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