CN104931699A - Bone marrow smear atypical lymphocyte staining kit and application method thereof - Google Patents
Bone marrow smear atypical lymphocyte staining kit and application method thereof Download PDFInfo
- Publication number
- CN104931699A CN104931699A CN201510289585.8A CN201510289585A CN104931699A CN 104931699 A CN104931699 A CN 104931699A CN 201510289585 A CN201510289585 A CN 201510289585A CN 104931699 A CN104931699 A CN 104931699A
- Authority
- CN
- China
- Prior art keywords
- reagent tube
- bone marrow
- reagent
- housed
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
Abstract
The invention provides a bone marrow smear atypical lymphocyte staining kit and an application method thereof, wherein the kit comprises a reagent tube filled with Polymer Helper, a reagent tube filled with PBS phosphate buffer solution, a reagent tube filled with antigen retrieval buffer, a reagent tube filled with hydrogen dioxide solution, a reagent tube filled with diaminobenzidine staining solution, a reagent tube filled with hematoxylin staining solution, a reagent tube filled with 1% acidulated alcohol, a reagent tube filled with 1% ammonium hydroxide, a reagent tube filled with xylene, a reagent tube filled with ethyl alcohol, a reagent tube filled with CD3 monoclonal antibody, a reagent tube filled with Pax-5 monoclonal antibody and a reagent tube filled with general type CD3 and Pax-5 immunohistochemical antibody and a packing box for separating and centrally packing the reagent tubes. According to the kit and the application method thereof, a bone marrow smear also can be used for carrying out the classification of lymphocyte, the value of bone marrow smear lymphoma diagnosis is greatly improved, and the technology is relatively simple, is easy to popularize and can be used for pathological diagnosis and therapeutic outcome judgment.
Description
Technical field
The invention belongs to biological technical field, relate to bone marrow smear staining technique, particularly relate to bone marrow smear abnormal lymphocytes staining kit and the using method thereof of the antibody combined mark of a kind of CD3 and PAX5.
Background technology
Malignant lymphoma (hereinafter referred to as lymthoma) is one group of malignant tumour originating from lymph node or ENT, organ, Hodgkin lymphoma (Hodgkin'sLymphoma is divided into according to tissue pathology checking, molecular biology behavior and clinical characters, and non-Hodgkin lymphoma (non Hodgkin lymphoma, NHL) HD).In western countries, the lymphadenomatous incidence of disease accounts for the 8th of whole tumor disease.In China, account for the 9th of male malignancy and the 11st of female malignant respectively.Bone Marrow Involvement is lymphadenomatous common manifestation, therefore accurately detects lymthoma with or without Bone Marrow Involvement, to determining that lymphadenomatous clinical stages is significant.Bone marrow examination in lymphadenomatous pathological diagnosis, by stages, treatment and prognostic evaluation play an important role, its positive findings is the important evidence that lymthoma whole body is sent out, and often directly affects disease by stages.Lymphadenomatous diagnosis and classification depends on Histopathology, and bone marrow examination is still the reliable approach judging to invade in lymthoma marrow so far, and the concrete grammar therefore detecting lymthoma Bone Marrow Involvement seems particularly important.
Bone marrow smear and biopsy be the reliable and method of safety as classics, has important using value.Bone marrow biopsy does immunohistochemical staining because tissue can add, involve in marrow in lymthoma and play very crucial effect always, but because of technical sophistication, diagnosis difficulty is high, more difficult popularization always, only has some senior pathology departments of minority three Ji Jia hospital to carry out.Bone marrow smear technology is relatively simple, promote more extensive at home, a lot of secondary first hospital has all carried out this project, but because bone marrow smear can not do immunohistochemical staining always, acute and chronic lymphocytic leukemia can only be separated, and can not separate T category-B type, bone marrow smear diagnoses comparatively biopsy diagnosis to be worth not enough.
Summary of the invention
In order to solve the problems of the technologies described above, the object of the present invention is to provide the bone marrow smear abnormal lymphocytes staining kit of the antibody combined mark of a kind of CD3 and PAX5, the invention also discloses the using method of this kit.
Present inventor uses immunohistochemical staining principle design bone marrow smear abnormal lymphocytes staining kit, by the combined mark of rational CD3 and PAX5 Antibody Combination, make bone marrow smear also can carry out lymphocytic classification, greatly improve the value of bone marrow smear Lymphoma Diagnosis.
According to embodiments of the invention, provide the bone marrow smear abnormal lymphocytes staining kit with the antibody combined mark of CD3 and PAX5, it mainly comprises: the Reagent Tube that Polymer Helper reagent is housed, the Reagent Tube of PBS phosphate buffer is housed, the Reagent Tube of antigen retrieval buffers is housed, the Reagent Tube of superoxol is housed, the Reagent Tube of diaminobenzidine dyeing liquor is housed, the Reagent Tube of haematoxylin dyeing liquid is housed, the Reagent Tube of 1% sour alcohol is housed, the Reagent Tube of 1% ammoniacal liquor is housed, the Reagent Tube of dimethylbenzene is housed, fill spirituous Reagent Tube, the Reagent Tube of CD3 monoclonal antibody is housed, the Reagent Tube of Pax-5 monoclonal antibody is housed, the Reagent Tube of universal CD3 and Pax-5 SABC antibody is housed, separate and concentrate the packing box packing these Reagent Tubes.
Wherein, described Polymer Helper reagent is the Polymer Helper reagent of Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge.
Wherein, described sour alcohol is hydrochloride alcohol.
Wherein, PBS phosphate buffer density is 0.01M, and pH value is 7.2 ~ 7.4.
Wherein, superoxol concentration is 3 ~ 3.5%, and further, described superoxol concentration is 3%.
The using method of kit of the present invention, comprises the following steps:
1), after using antigen retrieval buffers to carry out antigen retrieval to bone marrow smear, distilled water washing bone marrow smear is used;
2) use superoxol to close bone marrow smear, then use PBS phosphate buffer washing bone marrow smear;
3) first antibody is dripped, incubated at room 25 ~ 35 minutes, after the washing of PBS phosphate buffer, drip Polymer Helper reagent again, incubated at room 15 ~ 25 minutes, after the washing of PBS phosphate buffer, then drips second antibody, incubated at room 15 ~ 25 minutes, then wash with PBS phosphate buffer;
4) use the colour developing of diaminobenzidine (Diaminobenzidine, DAB) dyeing liquor, then use haematoxylin dyeing, use distilled water washing bone marrow smear;
5) in 1% sour alcohol, rinse 2 times, wash slide with distilled water, and then rinse 2 times with 1% ammoniacal liquor, wash slide with distilled water;
6) (each 30s) dehydration after (2min) does transparent processing in dimethylbenzene, just can pass through microscope sentence read result in the alcohol (75% ~ 100%) of gradient dilution.
Wherein, step 3) described in first antibody be CD3 monoclonal antibody and Pax-5 monoclonal antibody; Second antibody is universal CD3 and Pax-5 SABC antibody.
Wherein, step 3) in, hatch 30 minutes after dripping first antibody, hatch 20 minutes after dripping Polymer Helper reagent, after dripping second antibody, hatch 20 minutes.
Wherein, step 6) in, the alcohol gradient of dilution is 75% → 85% → 100%.
By above technical scheme, beneficial effect of the present invention is as follows:
The present invention carries out technological innovation, overcome the technical limitation in the past can only carrying out immunostaining detection to histotomy, by the process of bone marrow smear sample characteristic, use immunohistochemical staining principle, by the combined mark of rational CD3 and PAX5 Antibody Combination, make bone marrow smear also can carry out lymphocytic classification, greatly improve the value of bone marrow smear Lymphoma Diagnosis, and technology is relatively simple, be easy to promote, can be used for pathological diagnosis and treatment results judge.
Embodiment
Be further described the specific embodiment of the present invention below in conjunction with embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.
The experimental technique used in following embodiment, if no special instructions, is conventional method.
The material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The bone marrow smear abnormal lymphocytes staining kit of the antibody combined mark of embodiment CD3 and PAX5 and using method thereof
1. be equipped with in this kit:
The Reagent Tube of Polymer Helper reagent is housed, the Reagent Tube of PBS phosphate buffer is housed, the Reagent Tube of antigen retrieval buffers is housed, the Reagent Tube of superoxol is housed, the Reagent Tube of diaminobenzidine dyeing liquor is housed, the Reagent Tube of haematoxylin dyeing liquid is housed, the Reagent Tube of 1% sour alcohol is housed, the Reagent Tube of 1% ammoniacal liquor is housed, the Reagent Tube of dimethylbenzene is housed, fill spirituous Reagent Tube, the Reagent Tube of CD3 monoclonal antibody is housed, the Reagent Tube of Pax-5 monoclonal antibody is housed, the Reagent Tube of universal CD3 and Pax-5 SABC antibody is housed, separate and concentrate the packing box packing these Reagent Tubes.
The reagent such as above-mentioned Polymer Helper reagent, antigen retrieval buffers, diaminobenzidine (Diaminobenzidine, DAB) dyeing liquor, PBS phosphate buffer are all bought from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge.Their catalog number is respectively: Polymer Helper reagent (PV-6000), antigen retrieval buffers (TA-999-DHBH), diaminobenzidine (Diaminobenzidine, DAB) dyeing liquor (ZLI-9032), PBS phosphate buffer (ZLI-9062).
2. the using method of kit is as follows:
(1) get out bone marrow smear to be detected, after using antigen retrieval buffers to carry out antigen retrieval to bone marrow smear, use distilled water washing bone marrow smear;
(2) use superoxol to close bone marrow smear, then use PBS phosphate buffer washing bone marrow smear;
(3) first antibody (CD3 monoclonal antibody and Pax-5 monoclonal antibody) is dripped, incubated at room 30 minutes, after the washing of PBS phosphate buffer, drip Polymer Helper reagent again, incubated at room 20 minutes, after the washing of PBS phosphate buffer, then drips second antibody (universal CD3 and Pax-5 SABC antibody), incubated at room 20 minutes, then wash with PBS phosphate buffer;
(4) use the colour developing of diaminobenzidine (Diaminobenzidine, DAB) dyeing liquor, then use haematoxylin dyeing, use distilled water washing bone marrow smear;
(5) in 1% hydrochloride alcohol, rinse 2 times, wash slide with distilled water, and then rinse 2 times with 1% ammoniacal liquor, wash slide with distilled water;
(6) (each 30s) dehydration after (2min) does transparent processing in dimethylbenzene, just can pass through microscope sentence read result in the alcohol (75% → 85% → 100%) of gradient dilution.
It should be appreciated by those skilled in the art, can modify to the details of technical solution of the present invention and form lower without departing from the spirit and scope of the present invention or replace, but these amendments and replacement all fall within the scope of protection of the present invention.
Claims (10)
1., with a bone marrow smear abnormal lymphocytes staining kit for the antibody combined mark of CD3 and PAX5, it is characterized in that, this kit comprises:
The Reagent Tube of Polymer Helper reagent is housed, the Reagent Tube of PBS phosphate buffer is housed, the Reagent Tube of antigen retrieval buffers is housed, the Reagent Tube of superoxol is housed, the Reagent Tube of diaminobenzidine dyeing liquor is housed, the Reagent Tube of haematoxylin dyeing liquid is housed, the Reagent Tube of 1% sour alcohol is housed, the Reagent Tube of 1% ammoniacal liquor is housed, the Reagent Tube of dimethylbenzene is housed, fill spirituous Reagent Tube, the Reagent Tube of CD3 monoclonal antibody is housed, the Reagent Tube of Pax-5 monoclonal antibody is housed, the Reagent Tube of universal CD3 and Pax-5 SABC antibody is housed,
Separate and concentrate the packing box packing these Reagent Tubes.
2. according to kit according to claim 1, it is characterized in that: described Polymer Helper reagent is the Polymer Helper reagent of Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge.
3. according to kit according to claim 1, it is characterized in that: described sour alcohol is hydrochloride alcohol.
4. according to kit according to claim 1, it is characterized in that: the concentration of described PBS phosphate buffer is 0.01M, and pH value is 7.2 ~ 7.4.
5. according to kit according to claim 1, it is characterized in that: the concentration of described superoxol is 3 ~ 3.5%.
6. the using method of kit according to claim 1, is characterized in that, comprises the following steps:
After step 1, use antigen retrieval buffers carry out antigen retrieval to bone marrow smear, then wash bone marrow smear with distilled water;
Step 2, use superoxol are closed bone marrow smear, then use PBS phosphate buffer washing bone marrow smear;
Step 3, dropping first antibody, incubated at room 25 ~ 35 minutes, after the washing of PBS phosphate buffer, drip Polymer Helper reagent again, incubated at room 15 ~ 25 minutes, after the washing of PBS phosphate buffer, then drips second antibody, incubated at room 15 ~ 25 minutes, then wash with PBS phosphate buffer;
Step 4, the colour developing of use diaminobenzidine dyeing liquor, then use haematoxylin dyeing, use distilled water washing bone marrow smear;
Step 5, in 1% sour alcohol, rinse 2 times, wash slide with distilled water, and then rinse 2 times with 1% ammoniacal liquor, wash slide with distilled water;
Step 6, bone marrow smear to be dewatered in the alcohol of 75% ~ 100% gradient dilution, each serial dehydration 30s, then do 2min transparent processing in dimethylbenzene after, by microscope sentence read result.
7. according to using method according to claim 6, it is characterized in that: the first antibody described in step 3 is CD3 monoclonal antibody and Pax-5 monoclonal antibody; Second antibody is universal CD3 and Pax-5 SABC antibody.
8. according to using method according to claim 6, it is characterized in that: in step 3, after dripping first antibody, hatch 30 minutes, hatch 20 minutes after dripping Polymer Helper reagent, after dripping second antibody, hatch 20 minutes.
9. according to using method according to claim 6, it is characterized in that: in step 6, the gradient of alcohol dilution is 75% → 85% → 100%.
10. according to the arbitrary described application of kit in bone marrow smear abnormal lymphocytes staining examine of Claims 1 to 5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510289585.8A CN104931699A (en) | 2015-05-29 | 2015-05-29 | Bone marrow smear atypical lymphocyte staining kit and application method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510289585.8A CN104931699A (en) | 2015-05-29 | 2015-05-29 | Bone marrow smear atypical lymphocyte staining kit and application method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104931699A true CN104931699A (en) | 2015-09-23 |
Family
ID=54118965
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510289585.8A Pending CN104931699A (en) | 2015-05-29 | 2015-05-29 | Bone marrow smear atypical lymphocyte staining kit and application method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104931699A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108623683A (en) * | 2018-05-30 | 2018-10-09 | 福州迈新生物技术开发有限公司 | The monoclonal antibody and its cell strain, preparation method and application of anti-Pax-5 albumen |
| WO2019083383A1 (en) * | 2017-10-25 | 2019-05-02 | Uniwersytet Medyczny Im. Piastów Śląskich We Wrocławiu | Method for diagnosing neoplasms of lymphoid tissue |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101251533A (en) * | 2008-04-09 | 2008-08-27 | 范洪 | Medulla smear plasmalogen staining agent group and use method |
| CN101424691A (en) * | 2008-12-15 | 2009-05-06 | 北京大学第三医院 | Infiltrative breast carcinoma recurring risk assessment kit |
| WO2012078138A1 (en) * | 2010-12-07 | 2012-06-14 | Wei-Sing Chu | Cell array quality control device for pathological analysis |
| CN202710380U (en) * | 2012-08-21 | 2013-01-30 | 汕头大学医学院第一附属医院 | Histochemical staining dish of blood smears and bone marrow smears |
| EP2749642A1 (en) * | 2011-11-30 | 2014-07-02 | National Cancer Center | Induced malignant stem cells |
| CN103994913A (en) * | 2014-02-26 | 2014-08-20 | 武汉艾迪康医学检验所有限公司 | Fixed-decalcifying fluid for bone marrow biopsy and paraffin section method of bone marrow biopsy tissue |
| CN104407151A (en) * | 2014-11-19 | 2015-03-11 | 汕头大学医学院 | Kit integrating three proteins such as Kindlin-2, Myosin-9 and Annexin II for prognosis evaluation of patient suffering from esophageal squamous cell carcinoma |
| CN104640881A (en) * | 2012-09-21 | 2015-05-20 | 瑞泽恩制药公司 | Anti-CD3 antibodies, bispecific antigen-binding molecules that bind CD3 and CD20, and uses thereof |
-
2015
- 2015-05-29 CN CN201510289585.8A patent/CN104931699A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101251533A (en) * | 2008-04-09 | 2008-08-27 | 范洪 | Medulla smear plasmalogen staining agent group and use method |
| CN101424691A (en) * | 2008-12-15 | 2009-05-06 | 北京大学第三医院 | Infiltrative breast carcinoma recurring risk assessment kit |
| WO2012078138A1 (en) * | 2010-12-07 | 2012-06-14 | Wei-Sing Chu | Cell array quality control device for pathological analysis |
| EP2749642A1 (en) * | 2011-11-30 | 2014-07-02 | National Cancer Center | Induced malignant stem cells |
| CN202710380U (en) * | 2012-08-21 | 2013-01-30 | 汕头大学医学院第一附属医院 | Histochemical staining dish of blood smears and bone marrow smears |
| CN104640881A (en) * | 2012-09-21 | 2015-05-20 | 瑞泽恩制药公司 | Anti-CD3 antibodies, bispecific antigen-binding molecules that bind CD3 and CD20, and uses thereof |
| CN103994913A (en) * | 2014-02-26 | 2014-08-20 | 武汉艾迪康医学检验所有限公司 | Fixed-decalcifying fluid for bone marrow biopsy and paraffin section method of bone marrow biopsy tissue |
| CN104407151A (en) * | 2014-11-19 | 2015-03-11 | 汕头大学医学院 | Kit integrating three proteins such as Kindlin-2, Myosin-9 and Annexin II for prognosis evaluation of patient suffering from esophageal squamous cell carcinoma |
Non-Patent Citations (4)
| Title |
|---|
| 周小鸽: "淋巴瘤病理诊断中的抗体选择", 《诊断病理学杂志》 * |
| 官兵和周晓军: "淋巴瘤病理诊断常用免疫组化抗体的选择策略", 《临床与实验病理学杂志》 * |
| 王晋芬 等: "根据WHO淋巴造血***肿瘤新分类对山西省淋巴瘤分布特点的分析", 《中华病理学杂志》 * |
| 陈新颖: "40例骨髓转移癌的临床研究", 《中国优秀硕士学位论文全文数据库（医药卫生科技辑，2012年）》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019083383A1 (en) * | 2017-10-25 | 2019-05-02 | Uniwersytet Medyczny Im. Piastów Śląskich We Wrocławiu | Method for diagnosing neoplasms of lymphoid tissue |
| PL423266A1 (en) * | 2017-10-25 | 2019-05-06 | Univ Medyczny Im Piastow Slaskich We Wroclawiu | Method for diagnosing lymphatic system neoplasms |
| CN108623683A (en) * | 2018-05-30 | 2018-10-09 | 福州迈新生物技术开发有限公司 | The monoclonal antibody and its cell strain, preparation method and application of anti-Pax-5 albumen |
| CN108623683B (en) * | 2018-05-30 | 2021-06-04 | 福州迈新生物技术开发有限公司 | Monoclonal antibody for resisting Pax-5 protein, cell strain, preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106635995B (en) | Negative enrichment method for circulating tumor cells | |
| CN106093392B (en) | The integrated testing method and detection chip of a kind of urine excretion body separation, enrichment and detection | |
| Kim et al. | A microchip filter device incorporating slit arrays and 3-D flow for detection of circulating tumor cells using CAV1-EpCAM conjugated microbeads | |
| Kojika et al. | Immunohistochemical differential diagnosis between thymic carcinoma and type B3 thymoma: diagnostic utility of hypoxic marker, GLUT-1, in thymic epithelial neoplasms | |
| Gao et al. | Analysis of circulating tumor cells from lung cancer patients with multiple biomarkers using high-performance size-based microfluidic chip | |
| CN109187146B (en) | Human body cell full-form immunofluorescence staining method and kit | |
| Tan et al. | Continuous-flow cytomorphological staining and analysis | |
| Sheng et al. | Comparison of analytic performances of Cellsearch and iFISH approach in detecting circulating tumor cells | |
| Son et al. | Evaluation of urine cytology in urothelial carcinoma patients: a comparison of CellprepPlus® liquid-based cytology and conventional smear | |
| Yahya et al. | Evaluation of the sensitivity and specificity of immunohistochemical markers in the differential diagnosis of effusion cytology | |
| CN106989970A (en) | Integrated device and circulating tumor cell catching method are dyed in cell separation film-making | |
| Shidham et al. | Approach to diagnostic cytopathology of serous effusions | |
| CN104931699A (en) | Bone marrow smear atypical lymphocyte staining kit and application method thereof | |
| CN202482328U (en) | Circulating tumor cell detector | |
| Rao et al. | Detection of circulating tumour cells in patients with epithelial ovarian cancer by a microfluidic system | |
| CN104698193A (en) | Method for identifying blood group of rhesus monkey | |
| CN104359746B (en) | Cell mass collector and the method for collecting cell in humoral specimen | |
| CN212514620U (en) | P16MCM2 immunohistochemical double-staining detection kit | |
| Yang et al. | A cocktail of MCM2 and TOP2A, p16 INK4a and Ki-67 as biomarkers for the improved diagnosis of cervical intraepithelial lesion | |
| Zhao et al. | Separation and single-cell analysis for free gastric cancer cells in ascites and peritoneal lavages based on microfluidic chips | |
| CN108982839A (en) | Circulating tumor cell detection method based on immunomagnetic beads and flow cytometer | |
| CN106996975B (en) | Circulating tumor cell albumen parting kit | |
| CN104865377A (en) | Staining kit of micro megakaryocytes of bone marrow smear, and using method of staining kit | |
| US20160178630A1 (en) | Methods for predicting overall survival of cancer patients based on numbers of circulating tumor cells | |
| CN103399157B (en) | Lack the localization method of the PML albumen of nuclear localization signal |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150923 |
|
| RJ01 | Rejection of invention patent application after publication |