CN104502609A - Immunohistochemical SIMPLE same part marker diagnosis method and kit for pathologic autopsy anaphylactic shock - Google Patents
Immunohistochemical SIMPLE same part marker diagnosis method and kit for pathologic autopsy anaphylactic shock Download PDFInfo
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- CN104502609A CN104502609A CN201410819203.3A CN201410819203A CN104502609A CN 104502609 A CN104502609 A CN 104502609A CN 201410819203 A CN201410819203 A CN 201410819203A CN 104502609 A CN104502609 A CN 104502609A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention provides an immunohistochemical SIMPLE same part marker diagnosis method and kit for a pathologic autopsy anaphylactic shock. The kit comprises a first mastocyte (MC) antibody, a first IgE antibody, a universal second antibody reagent, an antigen repair reagent, a DAB developing solution, an AEC developing solution, a buffer liquid system and the like, wherein the first antibody is a mouse-anti-human monoclonal MC antibody; and the first IgE antibody is a mouse-anti-human monoclonal IgE antibody. The kit has the characterstics of being low in price, good in specificity, high in sensitivity, high in accuracy, good in stability, simple and general. The kit is mainly applied to identification of pathology and forensic pathology; and an intuitive pathological basis for the anaphylactic shock can be accurately provided.
Description
Technical field
The invention belongs to medical detection technique field, relate to a kind of SABC SIMPLE of pathologic autopsy anaphylactic shock with part mark diagnostic method and kit thereof.
Background technology
Anaphylactic shock (anaphylaxis, anaphylactic shock) is type Ⅰ allergy, is that after some antigenicity substance extraneous enters the body of sensitization, a kind of strong multi viscera occurred at short notice by immunologic mechanism involves disease group.
After MC and MC activation, retting conditions is the core that development occurs anaphylactic shock.After MC is activated, the particle being rich in trypsinlike enzyme of its inside and histamine particle are all released in blood, and in MC, the excessive situation of trypsinlike enzyme is by as one of the important indicator weighed with or without anaphylactic shock.Except the method for this enzyme of Virus monitory, immunohistochemical method can also be carried out for pathological tissues in patient body and detect trypsinlike enzyme content in MC born of the same parents and outside born of the same parents.Use related software to carry out analysis MC Activation again, and diagnose anaphylactic shock type further.
Anaphylactic shock is one of acute disease occurred frequently clinically, account for about 10 ﹪ of annual legal medical expert's dispute, but because disease incidence is unexpected, the non-specific performances such as internal organs extravasated blood oedema are organized when only seeing sudden death during postmortem, be difficult to find clear and definite morphological change, also need during medical jurisprudence judicial expertise in conjunction with the dead's allergies, medication history, and get rid of other all causes of the death and could diagnose, this brings great inconvenience to the diagnosis of the medical jurisprudence of anaphylactic shock, therefore finds simple and easy to do diagnosis index and has become problem demanding prompt solution.In anaphylactic shock mechanism, MC, retting conditions MC, IgE, EOS have played very big effect, but MC Degranulation ratio also seldom has report so far, IgE and EOS be the research being limited to animal model also more, and not yet occur their simultaneous determination and the discussion of relevance at human body.
Summary of the invention
The object of the invention is to, provide a kind of SABC SIMPLE of pathologic autopsy anaphylactic shock with part mark diagnostic method and kit thereof.Located altogether to observe IgE and MC retting conditions with part mark technology by SABC, can be used as the pathological diagnosis index of anaphylactic shock.
The SABC SIMPLE of a kind of pathologic autopsy anaphylactic shock of the present invention, with part mark diagnostic method, comprises the steps:
Step one: make tissue;
Step 2: antigen retrieval---IgE: first antigen retrieval reagent 1 is put into pressure cooker, after boiling, tissue is put into, after steaming, 3min closes fire, takes out tissue and antigen retrieval reagent, naturally cool to room temperature from pressure cooker;
Step 3: adopt PBS damping fluid to wash 3 times, each 5min; Tissue, in tissue, is put into wet box, 4 DEG C of process of spending the night by the diluted IgE first antibody of dropping PBS; 3 times are washed respectively, each 5min with PBS;
Step 4: all tissue are all added universal two anti-reagent, and hatch about 30min in 37 DEG C; Clean all tissue 3min by PBS solution, amount to 3 times;
Step 5: adopt AEC nitrite ion colour developing (this process will carry out dye levels control under an optical microscope), continues after flowing water color development stopping to rinse 10min;
Step 6: haematoxylin redyes 1min, flowing water rushes 2min, and put into 1% hydrochloride alcohol differentiation liquid and break up 6-8 second, flowing water rushes 5min;
Step 7: tissue is immersed in 70% alcohol, 80% alcohol, 90% alcohol, 1000% alcohol and be 5min; With dimethylbenzene, transparent 5min is carried out to tissue again, amount to 3 times; Use neutral gum mounting;
Step 8: with full automatic biological microscope, scanning is carried out to all tissue and to take pictures preservation;
Step 9: SIMPLE method tissue takes off sheet-by all tissue to be inserted among the hot water of more than 60 DEG C, washes 5min, amount to 3 times, to wash away mountant AEC with PBS liquid; Put it into subsequently in the dye vat containing concentration 80% alcohol and operate, and on shaking table, carry out jolting in 1 hour, wash 5min with PBS liquid, amount to 3 times; Afterwards tissue is inserted in Tris-HCl liquid and spend the night, wash 5min with PBS liquid, amount to 3 times;
Step 10: antigen retrieval again---MC: be added drop-wise in tissue by antigen retrieval reagent 2, puts into wet box by tissue, 37 DEG C of constant-temperature incubation 20min;
Step 11: adopt PBS damping fluid to wash 3 times, each 5min; The MC first antibody tissue dripped after dilution puts into wet box, 4 DEG C of process of spending the night; 3 times are washed respectively, each 5min with PBS;
Step 12: all tissue are all added universal two anti-reagent, and hatch about 30min in 37 DEG C; Clean all tissue 3min by PBS solution, amount to 3 times;
Wash 5min with PBS liquid, amount to 3 times;
Step 13: preparation DAB nitrite ion, carries out develop the color (this process will carry out dye levels control under an optical microscope) with DAB liquid, and continuous running water in this process;
Step 14: haematoxylin redyes 1min, flowing water rushes 2min, and put into 1% hydrochloride alcohol differentiation liquid and break up 6-8 second, flowing water rushes 5min; Carry out returning indigo plant in 10 seconds with unsaturated carbonate lithium liquid, carry out 3 min flushings with tap water subsequently;
Step 14: tissue is immersed in 70% alcohol, 80% alcohol, 90% alcohol, 1000% alcohol and be 5min; With dimethylbenzene, transparent 5min is carried out to tissue again, amount to 3 times; Use neutral gum mounting;
Step 15: carry out observing, take pictures and analyze relevant positive products with microscope afterwards.Observe the relation that in irritated group and control group, MC and IgE expresses.
Step 10 six: blank: contrast by the tissue adding PBS when instillation MC first is anti-, complete by wash-out to determine the primary antibodie instilled first.
Wherein, described PBS damping fluid is 0.01mol/L(pH7.2-7.4) PBS damping fluid;
Described first antibody is:
1) MC first antibody: mouse-anti people MC monoclonal antibody
2) IgE first antibody: mouse-anti human IgE monoclonal antibody
Described MC first antibody dilution is add mouse-anti people MC monoclonal antibody in PBS damping fluid simultaneously, and the ratio of first antibody and PBS damping fluid is 1:100;
Described IgE first antibody dilution is add mouse-anti human IgE monoclonal antibody in PBS damping fluid simultaneously, and the ratio of first antibody and PBS damping fluid is 1:100;
Described antigen retrieval reagent 1 is citrate 0.01M, Ph6.0;
Described antigen retrieval reagent 2 is the tryptic digestive juice of 0.125 ﹪;
Described DAB nitrite ion is ratio is that 1:50-1:100 configures fresh DAB nitrite ion;
Described AEC nitrite ion is ratio is that 1:50-1:100 configures fresh AEC nitrite ion;
Described universal two anti-reagent are two anti-(i.e. the Ig-HRP) that horseradish peroxidase carries out marking;
Described Tris-HCl liquid is the Tris-HCl solution of 0.01mol/L (pH1.5).
The SABC SIMPLE of a kind of pathologic autopsy anaphylactic shock described above with part mark diagnostic method, the method for making of tissue:
Step one: first by paraffin specimen connective tissue sheet (every sheet is 4-6 μm of thickness substantially), with distilled water, all tissue are carried out stand sheet, is placed on subsequently in 60 DEG C of baking boxs and bakes sheet 2 hours; Immerse 100% xylene solution 30min, totally 3 times; Immerse gradient 100% alcoholic solution successively, 95% alcoholic solution, 85% alcoholic solution, 75% alcoholic solution, each 5min; Distillation washing 3 times, each 5min;
Step 2: the superoxol 10min(immersing concentration 3% under room temperature can omit);
Step 3: tissue be placed in PBS again and clean 5min, amounts to 3 times, obtains the tissue preparing to carry out detecting.
The SABC SIMPLE of a kind of pathologic autopsy anaphylactic shock described above, with part mark diagnostic method, comprises the following steps:
Microscopic examination:
Step one: MC positive cell judges,
MC positive cell nuclear morphology: rounded or oval, regular shape, differs in size,
MC positive cell cytoplasmic form: endochylema is expressed obvious yellow or brown color positive particle or filled the air shape and is positive cell;
Step 2: the judgement of retting conditions mast cell: cell is irregular shape, visible yellow or brown color positive particle is around deviate from;
Step 3: the IgE positive judges, occurs that brownish red dyeing is for the positive with endochylema or after birth.
The SABC SIMPLE of a kind of pathologic autopsy anaphylactic shock described above, with part mark diagnostic method, comprises the following steps:
Its graphical analysis and result judge:
Observe the relation that in irritated group and control group, MC and IgE expresses; The tissue of carrying out gummy mounting process after immunohistochemical staining is observed, utilize all-round microscope (Leica DMRXA2) and image capturing system (Leica IM50), unduplicated 10 high power fields of random selecting carry out the collection of experimental result image; Image analysis software (Image-Pro Plus 6.0) is used to carry out the graphical analysis of experimental result: retting conditions MC number and MC sum in the every pictures of meter record, using the average of 10 visual field MC countings as the last average of this picture as statistic, and calculate the statistic of average MC Degranulation ratio (retting conditions MC number/10, the visual field, MC Degranulation ratio=10 visual field MC sum × 100 %) as last MC Degranulation ratio in every pictures 10 visuals field; IgE counts the positive cell in every pictures 10 visuals field, using the average of 10 visual field countings as the last average of this picture as statistic.
Method described above, is characterized in that, comprises the following steps:
Judge the MC retting conditions that SABC SIMPLE detects with part mark diagnostic method and IgE expression, observe its MC Degranulation ratio whether to be greater than 50% and MC Degranulation ratio and IgE and to express and whether be proportionate, especially with or without coexpression, if any, be then shown to be anaphylactic shock.
For the kit that the SABC SIMPLE of pathologic autopsy anaphylactic shock diagnoses with part mark, its content comprises:
Antigen retrieval reagent (citrate 0.01M, PH6.0);
MC first antibody: mouse-anti people MC monoclonal antibody;
IgE first antibody: mouse-anti human IgE monoclonal antibody.
The invention has the advantages that:
The SABC SIMPLE of a kind of pathologic autopsy anaphylactic shock of the present invention has with part mark diagnostic kit that price is low, specificity good, susceptibility is strong, accuracy is high, the characteristic of good stability, simple general-purpose.
accompanying drawing illustrates:
Fig. 1 is anaphylactic shock the dead gross specimen (the obvious oedema of a larynx, Closure states of glottis; The two pulmonary edema of b);
Fig. 2 is sample Histomorphological; (a allergy group larynx mucous membrane conveying oedema HE × 200; B allergy group lung tissue acute pulmonary venous pleonaemia oedema HE HE × 200; C allergy group intestinal tissue smooth muscle spasm HE × 200);
Fig. 3-1 is anaphylactic shock the dead intestinal tissue connective tissue sheet, the distribution of the same area MC and IgE, wherein, and the distribution (AEC development process) of A:MC; The distribution (DAB development process) (× 400) of B:IgE positive cell;
Fig. 3-2 anaphylactic shock the dead larynx organizes Simple with the dyeing of part mark method, the distribution of the same area MC and IgE, wherein, and the distribution (AEC development process) of A:MC; The distribution (DAB development process) of B:IgE positive cell.
Embodiment
Below in conjunction with drawings and Examples, the present invention will be described in detail.
The specific implementation process of the present embodiment:
1. suspect Anaphylactic Shock case pathologic autopsy and draw materials specification and assessment criteria
(1) by the complete taking-up tongue of specification postmortem method, larynx, tracheae and lung, postmortem field observation Taking Pictures recording larynx are with or without obvious oedema and glottis opening status, and lung is with or without bulge and weigh.Lung gross specimen after fixing is placed in be cut on lung plate, and by lung dorsal part close adhesion plate face, lung is fixed on plate by left hand, firmly evenly, makes lung large area be attached to plate face as far as possible.Make a horizontal section from lateral border to hilus pulumonis along lung major axis, observe and overflow with or without edematous fluid.
(2) histology is drawn materials: draw materials to larynx, tracheae, lung and intestines respectively, and lung must comprise each lobe of the lung, at least 1 piece, every leaf, and the thick 3mm ~ 4mm of every block, area about 2cm × 2cm, be placed on chopping block by intestines sample, draws materials respectively along the intestinal tube longitudinal axis, transverse axis.The tissue block of drawing materials will comprise pathology and suspicious lesions.Tissue block of drawing materials numbering will be substantially consistent.Histological tissue sheet adopts routine paraffin wax tissue and haematoxylin eosin stains.
(3) gross specimen observes pathology shooting recording method
Apparatus for making a video recording:
Rephotograph stand: corner is symmetrical, slants 45 ° of symmetrical lamp stand framves;
Camera and camera lens: single anti-digital camera configuration AF35-70mmf/2.8 automatic zoom camera lens; Pixel request is not less than M3216 × 2136.
Light source: 6400K color of sunshine
Method of operating: larynx, lung, intestines gross specimen situation after postmortem shooting is fixing.Fixed camera is on camera-shooting table elevating lever, and adjustment camera heights, camera lens is 800mm to the general distance of sample, can require suitably to be adjusted according to sample size composition.Lung preparation surface mucus, moisture are fully blotted, is placed on diagosis lamp opal glass, take pictures once to tangent plane before drawing materials, take pictures again once after mark of drawing materials.
2. the making of paraffin specimen and tissue
All samples are all fixed at normal temperatures with 10% neutral formalin solution, and the general time is 24 ~ 48 hours, then draws materials by the production standard of paraffin specimen.Normal experiment process is: this dehydration of advanced rower, then goes transparent, finally by sample waxdip and paraffin embedding.Finally standard compliant for making paraffin specimen is carried out connective tissue's sheet, slice, thin piece general thickness is 4 ~ 6 μm, 40 DEG C of distilled water carry out stand sheet to tissue, fish for the standard compliant complete tissue without tool marks and fold subsequently with the microslide after APES process.
3.HE dyes
Tissue conventional H E dyes, and determines anaphylactic shock and control group larynx, lung, the histological type of intestinal tissue and anaphylactoid Pathological thereof;
(1) they are placed on and carry out 1 hours in 60 DEG C of baking boxs and bake sheet by the standard compliant tissue of first picking;
(2) adopt xylene solution to dewax to sample, duration is 5min, amounts to 3 times;
(3) tissue is immersed in 100% alcohol, 90% alcohol, 80% alcohol, 70% alcohol successively subsequently and be about 5min, take out with tap water (timing: 3min);
(4) haematoxylin dye liquor 2min is immersed;
(5) tissue after dyeing is placed on flushing (duration is 5min) in tap water;
(6), about being 6-8 second with hydrochloride alcohol solution (general formulated by the 70% concentration alcohol liquid) divergaence time that concentration is 1%, tap water tissue sample 3min is used subsequently;
(7) with 0.5% eosin stain, dyeing in about 5 seconds is carried out to tissue sample;
(8) adopt 70% alcohol, 80% alcohol, 90% alcohol, 100% alcoholic solution to dewater to sample subsequently, each duration is 2min;
(9) with dimethylbenzene, transparent 5min is carried out to tissue again, amount to 3 times;
(10) finally use neutral gum mounting: first wipe dimethylbenzene liquid superfluous around sample tissue, do not allow it dry up, drip neutral gum rapidly subsequently, then carry out sealing with cover glass.
4.SIMPLE is with part mark method
SIMPLE is adopted to detect the distribution of MC, retting conditions MC, IgE in irritated group and control group larynx, intestines, lung tissue with part mark;
(1) first by experimental specimen connective tissue sheet (every sheet is 4 μm of thickness substantially), with distilled water, all tissue are carried out stand sheet, be placed on subsequently in 60 DEG C of baking boxs and bake sheet 3 hours;
(2) adopt xylene solution to dewax to sample, duration is 10min, amounts to 3 times;
(3) its tissue puts into immersion 100% alcohol successively subsequently, 90% alcohol, 80% alcohol, 70% alcohol is about 5min, takes out with tap water (timing: 3min);
(4) again sample tissue is placed in distilled water and cleans 5min, amount to 3 times;
(5) with the superoxol of concentration 3%, 12min is hatched to sample under room temperature;
(6) clean all sample tissue 3min with distilled water again, amount to 3 times;
(7) antigen retrieval---IgE: first antigen retrieval reagent 1 is put into pressure cooker, after boiling, tissue is put into, after steaming, 3min closes fire, takes out tissue and antigen retrieval reagent, naturally cool to room temperature from pressure cooker;
(8) PBS damping fluid is adopted to wash 3 times, each 5min; Tissue, in tissue, is put into wet box, 4 DEG C of process of spending the night by the diluted IgE first antibody of dropping PBS; 3 times are washed respectively, each 5min with PBS;
(9) all tissue are all added universal two anti-reagent, and hatch about 30min in 37 DEG C; Clean all tissue 3min by PBS solution, amount to 3 times;
(10) adopt AEC nitrite ion colour developing (this process will carry out dye levels control under an optical microscope), continue after flowing water color development stopping to rinse 10min;
(11) haematoxylin redyes 1min, and flowing water rushes 2min, and put into 1% hydrochloride alcohol differentiation liquid and break up 6-8 second, flowing water rushes 5min;
(12) tissue is immersed in 70% alcohol, 80% alcohol, 90% alcohol, 1000% alcohol and be 5min; With dimethylbenzene, transparent 5min is carried out to tissue again, amount to 3 times; Use neutral gum mounting;
(13) carry out scanning with full automatic biological microscope to all tissue to take pictures preservation;
(14) SIMPLE method tissue takes off sheet-by all tissue and inserts among the hot water of more than 60 DEG C, washes 5min, amount to 3 times, to wash away mountant AEC with PBS liquid; Put it into subsequently in the dye vat containing concentration 80% alcohol and operate, and on shaking table, carry out jolting in 1 hour, wash 5min with PBS liquid, amount to 3 times; Afterwards tissue is inserted in Tris-HCl liquid and spend the night, wash 5min with PBS liquid, amount to 3 times;
(15) antigen retrieval---MC again: antigen retrieval reagent 2 is added drop-wise in tissue, tissue is put into wet box, 37 DEG C of constant-temperature incubation 20min;
(16) PBS washes 5min × 3 time; The MC first antibody tissue dripped after dilution puts into wet box, 4 DEG C of process of spending the night; 3 times are washed respectively, each 5min with PBS;
(17) all tissue are all added universal two anti-reagent, and hatch about 30min in 37 DEG C; Clean all tissue 3min by PBS solution, amount to 3 times;
Wash 5min with PBS liquid, amount to 3 times;
(18) prepare DAB nitrite ion, carry out develop the color (this process will carry out dye levels control under an optical microscope) with DAB liquid, and continuous running water in this process;
(19) haematoxylin redyes 1min, and flowing water rushes 2min, and put into 1% hydrochloride alcohol differentiation liquid and break up 6-8 second, flowing water rushes 5min; Carry out returning indigo plant in 10 seconds with unsaturated carbonate lithium liquid, carry out 3 min flushings with tap water subsequently;
(20) tissue is immersed in 70% alcohol, 80% alcohol, 90% alcohol, 1000% alcohol and be 5min; With dimethylbenzene, transparent 5min is carried out to tissue again, amount to 3 times; Use neutral gum mounting;
(21) carry out observing, take pictures and analyze relevant positive products with microscope after.Observe the relation that in irritated group and control group, MC and IgE expresses.
(22) blank: contrast by the tissue adding PBS when instillation MC first is anti-, complete by wash-out to determine the primary antibodie instilled first.
5.SIMPLE, with part mark method, observes the relation that in irritated group and control group, MC and IgE expresses;
(1) cell positive coloration result judges: observe MC, IgE expression in sudden death from allergy the dead larynx, lung, intestinal tissue by carrying out;
A.MC positive cell: rounded or oval, regular shape, differs in size, and endochylema is expressed obvious yellow or brown color positive particle or filled the air shape and is positive cell; The judgement of retting conditions mast cell: cell is irregular shape, visible yellow or brown color positive particle is around deviate from;
B. IgE positive cell: occur that brownish red dyeing is for the positive with endochylema or after birth;
(2) mensuration of cell positive label index
The tissue of carrying out gummy mounting process after immunohistochemical staining is observed, utilize all-round microscope (Leica DMRXA2) and image capturing system (Leica IM50), unduplicated 10 high power fields of random selecting carry out the collection of experimental result image; Image analysis software (Image-Pro Plus 6.0) is used to carry out the graphical analysis of experimental result: retting conditions MC number and MC sum in the every pictures of meter record, using the average of 10 visual field MC countings as the last average of this picture as statistic, and calculate the statistic of average MC Degranulation ratio (retting conditions MC number/10, the visual field, MC Degranulation ratio=10 visual field MC sum × 100 %) as last MC Degranulation ratio in every pictures 10 visuals field; IgE counts the positive cell in every pictures 10 visuals field, using the average of 10 visual field countings as the last average of this picture as statistic.
6. graphical analysis and ImmunohistochemistryResults Results judge
Application Leica IM50 image capturing system and Image-Pro Plus 6.0 image analysis system are analyzed coloration result;
7. statistical analysis
Application SPSS13.0 analyzes statistics, adopts T inspection, Spearman correlation analysis and regression linear regression.MC, MC Degranulation ratio, the IgE difference be distributed between different group adopts T inspection; The mutual relationship of MC, MC Degranulation ratio, IgE and with the discussion of death time Spearman correlation analysis; Intestines IgE and lung MC Degranulation ratio are used regression linear regression by death time impact.Conspicuousness standard setting is: by result be
pthe difference that is considered as of < 0.05 has statistical significance, by result
p< 0.01 is considered as difference and has height statistical significance.
Larynx, lung, small intestine's sample of 72 examples are collected in this test altogether, all samples are all from University Of Shantou's judicial expertise center, wherein 20 examples are anaphylactic shock through the clinical pathology diagnosis cause of the death, and another 52 examples are control group, prove without anaphylactia through clinical pathology data.52 routine sample the statistics of cause of deaths are poisoning, the 3 routine cerebral hemorrhages of 6 example, 4 routine neonates' difficult labours suffocate, 5 routine respiratory tract infection to organ failure, 12 exception wounds, 12 routine heart diseases, 2 routine heatstrokes, 3 routine blood vessel embolisms, 1 example is committed suiside suffocates, 1 example shocks by electricity death, 1 routine septicemia, 2 routine postpartum hemorrhages.
MC, MC Degranulation ratio, IgE correlation analysis is carried out by the larynx to 72 examples, lung, small intestine's sample, it is correlation that result is presented at that MC and IgE in larynx tissue, MC Degranulation ratio and IgE express, in lung tissue, MC and IgE is proportionate, and in intestinal tissue, MC Degranulation ratio and IgE express is correlation.Observe IgE is positioned on MC film or MC endochylema, in thick liquid cell endochylema, the MC overwhelming majority located by IgE is in retting conditions state simultaneously.
The dead's tissue that this test is to Anaphylactic Shock and electric shock is dead, heart infarct is died carries out the Simple of MC and IgE with part mark paired observation, intuitively clearly to determine the relation of MC and IgE in tissue.Found that, in Anaphylactic Shock tissue MC and retting conditions phenomenon more, in the after birth that IgE is often positioned retting conditions MC and kytoplasm, in non-irritated group, the expression of IgE is almost feminine gender, and MC number is less and retting conditions phenomenon is few simultaneously.
Claims (7)
1. the SABC SIMPLE of pathologic autopsy anaphylactic shock, with a part mark diagnostic method, is characterized in that:
Step one: make tissue;
Step 2: antigen retrieval---IgE: first antigen retrieval reagent 1 is put into pressure cooker, after boiling, tissue is put into, after steaming, 3min closes fire, takes out tissue and antigen retrieval reagent 1, naturally cool to room temperature from pressure cooker;
Step 3: adopt PBS damping fluid to wash 3 times, each 5min; Drip IgE first antibody dilution in tissue, put into wet box, night incubation;
Step 4: all tissue are all added universal two anti-reagent, hatch in 37 DEG C;
Step 5: adopt AEC nitrite ion colour developing (this process will carry out dye levels control under an optical microscope);
Step 6: it is transparent that haematoxylin redyes rear dehydration, uses neutral gum mounting;
Step 7: with full automatic biological microscope, scanning is carried out to all tissue and to take pictures preservation;
Step 8: SIMPLE method tissue takes off sheet---among the hot water of all tissue being inserted more than 60 DEG C, wash 5min with PBS liquid, amount to 3 times, to wash away mountant AEC; Put it into subsequently in the dye vat containing concentration 80% alcohol and operate, and on shaking table, carry out jolting in 1 hour, wash 5min with PBS liquid, amount to 3 times; Afterwards tissue is inserted in Tris-HCl liquid and spend the night, wash 5min with PBS liquid, amount to 3 times;
Step 9: antigen retrieval again---MC: be added drop-wise in tissue by antigen retrieval reagent 2, puts into wet box by tissue, 37 DEG C of constant-temperature incubation 20min;
Step 10: adopt PBS damping fluid to wash 3 times, each 5min; Drip MC first antibody dilution in tissue, put into wet box, night incubation;
Step 11: all tissue are all added universal two anti-reagent, and hatch in 37 DEG C;
Step 12: preparation DAB nitrite ion, carries out develop the color (this process will carry out dye levels control under an optical microscope) with DAB liquid, and continuous running water in this process;
Step 13: it is transparent that haematoxylin redyes rear dehydration, uses neutral gum mounting;
Step 14: carry out observing, take pictures and analyze relevant positive products with microscope, observes the relation that in irritated group and control group, MC and IgE expresses;
Step 15: blank: contrast by the tissue adding PBS when instillation MC first is anti-, complete by wash-out to determine the primary antibodie instilled first.
2. a kind of SABC SIMPLE of pathologic autopsy anaphylactic shock, with part mark diagnostic method, is characterized in that as claimed in claim 1:
Described PBS damping fluid is 0.01mol/L(pH7.2-7.4) PBS damping fluid;
Described first antibody is:
1) MC first antibody: mouse-anti people MC monoclonal antibody
2) IgE first antibody: mouse-anti human IgE monoclonal antibody
Described MC first antibody dilution is add mouse-anti people MC monoclonal antibody in PBS damping fluid simultaneously, and the ratio of first antibody and PBS damping fluid is 1:100;
Described IgE first antibody dilution is add mouse-anti human IgE monoclonal antibody in PBS damping fluid simultaneously, and the ratio of first antibody and PBS damping fluid is 1:100;
Described antigen retrieval reagent 1 is citrate 0.01M, Ph6.0;
Described antigen retrieval reagent 2 is the tryptic digestive juice of 0.125 ﹪;
Described AEC nitrite ion is ratio is that 1:50-1:100 configures fresh AEC nitrite ion;
Described DAB nitrite ion is ratio is that 1:50-1:100 configures fresh DAB nitrite ion;
Described universal two anti-reagent are two anti-(i.e. the Ig-HRP) that horseradish peroxidase carries out marking;
Described Tris-HCl liquid is the Tris-HCl solution of 0.01mol/L (pH1.5).
3. a kind of SABC SIMPLE of pathologic autopsy anaphylactic shock, with part mark diagnostic method, is characterized in that as claimed in claim 1, the method for making of tissue:
Step one: first by paraffin specimen connective tissue sheet (every sheet is 4-6 μm of thickness substantially), with distilled water, all tissue are carried out stand sheet, is placed on subsequently in 60 DEG C of baking boxs and bakes sheet 2 hours; Immerse 100% xylene solution 30min, totally 3 times; Immerse gradient 100% alcoholic solution successively, 95% alcoholic solution, 85% alcoholic solution, 75% alcoholic solution, each 5min; Distillation washing 3 times, each 5min;
Step 2: the superoxol 10min(immersing concentration 3% under room temperature can omit);
Step 3: tissue be placed in PBS again and clean 5min, amounts to 3 times, obtains the tissue preparing to carry out detecting.
4. a kind of SABC SIMPLE of pathologic autopsy anaphylactic shock, with part mark diagnostic method, is characterized in that, comprises the following steps as claimed in claim 1:
Microscopic examination:
Step one: MC positive cell judges,
MC positive cell nuclear morphology: rounded or oval, regular shape, differs in size,
MC positive cell cytoplasmic form: endochylema is expressed obvious yellow or brown color positive particle or filled the air shape and is positive cell;
Step 2: the judgement of retting conditions mast cell: cell is irregular shape, visible yellow or brown color positive particle is around deviate from;
Step 3: the IgE positive judges, occurs that brownish red dyeing is for the positive with endochylema or after birth.
5. a kind of SABC SIMPLE of pathologic autopsy anaphylactic shock, with part mark diagnostic method, is characterized in that, comprises the following steps as claimed in claim 1:
Its graphical analysis and result judge:
Observe the relation that in irritated group and control group, MC and IgE expresses; The tissue of carrying out gummy mounting process after immunohistochemical staining is observed, utilize all-round microscope (Leica DMRXA2) and image capturing system (Leica IM50), unduplicated 10 high power fields of random selecting carry out the collection of experimental result image; Image analysis software (Image-Pro Plus 6.0) is used to carry out the graphical analysis of experimental result: retting conditions MC number and MC sum in the every pictures of meter record, using the average of 10 visual field MC countings as the last average of this picture as statistic, and calculate the statistic of average MC Degranulation ratio (retting conditions MC number/10, the visual field, MC Degranulation ratio=10 visual field MC sum × 100 %) as last MC Degranulation ratio in every pictures 10 visuals field; IgE counts the positive cell in every pictures 10 visuals field, using the average of 10 visual field countings as the last average of this picture as statistic.
6. a kind of SABC SIMPLE of pathologic autopsy anaphylactic shock, with part mark diagnostic method, is characterized in that, comprises the following steps as claimed in claim 5:
Judge the MC retting conditions that SABC SIMPLE detects with part mark diagnostic method and IgE expression, observe its MC Degranulation ratio whether to be greater than 50% and MC Degranulation ratio and IgE and to express and whether be proportionate, especially with or without coexpression, if any, be then shown to be anaphylactic shock.
7. the SABC SIMPLE of pathologic autopsy anaphylactic shock, with a part mark diagnostic kit, is characterized in that: content comprises:
Antigen retrieval reagent 1(citrate 0.01M, PH6.0);
The tryptic digestive juice of antigen retrieval reagent 2(0.125 ﹪)
MC first antibody: mouse-anti people MC monoclonal antibody;
IgE first antibody: mouse-anti human IgE monoclonal antibody;
Universal second antibody reagent;
DAB nitrite ion;
AEC nitrite ion.
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