1. Numbering 2. Fixation 3. Dehydration 4. Clearing 5. Infiltration 6. Embedding 7. Trimming 8. Section-Cutting 9. Staining 10. Mounting 11. Labeling

HISTOPATH TISSUE PREPARATION Flashcards by Sharmaine Madjid

is a process whereby a
selected tissue specimen is immersed in a watch
glass containing isotonic salt solution, carefully
dissected or separated, and examined under the
microscope, either unstained by phase contrast or
bright field microscopy, or stained with differential dyes

teasing or dissociation

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Small pieces of tissue not more than one mm. in
diameter are placed in a microscopic slide and
forcibly compressed with another slide or with a
cover glass

squash preparation (crushing)

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Process of examining sections or sediments, whereby cellular materials are spread lightly over a slide by means of a wireloop or applicator or by making an apposition smear with another slide.

smear preparation

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w/ an applicator stick or a platinum loop,
the material is rapidly and gently applied in a direct or zigzag line throughout the slide

streaking

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kind of smear that should be avoided

too thin and too thick smear

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a selected portion of the material is
transferred to a clean slide and gently spread into a moderately thick film by teasing the mucous strands apart with an applicator stick.

spreading

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For fresh sputum ____

bronchial aspirates and thick
mucoid secretions

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done by placing a drop of secretions
or sediment upon one slide and facing it to another clean slide

pull-apart

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pull-apart is used for

Serous fluid, conc. Sputum, enzymatic lavage
samples from GIT and blood smears

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the surface of a freshly cut piece of tissue is brought into contact and pressed on to the surface of a clean glass slide, allowing the cells to be transferred directly to the slide for examination by phase contrast microscopy or stained for light microscopic study

touch preparation (impression smear)

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utilized when a rapid diagnosis of
the tissue is required

frozen section

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frozen section is used for

For lipid, carbohydrates and nervous tissue elements
For rapid pathologic diagnosis during surgery
For diagnostic and research enzyme histochemistry
For silver stains (neuropathology) immunofluorescent and immunohistochemical staining

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Commonly Used Methods of
Freezing

Liquid nitrogen
Isopentane cooled by liquid nitrogen
Carbon dioxide gas
Aerosol sprays

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adequate for freezing small
pieces of tissue except muscle

aerosol sprays

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used for histochemistry and during operative procedures

Liquid nitrogen

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disadvantage of liquid nitrogen

soft tissue is liable to crack due to the
rapid expansion of the ice w/in the tissue, producing ice crystals or freeze artifacts.

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Overcools urgent biopsy blocks, causing damage to both..

block and blade if sectioning is done at -70 C or
below

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Non-fatty unfixed tissues are sectioned well at
temperatures between

-10 C and -25 C

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TISSUE PROCESSING

Numbering
Fixation
Dehydration
Clearing
Infiltration
Embedding
Trimming
Section-Cutting
Staining
Mounting
Labeling

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Identify properly all the specimens received without the need of writing the patient’s name to the accompanying specimen tag

Numbering

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where is the details written in numbering

Entering the details of the specimen in alog book

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how to do numbering

In numbering, the specimen number is preceded by either S (surgical), A (autopsy) or C (cytology). The year is also indicated. Example: S98-7677

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After numbering the pathologist will describe the
_____ of the specimen. The MT will
write down the ____ at the back of the
request

gross description and description

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Specimen size for processing:

3x2x0.5cm (length) and 3-5mmthick

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the function of fixation

Preserve the tissue- stop all cellular activities Prevent breakdown of cellular elements (prevents postmortem decomposition (autolysis) and prevents putrefaction) Coagulate or precipitate protoplasmic substances

for conventional bright field light microscopy, we follow the following steps:

0.5 x 0.5 cm tissue is added to a small jar containing the fixative

adding tissue to a small jar containing fixative is to:

prevent autolysis terminate cell metabolism kill bacteria harden tissue

fixative used is usually

Formalin 10% solution

tissues are fixed:

to preserve cells and tissues constituents to prevent their degredation

with fixative's volume 20x greater

Bouin's Fixative / Formalin for 24 hrs

TWO basic mechanisms in fixation

additive fixation Non- additive fixation

chemical constituent of the fixative is taken in and becomes part of the tissue by forming cross-links or molecular complexes and giving stability to the protein

additive fixation

example of additive fixatives

Formalin, mercury and osmium tetroxide

fixation-fixing agent is not incorporated into the tissue, but alters the tissue composition and stabilizes the tissue by removing the bound water attached to H bonds of certain groups w/in the CHON molecule.

non-additive fixation

non-additive fixatives example

alcohol fixatives

FACTORS INVOLVED IN FIXATION

pH Temperature Thickness of section Osmolality Concentration Duration of Fixation

the pH in fixation

6-8

temperature in fixation

Surgical spe - room temp. tissue processors- 40 deg celsius electron microscopy & histochem - 0-4 deg celsius

Formalin at 60 deg celsius is for

rapid fixation of urgent biopsy

Formalin 100 deg celsius is for

Thickness of section for EM

1-2 mm^2

Thickness of section for LM

2cm^2 or not more than 0.4 cm

Thickness of section for Brain

10% buffered formalin for 2-3 weeks

osmolality for slightly hypertonic solution

around 400-450mOsm

osmolality of isotonic solutions

340 mOsm

concentration in fixation

Formaldehyde- 10% Glutaraldehyde- 3% Glutaraldehyde - 0.25% immunoelectron microscopy

duration of fixation for buffered formalin

2-6 hours

duration of fixation for EM

fixation 3 hrs and then placed in a buffer

PRACTICAL CONSIDERATION OF FIXATIVES

SPEED PENETRATION VOLUME DURATION

formalin penetration

1mm/hr

volume of fixatives

10-25 times that of the tissue

the maximum effectiveness for volume of fixatives

20x the tissue volume

duration for fixatives

uterus & intestinal tract take longer time than small or loosely textured such as biopsies and scrapings

Characteristics of good fixatives

Cheap Stable Safe to handle Kill cell quickly with minimal distortion Inhibit bacterial decomposition and autolysis Minimum shrinkage Penetrate rapidly and permit application of stains Isotonic, insoluble to hypotonic

FIXATIVE TYPES

Simple Compound

what are the simple fixatives

Aldehydes Metallic Fixatives Lead Fixatives

what are the Aldehyde Fixatives

Formaldehyde, Glutaraldehyde

what are the Metallic Fixatives

Mercuric Chloride, Chromate (potassium dichromate, chromic acid)

what are the Lead Fixatives

Picric, Acetic, Acetone, Alcohol, Osmic Acid

2 or more fixatives

Compound Fixative

Fixative according to action

Microanatomical Fixatives and Cytologic Fixatives

permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissues in question.

Microanatomical Fixatives

types of Microanatomical Fixatives

1. 10% formal saline 2. 10% neutral buffered formalin 3. Heidenhain 's Susa 4. Formal sublimate (formal corrosive) 5. Zenker 's solution 6. Zenker-formal (Kelly 's solution) 7. Bouin's solution 8. Brasil's solution

Are those (fixatives) that preserve specific parts and particular microscopic elements of the cell itself

Cytologic Fixatives

the types of cytologic fixatives

1. Nuclear 2. Cytoplasmic 3. Histochemical

Preserves nuclear structures

Nuclear Fixatives

Fluids in Nuclear Fixatives

Flemming’s Fluid Carnoy’s Fluid Bouin’s Fluid Newcomer’s Fluid Heidenheins Susa

are those that preserve cytoplasmic structures in particular

Cytoplasmic Fixatives

Fluids in Cytoplasmic Fixatives

Flemming's fluid without acetic acid Kelly's fluid Formalin with "post-chroming" Regaud 's fluid (Muller 's fluid) Orth 's fluid

are those that preserve the chemical constituents of cells and tissues.

Histochemical Fixatives

fluids in Histochemical Fixatives

Formal Saline 10% Absolute Ethyl Alcohol Acetone Newcomer's Fluid

Bouin's Fluid composition

Picric Acid - 75 mL Formalin - 25mL Glacial Acetic Acid - 5mL

- it has rapid and even penetration - fixed tissue gives brilliant staining with trichome methods - used to demonstrate glycogen - good for GIT biopsies

Bouin's Fluid

Involves thermal coagulation of tissue proteins for rapid diagnosis

Heat Fixation

Heat Fixation is used for..

For frozen tissue sections For bacteriological smears For nuclear and cytoplasmic detail

Heat Fixation destroys

RBC

Heat Fixation dissolves

starch and glycogen

Is the process of placing an already fixed tissue in a second fixative

secondary fixation

secondary fixation Is the process of placing an already fixed tissue in a second fixative in order to:

Facilitate & improve demonstration of particular subs. Make special staining techniques possible Ensure further and complete hardening and preservation of tissues Done before dehydration and on deparaffinized sections before staining (10% formalin/10% formol saline as primary fixative)

Is a Form of secondary fixation whereby a primarily fixed is placed in aqueous solution of 2.5-3% potassium dichromate for 24 hours to act as mordant for better staining effects and in cytologic preservation of tissues

POST-CHROMATIZATION

Removing excess fixative from the tissue after fixation

Washing Out

remove excess chromates, formalin, osmic acid

Tap Water

wash excess amount of picric acid

50 to 70% Alcohol

remove excess mercuric fixatives

Alcoholic Iodine

Factors Affecting Fixation

Size and thickness Presence of mucus Presence of fat Presence of blood- Cold Temp

larger tissue requires more fixatives and longer fixation time

Size and thickness

maybe washed with saline solution

presence of mucus

cut in thin section and fixed longer

presence of fat

flushed out with saline

presence of blood

inactivates enzymes

cold temp

FIXATION IS ENHANCED BY

Size and thickness of tissues agitation Moderate heat (37-56 C) accelerates fixation but hastens autolytic changes and enzyme destruction

Principles and precautions in handling and fixation of Specimen

Fix Autopsy ASAP Fix surgical specimen ASAP Proper labeling and Identification Tissues size not more than 5mm thick except lung edema (1-2cm thick) Volume 20x except for osmium tetroxide 5-10x For prolong fixation vol not less than 50-100x Hollow organs (stomach) shld be completely opened before fixing Lungs may be covered with several layers of gauze Human brain (2 weeks) may be suspended by acord tied under circle of willis to prevent flattening Water should not be used for glycogen-containing tissue

Works as a physical agent similar in mechanism to vacuum, oven and agitation to increase the movement of molecules and accelerate fixation.

MICROWAVE TECHNIQUE

MICROWAVE TECHNIQUE used to..

Used to accelerate staining, decalcification, immunohistochemistry and electron microscopy

the tissue in microwave technique..

Tissue is heated right thru the block in a very short time.

For antibodies demonstration Formalin-fixed and paraffin embedded sections may be used Prepared as Cryostat section and fixation limited to a few seconds in absolute methanol or acetone.

IMMUNOFLUORESCENCE AND IMMUNOPEROXIDASE TECHNIQUES

preserve the maximum enzyme activity at its localization

ENZYME HISTOCHEMISTRY

Fixed in 4% formaldehyde or formol saline

ENZYME HISTOCHEMISTRY

Fresh frozen cryostat sections may be fixed in..

acetone or formaldehyde and washed in distilled water prior to enzyme staining

primary fiaxatives in EM

Osmium tetroxide, glutaraldehyde and paraformaldehyde

Osmium tetroxide, glutaraldehyde and paraformaldehyde is performed at

For electron histochemistry and electron immunocytochemistry ____ is useful

karnovsky’s paraformaldehyde-glutaraldehyde is useful