CN103675277B - Flow cytometry antidiastole rhabdomyosarcoma and neuroblastoma bone marrow neoplasms and leukemic fluorescence probe and kit - Google Patents

Abstract

The invention discloses a fluorescent probe and kit for the differential diagnosis of rhabdomyosarcoma, neuroblastoma, bone marrow metastasis and leukemia by flow cytometry. The fluorescent probe includes four fluorescently labeled antibodies, and the four fluorescently labeled antibodies These are GD2 antibody, CD90 antibody, CD45 antibody and CD56 antibody all with fluorescent labels, wherein different antibodies have different fluorescent labels. The kit includes a probe solution; a hemolytic agent; a phosphate buffer and a positive standard. The fluorescent probe of the present invention can specifically combine with rhabdomyosarcoma and neuroblastoma cells and acute leukemia blast cells in bone marrow respectively, and can quickly and accurately differentiate rhabdomyosarcoma and neuroblastoma bone marrow metastasis and leukemia by flow cytometry These three types of tumor cells, which are common in children's bone marrow, guide clinical treatment and have good application prospects.

Description

Fluorescent probes and kits for the differential diagnosis of rhabdomyosarcoma and neuroblastoma bone marrow metastasis and leukemia by flow cytometry

technical field

The invention relates to a medical detection reagent, in particular to a fluorescent probe and a kit for differential diagnosis of rhabdomyosarcoma and neuroblastoma bone marrow metastasis and leukemia by flow cytometry.

Background technique

Clinically, leukemia, rhabdomyosarcoma, and neuroblastoma are the most common tumor cells in children's bone marrow. Correct identification of the three has important guiding significance for the treatment of patients. Rhabdomyosarcoma and neuroblastoma are common malignant solid tumors in children, which pose a serious threat and harm to children's lives. Some patients can recover through surgical treatment. However, the disease is prone to bone marrow metastasis, which often endangers the life of the child. Treatment measures such as chemotherapy, radiotherapy and even autologous stem cell transplantation should be taken in time. Therefore, early detection, early diagnosis and early treatment of bone marrow metastasis are the key to improving the cure rate and reducing the mortality rate.

At present, bone marrow cell morphology is mainly used to diagnose rhabdomyosarcoma and neuroblastoma bone marrow metastasis at home and abroad, but the cell morphology is not sensitive to the diagnosis of patients with early metastasis, and it is easy to produce false negative results. Lymphocytes are similar to myeloblasts, so it is easy to be misdiagnosed as leukemia. At the same time, rhabdomyosarcoma and neuroblastoma have similar cell morphology in the bone marrow, making it difficult to distinguish.

It is precisely because the current various examination methods for the diagnosis and differential diagnosis of rhabdomyosarcoma and neuroblastoma bone marrow metastasis cannot meet the requirements of clinicians, so a fast, simple, accurate and sensitive method is sought to diagnose and differentiate Methods for diagnosing rhabdomyosarcoma and neuroblastoma bone marrow metastases and leukemia are extremely important. Flow cytometry (Flow Cytometry, FCM) is a high-tech technology developed in recent years. It integrates computer technology, laser technology, fluid mechanics, cytochemistry, and cellular immunology. It also has the functions of analyzing and sorting cells. It can not only measure cell size and internal particle properties, but also detect cell surface and cytoplasmic antigens, intracellular DNA, RNA content, etc. It can perform multi-parameter analysis on group cells at the single-cell level, that is, on the same cell The analysis of the co-expression of 2-3 or more antigens detects and analyzes a large number of cells in a short time, and collects, stores and processes data, and performs multi-parameter quantitative analysis, which has the characteristics of high sensitivity and accuracy. It is mostly used in the diagnosis of bone marrow samples of leukemia and lymphoma, but the diagnosis and differential diagnosis of rhabdomyosarcoma and neuroblastoma are rarely reported. Since there is no single specific antigen marker for the two cells, the key to the differential diagnosis of rhabdomyosarcoma and neuroblastoma bone marrow metastasis and leukemia by flow cytometry lies in the combination of multiple antibodies.

Contents of the invention

The invention provides a fluorescent probe for differential diagnosis of rhabdomyosarcoma, neuroblastoma bone marrow metastasis and leukemia by flow cytometry, and the diagnostic result can be obtained quickly and accurately by using the fluorescent probe.

A fluorescent probe for the differential diagnosis of rhabdomyosarcoma and neuroblastoma bone marrow metastasis and leukemia by flow cytometry, including four fluorescently labeled antibodies, the four fluorescently labeled antibodies are GD2 antibodies and CD90 antibodies with fluorescent labels , CD45 antibody and CD56 antibody, wherein different antibodies have different fluorescent labels.

Rhabdomyosarcoma is the most common soft tissue tumor in children, neuroblastoma is a neuroendocrine tumor, and CD56 is a class of membrane glycoprotein nerve cell adhesion molecules with immunoglobulin-like domains expressed on the surface of cells of neuroectodermal origin. CD90 and CD56 are expressed as adhesion molecules on rhabdomyosarcoma cells and neuroblastoma, and NK cells also mainly express this 140kDa CD56 isoform, while CD45 is a membrane protein molecule restricted to the blood cell series, expressed in red blood cells. CD90+CD56+/CD45- phenotype can avoid misdiagnosis as NK cell tumor and leukemia, and can be used as an indication for judging the existence of neuroendocrine tumors and rhabdomyosarcoma.

Neuroblastoma cells are derived from poorly differentiated ganglion cells, which express the nerve adhesion molecule CD56 antigen and ganglioside GD2 on their surface, but do not express the leukocyte common antigen CD45. A malignancy of differentiated mesenchymal cells that do not express the ganglioside GD2. Similarly, leukemia cells do not express GD2, but they express leukocyte common antigen CD45 and blast cell marker CD90. Therefore, the different expressions of these four antigens can be determined by multicolor flow cytometry to identify rhabdomyosarcoma, neuroblastoma, and leukemia blast cells. Three types of neoplastic cells commonly found in pediatric bone marrow specimens. The three have distinct immunophenotypes, namely:

Rhabdomyosarcoma cells: GD2 ^- CD90 ⁺ CD56 ⁺ CD45 ^- ;

Neuroblastoma cells: GD2 ⁺ CD90 ⁺ CD56 ⁺ CD45 ^- ;

Acute leukemia blasts: GD2 ^- CD90 ⁺ CD56 ^- CD45 ⁺ .

The present invention combines four fluorescently labeled antibodies GD2, CD90, CD56 and CD45 as probes, and uses the multicolor flow cytometer commonly used in hospitals to detect the immunophenotype of cells (one-tube method) to differentially diagnose rhabdomyosarcoma and neuroblastoma. Cell tumor bone marrow metastasis and leukemia, the specificity can reach 100%.

Specifically, the four fluorescently labeled antibodies are GD2-FITC antibody, CD90-PE antibody, CD45-PerCP antibody and CD56-APC antibody.

To ensure the specificity of detection, the four fluorescently labeled antibodies are monoclonal antibodies.

The present invention also provides a kit for the differential diagnosis of rhabdomyosarcoma and neuroblastoma bone marrow metastasis and leukemia by flow cytometry, comprising:

(1) Probe solution, containing four kinds of fluorescently labeled antibodies, among which, the four kinds of fluorescently labeled antibodies are GD2 antibody, CD90 antibody, CD45 antibody and CD56 antibody all with fluorescent labels, and different antibodies have different fluorescent labels ;

(2) Hemolytic agent;

(3) Phosphate buffer;

(4) Positive standard.

The probe solution uses phosphate buffer as a solvent.

The four fluorescently labeled antibodies are specifically GD2-FITC antibody, CD90-PE antibody, CD45-PerCP antibody and CD56-APC antibody. Among them, CD90-PE can be purchased from BD Company of the United States, article number: 555596, CD45-PerCP can be purchased from BD Company of the United States, article number: 347464, CD56-APC can be purchased from BD Company of the United States, article number: 341025, and the preparation method of GD2-FITC can be Refer to CANCER RESEARCH, Page 2988, Issue 46, 1986, Highly selective recognition of human neuroblastoma cells by mouse monoclonal antibody to a cytoplasmic antigen.

The concentration ratio of the four fluorescently labeled antibodies is 1:1:1:1.

The composition of the hemolytic agent is: 8.3% ammonium chloride and 15% paraformaldehyde. the

The probe solution contains a preservative which is sodium azide.

The phosphate buffer also contains a preservative, which is sodium azide.

The added amount of the sodium azide is 0.05-0.1%, preferably 0.1%.

The concentration of the phosphate buffer used in the kit is 0.8-1M, preferably 1M, and the pH of the phosphate buffer is 7.4.

It should be pointed out that since children do not have chronic lymphocytic leukemia and chronic myeloid leukemia is very rare, unless otherwise specified, the leukemia mentioned in this application refers to acute leukemia.

Compared with prior art, the beneficial effect of the present invention is:

The fluorescent probe of the present invention can specifically bind to tumor cells in bone marrow without cross-reaction, and can accurately and quickly detect whether rhabdomyosarcoma and neuroblastoma have bone marrow metastasis through flow cytometry, and has high specificity (up to 100%) and sensitivity, and can distinguish acute leukemia blasts at the same time, simple, fast and accurate, and has a good clinical application prospect.

Description of drawings

Fig. 1 is the result of Wright's staining of the bone marrow specimen of the patient in Example 1 according to the present invention; wherein, A is the bone marrow metastasis of rhabdomyosarcoma, and B is the bone marrow metastasis of neuroblastoma.

Fig. 2 is the flow cytometry detection result figure of the present invention to the bone marrow sample of patient with rhabdomyosarcoma in Example 1; wherein, A: CD45- and CD56+ cells set R1 gate, B: GD2- and CD90+ cells set R2 gate; C: FSC R3 gate was set for SSC cells; R2 was derived from R1, and R3 was derived from R2.

Fig. 3 is the result of flow cytometry detection of the bone marrow samples of patients with neuroblastoma in Example 1 of the present invention; wherein, A: R1 gate is set for CD45- and CD56+ cells, B: R2 gate is set for GD2+ and CD90+ cells; C: R3 gate was set for FSC and SSC cells; R2 was derived from R1, and R3 was derived from R2.

Fig. 4 is a graph showing the results of Wright's staining of acute leukemia blast cells in Example 1.

Fig. 5 is a diagram of the flow cytometry detection results of the bone marrow samples of patients with acute leukemia in Example 1 of the present invention; wherein, A: R1 gate is set for CD45+ and CD90+ cells, B: cells derived from R1 show GD2- and CD45+; C: Cells derived from R1 showed GD56- and CD45+.

Fig. 6a is a graph showing the sensitivity detection results of flow cytometry on the bone marrow specimen (dilution ratio: 1:100) of a patient with rhabdomyosarcoma in Example 1 according to the present invention.

Fig. 6b is a graph showing the sensitivity detection results of flow cytometry on the bone marrow specimen (dilution ratio: 1:1000) of a patient with rhabdomyosarcoma in Example 1 according to the present invention.

Fig. 6c is a graph showing the detection results of the flow cytometry sensitivity of the bone marrow specimen (dilution ratio: 1:10000) of the patient with rhabdomyosarcoma in Example 1 according to the present invention.

Fig. 7 is the result of Wright's staining of the cerebrospinal fluid sample of the patient with rhabdomyosarcoma in embodiment 2 according to the present invention.

Fig. 8 is a diagram of the flow cytometry detection results of the cerebrospinal fluid specimens of patients with rhabdomyosarcoma in Example 2, wherein, A: R1 gate is set for CD45- and CD56+ cells, B: R2 gate is set for GD2- and CD90+ cells; C: FSC R3 gate was set for SSC cells; R2 was derived from R1, and R3 was derived from R2.

Detailed ways

The present invention will be further explained below in combination with specific embodiments.

Example 1

GD2-FITC (self-prepared, BALB/c mice were immunized with neuroblastoma cell line LAN-1, mouse B lymphocytes were fused with mouse myeloma cell line NS1 to form hybridoma cells to prepare monoclonal antibody GD2 and labeled For FITC fluorescein, refer to the magazine CANCER RESEARCH for specific technical steps, page 2988, issue 46, 1986; Highly selective recognition of human neuroblastoma cells by mouse monoclonal antibody to a cytoplasmic antigen), CD90-PE (BD Company of the United States, article number: 555596), CD45 -PerCP (BD Company of the United States, article number: 347464), CD56-APC (BD Company of the United States, article number: 341025).

1. Kit composition:

1) Probe solution (2mL×1 bottle):

GD2-FITC: 2mL PBS (containing 0.1% sodium azide) containing 100μg antibody protein labeled with fluorescein isothiocyanate (50μg/mL);

CD90-PE: 2mL PBS (containing 0.1% sodium azide) containing phycoerythrin-labeled 100μg antibody protein (50μg/mL);

CD45-PerCP: 2mL PBS (containing 0.1% sodium azide) containing 100μg antibody protein labeled with paramecium chlorophyll protein (50μg/mL);

CD56-APC: 2mL PBS (containing 0.1% sodium azide) containing 100μg antibody protein labeled with isophycocyanin (50μg/mL);

The antibody concentration ratio of the composition GD2-FITC: CD90-PE: CD45-PerCP: CD56-APC is 1:1:1:1.

2) 10×hemolytic agent (100mL×1 bottle): 8.3% ammonium chloride, containing 15% paraformaldehyde;

3) 10× phosphate buffer saline (PBS, pH=7.4, 1M, containing 1% sodium azide) (100mL×1 bottle);

4) Positive standard (2mL×1 bottle): fixed and preserved cell lines (5×10 ⁷ cells/mL).

2. Application of kit

Research objects: Bone marrow samples from 15 children with non-leukemia and tumors, 21 rhabdomyosarcoma patients, 25 neuroblastoma patients and 22 patients with acute myeloid leukemia. The bone marrow samples were obtained from outpatients and inpatients of Children's Hospital Affiliated to Zhejiang University School of Medicine.

Operation steps: 100 μL of bone marrow fluid, add 20 μL each of monoclonal fluorescently labeled antibodies (GD2-FITC, CD90-PE, CD45-PerCP and CD56-APC), react at 4°C for 30 minutes in the dark, add 1 mL of hemolytic agent, and let stand for 5 minutes. Minutes, add 5 mL of PBS, centrifuge at 500G for 5 minutes, discard the supernatant, add 300 μL of PBS to the precipitate, mix well, and detect 50,000 cells on a flow cytometer.

Strategies for streaming image analysis:

Rhabdomyosarcoma: 1. Set R1 gate for CD45- and CD56+; 2. Set R2 gate for GD2- and CD90+ cells; 3. Set R3 gate for FSC and SSC cells; 4. Calculate the number of positive cells in R3 gate. (R2 is derived from R1; R3 is derived from R2).

Neuroblastoma: 1. Set R1 gate for CD45- and CD56+; 2. Set R2 gate for GD2+ and CD90+ cells; 3. Set R3 gate for FSC and SSC cells; 4. Calculate the number of positive cells in R3 gate. (R2 is derived from R1; R3 is derived from R2).

Acute leukemia blasts: 1. Set R1 gate for CD45+ and CD90+; 2. Analyze CD45+ and GD2- cells; 3. Analyze CD45+ and CD56- cells.

At the same time, a negative control was set up.

Positive results judge the cell type by immunophenotype as follows:

The phenotype is GD2 ^- CD90 ⁺ CD56 ⁺ CD45 ^- rhabdomyosarcoma cells;

The phenotype is GD2 ⁺ CD90 ⁺ CD56 ⁺ CD45 ^- neuroblastoma cells;

The phenotype was GD2 ^- CD90 ⁺ CD56 ^- CD45 ⁺ acute leukemia blasts.

Determination of detection sensitivity: Dilute rhabdomyosarcoma cells with normal bone marrow cells to a ratio of 1:100, 1:1000, and 1:10000, and use the same method for detection.

Statistical methods: Using the software SPSS11.5 (SPSS Inc., Chicago, IL, USA), the sensitivity and specificity of the diagnostic methods were analyzed and evaluated.

The detection results of various types of specimens are shown in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6a, Figure 6b, and Figure 6c.

The results showed that the above three types of immune phenotype cells were not detected in 15 children with non-leukemia and tumors; GD2 ^- CD90 ⁺ CD56 ⁺ CD45 ^- immunophenotype cells were detected in 5 of the 21 patients with rhabdomyosarcoma cells, and the final diagnosis of the disease was made clinically. 5 cases had rhabdomyosarcoma bone marrow metastasis; GD2 ⁺ CD90 ⁺ CD56 ⁺ CD45 ^- immunophenotype cells were detected in 11 of 25 neuroblastoma patients, and the 11 cases were finally clinically diagnosed as neuroblastoma bone marrow metastasis; 22 cases of acute Leukemic cells are all GD2 ^- CD90 ⁺ CD56 ^- CD45 ⁺ cell immunophenotype; the specificity of flow cytometry for the diagnosis of the three types of tumor cells in the bone marrow is 100%, and the sensitivity is 1/10 ^-4 (that is, in 10000 One tumor cell can be detected among normal cells).

Example 2

1. Kit composition:

1) Probe solution (2mL×1 bottle):

GD2-FITC: 2mL PBS (containing 0.1% sodium azide) containing 100μg antibody protein labeled with fluorescein isothiocyanate (50μg/mL);

CD90-PE: 2mL PBS (containing 0.1% sodium azide) containing phycoerythrin-labeled 100μg antibody protein (50μg/mL);

CD45-PerCP: 2mL PBS (containing 0.1% sodium azide) containing 100μg antibody protein labeled with paramecium chlorophyll protein (50μg/mL);

CD56-APC: 2mL PBS (containing 0.1% sodium azide) containing 100μg antibody protein labeled with isophycocyanin (50μg/mL);

The antibody concentration ratio of the composition GD2-FITC: CD90-PE: CD45-PerCP: CD56-APC is 1:1:1:1.

2) 10×hemolytic agent (100mL×1 bottle): 8.3% ammonium chloride, containing 15% paraformaldehyde;

3) 10× phosphate buffer saline (PBS, pH=7.4, 1M, containing 1% sodium azide) (100mL×1 bottle);

4) Positive standard (2mL×1 bottle): fixed and preserved cell lines (5×10 ⁷ cells/mL).

2. Application of kit

Research objects: 2 patients with suspected rhabdomyosarcoma cerebrospinal fluid metastasis punctured cerebrospinal fluid samples.

The operation steps, analysis strategies and statistical methods are the same as those in Example 1.

The results showed that: 2 patients with rhabdomyosarcoma clinically suspected of cerebrospinal fluid metastasis underwent puncture of cerebrospinal fluid and detected by this antibody combination flow cytometry, GD2 ^- CD90 ⁺ CD56 ⁺ CD45 ^- immunophenotype cells were found (Figure 7, Figure 8), and the final clinical diagnosis The two cases had cerebrospinal fluid metastasis from rhabdomyosarcoma, and received intrathecal chemotherapy and craniospinal radiation therapy. It shows that the fluorescent probe of the present invention can monitor the cerebrospinal fluid metastasis of rhabdomyosarcoma and guide clinical rational drug use.

Claims (3)

1. fluorescence probe is preparing the application in flow cytometry antidiastole rhabdomyosarcoma and neuroblastoma bone marrow neoplasms and leukemic kit, described fluorescence probe comprises four kinds of fluorescent-labeled antibody, described four kinds of fluorescent-labeled antibody are all with fluorescently-labeled GD2 antibody, CD90 antibody, CD45 antibody and CD56 antibody, wherein, different antibody is with different fluorescence labelings.

2. apply as claimed in claim 1, it is characterized in that, described four kinds of fluorescent-labeled antibody are GD2-FITC antibody, CD90-PE antibody, CD45-PerCP antibody and CD56-APC antibody.

3. apply as claimed in claim 1 or 2, it is characterized in that, four kinds of fluorescent-labeled antibody are monoclonal antibody.