Histotechnician: Fixation

Carnoy is best for:

*Cytology = removes RBCs so tumor cells can be seen. *Sentinal Lymph Node = make fat surrounding LN hard and clear so can be removed. *Amyloid = preserves amyloid *Nucleic acids = preserves nucleic acids *Glycogen = preserves glycogen

Bouins is good for (indications for use):

*GI Biopsies *Excellent nuclear and cytoplasmic staining

Hollande is best for:

*GI Biopsies *demonstrated granules of eosinophils and endocrine cells *decal *RBCs intact

Flemming chemical makeup

*osmium tetroxide *chromic acid *acetic acid

Flemming indications for use

*post fixation for EM (will show mitochondria and chromosomes) *fat

Properties of Osmium Tetroxide

*used mostly for EM (OT is the post fixative after zamboni in order to preserve lipids) *fixes and stains (black) lipids

Zinc salt indications for use:

-Preserves antigenicity (for IHC-no need for antigen retrieval) -great at fixing bone marrow and lymph node -DNA preserved better than formalin -Great nuclear staining (often used in place of Mercury)

What is the result of the addition of copper acetate to Hollande?

-RBCs will remain intact and not lysed -stabilizes eosinophil granules -but specimen will still be decalcified

What are the properties of zinc salts?

-best replacement for mercury -great nuclear staining -faster than formalin

Why is size/thickness important?

1. large tubes like colon need to be opened so the layers can be exposed to the fixative. Otherwise, improper fixation occurs 2. thickness of sections placed in the cassette need to be no greater than 3mm in order for fixatives to penetrate completely.

Safety for acetone

1000 ppm contact with skin = dermatitis Very flammable Narcotic in high concentrations

Flashpoint of glacial acetic acid

16.6 degrees celcius

osmium tetroxide requires specimen to be cut to _______ mm.

1mm because it penetrates poorly

Concentration of glutaraldehyde is

2-4%

When fixing in zenkers, do not exceed _______ hours

24

With Helly, do not exceed _________ hours in fixative

24

After fixation in picric acid, it is recommended to wash the tissue in _________.

50% alcohol + lithium carbonate -this is to remove the yellow color of picric acid and to remove excess picric acid -both would cause poor staining

B5 fixation should last no longer than _______ hours. beyond that brittle tissue will result

6

For 10% NBF, the time in fixative is recommended at?

6-8 hours in Formalin before processing begins

transport medium pH

7 - 7.2

Since Zamboni is used for EM, what is the best pH?

7.3

After fixation in B5 tissue must be placed in _________ for storage

70% alcohol

a good substitute (nuclear detail) for 95% alcohol would be _______% isopropanol or _______% methanol

80 isopropanol 100 methanol

Explain the refractive index in regards to fixation.

If air and the tissue had the same refractive index, then the tissue would be invisible. The further apart these numbers become, the more visible the elements become (think of increasing the contrast on a photo on Instagram). So fresh tissue, the elements are hard to see because the velocity of light through it is similar to that of air. After fixation, this number greatly increases which creates greater contrast.

When would you not want to use Carnoy?

If you want to see RBCS or if you want to see Acid Fast Bacteria (b/c carnoys is defattening and will make acid fast bacteria non acid fast)

When would you choose to not fix?

Immunofluorescence and enzyme studies (fixation stops enzymatic activity). You would do a frozen section on fresh tissue.

When you lay a ribbon out on the waterbath it immediately separates out. The problem is most likely?

Incomplete fixation.

Why is it so frequently used?

It is cheap, tolerant, and can be used with many different staining techniques.

Picric acid safety

It is explosive in its dry form and must always be saturated with 10% moisture. -for this reason keep it capped and add water if it appears too dry.

Why is penetration important in fixation?

It is important to note that fixatives penetrate at different rates. Penetration begins at the periphery and moves inward.

Why is this pigment undesirable?

It masks microorganisms and will reduce silver. It is also birefringent

What does it mean to render cell components insoluble? Which cell components are most effected?

It means that the fixative making the cell component stable so it cannot be changed during processing. Proteins are what are being denatured/stabilized during fixation. Other cell components, like carbs (glycogen) or lipids is preserved but not rendered insoluble, meaning processing can still effect/destroy it.

Does formaldehyde shrink or harden tissue?

It will harden the tissue. It will NOT shrink tissue. (but the subsequent alcohols and xylene will)

What are the safety concerns of formaldehyde?

Its a carcinogen *heavily monitored: any time the action level taken is above 0.5 ppm over two 8 hour periods, action must be taken.

Formaldehyde: A. Categorize it. B. In its 100% pure form it is a ________ C. The stock solution is _____________% formaldehyde D. 10% formalin is ____________% formaldehyde E. 10% formalin is made from _________ parts water and _________ parts stock solution.

A. Additive / Noncoagulant B. Gas C. 37-40% D. 3.7-4.0% E. 9 parts water and 1 part formaldehyde stock solution

Formalin penetrates ________A_________ and fixes ________B____________.

A. Fast B. Slow (this is because it is crosslinking proteins and building methylene bridges which takes much more TIME....why 6-8 hours is important).

Acetic Acid: A. Categorize it. B. Diluted it goes by the name of __________ C. Concentrated it is called __________ D. What it is used for: E. Safety

A. Nonadditive / coagulant for nucleoproteins B. Vinegar C. Glacial Acetic Acid D. +Preserving nuclei/DNA +Swells tissue (counteracts shrinking of other fixatives) +Lyses red blood cells E. *10ppm *can cause severe burns *store at room temp away from oxidizers *always add acid to water!

A. How does volume ratio affect fixation? B. What is the correct volume ratio?

A. Remember that most fixatives are additive (chemically added to the tissue), and therefore the amount of fixative you have is important. inadequate volume of fixative means fixation stops before the tissue is fully fixed. B. 15-20 times greater than the volume of the tissue.

A.What is considered the ideal temperature for fixation? B.What temperature should not be exceeded?

A. Room temperature is ideal B. Do not exceed 45 degrees Celsius (tissue morphology affected)

Mercuric Chloride Pigment A. color B. preventable? C. removable?

A. birefringent, crystalline brown pigment B. No C. Yes. Treat with iodine then sodium thiosulfate (removes the iodine)

B5 safety

MC = corrosive, nephritis, CNS damage, cradle to grave Formaldehyde = carcinogen

safety concerns with helly

MC = corrosive, nephritis, CNS damage, cradle to grave formaldehyde = carcinogen potassium dichromate = carcinogen

Safety concerns with Zenkers?

MC = corrosive, nephritis, cradle to grave, CNS damage PD = carcinogen, cradle to grave

Possible pigments for Haidenhains

MC and Formaldehyde

Safety concerns for Haidenhains

MC= corrosive, nephritis, CNS damage Form = carcinogen

Haidenhains fixative chemical makeup

Mercuric chloride trichloroacetic acid glacial acetic acid formaldehyde H2O

What do we add to stock formaldehyde and why?

Methanol! We add it because pure formaldehyde mixed with water (to create the stock form) has a tendency to polymerize. Methanol prevents that.

The best transport solution is ___________

Michels

Fixatives kill _____________ and _________.

Mold and bacteria = no putrefaction!

Bouins is both a fixative and a ____________ (think Massons)

Mordant

Could a pigment form when using Osmium Tetroxide?

No

B5 contraindications

No FS No silver stains

Will RBCs be preserved with Carnoy?

No it contains acetic acid which lyses them

Does formaldehyde fix lipids or carbohydrates?

No it only preserves them by stabilizing the surrounding proteins.

Is the chrome pigment removable?

No, not really. Small amounts can be removed with acid alcohol.

Is Haidenhains a tolerant fixative?

No, tissue should be left in it no longer than 24 hours. Then stored in 70% alcohol.

Is there a pigment associated with picric acid?

No. But the fixative is colored and that color must be removed before processing.

Explain noncoagulant fixatives.

Non coagulant fixatives are those that create a GEL-like network throughout the tissue. This gel is harder for reagents to penetrate than the mesh (coagulant) and is therefore less ideal .

Categorize Carnoy.

Nonadditive / Coagulant

What are the two compound fixatives of Potassium Dichromate?

Orth Muller

Marchi chemical makeup

Osmium Tetroxide Potassium Dichromate Potassium chlorate

Potassium Dichromate is categorized as what?

Additive Coagulant below pH 3.5 Non coagulant above pH 3.5

Zinc Salts are categorized as?

Additive Coagulant

Clarke fixative chemical makeup?

Alcohol Glacial acetic acid

The two zinc compound fixatives are?

Alcoholic zinc formalin Aqueous zinc formalin

What is the PEL and STEL for formaldehyde

PEL = 0.75 ppm (8 hours) STEL = 2 ppm (15 minutes)

What is the polymerized form of formaldehyde called?

Paraformaldehyde *it looks like white powder deposits in the solution

Why is temperature important in fixation?

An increase in temperature will increase the rate of fixation. BUT...it will also increase the rate of autolysis and can affect tissue morphology.

Why is tissue storage important?

Because tissue may be needed at a later date for further studies/slides.

B-5 indications for use

Bone Marrow Lymph Node great nuclear staining

What are the compound fixatives of picric acid

Bouins Alcoholic bouins (Gendre/Dubosque) Zamboni Hollande

Which tissues are rich in enzymes?

Brain, Liver, Pancreas

Muller is good for:

CNS tissue

What are the 6 Non aqueous fixatives?

Carnoy Clark Acetone Methanol Ethanol Acetic Acid

Additive fixatives work by?

Chemically adding themselves to the tissue; the tissue protein becomes chemically linked to the fixative molecule, making the protein insoluble. This linking causes the charge on the tissue protein to change, leaving either an excess of positive or an excess of negative charges (how tissue becomes either baso or acido-philic).

Safety of carnoy?

Chloroform = carcinogen repeated exposure to carnoy can cause damage to cns, eyes, liver, kidneys

Why is fixation choice important?

Choosing a fixative beyond Formalin becomes important when certain tissue elements need to be demonstrated (Zenkers for PTAH, alcohol for Urate crystals, Orths for chromaffin granules demonstrating pheochromocytosis). Use of another fixative destroys these elements.

Safety of potassium dichromate?

Chromium is a carcinogen It must be tracked Cradle to Grave

bloody cytology smears require this fixative

Clarke

Explain coagulant fixatives.

Coagulant fixatives are those that create a MESH-like network throughout the tissue when they fix. This mesh has small holes throughout and is easily penetrable by further reagents.

You should not use picric acid if you are staining for.....

DNA or RNA -they are left soluble

Potassium Dichromate should not be used if you are looking to demonstrate ____________.

DNA. It is dissolved by PD.

Glyoxal is a ______aldehyde

Di-

How is formaldehyde disposed?

Down the drain depending on state regulation

Modified Millonig: Indications for use:

Electron Microscopy **PH IS IMPORTANT for EM = pH 7.2 -7.4 this solution is isotonic which makes it great for EM.

List the non additive fixatives.

Ethanol Methanol Acetone

True or false, picric acid is tolerant?

False

Why is fixation such an important step?

Fixation is the only step that CANNOT be corrected or reversed. The finished tissue slide can therefore only be as good as this first step.

What are the compound fixatives of Osmium Tetroxide?

Flemming and Marchi

Which fixative pigments do you need to be cautious of when using Orth

Formalin and Potassium Dichromate

contraindications for use: Helly

Frozen section silver stains

Alcoholic formalin: -indications for use

Frozen sections -also used in processors to prevent phosphate salt precipitation

Acetic acid is not a fixative but rather an additive to fixatives. What is its concentrated form?

Glacial Acetic Acid

Disposal for Haidenhains?

Hazardous Waste

Disposal of Mercuric Chloride

Hazardous Waste

Will formaldehyde fixed tissue favor Hematoxylin or Eosin? Why?

Hematoxylin Formalin will attach itself to the amino group of the protein. This leaves an excess of negative charges on the tissue protein (because the other group left available to bind is the negatively charges COOH group). This excess negative charge will attract positively charged dyes like hematoxylin.

Hollande chemical makeup

Picric Acid formaldehyde acetic acid copper acetate

Bouins chemical makeup

Picric Acid (softens tissue and shrinks) Formaldehyde (hardens) acetic Acid (swells)

Chemical makeup of Zamboni

Picric acid (in H2O) paraformaldehyde

Alcoholic Bouins chemical makeup

Picric acid saturated in 95% alcohol stock formaldehyde acetic acid

Post-fixation

Post fixation is using a second fixative after a primary one has already been used. The best example of this is Bouins is used after formalin fixation when the tech is running a trichrome stain.

What fixation pigments should you look out for when using Muller

Potassium Dichromate

Muller chemical makeup:

Potassium Dichromate sodium sulfate water

Fixation works by altering which tissue element?

Proteins in the tissue are denatured (unraveled into a simpler form) and stabilized during fixation.

Between autolysis and putrefaction, which is preventable?

Putrefaction can be prevented all together. Autolysis is not preventable, and can only be stopped by fixation.

During physical fixation, heat is used to denature/fix the proteins much like the cooking of the whites of an egg (goopy and clear to hard and white). The temperature here is critical and must stay between 55-68 degrees C. What would you see if the temperature went above 68C?

Pyknotic, overstained nuclei = when heat causes the chromatin in the nuclei to fuse together into a small blue dot

Zenkers is chemically made up of

Stock: Mercuric Chloride Potassium Dichromate Sodium Sulfate distilled h2O working: stock + acetic acid

What is the chemical makeup of B-5 stock and working?

Stock: Mercuric Chloride Sodium Acetate (raises pH to prevent formalin pigment) H2O Working: Stock + formaldehyde

Helly chemical makeup:

Stock: Mercuric chloride Potassium dichromate sodium sulfate working: stock + formaldehyde (not a stable fixative)

Orth chemical makeup

Stock: Potassium Dichromate Sodium Sulfate water Working: Stock + Formaldehyde

Which group on the tissue protein reacts with formaldehyde?

The amino group

Which tissue element is typically lost with delayed fixation (autolysis), and when is this common?

The epithelium, and it is commonly found in autopsy because the tissue has been delayed from fixation.

What is the refractive index?

The ratio of the velocity of light through air to the velocity of light through a solid or liquid (tissue).

Dessication is defined as..... This is really only used for.....

The removal of water It is only used as air drying of touch preps for the Wright Stain.

How do fixatives effect the other cell components, meaning the nucleic acids, lipids, and carbohydrates. Which fixatives will fix lipids?

These components are only entrapped or preserved when the proteins are stabilized, but are not rendered insoluble themselves. This is why lipids and carbs are typically lost during processing, and why nuclear bubbling is thought to occur, because these components are never fully fixed The exception to this are the few fixatives that fix lipids, such as osmium tetroxide and chromic acid.

Non additive fixatives work by?

They do NOT add themselves to the tissue chemically. These fixatives are alcohols that disassociate the bound water molecules from the tissue protein, causing denaturation. *removing this water can cause shrinking and hardening of the tissue.

What is important to remember about compound fixatives?

They share the properties of their main fixative. So zenkers is intolerant because mercuric chloride is intolerant.

How do fixatives stabilize cell constituents? And why is this important?

This is done through the stabilizing and denaturing of the tissue proteins; it literally holds the cells in their life like shape. This is important because processing uses harsh reagents, by stabilizing the cell elements, you ensure they will be less effected during processing.

Why is it important that the fixative maintains the relationship between cells and extracellular structures?

This preserves the life like state of the tissue and prevents distortion through processing.

Explain how fixatives affect staining using the amino and carboxyl group of the tissue proteins.

Tissue proteins have a amino group (NH2) and a carboxyl group (COOH). Think of yourself as the protein, one of your arms is the positively charged amino group, the other is the negatively charged carboxyl group. If another person acts as your fixative molecule, and holds your (+) amino group hand, the only hand available to grab anything is your (-) carboxyl group. Since that hand in negatively charged, it will be attracted to a positively charged dye like hematoxylin. If the opposite happens, and the person (fixative) holds your negatively charged hand, these leaves your positively charged hand available. Since this hand is (+) charged, it will be attracted to negatively charged stains like Eosin.

How does fixation enhance staining?

Tissue that is fixed poorly will stain poorly. Fixatives alter the charges on tissue proteins. This results in an excess of either a (+) or (-) charges on the protein. Those charges will attract anionic (negatively charged) or catonic (positively charged) stains.

Fixatives are described as tolerant and intolerant, why?

Tolerant means tissue can be left/stored in the fixative. Intolerant means the tissue cannot be left/stored in that fixative because it will destroy the tissue.

looking at the tissue you notice the center of the tissue stained with eosin and the periphery with hematoxylin. What is the problem?

Too little time in fixative. The center of the tissue was never reached by the fixative

True or false, tissue fixed in mercuric chloride should be washed before processing and stored in 70% alcohol.

True

You notice a tissue sections touches both the top and bottom of the cassette, what can you assume will be a resulting problem?

Under processed tissue = mushy, poor nuclear staining

Osmium Tetroxide safety

Vapor will fix eyes and nasal mucosa PEL = 0.002ppm must be used under hood

Does Carnoy work quickly or slowly?

Very rapidly

So we know black acid hematin is preventable. Is it removable? How?

Yes alcoholic picric acid alkaline alcohol

Will a pigment form with Zenkers?

Yes Mercuric Chloride or Potassium Dichromate pigment

Does formaldehyde cause a pigment?

Yes, black acid hematin

Will paraformaldehyde form a pigment?

Yes, black acid hematin just like formaldehyde

Is the chrome pigment preventable?

Yes, by washing the tissue of the fixative prior to processing

Would there be a pigment formation with Bouins?

Yes, formaldehyde and the tissue must be washed after fixation because of the yellow color of picric acid.

Could Zamboni develop a pigment?

Yes, formalin pigment!

After using B5, will you need to decalcify?

Yes, it does not contain Acetic Acid, so RBCs remain intact

Will a pigment form with the Helly fixative?

Yes, mercuric chloride, formaldehyde, and potassium dichromate

Does Potassium Dichromate have a fixation pigment?

Yes, the chrome pigment. It is formed when the chrome pigment mixes with alcohol.

Glutaraldehyde: -does it have a pigment

Yes, the same as formaldehyde

Can using B5 result in a fixation pigment?

Yes- formaldehyde or Mercuric Chloride pigments

Is there a pigment associated with Mercuric Chloride

Yes.

If you choose a shorter protocol for processing, would this change how thick your tissue should be?

Yes. Shorter protocols call for thinner tissue sections because reagents have less time to penetrate.

Picric acid leaves tissue proteins receptive to ______ dyes

anionic /acid dyes (Eosin)

Mercuric Chloride: Positive properties/indications for use

best for nuclear staining preserves antigens leaves tissue receptive to dyes

Methanol is the best fixative for ___________ and ___________.

blood smears and touch preps

best fixative for phospholipids

calcium formalin

Disposal for acetone?

down drain

How should you dispose of zinc salts?

down drain

How should you dispose of picric acid?

down drain with lots of water

Glutaraldehyde: -indications for use:

electron microscopy (pH 7.2-7.4) preservation of ultrastructure of the cell

Paraformaldehyde -indications for use

electron microscopy (pH 7.2-7.4) Immunoflourescence

True /False Osmium Tetroxide is a tolerant fixative

false. OT is intolerant.

Formaldehyde penetrates ___________ and fixes ____________

fast slow (cross linking proteins/building methylene bridges)

Hollande does have a fixation pigment due to the addition of which chemical?

formaldehyde

What pigment could result from alcoholic zinc formalin?

formalin pigment

what pigment might arise with aqueous zinc formalin?

formalin pigment

Dubusque may develop this pigment

formalin pigment/black acid hematin

formaldehyde becomes acidic by interacting with atmospheric oxygen to produce _____________

formic acid

contraindications for use: Zenkers

frozen sections RBCs silver stains

avoid this fixative when staining for hpylori

glyoxal

Aqueous zinc formalin is good for:

great nuclear detail antigenicity preserved

Zenkers indications for use:

great nuclear staining needed for PTAH stain Will fix and decalcify small BONE needle biopsies

Picric acid is really good for (indications for use):

great nuclear staining preserves glycogen decalcifies small calcium deposits leaves tissue soft

disposal of zenkers?

hazardous waste

to prevent smudgy nuclei (no visible chromatin), you should ______________ time in fixative (p.26 of carson)

increase to the full amount of time.

Is Gendre intolerant or tolerant

intolerant

Is Potassium Dichromate intolerant or tolerant

intolerant

Is acetone tolerant or intolerant?

intolerant

Is mercuric chloride intolerant or tolerant

intolerant

is bouins intolerant or tolerant

intolerant fix for no more than 24 hours

Glutaraldehyde: -safety and PEL

irritant and sensitizer PEL = 0.05 ppm

bouins is not used to demonstrate DNA or RNA why?

it hydrolyzes nucleic acids

What are the safety concerns with mercuric chloride

its corrosive (no metal forceps) tracked CRADLE TO GRAVE Nephritis Poisonous to the CNS

safety precautions with ethanol

its toxic 1000 ppm

Acetone is a ____________.

ketone

Glyoxal: -contraindications

lyses RBC leeches iron will not show H-pylori if left in glyoxal, staining will be decreased

Ethanol must be denatured with ___________ or ___________ due to government regulations.

methanol or isopropanol

formaldehyde is a __________aldehyde

mono-

formalin ammonium bromide: -indications for use:

neuropathology--demonstration of astrocytes in the CAJAL method

Any fixation pigment with zinc salts?

no

Ethanol is categorized as......

non additive / coagulant

categorize methanol

non additive / coagulant

categorize acetic acid

non additive / coagulant for nucleoproteins

categorize acetone

nonadditive, coagulant

the best fixatives for ultrastructure preservation are ___________

noncoagulants

Alcoholic zinc formalin is good for:

nuclear detail antigenicity preserved **best as a post fixative

Helly: indications for use

nuclear staining RBC fixation (no acetic acid)

Fun fact about Clarke, it is the _____________ fixative

oldest

Why is osmolality important in fixation?

osmolality refers to the salt content of the solution (fixative). The solution can be hypo, hyper, or iso-tonic. This determines whether water moves in, out, or stays in balance between the inside and outside of the cell. This is important because fixatives that are hypertonic will cause the cells to shrink as water is drawn out of the cells. Hypotonic fixatives will cause the cells to swell as water in drawn into the cells. And isotonic solutions (saline/transporting solutions) will cause no movement of the water in the cells.

good preservation of tissue for light microscopy is least dependent on temp, pH, volume ratio, or penetration rate?

pH

The best pH of binding for formaldehyde is:

pH 7.5-8.0

Why is pH important in fixation?

pH is especially important in electron microscopy, and for this the pH should always be between 7.2-7.4 to preserve the ultrastructure of the cell. In formalin fixation, you want the pH to be neutral. If the pH <6 the formalin pigment will form.

Clarke is good for

paraffin embedding (routine)

Alcohlic Bouin has this safety concern

picric acid is explosive in its dry form formaldehyde is a carcinogen

safety of Bouins

picric acid is explosive in its dry form formaldehyde is a carcinogen

nuclear bubbling is a result of _______________. (p.26 of carson)

poor fixation specifically too little time in formalin

autolysis is a result of ______________.

poor fixation specifically too much time between interruption of the blood flow to placement in fixative

mercuric chloride penetrates _______ and _______ the tissue

poorly and shrinks

Gendre is best for:

preserving glycogen CNS tissue

Properties of methanol

rapid acting dehydrating = will shrink and harden the tissue INTOLERANT

Ethanol properties are

rapid acting dehydrating = will shrink and harden tissue INTOLERANT

Properties of acetone

rapid acting will shrink and harden the tissue will fix and dehydrate at the same time, decreasing processing time

disposal of alcohol

recycled

disposal of methanol

recycled

safety of acetic acid

severe burns 10 ppm

Glutaraldehyde penetrates _________ and fixes _________

slow and slow *2 hours for fixation. It penetrates slowly and overhardens if tissue is left for >2 hours.

PIcric acid leaves tissue _______ but also causes ________

soft but causes shrinkage (only second to ethanol)

Potassium Dichromate will both ______________ and _____________ the tissue.

soften and shrink

Fixation changes tissue elements from _______________ to ___________.

soluble (can be altered during processing) to insoluble (cannot be changed).

Picric acid is both a fixative and a ________

stain

What are the two zinc salts?

sulfate and chloride

Paraformaldehyde

the polymerized form of formaldehyde

Are zinc salts tolerant or intolerant

tolerant

Zamboni is best for

transporting tissue to be used for EM ***MUST then be post fixed with Osmium Tetroxide

B5 is a intolerant fixative, true or false?

true

true or false acetone preserves enzymes

true

True or false, zenkers both decalcifies as it fixes and lyses RBCs.

true, because it has acetic acid it will do both

zinc formalin fixatives are poor for cell _________

ultrastructure

Ethanol is best for ____________ and ___________.

urate crystals (dx gout) and glycogen

Very important if you fix in Osmium Tetroxide to gross the sections __________

very thin. < 3mm

After fixation in Hollande, the next step is....

washing out the fixative to prevent precipitation of the salts in the processor.

Non aqueous fixatives are chosen when?

when a fixative containing water would destroy the element you are demonstrating.

Safety of Methanol

will turn to formaldehyde in the liver cause blindness and death 200 ppm

With Helly, is decalcification needed?

yes (no acetic acid)

Alcoholic zinc formalin chemical makeup

zinc chloride isopropyl alcohol water formaldehyde

chemical makeup of aqueous zinc formalin?

zinc sulfate water formaldehyde

What are the safety concerns with zinc salts?

zinc sulfate = skin and respiratory irritation zinc chloride = severe burns, corrosive.

Once very important thing to remember after fixing in zinc salts?

zinc will form a precipitate in the processor, so you must follow fixation with 60% alcohol. -if the precipitate forms, then wash the processor with acetic acid.

Glutaraldehyde is a __________-aldehyde BUT only _________ aldehyde is involved in fixation.

di- only 1 aldehyde group participates in fixation

Why is acetic acid added to other fixatives?

-lyses RBCs -decalcifies -swells tissue -fixes nucleoproteins

Bouins contraindications for use:

-lyses red blood cells -Not good for EM -DNA left soluble -Removes iron

Mercuric Chloride: Negative properties

-no frozen sections because mercury inhibits freezing -no silver stains -will overharden and shrink tissue -safety

Potassium Dichromate indications for use:

-preserves mitochondria -chromium will fix small amount of fat -causes an increased affinity for Eosin

Formalin ammonium bromide: -chemical makeup

1. formaldehyde 2. distilled water 3. ammonium bromide

What are the common formalin solutions we use? these solutions share all the safety, pigments, and disposal properties of formaldehyde

1. 10% NBF 2. Modified Millonig 3. Alcoholic Formalin 4. Formalin Ammonium Bromide 5. 10% Formalin Saline 6. Acetate Formalin

What are the Aqueous Fixatives?

1. Acetic Acid 2. Formaldehyde 3. Glutaraldehyde 4. Glyoxal 5. Mercuric Chloride 6. Osmium Tetroxide 7. Picric Acid 8. Potassium Dichromate 9. Zinc Salts

What are the 2 postmortem activities fixation stops?

1. Autolysis = enzymatic attack. Enzymes present in the tissue will continue to metabolize even after the blood supply has stopped. Fixation stops them from carrying out their metabolism, which if allowed to continue would result in the break down of tissue. 2. Putrefaction = bacterial attack. Prevented by following antiseptic techniques.

What are the compound fixatives of Mercuric Chloride

1. B-5 2. Zenkers 3. Helly 4. Haidenhains

The 2 types of fixation?

1. Chemical fixation = uses a chemical solution to denature proteins, and render the tissue elements insoluble. 2. Physical fixation = Heat or Dessication (removal of water) is used to denature proteins, and render tissue elements insoluble.

What are the 4 actions of fixation?

1. Enzymes are rendered inactive 2. Bacteria and mold are killed 3. Tissue is made more receptive to dyes 4. Tissue elements are stabilized

List the noncoagulant fixatives

1. Formaldehyde 2. Osmium Tetroxide 3. Glutaraldehyde 4. Potassium Dichromate pH>3.5 5. Glyoxal

Alcoholic Formalin: -chemical makeup

1. Formaldehyde 2. distilled water 3. alcohol

What is the chemical makeup of modified millonig?

1. Formaldehyde 2. distilled water 3. monobasic sodium phosphate 4. sodium hydroxide

What is the chemical makeup of 10% NBF?

1. Formaldehyde (3.7-4%) 2. distilled water 3. Buffer salts: monobasic and dibasic sodium phosphate (buffered to pH 6)

What are the 5 Functions of Fixation?

1. Kill the tissue, stopping autolysis and putrefaction 2. maintain the relationship between cells and extracellular substances 3. Increase visibility by bringing out the difference in refractive index 4. Enhance staining 5. Render tissue elements insoluble

Name the additive fixatives

1. Mercuric Chloride 2. Potassium Dichromate 3. Zinc Salts 4. Picric Acid 5. Formaldehyde 6. Osmium Tetroxide 7. Glyoxal 8. Glutaraldehyde

List the coagulant fixatives

1. Mercuric Chloride 2. Potassium Dichromate pH < 3.5 3. Chromium Trioxide 4. Picric Acid 5. Zinc Salts 6. Alcohol 7. Methanol 8. Acetone *acetic acid is a coagulant for nucleoproteins & DNA

Glutaraldehyde: -contraindications for use

1. PAS stains 2. GMS stain *does not work with these because only one aldehyde group is involved in fixation, leaving another free to react with these stains. Both of these stains oxidize tissue elements to aldehydes to be demonstrated, therefore this fixative would result in a false positive.

What are the 9 factors that affect fixation?

1. Quantity of fixative /volume ratio 2. Time of fixation 3. Thickness/Size of tissue 4. Temperature of fixation 5. Penetration 6. Choice of Fixative 7. Tissue storage 8. pH of fixative 9. Osmolality

How does time affect fixation (two ways)?

1. The time between blood interruption and placing in fixative is important to prevent autolysis. Ideally, this would be immediate. 2. Time in fixative is important because it prevents distortion during subsequent processing steps. Poorly fixed tissue = poorly processed tissue = poorly stained tissue (smudgy nuclei p.119 of Freida)

Which circumstances will cause pigment formation?

1. When the pH drops below 6 2. When the volume ratio is incorrect (too little volume of fixative) 3. If tissue is too bloody (rare)

Define Fixation

A process of immersing tissue in fluid that will penetrate, harden, kill the tissue (stop autolysis and putrefaction), resulting in preservation of the structure and tissue elements in a life-like state.

So we know that fixation's main target are those tissue proteins and stabilizing them. So, what is an enzyme?

A protein! That is why fixation renders them inactive, because they are denatured just as the other tissue proteins are. This is important because enzymes are the cause of autolysis.

Acetone is best for diagnosis of: but is also used for:

Rabies Frozen sections that need to demonstrate surface antigens for IHC

Haidenhains: Indications for use

Routine Biopsies

What are indications for use for 10% NBF

Routine use

Glyoxal -indications for use

Routine- replaced Formalin in many labs because less toxic, fast, and can be used with many stains (including PAS)

Chemical fixatives are categorized as either ______a______ or _____b_______. And further categorized as either ______c________ or ____d_______.

a. Additive b. non-additive c. coagulant d. non-coagulant

this chemical in Hollande is responsible for decal

acetic acid

neutralized formalin is stored over calcium carbonate. it will gradually become ________. why?

acidic. because the solution has not been buffered

Glutaraldehyde: -categorized as:

additive (crosslinks proteins like formal) noncoagulant

Picric Acid is categorized as ___________ and ___________.

additive and coagulant

Osmium Tetroxide is categorized as....

additive, noncoagulant

Glutaraldehyde will break down when exposed to _______

air. keep in small vials under just before use

Methanol is an ____________.

alcohol

Carnoy chemical makeup

alcohol chloroform acetic acid

Do to the acids in this fixative, Haidenhains both fixes and _________.

decalcifies

Orth is the best fixative for:

demonstrating the chromaffin granules in the adrenal glands to diagnose pheochromocytosis.

Marchi indications for use

demonstration of degenerative myelin in CNS