CN118010601A - System for diagnosing primary cholangitis - Google Patents
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Abstract
The present disclosure provides a system for diagnosing primary cholangitis by molecular markers that are mitochondrial functional indicators including the proportion of cd3+cd8+ T lymphocytes with low mitochondrial membrane potential in peripheral blood to total cd3+cd8+ T lymphocytes in peripheral blood and/or the effective protein content on the mitochondrial inner membrane respiratory chain of cd3+cd8+ T lymphocytes in peripheral blood; by the technical scheme, the molecular marker and the model with high specificity and high sensitivity are provided for the personalized diagnosis of the primary cholangitis.
Description
Technical Field
The present disclosure relates to the field of biomedical technology, and in particular to a system for diagnosing primary cholangitis.
Background
Primary cholangitis (Primary biliary cholangitis, PBC) is an autoimmune disease mainly characterized by non-suppurative cholangitis with intrahepatic granuloma, and its pathological characteristics are characterized by intrahepatic cholestasis caused by intrahepatic small bile duct epithelial cell degeneration and necrosis, followed by induction of hepatic fibrosis and cirrhosis, eventually leading to hepatic failure of the patient, thus threatening the life of the patient. The pathogenesis of PBC is not known and may be due to a combination of environmental, immune and genetic factors. Epidemiological studies have shown that PBC has a annual incidence of (0.23-5.31)/10 ten thousand and a prevalence of (1.91-40.2)/10 ten thousand, and is well developed in middle-aged women over 40 years, with a prevalence of about 1:10 for men and women. The early stage of PBC has no specific clinical manifestation, and patients may have skin itching, debilitation, liver discomfort and other non-specific symptoms in early stage of disease. At present, the clinical diagnosis of PBC mainly comprises the following three criteria: (1) Patient serum can detect anti-mitochondrial antibodies (AMA) or anti-mitochondrial antibody M2 subtype (AMA-M2); (2) Serum alkaline phosphatase (ALP) or glutamyl transpeptidase (GGT) progressive elevation; (3) liver tissue penetration examination is consistent with PBC pathology; the PBC can be diagnosed by conforming to two of the three diagnosis standards. Clinically, some PBC patients only showed positive AMA or AMA-M2, whereas ALP or GGT levels were normal, and these patients were further assessed for PBC by liver puncture examination. However, liver puncture tests are invasive tests, which are often unacceptable to patients, resulting in delayed diagnosis and difficulty in early treatment. Therefore, there is a need to mine novel noninvasive examination markers for PBC diagnosis, which are helpful for early diagnosis of PBC and improve patient prognosis.
Disclosure of Invention
The present disclosure aims to provide a molecular marker and a diagnostic system with high specificity and high sensitivity for primary cholangitis.
To achieve the above object, in one aspect, the present disclosure provides a system for diagnosing primary cholangitis, the system comprising means for inputting a mitochondrial function index of a subject, calculating means, and means for outputting a primary cholangitis diagnosis result;
the mitochondrial function index comprises the proportion of the CD3+CD8+T lymphocytes with low mitochondrial membrane potential in peripheral blood to the total CD3+CD8+T lymphocytes in peripheral blood and/or the effective protein content on the mitochondrial inner membrane respiratory chain of the CD3+CD8+T lymphocytes in peripheral blood;
The computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to implement a modeling algorithm and an algorithm of a discriminant function as shown in formula (1) and/or formula (2); the modeling algorithm is a support vector machine algorithm and/or a least partial square algorithm;
(1),
Formula (2);
In the formula (1), the components are as follows, And/>Representing a primary cholangitis diagnosis result, wherein a return value of 1 represents support, and a return value of-1 represents rejection; c 1 is the proportion of cd3+cd8+t lymphocytes with low mitochondrial membrane potential in peripheral blood to total cd3+cd8+t lymphocytes in peripheral blood; c 2 is the effective protein content on the mitochondrial inner membrane respiratory chain of cd3+cd8+t lymphocytes in peripheral blood; f 1(c1) and f 2(c2) are kernel functions trained according to the modeling algorithm, and b 1 and b 2 are critical scoring values trained according to the modeling algorithm, respectively.
The Mitochondrial Membrane Potential (MMP) refers to a transmembrane potential crossing the inner mitochondrial membrane, and energy generated in the mitochondrial oxidative phosphorylation process is stored in the inner mitochondrial membrane in the form of electrochemical potential energy, so that the concentration distribution of protons and other ions on two sides of the inner mitochondrial membrane is asymmetric, thereby forming MMP, and the level of MMP has important significance for maintaining the normal function of mitochondria and can reflect the actual state of the cellular metabolism level. The lower percentage of cells with lower mitochondrial membrane potential (MMP-low%) refers to the percentage of cells with lower mitochondrial membrane potential in the total number of the cells, and the lower the value, the worse the cell activity and the more serious the mitochondrial dysfunction.
Mitochondrial Mass (MM) represents an upper limit on the metabolic capacity of cells, with an effective protein content on the respiratory chain of the mitochondrial inner membrane. Alterations in immune cell MMPs and MMs can lead to damage to their mitochondria, thereby affecting normal mitochondrial metabolic function, which in turn leads to deregulation of immune cell function.
Single Cell Mitochondrial Mass (SCMM) is the fluorescence intensity of mitochondria of each lymphocyte subpopulation detected by flow cytometry, and then divided by the count of the corresponding lymphocyte subpopulation, thereby obtaining SCMM of each lymphocyte subpopulation. SCMM can more sensitively reflect cellular mitochondrial function, and its elevated levels reflect abnormal mitochondrial metabolism, positively correlated with the extent of mitochondrial damage.
Optionally, the system further comprises a detection device for mitochondrial functionality index, the detection device being a flow cytometer.
Optionally, the detection device of mitochondrial function indicators comprises a signal reader of mitochondrial function indicators.
Optionally, the signal reader of the mitochondrial functionality index comprises: a CD3 signal value reader, a CD8 signal value reader, a CD45 signal value reader, a CD4 signal value reader, a mitochondrial membrane potential signal value reader, a mitochondrial respiratory chain protein signal value reader.
Optionally, the signal read by the CD3 signal value reader is a PE probe signal, the signal read by the CD8 signal value reader is a FITC probe signal, the signal read by the CD45 signal value reader is a PC5.5 probe signal, and the signal read by the CD4 signal value reader is a PC7 probe signal.
Optionally, the signal read by the mitochondrial membrane potential signal value reader is a Mito TRACKER DEEP RED probe signal.
Optionally, the signal read by the mitochondrial respiratory chain protein signal value reader is a Mito TRACKER GREEN FM probe signal.
Optionally, the processor is further configured to execute a computer program stored in the memory to implement the following calculations:
When the cell is read to a positive signal by the cell CD45 signal value reader and the CD4 signal value reader, the cell is a neutrophil, and meanwhile, the mitochondrial membrane potential signal value of the neutrophil is read, and the mitochondrial membrane potential signal value of the neutrophil is marked as V1;
When the cell is read to a positive signal by the cell CD3 signal value reader and the CD8 signal value reader, the cell is a CD3+CD8+T lymphocyte, the CD3+CD8+T lymphocyte is counted to obtain the number N1, meanwhile, the mitochondrial membrane potential signal value and the respiratory chain protein signal value of the CD3+CD8+T lymphocyte are read, if the mitochondrial membrane potential signal value of the CD3+CD8+T lymphocyte is smaller than V1, the CD3+CD8+T lymphocyte is a CD3+CD8+T lymphocyte with mitochondrial low membrane potential, and the CD3+CD8+T lymphocyte with mitochondrial low membrane potential is counted to obtain the number N2;
Dividing N2 by N1 to obtain c 1;
Dividing the respiratory chain protein signal value of each CD3+CD8+T lymphocyte by the CD3 signal value of the CD3+CD8+T lymphocyte to obtain the effective protein content on the mitochondrial inner membrane respiratory chain of the CD3+CD8+T lymphocyte, and averaging the effective protein content on the mitochondrial inner membrane respiratory chain of all CD3+CD8+T lymphocyte to obtain c 2.
Alternatively, f 1(c1) takes-1 Xc 1,b1 as 0.4419, f 2(c2) takes 1 Xc 2,b2 as-4.66.
Through the technical scheme, the mitochondrial functional index in the peripheral blood sample can be used as a molecular marker for diagnosing the primary cholangitis, a molecular marker and a diagnostic model with high specificity and high sensitivity are provided for the personalized diagnosis and treatment of the primary cholangitis, and a novel, efficient and noninvasive auxiliary tool is provided for clinically specifying the diagnostic scheme of the primary cholangitis.
Additional features and advantages of the present disclosure will be set forth in the detailed description which follows.
Drawings
The accompanying drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrate the disclosure and together with the description serve to explain, but do not limit the disclosure. In the drawings:
FIG. 1 is a view from the total cell cycle gate.
FIG. 2 is a gate view from lymphocyte circles.
FIG. 3 is a gate view from the total T cell loop.
Fig. 4 is a Tc gating diagram.
FIG. 5 is a graph of statistical analysis of Tc MMP-low%, SCMM-Tc.
FIG. 6 is an analysis of ROC curves for Tc MMP-low%, SCMM-Tc.
Detailed Description
The following describes specific embodiments of the present disclosure in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the disclosure, are not intended to limit the disclosure.
The present disclosure provides a system for diagnosing primary cholangitis, the system comprising means for inputting a mitochondrial functionality index of a subject, computing means, and means for outputting a primary cholangitis diagnostic result;
The mitochondrial function indexes comprise the proportion of the CD3+CD8+T lymphocytes with low mitochondrial membrane potential in peripheral blood to the total CD3+CD8+T lymphocytes in peripheral blood and/or the effective protein content on the mitochondrial inner membrane respiratory chain of the CD3+CD8+T lymphocytes in peripheral blood;
The computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to implement a modeling algorithm and an algorithm of a discriminant function as shown in formula (1) and/or formula (2); the modeling algorithm is a support vector machine algorithm and/or a least partial square algorithm;
(1),
Formula (2);
In the formula (1), the components are as follows, And/>Representing a primary cholangitis diagnosis result, wherein a return value of 1 represents support, and a return value of-1 represents rejection; c 1 is the proportion of cd3+cd8+t lymphocytes with low mitochondrial membrane potential in peripheral blood to total cd3+cd8+t lymphocytes in peripheral blood; c 2 is the effective protein content on the mitochondrial inner membrane respiratory chain of cd3+cd8+t lymphocytes in peripheral blood; f 1(c1) and f 2(c2) are kernel functions trained according to the modeling algorithm, and b 1 and b 2 are critical scoring values trained according to the modeling algorithm, respectively.
Optionally, the system further comprises a detection device for mitochondrial functionality index, the detection device being a flow cytometer.
Optionally, the detection device of mitochondrial function indicators comprises a signal reader of mitochondrial function indicators.
The signal reader of the mitochondrial functionality index comprises: a CD3 signal value reader, a CD8 signal value reader, a CD45 signal value reader, a CD4 signal value reader, a mitochondrial membrane potential signal value reader, a mitochondrial respiratory chain protein signal value reader.
Optionally, the signal read by the CD3 signal value reader is a PE probe signal, the signal read by the CD8 signal value reader is a FITC probe signal, the signal read by the CD45 signal value reader is a PC5.5 probe signal, and the signal read by the CD4 signal value reader is a PC7 probe signal.
Optionally, the signal read by the mitochondrial membrane potential signal value reader is a Mito TRACKER DEEP RED probe signal.
Optionally, the signal read by the mitochondrial respiratory chain protein signal value reader is a Mito TRACKER GREEN FM probe signal.
Optionally, the processor is further configured to execute a computer program stored in the memory to implement the following calculations:
When the cell is read to a positive signal by the cell CD45 signal value reader and the CD4 signal value reader, the cell is a neutrophil, and meanwhile, the mitochondrial membrane potential signal value of the neutrophil is read, and the mitochondrial membrane potential signal value of the neutrophil is marked as V1;
When the cell is read to a positive signal by the cell CD3 signal value reader and the CD8 signal value reader, the cell is a CD3+CD8+T lymphocyte, the CD3+CD8+T lymphocyte is counted to obtain the number N1, meanwhile, the mitochondrial membrane potential signal value and the respiratory chain protein signal value of the CD3+CD8+T lymphocyte are read, if the mitochondrial membrane potential signal value of the CD3+CD8+T lymphocyte is smaller than V1, the CD3+CD8+T lymphocyte is a CD3+CD8+T lymphocyte with mitochondrial low membrane potential, and the CD3+CD8+T lymphocyte with mitochondrial low membrane potential is counted to obtain the number N2;
Dividing N2 by N1 to obtain c 1;
Dividing the respiratory chain protein signal value of each CD3+CD8+T lymphocyte by the CD3 signal value of the CD3+CD8+T lymphocyte to obtain the effective protein content on the mitochondrial inner membrane respiratory chain of the CD3+CD8+T lymphocyte, and averaging the effective protein content on the mitochondrial inner membrane respiratory chain of all CD3+CD8+T lymphocyte to obtain c 2.
Alternatively, f 1(c1) takes-1 Xc 1,b1 as 0.4419, f 2(c2) takes 1 Xc 2,b2 as-4.66.
The present disclosure is further illustrated in detail by the following examples. The starting materials used in the examples are all available commercially.
The present disclosure detects mitochondrial functionality index in its peripheral whole blood by flow cytometry on 64 PBC patients and 32 gender age-matched healthy controls, analyzes the acquired flow software with NovoExpress software, compares the acquired mitochondrial functionality index with differences between PBC patients and healthy controls with t-test or Mann-Whitney U test, and further evaluates the diagnostic value of Tc MMP-low% and SCMM-Tc for PBC by subject work curve (ROC) analysis. Wherein Tc MMP-low% represents the proportion of CD3+CD8+T lymphocytes with low mitochondrial membrane potential in peripheral blood to total CD3+CD8+T lymphocytes in peripheral blood, and SCMM-Tc represents the effective protein content on the mitochondrial inner membrane respiratory chain of CD3+CD8+T lymphocytes in peripheral blood.
Example 1
The experimental process comprises the following steps:
1. screening of subject samples:
Inclusion criteria for PBC patients: (1) subject serum AMA and or AMA-M2 positive; (2) elevated serum ALP and GGT levels in the subject; (3) The subject liver tissue puncture examination accords with the pathological manifestations of PBC; the above 3 standards meet 2 standards to diagnose PBC.
Inclusion criteria for healthy controls: the collection of healthy human samples also requires matching of sex and age of PBC patients.
The research scheme of the embodiment has been obtained by Beijing co-ordination of China medical science center and the ethical committee of hospitals.
2. Flow cytometry staining:
Peripheral whole blood samples of all subjects were collected and tested for Tc MMP-low% and SCMM-Tc in the peripheral blood of the subjects by flow cytometry using a kit of CD8+19 FITC/CD3+56 PE/CD45 PC5.5/CD4 PC7/MitoDye (flow cytometry) test kit [ Pan peptide Biotechnology (Zhejiang Co.) ], comprising the following steps:
(1) Preparing a hemolysin working solution: taking 1 part of hemolysin recovered to room temperature, slowly adding 9 parts of purified water, uniformly mixing to prepare working solution, and standing at room temperature for later use;
(2) Taking 20 mu L of antibody detection reagent in a flow tube with marked at room temperature;
(3) 100 mu L of the well-mixed anticoagulated human peripheral blood sample is added to the bottom of a test tube;
(4) Gently shaking on a vortex mixer for 5 seconds, standing at room temperature in a dark place for 15 minutes;
(5) Adding 2mL of a hemolysin working solution placed at room temperature;
(6) Gently shaking on a vortex mixer for 5 seconds, standing at room temperature in a dark place for 15 minutes;
(7) The flow tube was placed in a centrifuge and centrifuged at 300g for 5 minutes to remove the supernatant;
(8) 200. Mu.L PBS was added, gently shaken on a vortex mixer for 5 seconds, and the resuspension was added to an 8-well tube pre-loaded with mitochondrial probes;
(9) Gently shaking on a vortex mixer for 5 seconds, and incubating for 30 minutes in a constant temperature incubator at 37 ℃ in the absence of light;
(10) Detection was performed on a Cytek TM NL-CLC full spectrum flow cytometer (3 seconds of low speed shake before going on-line), data was obtained, and analysis was performed using NovoExpress software.
Referring to the analytical strategy of FIGS. 1-4, the ratio of CD3+CD8+T lymphocytes with low mitochondrial membrane potential in peripheral blood to total CD3+CD8+T lymphocytes in peripheral blood (Tc MMP-low%) and/or the effective protein content on the mitochondrial inner membrane respiratory chain (SCMM-Tc) of CD3+CD8+T lymphocytes in peripheral blood was obtained in each peripheral blood sample.
3. Model construction:
Based on the mitochondrial functional index, a least partial square method and a support vector machine algorithm are adopted to construct a diagnosis model of the primary cholangitis, and the diagnosis model is specifically as follows: taking the proportion of the CD3+CD8+T lymphocytes with low mitochondrial membrane potential in the peripheral blood, which are detected by a flow cytometry detection technology, to the total CD3+CD8+T lymphocytes in the peripheral blood and/or the effective protein content on the mitochondrial inner membrane respiratory chain of the CD3+CD8+T lymphocytes in the peripheral blood as a data set; wherein the effective protein content on the mitochondrial inner membrane respiratory chain of CD3+CD8+T lymphocytes in peripheral blood is detected by a signal reader in the detection device: the device comprises a CD3 signal value reader, a CD8 signal value reader, a CD45 signal value reader, a CD4 signal value reader, a mitochondrial membrane potential signal value reader and a mitochondrial respiratory chain protein signal value reader, wherein the signals read by the CD3 signal value reader are PE (phycoerythrin) probe signals, the signals read by the CD8 signal value reader are FITC (Fluorescein Isothiocyanate ) probe signals, the signals read by the CD45 signal value reader are PC5.5 probe (PerCP-Cyanine 5.5 dye) signals, the signals read by the CD4 signal value reader are PC7 (PE-Cyanine 7 dye) probe signals, the signals read by the mitochondrial membrane potential signal value reader are Mito TRACKER DEEP RED 633 (CAS number: 873315-86-7) average fluorescence intensity values serving as probe signals, and the signals read by the mitochondrial respiratory chain protein signal value reader are Mito TRACKER GREEN FM (CAS number: 201860-17-5) probe signals.
Taking the mitochondrial membrane potential signal value of the neutrophil as a positive control, when the cell is read to a positive signal by the cell CD45 signal value reader and the CD4 signal value reader, the cell is the neutrophil, and simultaneously reading the mitochondrial membrane potential signal value of the neutrophil, and marking the mitochondrial membrane potential signal value of the neutrophil as V1;
when the cell is read to a positive signal by the cell CD3 signal value reader and the CD8 signal value reader, the cell is a CD3+CD8+T lymphocyte, the CD3+CD8+T lymphocyte is counted to obtain the number N1, meanwhile, the mitochondrial membrane potential signal value and the respiratory chain protein signal value of the CD3+CD8+T lymphocyte are read, if the mitochondrial membrane potential signal value of the CD3+CD8+T lymphocyte is smaller than V1, the CD3+CD8+T lymphocyte is a CD3+CD8+T lymphocyte with mitochondrial low membrane potential, and the CD3+CD8+T lymphocyte with mitochondrial low membrane potential is counted to obtain the number N2; dividing N2 by N1 to obtain c 1;
Dividing the respiratory chain protein signal value of each CD3+CD8+T lymphocyte by the CD3 signal value of the CD3+CD8+T lymphocyte to obtain the effective protein content on the mitochondrial inner membrane respiratory chain of the CD3+CD8+T lymphocyte, and averaging the effective protein content on the mitochondrial inner membrane respiratory chain of all CD3+CD8+T lymphocyte to obtain c 2.
Randomly dividing the data set into a training set and a testing set, and repeating for 3 times; using the calculated coefficients and thresholds for each component (mitochondrial functionality index) in the training set training model, the test set was used to evaluate, and the validation was repeated 3 times, the discriminant function of the model was as shown in equations (1) and (2):
(1),
Formula (2);
In the formula (1), the components are as follows, And/>Representing a primary cholangitis diagnosis result, wherein a return value of 1 represents support, and a return value of-1 represents rejection; f 1(c1) and f 2(c2) are kernel functions trained according to the modeling algorithm, and b 1 and b 2 are critical scoring values trained according to the modeling algorithm; f 1(c1) takes-1×c 1,b1 as 0.4419, f 2(c2) takes 1×c 2,b2 as-4.66, that is to say the discriminant function is specifically:
(1),
Formula (2).
Example 2
This example demonstrates the diagnostic model constructed in example 1.
This example further carried out statistical analysis of Tc MMP-low and SCMM-Tc levels in peripheral blood of PBC patients and healthy controls, and the results are shown in FIG. 5 (wherein HC represents healthy controls, PBC represents primary biliary cholangitis patients), MMP-low% levels of PBC patients are significantly reduced compared with healthy controls, while SCMM-Tc levels are significantly increased, and the difference is statistically significant, suggesting that changes in Tc MMP-low and SCMM-Tc levels can be used as potential diagnostic markers for PBC.
As shown in FIG. 6, further analysis of the diagnostic performance of Tc MMP-low%, SCMM-Tc levels (ROC curve analysis) revealed that Tc MMP-low% < 44.19%, when used for diagnosis of PBC, AUC was 0.754, sensitivity was 56.25%, specificity was 84.38%; when the SCMM-Tc is used for diagnosing PBC, the AUC is 0.707, the sensitivity is 56.25%, and the specificity is 81.25%, so that the system for diagnosing primary cholangitis, which is established by the present disclosure, has good clinical prediction value.
The preferred embodiments of the present disclosure have been described in detail above, but the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.
In addition, the specific features described in the above embodiments may be combined in any suitable manner without contradiction. The various possible combinations are not described further in this disclosure in order to avoid unnecessary repetition.
Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.
Claims (10)
1. A system for diagnosing primary cholangitis, the system comprising means for inputting a mitochondrial functionality index of a subject, computing means, and means for outputting a primary cholangitis diagnostic result;
The mitochondrial function indexes comprise the proportion of the CD3+CD8+T lymphocytes with low mitochondrial membrane potential in peripheral blood to the total CD3+CD8+T lymphocytes in peripheral blood and/or the effective protein content on the mitochondrial inner membrane respiratory chain of the CD3+CD8+T lymphocytes in peripheral blood;
The computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to implement a modeling algorithm and an algorithm of a discriminant function as shown in formula (1) and/or formula (2); the modeling algorithm is a support vector machine algorithm and/or a least partial square algorithm;
(1),
Formula (2);
In the formula (1), the components are as follows, And/>Representing a primary cholangitis diagnosis result, wherein a return value of 1 represents support, and a return value of-1 represents rejection; c 1 is the proportion of cd3+cd8+t lymphocytes with low mitochondrial membrane potential in peripheral blood to total cd3+cd8+t lymphocytes in peripheral blood; c 2 is the effective protein content on the mitochondrial inner membrane respiratory chain of cd3+cd8+t lymphocytes in peripheral blood; f 1(c1) and f 2(c2) are kernel functions trained according to the modeling algorithm, and b 1 and b 2 are critical scoring values trained according to the modeling algorithm, respectively.
2. The system of claim 1, wherein the system further comprises a detection device for a mitochondrial functionality index, the detection device being a flow cytometer.
3. The system of claim 2, wherein the means for detecting a mitochondrial functionality index comprises a signal reader of a mitochondrial functionality index.
4. A system according to claim 3, wherein the signal reader of the mitochondrial functionality index comprises: a CD3 signal value reader, a CD8 signal value reader, a CD45 signal value reader, a CD4 signal value reader, a mitochondrial membrane potential signal value reader, a mitochondrial respiratory chain protein signal value reader.
5. The system of claim 4, wherein the signals read by the CD3 signal value reader are PE probe signals, the signals read by the CD8 signal value reader are FITC probe signals, the signals read by the CD45 signal value reader are PC5.5 probe signals, and the signals read by the CD4 signal value reader are PC7 probe signals.
6. The system of claim 4, wherein the mitochondrial membrane potential signal value reader reads a signal that is a Mito TRACKER DEEP RED 633 probe signal.
7. The system of claim 4, wherein the signal read by the mitochondrial respiratory chain protein signal value reader is a Mito TRACKER GREEN FM probe signal.
8. The system of any of claims 4-7, wherein the processor is further configured to execute a computer program stored in the memory to perform the following calculations:
When the cell is read to a positive signal by the cell CD45 signal value reader and the CD4 signal value reader, the cell is a neutrophil, and meanwhile, the mitochondrial membrane potential signal value of the neutrophil is read, and the mitochondrial membrane potential signal value of the neutrophil is marked as V1;
When the cell is read to a positive signal by the cell CD3 signal value reader and the CD8 signal value reader, the cell is a CD3+CD8+T lymphocyte, the CD3+CD8+T lymphocyte is counted to obtain the number N1, meanwhile, the mitochondrial membrane potential signal value and the respiratory chain protein signal value of the CD3+CD8+T lymphocyte are read, if the mitochondrial membrane potential signal value of the CD3+CD8+T lymphocyte is smaller than V1, the CD3+CD8+T lymphocyte is a CD3+CD8+T lymphocyte with mitochondrial low membrane potential, and the CD3+CD8+T lymphocyte with mitochondrial low membrane potential is counted to obtain the number N2;
Dividing N2 by N1 to obtain c 1;
Dividing the respiratory chain protein signal value of each CD3+CD8+T lymphocyte by the CD3 signal value of the CD3+CD8+T lymphocyte to obtain the effective protein content on the mitochondrial inner membrane respiratory chain of the CD3+CD8+T lymphocyte, and averaging the effective protein content on the mitochondrial inner membrane respiratory chain of all CD3+CD8+T lymphocyte to obtain c 2.
9. The system of claim 1, wherein f 1(c1) is-1 xc 1,b1 is 0.4419.
10. The system of claim 1, wherein f 2(c2) is taken to be-4.66 for 1 xc 2,b2.
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