| CPC G01N 33/57492 (2013.01) [G01N 2333/70503 (2013.01); G01N 2333/7051 (2013.01); G01N 2333/70514 (2013.01); G01N 2333/70589 (2013.01); G01N 2333/70596 (2013.01)] | 4 Claims |
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1. A method for detecting a non-hematopoietic tumor by flow cytometry, the method comprising:
STEP I: preparing a flow cytometry loading sample for detection of the non-hematopoietic tumor by processing a cell sample using a reagent composition,
wherein the reagent composition comprises a first set of antibodies, a second set of antibodies, and a third set of antibodies, wherein the antibodies in each set are fluorescently-labeled monoclonal antibodies; wherein,
the first set of antibodies consists of: an anti-CD9 antibody labeled with FITC, an anti-disialoganglioside 2 (GD2) antibody labeled with PE, an anti-CD3 antibody labeled with PerCP-Cy5.5, an anti-CD4 antibody labeled with PE-Cy7, an anti-CD56 antibody labeled with APC, an anti-CD36 antibody labeled with APC-Cy7, an anti-CD81 antibody labeled with BV421, and an anti-CD45 antibody labeled with V500; and the first set of antibodies is to be added to a first flow cytometric tube in which the cell sample to be assayed is in the form of a single cell suspension,
the second set of antibodies consists of: an anti-human leukocyte antigen (HLA)-ABC antibody labeled with PE, an anti-CD38 antibody labeled with PerCP-Cy5.5, an anti-CD19 antibody labeled with PE-Cy7, an anti-CD56 antibody labeled with APC, an anti-CD36 antibody labeled with APC-Cy7, an anti-CD7 antibody labeled with BV421, and an anti-CD45 antibody labeled with V500; and the second set of antibodies is to be added to a second flow cytometric tube in which the cell sample to be assayed is in the form of a single cell suspension,
the third set of antibodies consists of: anti-cytoplasmic cytokeratin antibodies labeled with FITC; and the third set of antibodies is to be added to the second flow cytometric tube in which the second set of antibodies has been added and cell sample permeabilization has been performed;
wherein preparing the flow cytometry loading sample for detection of the non-hematopoietic tumor comprises the steps of:
(1) adding the cell sample to be assayed into two flow cytometric tubes, the first flow cytometric tube and the second flow cytometric tube, respectively, to form a single cell suspension in each tube and ensure a cell amount of 1×106 cells/tube to 1×107 cells/tube; wherein the cell sample to be assayed is bone marrow, peripheral blood or a tissue cell sample that can be prepared as single live cells suitable for flow cytometry assay;
(2) adding to the first flow cytometric tube obtained from step (1) the first set of antibodies in the reagent composition, adding to the second flow cytometric tube obtained from step (1) the second set of antibodies in the reagent composition, and incubating each flow cytometric tube at room temperature in the dark;
(3) adding a solution of a permeabilization reagent A to the second flow cytometric tube after the incubation in step (2) under conditions sufficient to permeabilize the cells in the cell sample, and continuing the incubation at room temperature in the dark;
(4) adding 1× hemolysin to the first flow cytometric tube after the incubation in step (2) under conditions sufficient to hemolyse the cells in the cell sample and adding 1× hemolysin to the second flow cytometric tube after the incubation in step (3) under conditions sufficient to hemolyse the cells in the cell sample, and continuing the incubation at room temperature in the dark;
(5) centrifuging each flow cytometric tube after the incubation in step (4) and removing the supernatant;
(6) adding to the second flow cytometric tube after removing the supernatant in step (5) a solution of a permeabilization reagent B under conditions sufficient to permabilize the cells in the cell sample and the third set of antibodies of the reagent composition, and incubating at room temperature in dark; and
(7) adding a phosphate buffered saline (PBS) buffer for washing to the first flow cytometric tube after removing the supernatant in step (5) and to the second flow cytometric 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; and
STEP II: performing a flow cytometry assay, wherein, in the flow cytometry assay:
(i) the gates for the first flow cytometric 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−/CD56+ cells, and a gate NH2 is set with CD45/GD2antibodies to detect CD45−/GD2+ cells; within the gate P2, a gate NH3 is set with CD45/CD36 antibodies to detect CD45−/CD36+ 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
ii) the gates for the second flow cytometric 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−/CD56+ cells, a gate NH5 is set with a CD45/cytoplasmic cytokeratin antibody to detect CD45−/cytokeratin+ cells; within the gate P2, a gate NH6 is set with CD45/CD36 to detect CD45−/CD36+ cells; the expressions of HLA-ABC/CD38/CD19/CD36/CD7 in the cells within the gates NH4 and NH5 are displayed, and the expressions of cytokeratin/HLA-ABC/CD38/CD56 in the cells within the gate NH6 are displayed;
wherein said non-hematopoietic tumor comprises cells of 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.
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