Multi-staining flaking method of cytopathology sample

A pathology and cell technology, applied in the field of cell pathology, can solve the problems of loss, false positive, conventional dye shedding, etc., and achieve the effect of improving accuracy and uniform distribution

Active Publication Date: 2020-12-22
王道祥
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 1) The dyes of conventional cytopathological staining methods will affect the binding of the first antibody or the first probe to the relevant target, so these stains need to be washed to better realize the immunohistochemical staining of biomarkers
2) After cleaning, a higher concentration of protein antibody or nucleic acid probe is often required for staining than conventional staining, which is likely to cause false positives and mislead clinical diagnosis, treatment and prognosis
With the continuous replacement and discarding of staining solution and washing solution, cell samples on pathological slides will fall off and be lost, affecting the accuracy of results
4) Even if routine staining does not need to be removed in individual cases, the process of immunohistochemical staining or chromogenic in situ hy

Method used

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  • Multi-staining flaking method of cytopathology sample

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Embodiment 1

[0059] Novel and Improved Immunochemical Protein Marker Staining Method for Cells and Diff Fast Cell Multiplex Staining

[0060] (All centrifugation is performed at 1500 r / min for 5 min, the supernatant is removed, and then PBS is used to prepare the suspension)

[0061] 1. 25ml of bladder cancer patient urine containing 4% paraformaldehyde cell fixative, centrifuged to pellet the cells, discarded the supernatant, then diluted and suspended in 1ml of PBS (pH7.4) buffer, placed in 10ml in plastic test tubes.

[0062] 2. Add 20 microliters of Triton-100 (to increase the permeability of the cell membrane, which is beneficial to the staining of the cytoplasm and nucleus).

[0063] 3. Heat the cell suspension at 92°C for 5 minutes for antigen retrieval.

[0064] 4. Add 100 microliters of Dual Endogenous Enzyme Block (Dako, Carpinteria, Ca) to remove endogenous peroxidase (peroxidase) and alkaline phosphatase (alkline phosphatase) activity in cells, and incubate for 5 minutes.

...

Embodiment 2

[0080] Based on the new and improved staining method of cell nucleic acid chromogenic in situ hybridization and multiple staining of Diff fast cells:

[0081] (All centrifugation is performed at 1500 r / min for 5 min, the supernatant is removed, and then PBS is used to prepare the suspension)

[0082] 1. 25ml of bladder cancer patient urine containing 4% paraformaldehyde cell fixative, centrifuged to pellet the cells, discarded the supernatant, then diluted and suspended in 1ml of PBS (pH7.4) buffer, placed in 10ml in plastic test tubes.

[0083] 2. Add 20 microliters of Triton-100 (to increase the permeability of the cell membrane, which is beneficial to the staining of the cytoplasm and nucleus).

[0084] 3. Purchase RNAscope® 2.5 HD Reagent Kit (ACD Bio, California, USA) immunohistochemical nucleic acid in situ hybridization kit, and perform nucleic acid marker staining according to the experimental procedures recommended by the manufacturer. Add the RNA nucleic acid probe...

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Abstract

The invention relates to a multi-staining flaking method of a cytopathology sample. Specifically, the invention relates to a multi-staining method for performing liquid-phase immunohistochemical staining and/or nucleic acid immune in-situ hybridization staining and conventional cytopathology staining on a cell sample. The invention also relates to isolated cells obtained by the method, pathological sections carrying the cells, and kits for use in the method.

Description

technical field [0001] The invention relates to a cell multi-staining film preparation method in the field of cytopathology. This method can simultaneously display detailed cell morphology characteristics and expression information of one or more specific biomarkers on a cell sample. The staining results are suitable for observation under an ordinary light microscope, and the expression of one or more specific biomarkers on the same cell can be simultaneously observed under the premise of meeting the requirements of pathologists for the diagnosis of cytopathological morphology. The present invention also relates to the cells obtained by the method and the pathological sections loaded with the cells. Background technique [0002] Cytopathology is mainly based on the abnormal conditions in cells to study the causes and pathogenesis of diseases, as well as the changes in the physiological functions of cells during the course of diseases, so as to propose the basis for diagnosi...

Claims

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Application Information

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IPC IPC(8): G01N1/30
CPCG01N1/30
Inventor 楚文江王剑
Owner 王道祥
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