ANTIBODY COMBINATION FOR SUBSTITUTING SIDE SCATTER SIGNAL IN MASS CYTOMETRY HEMATOLOGIC TUMOR IMMUNOPHENOTYPING AND USE THEREOF

Abstract

The present disclosure discloses an antibody combination for substituting a side scatter signal in mass cytometry hematologic tumor immunophenotyping, including a Lactoferrin antibody and a Lysozyme antibody. The present disclosure also discloses a gating method for mass cytometry hematologic tumor immunophenotyping. The present disclosure also discloses a kit for mass cytometry hematologic tumor immunophenotyping. According to the present disclosure, the Lactoferrin antibody and the Lysozyme antibody are used for the first time, are combined with a CD45 antibody for two-stage gating strategy, and are combined with a mass cytometer to substitute traditional flow cytometry CD45/SSC to distinguish mature granulocytes, monocytes, nucleated red blood cells, lymphocytes, primitive and juvenile cells, and abnormal cell subsets in bone marrow. Combined with the multi-parameter high-throughput characteristics of the mass cytometry, the present disclosure can improve the depth of the current hematologic tumor immunophenotyping.

Claims

1. An antibody combination for substituting a side scatter signal in mass cytometry hematologic tumor immunophenotyping, comprising a Lactoferrin antibody and a Lysozyme antibody, the Lactoferrin antibody and the Lysozyme antibody having metal tags respectively, and the metal tags of the Lactoferrin antibody and the Lysozyme antibody being different.

2. The antibody combination for substituting a side scatter signal in mass cytometry hematologic tumor immunophenotyping according to claim 1, wherein the metal tag is selected from 89Y, 115In, 139La, 141Pr, 142Nd, 143Nd, 144Nd, 145Nd, 146Nd, 147Sm, 148Nd, 149Sm, 150Nd, 151Eu, 152Sm, 153Eu, 154Sm, 155Gd, 156Gd, 157Gd, 158Gd, 159Tb, 160Gd, 161Dy, 162Dy, 163Dy, 164Dy, 165Ho, 166Er, 167Er, 168Er, 169Tm, 170Er, 171Yb, 172Yb, 173Yb, 174Yb, 175Lu, 176Yb, 195Pt, 197Au, 198Pt, and 209Bi.

3. Use of the antibody combination according to claim 1 in mass cytometry hematologic tumor immunophenotyping.

4. The use according to claim 3, comprising the following steps: (1) distinguishing a mature granulocyte subset, a monocyte subset and other cell subsets by the Lactoferrin antibody and the Lysozyme antibody; (2) distinguishing the other cell subsets by a CD45 antibody, comprising a primitive and juvenile cell subset or/and an abnormal cell subset, a nucleated red blood cell subset, and a lymphocyte subset; and (3) analyzing expression of antigens of related subsets by other common hematologic tumor immunophenotyping antibodies to determine whether there is abnormal expression of the antigens of related subsets, wherein the Lactoferrin antibody, the Lysozyme antibody, the CD45 antibody, and the other common hematologic tumor immunophenotyping antibodies have metal tags respectively, and the metal tags of the antibodies are different.

5. The use according to claim 4, wherein the metal tag is selected from 89Y, 115In, 139La, 141Pr, 142Nd, 143Nd, 144Nd, 145Nd, 146Nd, 147Sm, 148Nd, 149Sm, 150Nd, 151Eu, 152Sm, 153Eu, 154Sm, 155Gd, 156Gd, 157Gd, 158Gd, 159Tb, 160Gd, 161Dy, 162Dy, 163Dy, 164Dy, 165Ho, 166Er, 167Er, 168Er, 169Tm, 170Er, 171Yb, 172Yb, 173Yb, 174Yb, 175Lu, 176Yb, 195Pt, 197Au, 198Pt, and 209Bi.

6. Use of the antibody combination according to claim 2 in mass cytometry hematologic tumor immunophenotyping.

7. The use according to claim 6, comprising the following steps: (1) distinguishing a mature granulocyte subset, a monocyte subset and other cell subsets by the Lactoferrin antibody and the Lysozyme antibody; (2) distinguishing the other cell subsets by a CD45 antibody, comprising a primitive and juvenile cell subset or/and an abnormal cell subset, a nucleated red blood cell subset, and a lymphocyte subset; and (3) analyzing expression of antigens of related subsets by other common hematologic tumor immunophenotyping antibodies to determine whether there is abnormal expression of the antigens of related subsets, wherein the Lactoferrin antibody, the Lysozyme antibody, the CD45 antibody, and the other common hematologic tumor immunophenotyping antibodies have metal tags respectively, and the metal tags of the antibodies are different.

8. The use according to claim 7, wherein the metal tag is selected from 89Y, 115In, 139La, 141Pr, 142Nd, 143Nd, 144Nd, 145Nd, 146Nd, 147Sm, 148Nd, 149Sm, 150Nd, 151Eu, 152Sm, 153Eu, 154Sm, 155Gd, 156Gd, 157Gd, 158Gd, 159Tb, 160Gd, 161Dy, 162Dy, 163Dy, 164Dy, 165Ho, 166Er, 167Er, 168Er, 169Tm, 170Er, 171Yb, 172Yb, 173Yb, 174Yb, 175Lu, 176Yb, 195Pt, 197Au, 198Pt, and 209Bi.

9. A gating method for mass cytometry hematologic tumor immunophenotyping, comprising the following steps: (1) distinguishing a mature granulocyte subset, a monocyte subset and other cell subsets by the Lactoferrin antibody and the Lysozyme antibody; (2) distinguishing the other cell subsets by a CD45 antibody, comprising a primitive and juvenile cell subset or/and an abnormal cell subset, a nucleated red blood cell subset, and a lymphocyte subset; and (3) analyzing expression of antigens of related subsets by other common hematologic tumor immunophenotyping antibodies to determine whether there is abnormal expression of the antigens of related subsets, wherein the Lactoferrin antibody, the Lysozyme antibody, the CD45 antibody, and the other common hematologic tumor immunophenotyping antibodies have metal tags respectively, and the metal tags of the antibodies are different.

10. The gating method for mass cytometry hematologic tumor immunophenotyping according to claim 9, wherein the metal tag is selected from 89Y, 115In, 139La, 141Pr, 142Nd, 143Nd, 144Nd, 145Nd, 146Nd, 147Sm, 148Nd, 149Sm, 150Nd, 151Eu, 152Sm, 153Eu, 154Sm, 155Gd, 156Gd, 157Gd, 158Gd, 159Tb, 160Gd, 161Dy, 162Dy, 163Dy, 164Dy, 165Ho, 166Er, 167Er, 168Er, 169Tm, 170Er, 171Yb, 172Yb, 173Yb, 174Yb, 175Lu, 176Yb, 195Pt, 197Au, 198Pt, and 209Bi.

11. A kit for mass cytometry hematologic tumor immunophenotyping, consisting of 43 monoclonal antibodies with metal tags, as shown in the following table: TABLE-US-00003 No. Antibody Metal 1 cCD3 89Y 2 CD3 115ln 3 cIgM 139La 4 CD56 141Pr 5 CD22 142Nd 6 CD235ab 143Nd 7 CD61 144Nd 8 CD23 145Nd 9 CD5 146Nd 10 CD15 147Sm 11 CD33 148Nd 12 MPO 149Sm 13 CD14 150Nd 14 λ 151Eu 15 CD13 152Sm 16 CD41 153Eu 17 Lactoferrin 154Sm 18 CD123 155Gd 19 CD34 156Gd 20 CD71 157Gd 21 CD19 158Gd 22 CD9 159Tb 23 κ 160Gd 24 CD99 161Dy 25 CD10 162Dy 26 Lysozyme 163Dy 27 CD64 164Dy 28 CD2 165Ho 29 CD117 166Er 30 CD1a 167Er 31 CD11c 168Er 32 CD45 169Tm 33 CD7 170Er 34 CD79a 171Yb 35 CD38 172Yb 36 CD138 173Yb 37 CD20 174Yb 38 TdT 175Lu 39 HLA-DR 176Yb 40 CD300e 195Pt 41 CD4 197Au 42 CD8 198pt 43 CD11b 209Bi — — — where numbers 1, 3, 12, 14, 17, 23, 26, 34, and 38 are intracellular antibodies, and others are extracellular antibodies.

12. Use of the kit according to claim 11 in mass cytometry hematologic tumor immunophenotyping.

13. The use according to claim 12, comprising the following steps: (1) pre-treating a bone marrow sample to remove mature red blood cells in a bone marrow sample; (2) detecting, by a mass cytometer, expressive abundance of antigens corresponding to 43 antibodies in the bone marrow sample; and (3) analyzing, by flow cytometry software, according to the expressive abundance of the antigens corresponding to 43 antibodies in the bone marrow sample, the flow cytometry software comprising Flowjo analysis software, specifically as follows: (3.1) distinguishing a mature granulocyte subset, a monocyte subset and other cell subsets by the Lactoferrin antibody and the Lysozyme antibody; (3.2) distinguishing the other cell subsets by a CD45 antibody, comprising a primitive and juvenile cell subset or/and an abnormal cell subset, a nucleated red blood cell subset, and a lymphocyte subset; and (3.3) analyzing expression of antigens of related subsets by other antibodies to determine whether there is abnormal expression of the antigens of related subsets.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIGS. 1A-1I show bone marrow cell immunophenotyping of healthy human of Example 1, where:

[0040] in FIG. 1A, Lysozyme and Lactoferrin are used for plotting; the bone marrow sample is divided into three sets, where: the lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets, expressing mature granulocyte markers CD33, CD11b, and CD15; with medium-strength Lactoferrin, the Lysozyme+ cell set is a monocyte subset, expressing monocyte markers CD14 and CD64; and the cell sets with low expression of Lactoferrin and Lysozyme are a nucleated red blood cell subset and a lymphocyte subset;

[0041] in FIG. 1B, the Lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets, expressing mature granulocyte markers CD13 and CD11b;

[0042] in FIG. 1C, the Lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets, expressing mature granulocyte markers CD15 and CD33;

[0043] in FIG. 1D, with medium-strength Lactoferrin, the Lysozyme+ cell set is a monocyte subset, expressing monocyte markers CD14 and CD64;

[0044] in FIG. 1E, the cell set with low expression of Lactoferrin and Lysozyme is gated for a next level using CD45 to obtain a CD45+ lymphocyte subset and a CD45− nucleated red blood cell subset;

[0045] in FIG. 1F, CD45+ lymphocytes are grouped using CD19 and CD3 to obtain CD3+CD19− T cells, CD3−CD19+ B cells, and CD3−CD19− NK cells;

[0046] in FIG. 1G, CD3−CD19+ B cells are grouped using κ and λ to obtain κ+ B cells and λ+ B cells;

[0047] in FIG. 1H: CD3+CD19− T cells are grouped using CD4 and CD8 to obtain CD3+CD4+T cells and CD3+CD8+T cells; and

[0048] in FIG. 1I, CD3−CD19− NK cells are grouped using CD19 and CD56 to obtain CD3−CD19-CD56+ NK cells.

[0049] FIGS. 2A-2I show bone marrow cell immunophenotyping of patients with acute lymphoblastic leukemia of Example 2, where:

[0050] in FIG. 2A, Lysozyme and Lactoferrin are used for plotting; a bone marrow sample is divided into three sets, where: the lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets; with medium-strength Lactoferrin, the Lysozyme+ cell set is a monocyte subset; and the cell sets with low expression of Lactoferrin and Lysozyme are an abnormal cell subset and a lymphocyte subset;

[0051] in FIG. 2B, the Lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets, expressing mature granulocyte markers CD33 and CD11b;

[0052] in FIG. 2C, the Lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets, expressing mature granulocyte markers CD13 and CD33;

[0053] in FIG. 2D, with medium-strength Lactoferrin, the Lysozyme+ cell set is a monocyte subset, expressing monocyte markers CD14 and CD64;

[0054] in FIG. 2E, the cell set with low expression of Lactoferrin and Lysozyme is gated for a next level using CD45 to obtain a CD45 weakly positive abnormal subset and a CD45+ lymphocyte subset;

[0055] in FIG. 2F, CD45+ lymphocytes are grouped using CD19 and CD3 to obtain CD3+CD19− T cells, CD3−CD19+ B cells, and CD3−CD19− NK cells;

[0056] in FIG. 2G, the abnormal cell expresses CD34 and CD117, with primitive B cell characteristics;

[0057] in FIG. 2H, the abnormal cell expresses CD34 and HLA-DR; and

[0058] in FIG. 2I, the abnormal cell expresses CD19.

[0059] FIGS. 3A-3M show bone marrow cell immunophenotyping of patients with acute myelogenous leukemia of Example 3, where:

[0060] FIG. 3A, Lysozyme and Lactoferrin are used for plotting; a bone marrow sample is divided into three sets, where: the lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets; with medium-strength Lactoferrin, the Lysozyme+ cell set is a monocyte subset; and the cell sets with low expression of Lactoferrin and Lysozyme are a nucleated red blood cell subset, an abnormal cell subset and a lymphocyte subset;

[0061] in FIG. 3B, the Lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets, expressing mature granulocyte markers CD15 and CD33;

[0062] in FIG. 3C, the Lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets, expressing mature granulocyte markers CD13 and CD33;

[0063] in FIG. 3D, with medium-strength Lactoferrin, the Lysozyme+ cell set is a monocyte subset, expressing monocyte markers CD14 and CD64;

[0064] in FIG. 3E, the cell set with low expression of Lactoferrin and Lysozyme is gated for a next level using CD45 to obtain a CD45+ lymphocyte subset, a CD45 weakly positive abnormal subset and a CD45 negative nucleated red blood cell subset;

[0065] in FIG. 3F, the nucleated red blood cell subset expresses CD71 and CD235ab;

[0066] in FIG. 3G, CD45+ lymphocytes are grouped using CD19 and CD3 to obtain CD3+CD19− T cells, CD3−CD19+ B cells, and CD3−CD19− NK cells;

[0067] in FIG. 3H, the abnormal cell expresses CD34 and CD117;

[0068] in FIG. 3I, the abnormal cell expresses CD34 and HLA-DR;

[0069] in FIG. 3J, the abnormal cell expresses CD33 and CD13;

[0070] in FIG. 3K, the abnormal cell expresses CD33, but not express CD14;

[0071] in FIG. 3L, the abnormal cell expresses CD33, but not express CD15; and

[0072] in FIG. 3M, the abnormal cell expresses CD33 and CD123.

[0073] FIGS. 4A-4L show bone marrow cell immunophenotyping of patients with myelodysplastic syndrome of Example 4, where:

[0074] in FIG. 4A, Lysozyme and Lactoferrin are used for plotting; a bone marrow sample is divided into three sets, where: the lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets; with medium-strength Lactoferrin, the Lysozyme+ cell set is a monocyte subset; and the cell sets with low expression of Lactoferrin and Lysozyme are an abnormal cell subset and a lymphocyte subset;

[0075] in FIG. 4B, the Lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets, expressing mature granulocyte markers CD15 and CD33;

[0076] in FIG. 4C, the Lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets, expressing mature granulocyte markers CD13 and CD33;

[0077] in FIG. 4D, with medium-strength Lactoferrin, the Lysozyme+ cell set is a monocyte subset, expressing monocyte markers CD14 and CD64;

[0078] in FIG. 4E, the cell set with low expression of Lactoferrin and Lysozyme is gated for a next level using CD45 to obtain a CD45+ lymphocyte subset and a CD45 weakly positive abnormal subset;

[0079] in FIG. 4F, CD45+ lymphocytes are grouped using CD19 and CD3 to obtain CD3+CD19− T cells, CD3−CD19+ B cells, and CD3−CD19− NK cells;

[0080] in FIG. 4G, CD3−CD19− T cells are grouped using CD4 and CD8 to obtain CD3+CD4+T cells and CD3+CD8+T cells;

[0081] in FIG. 4H, the abnormal cell does not express CD34 and CD117;

[0082] in FIG. 4I, the abnormal cell expresses CD19, but not express CD79a;

[0083] in FIG. 4J, the abnormal cell expresses CD33 and CD15;

[0084] in FIG. 4K, the abnormal cell expresses CD64, but not express CD14; and

[0085] in FIG. 4L, the abnormal cell expresses CD13, with a small amount of CD11b.

[0086] FIGS. 5A-5M show bone marrow cell immunophenotyping of patients with multiple myeloma of Example 5, where:

[0087] in FIG. 5A, Lysozyme and Lactoferrin are used for plotting; a bone marrow sample is divided into three sets, where: the lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets; with medium-strength Lactoferrin, the Lysozyme+ cell set is a monocyte subset; and the cell sets with low expression of Lactoferrin and Lysozyme are an abnormal cell subset and a lymphocyte subset;

[0088] in FIG. 5B, the Lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets, expressing mature granulocyte markers CD15 and CD33;

[0089] in FIG. 5C, the Lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets, expressing mature granulocyte markers CD13 and CD33;

[0090] in FIG. 5D, with medium-strength Lactoferrin, the Lysozyme+ cell set is a monocyte subset, expressing monocyte markers CD14 and CD64;

[0091] in FIG. 5E, the cell set with low expression of Lactoferrin and Lysozyme is gated for a next level using CD45 to obtain a CD45+ lymphocyte subset and a CD45 negative abnormal subset;

[0092] in FIG. 5F, CD45+ lymphocytes are grouped using CD19 and CD3 to obtain CD3+CD19− T cells, CD3−CD19+ B cells, and CD3−CD19− NK cells;

[0093] in FIG. 5G, CD3−CD19− T cells are grouped using CD4 and CD8 to obtain CD3+CD4+T cells and CD3+CD8+T cells;

[0094] in FIG. 5H, the abnormal cell expresses CD38 and CD138;

[0095] in FIG. 5I, the abnormal cell expresses κ;

[0096] in FIG. 5J, the abnormal cell does not express CD19 or CD45;

[0097] in FIG. 5K, the abnormal cell does not express CD33 or CD117;

[0098] in FIG. 5L, the abnormal cell does not express CD45 or CD56; and

[0099] in FIG. 5M, the abnormal cell does not express CD13, with a small amount of cells expressing CD20.

DETAILED DESCRIPTION

[0100] The present disclosure will be further explained below in conjunction with the examples and drawings. The following examples are only used to illustrate the present disclosure, but cannot be used to limit the implementation scope of the present disclosure.

[0101] The antibodies involved in the following examples are as shown in Table 1:

TABLE-US-00002 TABLE 1 No. Antibody Metal Clone 1 cCD3 89Y UCHT1 2 CD3 115ln UCHT1 3 cIgM 139La MHM-88 4 CD56 141Pr NCAM16.2 5 CD22 142Nd HIB22 6 CD235ab 143Nd HIR2 7 CD61 144Nd VI-PL2 8 CD23 145Nd EBVC5-5 9 CD5 146Nd UCHT2 10 CD15 147Sm W6D3 11 CD33 148Nd WM53 12 MPO 149Sm 1B10 13 CD14 150Nd M5E2 14 λ 151Eu MHL-38 15 CD13 152Sm WM15 16 CD41 153Eu HIP-8 17 Lactoferrin 154Sm 1C6 18 CD123 155Gd 6H6 19 CD34 156Gd 581 20 CD71 157Gd CY1G4 21 CD19 158Gd HIB19 22 CD9 159Tb SN4 C3-3A2 23 κ 160Gd MHK-49 24 CD99 161Dy hec2 25 CD10 162Dy HI10a 26 Lysozyme 163Dy BGN/0696/5B1 27 CD64 164Dy  10.1 28 CD2 165Ho RPA-2.10 29 CD117 166Er 104D2 30 CD1a 167Er HI149 31 CD11c 168Er Bu15 32 CD45 169Tm HI30 33 CD7 170Er CD7-6B7 34 CD79a 171Yb HM47 35 CD38 172Yb HIT2 36 CD138 173Yb DL101 37 CD20 174Yb 2H7 38 TdT 175Lu 4B10A6 39 HLA-DR 176Yb L243 40 CD300e 195Pt UP-H2 41 CD4 197Au RPA-T4 42 CD8 198pt RPA-T8 43 CD11b 209Bi M1/70 — — —

[0102] where cCD3, cIgM, MPG, λ, Lactoferrin, κ, Lysozyme, CD79a, and TdT antibodies with numbers 1, 3, 12, 14, 17, 23, 26, 34, and 38 are intracellular antibodies, and others are extracellular antibodies.

Example 1: Bone Marrow Cell Immunophenotyping of Healthy Human

[0103] 1) Fresh bone marrow of healthy human was prepared, with mature red blood cells removed.

[0104] 2) 1-3×10{circumflex over ( )}6 cells were taken and re-suspended with PBS, the volume was adjusted to 1 mL, 50 μL−1 mL of 194Pt (0.1-1 μM) was added, and staining was carried out at room temperature for 2 min to determine whether the cells were dead or alive.

[0105] 3) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, and 50 μL of blocking buffers was added for blocking on ice for 20 min. The blocking buffer consisted of 0.5 μL of human immunoglobulin solutions (including 15-25 parts by mass of human immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of mouse immunoglobulin solutions (including 15-25 parts by mass of mouse immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of rat immunoglobulin solutions (including 15-25 parts by mass of rat immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of hamster immunoglobulin solutions (including 15-25 parts by mass of hamster immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), and 48 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers).

[0106] 4) 50 μL of extracellular antibody mixed liquid (0.5 μL of each of 34 extracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 33 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added, cells were resuspended, and staining was carried out on ice for 30 min.

[0107] 5) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, 1 mL of fixation/permeabilization solutions containing 0.5 v/v % c single-cell indicator 191/193 Ir was added and cells were re-suspended overnight at 4° C.

[0108] 6) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction, for the control group, 50 μL of fixation-permeabilization solutions was added as blank control, for the experimental group, 50 μL of intracellular antibody mixed liquid (0.5 μL of each of 9 intracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 45.5 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers)) was added, cells were suspended and placed on ice for 30 min.

[0109] 7) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0110] 8) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0111] 9) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0112] 10) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0113] 11) The sample was filtered, the cells were counted, the volume was adjusted, and preparation was carried out for on-machine mass cytometry detection.

[0114] The analysis results are shown in FIG. 1A. Lysozyme and Lactoferrin are used for plotting. The bone marrow sample is divided into three sets: the lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets, expressing mature granulocyte markers CD33, CD11b, and CD15 (FIGS. 1B and 1C); with medium-strength Lactoferrin, the Lysozyme+ cell set is a monocyte subset, expressing monocyte markers CD14 and CD64 (FIG. 1D); and the cell sets with low expression of Lactoferrin and Lysozyme are a nucleated red blood cell subset and a lymphocyte subset. As shown in FIG. 1E, cell set with low expression of Lactoferrin and Lysozyme is gated for a next level using CD45 to obtain a CD45+ lymphocyte subset and a CD45− nucleated red blood cell subset. As shown in FIG. 1F, CD45+ lymphocytes are grouped using CD19 and CD3 to obtain CD3+CD19− T cells (FIG. 1H), CD3−CD19+ B cells (FIG. 1G), and CD3−CD19− NK cells (FIG. 1I).

Example 2: Bone Marrow Cell Immunophenotyping of Patients with Acute Lymphoblastic Leukemia

[0115] 1) Fresh bone marrow of patients with acute lymphoblastic leukemia was prepared, with mature red blood cells removed.

[0116] 2) 1-3×10{circumflex over ( )}6 cells were taken and re-suspended with PBS, the volume was adjusted to 1 mL, 50 μL−1 mL of 194Pt (0.1-1 μM) was added, and staining was carried out at room temperature for 2 min to determine whether the cells were dead or alive.

[0117] 3) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, and 50 μL of blocking buffers was added for blocking on ice for 20 min. The blocking buffer consisted of 0.5 μL of human immunoglobulin solutions (including 15-25 parts by mass of human immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of mouse immunoglobulin solutions (including 15-25 parts by mass of mouse immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of rat immunoglobulin solutions (including 15-25 parts by mass of rat immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of hamster immunoglobulin solutions (including 15-25 parts by mass of hamster immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), and 48 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers).

[0118] 4) 50 μL of extracellular antibody mixed liquid (0.5 μL of each of 34 extracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 33 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added, cells were resuspended, and staining was carried out on ice for 30 min.

[0119] 5) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, 1 mL of fixation/permeabilization solutions containing 0.5 v/v % c single-cell indicator 191/193 Ir was added and cells were re-suspended overnight at 4° C.

[0120] 6) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction, for the control group, 50 μL of fixation-permeabilization solutions was added as blank control, for the experimental group, 50 μL of intracellular antibody mixed liquid (0.5 μL of each of 9 intracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 45.5 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers)) was added, cells were suspended and placed on ice for 30 min.

[0121] 7) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0122] 8) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0123] 9) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0124] 10) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0125] 11) The sample was filtered, the cells were counted, the volume was adjusted, and preparation was carried out for on-machine mass cytometry detection.

[0126] The analysis results are shown in FIG. 2A. Lysozyme and Lactoferrin are used for plotting. The bone marrow sample is divided into three sets: the lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets (FIGS. 2B and 2C); with medium-strength Lactoferrin, the Lysozyme+ cell set is a monocyte subset (FIG. 2D); and the cell sets with low expression of Lactoferrin and Lysozyme are an abnormal cell subset and a lymphocyte subset. As shown in FIG. 2E, the cell set with low expression of Lactoferrin and Lysozyme is gated for a next level using CD45 to obtain a CD45+ lymphocyte subset (FIG. 2F) and a CD45 weakly positive abnormal cell subset. The abnormal cell expresses CD34, CD117, HLA-DR, and CD19 (FIGS. 2G, 2H and 2I).

Example 3: Bone Marrow Cell Immunophenotyping of Patients with Acute Myelogenous Leukemia

[0127] 1) Fresh bone marrow of patients with acute myelogenous leukemia was prepared, with mature red blood cells removed.

[0128] 2) 1-3×10{circumflex over ( )}6 cells were taken and re-suspended with PBS, the volume was adjusted to 1 mL, 50 μL−1 mL of 194Pt (0.1-1 μM) was added, and staining was carried out at room temperature for 2 min to determine whether the cells were dead or alive.

[0129] 3) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, and 50 μL of blocking buffers was added for blocking on ice for 20 min. The blocking buffer consisted of 0.5 μL of human immunoglobulin solutions (including 15-25 parts by mass of human immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of mouse immunoglobulin solutions (including 15-25 parts by mass of mouse immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of rat immunoglobulin solutions (including 15-25 parts by mass of rat immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of hamster immunoglobulin solutions (including 15-25 parts by mass of hamster immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), and 48 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers).

[0130] 4) 50 μL of extracellular antibody mixed liquid (0.5 μL of each of 34 extracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 33 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added, cells were resuspended, and staining was carried out on ice for 30 min.

[0131] 5) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, 1 mL of fixation/permeabilization solutions containing 0.5 v/v % c single-cell indicator 191/193 Ir was added and cells were re-suspended overnight at 4° C.

[0132] 6) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction, for the control group, 50 μL of fixation-permeabilization solutions was added as blank control, for the experimental group, 50 μL of intracellular antibody mixed liquid (0.5 μL of each of 9 intracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 45.5 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers)) was added, cells were suspended and placed on ice for 30 min.

[0133] 7) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0134] 8) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0135] 9) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0136] 10) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0137] 11) The sample was filtered, the cells were counted, the volume was adjusted, and preparation was carried out for on-machine mass cytometry detection.

[0138] The analysis results are shown in FIG. 3A. Lysozyme and Lactoferrin are used for plotting. The bone marrow sample is divided into three sets: the lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets (FIGS. 3B and 3C); with medium-strength Lactoferrin, the Lysozyme+ cell set is a monocyte subset (FIG. 3D); and the cell sets with low expression of Lactoferrin and Lysozyme are a nucleated red blood cell subset, an abnormal cell subset and a lymphocyte subset. As shown in FIG. 3E, the cell set with low expression of Lactoferrin and Lysozyme is gated for a next level using CD45 to obtain a CD45+ lymphocyte subset (FIG. 3G), a CD45 weakly positive abnormal cell subset, and a CD45 negative nucleated red blood cell subset (FIG. 3F). The abnormal cell expresses CD34, CD117, HLA-DR, CD33, CD13, and CD123 (FIGS. 3H, 3I, 3J, 3K, 3L and 3M).

Example 4: Bone Marrow Cell Immunophenotyping of Patients with Myelodysplastic Syndrome (MDS)

[0139] 1) Fresh bone marrow of patients with myelodysplastic syndrome was prepared, with mature red blood cells removed.

[0140] 2) 1-3×10{circumflex over ( )}6 cells were taken and re-suspended with PBS, the volume was adjusted to 1 mL, 50 μL−1 mL of 194Pt (0.1-1 μM) was added, and staining was carried out at room temperature for 2 min to determine whether the cells were dead or alive.

[0141] 3) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, and 50 μL of blocking buffers was added for blocking on ice for 20 min. The blocking buffer consisted of 0.5 μL of human immunoglobulin solutions (including 15-25 parts by mass of human immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of mouse immunoglobulin solutions (including 15-25 parts by mass of mouse immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of rat immunoglobulin solutions (including 15-25 parts by mass of rat immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of hamster immunoglobulin solutions (including 15-25 parts by mass of hamster immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), and 48 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers).

[0142] 4) 50 μL of extracellular antibody mixed liquid (0.5 μL of each of 34 extracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 33 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added, cells were resuspended, and staining was carried out on ice for 30 min.

[0143] 5) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, 1 mL of fixation/permeabilization solutions containing 0.5 v/v % c single-cell indicator 191/193 Ir was added and cells were re-suspended overnight at 4° C.

[0144] 6) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction, for the control group, 50 μL of fixation-permeabilization solutions was added as blank control, for the experimental group, 50 μL of intracellular antibody mixed liquid (0.5 μL of each of 9 intracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 45.5 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers)) was added, cells were suspended and placed on ice for 30 min.

[0145] 7) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0146] 8) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0147] 9) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0148] 10) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0149] 11) The sample was filtered, the cells were counted, the volume was adjusted, and preparation was carried out for on-machine mass cytometry detection.

[0150] The analysis results are shown in FIG. 4A. Lysozyme and Lactoferrin are used for plotting. The bone marrow sample is divided into three sets: the lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets (FIGS. 4B and 4C); with medium-strength Lactoferrin, the Lysozyme+ cell set is a monocyte subset (FIG. 4D); and the cell sets with low expression of Lactoferrin and Lysozyme are an abnormal cell subset and a lymphocyte subset. As shown in FIG. 4E, cell set with low expression of Lactoferrin and Lysozyme is gated for a next level using CD45 to obtain a CD45+ lymphocyte subset (FIGS. 4F and 4G) and a CD45 weakly positive abnormal cell subset. The abnormal cell expresses CD33, CD15, CD13, CD11b, CD19, and CD64 (FIGS. 4H, 4I, 4J, 4K and 4L).

Example 5: Bone Marrow Cell Immunophenotyping of Patients with Multiple Myeloma

[0151] 1) Fresh bone marrow of patients with multiple myeloma was prepared, with mature red blood cells removed.

[0152] 2) 1-3×10{circumflex over ( )}6 cells were taken and re-suspended with PBS, the volume was adjusted to 1 mL, 50 μL−1 mL of 194Pt (0.1-1 μM) was added, and staining was carried out at room temperature for 2 min to determine whether the cells were dead or alive.

[0153] 3) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, and 50 μL of blocking buffers was added for blocking on ice for 20 min. The blocking buffer consisted of 0.5 μL of human immunoglobulin solutions (including 15-25 parts by mass of human immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of mouse immunoglobulin solutions (including 15-25 parts by mass of mouse immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of rat immunoglobulin solutions (including 15-25 parts by mass of rat immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), 0.5 μL of hamster immunoglobulin solutions (including 15-25 parts by mass of hamster immunoglobulin, 0.15-0.25 parts by mass of sodium azide, and 0.75-1.25 parts by volume of phosphate buffers), and 48 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers).

[0154] 4) 50 μL of extracellular antibody mixed liquid (0.5 μL of each of 34 extracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 33 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added, cells were resuspended, and staining was carried out on ice for 30 min.

[0155] 5) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 500 g/5 min, supernatant was removed by suction, 1 mL of fixation/permeabilization solutions containing 0.5 v/v % c single-cell indicator 191/193 Ir was added and cells were re-suspended overnight at 4° C.

[0156] 6) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction, for the control group, 50 μL of fixation-permeabilization solutions was added as blank control, for the experimental group, 50 μL of intracellular antibody mixed liquid (0.5 μL of each of 9 intracellular antibodies in Table 1, at an antibody concentration of 0.1-1 μg/μL respectively, and 45.5 μL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers)) was added, cells were suspended and placed on ice for 30 min.

[0157] 7) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0158] 8) 2 mL of bovine serum albumin solutions (including 375-625 parts by mass of bovine serum albumin, 15-25 parts by mass of sodium azide, and 75-125 parts by volume of phosphate buffers) was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0159] 9) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0160] 10) 2 mL of deionized water was added and centrifuged at 800 g/5 min, and supernatant was removed by suction.

[0161] 11) The sample was filtered, the cells were counted, the volume was adjusted, and preparation was carried out for on-machine mass cytometry detection.

[0162] The analysis results are shown in FIG. 5A. Lysozyme and Lactoferrin are used for plotting. The bone marrow sample is divided into three sets: the lactoferrin+ and Lysozyme+ cell sets are mature granulocyte subsets (FIGS. 5B and 5C); with medium-strength Lactoferrin, the Lysozyme+ cell set is a monocyte subset (FIG. 5D); and the cell sets with low expression of Lactoferrin and Lysozyme are an abnormal cell subset and a lymphocyte subset. As shown in FIG. 5E cell set with low expression of Lactoferrin and Lysozyme is gated for a next level using CD45 to obtain a CD45+ lymphocyte subset and a CD45 negative abnormal cell subset (FIGS. 5F and 5G). The abnormal cell expresses CD38, CD138, κ, and CD20; and CD56 and CD45 are negative (FIGS. 5H, 5I, 5J, 5K, 5L and 5M).