Application of sTREM2 in the disease diagnosis and clinical evaluation of neuromyelitis optica

Abstract

The present invention discloses the application of sTREM2 in the diagnosis or clinical symptom assessment of the acute attack phase of neuromyelitis optica spectrum disorder (NMOSD). The level of sTREM2 in the cerebrospinal fluid of NMOSD patients during the acute attack phase is significantly higher than that in the clinical remission phase of NMOSD patients and healthy control subjects. Moreover, there is a positive correlation between the level of sTREM2 in the cerebrospinal fluid and the degree of demyelination and neurological deficit in the acute attack phase of NMOSD. This indicates that the level of sTREM2 in the cerebrospinal fluid of NMOSD patients can serve as a biomarker for the acute attack phase of NMOSD patients and can reflect the severity of myelin loss and neurological deficit in NMOSD patients. At the same time, the present invention also determines that there is a positive correlation between sTREM2 in the cerebrospinal fluid and microglial activation and neuroinflammation in the acute attack phase of NMOSD. Therefore, as a biomarker, sTREM2 can provide a powerful auxiliary means for the early warning, clinical symptom assessment, and neuroinflammatory damage of NMOSD patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease detection, and particularly relates to the application of the biomarker sTREM2 in the diagnosis or clinical symptom assessment of the acute attack phase of neuromyelitis optica spectrum disorder. Background Art

[0002] Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune demyelinating disease mainly involving the optic nerve and spinal cord, and is mainly manifested as symptoms related to optic neuritis and acute transverse myelitis, such as visual impairment, limb numbness and weakness, movement disorders, and autonomic nervous system dysfunction. Pathologically, it is manifested as white matter demyelination, inflammatory cell infiltration, and astrocyte lesions. Existing epidemiological data show that the prevalence of NMOSD is relatively close in various regions of the world, but non-Caucasian (Asian, African) populations are more susceptible. The incidence rate of NMOSD in China is about 0.278 / (100,000 person-years), with children and adults being 0.075 / (100,000 person-years) and 0.347 / (100,000 person-years), respectively. As a highly recurrent and disabling disease, more than 90% of patients have a multi-phase course; about 60% of patients relapse within 1 year, and 90% of patients relapse within 3 years, which brings a great economic burden to both families and society.

[0003] Triggering receptor expressed on myeloid cells 2 (TREM2) is a transmembrane receptor on the cell surface of the immunoglobulin superfamily, mainly expressed in microglia of the central nervous system (CNS), regulating cytokine production and cell proliferation, and protecting nerve cells from inflammatory damage by promoting phagocytosis and clearing apoptotic neurons. TREM2 can be cleaved from the cell surface by a disintegrin and metalloprotease domain-containing protein 10 / 17 (ADAM10 / 17) and γ-secretase and released extracellularly as a soluble variant (soluble triggering receptor expressed on myeloid cells 2, sTREM2), which can be detected in cerebrospinal fluid, plasma, and serum. Currently, there are research reports on sTREM2 in cerebrospinal fluid and blood in some neurological diseases. It has been found that an increase in sTREM2 levels in Alzheimer's disease (AD) may be related to neuronal damage associated with phosphorylated tau protein in cerebrospinal fluid; in other neurodegenerative diseases, including Parkinson's disease and frontotemporal dementia, the correlation between sTREM2 levels in the CSF of patients and their specific clinical disease diagnoses is relevant, and studies have shown that sTREM2 levels in the CSF are related to nerve injury markers (total tau protein or phosphorylated tau in CSF, etc.). Currently, research on sTREM2 in cerebrospinal fluid mainly focuses on neurodegenerative diseases, while neuromyelitis optica spectrum disorder (NMOSD), as an autoimmune demyelinating disease mediated by autoantibodies, has significant differences in its pathogenesis, pathological characteristics, and clinical manifestations from the above-mentioned neurodegenerative diseases. There have been no relevant clinical studies showing the specific role of sTREM2 in NMOSD and its correlation with the clinical symptoms of NMOSD patients. Summary of the Invention

[0004] The present invention provides the use of the biomarker sTREM2 in the preparation of a diagnostic kit for neuromyelitis optica spectrum disorder (NMOSD).

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The use of the biomarker sTREM2 in the preparation of a kit includes the following aspects of use:

[0007] (1) sTREM2 in cerebrospinal fluid is used for diagnosing acute neuromyelitis optica spectrum disorder (NMOSD); specifically, it includes the following steps: a. measuring the level of sTREM2 in the cerebrospinal fluid of a subject; b. performing a significant difference analysis on the level of sTREM2 in the cerebrospinal fluid of the subject and the level of sTREM2 in healthy controls;

[0008] (2) sTREM2 in cerebrospinal fluid is used for evaluating the severity of demyelination or nerve function deficit in patients with neuromyelitis optica spectrum disorder;

[0009] (3) sTREM2 in cerebrospinal fluid is used for evaluating the activation level of microglia in patients with neuromyelitis optica spectrum disorder;

[0010] (4) sTREM2 in cerebrospinal fluid is used for evaluating the level of neuroinflammation in patients with neuromyelitis optica spectrum disorder.

[0011] There is a positive correlation between the level of sTREM2 in the cerebrospinal fluid of patients with neuromyelitis optica spectrum disorder (NMOSD) and the demyelination area, the degree of nerve function deficit, the degree of microglia activation, and the level of neuroinflammation. Therefore, compared with the sTREM2 level in healthy controls, the higher the level of sTREM2 in the cerebrospinal fluid of a subject, the more severe the demyelination or nerve function deficit, and the higher the activation level of microglia and the level of neuroinflammation.

[0012] Compared with the prior art, the present invention has the following advantages and beneficial effects: First, the present invention determines that the level of sTREM2 in the cerebrospinal fluid of NMOSD patients during the acute attack period is significantly higher than that of NMOSD patients during the clinical remission period and healthy control subjects. Further, the present invention determines that there is a positive correlation between the concentration of sTREM2 in the cerebrospinal fluid of NMOSD patients during the acute attack period and the degree of deterioration of the corresponding clinical symptom score (ΔEDSS score) and nerve injury-related indicators, and there is a positive correlation with the corresponding demyelination area. This indicates that the level of sTREM2 in the cerebrospinal fluid of NMOSD patients can be used as a biomarker for the acute attack period of NMOSD patients and can reflect the severity of demyelination, neuropathological damage, and nerve function deficit in NMOSD patients. At the same time, the present invention determines that there is a positive correlation between sTREM2 in the cerebrospinal fluid of NMOSD patients and microglia activation and neuroinflammation. Therefore, sTREM2, as a biomarker, can provide a powerful auxiliary means for the early warning and clinical symptom assessment of NMOSD patients. Description of the Drawings

[0013] Figure 1 Analysis of serum and cerebrospinal fluid sTREM2 levels in clinical research subjects (healthy controls, NMOSD patients during the acute attack period, NMOSD patients during the clinical remission period).

[0014] Figure 2 Comparison of cerebrospinal fluid sTREM2 levels in NMOSD model mice during the acute attack phase (7 days) and clinical remission phase (28 days).

[0015] Figure 3 Results of the correlation analysis between the cerebrospinal fluid sTREM2 level and the severity of demyelination in NMOSD model mice.

[0016] Figure 4 Results of the correlation analysis between the cerebrospinal fluid sTREM2 level and the ΔEDSS score in NMOSD patients.

[0017] Figure 5 Results of the correlation analysis between the cerebrospinal fluid sTREM2 level and nerve injury-related indicators in NMOSD patients.

[0018] Figure 6 Comparison of the activation status of microglia in NMOSD model mice during the acute attack phase (7 days) and clinical remission phase (28 days).

[0019] Figure 7 Results of the correlation analysis between the sTREM2 level in the cerebrospinal fluid of NMOSD mice and microglia activation indicators.

[0020] Figure 8 Results of the correlation analysis between the sTREM2 level in the cerebrospinal fluid of NMOSD mice and microglia activation markers.

[0021] Figure 9 Results of the correlation analysis between the cerebrospinal fluid sTREM2 level of NMOSD patients and the immune response, phagocytosis, and cytokine signaling pathways of macrophages / microglia. NMO represents NMOSD patients, and HC represents healthy controls.

[0022] Figure 10 Results of the correlation analysis between the cerebrospinal fluid sTREM2 level of NMOSD patients and inflammation-related cytokines. Detailed implementation methods

[0023] NMOSD mouse model: Serum samples of patients with aquaporin-4 antibody (AQP4-IgG) positive NMOSD were collected, purified with G-agarose, and the concentration was adjusted to 20 mg / mL. After anesthetizing the mice with isoflurane, they were fixed on a stereotaxic injection instrument. 4 μL of human complement and 6 μL of aquaporin-4 antibody AQP4-IgG and its control IgG were injected into the striatum of wild-type mice at a speed of 0.5 μL / min to establish an NMOSD mouse model. 7 days and 28 days were selected to represent the acute attack phase and clinical remission phase of NMOSD mice, respectively.

[0024] Clinical research subjects: In the following examples, the clinical research subjects included 35 control subjects who were matched in gender and age and negative for serum AQP4 antibody, and 53 NMOSD patients (positive for aquaporin 4 antibody - AQP4-IgG positive). The specific situation is shown in the following table:

[0025]

[0026] Example 1 sTREM2 in cerebrospinal fluid as a biomarker for acute exacerbation of NMOSD

[0027] (1) Sample collection and preparation

[0028] Collect blood specimens and cerebrospinal fluid specimens from NMOSD mouse models and clinical research subjects using EDTA anticoagulant blood collection tubes and sterile centrifuge tubes respectively. Centrifuge the collected blood specimens at a speed of 4000 rpm for 5 minutes at 4°C, aspirate the upper plasma, aliquot it into cryotubes, and store it in a -80°C refrigerator for later use. Centrifuge the collected cerebrospinal fluid at a speed of 400×g for 10 minutes at 4°C, aspirate the upper supernatant, aliquot it into cryotubes, and store it in a -80°C refrigerator.

[0029] (2) Detection method for sTREM2 concentration

[0030] Use a human TREM2 ELISA kit (Abcam, ab224881) to detect the expression levels of sTREM2 in serum and cerebrospinal fluid. Specifically, thaw the samples to 18 - 25°C before detection. Dilute the samples and prepare the standards according to the instructions. Add 100 μl of standards and samples to the well plate and incubate at room temperature for 2.5 hours. Then discard the liquid in the well plate, add 1× washing solution to wash the plate 3 times (300 μl per well), 3 - 5 minutes each time. Then add 100 μl of antibody and incubate at room temperature for 1 hour. Wash the plate 3 times with the washing solution as above. Then add 100 μl of HRP and incubate at room temperature for 45 minutes. After incubation, wash the plate again. Add 100 μl of substrate solution to each well and incubate at room temperature in the dark for 30 min. After color development, add 50 μl of stop solution (2 mol / L H 2 SO 4 ) to terminate the reaction. Use an enzyme-linked immunosorbent assay reader to read the OD value at 450 nm to determine the antibody levels in serum and cerebrospinal fluid.

[0031] (3) Analysis of sTREM2 levels in serum and cerebrospinal fluid in NMOSD

[0032] Specifically, the present invention detected the serum and cerebrospinal fluid of 53 NMOSD patients and 35 healthy control subjects, and divided the 53 NMOSD patients into acute exacerbation patients (36 cases) and relapse patients (17 cases). According to the detection results( Figure 1) In terms of this, there is no significant difference in the level of sTREM2 in the serum of NMOSD patients and that of healthy controls. The level of sTREM2 in the cerebrospinal fluid of NMOSD patients is significantly higher than that of healthy control subjects (p < 0.05), especially during the acute attack phase of NMOSD patients, the level of sTREM2 in the cerebrospinal fluid is significantly increased. The expression level of sTREM2 during the acute attack phase (7 days) of mice after NMOSD modeling is significantly increased, while the expression level of sTREM2 during the clinical remission phase (28 days) is down-regulated ( Figure 2 ). This indicates that sTREM2 in the cerebrospinal fluid can be used as a biomarker for the acute attack phase of NMOSD and assist in the early diagnosis of NMOSD with this.

[0033] Example 2: sTREM2 in cerebrospinal fluid as a biomarker for the degree of myelin loss and neurological deficit in NMOSD

[0034] (1) sTREM2 can indicate the severity of myelin loss in NMOSD

[0035] As an autoimmune demyelinating disease mediated by autoantibodies, the main pathological change of NMOSD is the loss of myelin in the central nervous system. Take the brain tissues of mice during the acute attack phase (7 days) and the clinical remission phase (28 days) after NMOSD modeling, and make frozen blue staining on the sections. Frozen blue staining is a staining method to show the morphological structure and pathological changes of nerve myelin, and the severity of myelin loss can be reflected by counting the area of the myelin loss region. The demyelinated area of mice after NMOSD modeling significantly increases at 7 days and decreases after 28 days ( Figure 3 ). To further clarify the relationship between the severity of demyelination in NMOSD mice and sTREM2 in the cerebrospinal fluid of mice, a correlation analysis was performed on the demyelinated area and the concentration of sTREM2 in the cerebrospinal fluid of mice. As Figure 3 shown, there is a positive correlation between the severity of demyelination in NMOSD mice and sTREM2 in the cerebrospinal fluid of mice (r = 0.7224, p < 0.0001). Therefore, sTREM2 can be used as a biomarker to indicate the severity of myelin loss in NMOSD.

[0036] (2) The relationship between the level of sTREM2 in the cerebrospinal fluid of NMOSD patients and neurological deficit

[0037] To determine the correlation between the level of sTREM2 in the cerebrospinal fluid of NMOSD patients and clinical characteristics, in this example, the Expanded Disability Status Scale (EDSS) was used to measure the degree of neurological disability in NMOSD patients. Specifically, the change in EDSS compared with the baseline (Δ score) was taken to evaluate the severity of neurological impairment brought by a single acute attack to NMOSD patients.

[0038] The EDSS score is the most commonly used and widely recognized clinical evaluation method for assessing the neurological dysfunction and disease severity of NMOSD patients in clinical practice. The scoring range is 0 - 10 points. The higher the score, the more severe the degree of neurological deficit. An EDSS score ≤ 2.5 is the low-score group, between 3 - 6 points is the medium-score group, and an EDSS score ≥ 6.5 is the high-score group. Specifically, 0 points: normal neurological function; 1.0: no disability, only mild abnormal signs in 1 functional system; 1.5: no disability, with mild abnormal signs in more than 1 functional system; 2.0: mild disability involving 1 functional system; 2.5: mild disability involving 2 functional systems; 3.0: moderate disability involving 1 functional system or mild disability involving 3 - 4 functional systems, with unrestricted walking; 3.5: unrestricted walking, moderate disability in 1 functional system, combined with a score of 2 in 1 - 2 systems, or a score of 3 in 2 functional systems, or a score of 2 in five functional systems; 4.0: unrestricted walking, even with relatively severe disability involving 1 functional system, and other systems with scores of 0 - 1, but able to take care of oneself and walk independently for more than 500m without rest; 4.5: unrestricted walking, mostly able to stand every day, can complete normal work, but activities are partially limited and require a little help, characterized by relatively severe disability involving 1 functional system, and other systems with scores of 0 - 1, walking independently for more than 300 meters without rest; 5.0: severe disability, affecting daily life and work, walking independently for 200 meters without rest, with a score of 5 in 1 functional system, or a performance more severe than 4.5, and other systems with scores of 0 - 1; 5.5: walking independently for 100 meters without rest, severe disability, affecting daily life and work, with a score of 5 in 1 functional system, or a performance more severe than 5, and other systems with scores of 0 - 1; 6.0: intermittent walking, or walking 100 meters with the assistance of others, with or without rest in the middle, or more than 2 neurological functional systems with scores greater than 3+; 6.5: able to walk 20 meters with bilateral assistance, without rest in the middle, and more than 2 neurological functional systems with scores greater than 3+; 7.0: able to walk no more than 5 meters with assistance, activities limited to a wheelchair, can independently push the wheelchair; 7.5: hardly able to walk, life limited to a wheelchair, can only move with assistance; 8.0: activities limited to bed, chair, and wheelchair, with a certain amount of time in the wheelchair every day, can take care of part of life, normal upper limb function, and more than one functional system with a score of 4+; 8.5: mostly in bed every day, can take care of part of life, with some remaining upper limb function, and several functional systems with a score of 4+; 9.0: bedridden, can communicate, eat, and most functional systems have a score of 4+; 9.5: completely bedridden, unable to communicate and eat normally, and most functional systems have a score of 4+; 10.0: died of multiple sclerosis, with the direct cause of death being respiratory paralysis, coma, or repeated seizures.

[0039] The ΔEDSS score was obtained by scoring according to the EDSS scoring scale at the pre-onset baseline period and the acute attack period respectively, and subtracting the two scores, which was used to evaluate the clinical severity / severity of neurological deficit of this acute attack.

[0040] Correlation analysis was performed on the cerebrospinal fluid sTREM2 concentration of NMOSD patients and their corresponding ΔEDSS scores, and a positive correlation was found between the cerebrospinal fluid sTREM2 concentration of NMOSD patients in the acute attack period and their corresponding ΔEDSS scores (r = 0.6440, p = 0.009) (see Figure 4 ). This indicates that the cerebrospinal fluid sTREM2 level of NMOSD patients can reflect the severity of neurological deficit in NMOSD patients. The higher the cerebrospinal fluid sTREM2 level, the more severe the neurological deficit in patients during the acute phase. Therefore, the cerebrospinal fluid sTREM2 level can be used as a biomarker to reflect the severity of neurological deficit in NMOSD patients.

[0041] (3) Correlation between the cerebrospinal fluid sTREM2 level and nerve injury-related indicators in NMOSD patients

[0042] In this example, nerve injury-related indicators were further detected, including glial fibrillary acidic protein (GFAP, a biomarker of astrocyte injury), neurofilament light protein (NFL, a biomarker of nerve injury), and albumin quotient (QALB, a marker of blood-cerebrospinal fluid barrier injury). Among them, GFAP was detected by enzyme-linked immunosorbent assay (ELISA) (human GFAP ELISA kit, Abcam, ab223867), NFL was detected using MSD (Meso Scale Discovery) based on electrochemiluminescence technology, and the calculation formula for QALB was (ALB in cerebrospinal fluid / ALB in blood) × 1000.

[0043] As Figure 5As shown, the cerebrospinal fluid sTREM2 level was positively correlated with GFAP (r = 0.671, p < 0.001), NFL (r = 0.484, p < 0.001), and QALB (r = 0.481, p < 0.001), suggesting that the cerebrospinal fluid sTREM2 level can reflect astrocyte injury, nerve injury, and blood-brain barrier injury. An increase in the cerebrospinal fluid sTREM2 level represents an exacerbation of astrocyte injury, nerve injury, and blood-brain barrier injury. Therefore, the cerebrospinal fluid sTREM2 level can be used as a biomarker for predicting neuropathological injuries such as astrocyte injury, nerve injury, and blood-brain barrier injury, playing a role in early warning and assisting in early diagnosis.

[0044] Example 3: sTREM2 in cerebrospinal fluid is related to microglial activation and inflammation in NMOSD

[0045] (1) The level of sTREM2 in cerebrospinal fluid reflects the activation degree of microglia in the central nervous system

[0046] Microglia are the most important immune cells in the CNS, mediating the innate immune response of the CNS and being an important source of CNS inflammatory mediators. Existing studies have shown that microglial activation exists in the pathological process of NMOSD, and the recognized activation manifestations include an increase in microglial density and morphological changes (such as cell body area, cell body volume, and firmness). However, the direct relationship between microglial activation and the expression level of sTREM2 in cerebrospinal fluid has not been clarified.

[0047] In this example, microglial activation was described from four aspects: microglial density, cell body area, cell body volume, and firmness. The microglial density of mice 7 days after NMOSD modeling (acute attack phase) was significantly increased, accompanied by a significant increase in cell body area and volume and an increase in firmness, indicating that microglia were activated and in an obvious activated state. At 28 days (clinical remission phase), the activated state of microglia was significantly reduced ( Figure 6 ).

[0048] Performing a correlation analysis on each index of microglial activation and the concentration of sTREM2 in the cerebrospinal fluid of NMOSD mice, the concentration of sTREM2 in the cerebrospinal fluid of mice was positively correlated with microglial density (r = 0.6845, p = 0.0002), cell body area (r = 0.7304, p < 0.0001), cell body volume (r = 0.6122, p = 0.0015), and firmness (r = 0.6635, p = 0.0004) ( Figure 7 ), suggesting that the concentration of sTREM2 in cerebrospinal fluid can reflect the activation degree of microglia in the pathological process of NMOSD.

[0049] In addition, during the activation of microglia, the expression levels of some related markers increase, such as Mac2 and TREM2. Immunofluorescence labeling of Mac2 and TREM2 was used in the lesion area of NMOSD model mice, and the proportion of double-positive cells with the microglia marker (Iba-1) was counted, which can also reflect the activation degree of microglia. Correlation analysis was performed between the two microglia activation markers and the concentration of sTREM2 in the cerebrospinal fluid of NMOSD mice. The concentration of sTREM2 in the cerebrospinal fluid was correlated with Mac2 + Iba-1 + The proportion of double-positive microglia (r = 0.6357, p = 0.0008), and TREM2 + Iba-1 + The proportion of double-positive microglia (r = 0.6217, p = 0.0012) showed a positive correlation ( Figure 8 ), further verifying that the concentration of sTREM2 in the cerebrospinal fluid can reflect the activation degree of microglia in the pathological process of NMOSD.

[0050] (2) The level of sTREM2 in cerebrospinal fluid reflects the immune-inflammatory response mediated by microglia

[0051] To verify the relationship between the level of sTREM2 in the cerebrospinal fluid of NMOSD patients and the immune-inflammatory response mediated by microglia, single-cell transcriptional analysis was performed on the cerebrospinal fluid cells of 6 AQP4-IgG-positive NMOSD patients and 3 control groups. By clustering and grouping the cerebrospinal fluid cell subsets, the macrophage / microglia subset in the cerebrospinal fluid was screened out. Further, correlation analysis was performed between the transcriptional profile changes of this cell subset for each patient and the matched cerebrospinal fluid sTREM2 level. Specifically, single-sample gene set variation analysis (ssGSVA) scores were used for transcriptional profile analysis. Finally, it was found that the cerebrospinal fluid sTREM2 level was positively correlated with the activation-related pathway scores of the immune response of macrophages / microglia (r = 0.870, p = 0.016), the phagocytosis-related pathway scores (r = 0.955, p = 0.001), and the immune system cytokine-related pathway scores (r = 0.861, p = 0.013) ( Figure 9 ). This indicates that cerebrospinal fluid sTREM2 can be used as a biomarker for the immune response, phagocytosis function, and cytokine signaling pathway mediated by macrophages / microglia in the CNS of NMOSD patients.

[0052] (3) The level of sTREM2 in cerebrospinal fluid reflects the neuroinflammatory level of the CNS

[0053] In this embodiment, the correlation between the levels of several inflammatory factors (including IL-6, IL-8, IL-12p70, interferon-γ, and tumor necrosis factor-α) in the cerebrospinal fluid of NMOSD patients and the level of cerebrospinal fluid sTREM2 was further analyzed. The results are as Figure 10 shown. Cerebrospinal fluid sTREM2 was significantly positively correlated with IL-6, IL-8, IL-12p70, interferon-γ, and tumor necrosis factor-α, respectively, indicating that cerebrospinal fluid sTREM2, as a biomarker in NMOSD patients, can strongly reflect the level of neuroinflammation in the CNS of NMOSD patients.

[0054] Taken together, the above analysis indicates that sTREM2 in NMOSD can reflect the activation of microglia, inflammation in the central nervous system, and damage to nerve function.

Claims

1. Use of the biomarker sTREM2 in the preparation of a kit, characterized in that, the sTREM2 is taken from cerebrospinal fluid, and the kit is used for diagnosing acute attacks of neuromyelitis optica spectrum disorder.

2. Use of the biomarker sTREM2 in the preparation of a kit, characterized in that, the sTREM2 is taken from cerebrospinal fluid, and the kit is used for evaluating the severity of myelin loss or neurological deficit in patients with neuromyelitis optica spectrum disorder.

3. Use of the biomarker sTREM2 in the preparation of a kit, characterized in that, the sTREM2 is taken from cerebrospinal fluid, and the kit is used for evaluating the activation level of microglia in patients with neuromyelitis optica spectrum disorder.

4. Use of the biomarker sTREM2 in the preparation of a kit, characterized in that, the sTREM2 is taken from cerebrospinal fluid, and the kit is used for evaluating the neuroinflammatory level in patients with neuromyelitis optica spectrum disorder.

5. The use according to claim 1, characterized in that, comprises the following steps: 1) measuring the level of sTREM2 in the cerebrospinal fluid of the subject; 2) performing a significant difference analysis on the level of sTREM2 in the cerebrospinal fluid of the subject and the level of sTREM2 in healthy controls.

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