CN109734797B - Glycosylated hemoglobin extraction kit and extraction method - Google Patents
Glycosylated hemoglobin extraction kit and extraction method Download PDFInfo
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- 102000017011 Glycated Hemoglobin A Human genes 0.000 title claims abstract description 63
- 238000000605 extraction Methods 0.000 title claims abstract description 39
- 108010014663 Glycated Hemoglobin A Proteins 0.000 title claims abstract description 35
- 239000000243 solution Substances 0.000 claims abstract description 61
- 238000004140 cleaning Methods 0.000 claims abstract description 52
- 239000003480 eluent Substances 0.000 claims abstract description 22
- 239000007788 liquid Substances 0.000 claims abstract description 13
- 239000007853 buffer solution Substances 0.000 claims abstract description 3
- 108091005995 glycated hemoglobin Proteins 0.000 claims description 28
- 239000002244 precipitate Substances 0.000 claims description 27
- 239000006228 supernatant Substances 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 15
- 238000005119 centrifugation Methods 0.000 claims description 12
- 210000004369 blood Anatomy 0.000 claims description 8
- 239000008280 blood Substances 0.000 claims description 8
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 4
- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 2
- 235000011009 potassium phosphates Nutrition 0.000 claims description 2
- 239000001488 sodium phosphate Substances 0.000 claims description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 abstract 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 abstract 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 abstract 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 abstract 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 abstract 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 abstract 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 abstract 2
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 abstract 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 abstract 2
- 235000019270 ammonium chloride Nutrition 0.000 abstract 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 abstract 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 abstract 1
- 235000011130 ammonium sulphate Nutrition 0.000 abstract 1
- 239000000872 buffer Substances 0.000 abstract 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 abstract 1
- 235000019341 magnesium sulphate Nutrition 0.000 abstract 1
- 239000001103 potassium chloride Substances 0.000 abstract 1
- 235000011164 potassium chloride Nutrition 0.000 abstract 1
- 239000011780 sodium chloride Substances 0.000 abstract 1
- 239000003381 stabilizer Substances 0.000 abstract 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 abstract 1
- 238000011084 recovery Methods 0.000 description 9
- 238000001514 detection method Methods 0.000 description 8
- 102000001554 Hemoglobins Human genes 0.000 description 4
- 108010054147 Hemoglobins Proteins 0.000 description 4
- 210000003743 erythrocyte Anatomy 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000003149 assay kit Methods 0.000 description 3
- 241000345998 Calamus manan Species 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000004816 latex Substances 0.000 description 2
- 229920000126 latex Polymers 0.000 description 2
- 235000012950 rattan cane Nutrition 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000009172 bursting Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 229920001971 elastomer Polymers 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a glycosylated hemoglobin extraction kit and an extraction method, and relates to the technical field of biology. The extraction kit consists of a cleaning solution A, a cleaning solution B, an eluent and a storage solution, wherein: the cleaning solution A comprises the following components: buffer solution PH7.0, sodium chloride, potassium chloride, sodium azide; the cleaning liquid B comprises the following components: HEPS buffer, lithium chloride, magnesium sulfate and TCEP; the eluent comprises the following components: ammonium sulfate, ammonium chloride, triton, ethylene glycol; the storage solution comprises the following components: glycerol, TCEP, a stabilizer and sodium azide. The present invention has low requirement on operation equipment, and the extracted glycosylated hemoglobin can be stored for a long period of time.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a glycosylated hemoglobin extraction kit and an extraction method.
Background
There are three forms of hemoglobin in normal human blood: HbA, HbF, HbA2, HbA being the main component in generally adult erythrocytes. By using chromatography for hemoglobin separation, three glycohemoglobin can be isolated: HbAla, HbAlb and HbAlc, which together constitute glycated hemoglobin. The glycosylated hemoglobin is produced by combining hemoglobin in erythrocytes in human blood with blood sugar, and when the value of the glycosylated hemoglobin is higher, the combination of the hemoglobin and the blood sugar is more, so that the diabetic condition is more serious. Erythrocytes generally persist for 120 days in humans, and glycated hemoglobin levels generally reflect the average blood glucose level at 120 scales prior to testing.
At present, the method for extracting the glycosylated hemoglobin mainly comprises the steps of separating and purifying the glycosylated hemoglobin by an electric focusing fusion technology such as a non-immobilized rubber strip and polyacrylamide, separating and extracting the glycosylated hemoglobin by ion exchange chromatography, extracting the glycosylated hemoglobin by a free flow electric focusing technology and the like. However, the above three techniques have high facility requirements, and the extracted glycated hemoglobin cannot be stored for a long time after recovery, which is not favorable for scientific research.
Disclosure of Invention
The invention provides a glycosylated hemoglobin extraction kit and an extraction method, which have low requirements on operating equipment and can store extracted glycosylated hemoglobin for a long time.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a glycosylated hemoglobin extraction kit, which consists of a cleaning solution A, a cleaning solution B, an eluent and a storage solution, wherein:
the cleaning solution A comprises the following components in percentage by weight:
the cleaning liquid B comprises the following components in percentage by weight:
the eluent comprises the following components in percentage by weight:
the storage liquid comprises the following components in percentage by weight:
further, the final PH of the cleaning solution a and the cleaning solution B is 6.95. Further, the cleaning liquid A comprises the following components in percentage by weight:
further, the buffer solution is sodium phosphate or potassium phosphate.
Further, the cleaning liquid B comprises the following components in percentage by weight:
the present invention also provides a method for extracting glycated hemoglobin using the glycated hemoglobin extraction kit according to any one of claims 1 to 5, the method comprising:
step 1: standing the collected blood at room temperature for half an hour;
step 2: adding the cleaning solution A into the collected plasma for 37-degree treatment, then adding the cleaning solution B, reversing and uniformly mixing, centrifuging to remove supernatant, and repeating the operation until the supernatant is colorless to obtain a first precipitate, wherein the volume ratio of the plasma to the cleaning solution A to the cleaning solution B is as follows: plasma: cleaning solution A: cleaning solution B is 1: 2: 3;
and step 3: adding a cleaning solution B into the first precipitate for uniformly mixing, centrifuging and discarding the supernatant to obtain a second precipitate, wherein the volume ratio of the first precipitate to the cleaning solution is 1: 2;
and 4, step 4: adding the eluent into the second precipitate, uniformly mixing, and centrifuging the supernatant to obtain a third precipitate, wherein the ratio of the second precipitate to the eluent is 1: 3;
and 5: adding a storage solution into the third precipitate for 37-degree treatment, centrifuging and retaining a supernatant, wherein the ratio of the second precipitate to the eluent is 1: 10;
step 6: and (5) uniformly mixing the supernatant obtained in the step (5), centrifuging and reserving the supernatant, and finally obtaining the supernatant which is the glycosylated hemoglobin solution.
Further, the centrifugation conditions in the step 2 and the step 3 are 5000 r/min and 15-25 min.
Further, the centrifugation condition in the step 4 is 2000 rpm for 8-15 min.
Further, the centrifugation condition in the step 5 is 8000 rpm, and 15-25 min.
Further, the centrifugation condition in the step 6 is 12000 r/min and 15-25 min.
Compared with the prior art, the invention has the following beneficial effects:
according to the glycosylated hemoglobin extraction kit and the extraction method, the detection kit for extracting the glycosylated hemoglobin is prepared, the glycosylated hemoglobin is released by bursting red blood cells through intermolecular pressure, the glycosylated hemoglobin is eluted by using eluent, and the storage solution is used for dissolving, so that the obtained glycosylated hemoglobin has uniform components and can meet the detection requirement.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.
Example 1:
the glycosylated hemoglobin extraction kit mainly comprises a cleaning solution A, a cleaning solution B, an eluent and a storage solution, wherein:
the cleaning solution A comprises the following components in percentage by weight:
the cleaning liquid B comprises the following components in percentage by weight:
the eluent comprises the following components in percentage by weight:
the components and contents of the storage liquid are as follows:
example 2:
the glycosylated hemoglobin extraction kit mainly comprises a cleaning solution A, a cleaning solution B, an eluent and a storage solution, wherein:
the cleaning solution A comprises the following components in percentage by weight:
the cleaning liquid B comprises the following components in percentage by weight:
the eluent comprises the following components in percentage by weight:
the components and contents of the storage liquid are as follows:
example 3:
the glycosylated hemoglobin extraction kit mainly comprises a cleaning solution A, a cleaning solution B, an eluent and a storage solution, wherein:
the cleaning solution A comprises the following components in percentage by weight:
the cleaning liquid B comprises the following components in percentage by weight:
the eluent comprises the following components in percentage by weight:
the components and contents of the storage liquid are as follows:
the kit of the three embodiments is adopted to extract the glycosylated hemoglobin, and the extraction method can adopt the following steps:
step 1: standing the collected blood at room temperature for half an hour;
step 2: adding the cleaning solution A into the collected plasma for 37-degree treatment, then adding the cleaning solution B, reversing and uniformly mixing, centrifuging to remove supernatant, and repeating the operation until the supernatant is colorless to obtain a first precipitate, wherein the volume ratio of the plasma to the cleaning solution A to the cleaning solution B is as follows: plasma: cleaning solution A: cleaning solution B is 1: 2: 3;
in the step, the centrifugal condition is preferably 5000 r/min, and the centrifugal time is 15-25 min, preferably 20 min. Step 2 is typically repeated twice, after which it may be increased if the supernatant is found to show a red color.
And step 3: adding a cleaning solution B into the first precipitate for uniformly mixing, centrifuging and discarding the supernatant to obtain a second precipitate, wherein the volume ratio of the first precipitate to the cleaning solution B is 1: 2;
in the step, the centrifugal condition is preferably 5000 r/min, and the centrifugal time is 15-25 min, preferably 20 min.
And 4, step 4: adding the eluent into the second precipitate, uniformly mixing, and centrifuging the supernatant to obtain a third precipitate, wherein the ratio of the second precipitate to the eluent is 1: 3;
the centrifugal condition in the step 4 is preferably 2000 r/min, and the centrifugal time is 8-15 min, preferably 10 min.
And 5: adding a storage solution into the third precipitate for 37-degree treatment, centrifuging and retaining a supernatant, wherein the ratio of the second precipitate to the eluent is 1: 10;
the centrifugation condition in the step 5 is preferably 8000 rpm, and the centrifugation time is 15-25 min, preferably 20 min.
Step 6: and (5) uniformly mixing the supernatant obtained in the step (5), centrifuging and reserving the supernatant, and finally obtaining the supernatant which is the glycosylated hemoglobin solution.
The centrifugation condition in the step 6 is preferably 12000 r/min, and the centrifugation time is 15-25 min, preferably 20 min.
And (3) detecting the uniformity of the glycosylated hemoglobin:
the glycated hemoglobin extracted by the extraction kit of the 3 embodiments was analyzed for homogeneity, and the procedure was performed using a fully automatic biochemical analyzer Hitachi 7180, and the glycated hemoglobin assay kit was a Japanese rattan storehouse glycated hemoglobin assay kit (latex enhancement method), and the detailed procedures were performed according to the instructions, and the results are shown in Table 1:
TABLE 1
As can be seen from Table 1, the glycated hemoglobin extracted in examples 1 to 3 has a variation coefficient of not more than 0.5%, and the glycated hemoglobin fraction thus extracted is homogeneous and can meet the requirements of the assay standards.
And (3) detecting the stability of the glycosylated hemoglobin:
the glycated hemoglobin extracted in the 3 examples was subjected to stability measurement, and the glycated hemoglobin extracted by chromatography as a control was placed at 2 to 8 degrees and was subjected to the operation using Hitachi 7180, which is a glycated hemoglobin assay kit (latex enhancement method) from Japan rattan storehouse, according to the instructions. The results are shown in table 2:
TABLE 2 stability test results of glycated hemoglobin extracted by the extraction kit of the present invention
As can be seen from Table 2, the glycated hemoglobin extracted in examples 1-3, when allowed to stand at 2-8 degrees, is substantially unchanged after being allowed to stand for 150 days, and the chromatographic extract is reduced by about 20% after being allowed to stand for less than 50 days, thereby indicating that the stability of the glycated hemoglobin extracted by the kit of the present invention exceeds that of the glycated hemoglobin extracted by other methods.
Stability detection
The extraction kit in examples 1-2 is put under different conditions for uniform treatment and operation through purchased glycated hemoglobin standard substances, the glycated hemoglobin standard substances after re-fusion are respectively extracted, in order to ensure the accuracy of detection results, the kit is calibrated according to the uniform calibration substances, and then the extraction recovery rate of the standard substances is detected, wherein the detection results are shown in tables 3 and 4:
TABLE 3 detection results of the extraction kit of the present invention after 37 deg.C and-20 deg.C treatment
TABLE 4 detection results of the extraction kit of the present invention after 2-8 degree treatment
As can be seen from tables 3 and 4, the recovery rate of the extraction kit of the invention is more than 96% after being treated for one week at 37 ℃, and the recovery rate of the extraction kit of the invention is more than 99% in example 2; after 10 days of treatment, the recovery rate is more than 94%, and the recovery rate in example 2 is more than 95%; the recovery rate reaches more than 98 percent when the membrane is treated for 10 days at the temperature of minus 20 ℃, and the recovery rate is not changed in the example 2; the recovery rate of the extraction kit is still controlled to be more than 95% when the extraction kit is placed for 500 days at the temperature of 2-8 ℃, and the data fully show that the extraction kit can cope with the adverse environmental conditions in the market.
In conclusion, the glycosylated hemoglobin extraction kit and the extraction method provided by the invention are in the form of the kit for the first time in the glycosylated hemoglobin extraction technology, and avoid the use of technical equipment with high technical requirements, and only need to adopt equipment such as a centrifuge in the prior art, so that the requirements on the equipment are reduced, the storage time of the extracted glycosylated hemoglobin can be prolonged on the premise that the extracted glycosylated hemoglobin meets the detection standard, and powerful help is provided for researching the glycosylated hemoglobin. The ability of the glycosylated hemoglobin extraction kit of the present invention to cope with extreme environments provides a guarantee for the transportation and circulation of products in the market.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (9)
1. The glycosylated hemoglobin extraction kit is characterized by comprising a cleaning solution A, a cleaning solution B, an eluent and a storage solution, wherein:
the cleaning solution A comprises the following components in percentage by weight:
the cleaning liquid B comprises the following components in percentage by weight:
the eluent comprises the following components in percentage by weight:
the storage liquid comprises the following components in percentage by weight:
the buffer solution is sodium phosphate or potassium phosphate.
2. The glycated hemoglobin extraction kit as set forth in claim 1, wherein the final pH of wash A and wash B is 6.95.
3. The glycated hemoglobin extraction kit according to claim 2, wherein the wash solution A comprises the following components in amounts:
4. the glycated hemoglobin extraction kit according to claim 1, wherein the wash solution B comprises the following components and contents:
5. a method for extracting glycated hemoglobin, which comprises using the glycated hemoglobin extraction kit according to any one of claims 1 to 4, the method comprising:
step 1: standing the collected blood at room temperature for half an hour;
step 2: adding the cleaning solution A into the collected plasma for 37-degree treatment, then adding the cleaning solution B, reversing and uniformly mixing, centrifuging to remove supernatant, and repeating the operation until the supernatant is colorless to obtain a first precipitate, wherein the volume ratio of the plasma to the cleaning solution A to the cleaning solution B is as follows: plasma: cleaning solution A: cleaning solution B is 1: 2: 3;
and step 3: adding a cleaning solution B into the first precipitate for uniformly mixing, centrifuging and discarding the supernatant to obtain a second precipitate, wherein the volume ratio of the first precipitate to the cleaning solution B is 1: 2;
and 4, step 4: adding the eluent into the second precipitate, uniformly mixing, and centrifuging the supernatant to obtain a third precipitate, wherein the ratio of the second precipitate to the eluent is 1: 3;
and 5: adding a storage solution into the third precipitate for 37-degree treatment, centrifuging and retaining a supernatant, wherein the ratio of the second precipitate to the eluent is 1: 10;
step 6: and (5) uniformly mixing the supernatant obtained in the step (5), centrifuging and reserving the supernatant, and finally obtaining the supernatant which is the glycosylated hemoglobin solution.
6. The method for extracting glycated hemoglobin according to claim 5, wherein the centrifugation conditions in steps 2 and 3 are 5000 rpm, 15-25 min.
7. The method of extracting glycated hemoglobin according to claim 5, wherein the centrifugation in step 4 is carried out at 2000 rpm for 8-15 min.
8. The method of extracting glycated hemoglobin according to claim 5, wherein the centrifugation in step 5 is carried out at 8000 rpm for 15 to 25 min.
9. The method of extracting glycated hemoglobin as set forth in claim 5, wherein the centrifugation in step 6 is carried out at 12000 rpm for 15-25 min.
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| WO2004104203A1 (en) * | 2003-05-21 | 2004-12-02 | Asahi Kasei Pharma Corporation | Method of measuring glycolated hemoglobin a1c, enzyme to be used therefor and process for producing the same |
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| CN104808006A (en) * | 2015-03-17 | 2015-07-29 | 卫生部北京医院 | Glycosylated hemoglobin reference material and preparation method thereof |
| CN105241728A (en) * | 2015-09-28 | 2016-01-13 | 华南理工大学 | Preparation method of human glycated hemoglobin |
| CN106198415A (en) * | 2016-07-12 | 2016-12-07 | 安徽伊普诺康生物技术股份有限公司 | A kind of test kit measuring glycolated hemoglobin and preparation method thereof |
| WO2018008447A1 (en) * | 2016-07-08 | 2018-01-11 | 東ソー株式会社 | Hemoglobin liquid preparation and liquid chromatography method for measuring hemoglobin component |
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2018
- 2018-12-30 CN CN201811649480.9A patent/CN109734797B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004104203A1 (en) * | 2003-05-21 | 2004-12-02 | Asahi Kasei Pharma Corporation | Method of measuring glycolated hemoglobin a1c, enzyme to be used therefor and process for producing the same |
| CN103076214A (en) * | 2012-12-26 | 2013-05-01 | 宁波美康生物科技股份有限公司 | Preparation method of glycosylated hemoglobin quality control |
| CN104808006A (en) * | 2015-03-17 | 2015-07-29 | 卫生部北京医院 | Glycosylated hemoglobin reference material and preparation method thereof |
| CN105241728A (en) * | 2015-09-28 | 2016-01-13 | 华南理工大学 | Preparation method of human glycated hemoglobin |
| WO2018008447A1 (en) * | 2016-07-08 | 2018-01-11 | 東ソー株式会社 | Hemoglobin liquid preparation and liquid chromatography method for measuring hemoglobin component |
| CN106198415A (en) * | 2016-07-12 | 2016-12-07 | 安徽伊普诺康生物技术股份有限公司 | A kind of test kit measuring glycolated hemoglobin and preparation method thereof |
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