CN118330216B

CN118330216B - Antibody combination, kit and system for detecting peripheral blood mononuclear cell subpopulation

Info

Publication number: CN118330216B
Application number: CN202410462911.XA
Authority: CN
Inventors: 韦杰; 李海艳; 董苹; 王青; 王佳佳; 范昌军; 卞孟姣
Original assignee: JINAN KINGMED CENTER FOR CLINICAL LABORATORY CO LTD
Current assignee: Jinan Jinyu Medical Laboratory Co.,Ltd.
Priority date: 2024-04-17
Filing date: 2024-04-17
Publication date: 2024-11-05
Anticipated expiration: 2044-04-17
Also published as: CN118330216A

Abstract

The invention discloses an antibody combination, a kit and a system for detecting peripheral blood mononuclear cell subpopulations, wherein the antibody combination comprises the following components: CD36 antibodies, CD64 antibodies, CD14 antibodies, CD16 antibodies, and CD45 antibodies. The invention reasonably applies various antibody indexes, has wide coverage range, can rapidly, simply, conveniently and rapidly detect monocyte subpopulations with high sensitivity by adopting the antibody combination through a multiparameter flow detection technology, and can assist the clinical accurate diagnosis of chronic granulomonocytic leukemia.

Description

Antibody combination, kit and system for detecting peripheral blood mononuclear cell subpopulation

Technical Field

The invention belongs to the technical field of immunological detection, and particularly relates to an antibody combination, a kit and a system for detecting peripheral blood mononuclear cell subpopulations.

Background

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic malignancy, with the manifestations of both myeloproliferative neoplasms (MPNs) and myelodysplastic syndromes (MDS), commonly seen in elderly patients, with an incidence of about 4/10 ten thousand, reported survival for CMML patients ranging from 1 month to >100 months, in most reports with a median survival of 20-40 months, with about 15-30% of cases progressing to AML, with early identification and accurate diagnosis important for accurate treatment and prognosis of the patient.

Peripheral blood mononucleosis is a prominent feature of CMML, and the first prerequisite for definition of its diagnostic criteria in WHO version 2016 is the persistent increase in peripheral blood mononucleosis: the absolute value is more than or equal to 1 multiplied by 10 ⁹/L, the relative value is more than or equal to 10%, and on the basis, a plurality of other detection indexes are combined to confirm CMML. The international guidelines state that chronic granulo-monocytic leukemia peripheral blood mononuclear cell phenotypes are: by classical (MO 1), phenotype is defined as CD14 ^bright/CD16^-, the proportion is equal to or greater than 94%; intermediate (MO 2), phenotype defined as CD14 ^bright/CD16⁺, proportion < 20%; non-classical (MO 3), phenotype defined as CD14 ^dim/CD16⁺, proportion < 5%.

The analysis of peripheral blood mononuclear cell subpopulations was included in the auxiliary diagnostic criteria in the guidelines for diagnosis and treatment of chronic myelomonocytic leukemia (2021 edition) and indicated that the sensitivity of > 90%, specificity > 95% and MO3 monocyte depletion was of equal diagnostic value even with circulating MO1 cytopenia in CMML cases using a threshold of > 94% by typical (MO 1) monocyte percentages.

The diagnostic criteria for CMML in the latest 2022 WHO modified the requirements for peripheral blood mononuclear cells to: the absolute value is more than or equal to 0.5X10 ⁹/L and the relative value is more than or equal to 10%, and the abnormal grouping of the peripheral blood mononuclear cell subgroup is included in the support standard, so that the diagnostic value of the peripheral blood mononuclear cell subgroup in CMML is improved. It is seen that peripheral blood mononuclear cell subpopulation analysis is increasingly important for the assistance of CMML patients and clinical applications are becoming more and more widespread.

However, there is currently no clear indication in international guidelines as to how to circle total monocytes and which specific antibody combinations to use to determine the immunophenotype.

Disclosure of Invention

Based on the above, the invention aims to provide an antibody combination, a kit and a system for detecting peripheral blood mononuclear cell subpopulations, and the use of the antibody combination for detecting peripheral blood mononuclear cells can accurately classify the peripheral blood mononuclear cells, and has the advantages of high sensitivity, strong specificity, simplicity and convenience.

The technical scheme for realizing the aim of the invention comprises the following steps.

In a first aspect of the invention, there is provided an antibody combination for detecting a subpopulation of peripheral blood mononuclear cells, said antibody combination comprising: CD36 antibodies, CD64 antibodies, CD14 antibodies, CD16 antibodies, and CD45 antibodies.

In a second aspect, the invention provides the use of an antibody combination for detecting a subpopulation of peripheral blood mononuclear cells as described above in the preparation of a kit for aiding in the diagnosis of chronic myelomonocytic leukemia.

In a third aspect of the invention, there is provided a kit for detecting a subpopulation of peripheral blood mononuclear cells, comprising an antibody combination for detecting a subpopulation of peripheral blood mononuclear cells as described above.

In a fourth aspect of the invention, there is provided a system for detecting a subpopulation of peripheral blood mononuclear cells, comprising: the device comprises a detection module for detecting the flow cytometry of the peripheral blood mononuclear cells to be detected, a data acquisition module for acquiring the detection result of the flow cytometry, and a data analysis module for analyzing the detection result of the flow cytometry.

In a fifth aspect of the invention, there is provided a method of detecting a subpopulation of peripheral blood mononuclear cells, comprising the steps of: detecting single cell suspension of peripheral blood to be detected by using the antibody combination and the system, and sequentially analyzing the expression conditions of the antibodies on CD45-SSC, CD36-CD64, CD14-CD64 and CD16-CD14 and the proportions of three monocyte subtypes to obtain an antibody expression pattern of the peripheral blood to be detected; and then comparing the expression pattern of the antibodies with that of normal control population.

The invention has the following beneficial effects:

In the present invention, the inventors screened CD36 antibody, CD64 antibody, CD14 antibody, CD16 antibody and CD45 antibody as antibody combinations based on the study experience of chronic myelomonocytic leukemia peripheral blood mononuclear cells to detect peripheral blood mononuclear cells, the antibody combinations can recognize the target mononuclear cell group, and by analyzing the target mononuclear cell group by sequentially carrying out CD36-CD64, CD14-CD64, CD16-CD14 fluorescent antibody expression, the percentage of the three sub-populations of the typical mononuclear cell (MO 1), the intermediate mononuclear cell (MO 2) and the atypical mononuclear cell (MO 3) can be rapidly obtained, so that the determination of the normal or suspicious tumor mononuclear cell of the immunophenotype can be made according to the immunophenotype and the percentage of the three sub-populations. The invention reasonably applies various antibody indexes, has wide coverage range, can rapidly, simply and conveniently detect monocyte subpopulations (taking 1 case of clinical normal samples and 1 case of clinical abnormal samples as examples, verifying that the detection of peripheral blood mononuclear cells by adopting the antibody combination of the invention accords with clinical detection results) with high sensitivity by adopting the antibody combination through a multiparameter flow detection technology (only 1-2 hours is needed for receiving detection results from samples), and can assist the accurate diagnosis of clinical chronic granulomonocytic leukemia (CMML). In addition, the invention fixes the analysis template, the towed data can be directly analyzed, the operation is simple, the requirement of the expertise level of the analyst is reduced, and the limitation of the existing detection technology is made up.

Drawings

FIG. 1 is a plot of immunophenotype CD45-SSC expression scatter of monocyte subpopulations of clinical normal samples tested in example 3 of the present invention.

FIG. 2 is a plot of immunophenotype CD36-CD64 expression scatter of monocyte subpopulations of clinical normal samples tested in example 3 of the present invention.

FIG. 3 shows the immunophenotype CD14-CD64 expression of monocyte subpopulations of clinical normal samples tested in example 3 of the present invention.

FIG. 4 shows the immunophenotype CD16-CD14 expression of monocyte subpopulations of clinical normal samples tested in example 3 of the present invention.

FIG. 5 is a plot of immunophenotype CD45-SSC expression scatter of monocyte subpopulations of clinical abnormality samples tested in example 3 of the present invention.

FIG. 6 is a plot of immunophenotype CD36-CD64 expression scatter of monocyte subpopulations of clinical abnormality samples tested in example 3 of the present invention.

FIG. 7 shows the immunophenotype CD14-CD64 expression of monocyte subpopulations of clinical abnormality samples to be tested in example 3 of the present invention.

FIG. 8 shows the immunophenotype CD16-CD14 expression of monocyte subpopulations of clinical abnormality samples to be tested in example 3 of the present invention.

FIG. 9 is a plot of immunophenotype CD45-SSC expression scatter of monocyte subpopulations of clinical normal samples tested in comparative example 1 according to the present invention.

FIG. 10 shows the immunophenotype CD16-CD14 expression of monocyte subpopulations of clinical normal samples tested in comparative example 1 according to the present invention.

FIG. 11 is a plot of immunophenotype CD45-SSC expression scatter of monocyte subpopulations of clinical abnormality samples tested in comparative example 1 of the present invention.

FIG. 12 shows the immunophenotype CD16-CD14 expression of monocyte subpopulations of clinical abnormality samples to be tested in comparative example 1 of the present invention.

Detailed Description

The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

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 term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

The experimental procedures, which do not address the specific conditions in the examples below, are generally followed by conventional conditions, such as those described in Green and Sambrook et al, molecular cloning, an experimental guideline (Molecular Cloning: A Laboratory Manual, 2013), or by the manufacturer's recommendations. The various chemicals commonly used in the examples are commercially available.

In the present invention, in view of the deficiencies of the prior art, an antibody combination, kit and system for detecting a subpopulation of peripheral blood mononuclear cells is provided. The inventor finds that the accurate diagnosis of clinical CMML can be assisted by selecting the combination of the CD36 antibody, the CD64 antibody, the CD14 antibody, the CD16 antibody and the CD45 antibody and detecting peripheral blood mononuclear cells, namely, the percentage of three sub-groups of typical mononuclear (MO 1), intermediate mononuclear (MO 2) and atypical mononuclear (MO 3) can be detected rapidly, simply, conveniently and accurately. In the detection process, firstly, CD45-SSC (side scattered light) is used for gating (CD 45 is a leukocyte common antigen, so that the CD45 is used as gating antibody, the expression intensity of CD45 of normal blood cells is that of lymphocytes > monocytes > granulocytes, SSC is used as side scattered light, the normal expression intensity of the side scattered light is that of granulocytes > monocytes > lymphocytes, the expression characteristics of CD45 and SSC are combined, the lymphocyte, the monocyte and the granulocyte can be partitioned, wherein the lymphocyte is positioned at the position with the strongest CD45 and the lowest SSC, the granulocyte CD45 is weaker than the monocyte and the SSC signal is highest), the cell population is divided into granulocyte population, monocyte population and lymphocyte population according to the expression condition, and then the fluorescence expression intensity and the proportion of antibodies in target cells (namely the monocyte population) to CD36-CD64, CD14-CD64 and CD16-CD14 are analyzed sequentially (the total monocyte population can be identified through skeleton antibodies CD36 and CD 64), and the monocyte population expressed by CD36+CD64+ is circled; looking again at the expression of CD16 and CD14 to determine the proportion of the subpopulations, monocytes with phenotype CD14+/CD 16-were classical monocytes (MO 1), monocytes with phenotype CD14+/CD16+ were intermediate mononuclear (MO 2), and monocytes with phenotype CD14dim/CD16+ were non-classical mononuclear (MO 3), showing the percentage of the three subpopulations to total monocytes; finally, judging the result according to a preset analysis logic and a judgment standard, and judging the cells to be detected as normal monocyte subpopulations if the expression mode and percentage of the antibodies of the cells to be detected fall into the antibody expression mode template of the normal control crowd; if the antibody expression pattern and percentage of the cells to be detected do not fall into the antibody expression pattern template of the normal control population, the cells to be detected are judged to be suspected tumor cell groups.

In some embodiments of the invention, an antibody combination for detecting a subpopulation of peripheral blood mononuclear cells is disclosed, comprising: CD36 antibodies, CD64 antibodies, CD14 antibodies, CD16 antibodies, and CD45 antibodies.

In some of these embodiments, the antibody of the antibody combination is a monoclonal antibody.

In some embodiments, the monoclonal antibody is a fluorescein-labeled antibody, so that the indicators can be detected simultaneously in a ten-color or more flow cytometry, and the method has certain convenience; the luciferin is selected from: FITC, PE, ECD, APC and KO.

In some of these embodiments, the CD36 antibody labels fluorescein FITC; the CD64 antibody marks fluorescein PE; the CD14 antibody labels fluorescein ECD; the CD16 antibody labels fluorescein APC; the CD45 antibody marks fluorescein KO.

In other embodiments of the invention, a kit for detecting a subpopulation of peripheral blood mononuclear cells is disclosed, the kit comprising an antibody combination for detecting a subpopulation of peripheral blood mononuclear cells as described above.

In other embodiments of the invention, the use of an antibody combination as described above for detecting a subpopulation of peripheral blood mononuclear cells for the preparation of a kit for aiding in the diagnosis of chronic myelomonocytic leukemia (CMML) is disclosed.

In other embodiments of the invention, a system for detecting a subpopulation of peripheral blood mononuclear cells is disclosed, comprising: the detection module is used for detecting the flow cytometry of the peripheral blood mononuclear cells to be detected, the data acquisition module is used for acquiring the detection result of the flow cytometry and the data analysis module is used for analyzing the detection result of the flow cytometry.

In some of these embodiments, the detection template includes a single cell suspension module (to prepare a single cell suspension), an incubation module (to incubate the single cell suspension with an antibody in combination in the absence of light for antigen-antibody reaction), a resuspension module (to add hemolysin to the incubated cells, centrifuge, resuspension the cells after washing to prepare a suspension), and an assay module (to perform flow cytometry assays on the suspension after resuspension).

In other embodiments of the invention, a method of detecting a subpopulation of peripheral blood mononuclear cells is disclosed, comprising the steps of: detecting single cell suspension of the peripheral blood to be detected by utilizing the antibody combination and the system, and sequentially analyzing the expression conditions of the antibodies on CD45-SSC, CD36-CD64, CD14-CD64 and CD16-CD14 and the proportions of three monocyte subtypes to obtain an antibody expression pattern of the peripheral blood to be detected; and then comparing with the antibody expression pattern template of the normal control population.

In some of these embodiments, the normal control population antibody expression pattern template is established by the following method: obtaining flow cytometry detection result data of a normal control population cell population, grouping nucleated cells by gating, circling a target cell population (namely mononuclear cells), and analyzing the ratio of three subgroups MO1, MO2 and MO 3.

In some of these embodiments, the test peripheral blood cell antibody expression pattern is established by: obtaining flow cytometry detection result data of peripheral blood cells to be detected, classifying nucleated cells according to a mode in a normal control crowd antibody expression mode template, circling a target cell group (namely mononuclear cells), and analyzing the ratio of three subgroups MO1, MO2 and MO3 in the target cell group according to the mode in the normal control crowd antibody expression mode template.

The technical process of the system for detecting the peripheral blood mononuclear cell subpopulation of the embodiment is just to refer to the conventional technology of flow cytometry, and equipment and consumable materials for flow cytometry analysis are selected from the equipment and consumable materials such as a flow special pipe, an oscillator, a liquid transfer device and the like. Specific sources of the partial antibodies used in the following examples are: CD36FITC (cat No. IM0766U, beckmanCoulter), CD64 PE (cat No. IM3601U, beckmanCoulter), CD14 ECD (cat No. B92391, beckmanCoulter), CD16 APC (cat No. 360706, beckmanCoulter), CD45 KO (cat No. B36294, biolegend).

The invention is described in detail below with reference to the drawings and the specific embodiments.

Example 1 antibody combinations for detecting peripheral blood mononuclear cell subpopulations

The present example provides an antibody combination for detecting a subpopulation of peripheral blood mononuclear cells, including CD36 antibody, CD64 antibody, CD14 antibody, CD16 antibody, and CD45 antibody; the fluorescent labeling and the amount of the related monoclonal antibody in the above antibodies are shown in Table 1.

TABLE 1

Fluorescent markers

FITC

PE

ECD

APC

KO

Antibody labelling

CD36

CD64

CD14

CD16

CD45

Total dosage of antibody

Sample addition amount

5μl

2.5μl

22.5μl

Note that: the commercially available monoclonal antibodies were subjected to concentration gradient verification to determine the optimal amounts, and the amounts of monoclonal antibodies were packed in flow-type tubes of No. 1.

Example 2 System and method for detecting peripheral blood mononuclear cell subpopulations

The present embodiment provides a system for rapid detection of a subpopulation of peripheral blood mononuclear cells, comprising:

1. detection module

Performing flow cytometry detection on a test cell, comprising:

(1) A single cell suspension module for preparing single cell suspension;

(2) An incubation module for incubating the single cell suspension with the antibody of example 1 in the absence of light for antigen-antibody reaction;

(3) A re-suspension module for adding hemolysin into the incubated cells, centrifuging, and re-suspending the cells after washing to prepare suspension;

(4) The measuring module is used for carrying out flow cytometry measurement on the resuspended suspension;

2. Data acquisition module

Acquiring data of a flow cytometry detection result of a cell to be detected; the percentage of total monocytes can be obtained directly from the three sub-populations classical mononuclei (MO 1, phenotype defined as CD14+/CD16+), intermediate mononuclei (MO 2, phenotype defined as CD14+/CD16+), atypical mononuclei (MO 3, phenotype defined as CD14 dim/CD16+).

3. Data analysis module

Analyzing the obtained data, and judging whether the cells to be detected are suspicious tumor monocytes or not according to preset analysis logic and judgment standards, wherein if the antibody expression mode and percentage of the cells to be detected fall into the antibody expression mode template of normal control crowd, the cells to be detected are judged to be normal monocyte subgroups; if the antibody expression pattern and percentage of the cells to be detected do not fall into the antibody expression pattern template of the normal control population, the cells to be detected are judged to be suspected tumor cell groups.

The embodiment also provides a method for rapidly detecting the peripheral blood mononuclear cell subgroup, which adopts the system, and the specific workflow for detecting the peripheral blood mononuclear cell subgroup is as follows:

1. Each antibody in the antibody combination of example 1 was formulated as shown in table 1.

2. Sample processing

The cell number of the sample to be tested (peripheral blood) was adjusted to 1X 10 ⁶ cells/ml to prepare a single cell suspension.

3. Sample detection

(1) The flow tube was taken, labeled 1, and a set of antibodies from example 1 was added in an amount of 22.5. Mu.l (Table 1), followed by 100. Mu.l of the single cell suspension from step 2, vortexing and mixing, and incubation at room temperature for 15min in the absence of light.

(2) 400 Μl Bc hemolysin is added into the incubated flow tube 1, vortex shaking and standing until hemolysis is clear. After the hemolysis was clear, flow tube 1 was centrifuged at 1500r/min for 5min, the supernatant was discarded, 2ml calf serum was added, vortexed, centrifuged at 1500r/min for 5min, and the supernatant was discarded. 400 μl 1% paraformaldehyde was added to resuspend.

(3) Detecting the flow tube 1 by BeckmanCoulterNavios ten-color flow cytometry to obtain the number of the mononucleosis of CD36+CD64+which is more than or equal to 5000, and analyzing the immunophenotype and the proportion of the mononucleosis.

4. Data analysis

(1) Building a healthy human antibody expression pattern template

According to the flow cytometry detection result data of 40 normal control population cell groups, the cells are clustered by gating, preferably by gating with CD45-SSC (side scattered light), the target cell group is circled, and then the cell group is analyzed for the expression condition of each fluorescent antibody and the proportion of three monocyte subtypes, and the following antibody pairs are sequentially selected: CD45-SSC, CD36-CD64, CD14-CD64, CD16-CD14.

(2) Establishing antibody expression mode of cell to be tested

Obtaining flow cytometry detection result data of the cells to be detected, setting a gate to divide the cells into groups according to the mode in the normal control crowd antibody expression mode, circling a target cell group, and analyzing the expression condition of each fluorescent antibody in the target cell group according to the mode in the normal control crowd antibody expression mode to obtain the antibody expression mode of the cells to be detected.

(3) Analysis of antibody expression patterns in test cells

If the antibody expression pattern of the cell to be detected falls into the antibody expression pattern template of the normal control population, judging the cell to be detected as a monocyte group with normal immunophenotype;

if the antibody expression pattern of the cell to be detected does not fall into the antibody expression pattern template of the normal control population, the cell to be detected is judged to be suspicious tumor mononuclear cells.

Example 3 consistency verification of the test results and clinical diagnostic results of the test method of the present invention

The detection method of example 2 was used to detect monocytes of two samples of test cells (1 clinical normal sample+1 clinical abnormal sample) and to perform antibody expression pattern and subpopulation analysis to verify whether the detection method of the present invention is consistent with the clinical results.

1. Normal sample

The sample is a clinical sample with normal physical examination and normal blood normal monocyte content, and the antibody expression pattern analysis is carried out according to the following steps:

1. And obtaining flow cytometry detection result data of the sample to be detected. The cell population was classified into 3 regions (shown in FIG. 1) based on the expression of CD45-SSC using a gate for CD45-SSC (side scattered light), and 3 regions of granulocytes (upper middle region in FIG. 1), monocytes (upper right region in FIG. 1), and lymphocytes (lower right region in FIG. 1), respectively, and the monocyte region was the target cell population, and immunophenotype analysis was performed on the cell population.

2. The immunophenotype scatter diagram of CD36-CD64 of all nucleated cells is shown in FIG. 2, and it can be seen from the figure that CD36+CD64+ mononuclear cells are 7.97% in normal proportion, the obtained mononuclear cell number is 10094, and the cell number meets the requirement (more than or equal to 5000).

3. The scatter plots for CD14-CD64 expression for all nucleated cells are shown in FIG. 3, where CD36+CD64+ monocytes CD14 are positive and mature monocytes are seen.

4. The scatter plot of CD16-CD14 expression for the monocyte population is shown in FIG. 4, and it can be seen that by dividing the expression of CD16 and CD14 into three subgroups, the typical mononuclear (MO 1, phenotype is defined as CD14+/CD16+) accounts for 90.24% of monocytes, the intermediate mononuclear (MO 2, phenotype is defined as CD14+/CD16+) accounts for 7.61% of monocytes, and the non-classical mononuclear (MO 3, phenotype is defined as CD14 dim/CD16+) accounts for 0.54% of monocytes.

Therefore, the cell population of the monocyte subgroup of the sample to be tested is in the normal control population antibody expression pattern template, and the classical mononuclear (MO 1) < 94%, which indicates that the patient is non-chronic myelomonocytic leukemia (non-CMML), and is consistent with the clinical diagnosis result.

2. Abnormal sample

The sample is a peripheral blood specimen of a clinically well-diagnosed CMML patient. The antibody expression pattern analysis was performed as follows:

1. And obtaining flow cytometry detection result data of the sample to be detected. The cell population was classified into 3 regions according to the expression of CD45-SSC using a gate for CD45-SSC (side scattered light), and as shown in FIG. 5, granulocytes (upper middle region in FIG. 5), monocytes (upper right region in FIG. 5), and lymphocytes (lower right region in FIG. 5), and the monocyte region was the target cell population, and immunophenotype analysis was performed as follows.

2. The immunophenotype scatter diagram of CD36-CD64 of all nucleated cells is shown in FIG. 6, and the proportion of CD36+CD64+ mononuclear cells is 15.84% and is increased, so that the obtained mononuclear cell number is 15772, and the cell number meets the requirement (more than or equal to 5000).

3. The scatter plots for CD14-CD64 expression for all nucleated cells are shown in FIG. 7, where CD36+CD64+ monocytes CD14 are positive and mature monocytes are seen.

4. The scatter plot of CD16-CD14 expression for the monocyte population is shown in FIG. 8, and it can be seen that, based on CD16 and CD14 expression, it is classified into three subgroups, typically one nucleus (MO 1, phenotype is defined as CD14+/CD16+) constituting 98.39% of monocytes, an intermediate one nucleus (MO 2, phenotype is defined as CD14+/CD16+) constituting 0.48% of monocytes, and a non-classical one nucleus (MO 3, phenotype is defined as CD14 dim/CD16+) constituting 0.21% of monocytes.

The immunophenotype indicates that the cell population of the sample to be tested is not in the antibody expression pattern template of the normal control population, the proportion of classical mononuclear (MO 1) is 98.39 percent and is more than 94 percent, and the cell population of the sample to be tested indicates that the patient is chronic myelomonocytic leukemia (CMML) and accords with the clinical diagnosis result.

Therefore, by combining the results, the detection method is adopted to detect the clinical samples, the obtained detection result has consistency with the clinical result, and the antibody combination and the detection method have very good value for CMML diagnosis.

Comparative example detection of monocytes of test cells Using other antibody combinations

The mononuclear cells of the two test cells of example 3 were examined by the detection method of example 2 and analyzed for antibody expression pattern and subpopulation, except that the antibody combinations used were different.

1. Normal sample

The sample was a clinical sample with normal physical examination and normal blood normal monocyte content (same as in example 3), and the obtained data was analyzed for antibody expression pattern using antibody combinations of CD64-PE, CD14-ECD, CD16-APC, CD45-KO according to the following steps:

1. And obtaining flow cytometry detection result data of the sample to be detected. Gating with CD45-SSC (side scatter light), and circling the target cell population according to the expression of CD 45-SSC: total monocytes (upper right region of fig. 9), with a total monocyte fraction of 7.92% were seen, and the population of cells was immunophenotyped as follows.

2. The scatter plot of CD16-CD14 expression for the total monocyte population is shown in FIG. 10, and it can be seen that, based on CD16 and CD14 expression, it is classified into three subgroups, with a typical mononuclear (MO 1, phenotype defined as CD14+/CD16+) accounting for 75.61% of monocytes, an intermediate mononuclear (MO 2, phenotype defined as CD14+/CD16+) accounting for 10.80% of monocytes, and a non-classical mononuclear (MO 3, phenotype defined as CD14 dim/CD16+) accounting for 6.45% of monocytes.

The proportion of mononuclear cells in the sample tested was 75.61% by using this antibody combination, and although it was < 94% by typical mononuclear cell (MO 1), it was suggested that the patient was non-chronic myelomonocytic leukemia (non-CMML) and the clinical diagnostic result was consistent, but as shown in FIG. 10, 7.13% of the cell population in the L-region, which was not expressed by CD16/CD14, suggested that the total mononuclear cell population, which was circled with CD45-SSC only, could be mixed with other cells (e.g., partially smaller granulocytes and partially larger lymphocytes), and that the total mononuclear cells were not pure, could directly affect the proportion of the subpopulations, resulting in inaccurate results.

2. Abnormal sample

The sample was a peripheral blood specimen of a clinically established CMML patient (as in example 3). The data obtained were analyzed for antibody expression patterns using antibody combinations as CD64-PE, CD14-ECD, CD16-APC, CD45-KO as follows:

1. And obtaining flow cytometry detection result data of the sample to be detected. Gating with CD45-SSC (side scatter light), and circling the target cell population according to the expression of CD 45-SSC: total monocytes (upper right region of fig. 11) were found to be 15.75% in proportion, and this population of cells was immunophenotyped as follows.

2. The scatter plot of CD16-CD14 expression for the total monocyte population is shown in FIG. 12, and it can be seen that the typical mononuclear (MO 1, phenotype is defined as CD14+/CD16+) accounts for 85.24% of monocytes, the intermediate mononuclear (MO 2, phenotype is defined as CD14+/CD16+) accounts for 1.11% of monocytes, and the non-classical mononuclear (MO 3, phenotype is defined as CD14 dim/CD16+) accounts for 3.79% of monocytes, divided into three subgroups based on CD16 and CD14 expression.

The immunophenotype indicates that the proportion of classical mononuclear (MO 1) in the cell population of the sample to be tested in an antibody expression mode template of normal control population is 85.24 percent and less than 94 percent, and indicates that the patient is non-chronic myelomonocytic leukemia (CMML) and does not accord with the clinical diagnosis result.

Thus, combining the results of one and two, it was found that increasing CD36 antibody based on the CD45-SSC loop gate, using CD36-CD64 to round out the total monocyte population, was more accurate, ensuring a more accurate percentage of the subsequent mononuclear subpopulation analysis.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described 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.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. An antibody combination for detecting peripheral blood monocyte subsets, characterized in that the antibody combination consists of CD36 antibody, CD64 antibody, CD14 antibody, CD16 antibody and CD45 antibody.

2 . The antibody combination for detecting peripheral blood monocyte subsets according to claim 1 , wherein the antibodies in the antibody combination are monoclonal antibodies.

3. The antibody combination for detecting peripheral blood monocyte subsets according to claim 2, characterized in that the monoclonal antibody is a fluorescein-labeled antibody; the fluorescein is selected from: FITC, PE, ECD, APC and KO.

4. The antibody combination for detecting peripheral blood monocyte subsets according to claim 3, characterized in that the CD36 antibody is labeled with fluorescein FITC; the CD64 antibody is labeled with fluorescein PE; the CD14 antibody is labeled with fluorescein ECD; the CD16 antibody is labeled with fluorescein APC; and the CD45 antibody is labeled with fluorescein KO.

5. Use of the antibody combination for detecting peripheral blood mononuclear cell subsets according to any one of claims 1 to 4 in the preparation of a kit for auxiliary diagnosis of chronic myelomonocytic leukemia.

6. A kit for detecting peripheral blood monocyte subpopulations, characterized in that it comprises the antibody combination for detecting peripheral blood monocyte subpopulations according to any one of claims 1 to 4.

7. The kit for detecting peripheral blood monocyte subsets according to claim 6, characterized in that the dosage of the CD36 antibody, CD64 antibody, CD14 antibody and CD16 antibody is 4 to 6 μl, and the dosage of the CD45 antibody is 2 to 3 μl.

8. A method for detecting peripheral blood monocyte subpopulations, characterized in that it comprises the following steps: using the antibody combination described in any one of claims 1 to 4 to detect a single cell suspension of peripheral blood to be tested, sequentially analyzing the expression of antibodies to CD45-SSC, CD36-CD64, CD14-CD64 and CD16-CD14 and the ratio of the three monocyte subtypes to obtain the antibody expression pattern of the peripheral blood to be tested; and then comparing the antibody expression pattern with that of a normal control population.