Fixation Flashcards
Define autolysis
Cells self-destruct using their own enzymes released by lysosomes.
Define fixation
Stabilizing cell proteins by making them insoluble. This way, they resist changes, such as autolysis and putrefaction.
Tissue, properly fixed, is preserved in a life-like manner, has improved refractive contrasts, and is more receptive to dyes.
What is fixation artifact?
A substance or tissue alteration from an outside source.
What is a fixation pigment?
An artifact chemically produced by cell component/fixative interaction
Explain the difference between buffered and neutralized formalin.
Formalin can be neutralized (with calcium or magnesium carbonate), but it will still become acidic (pH 5-5.7) if buffering salts are not added. Buffering salts bring the pH to 7.2-7.4.
What is black acid hematin?
Formalin pigment. An artifactual pigment that forms when hemoglobin reacts with formic acid and the solution drops below a pH of 6.0.
Which is formaldehyde’s physical state? Gas, liquid or solid?
Gas (HCHO).
What speeds the process of dissolving formaldehyde gas molecules in water?
Buffering salts.
In what physical state is formaldehyde received in the laboratory?
Liquid (HO-CH₂-OH), a 37%-40% aqueous solution chemically called methylene hydrate.
What is the difference between formaldehyde and formalin?
Formaldehyde is a colorless gas. Formalin is an aqueous solution (technically methylene hydrate).
What percentage formaldehyde is actually in 10% formalin?
3.7% to 4.0%
If a commercial solution labeled 37%-40% formaldehyde is received in the lab, is it really formalin?
Yes. It is considered 100% formalin by histotechs. Pure formaldehyde is technically a gas.
What is the most commonly used fixative in the histology laboratory?
10% Neutral Buffered Formalin
What does NBF stand for?
Neutral Buffered Formalin
Is alcohol a fixative?
Yes alcohol is one of two primary nonaqueous fixatives. The other is Acetone.
What are the 3 types of alcohol used as fixatives?
Ethyl, methyl, and isopropyl
Which alcohol is drinking alcohol?
Ethyl
How does paraformaldehyde differ from formaldehyde?
Formalin polymerizes into paraformaldehyde if left untreated with methyl alcohol.
What is done to methylene hydrate (formalin) to slow down the polymerization into paraformaldehyde?
10% to 14% methanol is added to commercial 37%-40% formaldehyde (which is correctly called methylene hydrate).
Is paraformaldehyde a fixative?
Yes. It is used at about 4% and is preferred to formalin for EM due to the absence of methanol, which tends to distort cell components. Although, Carson states that Modified Millonig Fixative (made with 37%-40% commercial formaldehyde) can be used for both light and electron microscopy.
What is a methylene bridge?
A cross link between aldehydes and nitrogen or proteins. A methylene bridge occurs when collagen is tanned to make leather.
Why do we say formalin penetrates quickly but fixes slowly?
Because it can bind with proteins within 24 hours but takes longer for methylene bridges to form. This is what leads to inadverdent alcohol fixation on the tissue processor.
What does absolute mean when referring to alcohol?
Pure. Less than 1% water.
What is the name for pure acetic acid?
Glacial acetic acid.
Why do we call concentrated acetic acid glacial?
Because it freezes at 16.6°C
True or False-RBC’s are lysed by acetic acid.
True
What are the 2 primary nonaqueous fixatives?
Alcohol and acetone.
What happens if you combine alcohol and acetic acid?
You get the fixative called Clarke Fluid.
What is the chemical definition of conformation?
The arrangement (especially bonding) of atoms in molecules.
What is the chemical definition of denature?
To disrupt the conformation of biomolecules. For example: To denature a protein is to unravel its 3 dimensional shape by breaking hydrogen bonds.
What is cytolysis?
Osmotic lysis, or cytolysis, happens when a cell swells until it bursts due to excess water moving into the cell.
What is the difference between a coagulative fixative and a non-coagulative fixative?
Coagulant fixatives create a mesh-like network which aids penetration of subsequent reagents. (such as alcohol). Non coagulative fixatives create a gel that makes penetration more difficult. (such as formalin).
What is ischemic time?
The time from the interruption of the blood supply to the beginning of fixation.
Name the major factors affecting fixation in general.
Time (both ischemic and duration), temperature, size, volume ratio, penetration, and pH.
Explain how temperature affects fixation and why it can be complicated.
As with other chemical processes, the rate of fixation increases as temperature increases and decreases as temperature falls. Choosing between cold fixation, room temperature fixation, and fixation involving heat can be a difficult decision because, of course, temperature affects autolysis in the same way.
What is the preferred volume ratio of tissue to fixative?
The fixative volume should be at least 15 to 20 times greater than the tissue volume.
How does denaturation work?
Proteins are highly ordered structures. Denaturing disrupts that structure by breaking hydrogen bonds.
What is the difference between additive and non-additive fixation.
In additive fixation, denaturation occurs when a fixative binds or adds to the proteins in order to make them insoluble. Non-additive fixatives do not combine with tissue proteins.
Which fixatives are included in the 3 As mnemonic? What do they have in common?
Alcohol, Acetone, and Acids. They are NON-additive.
What is the difference between hypertonic and hypotonic?
In aqueous solutions, HYPERtonic has more solute (water will flow into it to equalize), and HYPOtonic has less solute (water will flow out of it to equalize) In an isotonic solution, water is at equilibrium.
What are other names for acetic acid?
Glacial, if it’s pure. Vinegar, if it’s 3% to 5%.
What effect does acetic acid have on tissue?
Because it is an acid, it increases protein swelling, especially collagen. Lyses RBC’s. Does not fix carbohydrates or lipids. It is a coagulant, nonadditive fixative of nucleic acids.
The two primary nonaqeous fixatives are alcohol and acetone. How are they used in the anatomic pathology laboratory?
As coagulant, non-additive fixatives, they are mainly used in cytology. They are not used alone in histology as they overharden and shrink tissue. The exception to this is when processing urate crystals. They do preserve glycogen, and they preserve enzymes if cold.
What is gluteraldehyde?
The primary electron microscopy fixative. It is additive and should be stored cold. It has 2 aldehyde groups and can cross link more than formaldehyde. However, the free aldehyde groups will give false-positive results with Schiff’s reactions and cause non specific binding of antibodies.
What do Mercuric chloride and potassium dichromate have in common?
They are both metal compounds that leave tissue more eosinophilic, neither is used alone as a fixative, and they both produce artifactual pigments. Together, they are the stock solution for both Helly and Zenker fixative.
What 3 main fixative ingredients cause artifactual pigments?
Formaldehyde, Mercuric Chloride, and Potassium Dichromate. Formaldehyde and Mercuric Chloride will both polarize light.
Of the three artifactual pigment producing fixative ingredients, which can be used alone?
Formaldehyde, used as various percentages of formalin.
What do we mean by zinc salts, and how are they used?
Zinc sulfate or zinc chloride can be combined with formaldehyde to make a fixative that gives good morphology and preserves immunoreactivity.
What is the disadvantage to zinc formalin?
Precipitate can form in tissue processors and patient tissues. Precipitate in the tissue processor can be removed with dilute acetic acid.
What is picric acid?
It is an additive fixative ingredient. It fixes glycogen well. It is a strong enough acid to decalcify small calcium deposits. It must be washed out of tissue before processing and causes almost as much shrinkage as alcohol. It is highly explosive and has been replaced in most labs.
What tissue is fixed in 20% formalin?
Whole brains.
What is osmium tetroxide used for?
It is primarily used in electron microscopy labs due to its excellent preservation of ultrastructure. But it can be used to demonstrate fat in paraffin sections. It will stain tissue black.
What is B-5?
A fixative combination of Mercuric Chloride and Formaldehyde. It has traditionally been used to fix bone marrow biopsies.
What is Bouin Solution?
A combination of Picric acid, formaldehyde, and acetic acid that is preferred for GI biopsies due to nuclear preservation. It is used to postfix the trichrome stain. This is due to picric acid.
What is Hollande Solution?
A modification of Bouin Solution that adds copper acetate.
What is Gendre Solution?
Alcoholic Bouin Solution.
What should I know about Zenker and Helly solutions?
These are 2 fixatives that use the same stock solution composed of mercuric chloride, potassium dichromate, and water. Zenker adds acetic acid (mnemonic A to Z) and therefore lyses RBCs. Helly adds formaldehyde, making it the one that contains all 3 of the pigment forming fixatives. Neither is a good fixative for silver stains.
Which would autolyze fastest; part of a patient’s ear or a part of their pancreas?
The pancreas as it is enzyme-rich, and it is the enzymes that we want to inactivate with fixation.
Which would autolyze fastest; a placenta or a femur?
The placenta is more enzyme-rich and will begin autolysis within 15 minutes of the interruption of the blood supply. This is why the delivering physician does a quick examination of the placenta in the delivery room.
What is Davidson solution?
An aldehyde based fixative that contains ethanol and acetic acid. It can be substituted for Bouin Solution because it does not contain picric acid. It is used for eyes and testis. Tissue should be transferred to 70% alcohol for storage.
Find the artifactual pigment in this image and name it.
Black acid hematin, or formalin pigment, forms when a specimen containing hemoglobin is fixed in acidic formalin.
How is formalin pigment removed from tissue sections?
By treating with saturated, alcoholic picric acid or 10% ammonia in 70% alcohol.
What is fixation through perfusion?
Injecting fixative through normal body channels such as blood vessels. This is done so that internal structures may be fixed BEFORE dissection. Similar to embalming. Often done with lungs, eyes, and brains.
What is the difference between autolysis and putrefaction?
Autolysis is the enzymatic breakdown of tissue, and putrefaction is the bacterial breakdown of tissue. They both begin immediately after the interruption of the blood supply.
True or False: Because of the natural flora in your gut, putrefaction usually begins there.
True, it usually begins in the intestines and spreads from there. Autolysis also happens quickly in the stomach and small intestine due to the many digestive enzymes.
True or False: The liver is often the first organ to autolyze.
True, the liver is very enzymatic.
True or False: Acetone is a form of physical fixation.
False. Acetone is a chemical fixative. Heat and dessication are 2 physical means of fixation.
True or False: All chemical fixatives other than the 3 As (Alcohols, Acetone, or Acids) are additive fixatives.
True.
Does formalin fix quickly or slowly.
Slowly. Cross linking takes time.
True or False: If a cell is placed in a HYPERtonic solution, it will shrink.
True. The cell will plasmolyse.
Water will leave the cell to try to equalize.
What is plasmolysis?
The opposite of cytolysis. The protoplasm of the cell shrinks and the cell dies.
True or False: Picric Acid causes tissue to swell.
False: Picric Acid causes shrinkage and is often paired with acetic acid (which does cause swelling) for this reason.
True or False: The maximum saturation of formaldehyde gas bubbled into water is 40%.
True.
Why is alcoholic formalin a popular fixative?
It can prevent primary alcohol fixation, it adds a dehydration step, tissue can be stored in alcoholic formalin indefinitely, and it will not freeze when shipped.
True or False: Glyoxal is a type of alcohol.
False: Glyoxal is an aqueous fixative. It is an aldehyde and preserves tissue morphology similarly to NBF.
True or False: Zamboni solution is a dual purpose fixative. It can be used for both electron and light microscopy.
True. It is Buffered Picric Acid Formaldehyde which is made from paraformaldehyde.
Name 2 non-aqueous fixative mixtures.
Carnoy, Methacarn, and Clark
What are the ingredients in Carnoy solution?
Ethyl Alcohol, Chloroform, and Acetic Acid. Carnoy solution is used for the preservation of nucleic acids.
If I substitute Methanol for Ethanol in Carnoy solution, what do I get?
Methacarn.
Name a cause of nuclear bubbling.
Alcohol fixation or drying slides at a high temperature without draining them.
True or False: Microchatter is caused by poor microtomy.
False. Microchatter is a fixation and processing problem. It can be exacerbated by taking ribbons too quickly, but it is primarily caused by alcohol fixation.
True or False: Heavy metal fixatives work well with silver stains.
False.
How is Osmium Tetraoxide used in histology?
It the oldest fixative for EM. Its use as a fixative for FFPE is limited because it interferes with staining by acidic dyes. However, it does completely fix fat.
What 2 aldehyde fixatives are generally used in electron microscopy?
Paraformaldehyde and Glutaraldehyde.
Name 2 dual purpose fixatives.
Zamboni, Modified Millonig formalin, and gluteraldehyde are used in both light and electron microscopy.
True or False: Gluteraldehyde is not only used as a fixative in histology laboratories but also in tanning leather.
True. The tanning solution is technical grade and will not fix tissues adequately. Histology laboratories use an EM grade.
True or False: You can combine formaldehyde and gluteraldehyde to get faster penetration and faster fixation.
True. It is called Karnovsky’s fixative, and it is used in EM.
True or False: Osmium Tetroxide preserves lipids.
True.
