CN103018459A - Fluorescence probe for diagnosis and monitoring of meningeal metastasis of retinoblastoma through flow cytometry - Google Patents
Fluorescence probe for diagnosis and monitoring of meningeal metastasis of retinoblastoma through flow cytometry Download PDFInfo
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- CN103018459A CN103018459A CN2012105209705A CN201210520970A CN103018459A CN 103018459 A CN103018459 A CN 103018459A CN 2012105209705 A CN2012105209705 A CN 2012105209705A CN 201210520970 A CN201210520970 A CN 201210520970A CN 103018459 A CN103018459 A CN 103018459A
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Abstract
The invention discloses a fluorescence probe for diagnosis and monitoring of meningeal metastasis of retinoblastoma through flow cytometry. The fluorescence probe comprises three fluorescent-labeled antibodies, i.e., the GD2-FITC antibody, the CD56-PE antibody and the CD45-PerCP antibody. The fluorescence probe provided by the invention can specifically bind to retinal metrocytes in cerebrospinal fluid, can accurately and rapidly detect metastasis of tumor cells through flow cytometry and has a good application prospect.
Description
Technical field
The present invention relates to a kind of medical treatment and detect reagent, relate in particular to the fluorescence probe that a kind of flow cytometry diagnosis and monitoring retinoblastoma meninx shift.
Background technology
Retinoblastoma is the modal intraocular malignant of infant, and eyesight and life are had serious threat and harm.It betides the retina stratum nucleare, multiple being born in below 5 years old, can be simple eye, eyes successively or suffer from simultaneously, 90% patient can obtain rehabilitation by operative treatment.But encephalic cerebrospinal fluid easily occurs this disease shifts, and often jeopardizes infant life, need in time take the treatment measures such as chemotherapy, radiotherapy even autologous stem cell transplantation.Therefore the early detection of cerebrospinal fluid being shifted, early diagnosis and early stage treatment are the keys that improves cure rate, reduces mortality ratio.
Mainly check Diagnosis of Retinoblastoma cerebrospinal fluid to shift by MRI and CT and cerebrospinal fluid cytomorphology both at home and abroad at present, but MRI and CT are insensitive for the patient's of early stage transfer diagnosis, easily produce false negative result, the inspection of cerebrospinal fluid cytomorphology since the cerebrospinal fluid tumour cell be difficult to distinguish because of sex change or apoptosis and lymphocyte and inflammatory cell, and be difficult for less the reasons such as discovery shifting the infantile tumour cell number, the accuracy of its diagnosis and susceptibility are all relatively poor, and the monitoring for the treatment of curative effect is also had deficiency (N.R.Cohen.Cytopathology2009 (20) 256-260).
Also can't satisfy clinician's requirement just because of the present various inspection methods that Diagnosis of Retinoblastoma cerebrospinal fluid is shifted, seek a kind of fast and convenient and accuracy and the high method of susceptibility comes Diagnosis of Retinoblastoma cerebrospinal fluid to shift and the method for monitoring curative effect is very important.
Flow cytometry (Flow Cytometry, FCM) is the high science and technology that development in recent years is got up, and it integrates computer technology, laser technology, fluid mechanics, cytochemistry, cellular immunology, has simultaneously to analyze and the sorting cells function.
Flow cytometry not only can be measured the proterties of cell size, internal particle, also can detect DNA, rna content etc. in cell surface and cytoplasmic antigen, the cell, can carry out multi parameter analysis in unicellular level to colony's cell, namely carry out the analysis of the co expression of 2-3 kind or plurality of antigens at same cell, detect at short notice and analyze a large amount of cells, and collection, storage and deal with data, carry out the multiparameter quantitative test, have the high characteristics of susceptibility and accuracy.
The at present clinically diagnosis that apply to leukaemia and lymphadenomatous marrow sample more, and to the rare report of the diagnosis of retinoblastoma.Because Retinoblastoma Cells does not have single specific antigen mark at present, flow cytometry is the combination of Multiple Antibodies to the diagnosis key of retinoblastoma.
Summary of the invention
The invention provides the fluorescence probe that a kind of flow cytometry diagnosis and monitoring retinoblastoma meninx shift, utilize this fluorescence probe can obtain fast diagnostic result.
The fluorescence probe that a kind of flow cytometry diagnosis and monitoring retinoblastoma cerebrospinal fluid shift comprises three kinds of fluorescent-labeled antibody, and described three kinds of fluorescent-labeled antibody are GD2-FITC antibody, CD56-PE antibody and CD45-PerCP antibody.
Retinoblastoma cell derives from the neuroectodermal cell of multipotency, and CD56 is the membrane glycoprotein N-CAM in an immunoglobulin like protein sample zone, cell surface expression in the neuroderm source, the NK cell is mainly also expressed the CD56 isomeride of this 140kDa, and CD45 is the restrictive membrane protein molecule of haemocyte series, expression except the exo-erythrocytic candidate stem cell that comprises to mature blood cell (comprising the NK cell) film surface, therefore can to avoid mistaken diagnosis be the NK cell tumour to the CD56+/CD45-phenotype, and can be used as and judge accusing of that neuroendocrine tumor exists.
Chantada etc. detect the retinoblastoma [G.L.Chantada.J Pediatr Hematol Oncol 2006 (28) 369-373] of cellular neural joint glycosides fat GD2 expression progress in evaluation in the marrow with SABC.The report PCR such as Laurent detect the cerebrospinal fluid transfer [V.E.Laurent.Molec μ Lar MedicineReports2010 (3) 253-259] that cerebrospinal fluid cell GD2mRNA expresses diagnosable retinoblastoma.The present invention unites GD2, CD56 and three kinds of fluorescent-labeled antibody of CD45 as the Retinoblastoma Cells in the probe in detecting cerebrospinal fluid, uses the immunophenotype of polychrome Flow cytometry cell CD45-/CD56+/GD2+ to come Diagnosis of Retinoblastoma cell cerebrospinal fluid to shift.
Three kinds of fluorescent-labeled antibody are monoclonal antibody.
The kit that the present invention also provides a kind of Flow Cytometry diagnosis and monitoring retinoblastoma cerebrospinal fluid to shift comprises:
Probe solution take phosphate buffer as solvent, comprises G D2-FITC antibody, CD56-PE antibody and CD45-PerCP antibody;
Hemolytic agent;
Phosphate buffer;
The positive criteria product.
The concentration ratio of three kinds of fluorescent-labeled antibody is 1: 1: 1.
Described probe solution comprises antiseptic, and described antiseptic is Sodium azide.
Compared with prior art, beneficial effect of the present invention is:
Fluorescence probe of the present invention can with cerebrospinal fluid in the retinoblastoma cell specific binding, can accurately whether shift by the Flow cytometry tumour cell fast, have preferably application prospect.
Description of drawings
Fig. 1 is that the present invention is to the testing result figure of retinoblastoma patients samples of CSF;
Fig. 2 is that the present invention is to the testing result figure of the rear samples of CSF of retinoblastoma patients treatment;
Fig. 3 is that the present invention is to the testing result figure of Patients with Virus Meningitis samples of CSF;
Fig. 4 is that the present invention is to the testing result figure of purulent meningitis Cerebrospinal Fluid in Patients sample;
Fig. 5 is that the present invention is to the testing result figure of acute leukemic patient samples of CSF;
Among each figure, A: scattering and the two-parameter scatter diagram of sidescattering are established door (R1) before the cell; B: show that fluidic cell detects this group cell and is CD56
+/ CD45
-Phenotype (R2); C: show that this group cell is CD45
-CD56
+/ GD2
+Phenotype (R3).
Embodiment
Research object: the samples of CSF of 15 routine viral meningitises, 19 routine purulent meningitiss, 52 routine acute leukemias and 21 routine retinoblastoma patients, samples of CSF are all from Medical College of Zhejiang Univ.'s attached children's hospital outpatient service and inpatient.
Operation steps: 1mL cerebrospinal fluid 500G centrifugation 5 minutes, abandon supernatant, sediment adds PBS200 μ L, mixing adds respectively monoclonal fluorescent-labeled antibody (GD2-FITC, CD56-PE, CD45-PerCP) 20 μ L, 4 ℃ of lucifuges were reacted 30 minutes, added hemolytic agent 1mL, left standstill 5 minutes, and added PBS5mL, 500G centrifugation 5 minutes, abandon supernatant, sediment adds PBS300 μ L, mixing, and the up flow type cell instrument detects.
The streamed image analysis strategy: 1, FSC and SSC establish the R1 door; 2, CD56+ and CD45-cell are established the R2 door; 3, GD2+ and CD56+ cell are established the analysis of R3 door; 4, calculate the interior positive percentage of R3 door.(R2 derives from R1; R3 derives from R2).5, GD2+CD56+CD45-immunophenotype cell occurs take cerebrospinal fluid positive as meninx shifts, set up simultaneously negative control.
Statistical method: utilize software SPSS11.5 (SPSS Inc., Chicago, IL, USA), the susceptibility of assay diagnostic method and specificity.
The testing result of all types of samples is shown in Fig. 1,2,3,4,5.
The result shows: viral meningitis, purulent meningitis and acute leukemic patient cerebrospinal fluid all do not detect CD45-/CD56+/GD2+ immunophenotype cell; CD45-/CD56+/GD2+ immunophenotype cell appears in 6-13 month cerebrospinal fluid after the 3 routine retinoblastoma patients operations, being diagnosed as retinoblastoma cerebrospinal fluid shifts, to be that retinoblastoma cerebrospinal fluid shifts the result consistent with clinical final comprehensive diagnos, and susceptibility and the specificity of diagnosis are 100%.
Kit forms:
1) probe solution (2mL * 1 bottle): GD2-FITC:2mL PBS (containing 0.1% Sodium azide) contains the 100 μ g antibody proteins (50 μ g/mL) of marked by fluorescein isothiocyanate; CD56-PE:2mLPBS (containing 0.1% Sodium azide) contains the 100 μ g antibody proteins (50 μ g/mL) of phycoerythrin mark; CD45-PerCP:2mL PBS (containing 0.1% Sodium azide) contains the 100 μ g antibody proteins (50 μ g/mL) of paramecium phyllochlorin mark; Composition antibody concentration ratio GD2-FITC:CD56-PE:CD45-PerCP is 1: 1: 1.
2) 10 * hemolytic agent (100mL * 1 bottle): 8.3% ammonium chloride contains 15% paraformaldehyde;
3) 10 * phosphate buffer (1M contains 1% Sodium azide for PBS, pH=7.4) (100mL * 1 bottle);
4) positive criteria product (2mL * 1 bottle): fixing and preserve the cell line (5 * 10 that liquid is processed
7Individual cell/mL).
Research object: 2 routine cerebrospinal fluid of the complex treatments such as intrathecal chemotherapy and craniospinal radiation therapy shift patients' samples of CSF that punctures.
Operation steps, analysis strategy and statistical method are the same.
The result shows: 2 routine patients through complex treatments such as the intrathecal chemotherapies (methotrexate (MTX) 12.5mg and dexamethasone 5mg) of 8 courses for the treatment of and craniospinal radiation therapys after, puncture cerebrospinal fluid detects through this antibody combination flow cytometer again, wherein an example is not found CD45-/CD56+/GD2+ immunophenotype cell, and healing is left hospital.Another example still detects CD45-/CD56+/GD2+ immunophenotype cell, continues chemotherapy and radiotherapy, and cerebrospinal fluid detects still lasting masculin, and is final dead.Show that fluorescence probe of the present invention can monitor the treatment curative effect that retinoblastoma cerebrospinal fluid shifts, instruct clinical rational drug use.
Claims (6)
1. the fluorescence probe that shifts of flow cytometry diagnosis and monitoring retinoblastoma cerebrospinal fluid comprises three kinds of fluorescent-labeled antibody, it is characterized in that, described three kinds of fluorescent-labeled antibody are GD2-FITC antibody, CD56-PE antibody and CD45-PerCP antibody.
2. fluorescence probe as claimed in claim 1 is characterized in that, three kinds of fluorescent-labeled antibody are monoclonal antibody.
3. the kit that shifts of Flow Cytometry diagnosis and monitoring retinoblastoma cerebrospinal fluid comprises:
Probe solution take phosphate buffer as solvent, comprises GD2-FITC antibody, CD56-PE antibody and CD45-PerCP antibody;
Hemolytic agent;
Phosphate buffer;
The positive criteria product.
4. kit as claimed in claim 3 is characterized in that, the concentration ratio of three kinds of fluorescent-labeled antibody is 1: 1: 1.
5. kit as claimed in claim 3 is characterized in that, described probe solution comprises antiseptic.
6. kit as claimed in claim 5 is characterized in that, described antiseptic is Sodium azide.
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| CN2012105209705A CN103018459A (en) | 2012-12-03 | 2012-12-03 | Fluorescence probe for diagnosis and monitoring of meningeal metastasis of retinoblastoma through flow cytometry |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101620220A (en) * | 2008-06-30 | 2010-01-06 | 张晖 | Cytoperm capable of being used as hemolytic agent and using method thereof |
| CN103424540A (en) * | 2012-05-18 | 2013-12-04 | 嘉善加斯戴克医疗器械有限公司 | Leukocyte classification kit and classification method thereof |
-
2012
- 2012-12-03 CN CN2012105209705A patent/CN103018459A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101620220A (en) * | 2008-06-30 | 2010-01-06 | 张晖 | Cytoperm capable of being used as hemolytic agent and using method thereof |
| CN103424540A (en) * | 2012-05-18 | 2013-12-04 | 嘉善加斯戴克医疗器械有限公司 | Leukocyte classification kit and classification method thereof |
Non-Patent Citations (3)
| Title |
|---|
| HONGQIAO SHEN ET AL: "Detection of the GD2+/CD56+/CD45- Immunophenotype by Flow Cytometry in Cerebrospinal Fluids from a Patient with Retinoblastoma", 《PEDIATRIC HEMATOLOGY AND ONCOLOGY》 * |
| 姚长青等: "DOI在精品科技期刊全文数据库中的应用", 《中国科技资源导刊》 * |
| 肖婧等: "以流式细胞术建立中国汉族健康儿童外周血淋巴细胞亚群相对计数的正常参考值", 《中国循证儿科杂志》 * |
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Application publication date: 20130403 |