CN204008664U - For detection of PNH clone's kit - Google Patents
For detection of PNH clone's kit Download PDFInfo
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- CN204008664U CN204008664U CN201420429778.XU CN201420429778U CN204008664U CN 204008664 U CN204008664 U CN 204008664U CN 201420429778 U CN201420429778 U CN 201420429778U CN 204008664 U CN204008664 U CN 204008664U
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- 238000001514 detection method Methods 0.000 title claims abstract description 16
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 49
- 210000003743 erythrocyte Anatomy 0.000 claims abstract description 15
- 102100022002 CD59 glycoprotein Human genes 0.000 claims abstract description 12
- 101000897400 Homo sapiens CD59 glycoprotein Proteins 0.000 claims abstract description 12
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 claims abstract description 10
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 claims abstract description 10
- 101000884271 Homo sapiens Signal transducer CD24 Proteins 0.000 claims abstract description 9
- 102100038081 Signal transducer CD24 Human genes 0.000 claims abstract description 9
- 101000981881 Brevibacillus parabrevis ATP-dependent glycine adenylase Proteins 0.000 claims abstract description 8
- 101000981889 Brevibacillus parabrevis Linear gramicidin-PCP reductase Proteins 0.000 claims abstract description 8
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 claims abstract description 8
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 claims abstract description 8
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 claims abstract description 7
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 claims abstract description 7
- 102100034540 Adenomatous polyposis coli protein Human genes 0.000 claims abstract description 6
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 238000013016 damping Methods 0.000 claims abstract description 5
- 239000012530 fluid Substances 0.000 claims abstract description 5
- 238000013461 design Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 17
- 210000003714 granulocyte Anatomy 0.000 description 9
- 210000001616 monocyte Anatomy 0.000 description 9
- 238000010586 diagram Methods 0.000 description 8
- 230000035945 sensitivity Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 4
- 102000008102 Ankyrins Human genes 0.000 description 3
- 108010049777 Ankyrins Proteins 0.000 description 3
- 230000008034 disappearance Effects 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 210000000265 leukocyte Anatomy 0.000 description 3
- 102100025680 Complement decay-accelerating factor Human genes 0.000 description 2
- 101000856022 Homo sapiens Complement decay-accelerating factor Proteins 0.000 description 2
- 210000001728 clone cell Anatomy 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000000554 iris Anatomy 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 230000001568 sexual effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 241000607528 Aeromonas hydrophila Species 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 101000595489 Homo sapiens Phosphatidylinositol N-acetylglucosaminyltransferase subunit A Proteins 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 102100036050 Phosphatidylinositol N-acetylglucosaminyltransferase subunit A Human genes 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 201000001505 hemoglobinuria Diseases 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000000422 nocturnal effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The utility model discloses a kind of kit for detection of PNH clone, it comprises box body (1), lid (3) and be arranged on the liner (2) in box body (1), described liner (2) is provided with aperture, the Reagent Tube (4) of GlyA FITC antibody is housed, the Reagent Tube (5) of CD59 PE antibody is housed, the Reagent Tube (6) of FLAER antibody is housed, the Reagent Tube (7) of CD24 PE antibody is housed, the Reagent Tube (8) that CD33 APC antibody is housed is equipped with the Reagent Tube (9) of CD14 APC-CY7 antibody, the Reagent Tube (10) of CD45 PerCP antibody is housed, the Reagent Tube (11) of erythrocyte cracked liquid is housed, the Reagent Tube (12) that PBS damping fluid is housed is arranged in described aperture.The utility model is simple in structure, reasonable in design, and detecting step is easy, and detection speed is fast, highly sensitive, is convenient at clinical application.
Description
Technical field
The utility model belongs to technical field of molecular biology, relates to a kind of kit for detection of PNH clone.
Background technology
Paraoxysmal nocturnal hemoglobinuria (PNH) is a kind of special acquired clone's property candidate stem cell disease, and this disease is due to PIGA genosome cell mutation, causes connecting due to the relevant ankyrin disappearance of GPI of cell membrane.The relevant experiment of the main centralized detecting red blood cell of detection of initial PNH: as Ham experiment and SH experiment.These two kinds of detection meanss all point out red blood cell to increase the haemolysis susceptibility of complement-mediated.But the two is not specific detection, and inconvenient operation.Flow cytometry is one of method detecting the relevant ankyrin disappearance of GPI, is also the necessary detection index of diagnosis PNH simultaneously.Conventional detection method is the deletion condition of Flow cytometry red blood cell and leukocyte surface CD55 and CD59 at present.Although CD55 and CD59 are widely used in detecting the PNH clone cell in haemocyte, have data to show, the two use in conjunction also possibly cannot detect trace P NH clone cell.
Utility model content
In order to solve the problems of the technologies described above, the purpose of this utility model is to provide a kind of kit for detection of PNH clone, can quick, easy, accurate, high sensitivity detection.
For achieving the above object, the technical scheme that the utility model is taked is: a kind of for detection of PNH clone kit, it comprises box body (1), lid (3) and be arranged on the liner (2) in box body (1), described liner (2) is provided with aperture, the Reagent Tube (4) of GlyA FITC antibody is housed, the Reagent Tube (5) of CD59 PE antibody is housed, the Reagent Tube (6) of FLAER antibody is housed, the Reagent Tube (7) of CD24 PE antibody is housed, the Reagent Tube (8) that CD33 APC antibody is housed is equipped with the Reagent Tube (9) of CD14 APC-CY7 antibody, the Reagent Tube (10) of CD45 PerCP antibody is housed, the Reagent Tube (11) of erythrocyte cracked liquid is housed, the Reagent Tube (12) that PBS damping fluid is housed is arranged in described aperture.
Wherein, described liner (2) is provided with 9 apertures, and described aperture is divided into 3 rows and is arranged in parallel on described liner (2).
By above technical scheme, the beneficial effects of the utility model are as follows:
(1) kit of the present utility model is simple in structure, reasonable in design, is convenient for carrying, preserves, not vulnerable to pollution.
(2) kit of the present utility model detection sensitivity in the time detecting PNH clone is high, and interpretation of result method is simple, directly perceived, is convenient at clinical application.
Brief description of the drawings
Fig. 1 is structural representation of the present utility model;
In figure, 1-box body, 3-lid, 2-liner, 4-is equipped with the Reagent Tube of GlyA FITC antibody, and 5-is equipped with the Reagent Tube of CD59 PE antibody, and 6-is equipped with the Reagent Tube of FLAER antibody, 7-is equipped with the Reagent Tube of CD24 PE antibody, 8-is equipped with the Reagent Tube of CD33 APC antibody, and 9-is equipped with the Reagent Tube of CD14 APC-CY7 antibody, and 10-is equipped with the Reagent Tube of CD45 PerCP antibody, 11-is equipped with the Reagent Tube of erythrocyte cracked liquid, and 12-is equipped with the Reagent Tube of PBS damping fluid.
Fig. 2 is the interior normocytic schematic diagram of human body that adopts kit of the present utility model;
Fig. 3 is the interior normal leukocytic schematic diagram of human body that adopts kit of the present utility model;
Fig. 4 is the erythrocytic schematic diagram that adopts the PNH clone patient of kit of the present utility model;
Fig. 5 is the leukocytic schematic diagram that adopts the PNH clone patient of kit of the present utility model.
Embodiment
Below in conjunction with embodiment, embodiment of the present utility model is further described, advantage and disadvantage of the present utility model will be more clear along with description.But these embodiment are only exemplary, scope of the present utility model are not formed to any restriction.
The experimental technique using in following embodiment if no special instructions, is conventional method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
As shown in Figure 1, the utility model provides a kind of kit for detection of PNH clone, comprise box body 1, lid 3 and be arranged on the liner 2 in box body 1, described liner 2 is provided with 9 apertures, described aperture is divided into three rows and is arranged in parallel on described liner 2, the Reagent Tube 4 of GlyA FITC antibody is housed, the Reagent Tube 5 of CD59 PE antibody is housed, the Reagent Tube 6 of FLAER antibody is housed, the Reagent Tube 7 of CD24PE antibody is housed, the Reagent Tube 8 of CD33 APC antibody is housed, the Reagent Tube 9 of CD14 APC-CY7 antibody is housed, the Reagent Tube 10 of CD45 PerCP antibody is housed, the Reagent Tube 11 of erythrocyte cracked liquid is housed, the Reagent Tube 12 that PBS damping fluid is housed is arranged in described aperture.
The present invention adopts flow cytometry high sensitivity and detects PNH clone, thus auxiliary clinical diagnosis better.Data show, the best reagent that detects the relevant ankyrin disappearance of leucocyte GPI is the variant that contains fluorescently-labeled non-activity Aeromonas hydrophila lysin precursor, i.e. FLAER, can specific bond in GPI anchorin.Compared with all the other reagent, the sensitivity of FLAER is higher, and it can also combine use with other grouping by monoclonal reagents, improves specificity, thereby makes conclusion more accurate.For the raising of red blood cell detection sensitivity, we are establishing by cell size and graininess on the basis of door before, then with the further tagged blood cells of GlyA, utilize CD59 that susceptibility is relatively high to define without red blood cell PNH and clone.
Detecting step is as follows:
(1) get four streaming dedicated pipe, be labeled as respectively R-C, R-T, W-C, W-T, and dated sample numbering (R-C, W-C are control tube);
(2) in each pipe, antibody combination is as follows:
The combination of table 1 antibody
| Guan Hao | PerCP | FITC | PE | APC | APC-CY7 |
| R-C | GlyA | ||||
| R-T | GlyA | CD59 | |||
| Pipe 1 | CD45 | ||||
| Pipe 2 | CD45 | CD24 | CD33 | CD14 |
(3) these 10 μ l of label taking, be diluted to 100 μ l with PBS, in R-C, R-T pipe, add respectively sample 30 μ l, room temperature lucifuge is hatched 20 minutes, hatch in backward 2 pipes and added respectively PBS 1000 μ l, with 1500rpm rotating speed after centrifugal 5 minutes, supernatant discarded, repeat 2 times, then in 2 pipes, add respectively 0.2ml PBS resuspended rear on machine.
(4) sample is carried out to white blood cell count(WBC), control cell concentration in 4-6 × 10
6/ ml adds sample 70 μ l in W-C, W-T pipe, and low speed vortex mixes, and room temperature lucifuge is hatched 20 minutes;
(5) in W-C, W-T pipe, add erythrocyte cracked liquid 1000 μ l, low speed vortex mixes, room temperature lucifuge splitting erythrocyte 10 minutes;
(6) after cracking finishes, with 1500rpm rotating speed centrifugal 5 minutes;
In (7) two pipes, add 1000 μ lPBS washings, with 1500rpm rotating speed centrifugal 5 minutes.
(8) in W-T pipe, add FLAER antibody 2 μ l, room temperature lucifuge is hatched 15 minutes.
(9) add 1000 μ lPBS washings after having hatched, with 1500rpm rotating speed centrifugal 5 minutes.In two pipes, add respectively 0.2ml PBS resuspended, low speed vortex mixes, upper machine testing in 24h.
(10) upper machine testing.
(11) analyze in R-T pipe and have or not CD59 feminine gender or weak positive red blood cell, in W-T pipe, have or not FLAER-CD24 jack to jack adapter granulocyte and FLAER-CD14 jack to jack adapter monocyte.
Fig. 2 is the erythrocytic schematic diagram of normal human, and left figure is blank, in right figure P3 door, is III type cell, in P4 door, is II type cell, in P5 door, is I type cell.
Fig. 3 is the leukocytic schematic diagram of normal human, left figure slightly irises out respectively granulocyte and monocyte according to the fluorescence of CD33, in P5 door, it is granulocyte, in P3 door, it is monocyte, in Q2-1 door, it is normal granulocytes, CD24 and FLAER are all positive, in Q2 door, are normal monocyte, and CD14 and FLAER are all positive.
Fig. 4 is the schematic diagram of PNH erythrocyte, and left figure is blank, in right figure P3 door, is III type cell, in P4 door, is II type cell, in P5 door, is I type cell.
Fig. 5 is the leukocytic schematic diagram of PNH patient, left figure slightly irises out respectively granulocyte and monocyte according to the fluorescence of CD33, in P5 door, is granulocyte, in P3 door, is monocyte, in Q2-1 door, it is normal granulocytes, CD24 and FLAER are all positive, in Q3-1 door, are granulocyte PNH clone, and CD24 and FLAER are all negative, in Q2 door, it is normal monocyte, CD14 and FLAER are all positive, in Q3 door, are monocyte PNH clone, and CD14 and FLAER are all negative.
Result basis for estimation:
In R-T pipe, CD59 weak positive cell is II type cell, and CD59 negative cells is III type cell, and II type cell and III type cell sum are red blood cell PNH clone.In W-T pipe, FLAER and CD24 jack to jack adapter sexual cell are granulocyte PNH clone, and FLAER and CD14 jack to jack adapter sexual cell are monocyte PNH clone.
For example understand embodiment of the present utility model above; what those skilled in the art should understand that is; do not departing under spirit and scope of the present utility model and can the details of technical solutions of the utility model and form modified or be replaced, but these amendments and replacement all fall in protection domain of the present utility model.
Claims (2)
1. the kit for detection of PNH clone, it is characterized in that comprising box body (1), lid (3) and be arranged on the liner (2) in box body (1), described liner (2) is provided with aperture, the Reagent Tube (4) of GlyA FITC antibody is housed, the Reagent Tube (5) of CD59 PE antibody is housed, the Reagent Tube (6) of FLAER antibody is housed, the Reagent Tube (7) of CD24 PE antibody is housed, the Reagent Tube (8) that CD33 APC antibody is housed is equipped with the Reagent Tube (9) of CD14 APC-CY7 antibody, the Reagent Tube (10) of CD45 PerCP antibody is housed, the Reagent Tube (11) of erythrocyte cracked liquid is housed, the Reagent Tube (12) that PBS damping fluid is housed is arranged in described aperture.
2. according to kit claimed in claim 1, it is characterized in that, described liner (2) is provided with 9 apertures, and described aperture is divided into 3 rows and is arranged in parallel on described liner (2).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201420429778.XU CN204008664U (en) | 2014-07-31 | 2014-07-31 | For detection of PNH clone's kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201420429778.XU CN204008664U (en) | 2014-07-31 | 2014-07-31 | For detection of PNH clone's kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN204008664U true CN204008664U (en) | 2014-12-10 |
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ID=52048643
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201420429778.XU Expired - Lifetime CN204008664U (en) | 2014-07-31 | 2014-07-31 | For detection of PNH clone's kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN204008664U (en) |
-
2014
- 2014-07-31 CN CN201420429778.XU patent/CN204008664U/en not_active Expired - Lifetime
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP03 | Change of name, title or address |
Address after: 100176 building 4, building 3, No. 1, Desheng North Street, Beijing Economic and Technological Development Zone, Daxing District, Beijing Patentee after: BEIJING HAISITE MEDICAL EXAMINE LABORATORY CO.,LTD. Address before: 100176 A-3, A-4, 1 North Sheng street, Yizhuang economic and Technological Development Zone, Beijing, Daxing District Patentee before: BEIJING HIGHTRUST DIAGNOSTICS CO.,LTD. |
|
| CP03 | Change of name, title or address | ||
| CX01 | Expiry of patent term |
Granted publication date: 20141210 |
|
| CX01 | Expiry of patent term |