Tissue processing PDF

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Summary

Tissue processingMicroscopic examination- 4-6 μ tissue specimen o Size that permits the passage of the transmitted lightTissue processing- Treatment of tissue specimens to allow the paraffin wax embedding - Aim is to embed the tissue in a solid medium that is firm enough to support the tissue and gi...

Description

TISSUE PROCESSING (MIDTERMS)

Tissue processing Microscopic examination -

4-6 µ tissue specimen o Size that permits the passage of the transmitted light

Steps done fixation -

-

Tissue processing -

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Treatment of tissue specimens to allow the paraffin wax embedding Aim is to embed the tissue in a solid medium that is firm enough to support the tissue and give it sufficient rigidity to enable sections to be cut, which can be viewed under the microscope.

 Biopsy/ autopsy - source of tissue specimen  The specimen should be immersed immediately to prevent autolysis and bacterial putrefaction.  The tissue is preserves at its most natural form that can help in visualizing the structure and structural defects of the tissue.  Fixatives- solutions that are being used  Tissue processing can be manual or automatic Specimen that got to the lab should be examined for -

Relevant details Adequate size of the tissue specimen Labeling of the specimen bottle Fixative used

-

to

ensure

complete

Sufficient volume of fixative o 10:1 ratio fixative to tissue volume. Large tissue o Cut into smaller size tissues o Representative areas to be sent for processing  As paraffin wax is immiscible with water, tissues fixed in aqueous fixatives must be treated to remove water. This is achieved by various stages of tissue processing procedure used for soft tissue can be grouped under Fixation Dehydration Clearing Impregnation Embedding Sectioning Staining Mounting

Fixation -

First step and foundation Involve in series chemical events Makes tissue resistant to damage

Objective of fixation: -

To Preserve the living structure as closely as possible. To prevent autolysis of tissue To inhibit bacterial or fungal growth To make the tissueresistant to damage during subsequent stages of processing Page 1|6

TISSUE PROCESSING (MIDTERMS)

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To prevent alteration of tissue volume and distort any part of the tissue structure. To avoid interchanging specimen, a piece of paper with graphite pencil marking to be put in the tissue capsule  Formalin- most commonly used fixative  Increases the cross linking and results in the stabilization of proteins

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-

Other fixatives used: -

Glutaraldehyde Osmium tetroxide Chromic acid Methyl alcohol & ethyl alcohol Mercuric chloride Picric acid

Factors influencing the fixation/ fixation process.

Buffering

rate

Solutions used -

•

Removal of the dehydrant with a substance that will be miscible with the embedding medium. (Paraffin)

•

Paraffin and miscible.

•

So impregnation of tissue by paraffin is not possible unless alcohol is replaced by a fluid that is miscible with both alcohol & paraffin.

•

This process is called clearing

•

Xylene is one of the solutions that is miscible with both paraffin and alcohol

•

The term “clearing” comes from the fact that the clearing agents

of

Volume

Concentration Time Interval

Dehydration

Methyl & Ethyl alcohol Isopropyl alcohol Acetone

Clearing

Penetration

Temperature

Remove the water content from the tissue to allow penetration of paraffin wax. Done by passing through ascending grades of alcohol (to prevent sudden shrinkage of tissue) o E.g.- 50%, …..70%, 90% & 100% o 70% to 95% to 100% Acetone is very fast, but a fire hazard Dioxane can be used without clearing but has toxic fumes

alcohol

are

not

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TISSUE PROCESSING (MIDTERMS)

•

•

often have the same refractive index as proteins.

Toluene(But is 3 times more expensive than xylene)

As a result, when the tissue is completely infiltrated with the clearing agent, it becomes translucent/clear.

Benzene

The

presence of areas indicates dehydration.

CNP 30; Methyl Salicylate ( Rarely used because it is expensive but smells nice.) Oil of Wintergreen

opaque incomplete

Food oil derivatives Cedar wood oil

Ideal requirements solution

of

a

clearing

Clearing agents based on limolene (Volatile oil from citrus peels)

Speedy removal of alcohol Minimum toxicity

tissue

damage

& Impregnation

Cost factor Choice of a clearing agent depends on: The type processed

of

The type of undertaken

tissues

to

be

-

processing to be

Saturation of tissue cavities and cells Replaces xylene with paraffin by immersion in molten wax (60*C) Paraplastsontain plasticizers that makes the paraffin blocks easier to cut

The processor system to be used Processing conditions ( temp, vaccum & pressure) Safety factors Cost and convenience

•

Factors affecting impregnation -

Embedding/ blocking -

process by which tissues are surrounded by a medium such as agar, gelatin or wax.

-

Impregnated tissue transferred from wax bath to a mould filled with molten wax to get a block of Page 3|6

Reagents used Xylene (most commonly used) Chloroform (But is health hazard and is slow)

size and type of tissue clearing agent employed

TISSUE PROCESSING (MIDTERMS)

wax with the tissue specimen at the center with the cutting surface facing the base of the block Done using: -

Leukhart’s L shaped pieces Ice trays Paper boats Embedding cassette

quantify the presence of a specific compound. Commonly procedures

-

No air bubbles between the tissue and molten wax Wax filled in mold is allowed to cool Wax block is labeled for easy identification Wax hardened block removed from the mould and trimmed Wax block to be fixed on to a wooden/ metal block to prevent wax block from crumbling during sectioning.

Gramstaining

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Haematoxylinandeosin(H&E) staining

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Papanicolaoustaining(PAP)

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PASstaining

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Masson'strichromestaining

H and E stain/Haematoxylin and Eosin stain -

Most popular staining method

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Most widely used stain in medical diagnosis.

Sectioning -

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3-5 µ (cut of the embedded tissue done with microtome) Cut section is then to be float in the water bath for it helps to remove wrinkles and spread the specimen. Floated section is then picked up on an adhesive coated glass slide. Glass slide is then kept on a slide warmer at 58 Deg C temp for 15-20 min to ensure adhesion Egg albumin with additives commonly used adhesive

Staining -

Is a biochemical technique of adding a class- specific dye to a substrate (DNA, proteins, lipids, carbohydrates) to qualify or

staining

-

Remember: -

used

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The staining method involves application of the basic dye haematoxylin,which colors basophilic structures with bluepurple hue, and alcohol based acidic eosinY, which colors eosinophilic structures bright pink. Basophilic structure -

Usually the ones containing nucleic acids, such as the ribosomes and the chromatin- rich cell nucleus, and the cytoplasmatic regions rich in RNA.

Eosinophilic structures -

Generally composed of intracellular or extracellular protein. Most of the cytoplasmic is eosinophilic

RBC- stained intensely red. Page 4|6

TISSUE PROCESSING (MIDTERMS)

Hematoxylin

-

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Mounting/cover slipping

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Natural dye that is extracted from the heartwood (tree of hematoxylin campechianum) Dye id usually used in conjunction with a mordant which helps the stain to bind to the tissue.

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Clearing with xylene

The section on the slide is then covered with a thin glass coverslip to protect tissue from being scratched.

Mounting medium -

Is used to adhere the coverslip to the slide.

2 types of mounting media -

Water based mounting media Resinous mounting media

BPX and Canada balsam commonly used Eosin -

-

Second component of the H and E COUNTERSTAIN Red fluorescent dye resulting from the action of bromine on fluorescein Eosin Y- commonly used form of eosin. stain the cytoplasm, collagen and muscle fibers. Both water and ethanol soluble.

To mount a slide A. Apply drops of mounting medium upon tissue section. B. Hold coverslip at 45oallowing the drop to spread along the edge of the slip. C. Let go of slip and allow the medium to spread slowly. - Allow it to dry and section viewed under microscope

Procedure -

Manual Automated

Steps involved -

-

Removal of wax with xylene Rehydrating the tissue (bring section to water) o Descending grades of alcohol Staining H and E Dehydrate: ascending grade of alcohol Page 5|6

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