CN1687780A - Method for preparing negative film of antigen of neutrophilic granulocyte - Google Patents
Method for preparing negative film of antigen of neutrophilic granulocyte Download PDFInfo
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- CN1687780A CN1687780A CN 200510012492 CN200510012492A CN1687780A CN 1687780 A CN1687780 A CN 1687780A CN 200510012492 CN200510012492 CN 200510012492 CN 200510012492 A CN200510012492 A CN 200510012492A CN 1687780 A CN1687780 A CN 1687780A
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Abstract
This invention relates to the preparation of neutral granulocyte antigen negative used to detect cytoplasmic antigen of neutrophilic granulocyte. It uses heparin anticoagulant method to subside erythrocyte and preserve leucocyte. Then it uses Ficoll-Hypaque gradient reagent to separate neutrophilic granulocyte. Then put in NaCl solution and it becomes cell suspension. Put the suspension directly on to the slide and a neutrophilic granulocyte antigen negative is ready. This negative's detectable rate is obviously greater than other domestic negatives. It has little difference with the import reagent and the cells has only one single deck and is evenly distributed. The result is easy to observe ,so it can meet clinic demand.
Description
Affiliated technical field
The invention relates to the preparation method of neutrophils antigen egative film, this negative film of antigen is used for the detection of ANCA.
Background technology
ANCA (anti-neutrophil cytoplasmic antibody, ANCA) be a kind of be the autoantibody of target antigen with neutrophil leucocyte and monocyte endochylema composition, be a kind of very important serodiagnosis instrument that is used for the diagnosing primary polyangitis that development in recent years is got up.Primary small vessel vasculitis mainly refers to wegener granulomatosis (WG), the many vasculitises of microscopically (MPA) and gangrenosum acne crescentic glomerulonephritis (NCGN), their morbidity and ANCA are closely related, therefore clinically above-mentioned disease are called ANCA correlativity vasculitis.ANCA can be divided into cANCA and pANCA according to the difference of fluorescent dye model, present research is thought, cANCA is more common in WG, and pANCA is normal relevant with MPA and NCGN, they can reflect state of an illness activity, assist diagnosis, instruct treatment and judge recurrence, and are significant in the diagnosis and differential diagnosis of vasculitic disease.Aspects such as the external in recent years character to ANCA and target antigen, disease association, detection method are furtherd investigate, and ANCA has become systemic vasculitis and autoimmune disease important clinical conventional sense project.
Indirect immunofluorescence is to detect the method that ANCA adopts the earliest, also is the most frequently used, most popular at present prescreening method.But this method at first needs to possess superior in quality negative film of antigen, and the form of cell distributes, the displaying of endochylema composition and the antigen active of cell, these all directly affect testing result accurately whether.
Currently used neutrophils antigen egative film preparation method mainly is the method for being formulated according to first the ANCA international symposium in Copenhagen in 1988:
Get the fresh heparin anti-coagulating of normal person, add the blood cytolysate of 3～4 times of 4 ℃ of precoolings, placed 5～10 minutes for 4 ℃, with 1500rpm/min centrifugal 7～10 minutes, abandon supernatant; Cell precipitation is bought the blood cytolysate that adds 4 ℃ of precoolings after even again, placed 5～10 minutes, with 1500rpm/min centrifugal 7～10 minutes, abandon supernatant; With the phosphate buffer washing precipitation of 4 ℃ of precoolings 2 times, centrifugal 7～10 minutes of 1500rpm/min abandons supernatant; Add the RPMI-1640 that contains 5% bovine serum albumin(BSA), adjust cell number to 2 * 10
6/ ml; Get rid of sheet with cytospin, centrifugal 3 minutes of 1000rpm/min gets rid of cell to be individual layer on slide and evenly distributes, and behind the natural air drying, fixes 5 minutes with absolute ethyl alcohol under 4 ℃, air-dry after ,-20 ℃ of preservations are used for the detection of ANCA.
In the preparation process of above-mentioned neutrophils antigen egative film, it is a critical step that cytospin is got rid of sheet, do not reach expected effect after using homemade cytospin to get rid of sheet, the neutrophils antigen egative film of preparation often is the shrinkage state because of cell, the endochylema composition is showed insufficient and is directly influenced result's observation, and import cytospin price very expensive (surplus in the of about 10 ten thousand yuan), therefore, the commercialization neutrophils antigen egative film that uses mostly is imported product at present, cost an arm and a leg, it is too high to detect cost, has greatly restricted its routine clinically and has used.
Summary of the invention
The purpose of this invention is to provide that a kind of preparation cost is low, testing result accurately, stable performance, be fit to the preparation method of the neutrophils antigen egative film of routine clinical detection.
Neutrophils antigen egative film preparation method of the present invention comprises the steps:
Get the fresh heparin anti-coagulating of normal person, mix according to 4: 1 volume ratio with 1% methocel solution, sedimented red cell under the room temperature keeps the leucocyte plasma layer;
Under the uniform temp, in test tube, isopyknic Ficoll-Hypaque gradient agent I is superimposed upon on the Ficoll-Hypaque gradient agent II gently;
The leucocyte plasma layer is superimposed upon the upper strata of gradient agent gently, and balance is placed in the horizontal hydro-extractor, centrifugal 20～25 minutes of 1800～2000rpm/min;
Neutrophil leucocyte layer in the middle of drawing, the damping fluid of 4 ℃ of precoolings of adding, centrifugal 5～10 minutes of 10000～1200rpm/min abandons supernatant;
Add 0.1%～0.85%NaCl solution of 4 ℃ of precoolings in the cell precipitation, and adjust neutrophil leucocyte number to 5 * 10
5/ ml;
Cell suspension adds natural air drying on the cleaning wave carrier piece, and 4 ℃ of following absolute ethyl alcohols are fixed 5 minutes, the sealing of air-dry back.
Wherein, the concrete grammar of sedimented red cell is that heparin anti-coagulating is mixed with methocel solution, under the room temperature tilting 15～20 minutes, makes erythrocyte sedimentation; Perhaps at room temperature tilting 5～10 minutes, centrifugal 2～5 minutes of 1000～1200rpm/min made erythrocyte sedimentation.
Described damping fluid is the phosphate buffer of pH 7.4.
Compare with the International Standards Method of continuing to use now, neutrophils antigen egative film preparation method of the present invention has the following advantages.
At first, in the separation of neutrophil leucocyte, use Ficoll-Hypaque discontinuous density gradient one step separation, obtained the neutrophil leucocyte of high concentration, discarded need use ammonium chloride or dissolved in distilled water red blood cell in the past and may cause to the granulocytic damage of neutrality.
Secondly, after the neutrophil leucocyte washing of precipitate after will separating, add the NaCl solution that is fit to concentration and make cell suspension, make cell swelling under hypotonic state, form is full, endochylema is showed fully, drip and on wave carrier piece, to be individual layer and evenly to distribute, thereby solved the problem of negative film of antigen cell shrinkage, be beneficial to result's observation.
Once more, when preparing, do not use by egative film cytospin to get rid of sheet, but with after the cell swelling, adjusting cell number directly drips on wave carrier piece, solved and used the import cytospin to cost an arm and a leg and problem that homemade cytospin falls flat, greatly reduced the cost of egative film preparation.
Adopt the neutrophils antigen egative film of the inventive method preparation, seal behind the natural air drying, be stored in-20 ℃ and-70 ℃ respectively, through experimental observation, preserved 6 months for-20 ℃, cell sheet antigenicity does not have notable difference.Preserved 1 year for-70 ℃, 5 parts at random the positive sample testing result do not have difference.
Use the neutrophils antigen egative film of the inventive method preparation, with indirect immunofluorescence 325 routine primary and Secondary cases vasculitis patient are carried out the ANCA detection, comprised systemic vasculitis 58 examples: be respectively wegener granulomatosis (WG) 6 examples, aorto-arteritis 5 examples, Behcet's disease 22 examples, anaphylactoid purpura 10 examples, other unfiled vasculitis 15 examples; Secondary cases vasculitis 267 examples: be respectively renal glomerular disease patient 75 examples, systemic loupus erythematosus (SLE) 109 examples, rheumatoid arthritis (RA) 83 examples.Among the 325 routine patients, detect positive 36 examples of ANCA, positive 29 examples of pANCA wherein, positive 7 examples of cANCA are respectively positive 5 examples of WG group cANCA, and positive rate is 83.33% (5/6); Positive 1 example of other systemic vasculitis group pANCA, positive rate is 1.92% (1/52); Positive 2 examples of ephrosis group pANCA, positive rate is 2.67% (2/75); Positive 1 example of RA group cANCA, positive rate is 1.2% (1/83); Positive 26 examples of SLE group pANCA, positive 1 example of cANCA, positive rate is 24.77% (27/109).
Simultaneously, the ANCA detectable that adopts German Ou Meng company to provide has carried out rechecking and contrast to the part SLE patients serum of the ANCA positive serum among the 325 routine patients, 6 routine WG patients serums and picked at random.All the neutrophils antigen egative film testing result of ANCA positive serum and 6 routine WG patients serums' testing result and the inventive method preparation is identical, does not have difference.Just in SLE group testing result, there are 4 examples different with the neutrophils antigen egative film testing result of the present invention's preparation, wherein 1 routine the present invention prepares the negative film of antigen pANCA positive and Ou Meng company feminine gender, 2 routine the present invention prepare negative film of antigen pANCA feminine gender and the Ou Meng company positive, but titre is not high, 1 routine the present invention prepares the negative film of antigen cANCA positive and Ou Meng company feminine gender, and testing result is slightly variant.
The neutrophils antigen egative film of the present invention's preparation is applied to routine clinical detection simultaneously with homemade negative film of antigen, to compare.In clinical doubtful vasculitis patient 604 people that detect, use product of the present invention to detect positive 66 examples of pANCA, positive 10 examples of cANCA, positive 3 examples of the ANCA that is not true to type, positive rate is respectively 10.93%, 1.66% and 0.50%; And homemade negative film of antigen product detects positive 48 examples of pANCA, positive 8 examples of cANCA, and positive rate is respectively 7.94% and 1.32%.The recall rate of the neutrophils antigen egative film recall rate of the present invention's preparation apparently higher than homemade negative film of antigen is described, and the neutrophils antigen egative film cellular morphology of the present invention's preparation is full, the endochylema composition shows that fully the result is easy to observe, and can satisfy clinical demand fully.
Embodiment
Embodiment 1
Get the fresh heparin anti-coagulating of normal person, add 1% methylcellulose according to 4: 1 volume ratios, behind the mixing at room temperature tilting 20 minutes, make erythrocyte sedimentation, collect and be rich in leukocytic plasma layer.
Under the uniform temp, in test tube, add 3ml proportion and be 1.119 Ficoll-Hypaque gradient agent II, with 3ml proportion be again 1.077 Ficoll-Hypaque gradient agent I be superimposed upon gently gradient agent II above.
On gradient agent upper strata along tube wall 3～4ml leucocyte plasma layer that superposes gently, in test tube balance trailing water flat hydro-extractor, centrifugal 25 minutes of 1800rpm/min, the visible liquid in centrifugal back obviously is divided into 3 layers, and wherein the 1st layer is blood plasma-solution I contact bed, contains monocyte and blood platelet, the 2nd layer is solution-solution interface layer, contain neutrophil leucocyte, the 3rd layer is the beds of precipitation, contains red blood cell.
The neutrophil leucocyte layer of centre is sucked out, cell mass is opened gently, add pH=7.4 phosphate (PBS) damping fluid of 4 ℃ of precoolings, blow and beat gently with suction pipe, make the abundant mixing of cell, centrifugal 5 minutes of 1200rpm/min abandons supernatant.
With suction pipe cell mass is opened gently, in cell precipitation, added the 0.3%NaCl solution of 4 ℃ of precoolings, make cell suspension, make the cell swelling under the hypotonic state, and examine under a microscope the cellular morphology of neutrophil leucocyte, adjust neutrophil leucocyte number to 5 * 10
5/ ml.
Cell suspension is joined on the cleaning wave carrier piece, every hole 15 μ l, natural air drying is fixed 5 minutes with absolute ethyl alcohol under 4 ℃, the sealing of air-dry back, labelled antigen sheet preparation time, it is standby to put-20 ℃ of preservations.
Embodiment 2
Get the fresh heparin anti-coagulating of normal person, add 1% methylcellulose according to 4: 1 volume ratios, after at room temperature tilting 5 minutes, centrifugal 2.5 minutes of 1000rpm/min makes erythrocyte sedimentation behind the mixing, and leukocytic plasma layer is rich in sucking-off.
Under the uniform temp, in test tube, add 3ml Ficoll-Hypaque gradient agent II (proportion 1.119), again 3ml Ficoll-Hypaque gradient agent I (proportion 1.077) is superimposed upon gently on the sharp II of gradient.
On gradient agent upper strata along tube wall 3～4ml leucocyte plasma layer that superposes gently, in test tube balance trailing water flat hydro-extractor, centrifugal 20 minutes of 2000rpm/mm, the visible liquid in centrifugal back obviously is divided into 3 layers, and wherein the 1st layer is blood plasma-solution I contact bed, contains monocyte and blood platelet, the 2nd layer is solution-solution interface layer, contain neutrophil leucocyte, the 3rd layer is the beds of precipitation, contains red blood cell.
Neutrophil leucocyte layer in the middle of drawing out is opened cell mass gently with suction pipe, adds the PBS damping fluid (pH 7.4) of 4 ℃ of precoolings, blows and beats gently with suction pipe, makes the abundant mixing of cell, and centrifugal 10 minutes of 1000rpm/min abandons supernatant.
With suction pipe cell mass is opened gently, in cell precipitation, added the 0.6%NaCl solution of 4 ℃ of precoolings, make cell suspension, make the cell swelling under the hypotonic state, and examine under a microscope the cellular morphology of neutrophil leucocyte, adjust neutrophil leucocyte number to 5 * 10
5/ ml.
Cell suspension is joined on the cleaning wave carrier piece, every hole 15 μ l, natural air drying is fixed 5 minutes with absolute ethyl alcohol under 4 ℃, the sealing of air-dry back, labelled antigen sheet preparation time, it is standby to put-20 ℃ of preservations.
Claims (4)
1, a kind of preparation method of neutrophils antigen egative film is characterized in that comprising the steps:
Get the fresh heparin anti-coagulating of normal person, mix according to 4: 1 volume ratio with 1% methocel solution, sedimented red cell under the room temperature keeps the leucocyte plasma layer;
Under the uniform temp, in test tube, isopyknic Ficoll-Hypaque gradient agent I is superimposed upon on the Ficoll-Hypaque gradient agent II gently;
The leucocyte plasma layer is superimposed upon the upper strata of gradient agent gently, and balance is placed in the horizontal hydro-extractor, centrifugal 20～25 minutes of 1800～2000rpm/min;
Neutrophil leucocyte layer in the middle of drawing, the damping fluid of 4 ℃ of precoolings of adding, centrifugal 5～10 minutes of 1000～1200rpm/min abandons supernatant;
Add 0.1%～0.85%NaCl solution of 4 ℃ of precoolings in the cell precipitation, and adjust neutrophil leucocyte number to 5 * 10
5/ ml;
Cell suspension adds natural air drying on the cleaning wave carrier piece, and 4 ℃ of following absolute ethyl alcohols are fixed 5 minutes, the sealing of air-dry back.
2, neutrophils antigen egative film preparation method according to claim 1 is characterized in that described sedimented red cell is with at room temperature tilting 15～20 minutes of the mixed liquor of heparin anti-coagulating and methocel solution, makes erythrocyte sedimentation.
3, neutrophils antigen egative film preparation method according to claim 1, it is characterized in that described sedimented red cell is with at room temperature tilting 5～10 minutes of the mixed liquor of heparin anti-coagulating and methocel solution, centrifugal 2～5 minutes of 1000～1200rpm/min makes erythrocyte sedimentation.
4, neutrophils antigen egative film preparation method according to claim 1 is characterized in that described damping fluid is the phosphate buffer of pH7.4.
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| CNB2005100124927A CN1316250C (en) | 2005-05-01 | 2005-05-01 | Method for preparing negative film of antigen of neutrophilic granulocyte |
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|---|---|---|---|
| CNB2005100124927A CN1316250C (en) | 2005-05-01 | 2005-05-01 | Method for preparing negative film of antigen of neutrophilic granulocyte |
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| CN1687780A true CN1687780A (en) | 2005-10-26 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103293301A (en) * | 2013-06-17 | 2013-09-11 | 天津瑞华生物科技有限公司 | Diagnostic kit for autoimmune disease ulcerative colitis and preparation method thereof |
| CN106771117A (en) * | 2016-11-25 | 2017-05-31 | 王婧 | A kind of preparation method of negative film of antigen of neutrophilic granulocyte |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0615129B1 (en) * | 1993-03-10 | 2000-05-31 | Cedars-Sinai Medical Center | Methods for selectively detecting perinuclear anti-neutrophil cytoplasmic antibody of ulcerative colitis or primary sclerosing cholangitis |
| US5830675A (en) * | 1993-03-10 | 1998-11-03 | Cedars-Sinai Medical Center | Methods for selectively detecting perinuclear anti-neutrophil cytoplasmic antibody of ulcerative colitis, primary sclerosing cholangitis, or type 1 autoimmune hepatitis |
| EP1364002A2 (en) * | 2000-08-23 | 2003-11-26 | Pfizer Products Inc. | Process for the preparation of neutrophil inhibitory factor |
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2005
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103293301A (en) * | 2013-06-17 | 2013-09-11 | 天津瑞华生物科技有限公司 | Diagnostic kit for autoimmune disease ulcerative colitis and preparation method thereof |
| CN106771117A (en) * | 2016-11-25 | 2017-05-31 | 王婧 | A kind of preparation method of negative film of antigen of neutrophilic granulocyte |
| CN106771117B (en) * | 2016-11-25 | 2018-09-14 | 王婧 | A kind of preparation method of negative film of antigen of neutrophilic granulocyte |
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