REAGENT COMPOSITION FOR DETECTION OF NON-HEMATOPOIETIC TUMOR AND USE THEREOF

Abstract

The present invention provides a reagent composition for detection of a non-hematopoietic tumor and use thereof. Said reagent composition includes three sets of antibodies, with the first set of antibodies including an anti-CD9 antibody, an anti-GD2 antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD56 antibody, an anti-CD36 antibody, an anti-CD81, and an anti-CD45 antibody; the second set of antibodies including an anti-HLA-ABC antibody, an anti-CD38 antibody, an anti-CD19 antibody, an anti-CD56 antibody, an anti-CD36 antibody, an anti-CD7 antibody, and an anti-CD45 antibody; the third set of antibodies including anti-cytoplasmic cytokeratin antibodies; wherein the first set of antibodies and the second set of antibodies are respectively used for samples in separate tubes, and the third set of antibodies is used for the sample in the same tube as the second set of antibodies. The reagent composition of the present invention can be applied for flow cytometry screening, diagnosis and/or follow-up detection of a non-hematopoietic tumor.

Claims

1. A reagent composition that can be used for detection of a non-hematopoietic tumor by flow cytometry, wherein the reagent composition includes a first set of antibodies, a second set of antibodies, and a third set of antibodies, wherein: the first set of antibodies includes: an anti-CD9 antibody, an anti-GD2 antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD56 antibody, an anti-CD36 antibody, an anti-CD81 antibody, and an anti-CD45 antibody; and the first set of antibodies is to be added to a first flow cytometric tube in which a sample to be assayed is in the form of a single cell suspension; the second set of antibodies includes: an anti-HLA-ABC antibody, an anti-CD38 antibody, an anti-CD19 antibody, an anti-CD56 antibody, an anti-CD36 antibody, an anti-CD7 antibody, and an anti-CD45 antibody; and the second set of antibodies is to be added to a second flow cytometric tube in which a sample to be assayed is in the form of a single cell suspension; and the third set of antibodies includes: anti-cytoplasmic cytokeratin antibodies; and the third set of antibodies is to be added to the second flow cytometric tube to which the second set of antibodies has been added and permeabilization has been performed.

2. The reagent composition according to claim 1, wherein each antibody is a monoclonal antibody, and wherein the anti-cytoplasmic cytokeratin antibodies are complexes comprising an anti-CK8 monoclonal antibody, an anti-CK18 monoclonal antibody, and an anti-CK19 monoclonal antibody.

3. The reagent composition according to claim 1, wherein each antibody is a fluorescein-labeled antibody, and in the first set of antibodies, the anti-CD9 antibody, the anti-GD2 antibody, the anti-CD3 antibody, the anti-CD4 antibody, the anti-CD56 antibody, the anti-CD36 antibody, the anti-CD81 antibody, and the anti-CD45 antibody are fluorescein-labeled with FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, and V500, respectively; in the second set of antibodies, the anti-HLA-ABC antibody, the anti-CD38 antibody, the anti-CD19 antibody, the anti-CD56 antibody, the anti-CD36 antibody, the anti-CD7 antibody, and the anti-CD45 antibody are fluorescein-labeled with PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, and V500, respectively; and in the third set of antibodies, the anti-cytoplasmic cytokeratin antibodies are fluorescein-labeled with FITC.

4. The reagent composition according to claim 1, wherein the first set of antibodies is a mixture of an anti-CD9 antibody, an anti-GD2 antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD56 antibody, an anti-CD36 antibody, an anti-CD81 antibody, and an anti-CD45 antibody mixed in a volume ratio of 5:5:5:3:2:3:3:3, and the second set of antibodies is a mixture of an anti-HLA-ABC antibody, an anti-CD38 antibody, an anti-CD19 antibody, an anti-CD56 antibody, an anti-CD36 antibody, an anti-CD7 antibody, and an anti-CD45 antibody mixed in a volume ratio of 5:5:3:2:3:3:3.

5. A kit for detection of a non-hematopoietic tumor comprising a plurality of containers, wherein each container separately contains each set of antibodies of the reagent composition according to claim 1.

6. The kit according to claim 5, wherein each container separately contains each set of antibodies of the reagent composition, wherein each antibody is a monoclonal antibody, and wherein the anti-cytoplasmic cytokeratin antibodies are complexes comprising an anti-CK8 monoclonal antibody, an anti-CK18 monoclonal antibody, and an anti-CK19 monoclonal antibody.

7. The kit according to claim 5, wherein each container separately contains each set of antibodies of the reagent composition, wherein each antibody is a fluorescein-labeled antibody, and in the first set of antibodies, the anti-CD9 antibody, the anti-GD2 antibody, the anti-CD3 antibody, the anti-CD4 antibody, the anti-CD56 antibody, the anti-CD36 antibody, the anti-CD81 antibody, and the anti-CD45 antibody are fluorescein-labeled with FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, and V500, respectively; in the second set of antibodies, the anti-HLA-ABC antibody, the anti-CD38 antibody, the anti-CD19 antibody, the anti-CD56 antibody, the anti-CD36 antibody, the anti-CD7 antibody, and the anti-CD45 antibody are fluorescein-labeled with PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, and V500, respectively; and in the third set of antibodies, the anti-cytoplasmic cytokeratin antibodies are fluorescein-labeled with FITC.

8. The kit according to claim 5, wherein each container separately contains each set of antibodies of the reagent composition, wherein the first set of antibodies is a mixture of an anti-CD9 antibody, an anti-GD2 antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD56 antibody, an anti-CD36 antibody, an anti-CD81 antibody, and an anti-CD45 antibody mixed in a volume ratio of 5:5:5:3:2:3:3:3, and the second set of antibodies is a mixture of an anti-HLA-ABC antibody, an anti-CD38 antibody, an anti-CD19 antibody, an anti-CD56 antibody, an anti-CD36 antibody, an anti-CD7 antibody, and an anti-CD45 antibody mixed in a volume ratio of 5:5:3:2:3:3:3.

9. A method for detecting a non-hematopoietic tumor by flow cytometry, comprising preparing a flow cytometry loading sample after processing a sample to be tested using a reagent composition, wherein the reagent composition includes a first set of antibodies, a second set of antibodies, and a third set of antibodies, wherein, the first set of antibodies includes: an anti-CD9 antibody, an anti-GD2 antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD56 antibody, an anti-CD36 antibody, an anti-CD81 antibody, and an anti-CD45 antibody; and the first set of antibodies is to be added to a first flow cytometric tube in which a sample to be assayed is in the form of a single cell suspension, the second set of antibodies includes: an anti-HLA-ABC antibody, an anti-CD38 antibody, an anti-CD19 antibody, an anti-CD56 antibody, an anti-CD36 antibody, an anti-CD7 antibody, and an anti-CD45 antibody; and the second set of antibodies is to be added to a second flow cytometric tube in which a sample to be assayed is in the form of a single cell suspension, the third set of antibodies includes: anti-cytoplasmic cytokeratin antibodies; and the third set of antibodies is to be added to the second flow cytometric tube to which the second set of antibodies has been added and permeabilization has been performed.

10. The method according to claim 9, wherein each antibody is a monoclonal antibody, and wherein the anti-cytoplasmic cytokeratin antibodies are complexes comprising an anti-CK8 monoclonal antibody, an anti-CK18 monoclonal antibody, and an anti-CK19 monoclonal antibody.

11. The method according to claim 9, wherein each antibody is a fluorescein-labeled antibody, and in the first set of antibodies, the anti-CD9 antibody, the anti-GD2 antibody, the anti-CD3 antibody, the anti-CD4 antibody, the anti-CD56 antibody, the anti-CD36 antibody, the anti-CD81 antibody, and the anti-CD45 antibody are fluorescein-labeled with FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, and V500, respectively; in the second set of antibodies, the anti-HLA-ABC antibody, the anti-CD38 antibody, the anti-CD19 antibody, the anti-CD56 antibody, the anti-CD36 antibody, the anti-CD7 antibody, and the anti-CD45 antibody are fluorescein-labeled with PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, and V500, respectively; and in the third set of antibodies, the anti-cytoplasmic cytokeratin antibody is fluorescein-labeled with FITC.

12. The method according to claim 9, wherein the first set of antibodies is a mixture of an anti-CD9 antibody, an anti-GD2 antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD56 antibody, an anti-CD36 antibody, an anti-CD81 antibody, and an anti-CD45 antibody mixed in a volume ratio of 5:5:5:3:2:3:3:3, and the second set of antibodies is a mixture of an anti-HLA-ABC antibody, an anti-CD38 antibody, an anti-CD19 antibody, an anti-CD56 antibody, an anti-CD36 antibody, an anti-CD7 antibody, and an anti-CD45 antibody mixed in a volume ratio of 5:5:3:2:3:3:3.

13. The method according to claim 9, wherein said non-hematopoietic tumor includes one or more of: a malignant tumor of epithelial origin, an embryonal tumor, a soft tissue tumor, an extraosseous sarcoma, a bone tumor, a cartilage tumor, a malignant kidney tumor, and a malignant melanoma.

14. The method according to claim 9, wherein a process for preparing the flow cytometry loading sample for detection of the non-hematopoietic tumor includes the steps of: (1) adding samples to be assayed into two flow cytometric tubes, a first tube and a second tube, respectively, to form a single cell suspension and ensure a cell amount of 110.sup.6 cells/tube to 110.sup.7 cells/tube, (2) adding to the first tube obtained from the treatment in step (1) the first set of antibodies in the reagent composition, adding to the second tube obtained from the treatment in step (1) the second set of antibodies in the reagent composition, and incubating each flow cytometric tube at room temperature in dark, (3) adding a solution of a permeabilization reagent A to the second tube after the incubation in step (2), and continuing the incubation at room temperature in dark, (4) adding 1 hemolysin to the first flow cytometric tube after the incubation in step (2) and adding 1 hemolysin to the second flow cytometric tube after the incubation in step (3), and continuing the incubation at room temperature in dark, (5) centrifuging each flow cytometric tube after the incubation in step (4) and removing the supernatant, (6) adding to the second tube after removing the supernatant in step (5) a solution of a permeabilization reagent B and the third set of antibodies of the reagent composition, and incubating at room temperature in dark, and (7) adding a PBS buffer for washing to the first tube after removing the supernatant in step (5) and to the second tube after the incubation in step (6), respectively, followed by centrifugation, removal of supernatant, and resuspension of cells with a PBS buffer, to obtain the flow cytometry loading sample.

15. The method according to claim 9, wherein the detection of the non-hematopoietic tumor includes screening, diagnosis and/or follow-up detection, and the device comprises a detection unit and an analysis unit.

16. The method according to claim 11, further comprising: wherein, in the flow cytometry assay, the gates for the first tube are set as follows: an adherent cell removal gate P1 is set, and a live cell gate P2 is set within P1 to obtain single live cells; blood cells are each gated within the gate P2 with CD45/SSC antibodies; within the gate P2, a gate NH1 is set with CD45/CD56 antibodies to detect CD45.sup./CD56.sup.+ cells, and a gate NH2 is set with CD45/GD2 antibodies to detect CD45.sup./GD2+ cells; within the gate P2, a gate NH3 is set with CD45/CD36 antibodies to detect CD45.sup./CD36.sup. cells; the expressions of CD3/CD4/CD36/CD9/CD81 in the cells within the gates NH1 and NH2 are displayed; and the expressions of CD9/GD2/CD56/CD81 in the cells within the gate NH3 are displayed; and wherein, in the flow cytometry assay, the gates for the second tube are set as follows: an adherent cell removal gate P1 is set, and a live cell gate P2 is set within P1 to obtain single live cells; blood cells are each gated within the gate P2 with CD45/SSC antibodies; within the gate P2, a gate NH4 is set with CD45/CD56 antibodies to detect CD45.sup./CD56.sup.+ cells, a gate NH5 is set with a CD45/cytoplasmic cytokeratin antibody to detect CD45.sup./cytokeratin.sup.+ cells; within the gate P2, a gate NH6 is set with CD45/CD36 to detect CD45.sup./CD36.sup. cells; the expressions of HLA-ABC/CD38/CD19/CD36/CD7 in the cells within the gates NH4 and NH5 are displayed, and theexpressions of cytokeratin/HLA-ABC/CD38/CD56 in the cells within the gate NH6 are displayed.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIGS. 1 to 2 show the results of flow cytometry gating and analysis of normal bone marrow samples in a specific embodiment of the present invention.

[0067] FIGS. 3 to 4 show the results of flow cytometry gating and analysis of bone marrow samples from neuroblastoma in a specific embodiment of the present invention.

[0068] FIGS. 5 to 6 show the results of flow cytometry gating and analysis of bone marrow samples from epithelial-derived tumors (lung cancer) in a specific embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0069] For better understanding of the technical features, objectives and beneficial effects of the present invention, the technical solutions of the present invention are hereinafter described in details, but it is not to be construed as limitation to the implementable scope of the present invention.

Example 1. Preparation of Reagents

[0070] The combination of antibodies used in this example was:

[0071] A first set of antibody components of: an anti-CD9 antibody, an anti-GD2 antibody, an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD56 antibody, an anti-CD36 antibody, an anti-CD81 antibody, and an anti-CD45 antibody, with each antibody being fluorescein labeled with FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, and V500, respectively; the above eight monoclonal antibody reagents were mixed in a first container in a volume ratio of 5:5:5:3:2:3:3:3.

[0072] A second set of antibody components of: an anti-HLA-ABC antibody, an anti-CD38 antibody, an anti-CD19 antibody, an anti-CD56 antibody, an anti-CD36 antibody, an anti-CD7 antibody, and an anti-CD45 antibody, with each antibody being fluorescein labeled with PE, PerCP-Cy5.5, PE-Cy7, APC, APC-Cy7, BV421, and V500, respectively; the above seven monoclonal antibody reagents were mixed in a second container in a volume ratio of 5:5:3:2:3:3:3.

[0073] A third set of antibody components of: anti-cytoplasmic cytokeratin (CK) antibodies, fluorescein-labeled with FITC, which was contained in a third container.

[0074] Each of the antibodies in this example is commercially available; among them, the anti-cytoplasmic cytokeratin (CK) antibodies are a product of Miltenyi Biotec, Germany (clone number: CK3-6H5), which is a complex composed mainly of CK8, CK18 and CK19 monoclonal antibodies, and the rest of the directly fluorescein-labeled antibodies are products of Becton Dickinson, USA.

[0075] Optionally, haemolysin was prepared and contained in a fourth container, solution A of a permeabilization reagent in a fifth container, solution A of the permeabilization reagent in a sixth container, and PBS buffer in a seventh container. The haemolysin, permeabilization reagent and PBS buffer are commercially available; among them, the cell lysing solution and permeabilization reagent are products from Becton Dickinson, USA, and the PBS buffer is from Beckman Coulter, Inc.

Example 2. Sample Processing

[0076] The samples were processed by using the sets of antibodies of Example 1.

[0077] According to the cell counting results, a sample was added to a first flow cytometric tube to ensure that the amount of cells added was about 2'10.sup.6. Then 29 L of eight different fluorescein-labeled cytosolic monoclonal antibody reagents were added to the flow cytometric tube according to Table 1, mixed thoroughly with the cell suspension and incubated for 15 minutes at room temperature in dark. 3 ml of 1 hemolysin was added and incubated for 10 minutes in dark to lyse the red blood cells. After centrifugation at 1500 rpm for 5 minutes to remove the supernatant, 3 ml of PBS buffer was added and mixed evenly, and the supernatant was removed by centrifugation at 1500 rpm for 5 minutes. Then 0.5 ml PBS buffer was added to resuspend the cells to obtain a processed sample ready for machine assay.

[0078] According to the cell counting results, a sample was added to a second flow cytometric tube to ensure that the amount of cells added was about 210.sup.6. Then 24 L of seven different fluorescein-labeled cytokeratin monoclonal antibody reagents were added to the flow cytometric tube according to Table 1, mixed thoroughly with the cell suspension and incubated for 15 minutes at room temperature in dark. 100 L of a solution A of a permeabilization reagent was added, and incubated for 5 minutes at room temperature in dark. 3 ml of 1 hemolysin was added and incubated for 10 minutes in dark to lyse the red blood cells. After centrifugation at 1500 rpm for 5 minutes to remove the supernatant, 50 L of a solution B of a permeabilization reagent and 50 L, of the cytoplasmic monoclonal antibody reagent cytokeratin (CK)-FITC were added, incubated for 15 minutes at room temperature in dark. Finally, 3 ml of PBS buffer was added and mixed evenly, and the supernatant was removed by centrifugation at 1500 rpm for 5 minutes. Then 0.5 ml PBS buffer was added to resuspend the cells to obtain a processed sample ready for machine assay.

TABLE-US-00001 TABLE 1 Antibody composition for screening/diagnosis and follow-up of non-hematopoietic tumors First tube Second tube First Amount Second Third Amount Fluorescein container (L) container container (L) FITC CD9 5 \ Cytokeratin 5 (CK) PE GD2 5 HLA-ABC \ 5 PerCP-Cy5.5 CD3 5 CD38 \ 5 PE-CY7 CD4 3 CD19 \ 3 APC CD56 2 CD56 \ 2 APC-Cy7 CD36 3 CD36 \ 3 BV421 CD81 3 CD7 \ 3 V500 CD45 3 CD45 \ 3

Example 3. Sample Assaying

[0079] Samples processed according to the method of Example 2 were assayed on a 3-laser 10-color FACS Canto plus flow cytometer from Becton Dickinson, USA, with preferably 1 million cells per tube (at least 300,000 recommended) acquired before the data were analyzed by using a Diva 2.8 software or other software such Kaluza.

[0080] Here, gates for the flow cytometry assay were set as follows: [0081] 1) fixed gating: a single cell gate, a live cell gate, and blood cell gates were sequentially set up; 2) multi-marker combination gating: starting from single live cells, all cells needed to be gated and defined in parallel with blood cell gates; 3) within the gates set with the multi-marker combination, tumor cells were identified when cells that were not normally present appeared; 4) other CD45-negative, CD56-positive or negative lymphohematopoietic tumors, and other possible non-specific staining were excluded, and a diagnosis was made; and 5) differentiation of common subtypes was done based on the expression of GD2 and cytokeratin.

[0082] 1. Fixed gating: consisted of an adherent cell removal gate, live cell gate, and blood cell gates, which were present in a tandem fashion. [0083] Adherent cell removal gate: an adherent cell removal gate (often denoted as P1) was first set by using the area (A) and height (H) from forward scattering (FSC), and the adherent cells could be removed by FSC-Area (A)/Height (H), based on the principle that A and H are positively correlated for a spherical cell (See the first panel in FIGS. 1 to 6). [0084] Live cell gate: for cells within P1, live cell gates (often denoted as P2) were set up by using FSC/side scattering (SSC) to obtain single live cells. The principle of FSC/SSC is that live cells are sub-normally distributed in size and granularity, clustered around a center with clear boundaries from dead cells, apoptotic cells, debris and background noise (see the second panel in FIGS. 1 to 6). [0085] Blood cell gates: Within the single live cell gates (P2 gates), blood cell gates were normally set first with CD45/SSC, for a preliminary observation of lymphocytes, monocytes, granulocytes, nucleated red blood cells, and the presence of obvious tumor cells or abnormal cells. Various populations of blood cells were roughly differentiated by CD45/SSC, based on the principle of the difference in fluorescence intensity of CD45 expression of hematopoietic cells (mature lymphocytes>monocytes>granulocytes>nucleated erythrocytes) and the difference in SSC size (eosinophils>granulocytes>monocytes>mature lymphocytes>nucleated erythrocytes) (see the third panel in FIGS. 1 to 6). The purpose was to prevent missing large tumor cell populations after missed diagnosis or tumor evolution, and also to set up internal control.

[0086] 2. Initial targeting of suspicious cells by multi-marker combination gating: performed in parallel with the CD45/SSC gating, starting with a single live cell (P2). For the first tube, a gate NH1 was set with CD45/CD56 antibodies to detect CD45.sup./CD56.sup.+ cells, which could screen for the vast majority of non-hematopoietic tumors (96.65% based on statistics of the present invention); a gate NH2 was set with CD45/GD2 antibodies to detect CD45.sup./GD2.sup.+ cells, which were mainly found in neuroblastoma and most types of melanomas and were expressed to a varying extent in other bone and soft tissue tumors, small cell lung cancer, and brain tumors. Within the gate P2, for the second tube, a gate NH4 was set with CD45/CD56 antibodies to detect CD45.sup./CD56.sup.+ cells, which could screen for the vast majority of non-hematopoietic tumors, and a gate NH5 was set with CD45/cytoplasmic cytokeratin antibodies detects CD45.sup./cytokeratin.sup.+ cells, which were mainly found in epithelial-derived tumor cells (based on the investigation of cell lines and clinical sample assaying according to the present invention, there was a CD45.sup./cytokeratin.sup.+ positivity rate of 94.59% in tumor cells of epithelial origin).

[0087] 3. Prevention of missed diagnosis by using further universal markers for lymphohematopoietic cells within gate P2: within the gate P2, for both the first and second tubes, CD45/CD36 was used to set a gate NH3 (the first tube) and a gate NH6 (the second tube), respectively, to determine the presence of CD45.sup./CD36.sup. cells; the expressions of CD9/GD2/CD56/CD81 cells in the gate NH3 (the first tube) and cytokeratin/HLA-ABC/CD38/CD56 cells in the gate NH6 (the second tube) were displayed, and the omission of CD45.sup./GD2.sup./CD56.sup./cytokeratin.sup. non-hematopoietic tumor cells was prevented, with the majority of these tumors showing CD81.sup.+ and/or CD9.sup.+ as well as HLA-ABC.sup./CD38.sup./CD36.sup..

[0088] 4. Further exclusion of CD45-negative lymphohematopoietic tumor cells and subtype diagnosis: in the first tube, the expressions of CD3/CD4/CD36/CD9/CD81 in the cells within the gates NH1 and NH2 were displayed, and the following cells were excluded: CD3.sup.+ T cells, CD4.sup.+ T cells, CD4dim monocytes or blastic plasmacytoid dendritic cells, CD36.sup.+ nucleated red blood cells, megakaryocytes and monocytes; in the second tube, the expressions of HLA-ABC/CD38/CD19/CD36/CD7 in the cells within the gates NH4 and NH5 were displayed, and CD38-positive benign and malignant plasma cells, various acute leukemia cells and the like, CD19.sup.+ B cells and/or plasma cells, CD36.sup.+ nucleated erythrocytes, megakaryocytes and monocytes, CD7.sup.+ T cells, NK cells and other dendritic cell tumors with abnormal CD7 expression and acute myeloid leukemia and the like were excluded; all of these lymphohematopoietic cells with 99% or more as shown by experimental data of the present invention, express HLA-ABC almost exclusively, except for nucleated erythrocytes where 95% or more expresses CD36. Determination of subtypes was made based on the GD2 expression in the first tube and the cytokeratin expression in the second tube: GD2 was mainly found in neuroblastoma as well as some melanomas and other embryonal tumors, and cytokeratin was mainly found in non-hematopoietic tumors of epithelial origin.

[0089] In the present invention, by comparing each cell population as shown with the corresponding normal cells within the multi-marker combination gates, tumor cells are identified, lymphohematopoietic tumors are excluded, and the nature of the non-hematopoietic tumors was determined.

[0090] In short, the present invention covers as many common and rare non-hematopoietic tumors of all ages as possible with the multi-marker combination gating, and universal markers for lymphohematopoietic tumors are identified, and lymphohematopoietic tumors and non-specific staining are ingeniously excluded by using these markers, and missed diagnosis of non-hematopoietic tumors that do not express GD2 and cytokeratin is prevented. Here, the non-hematopoietic tumors are most types of solid tumors other than lymphohematopoietic tumors as published by the World Health Organization, including: tumors of malignant epithelial origin (e.g., lung cancer, breast cancer, gallbladder cancer, colon cancer, prostate cancer, esophageal cancer, fallopian tube cancer, head and neck cancer, liver blastoma, pancreaticoblastoma, pancreatic cancer, etc.), embryonal tumors (neuroblastoma, retinoblastoma, germ cell tumor, medulloblastoma, primitive neuroectodermal tumor (PNET), etc.), soft tissue tumors and extraosseous sarcomas (Ewing sarcoma, rhabdomyosarcoma, etc.), bone tumors and cartilage tumors (osteosarcoma, soft tissue sarcoma, etc.), malignant kidney tumors (nephroblastoma, adrenal carcinoma, renal clear cell carcinoma, etc.), malignant melanoma and the like.

[0091] Specifically, FIGS. 1-2: normal bone marrow sample from the same case, with the 2 tubes analyzed together.

[0092] More specifically, FIG. 1 shows the analysis of the normal bone marrow sample in the first tube. The following was done sequentially: 1) P1 was set as an adherent cell removal gate with FSC-A/H to obtain single cells in P1; 2) P2 was set as a live cell gate within P1 by showing FSC/SSC to obtain single live cells in P2; 3) within the gate P2, blood cell gates were set with CD45/SSC to obtain lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NRBC), and eosinophil (eo) gates; 4) within the gate P2, a gate NH1 was set with CD45/CD56 to screen CD45.sup./CD56.sup.+ tumor cells, which were absent in this normal sample; 5) a gate NH2 was set with CD45/GD2 antibodies to detect CD45.sup./GD2.sup.+ cells, which were absent in this normal sample; 6) within the gate P2, a gate NH3 was set with CD45/CD36 to determine the presence of CD45.sup./CD36.sup. cells, which were absent in this normal sample; 7) the expressions of CD3/CD4/CD36/CD9/CD81 in the cells within the gates NH1 and NH2 were displayed, and CD3.sup.+ T cells, CD4.sup.+ T cells, CD4dim monocytes or blastic plasmacytoid dendritic cells, CD36.sup.+ nucleated erythrocytes, megakaryocytes and monocytes were excluded, which were absent in this normal sample; 8) the expressions of CD9/GD2/CD56/CD81 in the cells within the gate NH3 were displayed, the omission of CD45.sup./GD2.sup./CD56.sup./CD36.sup. non-hematopoietic tumor cells was prevented; most of these tumors express CD81.sup.+ and/or CD9.sup.+, which was absent in this normal sample.

[0093] Specifically, FIG. 2 shows the analysis of the normal bone marrow sample in the second tube (in the figure, cytokeratin is abbreviated as CK). The following was done sequentially: 1) P1 was set as an adherent cell removal gate with FSC-A/H to obtain single cells in P1; 2) P2 was set as a live cell gate within P1 by showing FSC/SSC to obtain single live cells in P2; 3) within the gate P2, blood cell gates were set with CD45/SSC to obtain lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NRBC), and eosinophil (eo) gates; 4) within the gate P2, a gate NH4 was set with CD45/CD56 antibodies to detect CD45.sup./CD56.sup.+ tumor cells, which were absent in this normal sample; 5) within the gate P2, a gate NH5 was set with CD45/cytoplasmic cytokeratin antibodies to detect CD45.sup./cytokeratin.sup.+ cells, which were absent in this normal sample; 6) within the gate P2, a gate NH6 was set with CD45/CD36 to determine the presence of CD45.sup./CD36.sup. cells, which were absent in this normal sample; 7) the expressions of HLA-ABC/CD38/CD19/CD36/CD7 in the cells within the gates NH4 and NH5 were displayed, and CD38-positive benign and malignant plasma cells, various acute leukemia cells and the like, CD19.sup.+ B cells and/or plasma cells, CD36.sup.+ nucleated erythrocytes, megakaryocytes and monocytes, CD7.sup.+ T cells, NK cells and other dendritic cell tumors with abnormal CD7 expression and acute myeloid leukemia and the like were excluded, which were absent in this normal sample; 8) the expressions of cytokeratin/HLA-ABC/CD38/CD56 in the cells within the gate NH6 were displayed, the omission of CD45.sup./cytokeratin.sup./CD56.sup./CD36.sup. non-hematopoietic tumor cells was prevented, which was absent in this normal sample.

[0094] Specifically, FIGS. 3-4 show the immunotyping in the first and second tubes of the bone marrow sample from the same neuroblastoma patient.

[0095] Specifically, FIG. 3 shows the analysis of the first tube of the bone marrow sample from the neuroblastoma patient. The following was done sequentially: 1) P1 was set as an adherent cell removal gate with FSC-A/H to obtain single cells in P1; 2) P2 was set as a live cell gate within P1 by showing FSC/SSC to obtain single live cells in P2; 3) within the gate P2, blood cell gates were set with CD45/SSC to obtain lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NRBC), and eosinophil (eo) gates; 4) within the gate P2, a gate NH1 was set with CD45/CD56 to screen CD45.sup./CD56.sup.+ tumor cells, and 13.2% of the cells (in nucleated cells, dark gray cell population) expressing CD45.sup./CD56.sup.+ were found in this sample; 5) a gate NH2 was set with CD45/GD2 antibodies to detect CD45.sup./GD2.sup.+ cells, and 13.2% of the cells (in nucleated cells, dark gray cell population) expressing CD45.sup./GD2.sup.+ were found in this sample; 6) within the gate P2, a gate NH3 was set with CD45/CD36 to determine the presence of CD45.sup./CD36.sup. cells, and 13.2% of the cells (in nucleated cells, dark gray cell population) was found CD45.sup./CD36.sup. in this sample; 7) the expressions of CD3/CD4/CD36/CD9/CD81 in the cells within the gates NH1 and NH2 were displayed, and CD3.sup.+ T cells, CD4.sup.+ T cells, CD4dim monocytes or blastic plasmacytoid dendritic cells, CD36.sup.+ nucleated erythrocytes, megakaryocytes and monocytes were excluded, and 13.2% of the cells (in nucleated cells, dark gray cell population) not expressing CD3, CD4, and CD36, but expressing CD9 and CD81, was found in this sample; 8) the expressions of CD9/GD2/CD56/CD81 in the cells within the gate NH3 were displayed, and the omission of CD45.sup./GD2.sup./CD56.sup./CD36.sup. non-hematopoietic tumor cells was prevented; 13.2% of cells (in nucleated cells, dark gray cell population) not expressing CD3, CD4, CD36, and CD45, but expressing CD9, CD81, GD2, and CD56, were found in this sample as non-hematopoietic tumor cells, with a high possibility of neuroblastoma, and the diagnosis was confirmed in combination with clinical and other laboratory assays.

[0096] Specifically, FIG. 4 shows the analysis of the second tube of the same bone marrow sample as in FIG. 3 (in the figure, cytokeratin is abbreviated as CK). The following was done sequentially: 1) P1 was set as an adherent cell removal gate with FSC-A/H to obtain single cells in P1; 2) P2 was set as a live cell gate within P1 by showing FSC/SSC to obtain single live cells in P2; 3) within the gate P2, blood cell gates were set with CD45/SSC to obtain lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NRBC), and eosinophil (eo) gates; 4) within the gate P2, a gate NH4 was set with CD45/CD56 antibodies to detect CD45.sup./CD56.sup.+ tumor cells, and 13.2% of the cells (in nucleated cells, dark gray cell population) expressing CD45.sup./CD56.sup.+ were found in this sample; 5) within the gate P2, a gate NH5 was set with CD45/cytoplasmic cytokeratin antibodies to detect CD45.sup./cytokeratin.sup.+ cells, which were absent in this sample; 6) within the gate P2, a gate NH6 was set with CD45/CD36 to determine the presence of CD45.sup./CD36.sup. cells, and 13.2% of the cells (in nucleated cells, dark gray cell population) expressing CD45.sup./CD36.sup. were found in this sample; 7) the expressions of HLA-ABC/CD38/CD19/CD36/CD7 in the cells within the gates NH4 and NH5 were displayed, and CD38-positive benign and malignant plasma cells, various acute leukemia cells and the like, CD19.sup.+ B cells and/or plasma cells, CD36.sup.+ nucleated erythrocytes, megakaryocytes and monocytes, CD7.sup.+ T cells, NK cells and other dendritic cell tumors with abnormal CD7 expression and acute myeloid leukemia and the like were excluded; 13.2% of the cells (in nucleated cells, dark gray cell population) not expressing HLA-ABC, CD38, CD19, CD36, and CD7 were found in this sample; 8) the expressions of cytokeratin/HLA-ABC/CD38/CD56 in the cells within the gate NH6 were displayed, and the omission of CD45.sup./cytokeratin.sup./CD56.sup./CD36.sup. non-hematopoietic tumor cells was prevented; 13.2% of the cells (in nucleated cells, dark gray cell population) not expressing cytokeratin, HLA-ABC, CD38, CD19, CD36, CD7, and CD45, but expressing CD56, were found in this sample as non-hematopoietic tumor cells, with a high possibility of neuroblastoma, and the diagnosis was confirmed in combination with clinical and other laboratory assays.

[0097] Specifically, FIGS. 5-6 show the immunotyping in the first and second tubes of the bone marrow sample from the same patient with non-hematopoietic tumor of epithelial origin (lung cancer).

[0098] Specifically, FIG. 5 shows the analysis of the bone marrow sample from the patient with non-hematopoietic tumor of epithelial origin (lung cancer) in the first tube. The following was done sequentially: 1) P1 was set as an adherent cell removal gate with FSC-A/H to obtain single cells in P1; 2) P2 was set as a live cell gate within P1 by showing FSC/SSC to obtain single live cells in P2; 3) within the gate P2, blood cell gates were set with CD45/SSC to obtain lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NRBC), and eosinophil (eo) gates; 4) within the gate P2, a gate NH1 was set with CD45/CD56 to screen CD45.sup./CD56.sup.+ tumor cells, and 94.10% of the cells (in nucleated cells, dark gray cell population) expressing CD45.sup./CD56.sup.+ were found in this sample; 5) a gate NH2 was set with CD45/GD2 antibodies to detect CD45.sup./GD2.sup.+ cells, which was not present in this sample; 6) within the gate P2, a gate NH3 was set with CD45/CD36 to determine the presence of CD45.sup./CD36.sup. cells, and 94.10% of the cells (in nucleated cells, dark gray cell population) was found CD45.sup./CD36.sup. in this sample; 7) the expressions of CD3/CD4/CD36/CD9/CD81 in the cells within the gates NH1 and NH2 were displayed, and CD3.sup.+ T cells, CD4.sup.+ T cells, CD4dim monocytes or blastic plasmacytoid dendritic cells, CD36.sup.+ nucleated erythrocytes, megakaryocytes and monocytes were excluded, and 94.10% of the cells (in nucleated cells, dark gray cell population) not expressing CD3, CD4, and CD36, but expressing CD9 and CD81, was found in this sample; 8) the expressions of CD9/GD2/CD56/CD81 in the cells within the gate NH3 were displayed, and the omission of CD45.sup./GD2.sup./CD56.sup./CD36.sup. non-hematopoietic tumor cells was prevented; 94.10% of cells (in nucleated cells, dark gray cell population) not expressing CD3, CD4, CD36, GD2, and CD45, but expressing CD9, CD81, and CD56, were found in this sample as non-hematopoietic tumor cells.

[0099] Specifically, FIG. 6 shows the analysis of the bone marrow sample from the same patient with non-hematopoietic tumor of epithelial origin (lung cancer) as FIG. 5 in the second tube (in the figure, cytokeratin is abbreviated as CK). The following was done sequentially: 1) P1 was set as an adherent cell removal gate with FSC-A/H to obtain single cells in P1; 2) P2 was set as a live cell gate within P1 by showing FSC/SSC to obtain single live cells in P2; 3) within the gate P2, blood cell gates were set with CD45/SSC to obtain lymphocytes (lym), granulocytes (gra), monocytes (mono), nucleated erythrocytes (NRBC), and eosinophil (eo) gates; 4) within the gate P2, a gate NH4 was set with CD45/CD56 antibodies to detect CD45.sup./CD56.sup.+ tumor cells, and 94.10% of the cells (in nucleated cells, dark gray cell population) expressing CD45.sup./CD56.sup.+ were found in this sample; 5) within the gate P2, a gate NH5 was set with CD45/cytoplasmic cytokeratin antibodies to detect CD45.sup./cytokeratin.sup.+ cells, and 94.10% of the cells (in nucleated cells, dark gray cell population) expressing CD45.sup./cytokeratin.sup.+ were found in this sample; 6) within the gate P2, a gate NH6 was set with CD45/CD36 to determine the presence of CD45.sup./CD36.sup. cells, and 94.10% of the cells (in nucleated cells, dark gray cell population) expressing CD45.sup./CD36.sup. were found in this sample; 7) the expressions of HLA-ABC/CD38/CD19/CD36/CD7 in the cells within the gates NH4 and NH5 were displayed, and CD38-positive benign and malignant plasma cells, various acute leukemia cells and the like, CD19.sup.+ B cells and/or plasma cells, CD36.sup.+ nucleated erythrocytes, megakaryocytes and monocytes, CD7.sup.+ T cells, NK cells and other dendritic cell tumors with abnormal CD7 expression and acute myeloid leukemia and the like were excluded; 94.10% of the cells (in nucleated cells, dark gray cell population) not expressing HLA-ABC, CD38, CD19, CD36, and CD7 were found in this sample; 8) the expressions of cytokeratin/HLA-ABC/CD38/CD56 in the cells within the gate NH6 were displayed, and the omission of CD45.sup./cytokeratin.sup./CD56.sup./CD36.sup. non-hematopoietic tumor cells was prevented; 94.10% of the cells (in nucleated cells, dark gray cell population) not expressing HLA-ABC, CD38, CD19, CD36, CD7, and CD45, but expressing cytokeratin and CD56, were found in this sample as non-hematopoietic tumor cells, with a high possibility of epithelial origin, and a bone marrow involvement of lung cancer was contemplated in combination with clinical history.

[0100] The process of development of this protocol and clinical validation by using the method of this example: this study was started at the Hebei Yanda Lu Daopei Hospital in 2008, and by Mar. 31, 2022, 858 samples from 850 individuals were assayed: 476 males and 374 females, with a median age of 4 (aged 0 to 80); 792 of them were of age 14 or younger, and 58 were older than 14 years old. Of the 858 samples, 617 were bone marrow samples, 3 were peripheral blood samples, 196 were cerebrospinal fluid samples, 9 were thoracoabdominal fluid samples, 3 were lymph node dissection samples, and 30 were various tissue puncture samples. Among them, 575 showed positive results and 283 showed negative results, which were 90% or more consistent with other laboratory assays and clinical diagnosis.

[0101] The assay results of the 575 positive samples were analyzed, of which 8 were follow-up samples at different time points, and the one with highest percentage was selected for analysis. Thus, a total of 567 samples from 567 individuals were analyzed, 319 males and 248 females, with a median age of 4 (aged 0-80). The median tumor cell ratio was 0.16% (0.01%-96.23%), for 442 bone marrow samples, 2 peripheral blood samples, 84 cerebrospinal fluid samples, 9 thoracoabdominal fluid samples, 2 lymph node dissection samples, and 28 of various tissue puncture materials. Almost all tumor types indicated in WHO diagnostic criteria were covered (see Table 2).

TABLE-US-00002 TABLE 2 Types of diseases involved in assayed positive samples for non-hematopoietic tumors at Lu Dao Pei Hospital from 2008 to March 2022 Number Type Subtype of cases Malignant tumors of Lung Cancer 10 epithelial origin Breast cancer, gallbladder cancer, 36 colon cancer, prostate cancer, esophageal cancer, fallopian tube cancer, head and neck cancer, hepatoblastoma, pancreatoblastoma, pancreatic cancer, etc. Embryonal tumors Neuroblastoma 381 Retinoblastoma 71 Germ cell tumor 1 Medulloblastoma 9 Primitive neuroectodermal tumor 7 (PNET), etc. Soft tissue tumors and Ewing Sarcoma 6 extraosseous sarcomas Rhabdomyosarcoma 16 Other 3 Bone and cartilage 3 tumors Malignant kidney tumors 16 Malignant melanoma 8

[0102] Immunophenotypic analysis of the 567 samples (Table 3): the predominant phenotypes were CD45 negative (100%), CD56 positive (96.65%), CD36 negative (100%), HLA-ABC negative (93.10%), CD81 positive (98.52%), and CD38 negative (95.40%), with relatively poor sensitivity of CD9 and CD99 of 75% and 58.86%, respectively. The rate of expression of other specific lymphohematopoietic markers, CD4, CD3, CD19, and CD7, was all zero. CD45.sup./CD56.sup.+ had a 96.65% coverage, with CD56-negative cases mainly seen in tumor cells of epithelial origin, and the expression rate of cytokeratin (CK) in this type was up to 94.59%. GD2 expression in neuroblastoma was 93.21%, but the specificity for the differentiation of subtypes was relatively poor, with a positive rate of 76.7% in other subtypes.

TABLE-US-00003 TABLE 3 Immunophenotypic analysis of non-hematopoietic tumors of 567 cases HLA-ABC CD56 CD45 CD81 CD9 Total positive 6.9% 96.65% 0 98.52% 75% rate (30/434) (548/567) (0/567) (533/541) (384/512) Positive rate of 6.13% 99.21% 0 98.66% 78.93% neuroblastoma (20/326) (378/381) (0/381) (368/373) (281/356) Positive rate of 8.82% 55.88% 0 88.89% 70.59% tumors of (3/34) (19/34) (0/46) (16/18) (12/17) epithelial origin Positive rate of 9.46% 99.34% 0 99.33% 65.47% other subtypes (7/74) (151/152) (0/140) (149/150) (91/139) GD2 cytokeratin CD38 CD99 CD36 Total positive 85.71% 8.95% 4.6% 58.86% 0 rate (342/399) (35/391) (6/130) (206/350) (0/144) Positive rate of 93.21% 0 5.62% 557.03% 0 neuroblastoma (261/280) (0/284) (5/89) (142/249) (0/77) Positive rate of 12.5% 94.59% 0 75% 0 tumors of (2/16) (35/37) (0/12) (6/8) (0/13) epithelial origin Positive rate of 76.7% 0 3.45% 62.37% 0 other subtypes (79/103) (0/71) (1/29) (58/93) (0/54)

[0103] To further analyze the expression of these markers in the lymphohematopoietic system, 72 additional non-tumor bone marrow samples were additionally selected for the detection of the above markers, and CD45.sup./CD56.sup.+ or GD2.sup.+ or cytokeratin.sup.+ or CD45.sup./CD56.sup./GD2.sup./cytokeratin.sup./CD36.sup./HLA-ABC.sup. cells were not present in any of these samples.

[0104] Compared with the studies of the prior art, an important breakthrough of the present invention is the finding of non-lineage-specific markers that are very highly expressed in lymphohematopoietic tumors but rarely expressed in non-hematopoietic ones for screening and differential diagnosis, mainly including HLA-ABC, CD36, and CD38, based on the wide variety of types and big phenotypic differences in the diagnosis of non-hematopoietic tumors.

[0105] In order to further validate the efficiency of HLA-ABC identification among the specific markers of the present invention, the present inventors selected samples that had been immunophenotyped in the flow cytometry laboratory at the Hebei Yanda Lu Daopei Hospital from Aug. 1, 2016 to Oct. 30, 2021, for an HLA-ABC expression rate study. The samples included: 272 non-tumor samples, with 146 males and 126 females having a median age of 31 (aged 0-87), in which 268 were bone marrow samples, 1 was a peripheral blood sample, and 3 was tissue samples; 869 lymphohematopoietic tumors, with 544 males and 325 females having a median age of 28 (aged 0-85), in which 845 were bone marrow samples, 19 were peripheral blood samples, 7 were tissue puncture samples, and 1 was a thoracoabdominal fluid sample. All the diseases in the WHO lymphohematopoietic tumor category were covered (1 case of acute undifferentiated leukemia, 14 cases of mixed phenotype acute leukemia, 258 cases of acute B-lymphoblastic leukemia, 417 cases of acute myeloid leukemia, 83 cases of acute T-lymphoblastic leukemia, 10 cases of myeloproliferative neoplasm, 44 cases of myelodysplastic syndrome, 6 cases of myelodysplastic syndrome/myeloproliferative neoplasms, 11 cases of multiple myeloma, 18 cases of mature B-cell lymphoma, 5 cases of mature T-cell lymphoma, and 2 cases of mature NK-cell lymphoma. The HLA-ABC expression rate was found to be 100% in normal hematopoietic cells, except for nucleated red blood cells which did not express HLA-ABC, and the intensity of the expression was consistently high; in lymphohematopoietic tumors, the HLA-ABC loss rate was 0.12% (1/869).

[0106] Exploration was started in 2008 for the present invention, and a definitive protocol was established in October 2020. 192 patients have been assayed so far, with 130 positive and 62 negative cases, and it was found that screening/diagnosis and follow-up of non-hematopoietic tumors by using the present invention had a coverage and specificity of 99% or more and a sensitivity of 0.01%. The false-positive and false-negative rates were <1%. The non-hematopoietic tumors covered by the present invention refer to the vast majority of types of solid tumors other than lymphohematopoietic tumors published by the World Health Organization, including: tumors of malignant epithelial origin (such as lung cancer, breast cancer, gallbladder cancer, colon cancer, prostate cancer, esophageal cancer, fallopian tube cancer, head and neck cancer, hepatoblastoma, pancreatoblastoma, pancreatic cancer, etc.), embryonal tumors (neuroblastoma, retinoblastoma, germ cell tumor, medulloblastoma, primitive neuroectodermal tumor (PNET), etc.), soft tissue tumors and extraosseous sarcoma (Ewing sarcoma, rhabdomyosarcoma, etc.), bone tumors and cartilage tumors (osteosarcoma, soft tissue sarcoma, etc.), malignant kidney tumors (nephroblastoma, adrenal carcinoma, renal clear cell carcinoma, etc.), malignant melanoma and the like. Above all, with regard to the general field of clinical diagnosis of tumors by flow cytometry, the present invention provides an important protocol of detection and analysis that has a high coverage, great practicality, high sensitivity and high specificity, and can reduce the rate of missed diagnosis.