A kind of removal red blood cell treatment fluid and purposesTechnical 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, abdomenWhen water cytolgical examination, a large amount of blood are necessarily had extracting ascites process, also will form hemorrhagic sample.The positions such as bronchus fillWash sampling process, it is also possible to which bleeding also will form hemorrhagic sample, and hemorrhagic sample seriously affects observation in film-making process, mainlyShow: erythrocyte is largely attached to film-making area, or even covering pathological cells, influences interpretation, the erythrocyte of pathological cellsThe 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 redAlbumen is adhered on slide.It causes background complicated, influences pathological cells interpretation.Therefore erythrocyte can be handled into hemorrhagic sampleThis 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 guarantorA 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 removalThe 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 usedAgent 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 addedEffect, 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 crackingCracking, therefore while removing red blood cell missing inspection may be caused because sick cell is also cleaved.Different samplings simultaneouslyThe 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, meshPreceding commercially available hemolytic agent has stronger hemolyzing effect, but contains potassium cyanide severe toxicity ingredient, is all a kind of to operator and environmentVery 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, chlorinationSodium, 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 buffer0.01-0.1M, anticoagulant agent content are 1-5g/L, surface-active contents 1-6g/L, glacial acetic acid 1-5g/L, and distilled water supplements notThe treatment fluid volume of foot.
Further, the alcohols accounts for treatment fluid total volume 20%-25%, sodium chloride content 8-9g/L, phosphate buffer0.05-0.1M, anticoagulant agent content are 2-4g/L, surface-active contents 3-5g/L, glacial acetic acid 3-5g/L, and distilled water supplements notThe treatment fluid volume of foot.
Further, the alcohols be one of methanol, ethyl alcohol, propyl alcohol, isopropanol or any one more than, mass concentrationFor 95%-100%.
Further, the phosphate buffer is that 2.2g disodium hydrogen phosphate and 0.1g sodium dihydrogen phosphate constant volume 1L are formed0.1MPB 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 diformazanOne 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 atThe 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 removedIt forms sediment and 6ml removal red blood cell treatment fluid is added, mix 1min again, then 2000r/min is centrifuged 5min, removes supernatant；If faceColor is not decorporated, repeats above step until color fade；Preferably, the blood containing volume fraction >=10% in hemorrhagic sampleAmount.
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 hemoglobinDenaturation rapidly is conducive to maintain cell shape and removes hemoglobin.Lower than concentration of the invention, cell cannot obtain effectively solidIt 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 lesionCell and normal cell are harmless, utmostly retain sick cell for doctor's interpretation.Cell can be made swollen if being higher than upper concentrationSwollen, 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 positionsCell normal shape.
Surfactant of the present invention is Tween-20, Tween-80, hexadecyltrimethylammonium chloride, hexadecane dimethylOne or more than one kinds of combinations of benzyl ammonium chloride, cetyl trimethylammonium bromide, tetradecyltrimethylammonium bromideObject, surfactant can play the role of lysed erythrocyte, and can take out stains, and increase the solubility of solution, be not easy to form bloodLactoferrin compound, it is easier to remove erythrocyte.Surfactant concentration used in the present invention preferably in 1-6g/L, is higher than upperLimit 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 staffThe 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, backgroundSimply, 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-makingFigure；
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 endSame or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attachedThe 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.EmbodimentIn 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 artBook 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 comparesGood, 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 comparesGood, 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 embodimentPreferably, 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 embodimentRelatively 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 embodimentRelatively 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 toCervical 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 filteringIt 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 automaticallyPiece, 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 precipitatingLiquid is managed, mixes 1min again, then 2000r/min is centrifuged 5min, removes supernatant, repetitive operation is primary, removes supernatant, remaining thinBorn 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, 0.1Bulking 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 mixedLiquid 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 bloodLactoferrin covers most film-making region, seriously affects diagnosis；And pass through hemolytic agent (see figure 3) and blood removal of the inventionRed 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 knotFruit.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. 4It 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 exampleProperty, it is not considered as limiting the invention, those skilled in the art are not departing from the principle of the present invention and objectiveIn the case where can make changes, modifications, alterations, and variations to the above described embodiments within the scope of the invention.