A blastic plasmacytoid dendritic cell tumor residue detection composition and its application

Abstract

The present invention provides a kind of blastic plasmacytoid dendritic cell tumor residual detection composition, blastic plasmacytoid dendritic cell tumor residual detection composition, including composition one and composition two, wherein, the composition one at least includes CD56 antibody, CD123 antibody, CD34 antibody, CD4 antibody, CD304 antibody, HLA-DR antibody, CD303 antibody and CD45 antibody, and the composition two at least includes CD64 antibody, CD123 antibody, CD33 antibody, CD11b antibody, HLA-DR antibody, CD15 antibody and CD45 antibody. The composition provided by the present invention can not only distinguish abnormal tumor cells from normal cells, but also distinguish dendritic cells from myeloid cells, with high detection accuracy, and can effectively avoid misdiagnosis caused by missed detection or misdetection.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a composition for detecting residual blastic plasmacytoid dendritic cells in tumors and an application thereof. Background Art

[0002] Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a hematological malignancy that originates from immature plasmacytoid dendritic cells and is often clinically invasive. The histopathology of BPDCN lesions is mostly pleomorphic cells, with single, diffusely distributed tumor cells, usually dispersed chromatin without granules, irregular nuclear membranes, prominent nucleoli, and enhanced mitosis. There is no vascular infiltration and coagulative necrosis. It often involves the dermis, can invade subcutaneous fat tissue, and can also cause damage to skin appendages, but generally does not involve the epidermis.

[0003] BPDCN usually expresses CD4, CD56, CD43, CD45RA and at least one pDCs-related antigen, such as CD123, TCL-1, CD2AP, CD303 (BDCA-2), CD304 (BDCA-4), etc. One-third express TdT, and usually do not express T and B cell immunophenotypes and CD34 and CD117. Some BPDCN also express other cell lineage antigens, such as CD2, CD33, CD79a, etc.

[0004] BPDCN responds well to chemotherapy regimens for acute leukemia or aggressive lymphoma, with an overall complete remission rate (CR) of 40%-90%, but 50%-90% relapse quickly. In summary, BPDCN is a malignant hematopoietic tumor derived from plasmacytoid dendritic cells, with a low incidence and common skin involvement. Diagnosis depends on immunohistochemistry, etc. HSCT, conventional chemotherapy and targeted therapy can prolong survival, but the overall prognosis is still poor.

[0005] Currently, the best way to prolong the patient's survival time is to monitor the residual tumors in the body after chemotherapy and perform bone marrow transplantation after sustained remission. However, there is currently a lack of detection methods and means for the tiny residual tumors of blastic plasmacytoid dendritic cells in patients. Summary of the invention

[0006] Based on the above background technology, the technical problem to be solved by the present invention is to provide a composition for detecting residual blastic plasmacytoid dendritic cell tumors, which can distinguish abnormal tumor cells from normal dendritic cells, effectively detect tiny residual blastic plasmacytoid dendritic cell tumors in patients, and avoid diagnostic errors caused by missed detection.

[0007] The present invention provides a composition for detecting residual blastic plasmacytoid dendritic cell tumors, comprising composition one and composition two, wherein composition one comprises CD56 antibody, CD123 antibody, CD34 antibody, CD4 antibody, CD304 antibody, HLA-DR antibody, CD303 antibody and CD45 antibody, and composition two comprises CD64 antibody, CD123 antibody, CD33 antibody, CD11b antibody, HLA-DR antibody, CD15 antibody and CD45 antibody.

[0008] Composition 1 can distinguish abnormal dendritic cells from normal dendritic cells, while composition 2 can distinguish dendritic cells from myeloid cells. Composition 1 and composition 2 can quickly and accurately distinguish normal dendritic cells from abnormal dendritic cells, and effectively reduce misdiagnosis caused by interference from myeloid cells.

[0009] Preferably, in the composition 1, the mass ratio of HLA-DR antibody to CD123 antibody is 2:1 to 1:1, the mass ratio of CD303 antibody to CD304 antibody is 1:1 to 1:3, and the mass ratio of CD56 antibody to CD4 antibody is 2:1 to 4:1.

[0010] Preferably, in the second composition, the mass ratio of HLA-DR antibody to CD123 antibody is 2:1 to 1:1, the mass ratio of CD15 antibody to CD11b antibody is 3:1 to 5:1, and the mass ratio of CD64 antibody to CD33 antibody is 2:1 to 1:1.

[0011] Preferably, the mass ratio of CD56 antibody, CD123 antibody, CD34 antibody, CD4 antibody, CD304 antibody, HLA-DR antibody, CD303 antibody and CD45 antibody in the composition is (6-12):25:100:(3-4):(30-60):(25-50):(30-90):80.

[0012] Preferably, the mass ratio of CD64 antibody, CD123 antibody, CD33 antibody, CD11b antibody, HLA-DR antibody, CD15 antibody and CD45 antibody in the second composition is 20:5:(10-20):3:(5-10):(9-15):16.

[0013] Preferably, the antibody is a fluorescently labeled antibody.

[0014] The present invention uses a composition consisting of HLA-DR antibody, CD4 antibody, CD11b antibody, CD15 antibody, CD56 antibody, CD64 antibody, CD123 antibody, CD303 antibody, CD304 antibody, CD34 antibody, CD45 antibody and CD33 antibody for the detection of residual blastic plasmacytoid dendritic cell tumors, which can not only distinguish abnormal tumor cells from normal cells, but also distinguish dendritic cells from myeloid cells, has a high detection accuracy, and can effectively avoid diagnostic errors caused by missed detection or false detection.

[0015] Another object of the present invention is to provide a blastic plasmacytoid dendritic cell tumor residue detection kit, comprising the above-mentioned composition, red blood cell lysate and PBS buffer.

[0016] The beneficial effects of the blastic plasmacytoid dendritic cell tumor residue detection kit provided by the present invention are consistent with those of the blastic plasmacytoid dendritic cell tumor residue detection composition, which will not be described in detail here.

[0017] Another object of the present invention is to provide a method for detecting residual blastic plasmacytoid dendritic cell tumors, which uses the above-mentioned kit for detection, comprising the following steps:

[0018] Step S1, respectively adding CD56 antibody, CD123 antibody, CD34 antibody, CD4 antibody, CD304 antibody, HLA-DR antibody, CD303 antibody and CD45 antibody combination and CD64 antibody, CD123 antibody, CD33 antibody, CD11b antibody, HLA-DR antibody, CD15 antibody and CD45 antibody combination into two different test tubes, then adding the sample to be tested into both test tubes and incubating in the dark;

[0019] Step S2, adding red blood cell lysis solution to each of the two test tubes, mixing evenly, and then standing at room temperature in the dark to obtain a lysis mixture;

[0020] Step S3, centrifuging the lysis mixture, removing the supernatant, and obtaining a lysis precipitate;

[0021] Step S4, adding PBS to the lysed precipitate and mixing evenly, then centrifuging, and then re-adding PBS to resuspend, to obtain a resuspended solution;

[0022] Step S5: Detect the resuspended solution on a flow cytometer and analyze the results using BDDiva Software.

[0023] Preferably, abnormal dendritic cells are identified by:

[0024] First, circle the cells that are co-positive for HLA-DR and CD123;

[0025] Secondly, further analysis is performed on this group of cells. If they are CD56 positive and / or CD4 positive, and CD303 or CD304 positive, they are abnormal dendritic cells.

[0026] If CD56 is negative, and CD304 and CD304 are co-positive, it is a normal dendritic cell;

[0027] If CD64, CD33, CD15 and CD123 are positive, they are myeloid cells.

[0028] The present invention reacts the sample to be tested with two groups of antibody compositions, and after detection and analysis by a flow cytometer, can accurately distinguish abnormal dendritic cells in the sample to be tested according to the expression of different antibodies, thereby detecting residual blastic plasmacytoid dendritic cell tumors, and can distinguish dendritic cells and myeloid cells in the sample to be tested, effectively reducing missed detection and false detection, and ensuring the accuracy of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the location of lymphocyte groups during machine testing;

[0030] Figure 2 This is a diagram showing the results of the analysis of samples from patients with blastic plasmacytoid dendritic cell tumors;

[0031] Figure 3 This is a sample test analysis result diagram for normal testers;

[0032] Figure 4 Comparative analysis of test results from different kits Figure 1 ;

[0033] Figure 5 Comparative analysis of test results from different kits Figure 2 . DETAILED DESCRIPTION

[0034] The principles and features of the present invention are described below in conjunction with specific embodiments. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein belong to the meanings commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0035] The technical concept of the present invention is as follows: the inventors detected and analyzed the expression of leukocyte differentiation antigens in 216 BPDCN patients after chemotherapy, and compared them with the leukocyte phenotypes of normal testers, and found that the leukocyte differentiation antigens of BPDCN patients had regular immune phenotypes. Further, after screening a large number of differentiation antigen detection antibody combinations, the optimal combination for residual detection of blastic plasmacytoid dendritic cell tumors was obtained, which includes composition one and composition two, wherein composition one includes CD56 antibody, CD123 antibody, CD34 antibody, CD4 antibody, CD304 antibody, HLA-DR antibody, CD303 antibody and CD45 antibody, and composition two includes CD64 antibody, CD123 antibody, CD33 antibody, CD11b antibody, HLA-DR antibody, CD15 antibody and CD45 antibody.

[0036] The above composition can be used to evaluate the test results in the following ways:

[0037] First, circle the cells that are co-positive for HLA-DR and CD123;

[0038] Secondly, further analysis is performed on this group of cells. If they are CD56 positive and / or CD4 positive, and CD303 or CD304 positive, they are abnormal dendritic cells.

[0039] If CD56 is negative, and CD304 and CD304 are co-positive, it is a normal dendritic cell;

[0040] If CD64, CD33, CD15 and CD123 are positive, they are myeloid cells.

[0041] Further research found that when the above antibodies are mixed in specific proportions, that is, in composition one, the mass ratio of HLA-DR antibody and CD123 antibody is 2:1-1:1, the mass ratio of CD303 antibody and CD304 antibody is 1:1-1:3, and the mass ratio of CD56 antibody and CD4 antibody is 2:1-4:1; in composition two, the mass ratio of HLA-DR antibody and CD123 antibody is 2:1-1:1, the mass ratio of CD15 antibody and CD11b antibody is 3:1-5:1, and the mass ratio of CD64 antibody and CD33 antibody is 2:1-1:1, the detection is less likely to have false negatives.

[0042] The composition and evaluation method provided by the present invention can quickly and accurately detect the residual BPDCN in the patient's body, which is helpful for guiding clinical medication and prolonging the patient's survival time.

[0043] The sources of the raw materials involved in the following examples are shown in Table 1 below:

[0044] Table 1 Antibody sources in the examples

[0045]

[0046]

[0047] Example 1

[0048] The present embodiment provides a blastic plasmacytoid dendritic cell tumor residue detection kit, comprising HLA-DRAPC-cy7, CD4 APC, CD11b PE-cy7, CD15 BV450, CD56 FITC, CD64 FITC, CD123 PE, CD303BV421, CD304 PE-cy7, CD34 PerCP, CD33APC and CD45 V500 antibodies, red blood cell lysate, and PBS buffer, wherein composition one comprises CD56 FITC, CD123 PE, CD34 PerCP, CD4 APC, CD304 PE-cy7, HLA-DRAPC-cy7, CD303 BV421 and CD45 V500 antibodies, and composition two comprises CD64 FITC, CD123 PE, CD33 APC, CD11b PE-cy7, HLA-DRAPC-cy7, CD15 BV450 and CD45 V500 antibodies.

[0049] Red blood cell lysis buffer was purchased from BD, and included the following components: ammonium chloride, potassium dihydrogen phosphate, disodium ethylenediaminetetraacetic acid, and paraformaldehyde;

[0050] PBS buffer (phosphate buffer) has a concentration of 0.01 M, a pH of 7.2 to 7.4, and includes the following components: 1.8 mmol / L potassium dihydrogen phosphate, 10 mmol / L disodium hydrogen phosphate, 137 mmol / L sodium chloride, and 2.7 mmol / L potassium chloride;

[0051] In composition one, the mass ratio of CD56 FITC, CD123 PE, CD34 PerCP, CD4 APC, CD304 PE-cy7, HLA-DRAPC-cy7, CD303 BV421 and CD45 V500 antibodies is 12:25:100:3.6:60:30:30:80; in composition two, the mass ratio of CD64 FITC, CD123 PE, CD33 APC, CD11b PE-cy7, HLA-DRAPC-cy7, CD15 BV450 and CD45V500 antibodies is 20:5:12:3:6:12:16.

[0052] Example 2

[0053] The difference between this embodiment and embodiment 1 is that in composition 1, the mass ratio of CD56 FITC, CD123 PE, CD34 PerCP, CD4 APC, CD304 PE-cy7, HLA-DR APC-cy7, CD303 BV421 and CD45 V500 antibodies is 12:24:100:3.6:60:30:30:80, and the other conditions are the same as those in embodiment 1.

[0054] Comparative Example 1

[0055] The difference between this embodiment and embodiment 1 is that in composition 1, the mass ratio of CD56 FITC, CD123 PE, CD34 PerCP, CD4 APC, CD304 PE-cy7, HLA-DR APC-cy7, CD303 BV421 and CD45 V500 antibodies is 12:25:100:3.6:60:75:30:80, and the other conditions are the same as those in embodiment 1.

[0056] Comparative Example 2

[0057] The difference between this embodiment and embodiment 1 is that in composition 1, the mass ratio of CD56 FITC, CD123 PE, CD34 PerCP, CD4 APC, CD304 PE-cy7, HLA-DR APC-cy7, CD303 BV421 and CD45 V500 antibodies is 12:24:100:3.6:30:30:60:80, and the other conditions are the same as those in embodiment 1.

[0058] Experimental Example 1

[0059] The kit in Example 1 was used to detect and analyze samples from patients with blastic plasmacytoid dendritic cell tumors and samples from normal test subjects. The detection steps are as follows:

[0060] S1. Add 23 μL of composition 1 and 18 μL of composition 2 to two different test tubes respectively, and then add 200 μL of the sample to be tested (the cell solubility is adjusted to 10 7 / mL), incubate in dark for 15min;

[0061] S2. Add 1 mL of red blood cell lysis solution to each of the two test tubes, mix well, and then stand at room temperature in the dark for 8 minutes to obtain a lysis mixture;

[0062] S3, centrifuging the lysate mixture at 300 rct / min for 5 min, removing the supernatant to obtain a lysate precipitate;

[0063] Step S4, adding 1 mL of PBS to the lysed precipitate and mixing evenly, then centrifuging at 150 rct / min for 5 min, and then re-adding 150 μL of PBS to resuspend the precipitate to obtain a resuspended solution;

[0064] S5. Detect the resuspended solution on a flow cytometer and adjust the instrument conditions using quality control products so that the lymphocyte population is located in the Figure 1 One million signals were acquired at the position of P1 as shown, and the results were analyzed using BD Diva Software.

[0065] Abnormal dendritic cells are identified by:

[0066] First, circle the cells that are co-positive for HLA-DR and CD123;

[0067] Secondly, further analysis is performed on this group of cells. If they are CD56 positive and / or CD4 positive, and CD303 or CD304 positive, they are abnormal dendritic cells.

[0068] If CD56 is negative, and CD304 and CD304 are co-positive, it is a normal dendritic cell;

[0069] If CD64, CD33, CD15 and CD123 are positive, they are myeloid cells.

[0070] Test results such as Figure 2 and Figure 3 As shown, Figure 2 Results for patients with blastic plasmacytoid dendritic cell neoplasm. Figure 2 P6 in (a) is a CD123 and HLA-DR co-positive cell. Further analysis of the cells in P6 revealed that Figure 2 (b) and Figure 2 As can be seen in (c), these cells simultaneously expressed CD303 and CD56, and were thus identified as abnormal dendritic cells; Figure 3 The test results for normal testers are: Figure 3 P8 in (a) is a CD123 and HLA-DR co-positive cell. Further analysis of the cells in P8 revealed that Figure 3 (b) and Figure 3 As can be seen in (c), these cells expressed both CD303 and CD304, and were thus determined to be normal dendritic cells.

[0071] Experimental Example 2

[0072] The samples of patients with blastic plasmacytoid dendritic cell tumors were tested using the kits in Example 1, Example 2, Comparative Example 1 and Comparative Example 2, respectively. The testing steps are as follows:

[0073] S1. Add 23 μL of composition 1 and 18 μL of composition 2 to two different test tubes respectively, and then add 200 μL of the sample to be tested (the cell solubility is adjusted to 10 7 / mL), incubate in dark for 15min;

[0074] S2. Add 1 mL of red blood cell lysis solution to each of the two test tubes, mix well, and then stand at room temperature in the dark for 8 minutes to obtain a lysis mixture;

[0075] S3, centrifuging the lysate mixture at 300 rct / min for 5 min, removing the supernatant to obtain a lysate precipitate;

[0076] Step S4, adding 1 mL of PBS to the lysed precipitate and mixing evenly, then centrifuging at 150 rct / min for 5 min, and then re-adding 150 μL of PBS to resuspend the precipitate to obtain a resuspended solution;

[0077] S5. Detect the resuspended solution on a flow cytometer and adjust the instrument conditions using quality control products so that the lymphocyte population is located in the Figure 1 One million signals were acquired at the position of P1 as shown, and the results were analyzed using BD Diva Software.

[0078] The results are as follows Figure 4 and Figure 5 As shown, Figure 4 (a) is the test result using the kit in Example 2, Figure 4 (b) is the detection result of the kit in Comparative Example 1. It can be seen from the figure that when the kit in Comparative Example 1 is used for detection, the fluorescence intensity of CD123 is reduced, which leads to false negatives in the detection process; Figure 5 (a) is the test result using the kit in Example 1, Figure 5 (b) is the detection result of the kit in Comparative Example 2. It can be seen from the figure that when the kit in Comparative Example 2 is used for detection, the fluorescence intensity of CD304 is reduced, which easily leads to false negatives in the detection process.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a composition in the preparation of a blastic plasmacytoid dendritic cell tumor residue detection reagent, characterized in that: The composition includes composition one and composition two, wherein the composition one includes at least CD56, CD123 antibody, CD34 antibody, CD4 antibody, CD304 antibody, HLA-DR antibody, CD303 antibody and CD45 antibody, and the composition two includes at least CD64 antibody, CD123 antibody, CD33 antibody, CD11b antibody, HLA-DR antibody, CD15 antibody and CD45 antibody, wherein, Abnormal dendritic cells are identified by: First, circle the cells that are co-positive for HLA-DR and CD123; Secondly, further analysis is performed on this group of cells. If they are CD56 positive and / or CD4 positive, and CD303 or CD304 positive, they are abnormal dendritic cells; If CD56 is negative, and CD303 and CD304 are both positive, it is a normal dendritic cell; If CD64, CD33, CD15, and CD123 are positive, they are myeloid cells; The mass ratio of CD56 antibody, CD123 antibody, CD34 antibody, CD4 antibody, CD304 antibody, HLA-DR antibody, CD303 antibody and CD45 antibody in the composition 1 is (6-12):25:100:(3-4):(30-60):(25-50):(30-90):80; The mass ratio of CD64 antibody, CD123 antibody, CD33 antibody, CD11b antibody, HLA-DR antibody, CD15 antibody and CD45 antibody in the second composition is 20:5:(10-20):3:(5-10):(9-15):

16.

2. The use according to claim 1, characterized in that: The antibody is a fluorescent labeled antibody.

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