CN113933511A - Antibody composition and method for detecting minimal residues of acute B lymphocyte leukemia - Google Patents
Antibody composition and method for detecting minimal residues of acute B lymphocyte leukemia Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
Abstract
The invention discloses an antibody composition and a method for detecting acute B lymphocyte leukemia minimal residual, wherein the antibody composition comprises an anti-CD 19 antibody, an anti-CD 10 antibody, an anti-CD 34 antibody and an anti-CD 22 antibody. The invention can realize that the detection of 17 indexes can be finished by only one tube, the sample amount requirement is less, the skeleton antibody does not need to be reused, the reagent cost is low, the designed 17 detection indexes cover the expression modes of various abnormal cells, the detection rate of leukemia tiny residual cells is improved, and the invention not only can be used for detecting tiny residual focuses after treatment, but also can be used for screening the tumor markers of patients in initial diagnosis.
Description
Technical Field
The invention belongs to the technical field of biomedical detection, and particularly relates to an antibody composition, a kit and a detection method for detecting acute B lymphocyte leukemia minimal residual.
Background
Acute Lymphoblastic Leukemia (ALL) is a clonal proliferative disease with malignant lymphocytes in early differentiation stage, and is also a group of highly heterogeneous diseases, wherein the incidence rate of the acute lymphoblastic leukemia (B-ALL) accounts for about 85% of the acute lymphoblastic leukemia, and is mainly characterized by abnormal proliferation of primitive and juvenile myeloid cells in bone marrow and peripheral blood, clinically manifested as anemia, hemorrhage, infection and fever, organ infiltration, metabolic abnormality and the like, and most cases are acute and serious, and the early and late cases are dangerous, and the life can be threatened if the treatment is not performed in time.
At present, the complete remission rate of leukemia is greatly improved by combined chemotherapy, but leukemia relapse is a main problem of the current leukemia treatment, and the source of relapse mainly comes from residual leukemia cells in the body. Generally, the state in which a small amount of leukemia cells remain in vivo after the treatment remission of such leukemia is defined as Minimal Residual Disease (MRD).
Clinically, the treatment scheme and the dosage are adjusted according to the level of MRD (total residual metal deficiency) so as to achieve the aim of curing. Therefore, it is of great clinical value whether MRD can be accurately measured. At present, the method can be used for detecting the minimal residual of the acute lymphocytic leukemia by several methods such as cell morphology, chromosome karyotype analysis, molecular biology related gene detection, multi-parameter flow cytometry analysis and the like.
The sensitivity of cell morphology analysis is low, residual tumor cells after treatment are possibly few, and the morphology has certain limitation; the period of the karyotype analysis experiment is long; although the molecular biological method has high sensitivity, it cannot be used in cases with normal karyotype. The method for detecting the tiny residues based on the leukemia related immunophenotyping multiparameter Flow Cytometry (FCM) has the characteristics of rapidness, simplicity and convenience, and is a currently accepted effective method for detecting the tiny residues.
The analysis method for detecting residual leukemia cells by using multi-parameter flow cytometry is mainly used for monitoring leukemia-associated immunophenotype (LAIP) or 'expression pattern different from normal' (DFN), the LAIP method needs to carry out screening of indexes in initial diagnosis immunophenotyping, otherwise, missed diagnosis is easy, and the DFN method has high requirements on the experience and professional knowledge level of analysts. The multi-parameter flow cytometry can be used for detection by using a traditional flow cytometer, but is influenced by a detection channel, a detection scheme with 6-10 colors is frequently used at present, each sample needs to be subjected to multiple different detections, not only is the skeleton antibody repeatedly detected, but also the required sample amount is large, and the sensitivity is not high.
The key point of detecting residual leukemia cells by using multi-parameter flow cytometry is to identify normal differentiation precursor cells and abnormal leukemia cells, and only one single leukocyte differentiation antigen is difficult to be used as an immune marker for detecting the specificity of minimal residual disease, so that the combination of antigens differentially expressed in acute lymphoblastic leukemia cells and normal bone marrow cells is mostly adopted for cell typing and classification at present.
In the prior researches, MRD detection generally uses a cell membrane pan B cell marker CD19 assisted by SSC or CD45(CD19/SSC or CD45/CD19 double parameters) to gate and find target B cells, but for a patient treated by CD19-CAR-T cell infusion, partial normal or abnormal B cell surface CD19 antigen is lost or weakened after treatment, and immune escape occurs, so that missed diagnosis caused by the reduction or loss of CD19 expression can be generated. At present, immune markers with high sensitivity and specificity are still lacking for efficient and accurate detection of minimal residual levels.
In light of the above, there is a clinical need for a detection technique with high sensitivity, high accuracy, high detectable rate, simplicity and rapidity, and for monitoring minute residual lesions of acute B-lymphocyte leukemia.
Disclosure of Invention
Based on the above, one of the objectives of the present invention is to provide an antibody composition for detecting minimal residual acute B-lymphoblastic leukemia, wherein the detection index of the antibody composition covers the expression patterns of various abnormal cells of acute B-lymphoblastic leukemia, and can overcome the defect of easy missed diagnosis.
The specific technical scheme for realizing the aim of the invention is as follows:
an antibody composition for detecting minimal residual acute B-lymphocyte leukemia, the antibody composition comprising: anti-CD 19 antibodies, anti-CD 10 antibodies, anti-CD 34 antibodies, and anti-CD 22 antibodies.
In some of these embodiments, the antibody composition further comprises an anti-CD 45 antibody, an anti-CD 20 antibody, an anti-CD 24 antibody, an anti-CD 38 antibody, an anti-CD 81 antibody, an anti-CD 58 antibody, an anti-CD 13 antibody, an anti-CD 33 antibody, an anti-CD 66c antibody, an anti-CD 73 antibody, an anti-CD 123 antibody, and an anti-CD 86 antibody.
In some of these embodiments, the anti-CD 19 antibody, anti-CD 10 antibody, anti-CD 34 antibody, anti-CD 20 antibody, anti-CD 45 antibody, anti-CD 22 antibody, anti-CD 24 antibody, anti-CD 38 antibody, anti-CD 81 antibody, anti-CD 58 antibody, anti-CD 13 antibody, anti-CD 33 antibody, anti-CD 66c antibody, anti-CD 73 antibody, anti-CD 123 antibody, and anti-CD 86 antibody have the clone numbers in the order: j3-119, H10a, 581, 2H7, H130, S-HCL-1, ML5, HIT2, JS-81, AICD58, L138, D3HL60.251, B6.2/CD66, AD2, 9F5 and 2331.
In some embodiments, the antibodies are fluorescein-labeled antibodies, and the fluorescein labeled antibodies of the anti-CD 19 antibody, anti-CD 10 antibody, anti-CD 34 antibody, anti-CD 20 antibody, anti-CD 45 antibody, anti-CD 22 antibody, anti-CD 24 antibody, anti-CD 38 antibody, anti-CD 81 antibody, anti-CD 58 antibody, anti-CD 13 antibody, anti-CD 33 antibody, anti-CD 66c antibody, anti-CD 73 antibody, anti-CD 123 antibody, and anti-CD 86 antibody are, in that order: PC7, BV786, ECD, BV421, BV510, APC-CY7, PC5.5, BV480, FITC, PE, BV650, BV605, BV750, and V450.
In some embodiments, the anti-CD 19 antibody, anti-CD 10 antibody, anti-CD 34 antibody, anti-CD 20 antibody, anti-CD 45 antibody, anti-CD 22 antibody, anti-CD 24 antibody, anti-CD 38 antibody, anti-CD 81 antibody, anti-CD 58 antibody, anti-CD 13 antibody, anti-CD 33 antibody, anti-CD 66c antibody, anti-CD 73 antibody, anti-CD 123 antibody, and anti-CD 86 antibody have titers of 2.5 μ L, 0.625 μ L, 5 μ L, 1.25 μ L, 0.625 μ L, 4 μ L, 2.5 μ L, 2.25 μ L, 2.5 μ L, 4 μ L, respectively.
In some of these embodiments, the antibody is a monoclonal antibody.
The invention also provides application of the antibody composition in preparation of a kit for detecting acute B lymphocyte leukemia minimal residual.
The invention also provides a kit for detecting the acute B lymphocyte leukemia minimal residual, and the kit can be used for detecting the acute B lymphocyte leukemia minimal residual, and is rapid, sensitive and accurate.
The specific technical scheme for realizing the aim of the invention is as follows:
a kit for detecting minimal residual acute B lymphocyte leukemia, which comprises the antibody composition.
In some of these embodiments, the kit further comprises a nucleic acid dye 7-AAD.
In some embodiments, the kit further comprises hemolysin, a washing solution and a fixing solution, wherein the washing solution is a PBS solution containing 0.8-1.2% fetal bovine serum, and the fixing solution is a PBS solution containing 0.8-1.2% paraformaldehyde.
The invention also provides a method for detecting the acute B lymphocyte leukemia tiny residue, the method can realize the detection of 17 indexes only by one tube, and the sample size requirement is less, sensitive and rapid.
The specific technical scheme for realizing the aim of the invention is as follows:
a method for detecting minimal residual acute B-lymphocyte leukemia, said method obtaining the expression pattern and intensity of each fluorescein-labeled antibody of non-diagnostic interest, comprising the steps of:
(1) adding nucleic acid dye 7-ADD and fluorescein labeled antibody composition into flow tube, and adding the mixture containing 1 × 108～5×108Uniformly mixing single cell suspensions of sample cells to be detected, and incubating for 15-20 minutes at room temperature in a dark place;
the fluorescein-labeled antibody composition is: fluorescein PC7, BV786, ECD, BV421, BV510, APC-CY7, PC5.5, BV480, FITC, PE, BV650, BV605, BV750, and V450 labeled anti-CD 19 antibody, anti-CD 10 antibody, anti-CD 34 antibody, anti-CD 20 antibody, anti-CD 45 antibody, anti-CD 22 antibody, anti-CD 24 antibody, anti-CD 38 antibody, anti-CD 81 antibody, anti-CD 58 antibody, anti-CD 13 antibody, anti-CD 33 antibody, anti-CD 66c antibody, anti-CD 73 antibody, anti-CD 123 antibody, and anti-CD 86 antibody are used in sequence;
the titer of the antibody is 2.5 muL, 0.625 muL, 5 muL, 1.25 muL, 0.625 muL, 4 muL, 2.5 muL, 5 muL, 2.5 muL, 1.25 muL, 2.5 muL and 4 muL in sequence;
(2) adding 380-420 mul of hemolysin into a flow tube, uniformly mixing, standing in a dark place, centrifuging and discarding the supernatant after hemolysis is transparent, washing with PBS (phosphate buffer solution) containing 0.8-1.2% fetal calf serum, centrifuging and discarding the supernatant, and resuspending cells with PBS containing 0.8-1.2% paraformaldehyde to obtain cell suspension of a sample to be detected;
(3) detecting the cell suspension of the sample to be detected in the step (2) by using a full-spectrum flow cytometer, and circling out a target cell group in a cell scatter diagram of the sample to be detected; the gate setting mode of the full-spectrum flow cytometer is as follows: removing dead cells, fragments and adherent cells in sequence, gating by using an anti-CD 45 antibody-SSC, an anti-CD 19 antibody-anti-CD 10 antibody, an anti-CD 19 antibody-anti-CD 34 antibody, an anti-CD 22 antibody-anti-CD 34 antibody and an anti-CD 22 antibody-anti-CD 10 antibody, and accurately delineating a target cell population, namely original B cells and immature B cells by using a two-parameter scatter diagram;
(4) analyzing the expression pattern and the intensity of each fluorescein-labeled antibody in the target cell population of the sample to be detected, comparing the expression pattern and the intensity with an expression pattern template of a normal control B progenitor cell antibody, and detecting whether each fluorescein-labeled antibody of the sample to be detected falls on the expression pattern template of the normal control B progenitor cell antibody;
the method for establishing the expression pattern template of the normal control B progenitor cell antibody comprises the following steps: detecting cell suspensions of 20-40 normal control samples by using a full-spectrum flow cytometer, circling out B progenitor cells in a cell scatter diagram of the normal control sample, analyzing the expression pattern and the intensity of each fluorescein labeled antibody in the B progenitor cells of the normal control sample to obtain the expression pattern of the normal cells, and establishing an expression pattern template of a normal control B progenitor cell antibody; the gate setting method of the full-spectrum flow cytometer is as described in step (3).
In some embodiments, the fluorescein labeled antibody in step (4) is one or more of the pair of anti-CD 19 antibody-anti-CD 34 antibody, anti-CD 19-antibody CD38, anti-CD 19 antibody-anti-CD 13 antibody, anti-CD 19 antibody-anti-CD 33 antibody, anti-CD 19 antibody-anti-CD 58 antibody, anti-CD 19 antibody-anti-CD 66c antibody, anti-CD 19 antibody-anti-CD 123 antibody, anti-CD 19 antibody-anti-CD 81 antibody, anti-CD 19 antibody-anti-CD 86 antibody, anti-CD 19 antibody-anti-CD 73 antibody, anti-CD 19 antibody-anti-CD 20 antibody, anti-CD 19 antibody-anti-CD 22 antibody, anti-CD 19 antibody-anti-CD 24 antibody.
In some of these embodiments, the centrifugation in step (2) is a 500g centrifugal force, centrifugation 5 min.
Compared with the prior art, the invention has the following beneficial effects:
(1) the antibody composition for detecting the acute B lymphocyte leukemia tiny residue at least comprises an antibody composition consisting of an anti-CD 19 antibody, an anti-CD 10 antibody, an anti-CD 34 antibody and an anti-CD 22 antibody, and the four antibodies, namely the anti-CD 19 antibody, the anti-CD 10 antibody, the anti-CD 34 antibody and the anti-CD 22 antibody, are used as mark skeleton antibodies for gating, so that target cells can be accurately locked, the influence of the immune escape of the target cells of CAR-T targeted therapeutic drugs on detection results can be effectively avoided, and the phenomenon of missed diagnosis caused by CAR-T targeted therapy can be prevented.
(2) The antibody composition for detecting the minimal residual acute B lymphocytic leukemia of the invention also comprises 12 antibodies and a nucleic acid dye 7-AAD, and the 17 detection indexes cover the expression patterns of various abnormal cells of the acute B lymphocytic leukemia, such as over-expression (observing the expression of CD58 antigen, CD66c antigen, CD73 antigen, CD123 antigen, CD86 antigen), deletion expression (observing the expression of CD38 antigen, CD81 antigen, CD24 antigen), cross-lineage expression (observing the expression of CD13/CD33 antigen, because the CD13/CD33 antigen belongs to a common myeloid lineage antigen and should not be expressed in a B cell lineage), and spatio-temporal disorganization expression (observing the expression of CD20 antigen and CD34 antigen); the antibodies are matched with the antibody of specific fluorescein respectively, and different antibodies are matched with the specific fluorescein, so that when the method is applied to flow cytometry for detecting the minimal residues of the acute B lymphocyte leukemia, all the fluorescein in each channel can achieve excellent dyeing effect.
(3) The method for detecting the acute B lymphocyte leukemia tiny residue by using the full-spectrum flow cytometer can realize that the detection of 17 clinical high-frequency generation indexes can be completed simultaneously by only one tube, the sample amount requirement is less, the skeleton antibody does not need to be reused, the reagent cost is low, the detection rate of the leukemia tiny residue cells is improved, and the defects that the prior art has no unified standard and is easy to miss diagnosis are effectively overcome; the standard detection method and analysis process can effectively improve the accuracy of MRD detection, and the detection sensitivity is further improved to 10 by increasing the number of cells obtained from a single tube sample from 50 ten thousand to 500 ten thousand-5(sensitivity is mainly influenced by cell number, the traditional flow cytometer has limited detection channels, if the same many indexes are detected, the detection needs to be carried out by separating a plurality of tubes, so that the cell number of each tube is small, the full-spectrum flow cytometer can complete the detection only by one tube, all collected samples are used in one tube, and the cell number can be obtained is large), so that the method can be used for detecting tiny residual focuses after treatment, can also be used for screening the neoplastic markers of patients in initial diagnosis, and provides a basis for clinical diagnosis and treatment.
Drawings
FIG. 1 is a 7AAD-SSC two-parameter scattergram in example 3 of the present invention.
FIG. 2 is a FSC-SSC two-parameter scattergram in example 3 of the present invention.
FIG. 3 is a FSC-A-FSC-H two-parameter scattergram in example 3 of the present invention.
FIG. 4 shows the distribution of lymphocytes, monocytes, granulocytes, weakly positive CD45 and negative CD45 after gating with anti-CD 45 antibody-SSC in example 3 of the present invention.
FIG. 5 shows the desired cell population found in example 3 of the present invention.
FIGS. 6 to 17 are graphs showing the expression intensity of each fluorescent antibody in the target cell population of the test sample in example 3 of the present invention.
FIG. 18 is a 7AAD-SSC two-parameter scattergram in example 4 of the present invention.
FIG. 19 is a FSC-SSC two-parameter scattergram in example 4 of the present invention.
FIG. 20 is a FSC-A-FSC-H two-parameter scattergram in example 4 of the present invention.
FIG. 21 shows the distribution of lymphocytes, monocytes, granulocytes, weakly positive CD45 and negative CD45 after gating with anti-CD 45 antibody-SSC in example 4 of the present invention.
FIG. 22 shows the desired cell population found in example 4 of the present invention.
FIGS. 23 to 34 are graphs showing the expression intensity of each fluorescent antibody in the target cell population of the test sample in example 4 of the present invention.
FIG. 35 is a graph showing the sensitivity analysis of the conventional flow-type detection method.
FIG. 36 is a graph showing the operation of the sensitivity analysis in example 5 of the present invention.
FIG. 37 is a FSC-SSC two-parameter scatterplot of comparative example 1.
FIG. 38 is a FSC-A-FSC-H two-parameter scattergram of comparative example 1.
FIG. 39 is a graph showing the distribution of lymphocytes, monocytes, granulocytes, CD45 weakly positive and CD45 negative after gating with anti-CD 45 antibody-SSC in comparative example 1.
FIG. 40 is a drawing of the cell population of interest found in comparative example 1.
FIGS. 41 to 45 are graphs showing the expression intensity of each fluorescent antibody in the target cell population of the test sample in the comparative example.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. The present 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. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Unless otherwise specified, this can be done according to protocols familiar to those skilled in the art and listed in the references cited herein. Wherein, the used reagent raw materials are all commercial products and can be purchased and obtained through public channels.
In the present invention, antibodies per se are well known to those skilled in the art, which specifically bind (anti-) the corresponding antigen. The antibody may be a monoclonal antibody or a polyclonal antibody, and in the embodiment of the present invention, a monoclonal antibody is preferred. The antibody may be of murine or rabbit origin, etc. In the present invention, antigens of each antibody, i.e., each cell surface antigen (CD19, CD10, CD34, CD20, CD45, CD22, CD24, CD38, CD81, CD58, CD13, CD33, CD66c, CD73, CD123, and CD86), are human cell surface antigens.
The present invention will be described in further detail with reference to the following specific embodiments and the accompanying drawings.
Example 1 antibody composition for detecting minimal residual acute B-lymphoblastic leukemia
An antibody composition for detecting minimal residual acute B-lymphoblastic leukemia of this example comprises (each antibody is named by antigen and fluorescent label): CD19-PC7, CD10-BV786, CD34-ECD, CD20-BV421, CD45-BV510, CD22-APC, CD24-APC-CY7, CD38-PC5.5, CD81-BV480, CD58-FITC, CD13-PE, CD33-PE, CD66c-BV650, CD73-BV605, CD123-BV750 and CD 86-V450. All of these antibodies having fluorescein attached thereto were purchased through public channels, and the product numbers and the manufacturers of the antibodies of the examples of the present invention are shown in Table 1.
Commercially available monoclonal antibodies as described above were sampled separately and subjected to concentration gradient verification to determine the optimum amount of each antibody, as shown in table 1.
TABLE 1 antibody information and amounts
Example 2 kit for detecting minimal residual acute B-lymphocyte leukemia
The kit for detecting the minimal residual acute B lymphocyte leukemia of the embodiment comprises:
(1) the antibody composition of example 1, wherein the titer of each antibody in the antibody composition is shown in table 1.
(2) Nucleic acid dye 7-AAD
(3) Hemolysin (H-alpha
(4) Washing solution (PBS + 1% fetal bovine serum)
(5) Fixing solution (PBS + 1% paraformaldehyde)
(6) And a flow tube used in cooperation with the full-spectrum flow cytometer.
Example 3 method for detecting minimal residual acute B-lymphocytic leukemia
The kit of example 2 was used to detect minimal residual of acute B lymphocyte leukemia in bone marrow sample cells (from gold field medical flow cytometry laboratories, bone marrow aspirate collected from treated B-ALL patients, heparin added for anticoagulation). The method specifically comprises the following steps:
1. establishing an expression pattern template of a normal control antibody
(1) The nucleic acid dye 7-ADD and the fluorescein-conjugated monoclonal antibody used in the amount shown in Table 1 in example 1 were added to a flow tube.
(2) The concentrations of the bone marrow cells (bone marrow of non-B-ALL patients, sample containing B progenitor cells, derived from gold field medical flow cytometry laboratory) of 40 normal control populations were adjusted to 1X 108～5×108At each ml, 40 normal control single cell suspensions were prepared.
(3) And adding 100 mul of normal control single cell suspension into the flow tube, vortexing, shaking and mixing uniformly, and incubating for 15min at room temperature in a dark place.
(4) After incubation, adding 400 mu l of hemolysin into the flow tube, carrying out vortex oscillation and standing in a dark place, after hemolysis is transparent, centrifuging the flow tube for 5min by using 500g of centrifugal force, discarding the supernatant, adding 2ml of washing solution (PBS + 1% fetal calf serum), carrying out vortex oscillation, 500g of centrifugal force, centrifuging for 5min, discarding the supernatant, and then adding 400 mu l of fixing solution (PBS + 1% paraformaldehyde) to resuspend cells to obtain a cell suspension for normal control.
(5) Using a CYTEK-NL-CLC full spectrum flow cytometer (instrument model:
NL-CLCV 16B 14R8(SN: NL-1044)) detects cell suspensions of 40 normal controls, 500-1000 ten thousand cell populations are obtained, 40 normal control B progenitor cells are circled, the expression pattern and the intensity of each fluorescein labeled antibody in the 40 normal control sample B progenitor cells are analyzed, the expression pattern of normal cells is obtained, and an expression pattern template of normal control B progenitor cell antibodies is established;
the gate setting mode of the full-spectrum flow cytometer is as follows:
(a) removing dead cells by using a 7AAD-SSC two-parameter scatter diagram;
(b) removing debris by using an FSC (Forward scatter) -SSC (Forward scatter) -two-parameter scattergram;
(c) using FSC-A (area, A) -FSC-H (height, H) two-parameter scatter diagram to remove adherent cells;
(d) each of the hematocytes gates was set using anti-CD 45 antibody-SSC, and lymphocytes, monocytes, granulocytes, and the presence or absence of significant tumor cells or abnormal cells were roughly observed. According to the expression of anti-CD 45 antibody-SSC, the cell population can be divided into 5 regions, namely 5 regions of granulocyte (Gran), monocyte (Mono), lymphocyte (Lym), CD45 weak positive (CD45 DIM) and CD45 negative (CD45 neg);
(e) b progenitor cells were gated using anti-CD 19 antibody-anti-CD 10 antibody, anti-CD 19 antibody-anti-CD 34 antibody, anti-CD 22 antibody-anti-CD 34 antibody, and anti-CD 22 antibody-anti-CD 10 antibody, and the two-parameter scattergram was used to accurately circle out B progenitor cells.
2. Detecting and analyzing antibody expression pattern of sample cell to be detected
The steps (1) to (4) are the same as above, wherein the concentration of the product obtained in the step (2) is 1X 108～5×108Single cell suspension of individual/ml bone marrow sample cells to be tested.
(5) Detecting the cell suspension of the sample to be detected by using a CYTEK-NL-CLC full-spectrum flow cytometer to obtain 500-1000 ten thousand cell groups, and circling out the target cell group in the sample scatter diagram to be detected;
the gating method and results of the full spectrum flow cytometer are as follows:
(a) dead cells were removed using a 7AAD-SSC two-parameter scatter plot (figure 1);
(b) debris removal using FSC (Forward scatter) -SSC two parameter scattergram (figure 2);
(c) using FSC-a (area, a) -FSC-H (height, H) two-parameter scattergram to remove adherent cells (fig. 3);
(d) each of the hematocytes gates was set using anti-CD 45 antibody-SSC, and lymphocytes, monocytes, granulocytes, and the presence or absence of significant tumor cells or abnormal cells were roughly observed. According to the expression of the anti-CD 45 antibody-SSC, the cell population can be divided into 5 regions (fig. 4), which are 5 regions of granulocyte (Gran) (middle upper region in fig. 4), monocyte (Mono) (upper right region in fig. 4), lymphocyte (Lym) (lower right region in fig. 4), CD45 weak positive (CD45 DIM) (middle lower region in fig. 4), CD45 negative (CD45 neg) (left region in fig. 4); b progenitor cells are generally located within the CD45 DIM region; whereas tumor cells may be present in CD45 DIM or in CD45 negative regions.
(e) And using anti-CD 19 antibody-anti-CD 10 antibody, anti-CD 19 antibody-anti-CD 34 antibody, anti-CD 22 antibody-anti-CD 34 antibody and anti-CD 22 antibody-anti-CD 10 antibody to set a gate, and accurately circling out the target cell population, namely, the original B cells and the naive B cells by using the two-parameter scatter diagram.
In this example, the target cell population of the test sample is CD19+ CD10+ cells, which are located in the CD45 DIM region at a ratio of 16.83% (fig. 5).
(6) Analyzing the expression intensity of each fluorescent antibody in the target cell population of the test sample (preferably, the following antibody pairs: anti-CD 19 antibody-anti-CD 34 antibody, anti-CD 19-antibody CD38, anti-CD 19 antibody-anti-CD 13 antibody, anti-CD 19 antibody-anti-CD 33 antibody, anti-CD 19 antibody-anti-CD 58 antibody, anti-CD 19 antibody-anti-CD 66c antibody, anti-CD 19 antibody-anti-CD 123 antibody, anti-CD 19 antibody-anti-CD 81 antibody, anti-CD 19 antibody-anti-CD 86 antibody, anti-CD 19 antibody-anti-CD 73 antibody, anti-CD 19 antibody-anti-CD 20 antibody, anti-CD 19 antibody-anti-CD 22 antibody, anti-CD 19 antibody-anti-CD 24 antibody) and comparing the expression pattern template of the normal control B cell antibody established in the step 1, if each fluorescent antibody in the target cell population of the test sample is on the expression pattern template of the normal control B progenitor B cell antibody, the cell population is then a normal cell population, whereas a tumor cell is suspected.
The results are shown in FIGS. 6 to 17.
As can be seen from FIGS. 6 to 17, the expression patterns of the antibodies in the cell population of the test sample fall into the expression pattern template of the antibody in the normal control B progenitor cell, each antibody has a normal expression pattern, and the majority of the primitive B cells and the naive B cells fall within the gates of the expression pattern template in the normal control, which indicates that the cells are normal primitive B cells and naive B cells, and the immunophenotypes thereof are CD58-, CD33-, CD13-, CD20-, CD22+, CD34+, a small amount, CD123-, CD24+, CD38+, CD81+, CD66c-, CD73-, and CD86-, and the expression intensities and the expression patterns thereof are normal. Namely, the target cell population of the test sample is determined to be a cell population with normal immunophenotype in the antibody expression pattern template of the normal control B progenitor cells.
By adopting the morphological analysis of a bone marrow smear, no obvious residual tumor cells are seen, which indicates that the bone marrow is completely relieved and is consistent with the results.
Example 4 method for detecting minimal residual acute B-lymphocytic leukemia
The method for detecting the minimal residual acute B lymphocyte leukemia by using the kit for detecting the minimal residual acute B lymphocyte leukemia in the embodiment 2 and by using a full spectrum flow cytometer to detect the minimal residual acute B lymphocyte leukemia of the bone marrow sample cells to be detected (abnormal cells, namely bone marrow puncture substances of patients confirmed to be B-ALL and heparin anticoagulated during collection, which are from gold field medical flow cytometry laboratories) comprises the following steps:
steps (1) to (4) were the same as steps (1) to (4) in example 3, and resuspended cells in the sample to be tested were obtained.
(5) Detecting the cell suspension of the sample to be detected by using a CYTEK-NL-CLC full-spectrum flow cytometer to obtain 500-1000 ten thousand cell groups, and circling out the target cell group in the sample scatter diagram to be detected;
the gating method and results of the full spectrum flow cytometer are as follows:
(a) dead cells were removed using a 7AAD-SSC two-parameter scattergram (fig. 18);
(b) debris was removed using FSC (Forward scatter) -SSC two parameter scattergram (figure 19);
(c) adherent cells were removed using an FSC-A (area, A) -FSC-H (height, H) two-parameter scattergram (FIG. 20);
(d) each of the hematocytes gates was set using anti-CD 45 antibody-SSC, and lymphocytes, monocytes, granulocytes, and the presence or absence of significant tumor cells or abnormal cells were roughly observed. According to the expression of the anti-CD 45 antibody-SSC, the cell population can be divided into 5 regions (fig. 21), which are 5 regions of granulocyte (Gran) (middle upper region in fig. 21), monocyte (Mono) (upper right region in fig. 21), lymphocyte (Lym) (lower right region in fig. 21), CD45 weak positive (CD45 DIM) (middle lower region in fig. 21), CD45 negative (CD45 neg) (left region in fig. 21); b progenitor cells are generally located within the CD45 DIM region; whereas tumor cells may be present in CD45 DIM or in CD45 negative regions.
(e) And using anti-CD 19 antibody-anti-CD 10 antibody, anti-CD 19 antibody-anti-CD 34 antibody, anti-CD 22 antibody-anti-CD 34 antibody and anti-CD 22 antibody-anti-CD 10 antibody to set a gate, and accurately circling out the target cell population, namely, the original B cells and the naive B cells by using the two-parameter scatter diagram.
In this example, the target cell population of the test sample is CD19+ CD10+ cells, which are located in the CD45 DIM region at a ratio of 77.55% (fig. 22).
(6) Analyzing the expression intensity of each fluorescent antibody in the target cell population of the test sample (preferably, the following antibody pairs: anti-CD 19 antibody-anti-CD 34 antibody, anti-CD 19-antibody CD38, anti-CD 19 antibody-anti-CD 13 antibody, anti-CD 19 antibody-anti-CD 33 antibody, anti-CD 19 antibody-anti-CD 58 antibody, anti-CD 19 antibody-anti-CD 66c antibody, anti-CD 19 antibody-anti-CD 123 antibody, anti-CD 19 antibody-anti-CD 81 antibody, anti-CD 19 antibody-anti-CD 86 antibody, anti-CD 19 antibody-anti-CD 73 antibody, anti-CD 19 antibody-anti-CD 20 antibody, anti-CD 19 antibody-anti-CD 22 antibody, anti-CD 19 antibody-anti-CD 24 antibody) and comparing the expression pattern template of the normal control B cell antibody established in step 1 of example 3, if each fluorescent antibody in the target cell population of the test sample falls on the expression pattern of the progenitor B cell antibody of the normal control B cell population, the desired cell population is then a normal cell population, whereas tumor cells are suspected.
The results are shown in FIGS. 23 to 34.
As can be seen from FIGS. 23 to 34, the antibody expression pattern of the target cell population in the test sample partially fell inside the gate and partially fell outside the gate, and the immunophenotypes of the CD 19-positive cells were CD58+, CD33-, CD13-, CD20-, CD22+, CD34+, CD123+, CD24+, CD38-, CD81+, CD66c +, CD73+ and CD86-, indicating that the cell population was B-ALL tumor cells, which was consistent with the clinical test results.
Example 5 methodological validation of the detection method of the invention
1. Accuracy (Specificity)
To evaluate the accuracy of the assay of the invention, 5 each of the clinically confirmed positive and negative bone marrow sample cells (samples from gold field medical flow cytometry laboratories) were selected, tested using the method of example 3 of the invention, and compared to a conventional flow assay (using the same antibody composition as in example 3 of the invention and using the same gating method as in example 3 of the invention) to evaluate the accuracy of the method of the invention. The results are shown in Table 2.
TABLE 2 accuracy check results
As can be seen from Table 2, the antibody composition and gating method of the present invention can be used to detect CD molecules with good consistency between the positive and negative expression patterns of CD molecules and the traditional flow detection method.
2. Precision (Precision)
Because a large number of bone marrow samples are difficult to obtain, 2 clinical MRD positive samples (all samples are from gold field medical flow cytometry laboratories) are selected for precision evaluation in the method of the invention, and are repeatedly determined for 3 times in a short time and under a stable condition according to a specified operation method, and the variation of MRD results and antigen expression patterns is evaluated so as to investigate the random error of the method of the invention.
The measurement results are shown in tables 3 and 4.
TABLE 3 results of precision measurement of the first sample
The results in Table 3 show that the CV value of the MRD detection result in batch measurement is 0.01%, the antigen expression patterns are consistent, and the result of the precision in batch measurement is 'pass'.
TABLE 4 results of precision measurement of the second example sample
The results in Table 4 show that the CV value of the MRD detection result in batch measurement is 0.49%, the antigen expression patterns are consistent, and the result of the precision in batch measurement is 'pass'.
3. Sensitivity (Assay Sensitivity)
1 clinical B-ALL MRD positive specimen (sample from gold field medical flow cytometry) was selected, and the ratio of positive to normal samples (sample from gold field medical flow cytometry) was 1:1, 1:9, 1:99, 1:999, 1: 9999, diluting the positive sample, detecting by adopting a traditional flow detection method, recording the proportion of the minimum MRD which can be detected, and determining the sensitivity of the traditional flow detection method.
The results of the sensitivity verification of the conventional flow-based detection method are shown in table 5.
TABLE 5 verification of sensitivity of conventional flow-type detection method
Using the actual detection value as the abscissa and the expected value as the ordinate, a working curve is drawn, as shown in fig. 35, where y is 0.99964x +0.05288, and R is2The sensitivity of the traditional flow-through detection method was determined to be 0.01%, 0.9999%.
Selecting the same clinical B-ALL MRD positive sample (the sample is from a gold field medical flow cytometry laboratory), and carrying out the following steps of 1:1, 1:79, 1:799 and 1: 7999. 1: 79999. 1: 799999, 0:1, the positive samples were diluted and tested using the method of example 3, and the proportion of the least MRD detectable was recorded to determine the sensitivity of the method of the invention.
The results are shown in Table 6.
TABLE 6 results of the verification of the sensitivity of the method of the invention
| Dilution ratio | MRD expected value (%) | Actual detection value of MRD (%) | Difference (% -) |
| 1:1 | 93.4000 | 91.5126 | -2.02 |
| 1:79 | 1.0378 | 0.8194 | -21.04 |
| 1:799 | 0.1037 | 0.0950 | -8.39 |
| 1：7999 | 0.0104 | 0.0105 | 0.96 |
| 1：79999 | 0.0010 | 0.0011 | 10.00 |
| 1：799999 | 0.0001 | 0.0001 | 0.00 |
| 0:1 | 0 | 0.0000 | N/A |
Using the actual detection value as the abscissa and the expected value as the ordinate, a working curve is drawn, as shown in fig. 36, where y is 0.9831x-0.7454, R2The correlation was better when 0.99974, and the sensitivity of the method of the invention was determined to be 0.001%.
It follows that the sensitivity of the method of the invention far exceeds that of conventional flow-based detection methods.
Comparative example 1
Referring to the method for detecting acute B lymphocytic leukemia cells of example 4, the test sample cells of example 4 were subjected to acute B lymphocytic leukemia minimal residual detection (gated with anti-CD 45 antibody-SSC, different from example 4 in that the antibody composition used in comparative example 1 was anti-CD 45 antibody, anti-CD 66c antibody, anti-CD 34 antibody, anti-CD 19 antibody, anti-CD 10 antibody and anti-CD 58 antibody, and the detection equipment used was a conventional flow cytometer). The expression intensities of the fluorescent antibody of the objective cell population, anti-CD 34 antibody-anti-CD 19 antibody, anti-CD 19 antibody-anti-CD 58 antibody, anti-CD 19 antibody-anti-CD 66c antibody, anti-CD 19 antibody-anti-CD 10 antibody were analyzed and compared with the expression pattern template of the normal control B progenitor cell antibody in example 3.
The results are shown in FIGS. 36 to 42. The results show that the target cells are within the normal expression gate and cannot be judged to be abnormal tumor cells by using the antibody composition in the comparative example.
The results of the detection of the anti-CD 81 antibody by addition of the anti-CD 24 antibody are shown in fig. 43 and 44, and the results indicate that the expression pattern of the target cells is different from the expression pattern template of the normal control B progenitor antibody, and therefore, it can be determined that the target cells are abnormal tumor cells.
The comparative example results show that there may be a missed diagnosis due to fewer antibody pairs being detected.
Comparative example 2
Referring to the method for detecting acute B lymphocytic leukemia cells of example 4, the test sample cells of example 4 were subjected to acute B lymphocytic leukemia minimal residual detection (gated with anti-CD 45 antibody-SSC, anti-CD 34 antibody, differing from example 4 in that the antibody composition used in comparative example 2 was anti-CD 117 antibody, anti-CD 45 antibody, anti-CD 34 antibody, anti-CD 13 antibody, anti-CD 333 antibody, anti-HLA-DR antibody, anti-CD 7 antibody, anti-CD 11B antibody, anti-CD 15 antibody and anti-CD 38 antibody). The expression intensity of the fluorescent antibody of the desired cell population, anti-CD 34 antibody-anti-CD 117 antibody, anti-CD 34 antibody-anti-CD 13 antibody, anti-CD 34 antibody-anti-CD 33 antibody, anti-CD 34 antibody-anti-CD 7 antibody, anti-CD 34 antibody-anti-CD 15 antibody, anti-CD 34 antibody-anti-CD 11B antibody, anti-CD 34 antibody-anti-CD 38 antibody, was analyzed and compared with the expression pattern template of the normal control B progenitor cell antibody in example 3.
As a result, it was found that: the detected antibody pair is mainly expressed in myeloid primitive cells and used for monitoring myeloid leukemia, so that the detected antibody pair cannot effectively identify primitive B cells and naive B cells and cannot be used for detecting the minimal residual of acute B lymphocyte leukemia.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. An antibody composition for detecting minimal residual acute B-lymphocyte leukemia, wherein the antibody composition comprises: anti-CD 19 antibodies, anti-CD 10 antibodies, anti-CD 34 antibodies, and anti-CD 22 antibodies.
2. The antibody composition for detecting the minimal residual of acute B lymphocytic leukemia of claim 1, wherein the antibody composition further comprises an anti-CD 20 antibody, an anti-CD 45 antibody, an anti-CD 24 antibody, an anti-CD 38 antibody, an anti-CD 81 antibody, an anti-CD 58 antibody, an anti-CD 13 antibody, an anti-CD 33 antibody, an anti-CD 66c antibody, an anti-CD 73 antibody, an anti-CD 123 antibody and an anti-CD 86 antibody.
3. The antibody composition for detecting the minimal residual of acute B lymphocyte leukemia according to claim 2, wherein the clone numbers of the anti-CD 19 antibody, the anti-CD 10 antibody, the anti-CD 34 antibody, the anti-CD 20 antibody, the anti-CD 45 antibody, the anti-CD 22 antibody, the anti-CD 24 antibody, the anti-CD 38 antibody, the anti-CD 81 antibody, the anti-CD 58 antibody, the anti-CD 13 antibody, the anti-CD 33 antibody, the anti-CD 66c antibody, the anti-CD 73 antibody, the anti-CD 123 antibody and the anti-CD 86 antibody are in sequence: j3-119, H10a, 581, 2H7, H130, S-HCL-1, ML5, HIT2, JS-81, AICD58, L138, D3HL60.251, B6.2/CD66, AD2, 9F5 and 2331.
4. The antibody composition for detecting minimal residual acute B lymphocyte leukemia according to claim 2, wherein the titers of the anti-CD 19 antibody, anti-CD 10 antibody, anti-CD 34 antibody, anti-CD 20 antibody, anti-CD 45 antibody, anti-CD 22 antibody, anti-CD 24 antibody, anti-CD 38 antibody, anti-CD 81 antibody, anti-CD 58 antibody, anti-CD 13 antibody, anti-CD 33 antibody, anti-CD 66c antibody, anti-CD 73 antibody, anti-CD 123 antibody and anti-CD 86 antibody are 2.5. mu.L, 0.625. mu.L, 5. mu.L, 1.25. mu.L, 0.625. mu.L, 4. mu.L, 2.5. mu.L, 5. mu.L, 2.5. mu.L, 4. mu.L.
5. The antibody composition for detecting the minimal residual of acute B lymphocyte leukemia according to claim 2, wherein the antibodies are all fluorescein-labeled antibodies, and the anti-CD 19 antibody, anti-CD 10 antibody, anti-CD 34 antibody, anti-CD 20 antibody, anti-CD 45 antibody, anti-CD 22 antibody, anti-CD 24 antibody, anti-CD 38 antibody, anti-CD 81 antibody, anti-CD 58 antibody, anti-CD 13 antibody, anti-CD 33 antibody, anti-CD 66c antibody, anti-CD 73 antibody, anti-CD 123 antibody and anti-CD 86 antibody are sequentially labeled with fluorescein: PC7, BV786, ECD, BV421, BV510, APC-CY7, PC5.5, BV480, FITC, PE, BV650, BV605, BV750, and V450.
6. The antibody composition for detecting minimal residual acute B lymphocyte leukemia according to any one of claims 1 to 5, wherein the antibody is a monoclonal antibody.
7. A kit for detecting minimal residual acute B-lymphocyte leukemia, which comprises the antibody composition according to any one of claims 1 to 6.
8. The kit for detecting the minimal residual acute B lymphocyte leukemia according to claim 7, characterized in that the kit further comprises a nucleic acid dye 7-AAD, hemolysin, a washing solution and a fixing solution, wherein the washing solution is a PBS solution containing 0.8-1.2% fetal bovine serum, and the fixing solution is a PBS solution containing 0.8-1.2% paraformaldehyde.
9. A method for detecting minimal residual acute B-lymphocyte leukemia, which comprises the steps of using the kit of claim 7 or 8 to detect and obtain the expression pattern and intensity of each fluorescein-labeled antibody for non-diagnostic purposes, wherein the method comprises the following steps:
(1) adding nucleic acid dye 7-ADD and fluorescein labeled antibody composition into flow tube, and adding the mixture containing 1 × 108～5×108Uniformly mixing single cell suspension of sample cells to be detected, and incubating for 15-20 minutes at room temperature in a dark place;
the fluorescein-labeled antibody composition is: fluorescein PC7, BV786, ECD, BV421, BV510, APC-CY7, PC5.5, BV480, FITC, PE, BV650, BV605, BV750, and V450 labeled anti-CD 19 antibody, anti-CD 10 antibody, anti-CD 34 antibody, anti-CD 20 antibody, anti-CD 45 antibody, anti-CD 22 antibody, anti-CD 24 antibody, anti-CD 38 antibody, anti-CD 81 antibody, anti-CD 58 antibody, anti-CD 13 antibody, anti-CD 33 antibody, anti-CD 66c antibody, anti-CD 73 antibody, anti-CD 123 antibody, and anti-CD 86 antibody are used in sequence; the titer of the antibody is 2.5 muL, 0.625 muL, 5 muL, 1.25 muL, 0.625 muL, 4 muL, 2.5 muL, 5 muL, 2.5 muL, 1.25 muL, 2.5 muL and 4 muL in sequence;
(2) adding 380-420 mul of hemolysin into a flow tube, uniformly mixing, standing in a dark place, centrifuging and discarding the supernatant after hemolysis is transparent, washing with PBS (phosphate buffer solution) containing 0.8-1.2% fetal calf serum, centrifuging and discarding the supernatant, and resuspending cells with PBS containing 0.8-1.2% paraformaldehyde to obtain cell suspension of a sample to be detected;
(3) detecting the cell suspension of the sample to be detected in the step (2) by using a full-spectrum flow cytometer, and circling out a target cell group in a cell scatter diagram of the sample to be detected; the gate setting mode of the full-spectrum flow cytometer is as follows: removing dead cells, fragments and adherent cells in sequence, gating by using anti-CD 45 antibody-SSC, anti-CD 19 antibody-anti-CD 10 antibody, anti-CD 19 antibody-anti-CD 34 antibody, anti-CD 22 antibody-anti-CD 34 antibody and anti-CD 22 antibody-anti-CD 10 antibody, and accurately delineating a target cell population by using a two-parameter scatter diagram;
(4) analyzing the expression pattern and the intensity of each fluorescein-labeled antibody in the target cell population of the sample to be detected, comparing the expression pattern and the intensity with an expression pattern template of a normal control B progenitor cell antibody, and detecting whether each fluorescein-labeled antibody of the sample to be detected falls on the expression pattern template of the normal control B progenitor cell antibody;
the method for establishing the expression pattern template of the normal control B progenitor cell antibody comprises the following steps: detecting cell suspensions of 20-40 normal control samples by using a full-spectrum flow cytometer, circling out B progenitor cells in a cell scatter diagram of the normal control sample, analyzing the expression pattern and the intensity of each fluorescein labeled antibody in the B progenitor cells of the normal control sample to obtain the expression pattern of the normal cells, and establishing an expression pattern template of a normal control B progenitor cell antibody; the gate setting method of the full-spectrum flow cytometer is as described in step (3).
10. The method for detecting the minimal residual of acute B lymphocyte leukemia according to claim 9, wherein the fluorescein labeled antibody in step (4) is one or more of the pairs of anti-CD 19 antibody-anti-CD 34 antibody, anti-CD 19-antibody CD38, anti-CD 19 antibody-anti-CD 13 antibody, anti-CD 19 antibody-anti-CD 33 antibody, anti-CD 19 antibody-anti-CD 58 antibody, anti-CD 19 antibody-anti-CD 66c antibody, anti-CD 19 antibody-anti-CD 123 antibody, anti-CD 19 antibody-anti-CD 81 antibody, anti-CD 19 antibody-anti-CD 86 antibody, anti-CD 19 antibody-anti-CD 73 antibody, anti-CD 19 antibody-anti-CD 20 antibody, anti-CD 19 antibody-anti-CD 22 antibody, anti-CD 19 antibody-anti-CD 24 antibody.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104471393A (en) * | 2012-06-14 | 2015-03-25 | 鹿特丹伊拉斯姆斯大学医疗中心 | Methods, reagents and kits for detecting minimal residual disease |
| CN109655616A (en) * | 2018-12-19 | 2019-04-19 | 广州金域医学检验中心有限公司 | Detect the composite reagent and system of acute myeloid leukemia cell |
| CN109884313A (en) * | 2019-02-22 | 2019-06-14 | 武汉康圣达医学检验所有限公司 | The detection kit of B-lineage Acute Lymphocyte Leukemia minimal residual |
-
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104471393A (en) * | 2012-06-14 | 2015-03-25 | 鹿特丹伊拉斯姆斯大学医疗中心 | Methods, reagents and kits for detecting minimal residual disease |
| US20150160226A1 (en) * | 2012-06-14 | 2015-06-11 | Erasmus University Medical Center Rotterdam | Method, reagents, and kits for detecting minimal residual disease |
| CN107422122A (en) * | 2012-06-14 | 2017-12-01 | 鹿特丹伊拉斯姆斯大学医疗中心 | For detecting method, reagent and the kit of minimal residual disease |
| CN109655616A (en) * | 2018-12-19 | 2019-04-19 | 广州金域医学检验中心有限公司 | Detect the composite reagent and system of acute myeloid leukemia cell |
| CN109884313A (en) * | 2019-02-22 | 2019-06-14 | 武汉康圣达医学检验所有限公司 | The detection kit of B-lineage Acute Lymphocyte Leukemia minimal residual |
Non-Patent Citations (5)
| Title |
|---|
| THEUNISSEN P 等: "Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia", BLOOD, vol. 129, no. 3, pages 347 - 357, XP086677254, DOI: 10.1182/blood-2016-07-726307 * |
| 张丽;罗文达;陈葆国;郭群依;: "急性淋巴细胞白血病免疫表型与微小残留病检测的意义", 现代中西医结合杂志, no. 09, pages 1147 - 1150 * |
| 张静;李芹;陈辉;朴文花;: "流式细胞术检测急性白血病微小残留病变68例临床分析", 宁夏医科大学学报, no. 12, pages 106 - 108 * |
| 方宗宇 等: "CD81联合CD10及CD34在流式细胞术监测儿童B-ALL微小残留病变中的应用价值", 山东医药, vol. 59, pages 75 - 78 * |
| 王月芳;张鸽;江咏梅;高举;: "免疫分型分化抗原与儿童B-ALL早期微小残留病的相关性研究", 中国实验血液学杂志, no. 05, pages 57 - 64 * |
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