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Photographing and analysis

Photographing and analysis

First, the analysis software currently used for slice analysis:

① Image-Pro Plus 6.0 (Media Cybemetics, INC., Rockville, MD, USA), analysis format: a single picture (jpg format) taken from a microscope or scanned file.

② 3D Histech Quant Center2.1 (3D Histech, Hungary), analysis format: files scanned by 3D Histech scanner.

③ Halo 2.32 (Indica labs, USA), analysis format: 3D Histech (MRXS) scanned files from different manufacturers; Leica (SCN, LIF); Zeiss (CZI); Olympus (VSI); Aperio (SVS, AFI) ); Ventana (BIF); Hamamatsu (NDPI, NDPIS)); Confocal nd2 format picture; Bright field tiff, jpg format picture; PE (Perkin Elmer) company's fluorescent multicolor separable Tiff format picture.

Second, the analysis content (if there are other analysis requirements other than the following, please consult directly)

1. Histochemistry/fluorescence analysis [price: 20 yuan/item/index/slice for histochemistry/fluorescence ordinary slices (scanning is to analyze the whole slice or a specific area, and taking pictures is to analyze 3 photos in each slice); chipset Analysis of chemical scanning results 200 yuan/item/chip (independent analysis of each point, the total number of points is less than 200 points); chip fluorescence scanning result analysis of 500 yuan/item/index/chip (independent analysis of each point, the total number of points is less than 200 points); If you want to analyze a specific area in each point separately in the chip analysis, you need to explain in advance, and you will add organization classification or artificial delineation as required, and charge related fees according to the actual difficulty.]

□Area Density, IOD/Tissue Area of the Test Area (IPP): measure the positive cumulative optical density value of IOD and the tissue area of the test area at the same time.

The number and depth of the positive reaction, the greater the value, the stronger the positive. Generally, this option is only used in the analysis of the image field of view, and not used in the full-scan analysis. The advantage takes into account both the area of the positive and the depth of the positive, and the tissue to be tested in the visual field does not fill the entire visual field and is not affected.

□Positive rate, number of positive cells/total number of cells (IPP/HALO): analyze the ratio of the number of positive cells to the total number of cells, and the ratio of the number of positive cells. The analysis software cannot distinguish between different cell types. It calculates the number and proportion of all positive cells and total cells in the field of view or in the entire scan. So this item is suitable for the case of a single cell type. For example, Tunel analyzes the ratio of apoptotic tumor cells in tumor tissue, and the ratio of CD3 positive T cells in spleen tissue.

□Positive cell density, number of positive cells/tissue area of the test area (IPP/HALO): Analyze the ratio of the number of positive cells to the tissue area of the test area, and reflect the number of positive cells per unit area. For example, the proportion of certain types of inflammatory cells in liver tissue, representative indicators CD3, CD68, etc. This item is suitable for complex cell types, compare the number of positive cells, and is often used in infiltrating inflammatory cells.

□Positive intensity, the average value of the color intensity of each positive spot (HALO): reflects the average intensity of the positive of the whole film or a certain area. Bright field is the average optical density (average optical density), fluorescence is the average intensity (average intensity), the larger the value, the stronger the positive. The applicability is wide. Because the positive area is not considered, in extreme cases, the data is larger when there are few positive spots and the color is dark, and the data is smaller when there are many positive spots but the color is light. It can be evaluated together with the positive area.

□Positive area ratio, positive area/to-be-tested area tissue area (IPP/HALO): It is less used, regardless of the impact of positive deep staining, and simply compares the proportion of positive area.

□MVD, the number of microvessels/field of view (IPP): CD31, CD34 microvessel density analysis, measuring the number of microvessels per unit area. This item is only used in the analysis of the image field of view, and not used in the full-scan analysis.

□0-12 score: the experimenter scores the staining degree (0-3 points) and the positive rate (0-4 points) of the immunohistochemical slices under the microscope or scanned documents respectively, and then multiplies them to obtain a comprehensive score (0-12) Minute). The details are as follows. The staining intensity is scored based on the staining characteristics of the target cells: 0 points for no staining, 1 point for light yellow, 2 points for brown, and 3 points for brown, as shown in the figure below. Scoring is based on the positive rate of cells: 0 to 5%, 1 to 6% to 25%, 2 to 26 to 50%, 3 to 51% to 75%, and 4 to >75%. The comprehensive score can be used to classify the positive level, which is convenient for statistics, and is often used in the analysis of cancer and adjacent survival.

□H-Score, 0-300 points (3D): Use the analysis software Quant center, which is provided with the 3D scanner, to score the depth and number of positives. Only the brown-yellow color developed by DAB can be quantified. The larger the value, the stronger the positive. H-SCORE=∑(PI×I)=(percentage of cells of weak intensity ×1)+(percentage of cells of moderate intensity ×2)+percentage of cells of strong intensity ×3), where pi represents the number of positive cells It is the percentage of all cells in the slice; i represents the intensity of staining. Wide applicability, scan slices or chips can be used.

□Fluorescence multi-standard co-localization analysis (HALO): Measure the data of the entire film or the individual channels of multiple indicators in the specified area and the number and ratio of co-localized cells (or co-localization area and ratio, choose one).

Different cell spatial distance analysis (HALO): mainly used to study the tumor microenvironment, the distance relationship between inflammatory cells and tumor cells, etc.

□Analyze the number of cells marked by another color within the micron range around the cells marked by one color;

□Measure the distance between cells marked with different colors;

□Analyze the number of cells marked with different colors within a micrometer range (such as tumor necrosis area boundary, or CD31-labeled positive blood vessel) based on a certain location (tumor CD3, CD4, CD8 inflammatory cell infiltration analysis is more)

2. Pathological analysis [Price: 40 yuan/slice (one type of tissue on a slice is one sample)]

Observe the slices under a microscope or browse the scanned files on the computer, and describe the common pathological changes of the slices such as inflammation, necrosis, degeneration, hyperplasia, and fibrosis, and reflect the differences between the slices. Reports are roughly divided into four types.

□Pathological description: each slice provides a corresponding typical visual field picture, describes the pathological changes, and uses different color arrows to mark different typical lesion features in the word document. It is suitable for customers who have fewer groups and a small number of films in each group, and customers who do not need to score or have no scoring standards.

□ Pathology score: each slice is scored for different pathological aspects (the scoring criteria need to be provided in advance, some scores will need to provide corresponding special stained sections), and provide corresponding typical visual field pictures by group, describing the main pathological changes of the group, in the word document Different color arrows are used to mark different typical lesion features. It is suitable for customers who have more groups and more films in each group.

□ Pathological diagnosis: You need to fill in the sample delivery form, and provide relevant information such as experimental information, general pictures and other relevant information. Give specific judgment results. It is suitable for the characterization of animal tumors and poultry diseases.

□ Pathological examination report: It is necessary to fill in the sample delivery form, and provide the analysis plan and relevant information of the experiment, the general picture, and the overall cost will increase by 30%. The signing of formal contracts, confidentiality agreements, etc., from the beginning of the acquisition and embedding are completed by the company. As a result, a formal report, representative graphs, statistical results, conclusions, original results, etc. are issued, and signed by the doctor of veterinary pathology, animal pathologist, and attached Qualification certificate. Mainly applicable to toxicity safety assessment or CRO projects.

3. Morphometric analysis [price: unit price of each item is 20 yuan/sample (3-5 data according to different content); when measuring several parameters at the same time, the first item is 20 yuan/sample, and the other items are 10 yuan/sample 】

Measure the length, width, area, quantity and density of various structures in HE dyeing.

Intestine: □ small intestine villi height; □ small intestine villi width; □ small intestinal crypt depth; □ number of crypts per unit area; □ large intestinal gland depth; □ intestinal mucosal layer thickness; □ intestinal muscular layer thickness; □ total intestinal wall thickness;

Lung: □ small pulmonary artery lumen area; □ small pulmonary artery diameter; □ average thickness of small pulmonary artery wall thickness; □ number of alveoli per unit field of view NA; □ area of lung parenchyma per unit field of view PA; □ alveoli per unit field of view Interval number NS; □Bronchial base circumference Pbm; □Bronchial lumen area; □Bronchial wall area WAt; □Bronchi inner wall area WAi; □Bronchial smooth muscle area WAm.

Ovary: □Ovarian area; □Number of primary follicles; □Number of secondary follicles; □Number of antral follicles; □Number of mature follicles; □Number of corpus luteum; □Number of white bodies. The analysis is a count of the entire ovarian slice after scanning.

Skin: □ Number of hair follicles per unit area; □ Epidermal thickness; □ Dermal thickness; □ Damage depth; □ Damage width.

Blood vessels: □vascular area; □vascular diameter; □luminal area; □luminal diameter; □intimal hyperplasia area; □media thickness; □plaque area.

Brain: □ Number of pyramidal cells per unit area of hippocampus; □ Area of ventricle.

Testis: the length and diameter of the seminiferous tubules; the circumference of the seminiferous tubules; the area of the seminiferous tubules; the number of interstitial cells per unit of visual field.

Heart: □Transverse cardiomyocyte area; □Transverse cardiomyocyte diameter; □Transverse cardiomyocyte density; □Ventricular wall thickness; □Heart cavity area.

Kidney: □ glomerular area; □ number of cells in the glomerulus.

Muscle: □ Average area of transverse muscle fibers; □ Diameter of transverse muscle fibers; □ Density of transverse muscle fibers.

Fat: □ Average area of fat cells; □ Diameter of fat cells; □ Number of fat cells per unit area.

Special staining mainly analyzes the positive area and the proportion or the number of positive cells in the field of view

Masson: □ collagen fiber area/tissue area of the area to be tested (HALO/IPP); □ collagen fiber IOD value (IPP).

Sirius red: □Collagen fiber area/Tissue area of the test area (HALO/IPP); □The area ratio of type I to type III collagen (IPP) under polarized light.

Oil red O: □ lipid droplet area/tissue area of the area to be tested (HALO/IPP).

Toluidine blue (IPP): □ density of mast cells per unit field of view; □ diameter of myelin sheath in semi-thin sections; □ thickness of myelin sheath in semi-thin sections.

Trap (IPP): □ The number of osteoclasts per trabecular bone length.

PAS (IPP): □Number of goblet cells per unit length of villi; □PAS-positive area of glomerular matrix/glomerular area.

ATP (IPP): □ Number of type I and type II muscle fibers per unit area.

Golgi (IPP): □ The number of intersections between dendrites and concentric circles, the density of dendritic spine per unit length (1000 times) [Photograph: 60 yuan/3 fields of view include 200× and 1000× pictures; analysis: 60 yuan/3 fields of view ].

TTC (IPP): □ Positive area of infarct area/Tissue area of area to be tested

Aortic oil red (IPP): □Positive area of fatty plaque/area of blood vessel tissue.

Goldner (IPP) osteoporosis related: □total tissue area T.Ar; □trabecular bone area Tb.Ar; □trabecular bone circumference Tb.Pm; □trabecular bone area percentage Tb.Ar; □trabecular bone area Thickness Tb.Th; □Number of trabecular bones Tb.N; □Trabecular bone separation Tb.Sp.

Safranin Green (IPP):

Plant roots: □ root area; □ root diameter; □ outer cortex thickness; □ cortical cell thickness; □ endothelial cell thickness; □ central column area; □ number of vessels per unit area; □ percentage of xylem area in central column.

Plant leaves: □ leaf thickness; □ mesophyll thickness; □ stratum corneum thickness; □ upper epidermal cell thickness; □ epidermal cell thickness; □ fence tissue thickness; □ sponge tissue thickness; □ number of vesicle cells; □ number of veins per unit length □ Thickness of midrib center.

Plant fruits: □cuticle thickness; □epidermal thickness; □sub-epidermal thickness; □average area of epidermal cells; □average area of sub-epithelial cells; □length and width of epidermal cells; □length and width of sub-epithelial cells; □average area of pulp cells.

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