CN104087660A - Method for deducting postmortem interval of human body - Google Patents
Method for deducting postmortem interval of human body Download PDFInfo
- Publication number
- CN104087660A CN104087660A CN201410269524.0A CN201410269524A CN104087660A CN 104087660 A CN104087660 A CN 104087660A CN 201410269524 A CN201410269524 A CN 201410269524A CN 104087660 A CN104087660 A CN 104087660A
- Authority
- CN
- China
- Prior art keywords
- sample
- gallbladder
- mucous membrane
- pmi
- postmortem interval
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 33
- 210000000232 gallbladder Anatomy 0.000 claims abstract description 55
- 210000004400 mucous membrane Anatomy 0.000 claims abstract description 45
- 230000003287 optical effect Effects 0.000 claims abstract description 33
- 238000012417 linear regression Methods 0.000 claims abstract description 25
- 238000004043 dyeing Methods 0.000 claims abstract description 13
- 238000004458 analytical method Methods 0.000 claims abstract description 11
- 210000002919 epithelial cell Anatomy 0.000 claims abstract description 11
- 238000010191 image analysis Methods 0.000 claims abstract description 10
- 238000005259 measurement Methods 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 230000000007 visual effect Effects 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 238000000576 coating method Methods 0.000 claims description 5
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims description 4
- 229930182566 Gentamicin Natural products 0.000 claims description 4
- 239000011248 coating agent Substances 0.000 claims description 4
- 229960002518 gentamicin Drugs 0.000 claims description 4
- 230000001575 pathological effect Effects 0.000 claims description 4
- 239000002504 physiological saline solution Substances 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- JUQPZRLQQYSMEQ-UHFFFAOYSA-N CI Basic red 9 Chemical compound [Cl-].C1=CC(N)=CC=C1C(C=1C=CC(N)=CC=1)=C1C=CC(=[NH2+])C=C1 JUQPZRLQQYSMEQ-UHFFFAOYSA-N 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 claims description 3
- 229940001584 sodium metabisulfite Drugs 0.000 claims description 3
- 235000010262 sodium metabisulphite Nutrition 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 description 15
- 238000012360 testing method Methods 0.000 description 13
- 238000010586 diagram Methods 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 7
- 238000010219 correlation analysis Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 210000000941 bile Anatomy 0.000 description 2
- 210000004958 brain cell Anatomy 0.000 description 2
- 210000002808 connective tissue Anatomy 0.000 description 2
- 238000011840 criminal investigation Methods 0.000 description 2
- 210000003890 endocrine cell Anatomy 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000000630 rising effect Effects 0.000 description 2
- 210000004988 splenocyte Anatomy 0.000 description 2
- 208000001889 Acid-Base Imbalance Diseases 0.000 description 1
- VWEWCZSUWOEEFM-WDSKDSINSA-N Ala-Gly-Ala-Gly Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(=O)NCC(O)=O VWEWCZSUWOEEFM-WDSKDSINSA-N 0.000 description 1
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229960001701 chloroform Drugs 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 210000001096 cystic duct Anatomy 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 210000003677 hemocyte Anatomy 0.000 description 1
- 229940000351 hemocyte Drugs 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000001365 lymphatic vessel Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000029052 metamorphosis Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/02—Investigating particle size or size distribution
- G01N15/0205—Investigating particle size or size distribution by optical means
- G01N15/0227—Investigating particle size or size distribution by optical means using imaging; using holography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Dispersion Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a method for deducting the postmortem interval of a human body, and belongs to the medical science examination field. The method comprises the following steps: extracting human gallbladder mucous membrane epithelial cells at different death times as samples, and smearing the samples; dyeing the smeared samples; carrying out image analysis on the dyed samples, wherein analysis parameters comprise index of density, gray scale parameters and geometric parameters of the nuclei of the gallbladder mucous membrane epithelial cells, and the gray scale parameters comprise average optical density, average gray scale and integral optical density; and carrying out statistics on the parameters to obtain linear regression equations of the parameters, and determining the postmortem interval according to one or more of the linear regression equations. The index of density, gray scale parameters and geometric parameters of the nuclei of the gallbladder mucous membrane epithelial cells are analyzed within 5-36h after human body death through method for deducting the postmortem interval of the human body to obtain a relation between each of the index of density, gray scale parameters and geometric parameters and PMI in order to deduct the postmortem interval of the human body.
Description
Technical field
The present invention relates to field of medical examination, particularly a kind of method of inferring human body postmortem interval.
Background technology
Human body postmortem interval (postmortem interval, PMI) infer it is one of emphasis, difficult point and focus of forensic study always, especially the deduction of early stage PMI is investigated suspect, is arbitrated in the juridical practices such as dead medical tangle and have more realistic meaning in criminal investigation.Objective, early stage PMI deduction accurately, can be public security organ's criminal investigation important objective clue be provided, and is conducive to public security organ and distinguishes whether suspect has crime time, determines or dwindle scope of investigation, improves investigation efficiency and accuracy.In addition, the deduction of postmortem interval is sometimes also most important to the analysis and assessment of natural disaster, insurance and the inheritance of property.
DNA is the genetic material with remarkable biochemical stability, with the somatic DNA content of species different tissues, is constant.After body death, cell tissue generation ischemic, anoxic, along with generating, increases oxyradical, cell autodigestion, its structure is disintegrated, DNA also degrades gradually, and the content of its DNA and nuclear metamorphosis also tool are regular, shows as that core solidifies, the change such as limit collection, cracked, dissolving.Researchist is when research postmortem interval, and its research object is brain cell, splenocyte or liver cell, and researchist all thinks that only the DNA in brain cell, splenocyte or liver cell can accurately reflect postmortem interval.
Summary of the invention
In order to solve the problem that does not reflect postmortem interval in prior art for gall-bladder, the embodiment of the present invention provides a kind of method of inferring human body postmortem interval.Described technical scheme is as follows:
The invention provides a kind of method of inferring human body postmortem interval, described method comprises:
Extract people's the mucous membrane of gallbladder epithelial cell of different death times as sample, and by described sample smear;
Described sample after smear is dyeed;
Described sample after dyeing is carried out to image analysis, the parameter of described analysis comprises: the epithelial nuclear special-shaped index of described mucous membrane of gallbladder, grey parameter and geometric parameter, and described grey parameter comprises average optical, average gray and integral optical density;
Described parameter is added up, obtain the equation of linear regression of described parameter, described equation of linear regression is:
Y
iD=11.333X
iD-33.338, wherein, Y
iDfor the postmortem interval of inferring, X
iDspecial-shaped index for correspondence measurement;
Y
aG=0.679X
aG-54.277, wherein, Y
aGfor the postmortem interval of inferring, X
aGaverage gray for correspondence measurement;
Y
aOD=-178.868X
aOD+ 84.222, wherein, Y
aODfor the postmortem interval of inferring, X
aODthe corresponding average optical of measuring;
Y
iOD=-6.422X
iOD+ 91.398, wherein, Y
iODfor the postmortem interval of inferring, X
iODintegral optical density for correspondence measurement;
According to the one or more definite postmortem interval in described equation of linear regression.
Particularly, described sample clip is from the described people described mucous membrane of gallbladder epithelial cell of 5～36h after death.
Further, the method of described smear is: the physiological saline that the described sample taking out is placed in to the gentamicin that contains 400mg/L, first described in clip the time of sample described people after death 5h complete, in 18 ℃ of thermostat containers, deposit 0.5h, then in moisture preservation box, sample is 1 part described in clip 0.5cm * 0.5cm * 1cm, and every 1h clip once, the described sample of clip is dipped in clear water and is washed, take out described sample and blot with filter paper, by the described sample coating after blotting.
Further, the described sample employing after coating being inspired confidence in to your root dyeing method dyes.
Particularly, described your root dyeing method that inspires confidence in comprises: the standby described smear dying is immersed in stationary liquid to fixedly 0.5h, taking-up is placed in 6N hydrochloric acid soln and is hydrolyzed 30min, taking-up is placed in distilled water and washs, immerse in the Xi Fushi reagent of being prepared by pararosaniline chloride again and place 30min, the Sodium Metabisulfite solution that is 0.5% by concentration embathes 2min, repeats to embathe three times, flowing water rinses after 5min, carries out mounting.
Further, described stationary liquid is Ka Nuoshi stationary liquid.
Particularly, described image analysis adopts color pathological image analysis system.
Further, the field of view of the described smear under microscope high power objective after picked-up dyeing, usings the shape of " bow " font as gathering circuit, guarantees that the image that gathers is without repeating, without intersection, and every described smear is got 10 visuals field at random, each visual field gathers piece image.
Further, described geometric parameter comprises area, mean diameter, equivalent diameter and girth.
The beneficial effect that the technical scheme that the embodiment of the present invention provides is brought is: the method for deduction human body postmortem interval provided by the invention, by analyzing the epithelial nuclear special-shaped index of mucous membrane of gallbladder, grey parameter and geometric parameter, obtain special-shaped index, grey parameter and geometric parameter and PMI relation, thereby infer human body postmortem interval, in addition, be compared to other histocytes, the epithelial nucleus of mucous membrane of gallbladder is easy to extract and preserve, the easier impact of having avoided individual difference and extraneous factor of processing of sample, overcome certain subjectivity, empirical, guaranteed the accuracy that the death time infers, reliability and repeatability.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing of required use during embodiment is described is briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skills, do not paying under the prerequisite of creative work, can also obtain according to these accompanying drawings other accompanying drawing.
Fig. 1 is that the epithelial nuclear area of mucous membrane of gallbladder that provides of the embodiment of the present invention is with the variation diagram of PMI;
Fig. 2 is that the epithelial nuclear mean diameter of mucous membrane of gallbladder that provides of the embodiment of the present invention is with the variation diagram of PMI;
Fig. 3 is that the epithelial nuclear equivalent diameter of mucous membrane of gallbladder that provides of the embodiment of the present invention is with the variation diagram of PMI;
Fig. 4 is the variation diagram of the epithelial nuclear all personal attendant PMI of mucous membrane of gallbladder that provide of the embodiment of the present invention.
Fig. 5 is that the epithelial nuclear special-shaped index of mucous membrane of gallbladder that provides of the embodiment of the present invention is with the variation diagram of PMI
Fig. 6 is that the epithelial nuclear average gray of mucous membrane of gallbladder that provides of the embodiment of the present invention is with the variation diagram of PMI;
Fig. 7 is that the epithelial nuclear average optical of mucous membrane of gallbladder that provides of the embodiment of the present invention is with the variation diagram of PMI;
Fig. 8 is that the epithelial nuclear integral optical density of mucous membrane of gallbladder that provides of the embodiment of the present invention is with the variation diagram of PMI.
Embodiment
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with accompanying drawing, embodiment of the present invention is described further in detail.
Gall-bladder is positioned at the pyriform pouch structure at liver rear under right-hand rib, has effect concentrated and storage bile.Gall-bladder divides four, the end, body, neck, pipe, and neck connects cystic duct.Gallbladder wall is comprised of three layers of mucous membrane, flesh layer and adventitias.This mucous membrane has the feature of typical absorption-type cell, has stronger absorption and concentrating function, and this mucous membrane also has muciparous function, in mucous membrane, except general extracellular, also has endocrine cell.Contriver, in realizing process of the present invention, finds that this organ of gall-bladder also can be used for inferring human body postmortem interval.The present invention is described in detail in conjunction with specific embodiments in employing below.
Embodiment
Select Hubei province within 2013, to have gallbladder tissue's 30 examples of the in vitro people of definite death time, the dead's age is in the majority with 30 ± 10 years old, wherein, and the male sex's 28 examples (account for sum 93.33%), women's 2 examples (account for sum 6.67%).Embalming before drawing materials is in the environment of 16～25 ℃, and the experimental situation temperature of drawing materials is at 14.5～25 ℃, draw materials first the time all human body after death 5h complete.
Under aseptic operation condition, take out complete gall-bladder, and be placed in the physiological saline containing 400mg/L gentamicin, in 18 ℃ of thermostat containers, deposit 0.5h, then in the moisture preservation box of aseptic cleaning, 1 part, clip 0.5cm * 0.5cm * 1cm sample, and every 1h clip once, the sample of clip is immersed in clear water and swings and wash, clean blood plasma components and hemocyte, take out this sample and blot with filter paper, with the sample that tweezers are clamped after blotting, carry out smear, and be coated with according to uniform straight line coating method.The present invention, in sample preparation process, need not destroy the epithelial morphological structure of mucous membrane of gallbladder, also need not make the unicellular homogenate suspension of gallbladder tissue.
Smear is unified Measurement and analysis by special messenger, before each collection, identical environment of system is all set, and before image analyzer reopens afterwards at every turn, measures, all will carry out yardstick calibration, optical density(OD) calibration.Step, the order of all operations program are consistent.
Wherein, gall-bladder inner face is by mucous membrane, three layers of composition of flesh layer and adventitia, moiety is except mucomembranous cell and endocrine cell, also comprise plastosome in kytoplasm, rough surfaced endoplasmic reticulum, MG, blood vessel, lymphatic vessel and nerve etc., gall-bladder is in collection and sample fritter cutting procedure, capital in various degree by bacterium or microbial contamination, itself just carries microorganism or bacterium some composition, so wherein any part is contaminated, capital impact other compositions around, the present invention is placed in the complete gall-bladder of taking-up in the physiological saline of 400mg/L gentamicin, can dispel more thoroughly like this material of the possibility interference experiment results such as bacterium of sample surface, can not destroy cell interior structure again simultaneously, do not affect cell extraction and smear effect.
Mucous membrane of gallbladder epithelial cell kytoplasm acidophilia, and absorb the water and minerals (being mainly Na+) in bile, be placed in 18 ℃ of thermostat containers and deposit 0.5h, can guarantee cell potential of hydrogen and electrolytical balance on the one hand, make cell acid base imbalance can not occur; Can guarantee the freshness of sample on the other hand, make cell surface and inner moistening, more serious dehydration phenomenon does not occur, farthest guarantee the integrity of sample cell.
According to aforesaid operations method, sample sterilization is more thorough, and cell extraction process is smooth, and cellularstructure is complete, and extraction effect is good, and smear effect reaches the requirement that experiment is set, and has guaranteed objectivity, science and the accuracy of experimental result.
After sample smear, adopt and inspire confidence in the epithelial nucleus of your root (Feulgen-Vans) dyeing process demonstration mucous membrane of gallbladder, for the quantitative measurment of nucleus DNA.
It is as follows that employing inspires confidence in your concrete steps of root dyeing method:
The standby smear dying is immersed to fixedly 0.5h of Ka Nuoshi (Carnoy) stationary liquid, taking-up is placed in 6N hydrochloric acid soln and is hydrolyzed 30min, taking-up is placed in distilled water and washs, immerse again in Xi Fushi (Schiff) reagent of being prepared by pararosaniline chloride and place 30min, the Sodium Metabisulfite solution that is 0.5% by concentration embathes 2min, repeat to embathe three times, flowing water rinses after 5min, for example, with conventional neutral gum (resinene), carry out mounting, the all smears of each gallbladder tissue in 5～36h after death, all, from same individuality, must disposablely concentrate dyeing.
Mucous membrane of gallbladder epithelial cell is smear time 3～5h after death first, therefore, the valid data unification that the present invention selects from 5h, by early stage of the present invention animal experiment study show, in 0～6h, DNA degradation is almost plateau phenomenon.Meanwhile, due under experimental situation of the present invention, the self-dissolving of dead 36h descendant gallbladder tissue is seriously and difficult by thin smear, therefore search time is chosen in after death, in 36h, completes.
Wherein, the preparation of Carnoy stationary liquid is raw spirit according to volume ratio: trichloromethane: Glacial acetic acid=6:3:1.
Adopt Motic Med6.0CMIAS color pathological image analysis system (Beijing Mike Audi image technique company limited provides), the field of view of the smear after the lower picked-up dyeing of microscope high power objective (400 *), using the shape of " bow " font as gathering circuit, guarantee that the image gathering is without repetition, without intersecting, every smear is got 10 visuals field at random, and each visual field gathers piece image.
Particularly, guaranteeing, under the prerequisite of coincidence statistics, to require to increase sample size as far as possible.Under the prerequisite of coincidence statistics measurement data test of significance result, the present invention at least needs 11 routine individual specimen data could react overall situation, need simultaneously by every smear be divided into 9 visuals field, and get at random 6 visuals field, every two field picture (two field picture is adopted in each visual field at random) is got 10 cells at random, can meet the needs to overall inference.
The parameter of analyzing comprises: epithelial nuclear special-shaped index (the Index of density of mucous membrane of gallbladder, ID), grey parameter and geometric parameter, wherein, grey parameter comprises: average optical (Average Optical Density, AOD), average gray (Average Gray, AG) and integral optical density (Integral Optical Density, IOD); Geometric parameter comprises: area (Area, A), mean diameter (Average Diameter, AD), girth (Perimeter, P) and equivalent diameter (Equivalent Diameter, ED)
Above-mentioned parameter is measured, then selected respectively stereoscopic analytical system and DNA analysis system automatically to measure, obtain corresponding related data.
Data acquisition carries out dependency and linear regression analysis with SPSS10.0 statistical software, specific as follows:
The epithelial nuclear geometric parameter of mucous membrane of gallbladder
For each final geometric parameter values of adding up, be the average of a certain parameter of all detected cell of the time point 30 routine samples of same PMI.
The average of epithelial each geometric parameter measurement of nucleus of the different PMI mucous membrane of gallbladder of table 1
Experimental result: known by the parameter in table 1, geometric parameter and postmortem interval are without statistics dependency.
Geometric parameter is added up, obtained geometric parameter and postmortem interval without statistical dependency.
The epithelial nuclear area of mucous membrane of gallbladder, human body after death 5～15h be obvious ascendant trend, at 18～30h, be a platform fluctuation, on a declining curve at 30～36h, referring to Fig. 1, and it changes with corresponding PMI without statistics dependency in 5～36h after death.
The observed value of the epithelial nuclear mean diameter of mucous membrane of gallbladder fluctuates up and down in normal size (6～8 μ m) scope of mature lymphocyte, referring to Fig. 2, its in 5～36h after death with corresponding PMI without statistics dependency.
The epithelial nuclear equivalent diameter of mucous membrane of gallbladder, human body after death 5～15h be obvious ascendant trend, 18～30h is a platform, 30～36h is on a declining curve, referring to Fig. 3, its equivalent diameter overall variation trend and area parameters are similar, and in 5～36h after death, it changes with corresponding PMI without statistics dependency.
The epithelial nuclear girth of mucous membrane of gallbladder, in 5～36h after death with corresponding PMI without statistics dependency, the average of its girth fluctuates up and down at 29 μ m, referring to Fig. 4.
The epithelial nuclear special-shaped index of mucous membrane of gallbladder
Human body is after death in 5～36h, and the epithelial nuclear special-shaped index of mucous membrane of gallbladder (ID) of human body is in rising trend with the prolongation of PMI, referring to Fig. 5.The corresponding relation conefficient of concrete statistics square, proofread and correct relation conefficient square, standard error, equation of linear regression and fit conformity test thereof and the results are shown in Table 2 and table 3.Result shows: between special-shaped index and PMI, have very strong statistics dependency, and have linear relationship.
The correlation analysis of the special-shaped index of table 2 and PMI
The linear regression analysis of the special-shaped index of table 3 and PMI
From table 2 and table 3, between special-shaped index and PMI, there is very strong statistics dependency, and R
2be 0.967, R
2for coefficient of determination, the linear relationship of abnormity index and PMI, through variance test, there is statistical significance (p<0.01, p value is a declining indicator of credible result degree), show that this abnormity index has to PMI the contribution of recurrence, there is clear and definite quantity dependence in this abnormity index and PMI.Wherein, equation of linear regression is Y
iD=11.333X
iD-33.338 (Y
iDfor the PMI inferring; X
iDiD average for corresponding PMI measurement), partial regression coefficient in its regression equation, learning by statistics null hypothesis check (T test) shows: in the level of ɑ=0.01, refuse null hypothesis (P<0.001), illustrate between special-shaped index and PMI and have linear relationship.
The epithelial nuclear grey parameter of mucous membrane of gallbladder
For each final grey parameter value of adding up, be the average of a certain parameter of all detected cell of the time point 30 routine samples of same PMI.
Each grey parameter of the epithelial nucleus of the different PMI mucous membrane of gallbladder of table 4 is measured average
Motic Med6.0CMIAS color pathological image analysis system is only required and is accurate to one decimal place number, makes the nearly one decimal place that retains of AG value.
Human body is after death in 5～36h, and the epithelial nuclear average gray of mucous membrane of gallbladder extends with PMI and in rising trend, referring to Fig. 6.The corresponding relation conefficient square of statistics, proofread and correct relation conefficient square, standard error, equation of linear regression and fit conformity test thereof and the results are shown in Table 5 and table 6.
The correlation analysis of table 6 average gray and PMI
The linear regression analysis of table 7 average gray and PMI
From table 5 and table 6, between average gray and PMI, there is very strong statistics dependency (R
2be 0.921), the linear relationship of average gray and PMI, has statistical significance (p<0.01) through variance test, shows that this average gray has to PMI the contribution of recurrence, there is clear and definite quantity dependence in this parameter and PMI, referring to table 7.The equation of linear regression drawing is: Y
aG=0.679X
aG– 54.277 (Y
aGfor the PMI inferring; X
aGaG average for the corresponding PMI measurement of average gray), the partial regression coefficient of this regression equation, learning by statistics null hypothesis check (T test) shows: in the level of ɑ=0.01, refuse null hypothesis (P<0.001), illustrate between average gray and PMI and have linear relationship.
Human body is after death in 5～36h, extending and on a declining curve with PMI of the average optical of mucous membrane of gallbladder epithelial nucleus, referring to Fig. 7, the corresponding relation conefficient of statistics square, proofread and correct that relation conefficient square, standard error, equation of linear regression and fit conformity test thereof the results are shown in Table 7, table 8.
The correlation analysis of table 7 average optical and PMI
The linear regression analysis of table 8 average optical and PMI
From table 7 and table 8, between average optical and PMI, there is very strong statistics dependency (R
2be 0.921).The linear relationship of average optical and PMI, has statistical significance (p<0.01) through variance test, shows that this parameter has to PMI the contribution of recurrence, and this parameter and PMI exist clear and definite quantity dependence, referring to table 7.The equation of linear regression drawing is: Y
aOD=-178.868X
aOD+ 84.222 (Y
aODfor the PMI inferring; X
aODaverage optical average for the corresponding PMI measurement of average optical), partial regression coefficient in its regression equation, learning by statistics null hypothesis check (T test) shows: in the level of ɑ=0.01, refuse null hypothesis (P<0.001), illustrate between average optical and PMI and have linear relationship.
Human body is after death in 5～36h, the integral optical density of mucous membrane of gallbladder epithelial nucleus is on a declining curve with the prolongation of PMI, referring to Fig. 8, the corresponding relation conefficient of concrete statistics square, proofread and correct that relation conefficient square, standard error, equation of linear regression and fit conformity test thereof the results are shown in Table 9, table 10.
The correlation analysis of table 9 integral optical density and PMI
The linear regression analysis of table 10 integral optical density and PMI
From table 7 and table 8, between integral optical density and PMI, there is very strong statistics dependency (R
2be 0.892).The linear relationship of integral optical density and PMI, has statistical significance (p<0.001) through variance test, shows that this parameter has to PMI the contribution of recurrence, and this integral optical density and PMI exist clear and definite quantity dependence, referring to table 9.Equation of linear regression is Y
iOD=-6.422X
iOD+ 91.398 (Y
iODfor the PMI inferring; X
iODiOD average for the corresponding PMI measurement of integral optical density), the partial regression coefficient of its regression equation is learned by statistics null hypothesis check (T test) and is shown: in the level of ɑ=0.01, refuse null hypothesis (P<0.001), illustrate between integral optical density and PMI and have linear relationship.
According to the one or more definite postmortem interval in equation of linear regression, particularly, when obtaining the postmortem interval of inferring by equation of linear regression, can adopt an equation of linear regression, also can adopt a plurality of equations of linear regression, when if the postmortem interval of a plurality of deductions of gained is inconsistent, get the mean value of the postmortem interval of a plurality of deductions, the postmortem interval of this mean value for inferring.
The method of deduction human body postmortem interval provided by the invention, aspect the selection of the kind of sample, it is first-selected research object that people's mucous membrane of gallbladder epithelial cell is take in the present invention, it is mainly mucomembranous epithelial cell that gall-bladder forms cell, tissue-derived relatively single, its corresponding iuntercellular difference and interference are less, and for other internal organs, its pathology pathology is few.Simultaneously experimentation on animals in early stage of the present invention also confirms, gall-bladder is one of best selected organ of image analysis.At human body after death in 5～36h, by analyzing the epithelial nuclear special-shaped index of mucous membrane of gallbladder, grey parameter and geometric parameter, obtain special-shaped index, grey parameter and geometric parameter and PMI relation, thereby infer human body postmortem interval, in addition, be compared to other histocytes, the epithelial nucleus of mucous membrane of gallbladder is easy to extract and preserve, the easier impact of having avoided individual difference and extraneous factor of processing of sample, overcome certain subjectivity, empirical, guaranteed accuracy, reliability and repeatability that the death time infers.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (9)
1. a method of inferring human body postmortem interval, is characterized in that, described method comprises:
Extract people's the mucous membrane of gallbladder epithelial cell of different death times as sample, and by described sample smear;
Described sample after smear is dyeed;
Described sample after dyeing is carried out to image analysis, the parameter of described analysis comprises: the epithelial nuclear special-shaped index of described mucous membrane of gallbladder, grey parameter and geometric parameter, and described grey parameter comprises average optical, average gray and integral optical density;
Described parameter is added up, obtain the equation of linear regression of described parameter, described equation of linear regression is:
Y
iD=11.333X
iD-33.338, wherein, Y
iDfor the postmortem interval of inferring, X
iDspecial-shaped index for correspondence measurement;
Y
aG=0.679X
aG-54.277, wherein, Y
aGfor the postmortem interval of inferring, X
aGaverage gray for correspondence measurement;
Y
aOD=-178.868X
aOD+ 84.222, wherein, Y
aODfor the postmortem interval of inferring, X
aODthe corresponding average optical of measuring;
Y
iOD=-6.422X
iOD+ 91.398, wherein, Y
iODfor the postmortem interval of inferring, X
iODintegral optical density for correspondence measurement;
According to the one or more definite postmortem interval in described equation of linear regression.
2. method according to claim 1, is characterized in that, described sample clip is from the described people described mucous membrane of gallbladder epithelial cell of 5～36h after death.
3. method according to claim 2, it is characterized in that, the method of described smear is: the physiological saline that the described sample taking out is placed in to the gentamicin that contains 400mg/L, first described in clip the time of sample described people after death 5h complete, in 18 ℃ of thermostat containers, deposit 0.5h, then in moisture preservation box, described in clip 0.5cm * 0.5cm * 1cm, sample is 1 part, and every 1h clip once, the described sample of clip is dipped in clear water and is washed, take out described sample and blot with filter paper, by the described sample coating after blotting.
4. method according to claim 3, is characterized in that, the described sample employing after coating is inspired confidence in to your root dyeing method and dye.
5. method according to claim 4, it is characterized in that, described your root dyeing method that inspires confidence in comprises: the standby described smear dying is immersed in stationary liquid to fixedly 0.5h, taking-up is placed in 6N hydrochloric acid soln and is hydrolyzed 30min, takes out to be placed in distilled water and washs, then immerse in the Xi Fushi reagent of being prepared by pararosaniline chloride and place 30min, the Sodium Metabisulfite solution that is 0.5% by concentration embathes 2min, repeat to embathe three times, flowing water rinses after 5min, carries out mounting.
6. method according to claim 5, is characterized in that, described stationary liquid is Ka Nuoshi stationary liquid.
7. method according to claim 1, is characterized in that, described image analysis adopts color pathological image analysis system.
8. method according to claim 7, it is characterized in that, the field of view of the described smear under microscope high power objective after picked-up dyeing, using the shape of " bow " font as gathering circuit, guarantee that the image gathering is without repetition, without intersecting, and every described smear is got 10 visuals field at random, each visual field gathers piece image.
9. method according to claim 1, is characterized in that, described geometric parameter comprises area, mean diameter, equivalent diameter and girth.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410269524.0A CN104087660B (en) | 2014-06-17 | 2014-06-17 | Method for deducting postmortem interval of human body |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410269524.0A CN104087660B (en) | 2014-06-17 | 2014-06-17 | Method for deducting postmortem interval of human body |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104087660A true CN104087660A (en) | 2014-10-08 |
| CN104087660B CN104087660B (en) | 2017-05-10 |
Family
ID=51635419
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410269524.0A Active CN104087660B (en) | 2014-06-17 | 2014-06-17 | Method for deducting postmortem interval of human body |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104087660B (en) |
-
2014
- 2014-06-17 CN CN201410269524.0A patent/CN104087660B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| 罗国厂: "比较人和兔细胞DNA降解来推断人死亡时间的可行性研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 舒细记等: "人脾细胞核几何参数变化与早期死亡时间的相关性研究", 《法医学杂志》 * |
| 舒细记等: "死后5～36 h 人脑细胞DNA降解含量的图像分析", 《第三军医大学学报》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104087660B (en) | 2017-05-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Buskey et al. | Use of the FlowCAM for semi-automated recognition and enumeration of red tide cells (Karenia brevis) in natural plankton samples | |
| Verma | Role of diatoms in the world of forensic science | |
| CN105548138B (en) | The detection method of brewing fermentation liquid yeast activity on-line measuring device based on Raman spectrum | |
| Martsenyuk et al. | Multispectral control of water bodies for biological diversity with the index of phytoplankton | |
| Fucci et al. | Evaluation of two methods for the use of diatoms in drowning cases | |
| CN112213172A (en) | Stable vaginal secretion visible component staining solution and preparation method thereof | |
| Avilov | Navigating across multi-dimensional space of tissue clearing parameters | |
| Jensen et al. | Quantification of subcellular glycogen distribution in skeletal muscle fibers using transmission electron microscopy | |
| CN104087660A (en) | Method for deducting postmortem interval of human body | |
| CN110794024B (en) | Standardized determination method for shellfish blood physiological indexes | |
| Gunasingh et al. | Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids | |
| CN105510543A (en) | Method for detecting genotoxic agent in water by utilization of Pseudorasbora parva | |
| CN109187367B (en) | Device and method for detecting activity of microalgae in ballast water by using polarized light flow control sheet | |
| CN108982192A (en) | A kind of nuclei suspension fast preparation method suitable for the measurement of jujube ploidy | |
| CN113899709B (en) | Method for quantitatively detecting deposition amount of fish mesentery fat and application | |
| Zhao et al. | Types of diatoms in China’s three major rivers and the possible application for an automatic forensic diatom test | |
| CN105504873A (en) | Dye composition for nervous tissue nissl body dyeing, dye liquor and dyeing method | |
| CN115227248A (en) | Meridian point measurement analyzer and health analysis method | |
| CN113533330A (en) | Cervical cancer detection screening management method based on artificial intelligence technology | |
| Staine et al. | Automated identification of rare sperm becomes possible: Is sperm selection the next frontier in male infertility? | |
| Pendarvis et al. | Exploring Biology in the Laboratory, 3e | |
| CN109239289A (en) | A kind of kit for water body detection | |
| CN205982284U (en) | Marine organism tetrodotoxin's quick detect reagent box of colloidal gold | |
| Chakraborty | Evaluation of the Effectiveness of an Alternate Diatom Extraction Procedure in Diatomological Study | |
| WO2024131012A1 (en) | Method for analyzing content and distribution of microplastics in marine cnidarian |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |