CN105092328B - A kind of removal red blood cell treatment fluid and purposes - Google Patents
A kind of removal red blood cell treatment fluid and purposes Download PDFInfo
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- CN105092328B CN105092328B CN201510456469.0A CN201510456469A CN105092328B CN 105092328 B CN105092328 B CN 105092328B CN 201510456469 A CN201510456469 A CN 201510456469A CN 105092328 B CN105092328 B CN 105092328B
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Abstract
The invention discloses a kind of removal red blood cell treatment fluid and purposes.Alcohols, 5-10g/L sodium chloride, 0.01-0.1M phosphate buffer, 1-5g/L glacial acetic acid, 1-5g/L anti-coagulants, 1-6g/L surfactant and distilled water by accounting for total volume 15%-30% form.Removal red blood cell treatment fluid of the present invention, low in cost, safety can reduce the damage to cell as far as possible, can guarantee the integrality of pathological cells while removing red blood cell.Cell, splitting erythrocyte and removing hemoglobin are handled using removal red blood cell treatment fluid of the present invention, it is harmless to sick cell and normal cell, utmostly retain sick cell, maintain cell shape, production effect is good, background is simple, clearly, it is easy to judging result.
Description
Technical field
The present invention relates to technical field of cell biology more particularly to a kind of removal red blood cell treatment fluid and purposes.
Background technique
When clinical epithelial tissue samples, sampling is accidentally or sampling point damage leads to bleeding, just forms hemorrhagic sample, abdomen
When water cytolgical examination, a large amount of blood are necessarily had extracting ascites process, also will form hemorrhagic sample.The positions such as bronchus fill
Wash sampling process, it is also possible to which bleeding also will form hemorrhagic sample, and hemorrhagic sample seriously affects observation in film-making process, mainly
Show: erythrocyte is largely attached to film-making area, or even covering pathological cells, influences interpretation, the erythrocyte of pathological cells
The small agglomerate of fragment agglutination is attached to film-making area by filter screen, influence the interpretation of pathological cells, dyed by dye liquor it is blood red
Albumen is adhered on slide.It causes background complicated, influences pathological cells interpretation.Therefore erythrocyte can be handled into hemorrhagic sample
This film-making is successfully crucial.Generally use the glacial acetic acid of high concentration clinically at present to handle, major way has: 1, directly to guarantor
A certain proportion of glacial acetic acid is added in liquid storage, is centrifuged after cracking, then glacial acetic acid is added into sediment, repeated centrifugation, reaches removal
The effect of red blood cell;2, with alcohol and glacial acetic acid mixture come splitting erythrocyte, the same first way of processing method;3, haemolysis is used
Agent carrys out splitting erythrocyte, and using the lysate for having potassium cyanide, processing method is the same as the first.
In three kinds of methods, method 1 and 2 main feature of method are all to reach splitting erythrocyte by a large amount of glacial acetic acid is added
Effect, completely, but a large amount of high concentration glacial acetic acid can be by the normal cell of a part and sick cell also together for cracking
Cracking, therefore while removing red blood cell missing inspection may be caused because sick cell is also cleaved.Different samplings simultaneously
The cell at position may be because that the glacial acetic acid of high concentration causes peracid and cracks.Method 3 is using hemolytic agent come splitting erythrocyte, mesh
Preceding commercially available hemolytic agent has stronger hemolyzing effect, but contains potassium cyanide severe toxicity ingredient, is all a kind of to operator and environment
Very big threat.
Summary of the invention
The purpose of the present invention is to provide a kind of efficient, nontoxic, single-minded and at low cost removal red blood cell treatment fluids.
To achieve the above object, the present invention provides a kind of removal red blood cell treatment fluid, which is characterized in that by alcohols, chlorination
Sodium, phosphate buffer, anti-coagulants, surfactant, glacial acetic acid and distilled water composition.
Further, the alcohols accounts for treatment fluid total volume 15%-30%, sodium chloride content 5-10g/L, phosphate buffer
0.01-0.1M, anticoagulant agent content are 1-5g/L, surface-active contents 1-6g/L, glacial acetic acid 1-5g/L, and distilled water supplements not
The treatment fluid volume of foot.
Further, the alcohols accounts for treatment fluid total volume 20%-25%, sodium chloride content 8-9g/L, phosphate buffer
0.05-0.1M, anticoagulant agent content are 2-4g/L, surface-active contents 3-5g/L, glacial acetic acid 3-5g/L, and distilled water supplements not
The treatment fluid volume of foot.
Further, the alcohols be one of methanol, ethyl alcohol, propyl alcohol, isopropanol or any one more than, mass concentration
For 95%-100%.
Further, the phosphate buffer is that 2.2g disodium hydrogen phosphate and 0.1g sodium dihydrogen phosphate constant volume 1L are formed
0.1MPB buffer solution, concentration used below can be diluted with above-mentioned preparation solution.
Further, the anti-coagulants is disodium ethylene diamine tetraacetate.
Further, the surfactant is Tween-20, Tween-80, hexadecyltrimethylammonium chloride, hexadecane diformazan
One or more kinds of compositions of base benzyl ammonium chloride, cetyl trimethylammonium bromide.
On the other hand, the present invention protect the removal red blood cell treatment fluid be used to remove erythrocyte in cell sample at
The purposes divided.
Further, the volume ratio of 10ml cell sample and removal red blood cell treatment fluid is 10:6.
On the other hand, the method that the present invention protects the removal red blood cell treatment fluid to remove red blood cell in hemorrhagic cell,
It is characterized in that, the steps include: to mix hemorrhagic sample 10ml, by 800g, after 5 minutes centrifugal concentratings, supernatant, Xiang Chen are removed
It forms sediment and 6ml removal red blood cell treatment fluid is added, mix 1min again, then 2000r/min is centrifuged 5min, removes supernatant;If face
Color is not decorporated, repeats above step until color fade;Preferably, the blood containing volume fraction >=10% in hemorrhagic sample
Amount.
It is 15%-30% that alcohols of the present invention, which accounts for treatment fluid total volume, can not only fix cell, but also will not make hemoglobin
Denaturation rapidly is conducive to maintain cell shape and removes hemoglobin.Lower than concentration of the invention, cell cannot obtain effectively solid
It is fixed, deformation.Higher than the concentration, it is easy the hemoglobin for releasing cracking denaturation, it is difficult to redissolve, be difficult exclusive PCR.
Glacial acetic acid content of the present invention is 1-5g/L, suitable glacial acetic acid concentration, not only cleavable red blood cell, but also to lesion
Cell and normal cell are harmless, utmostly retain sick cell for doctor's interpretation.Cell can be made swollen if being higher than upper concentration
Swollen, cell in high concentration, can rupture for a long time, lose cell.It, can not effective splitting erythrocyte if being lower than lower limit.
The phosphate is buffer system, and treatment fluid is integrally weakly acidic, and buffering range is wide, can maintain multiple materials positions
Cell normal shape.
Surfactant of the present invention is Tween-20, Tween-80, hexadecyltrimethylammonium chloride, hexadecane dimethyl
One or more than one kinds of combinations of benzyl ammonium chloride, cetyl trimethylammonium bromide, tetradecyltrimethylammonium bromide
Object, surfactant can play the role of lysed erythrocyte, and can take out stains, and increase the solubility of solution, be not easy to form blood
Lactoferrin compound, it is easier to remove erythrocyte.Surfactant concentration used in the present invention preferably in 1-6g/L, is higher than upper
Limit influences cellular prion protein.Desired effect is unable to reach lower than lower limit.
The beneficial effect of removal red blood cell treatment fluid of the present invention is:
1, agents useful for same is low in cost, is all made of common chemicals;
2, nontoxic, without the hypertoxic chemicals such as potassium cyanide, the pollution to environment is avoided, is also provided to technical staff
The operating environment of one safety;
3, the damage to cell is reduced as far as possible, can guarantee the integrality of pathological cells while removing red blood cell;
4, cell, splitting erythrocyte and removing hemoglobin are handled using removal red blood cell treatment fluid of the present invention,
It is harmless to sick cell and normal cell, utmostly retain sick cell, maintains cell shape, production effect is good, background
Simply, clearly, it is easy to judging result.
Detailed description of the invention
Fig. 1 is 10 times of light microscopic figures of untreated hemorrhagic cervical cell film-making;
Fig. 2 is 10 times of light microscopics by removal red blood cell treatment fluid of the invention treated hemorrhagic cervical cell film-making
Figure;
Fig. 3 is 10 times of light microscopic figures by treated the hemorrhagic cervical cell film-making of commercially available hemolytic agent;
Fig. 4 is 10 times of light microscopic figures through treated the hemorrhagic cervical cell film-making of overrich glacial acetic acid and alcohol mixture.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end
Same or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attached
The embodiment of figure description is exemplary, it is intended to is used to explain the present invention, and is not considered as limiting the invention.Embodiment
In particular technique or condition person is not specified, described technology or conditions or according to the description of product according to the literature in the art
Book carries out.Reagents or instruments used without specified manufacturer, being can be with conventional products that are commercially available.
Embodiment 1: the preparation of removal red blood cell treatment fluid
Raw material:
Methanol 150ml;
Sodium chloride 5g;
1.0 ml of glacial acetic acid;
Disodium ethylene diamine tetraacetate 1.0g;
Tween-20 1.0ml;
PB 0.01M;
Distilled water is settled to 1000 ml.
Preparation method:
After being weighed and measured by mentioned component, stirring and dissolving.
Cervical cell sample is handled with the preparation-obtained removal red blood cell treatment fluid of the present embodiment, production effect compares
Good, background is fairly simple, clearly, it is easy to judging result.
Embodiment 2: the preparation of removal red blood cell treatment fluid
Raw material:
Ethyl alcohol 300ml;
Sodium chloride 10g;
Glacial acetic acid 5.0ml;
Disodium ethylene diamine tetraacetate 5.0g;
Tween-20 6.0ml;
PB 0.1M;
Distilled water is settled to 1000 ml.
Preparation method: with embodiment 1.
Phlegm Cells sample is handled with the preparation-obtained removal red blood cell treatment fluid of the present embodiment, production effect compares
Good, background is fairly simple, clearly, it is easy to judging result.
Embodiment 3: the preparation of removal red blood cell treatment fluid
Raw material:
Ethyl alcohol 225ml;
Sodium chloride 7.5g;
Glacial acetic acid 3.0ml;
Disodium ethylene diamine tetraacetate 3.0g;
Tween-20 3.5ml;
PB 0.05M;
Distilled water is settled to 1000 ml.
Preparation method: with embodiment 1.
Mouth desquamated cells sample, production effect ratio are handled with the preparation-obtained removal red blood cell treatment fluid of the present embodiment
Preferably, background is fairly simple, clearly, it is easy to judging result.
Embodiment 4: the preparation of removal red blood cell treatment fluid
Raw material:
Methanol 200ml;
Ethyl alcohol 100ml;
Sodium chloride 8g;
Glacial acetic acid 3.5ml;
Disodium ethylene diamine tetraacetate 4g;
Tween-20 3.0ml;
PB 0.05M;
Distilled water is settled to 1000 ml.
Preparation method: with embodiment 1.
Cell specimen, production effect are punctured with the preparation-obtained removal red blood cell treatment fluid processing Pleural effusions of the present embodiment
Relatively good, background is fairly simple, clearly, it is easy to judging result.
Embodiment 5: the preparation of removal red blood cell treatment fluid
Raw material:
Ethyl alcohol 200ml
Sodium chloride 8.5g
Glacial acetic acid 5.0ml
Disodium ethylene diamine tetraacetate 3.5g;
Hexadecyltrimethylammonium chloride 2g;
Tween-80 3.0ml;
PB 0.06M;
Distilled water is settled to 1000 ml.
Preparation method: with embodiment 1.
Respiratory tract bal cell sample, production effect are handled with the preparation-obtained removal red blood cell treatment fluid of the present embodiment
Relatively good, background is fairly simple, clearly, it is easy to judging result.
Embodiment 6: the preparation of removal red blood cell treatment fluid and compliance test result
Raw material:
Ethyl alcohol 250ml;
Sodium chloride 9.0g;
Glacial acetic acid 4.0ml;
Disodium ethylene diamine tetraacetate 2.0g;
Hexadecyltrimethylammonium chloride 2.5g;
Tween-80 2.0ml;
PB 0.08M;
Distilled water is settled to 1000 ml.
Preparation method: with embodiment 1.
Enough cell samples are added in the formula described in embodiment 6, and the present embodiment uses cervical cell sample, but is not limited to
Cervical cell, can use sputum sample, mouth desquamated cells sample, and Pleural effusions puncture cell specimen, respiratory tract bal cell sample.
Normal person's venous whole of 4ml Hospital Physical Examination is added to 40ml cell sample, by supernatant after mixing well and filtering
It is divided into 4 parts.It is 2., 3., 4. spare labeled as 1..
After above-mentioned 1. sample blending centrifugal concentrating, direct film-making, film-making can take Pap smear, and membrane type machine is made automatically
Piece, centrifugal settling method film-making, natural sedimentation film-making, it is preferable that the present invention uses natural sedimentation film-making, after film-making,
Dry 1-2 minutes, can dying operation and microscopy, microscopy obtains Fig. 1 production effect.
After above-mentioned 2. sample blending centrifugal concentrating, supernatant is removed, is added at 6ml removal red blood cell of the invention to precipitating
Liquid is managed, mixes 1min again, then 2000r/min is centrifuged 5min, removes supernatant, repetitive operation is primary, removes supernatant, remaining thin
Born of the same parents' precipitating can be used to pathology film-making.Film-making obtains Fig. 2 production effect.
After above-mentioned 3. sample blending centrifugal concentrating, commercially available hemolytic agent is added and (contains 0.2 bulking value % potassium cyanide, 0.1
Bulking value % dodecyl trimethyl ammonium chloride, 0.1 bulking value % tetradecyltrimethylammonium bromide, 0.05 bulking value %
Disodium ethylene diamine tetraacetate and purified water), it is operated by operational manual, after film-making.Dyeing microscopic examination obtains Fig. 3 production effect.
Addition glacial acetic acid and dehydrated alcohol are that the mixing of 1::18 ratio is split by volume after above-mentioned 4. sample solution is mixed
Liquid is solved, operating method is the same as 2..Fig. 4 production effect obtained by film-making.
Contrast on effect: wherein Fig. 1 is without any processing, and production effect background is extremely complex, erythrocyte and blood
Lactoferrin covers most film-making region, seriously affects diagnosis;And pass through hemolytic agent (see figure 3) and blood removal of the invention
Red blood cell treatment fluid handles the sample of (see figure 2), and production effect is relatively good, and background is fairly simple, clearly, it is easy to judgement knot
Fruit.Cost is relatively low for blood removal red blood cell treatment fluid of the invention, and safety is free of hypertoxic chemicals ingredient (potassium cyanide),;Fig. 4
It is then the production effect figure with the glacial acetic acid of high concentration, it can be seen that there is part cell to be cleaved, although easy to operate,
It is more to be that cell can lose, and may cause missing inspection.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art are not departing from the principle of the present invention and objective
In the case where can make changes, modifications, alterations, and variations to the above described embodiments within the scope of the invention.
Claims (9)
1. a kind for the treatment of fluid for removing red blood cell in hemorrhagic cell, which is characterized in that by alcohols, sodium chloride, phosphate buffer,
Anti-coagulants, surfactant, glacial acetic acid and distilled water composition;The alcohols accounts for treatment fluid total volume 15%-30%, and sodium chloride contains
Amount be 5-10g/L, phosphate buffer 0.01-0.1M, anticoagulant agent content be 1-5g/L, surface-active contents 1-6g/L,
Glacial acetic acid 1-5g/L, distilled water supplement insufficient treatment fluid volume;
The phosphate buffer is that the 0.1MPB buffering that 2.2g disodium hydrogen phosphate and 0.1g sodium dihydrogen phosphate constant volume 1L are formed is molten
Liquid.
2. the treatment fluid of red blood cell in the hemorrhagic cell of removal described in claim 1, which is characterized in that it is total that the alcohols accounts for treatment fluid
Volume 20%-25%, sodium chloride content 8-9g/L, phosphate buffer 0.05-0.1M, anticoagulant agent content are 2-4g/L, surface
Active agent content is 3-5g/L, glacial acetic acid 3-5g/L, the insufficient treatment fluid volume of distilled water supplement.
3. the treatment fluid of red blood cell in the hemorrhagic cell of removal described in claim 1, which is characterized in that the alcohols is methanol, second
One of alcohol, propyl alcohol, isopropanol or any one more than, mass concentration 95%-100%.
4. the treatment fluid of red blood cell in the hemorrhagic cell of removal described in claim 1, which is characterized in that the anti-coagulants is ethylenediamine
Tetraacethyl disodium.
5. the treatment fluid of red blood cell in the hemorrhagic cell of removal described in claim 1, which is characterized in that the surfactant is to spit
Warm -20, Tween-80, hexadecyltrimethylammonium chloride, hexadecane dimethyl benzyl ammonium chloride, cetyl trimethylammonium bromide
One or more kinds of compositions.
6. any one of the claim 1-5 treatment fluid for removing red blood cell in hemorrhagic cell is used to remove the blood in cell sample
The purposes of red blood cell component.
7. in the hemorrhagic cell of removal described in claim 6 treatment fluid of red blood cell be used to remove the erythrocyte in cell sample at
The purposes divided, which is characterized in that the volume ratio of 10ml cell sample and removal red blood cell treatment fluid is 10:6.
8. a kind of remove hemorrhagic cell with any one of the claim 1-5 treatment fluid for removing red blood cell in hemorrhagic cell
The method of middle red blood cell, which is characterized in that the steps include: to mix hemorrhagic sample 10ml, by 800g, 5 minutes centrifugal concentratings
Afterwards, supernatant is removed, 6ml is added to precipitating and removes red blood cell treatment fluid, mixes 1min again, then 2000r/min is centrifuged 5min, goes
Except supernatant;If color is not decorporated, above step is repeated until color fade.
9. the method for removing the treatment fluid of red blood cell in hemorrhagic cell described in claim 8 to remove red blood cell in hemorrhagic cell,
It is characterized in that, the blood volume containing volume fraction >=10% in hemorrhagic sample.
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CN106234354B (en) * | 2016-09-30 | 2019-05-21 | 上海市奉贤区中心医院 | A kind of cervix cell preservation solution |
CN106855475A (en) * | 2017-03-18 | 2017-06-16 | 王瑞才 | A kind of rapid tissue reagent treatment for pathological section |
CN107219110A (en) * | 2017-07-24 | 2017-09-29 | 中国人民解放军第三军医大学第二附属医院 | Suitable for the MALDI TOF microculture liquid processing methods detected and rapid identification method |
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CN113218847A (en) * | 2021-04-12 | 2021-08-06 | 武汉凯普瑞生物技术有限公司 | Hemolytic agent for flow cytometry analysis and preparation method and application method thereof |
CN115184111A (en) * | 2022-07-06 | 2022-10-14 | 首都医科大学附属北京朝阳医院 | A kind of pathological cytology blood removal kit and using method thereof |
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CN101988875A (en) * | 2010-09-03 | 2011-03-23 | 江苏省原子医学研究所 | Sputum liquid-based treating fluid |
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