for example, 1:3. there is.

[0031] In the present invention, the homogenization step may additionally include an extrusion step of sucking a mixture of a sample and a sample homogenization composition through a suction device and then discharging it.

[0032] In the present invention, the suction device may be a syringe or a dropper.

[0033] In the present invention, the extrusion step may be a step of inhaling a mixture of a sample and a sample homogenization composition through a syringe or a dropper at least once and then discharging it, and for example, it may be a step of inhaling 1 to 30 times, 1 to 20 times, 5 to 30 times, or 5 to 20 times and then discharging it, but is not limited thereto.

[0034] In the present invention, the needle of the syringe may have a diameter of 21 to 34G, 21 to 30G, or 21 to 26G, for example, 22G, but is not limited thereto.

[0035] In the present invention, the extrusion step may be performed at a temperature of, but is not limited to, 2 to 8°C, 2 to 7°C, or 2 to 6°C. When the extrusion step is performed within the above temperature range, the long-term storage of the specimen becomes easier, and when testing is to be performed immediately without storage, the extrusion step can be performed at room temperature.

[0036] In the present invention, the analysis step may be analyzing one or more selected from the group consisting of microbiome, protein, genome, and exosome in a homogenized sample, but is not limited thereto.

[0037] In the present invention, the analysis step may be performed by one or more analysis methods selected from the group consisting of Western blot, ELISA analysis, Next Generation Sequencing (NGS) technique, lateral flow analysis, immunochromatography, and protein mass spectrometry, but is not limited thereto.

[0038] Another aspect of the present invention relates to a method for diagnosing and predicting a disease in a newborn or mother, comprising: a collection step of non-invasively collecting a sample isolated from a newborn; a homogenization step of homogenizing the sample by mixing the sample with a sample homogenization composition; and an analysis step of analyzing the homogenized sample.

[0039] In the present invention, the disease may be at least one selected from the group consisting of intrauterine infection, necrotizing enterocolitis, neonatal pulmonary hypertension, neonatal respiratory distress syndrome, and chorioamnionitis, but is not limited thereto.

[0040] In the present invention, the non-invasively separated specimen may be at least one selected from the group consisting of gastric aspirate, saliva gastric aspirate, saliva, amniotic fluid, and lung aspirate, and may be, for example, gastric aspirate, but is not limited thereto.

[0041] In the present invention, the specimen homogenization composition may include at least one selected from the group consisting of PBS and saline solution.

[0042] In the present invention, the homogenization step may be mixing the sample and the sample homogenization composition in a volume ratio of 1:0.01 to 10, 1:0.01 to 9, 1:0.01 to 8, 1:0.01 to 7, 1:0.01 to 6, 1:0.01 to 5, 1:0.01 to 4, 1:0.1 to 10, 1:0.1 to 9, 1:0.1 to 8, 1:0.1 to 7, 1:0.1 to 6, 1:0.1 to 5, 1:0.1 to 4, 1:1 to 10, 1:1 to 9, 1:1 to 8, 1:1 to 7, 1:1 to 6, 1:1 to 5, or 1:1 to 4, for example, 1:3. there is.

[0043] In the present invention, the homogenization step may additionally include an extrusion step of sucking a mixture of a sample and a sample homogenization composition through a suction device and then discharging it.

[0044] In the present invention, the suction device may be a syringe or a dropper.

[0045] In the present invention, the extrusion step may be a step of inhaling a mixture of a sample and a sample homogenization composition through a syringe or a dropper at least once and then discharging it, and for example, it may be a step of inhaling 1 to 30 times, 1 to 20 times, 5 to 30 times, or 5 to 20 times and then discharging it, but is not limited thereto.

[0046] In the present invention, the needle of the syringe may have a diameter of 21 to 34G, 21 to 30G, or 21 to 26G, for example, 22G, but is not limited thereto.

[0047] In the present invention, the extrusion step may be performed at a temperature of, but is not limited to, 2 to 8°C, 2 to 7°C, or 2 to 6°C. When the extrusion step is performed within the above temperature range, the long-term storage of the specimen becomes easier, and when testing is to be performed immediately without storage, the extrusion step can be performed at room temperature.

[0048] In the present invention, the analysis step may be analyzing one or more selected from the group consisting of microbiome, protein, genome, and exosome in a homogenized sample, but is not limited thereto.

[0049] In the present invention, the analysis step may be performed by one or more analysis methods selected from the group consisting of Western blot, ELISA analysis, Next Generation Sequencing (NGS) technique, lateral flow analysis, immunochromatography, and protein mass spectrometry, but is not limited thereto.

[0050] The present invention relates to a method for analyzing neonatal specimens using specimens noninvasively isolated from the newborn. It is expected that the present invention will enable the noninvasive, rapid, and accurate analysis of neonatal specimens, thereby readily providing information necessary for the diagnosis and prediction of diseases in the newborn or mother.

[0051] Figure 1 is a diagram schematically illustrating a method for analyzing a newborn sample according to one embodiment of the present invention.

[0052] FIG. 2 is a photograph showing a process of isolating exosomes from homogenized neonatal gastric aspirate according to one embodiment of the present invention.

[0053] FIG. 3 is a graph showing the distribution of the size and number of exosomes isolated from homogenized neonatal gastric aspirate according to one embodiment of the present invention.

[0054] FIG. 4 is a graph showing that the amount of a specific protein in exosomes isolated from homogenized neonatal gastric aspirate varies from patient to patient according to one embodiment of the present invention.

[0055] FIG. 5 is a graph showing that the diversity of the microbiome in homogenized gastric aspirate of a newborn differs depending on whether the newborn has pulmonary hypertension, according to one embodiment of the present invention.

[0056] FIG. 6 is a graph showing that the amount of cytokine IL-6 in homogenized neonatal gastric aspirate varies depending on the presence or absence of histological chorioamnionitis in the maternal placenta according to one embodiment of the present invention.

[0057] A collection step for non-invasively collecting a sample isolated from a newborn;

[0058] A homogenization step of homogenizing the sample by mixing the sample and the sample homogenization composition; and

[0059] A method for analyzing a newborn sample, comprising an analysis step of analyzing a homogenized sample.

[0060] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only intended to illustrate the present invention, and the scope of the present invention is not limited by these examples.

[0061] Example 1. Homogenization of gastric aspirate from a newborn

[0062] Immediately after birth, a nasogastric tube was inserted into the newborn, and the newborn's gastric aspirate was collected noninvasively through this tube. The noninvasively collected gastric aspirate was homogenized using the extrusion method. Specifically, PBS (hereinafter referred to as the specimen homogenization composition) was placed in a tube at a volume ratio of 1:3 with the gastric aspirate, and the gastric aspirate was homogenized by aspirating 5 times through a needle (22G) of a commercially available syringe at 2°C and then expelling the solution. The homogenized solution was divided into 1.5 ml tubes using a dropper and stored at -70°C until analysis. A schematic diagram of the method for homogenizing and analyzing the newborn's gastric aspirate as described above is shown in Figure 1.

[0063]

[0064] Example 2. Analysis of homogenized neonatal gastric aspirate.

[0065] 2-1. Exosome analysis

[0066] Exosomes were isolated from the homogenized solution of Example 1. Specifically, the homogenized solution of Example 1 was centrifuged at 1000g for 30 minutes, and the supernatant was carefully collected to avoid collecting sediment. 10% by volume of polyethylene glycol (PEG) of the supernatant was added, mixed, and centrifuged again at 6000g for 30 minutes. The supernatant was discarded, and the exosomes that had settled at the bottom were collected and stored at -70℃ until analysis. The process of isolating exosomes from the homogenized neonatal gastric aspirate as described above is shown in Fig. 2.

[0067] The size and number of the obtained exosomes were analyzed using a nanoparticle tracking analysis (NTA) device, and the results are shown in Fig. 3. The x-axis of Fig. 3 represents the size of the exosomes, and the y-axis represents the number of exosomes. The photo next to the graph is a captured image of the exosome measurement screen using an actual NTA. The results of Fig. 3 allowed us to confirm the size and distribution of the exosomes.

[0068] Additionally, exosomal factors were analyzed using ELISA for nine subjects (newborns), and the results are shown in Fig. 4. The x-axis of Fig. 4 represents the patient number, and the y-axis represents the relative amount of IL-6 in the exosomes. The results of Fig. 4 confirmed that the amount of IL-6 varied depending on the subject.

[0069]

[0070] 2-2. Analysis of correlations with specific diseases

[0071] We analyzed the microbiome in the gastric aspirate of newborns through genome analysis, and measured the diversity of the microbiome through 16s rRNA analysis. In addition, we measured the correlation between the diversity of the microbiome in the gastric aspirate of newborns and the presence or absence of pulmonary hypertension in newborns using the Pearson correlation analysis method, and the results are shown in Figure 5. The x-axis of Figure 5 indicates the presence or absence of pulmonary hypertension in newborns, and the y-axis indicates the Shannon's diversity index of the microbiome (bacterial communities) in the gastric aspirate of newborns. From the results of Figure 5, it was confirmed that there was a difference in the diversity of the microbiome in the gastric aspirate of newborns depending on the presence or absence of pulmonary hypertension.

[0072] Using a human IL-6 cytokine kit (R and D), the amount of cytokine IL-6 in 100-200 μl of gastric aspirate of newborns was measured using ELISA according to the standard experimental method suggested by the manufacturer. In addition, the correlation between the amount of cytokine IL-6 in the gastric aspirate of newborns and the presence or absence of histological chorioamnionitis in the maternal placenta was measured using Pearson's correlation analysis, and the results are shown in Fig. 6. The x-axis of Fig. 6 represents the presence or absence of histological chorioamnionitis in the maternal placenta of newborns, and the y-axis represents the amount of cytokine IL-6 in the gastric aspirate of newborns. From the results of Fig. 6, it was confirmed that there was a difference in the amount of cytokine IL-6 in the gastric aspirate of newborns depending on the presence or absence of histological chorioamnionitis in the maternal placenta.

[0073] Through the above results, it was confirmed that by homogenizing and analyzing gastric aspirate collected noninvasively from a newborn, diseases of the newborn or mother can be diagnosed noninvasively, quickly, and accurately.

Claims

1. Collection step for non-invasively collecting a sample isolated from a newborn; A homogenization step of homogenizing the sample by mixing the sample and the sample homogenization composition; and A method for analyzing a newborn sample, comprising an analysis step of analyzing a homogenized sample.

2. A method for analyzing a newborn's specimen in paragraph 1, wherein the non-invasively separated specimen is at least one selected from the group consisting of gastric aspirate, saliva, amniotic fluid, and lung aspirate.

3. A method for analyzing a newborn's sample in paragraph 1, wherein the sample homogenization composition comprises at least one selected from the group consisting of PBS and saline solution.

4. A method for analyzing a newborn's sample in paragraph 1, wherein the homogenization step is to homogenize the sample by mixing the sample and the sample homogenization composition in a volume ratio of 1:0.01 to 10.

5. A method for analyzing a newborn's sample in accordance with claim 1, wherein the homogenization step further includes an extrusion step of suctioning and then discharging a mixture of the sample and the sample homogenization composition through a suction device.

6. A method for analyzing a newborn's sample in paragraph 5, wherein the suction device is a syringe or a dropper.

7. A method for analyzing a newborn sample in paragraph 1, wherein the analysis step analyzes at least one selected from the group consisting of microbiome, protein, genome, and exosome in the homogenized sample.

8. A method for analyzing a newborn sample, wherein the analysis step is performed by at least one analysis method selected from the group consisting of Western blot, ELISA analysis, Next Generation Sequencing (NGS), and protein mass spectrometry.

9. Collection step for non-invasively collecting isolated samples from newborns; A homogenization step of homogenizing the sample by mixing the sample and the sample homogenization composition; and A method for providing information necessary for diagnosing and predicting diseases in a newborn or mother, comprising an analysis step of analyzing a homogenized sample.

10. A method for providing information necessary for diagnosing and predicting a disease in a newborn or mother, wherein the disease is at least one selected from the group consisting of intrauterine infection, necrotizing enterocolitis, neonatal pulmonary hypertension, neonatal respiratory distress syndrome, and chorioamnionitis.

11. A method for providing information necessary for diagnosing and predicting a disease in a newborn or mother, wherein the non-invasively separated sample is at least one selected from the group consisting of gastric aspirate, saliva, amniotic fluid, and lung aspirate.

12. A method for providing information necessary for diagnosing and predicting a disease in a newborn or mother, wherein the specimen homogenization composition comprises at least one selected from the group consisting of PBS and saline solution.

13. A method for providing information necessary for diagnosing and predicting a disease in a newborn or mother, wherein the homogenization step comprises mixing the sample and the sample homogenization composition in a volume ratio of 1:0.01 to 10 to homogenize the sample.

14. A method for providing information necessary for diagnosing and predicting a disease in a newborn or mother, wherein the homogenization step further includes an extrusion step of inhaling and then expelling a mixture of the sample and the sample homogenization composition through a suction device.

15. A method for providing information necessary for diagnosing and predicting diseases of a newborn or mother, wherein the suction device is a syringe or a dropper in the 14th paragraph.

16. A method for providing information necessary for diagnosing and predicting a disease in a newborn or mother, wherein the analysis step analyzes at least one selected from the group consisting of microbiome, protein, genome, and exosome in the homogenized sample.

17. A method for providing information necessary for diagnosing and predicting a disease in a newborn or mother, wherein the analysis step is performed by at least one analysis method selected from the group consisting of Western blot, ELISA analysis, Next Generation Sequencing (NGS), and protein mass spectrometry.

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