CN104865377A - Staining kit of micro megakaryocytes of bone marrow smear, and using method of staining kit - Google Patents
Staining kit of micro megakaryocytes of bone marrow smear, and using method of staining kit Download PDFInfo
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- CN104865377A CN104865377A CN201510289582.4A CN201510289582A CN104865377A CN 104865377 A CN104865377 A CN 104865377A CN 201510289582 A CN201510289582 A CN 201510289582A CN 104865377 A CN104865377 A CN 104865377A
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- 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
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Abstract
The invention provides a staining kit of micro megakaryocytes of a bone marrow smear, and a using method of the staining kit. The staining kit comprises a reagent tube filled with a Polymer Helper reagent, a reagent tube filled with a PBS (Phosphate Buffer Solution), a reagent tube filled with an antigen repairing solution, a reagent tube filled with a hydrogen dioxide solution, a reagent tube filled with diaminobenzidine, a reagent tube filled with hematoxylin, a reagent tube filled with acid alcohol of 1%, a reagent tube filled with ammonium hydroxide of 1%, a reagent tube filled with xylene, a reagent tube filled with alcohol, a reagent tube filled with CD41 monoclonal antibodies, a reagent tube filled with CD61 monoclonal antibodies, a reagent tube filled with universal CD41 and CD61 immunohistochemical antibodies, and a packing box for packing the reagent tubes in a separated and concentrated manner. The staining kit disclosed by the invention overcomes the defect that form cell smears are judged according to subjective experiences, and the interference of false positive of single CD antibody staining to results is avoided, so that the detection accuracy is improved.
Description
Technical field
The invention belongs to biological technical field, relate to bone marrow smear staining technique, particularly relate to the small megacaryocyte staining kit of bone marrow smear and the using method thereof of the antibody combined mark of a kind of CD41 and CD61.
Background technology
The megacaryocyte of normally a kind of morbid state of small macronucleus, small megacaryocyte diameter is less than 15 μm, this cell and lymphocyte sizableness, and Cytoplasmic inclusions is less, often with hematoblastic generation.Small polymerization cell sees multiple blood disease, is one of feature of marrow DH.Clinical conventional Rui Shi-Ji's nurse Sa decoration method carries out dyeing qualification to small megacaryocyte, but this colouring method is passed judgment on subjective experience by morphological cellular smear, be easy to small megacaryocyte and Megakaryoblast and lymphocyte to obscure, the accuracy detected so clinical is lower.At present, also have the detection technique of CD41 monoclonal antibody body bone marrow cell, but there is false-positive drawback in this technology, make the accuracy of testing result also lower.
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 small megacaryocyte staining kit of the bone marrow smear of the antibody combined mark of a kind of CD41 and CD61, the invention also discloses the using method of this kit.
The small megacaryocyte staining kit of bone marrow smear of the application, by the combined mark of rational CD41 and CD61 Antibody Combination, overcome the interference of the single antibody staining Jia positive, also can distinguish micromegakaryocyte, many circle micromegakaryocytes and large Dan Yuanhe megacaryocyte further, greatly improve clinical diagnosis and antidiastole.
According to embodiments of the invention, provide the small megacaryocyte staining kit of bone marrow smear with the antibody combined mark of CD41 and CD61, 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 CD41 monoclonal antibody is housed, the Reagent Tube of CD61 monoclonal antibody is housed, the Reagent Tube of universal CD41 and CD61 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 CD41 monoclonal antibody and CD61 monoclonal antibody; Second antibody is universal CD41 and CD61 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 concentration of gradient 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, groped by long-term, test, inquire into out a kind of brand-new CD41 and CD61 combined immunization histochemical staining method, both the shortcoming that morphological cellular smear is passed judgment on subjective experience had been overcome, combine colour developing again with antigen-antibody and find the positive cell effect that same histotomy SABC is similar for diagnosis basis reaches, turn avoid in the past single CD41 antibody staining false positive to the interference of result, improve the accuracy of detection, and sample is easy to obtain, the karyocyte collected is many, substantially increase positive rate, there is the advantage of oneself uniqueness, meet clinical demand.
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.
Embodiment
By the small megacaryocyte staining kit of the bone marrow smear of the antibody combined mark of CD41 and CD61 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 CD41 monoclonal antibody is housed, the Reagent Tube of CD61 monoclonal antibody is housed, the Reagent Tube of universal CD41 and CD61 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, antibody 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 (CD41 monoclonal antibody and CD61 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 CD41 and CD61 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 the small megacaryocyte staining kit of the bone marrow smear of the antibody combined mark of CD41 and CD61, 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 CD41 monoclonal antibody is housed, the Reagent Tube of CD61 monoclonal antibody is housed, the Reagent Tube of universal CD41 and CD61 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 CD41 monoclonal antibody and CD61 monoclonal antibody; Second antibody is universal CD41 and CD61 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.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108387745A (en) * | 2018-03-02 | 2018-08-10 | 首都医科大学附属北京胸科医院 | Application of the CD4+T lymphocytes characteristic protein in tuberculosis infection and active tuberculosis are hidden in identification |
| CN111004838A (en) * | 2019-12-30 | 2020-04-14 | 武汉大学 | Application of bone marrow smear fluorescence in situ hybridization technology in multiple myeloma |
Citations (3)
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| US20070077654A1 (en) * | 2004-11-01 | 2007-04-05 | Thomson James A | Platelets from stem cells |
| CN101200708A (en) * | 2007-11-16 | 2008-06-18 | 协和干细胞基因工程有限公司 | Mouse anti-human CD14 monoclonal antibody hybridoma cell line, monoclonal antibody, immunohistochemical reagent kit and uses thereof |
| 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 |
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2015
- 2015-05-29 CN CN201510289582.4A patent/CN104865377A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070077654A1 (en) * | 2004-11-01 | 2007-04-05 | Thomson James A | Platelets from stem cells |
| CN101200708A (en) * | 2007-11-16 | 2008-06-18 | 协和干细胞基因工程有限公司 | Mouse anti-human CD14 monoclonal antibody hybridoma cell line, monoclonal antibody, immunohistochemical reagent kit and uses thereof |
| 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 (3)
| Title |
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| JÜRGEN THIELE 等: "Atypical micromegakaryocytes, promegakaryoblasts and megakaryoblasts: a critical evaluation by immunohistochemistry, cytochemistry and morphometry of bone marrow trephines in chronic myeloid leukemia and myelodysplastic syndromes", 《VIRCHOWS ARCHIV B CELL PATHOL》 * |
| 冯志军 等: "CD41标记微小巨核细胞染色法及其临床应用价值探讨", 《医药论坛杂志》 * |
| 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（电子期刊） 医药卫生科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108387745A (en) * | 2018-03-02 | 2018-08-10 | 首都医科大学附属北京胸科医院 | Application of the CD4+T lymphocytes characteristic protein in tuberculosis infection and active tuberculosis are hidden in identification |
| CN111004838A (en) * | 2019-12-30 | 2020-04-14 | 武汉大学 | Application of bone marrow smear fluorescence in situ hybridization technology in multiple myeloma |
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