CN116298275A - NK cell subset of hematophagous lymphocytosis diagnostic marker and application thereof - Google Patents
NK cell subset of hematophagous lymphocytosis diagnostic marker and application thereof Download PDFInfo
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Abstract
The invention relates to a medical test technology, in particular to a hematophagous lymphocytosis diagnostic marker NK cell subset and application thereof. The NK cell subset includes CD56 + CD16 ‑ NK bri Cell, CD56 ‑ CD16 + NK dim Cells and CD56 ‑ CD16 ‑ NK uc At least one of the cells. It has been found that CD56 in peripheral blood of patient suffering from hemophagocytic lymphocytosis + CD16 ‑ NK bri Cell, CD56 ‑ CD16 ‑ NK uc Cell expression was significantly up-regulated, CD56 ‑ CD16 + NK dim The cell expression is obviously down-regulated, has statistical significance, and the NK cell subset can be used as a marker for assisting diagnosis of hemophagocytic lymphocytosis, thereby being beneficial to realizing early diagnosis and early treatment of HLH.
Description
Technical Field
The invention relates to a medical test technology, in particular to a hematophagous lymphocytosis diagnostic marker NK cell subset and application thereof.
Background
Hemophagocytic lymphocytosis (hemophagocytic lymphohistiocytosis, HLH), also known as hemophagocytic syndrome, is a clinical syndrome characterized by pathological inflammatory responses due to genetic or acquired immune dysfunction. It is mainly a series of inflammatory reactions caused by abnormal activation, proliferation, secretion of a large number of inflammatory cytokines by lymphocyte, monocyte and phagocyte systems. Clinically, it is mainly characterized by persistent fever, hepatosplenomegaly, whole blood cytopenia, and hemophagia found in bone marrow, liver, spleen and lymph node tissues. Because of the complicated clinical manifestations, clinicians are easy to delay diagnosis and treatment due to insufficient knowledge, and the disease itself progresses rapidly, so that the patient has higher fatality rate. Timely and correct diagnosis is therefore the first step in the success of HLH therapy.
Natural killer cells (natural killer cell, NK) are important immune cells of the body, accounting for about 15% of the total number of lymphocytes, and NK cells exert important immune functions in anti-infection, anti-tumor, immune regulation, hematopoietic regulation, etc. of the body by exerting cytotoxic effects and secreting cytokines.
There is no current study reporting the association of NK cell subpopulations with hematophagous lymphocytosis.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a NK cell subset of a diagnosis marker of hemophagocytic lymphocytosis and application thereof, and researches show that CD56 in peripheral blood of hemophagocytic lymphocytosis patients + CD16 - NK bri Cells, CD56-CD16-NK uc Cell expression was significantly up-regulated, CD56-CD16 + NK dim The cell expression is obviously down-regulated, and the NK cell subset can be used as a marker for assisting diagnosis of hemophagocytic lymphocytosis.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides an application of NK cell subset in preparing reagent for detecting hemophagocytic lymphocytosis, wherein the NK cell subset comprises CD56 + CD16 - NK bri Cell, CD56 - CD16 + NK dim Cells and CD56 - CD16 - NK uc At least one of the cells.
The invention provides an application of NK cell subsets in screening of hematophagous lymphocytosis detection reagent, wherein the NK cells are used for detecting hematophagous lymphocytosisCell subsets including CD56 + CD16-NK bri Cells, CD56-CD16 + NK dim Cells and CD56-CD16 - NK uc At least one of the cells.
The invention provides an application of a reagent for detecting the content of NK cell subsets in biological samples in the preparation of a hematophagous lymphocytosis detection product, wherein the NK cell subsets comprise CD56 + CD16-NK bri Cells, CD56-CD16 + NK dim Cells and CD56-CD16-NK uc At least one of the cells.
The invention is obtained through a plurality of experimental researches, and comprises CD56 + CD16-NK bri Cells, CD56-CD16 + NK dim Cells and CD56-CD16-NK uc The NK cell subset including cells can be used as a diagnosis marker of hemophagocytic lymphocytosis, can conveniently and rapidly carry out diagnosis of auxiliary Hemophagocytic Lymphocytosis (HLH), guides clinical medication, and is beneficial to early diagnosis and early treatment of HLH.
As a preferred embodiment of the use according to the invention, the reagent is a flow cytometry detection reagent.
Preferably, the detection reagent is a flow cytometry detection antibody.
As a preferred embodiment of the use according to the invention, the product comprises a kit.
In some preferred embodiments, the product includes not only a kit, but also a test product conventional in the art, the type of test product being encompassed within the scope of the claimed invention.
As a preferred embodiment of the use according to the invention, the CD56 + CD16-NK bri Expression of cells was significantly up-regulated in hemophagocytic lymphocytosis patients, CD56-CD16 + NK dim Expression of cells was significantly down-regulated in hemophagocytic lymphocytosis patients, CD56-CD16-NK uc The expression of cells was significantly up-regulated in hemophagocytic lymphocytosis patients.
Healthy volunteersIn contrast, CD56 in peripheral blood of patients with hemophagocytic lymphocytosis + CD16-NK bri Cells, CD56-CD16-NK uc The proportion of cells is obviously increased, and CD56-CD16 in peripheral blood of hemophagocytic lymphocytosis patient + NK dim The proportion of cells is significantly reduced, and the difference is statistically significant.
The invention provides a hematophagous lymphocytosis detection kit, which comprises a reagent for detecting the content of NK cell subpopulations in a biological sample, wherein the NK cell subpopulations comprise CD56 + CD16 - NK bri Cell, CD56 - CD16 + NK dim Cells and CD56 - CD16 - NK uc At least one of the cells.
NK cell subsets (CD 56) related to the invention + CD16 - NK bri Cell, CD56 - CD16 + NK dim Cells and CD56 - CD16 - NK uc Cells) as a marker for diagnosing hemophagocytic lymphocytosis, the detection sensitivity is high, the specificity is high, and NK cell subset (CD 56) + CD16 - NK bri Cell, CD56 - CD16 + NK dim Cells and CD56 - CD16 - NK uc Cell) detection reagent can be used as an early diagnosis reagent of hemophagocytic lymphocytosis, and can be used for preparing an early detection kit of hemophagocytic lymphocytosis.
As a preferred embodiment of the detection kit of the present invention, the reagent for detecting NK cell subsets in biological samples comprises Anti-CD 3 (PE-Cyanine 7 Anti-Human CD3 (OKT 3)) antibody, anti-CD 14 (APC Anti-Human CD14 (M5E 2)) antibody, anti-CD 19 (PE-Cy) TM 5Anti-Human CD 19) antibodies, anti-CD 56 (PE/Dazzle) TM 594Anti-Human CD56 (NCAM)) antibodies and Anti-CD 16 (BV 786Anti-Human CD 16) antibodies.
As a preferred embodiment of the detection kit, the kit further comprises a peripheral blood mononuclear cell separation reagent, a red blood cell lysate and a PBS buffer. In some embodiments, the detection kit further comprises components common to the art of kits, all of which are encompassed by the present invention.
In the present invention, CD56 + CD16 - NK bri Cell, CD56 - CD16 + NK dim Cells and CD56 - CD16 - NK uc "in cells" + "represents positive.
The invention for the first time confirms NK cell subgroup (CD 56) + CD16 - NK bri Cell, CD56 - CD16 + NK dim Cells and CD56 - CD16 - NK uc The cell) can be used as an auxiliary diagnosis marker of hemophagocytic lymphocytosis, the detection sample is convenient to collect, the detection method is simple and quick, an operator does not need to be trained in a long and professional way, and the operator only needs to master a basic cell analysis technology, so that the method has the advantages of high practicability, easiness in popularization and the like.
Compared with the prior art, the invention has the following beneficial effects:
the invention discovers that compared with healthy volunteers, CD56 in peripheral blood of a patient suffering from hemophagocytic lymphocytosis by analyzing the peripheral blood sample of the patient suffering from hemophagocytic lymphocytosis + CD16 - NK bri Cell, CD56 - CD16 - NK uc The proportion of cells is obviously increased, and CD56 in peripheral blood of hemophagocytic lymphocytosis patient - CD16 + NK dim The proportion of cells is significantly reduced, and the difference is statistically significant. NK cell subset (CD 56) + CD16 - NK bri Cell, CD56 - CD16 + NK dim Cells and CD56 - CD16 - NK uc The cells) have better accuracy in diagnosing hemophagocytic lymphocytosis, and the NK cell subset provided by the invention is an early marker for diagnosing hemophagocytic lymphocytosis with good diagnostic effect, has high clinical application value, can guide clinical medication in time, and reduces the burden and pain of patients. The present invention provides NK cell subsets (CD 56) + CD16 - NK bri Cell, CD56 - CD16 + NK dim Cells and CD56 - CD16 - NK uc Cells) in diagnosis of HLH, can realize early diagnosis and early treatment of HLH.
Drawings
FIG. 1 is a schematic representation of flow gate analysis of different NK cell subsets;
FIG. 2 is a graph of statistical analysis of different NK cell subsets (wherein HDs stands for healthy volunteers; HLH stands for hemophagocytic lymphocytosis patients);
FIG. 3 is a graph of ROC curve analysis of different NK subgroups.
Detailed Description
The experimental procedure of the present invention, in which no specific conditions are noted in the following examples, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having" and any variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device.
Example 1
This example shows the use of flow cytometry on NK cell subsets (CD 56) in peripheral blood samples from healthy volunteers and hemophagocytic lymphocytosis patients + CD16 - NK bri Cell, CD56 - CD16 + NK dim Cells and CD56 - CD16 - NK uc Cells) is measured. The method comprises the following steps:
1. sample information sample sources:
1) Inclusion criteria for hemophagocytic lymphocytosis patients: all HLH patients met HLH-2004 diagnostic criteria, and the data provided were from pre-treated samples. Pretreatment of HLH is defined as corticosteroid treatment for less than 2 weeks without immunosuppressant against HLH. Samples were withdrawn 48 hours after the group entry for subsequent testing analysis.
2) Healthy volunteers (HDs) were included in the standard: 11 healthy human samples were also collected as control samples, both sex and age matched with the HLH patient group, and the study protocol of this example had been passed by the applicant's ethics committee. Clinical data for patients incorporated into the study are shown in table 1.
According to the inclusion criteria described above:
table 1: clinical information table for group-entered patients and health controls
2. Experimental method
1. Peripheral Blood Mononuclear Cell (PBMC) isolation
(1) Adding PBS solution with the volume of 3 times of that of blood into the collected peripheral blood sample, and uniformly mixing to obtain a diluted blood sample;
(2) Adding lymphocyte separation liquid with the same volume as the diluted blood sample into a centrifuge tube, slowly adding the diluted blood sample into the upper layer of the lymphocyte separation liquid along the tube wall of the centrifuge tube by using a straw, and centrifuging for 25min at 18 ℃ under 600g (5 g and 0 g);
(3) After centrifugation, a white film layer can be seen, the middle white film layer is carefully sucked out and placed in a clean centrifuge tube, 10ml of precooled PBS solution is added, and the mixture is uniformly mixed; centrifuging at 2000rpm/min for 5min at room temperature, discarding supernatant, and collecting cell precipitate;
(4) 3ml of erythrocyte lysate (ACK) is added into the image cell sediment, after 5min of room temperature lysis, 10ml of precooled PBS is added for stopping the lysis; centrifuging at room temperature of 2000rpm/min for 5min, discarding supernatant, and collecting cell precipitate;
(5) Cell pellet was resuspended in PBS and counted for later use.
2. Flow cytometry staining
(1) Adding 1million PBMC single cell suspension into a flow tube, adding 5ml PBS, mixing, centrifuging at 2000rpm/min for 5min, discarding the supernatant, and collecting cell precipitate;
(2) Flow cell antibodies were taken for analysis of human NK: anti-CD 3 (PE-Cyanine 7 Anti-Human CD3 (OKT 3)) antibody, anti-CD 14 (APC Anti-Human CD14 (M5E 2)) antibody, anti-CD 19 (PE-Cy) TM 5Anti-Human CD 19) antibodies, anti-CD 56 (PE/Dazzle) TM 594Anti-Human CD56 (NCAM)) antibodies and Anti-CD 16 (BV 786Anti-Human CD 16) antibodies, diluting the antibodies with PBS buffer at a ratio of 1:200 to obtain an antibody diluent (the antibodies are simultaneously added into the same centrifuge tube for dilution to obtain an antibody diluent);
(3) Respectively adding 100 μl of antibody diluent into the flow tube corresponding to each sample, placing the flow tube on a vortex oscillator for oscillation, fully mixing, and then dyeing at 4deg.C for 30min in the absence of light;
(4) 4ml of precooled PBS buffer was added to the flow tube, and then centrifuged at 2000rpm/min for 5min, and the supernatant was discarded; mu.l of pre-chilled PBS was added to resuspend the cells, which were then checked on-line using a flow analyser (BD Canto II, USA), data obtained and analysed using FlowJo10 software.
3. Statistical analysis
The median of each set of data was calculated using GraphPad 9.2.0 and the P value was calculated, with P < 0.05 being considered statistically significant.
3. Experimental results
All samples were analyzed, and reference was made to the analytical strategy of FIG. 1, to obtain NK cell subsets (CD 56 + CD16 - NK bri Cell, CD56 - CD16 + NK dim Cells and CD56 - CD16 - NK uc Cells) and to healthy stateNK cell subsets (CD 56) in peripheral blood of volunteers (HDs) and Hemophagocytic Lymphocytosis (HLH) + CD16 - NK bri Cell, CD56 - CD16 + NK dim Cells and CD56 - CD16 - NK uc Cells) and the results are shown in FIG. 2, CD56 in peripheral blood of Hemophagocytic Lymphocytosis (HLH) patients compared with healthy volunteers + CD16 - NK bri Cell, CD56 - CD16 - NK uc The proportion of cells is obviously increased, and CD56 in peripheral blood of hemophagocytic lymphocytosis patient - CD16 + NK dim The proportion of cells decreased significantly, the difference was statistically significant, suggesting a subpopulation of NK cells (CD 56 + CD16 - NK bri Cells, CD56-CD16 + NK dim Cells and CD56-CD16-NK uc Cells) can be used as potential diagnostic markers for Hemophagocytic Lymphocytosis (HLH).
As shown in FIG. 3, the diagnostic performance of NK cell subset ratio was further analyzed (ROC curve analysis), and the results showed that NK bri >5.495% when used to diagnose HLH, AUC 0.9437, sensitivity 90.48%, specificity 100%; NK (Natural killer) Dim <85.90 percent of the kit has better clinical predictive value when being used for diagnosing HLH, wherein AUC is 0.9827, sensitivity is 90.48 percent, specificity is 100 percent.
Taken together, the present invention demonstrates for the first time that NK cell subsets (CD 56 + CD16-NK bri Cells, CD56-CD16 + NK dim Cells and CD56-CD16-NK uc The cells) can be used as auxiliary diagnostic markers of Hemophagocytic Lymphocytosis (HLH), has high clinical application value, can guide clinical medication in time, reduces the related morbidity and mortality of the hemophagocytic lymphocytosis, and reduces the burden and pain of patients. The invention has the advantages of convenient sample collection, simple and quick detection method, no longer needs long and professional training for operators, only needs to master basic cell analysis technology, and has strong practicability, easy popularization and the like. NK fineCell subset (CD 56) + CD16-NK bri Cell, CD56 - CD16 + NK dim Cells and CD56 - CD16 - NK uc Cell) detection reagent can be used as an early diagnosis reagent of hemophagocytic lymphocytosis, and can be used for preparing an early diagnosis kit of hemophagocytic lymphocytosis so as to realize early diagnosis and early treatment.
The technical features of the above embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
- Use of a subpopulation of NK cells comprising CD56 for the preparation of a reagent for detection of hemophagocytic lymphocytosis + CD16 - NK bri Cell, CD56 - CD16 + NK dim Cells and CD56 - CD16 - NK uc At least one of the cells.
- Use of a subpopulation of NK cells comprising CD56 for the selection of a reagent for the detection of lymphocytophagy + CD16 - NK bri Cell, CD56 - CD16 + NK dim Cells and CD56 - CD16 - NK uc At least one of the cells.
- 3. Reagent for detecting NK cell subgroup content in biological sample for preparing hemophagocytosisUse of a product for detecting cytolymphocytosis, wherein said NK cell subset comprises CD56 + CD16 - NK bri Cell, CD56 - CD16 + NK dim Cells and CD56 - CD16 - NK uc At least one of the cells.
- 4. The use of claim 3, wherein the reagent is a flow cytometry detection reagent.
- 5. The use of claim 3, wherein the product comprises a kit.
- 6. The use of claim 3, wherein said CD56 + CD16 - NK bri Expression of cells was significantly up-regulated in hemophagocytic lymphocytosis patients, CD56 - CD16 + NK dim Cell expression was significantly down-regulated in hemophagocytic lymphocytosis patients, CD56 - CD16 - NK uc The expression of cells was significantly up-regulated in hemophagocytic lymphocytosis patients.
- 7. A kit for detecting hemophagocytic lymphocytosis is characterized by comprising a reagent for detecting the content of NK cell subpopulations in a biological sample, wherein the NK cell subpopulations comprise CD56 + CD16 - NK bri Cell, CD56 - CD16 + NK dim Cells and CD56 - CD16 - NK uc At least one of the cells.
- 8. The assay kit of claim 7, wherein the reagents for detecting the level of NK cell subpopulations in a biological sample comprise anti-CD 3 antibodies, anti-CD 14 antibodies, anti-CD 19 antibodies, anti-CD 56 antibodies and anti-CD 16 antibodies.
- 9. As claimed inThe detection kit of 8, wherein the Anti-CD 3 antibody is PE-Cyanine7 Anti-Human CD3 (OKT 3); the Anti-CD 14 antibody is APC Anti-Human CD14 (M5E 2); the anti-CD 19 antibody is PE-Cy TM 5Anti-Human CD19; the anti-CD 56 antibody is PE/Dazzle TM 594Anti-human CD56 (NCAM); the Anti-CD 16 antibody is BV786Anti-Human CD16.
- 10. The test kit of claim 7, further comprising peripheral blood mononuclear cell separation reagent, red blood cell lysate, and PBS buffer.
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| CN116515995A (en) * | 2023-06-29 | 2023-08-01 | 迈杰转化医学研究(苏州)有限公司 | Serum microRNA marker for detecting hemophagocytic lymphocytohyperplasia and application thereof |
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| CN116515995B (en) * | 2023-06-29 | 2023-09-19 | 迈杰转化医学研究(苏州)有限公司 | Serum microRNA marker for detecting hemophagocytic lymphocytohyperplasia and application thereof |
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