Chapter 1: Fixation Flashcards
An example of an additive fixative is:
Picric Acid. The reaction with proteins is not entirely understood
When compared with tissue fixed in formalin, tissue fixed in zinc-formalin will show:
Superior nuclear detail. Zinc is also a less toxic additive to formalin than mercury.
Microscopic examination of an H&E stained section in formalin shows marked nuclear bubbling. One would most often see this artefact if:
The tissue has been incompletely fixed before processing.
Microscopic examination of an H&E stained section of GI shows marked absence of epithelium in otherwise normal tissue. This is likely resulted from:
Delayed fixation. GI needs to be instantly put in formalin to prevent post-mortem changes occuring.
A specimen of kidney must be shipped to another city for Immunoflourescence (IF) studies. Which fixative would you use?
Michel solution.
A certain project requires a fixative that contains acetic acid yet stabilizes erythrocytic membranes. Which fixative would you use?
Hollande solution. The cupric acetate in this solution stabilizes RBC membranes.
When the microwave oven is used for fixation, what is the most critical factor?
Temperature control.
To adequately remove the calcium from a specimen containing areas of microcalcifications, the tissue should be fixed in:
Hollande solution because it is acidic.
What fixative contains copper acetate?
Hollande solution which stabilized RBS membranes
A specimen is submitted with a note that it was fixed in formalin. Microscopic sections show marked RBC lysis. What happened?
The tissue was acidified with acetic acid.
Fixatives are classified as an additive because they:
Addition, or binding of the fixative to tissue proteins.
Kidney biopsy tissue has been fixed in phosphate buffered glutaraldehyde for 2 hours then placed in phosphate buffer solution. If a portion of this tissue is processed for light microscopy, sections would most likely show:
Nonspecific PAS staining which would lead to a false positive.
Uric acid crystals are preserved ONLY when tissue is fixed in:
Absolute (100%) alcohol.
Improper preservation of tissue will result if there is:
A delay in fixation
A good fixative will:
Protect tissue against alteration during subsequent processing.
The function of methanol in commercial formalin is to:
Prevent the polymerization of formaldehyde.
What does incomplete fixation look like?
Excessive cracks in tissue and smudging nucleii
In Electron Microscopy (EM), Zamboni fluid, glutaraldehyde and osmium tetroxide function as:
Fixative solutions
Tissue will remain unfixed if placed in:
Sodium borate.
Bouin solution is recommended for:
Tissue to be stained for the Feulgen reaction.
Formalin pigment can be removed by treatment with 10%
Ammonium hydroxide in 70% alcohol.
Stock neutralized formalin is prepared in the lab by storing the solution over a layer of calcium carbonate. The solution in this carboy will be:
Acidic.
Microscopic evaluation reveals a poorly stained H&E spleen section. This will be difficult to fix if the issue is involving:
Poor fixation.
To make a 10% formalin solution, how many mL of water should be added to 300 mL of 37%-40% formalin solution?
2700mL
One action of acetic acid is to:
Coagulate nucleoproteins and to lyse RBCs.
Aldehyde fixatives are used for EM preparations because:
They prepare cell infrastructure.
What is the main function of using a fixative containing potassium dichromate?
Will make tissue more receptive to eosin staining.
If mercuric chloride is used alone for fixation, what will be the main effect on the tissue?
Poor tissue penetration and excessive shrinkage.
Tissue stored for long periods of time in unbuffered formalin or in acetate formalin may show brown, crystalline pigment in stained section. To remove this pigment prior to staining, the sections need to be treated with:
Saturated alcohol picric acid.
For good fixation of tissue with osmium tetroxide for EM, it is recommended that the tissue segment be no larger than:
1mm.
Following fixation with Bouin solution, tissue should be washed with:
50%-70% alcohol.
The PTAH staining technique would require postfixation or mordanting if the tissue were originally fixed in:
Zenker solution.
Absolute ethanol is a poor choice for the fixation of:
Lipids because alcohol will dissolve the cells.
Which of the following fixatives may give false positive results in some carbohydrate techniques?
Glutaraldehyde. It will cause a false PAS reading.
It is necessary to adjust the pH of most formalin solutions because of the presence of:
Formic acid.
The rate of fixation varies with the fixative and also with the:
Temperature of the fixative solution.
Carnoy solution is recommended for the preservation of:
Nucleic acids.
What common fixation factor affects fixation for light microscopy the LEAST?
pH
Formalin pigment is generally created in tissues fixed in formalin when the pH
Falls below 6
Very bloody cytology smears are often treated with:
Clark solution because Clark lyses RBCs and can unobscure the cells
Glyoxal is one of the newer fixative which has the added advantage of:
Faster action time.
If a tissue section was fixed in a solution different from that required for a staining procedure, microscopic sections frequently can be stained anyway if they are:
Post fixed in the correct fixative prior to staining.
How can you identify “smudgy nucleii”
A lack of Chromatin pattern.
Fixation in Bouin solution is commonly used for what kind of tissue?
Endocrine tissues such as GI.
B-5 fixative contains
Mercuric chloride, sodium acetate and 37%-40% formaldehyde.
Pigment caused by mercury-containing fixatives can be removed by:
Iodine-sodium thiosulfate.
Which fixative has a mordanting effect on tissues:
Bouin solution for Trichrome if the tissue was not originally fixed in that solution.
What is mordanting?
A chemical that acts as a binding agent.
Tissue should be placed in a fixative immediately after removal from the body in order to:
Prevent decomposition due to enzymatic activity.
Calcium-formalin fixative is recommended for the BEST preservation and demonstration of:
Phospholipids. The calcium can prevent them from soaking up excess water.
For most fixatives, the volume of fixing fluid in relation to the volume of tissue should be:
15 to 20 times.
Ultrastructural preservation will be very poor following fixation in:
10% aqueous formalin.
Glyoxal is a
Dialdehyde
How are Zinc-formalin fixatives different than “normal” formalin?
Give poor ultrastructural preservation because Zinc is a coagulant.
Zamboni PAF refers to a fixative containing:
Buffered picric acid and formaldehyde, remember PAF= picric acid formaldehyde.
The preferred fixative when tissue is to be stained for the presence of simple fats is:
NBF because simple fats are always cut with frozen sections.
Fixation of cytology smears should occur within:
1-2 seconds to avoid an air drying artefact.
When osmium tetroxide is used as a fixative in histology, how does it do?
It interferes with staining
The breakdown of tissue due to enzyme activity is called:
Autolysis.
The fixative of choice for the demonstration of a gouty trophus is:
Absolute alcohol because water will melt the gout crystals.
A good fixative for routine use would be:
One that makes tissue more permeable to fluids so that all subsequent processes occur readilly.
When fixing tissue for EM with formaldehyde or gluteraldehyde, the preservation of ultrastructure does NOT depend on:
The type of tissue.
Formic acid present in premade formalin solutions might lead to:
Lead to the formation of black acid hematin (formalin pigment).
What chemicals make up Carnoy solution?
Chloroform, glacial acetic acid and absolute alcohol.
When using a nonimmunologic solution for chromaffin granules, it is necessary to fix the tissue in:
Primary chromate fixative.
What is the main advantage of liver tissue that is fixed with 2%-3% glutaraldehyde?
The ultrastructure is preserved.
What is ultrastructure?
The fine structures within a cell that can only be seen under the highest microscopic power.
A poor fixative is characterized by:
Slow tissue penetration.
Does Bouin solution contain absolute alcohol?
NO GIRL
What do coagulant fixatives do?
Change the spongework of proteins into a mesh-like network.
The breakdown of tissue by bacterial action is called:
Putrefaction.
When ultrastructure preservation is of the upmost importance, the fixative used should have a pH of:
7.2-7.4
A fixative component that produces a diffuse brownish black pigment is:
Mercuric chloride.
For the BEST preservation of staining properties during long term storage, tissues should be stored in:
70% ethanol for best IHC results.
Ethanol is useful as a fixative because it:
Preserved glycogen quite well.
To prevent the formation of formalin pigment in tissues, formalin should be:
Buffered
Which of the following fixatives should be used for the specimens that will NOT be processed for several days?
10% NBF
A biopsy that was placed in water by mistake is submitted to the lab. What will happen?
The cells will become swollen and will rupture.
Sections of a breast carcinoma were fixed in a saline solution in the microwave. Microscopic evaluation shows marked pyknotic overstained nuclei. What happened?
The solution temperature exceeded 68C.
What is pyknosis?
The irreversible condensation of Chromatins in nucleii.
An unknown pigment in a tissue section that can be bleached with a saturated alcoholic solution of picric acid is most likely:
Formalin pigment.
What is special about the formaldehyde in Helly solution?
It causes reduction of some chemicals in the solution, creating a dark turbid solution.
Formaldehyde solutions for routine cases are most commonly buffered by:
Monobasic and dibasic phosphates.
What is the most significant characteristic of Zamboni fixative?
It can be used for both light microscopy and EM.
The BEST fixative for blood smears would be:
Methanol
When used as a secondary fixative, osmium tetroxide should be:
Used under a chemical hood because of how quickly it vapors.
Which of the following fixatives is recommended for lipid IHC?
Calcium-formalin.
In the Cajal method for demonstrating astrocytes, sections of brain should be fixed in formalin containing:
Ammonium bromide.
Hollande solution is a modification of which of the following fixatives?
Bouin solution.
Tissue fixed in which of the following solutions must be posttreated for mercuric chloride pigment?
B-5
Acetone is recommended for the primary fixation of:
Brain tissue for the diagnosis of rabies
Fresh, unfixed tissue can be safely stored for a short time by:
Wrapping the tissue in saline soaked gauze and refrigerating it.
What is the main characterization of Bouin solution?
It mordants connective tissue stains and can stain delicate structures with ease.
The fixation of tissue by physical methods can be accomplished by:
Microwaves
Fixation in Canoy solution will result in:
Superior staining of amyloid tissue in the Congo red stain. Remember: Canoy=Congo.
The recommended fixative for tissue suspected of containing spirochetes is:
10% NBF because spirochetes stain with silver (Steiner).
Which of the following is frequently added to formalin solutions to help preserve immunoreactivity?
Zinc salts.
What is antigenicity?
The capacity of chemicals to bind to certain structures that have adaptive immunity. Basically how well a IHC would stain.
Which of the following fixatives is contraindicated (made useless) when silver stains are to be used for H Pylori?
They will not stain after treatment with glyoxal fixative.
A good fixative used for the preservation of some enzymes is:
Acetone.
A common reason for adding acetic acid to fixatives is to:
It is used to counteract cellular shrinkage.
Formaldehyde acts as a fixative by:
It crosslinks proteins by forming methylene bridges.
If it is known prior to fixation that a distinction must be made between collagen and muscle, the preferred fixative would be:
Bouin solution. This is commonly done with Trichrome stains (that’s the point of that stain).
Bouin fixation contradicts Feulgen stains because:
The nucleii would be excessively hydrolyzed so it is not recommended for DNA or RNA staining.
When using Bouin fixative, the shrinking effect produced by one component is balanced by the swelling effect of:
Acetic Acid.
10% formalin is equivalent to what percent paraformaldehyde solution?
Since 10% formalin is a 1:10 dilution of 37%-40% formaldehyde, it would be 4% paraformaldehyde.
To obtain the same nuclear detail in cytology preparations, what percentage concentration of isopropyl alcohol can be subbed out for the 95%?
80% would yield the same nuclear detail.
What fixative renders fat insoluble for subsequent processing?
Osmium tetroxide.
The presence of acetic acid in fixatives causes what to happen to tissue?
Marked RBC destruction.
Microscopic review of formalin fixed tissue demonstrates a fine, brown-black artefactual pigment. This could have been prevented by:
Preparing the solution just before use. This is formalin pigment and is caused by the pH below 6.0
When osmium tetroxide is used as a fixative for paraffin embedding, it:
Should be cut very thinly because osmium tetroxide always penetrates very poorly.
What fixative promotes shrinkage and penetrated poorly?
Mercuric chloride, mercury salts in general too.
The component in Bouin solution that causes shrinkage is:
Picric acid.
Which fixative would be the best one to use to preserve RBCs in tissue?
B-5 because it doesn’t contain acetic acid.
A tissue section reveals a dark brown microcrystalline pigment that is birefringent. How should this pigment be removed?
Picric acid. This pigment was most likely caused by the formalin being too acidic (<6).
Shrinkage and distortion of tissue is the worst following fixation in:
Absolute alcohol. This fixative should only be used with gout/ when non-aqueous fixative is needed.
Tissue to be stained by the Warthin-Starry technique should be fixed in:
Formalin ONLY.
When fat is to be preserved, the fixative of choice would be:
Formalin, so it can be used for a formalin section and it can be easily processed out later.
Picric acid was used solely for the fixation of liver tissue. What will microscopic examination show?
Hydrolyzing of nucleic acids so it cannot be used for examining DNA or RNA.
What does picric acid do to tissue?
Shrinks tissue, increases eosin uptake and leaves tissue soft.
The primary purpose of fixation is to:
Stabilize proteins.
Clark solution lyses RBCs because it contains:
Acetic acid. It is used in cytology smears because it can show the other cellular components without the RBCs getting in the way.
microscopic evaluation of a PAS stained section reveals nonspecific staining. This was caused by fixation in:
Glutaraldehyde. Since it is a dialdehyde, one aldehyde group is left “free” after the reaction and that can cause a false PAS positive.
The pathologist was sure that there were urate crystals present in the tissue prior to processing, but nothing shows up under the polarized scope. What happened?
The use of a water-based fixative destroyed the crystals.
Formaldehyde reacts primarily with what protein group?
NH2 and it forms crosslinks.
During embedding, a white deposit is noted on several tissues. The tissue started in zinc-formalin, was transferred to phosphate-buffered formalin was dehydrated with 65%, 95%, absolute and finally cleared with xylene. What is the cause of this white deposit?
Improper washing between the zinc-formalin and the phosphate NBF. The white substance is phosphate crystal buildup.
Electron micrographs of a tissue section reveals electron-lucent membranes. This most likely indicates that:
The specimen was not post-fixed in osmium tetroxide. The fat in the tissue was not properly fixed so they become electron dense.
What is Michel medium used for?
Transporting unfixed tissue, usually through mail *ex. kidneys).
Microscopic evaluation of H&E GI tissue shows marked absence of epithelium. What happened?
The tissue was not immediately put in formalin upon removal from the patient. Post-mortem changes have begun to occur.
A lab changed their primary fixative from NBF to an alcohol-based fixative to preserve antigens and a marked eosin uptake was noted. Why?
A different tissue isoelectric point has been established.
What is an isoelectric point?
The point at which a substance is electrically neutral, i.e. contains no electric charge.
Besides an absence of epithelium in GI tissue, what else can happen to the tissue during post-mortem changes?
Poor cellular detail of the lamina propria.
What is the lamina propria?
The thin band of tissue right next to the epithelium.
When sucrose is used to treat tissue for enzyme histochemistry, how should the solution be treated?
The solution should contain 30% sucrose and 1% gum acacia. It may be stored at 4C for several weeks.
Kidney tissue was submitted in Michel medium for IF studies. How should the tissue be fixed before studied?
It should be washed in 3 8 minute changes of Phosphate buffered saline containing 10% sucrose.
What is the osmolality of isotonic human tissue?
340 milliosmols. This is “normal” human tissue.
Marked distortion of architecture and pyknotic nuclei are noted on H&E stained kidney tissue. The tissue was fixed in a microwave with saline. How could this have been prevented?
Carefully controlling the temperature.
Paraffin tissue blocked fixed in Bouin solution were taken from storage after several years. There is no nuclear staining shown even though the original sections were stained just fine. How could this have been prevented?
The picric acid was not neutralized prior to processing so it remained in the tissue and degraded it over time.
Microscopic evaluation of formalin fixed tissue shows marked nuclear bubbling. How could this have been prevented?
Extending the length of fixation time. Nuclear bubbling is most commonly caused by incomplete fixation prior to processing.
During microscopic evaluation of an EM kidney print, it is noted that normal cellular morphology seems disrupted. What happened?
The tissue was put in osmium tetroxide for anything beyond 1-2 hours.
Is Zamboni PAF stable at room temperature?
Yes.
Why should the paraformaldehyde in Zamboni PAF solution be heated to 60C prior to adding the 1 M NaOH?
The paraformaldehyde needs to be slightly alkaline in order to dissociate it into formaldehyde.
Prior to processing, tissue fixed in glutaraldehyde for EM should be:
Postfixed in osmium tetroxide.
Poor fixation of EM specimens is often indicated by the appearence of:
Disrupted mitochondrial membranes.
Formalin pigment may be removed from microscopic sections by treating them with:
Potassium hydroxide in 70% alcohol.
Fixatives that tend to mask antigenic sites and hamper IHC localization of antigens contain:
Aldehydes.
Microscopic evaluation of H&E stained fallopian tube reveals muddy-appearing nuclei with no visible chromatin patterns. What happened?
Incomplete fixation.
In immuno-electron microscopy, osmium tetroxide should NOT be used because:
It has a harsh effect on the antigenicity of the sample.
The difference between Orth and Helly solutions is that ONLY Helly contains:
B-5 fluid.
A small biopsy is submitted with STAT orders for AFB and fungal stains. Which fixative should you use?
NBF.
Why can a microwave be used for fixation?
It creates instantaneous heat and that is a common physical fixation technique.
Unstained slides from B-5 fixed tissue should be treated with what before staining?
Iodine to remove the mercury, B-5 has mercuric chloride in it.
Microscopic evaluation of tissue reveals a brown pigment lying on top of the tissue. It was treated with:
1. Sodium Iodine thiosulfate
2. Potassium ferrocyanide and HCl
3. Saturated picric alcohol solution
Everything is still fuck. What happened?
The section were fixed in Zenker fluid and were not properly washed. The chromium trioxide will create an insoluble pigment in the tissue.
A possible rhabdomysarcoma has been fixed in 10% NBF. For the non-IHCs needed to confirm the tumor, microscopic sections should be:
Mordanted in another fixative before proceeding because the PTAH IHC for rhabdomysarcoma needs to be mordanted.
When osmium tetroxide is used on a kidney biopsy for ultrastructural studies, how long should it be fixed?
Should be one hour or less.
Helly fixative prepared 3 weeks ago is now discolored and turbid. Why?
The formaldehyde was added prematurely which breaks down the chromium and forms an insoluble pigment.
A stained microscope section shows marked lysis of RBCs and the presence of a brown crystalline pigment. Which fixative was used?
Acetic acid- formalin. The acetic acid will cause the lysing and the formalin will cause the pigment.
A small amount of white precipitate is found in the bottom of a lab’s stock 37-40% formaldehyde. What should be done with the stock?
The precipitate is most likely paraformaldehyde and should be disregarded, this is normal.
Why would acetone be used to fix tissue?
It demonstrates cell surface antigens by dissolving the surrounding fat and phospholipids.
A tissue block of hemorrhagic spleen is fixed for 24 hours in 6 month old formalin and a granular black pigment is seen on the surface of the tissue. Why?
The formalin had become acidic over that 6 months and wasn’t buffered properly.
If the tissue is fixed in formalin, postfixation is critical to perform the non-IHC special stain for which kind of tumor?
Rhabdomysarcoma. The PTAH stain will be used and it requires postfixation.
A gouty trophus is seen in a fresh tissue specimen. Which fixative would be appropriate?
Absolute alcohol.
To ensure adequate fixation, a 2 X 2 X 3 cm colon polyp should be placed in how much fixative?
240mL (20X the volume of the specimen).
What is the main characteristic of acetone as a fixative?
It overhardens tissue but preserves glycogen and surface antigens.
A wet tissue sample has been requested for EM. All available tissue has already been placed in Bouin fixative and stored in 70% alcohol. What would EM show?
It would show inadequately preserved ultrastructures. Bouin is not recommended for EM.
A surgical specimen is obtained from a patient with probable metastatic melanoma. What kinds of tests should the fixative be compatible with?
IHC. A S100, Melan-A or other melanoma stains will be ordered.
A section of tendon sheath has been fixed in 10% NBF for 24 hours, routinely processed, embedded and cut. The demonstration of urate crystals in this section are?
Gone. They will not be present due to improper fixation.
Clark solution differs from Carnoy solution because only Carnoy contains:
Carnoy contains chloroform and Clark does not.
A section of healthy adrenal gland is fixed in osmium tetroxide for 8 hours and is planned to be used as a control for chromaffin granules. When examined microscopically, there are no chromaffin granules present. What happened?
Osmium tetroxide will not preserve chromaffin granules, a Chromate fixative (Orth solution) should have been used instead.
If lipids are to be preserved, what kind of fixative MUST be used?
An aqueous fixative so that fat will not dissolve.
What is the relative fixation window for HER2 studies?
6-48 hours.
Tissues that undergo the most rapid autolysis are rich in:
Enzymes. Ex. liver, pancreas and brain.
PAP smears that have been fixed with a spray fixative are submitted to the lab. The smears are processed without further treatment. What will happen?
The nuclei will appear foggy and lack detail because the smear should have been placed in 95%.
Is gum sucrose used during glutaraldehyde fixation for EM?
No.
What is the best Ab for determining if a tissue has been overfixed?
Vimentin.
What is the recommended fixative for HER2 testing?
NBF.
Tissue antigens are irreversibly blocked by fixation in:
Gluteraldehyde.
Microscopic evaluation of a PAP smear shows impaired nuclear staining. What happened?
The smears were allowed to airdry. This will show a loss of chromatin patterns, nuclear swelling, eosinophillic cytoplasm and loss of cytoplasmic density.
Which fixative is BEST when used at an approximate neutrality?
NBF.
Relatively unnoted formalin pigment may be visualized if the slide is examined under what kind of scope?
A polarized microscope because formalin is birefringent.
What does birefringent mean?
A pigment that when hit with light, shows two different colors under refraction. Ex. Congo red under a polarized scope shows dual refraction of light and the colors seem to “split”.
The routine fixative has been changed, and the pathologists aren’t happy with the new staining. Why would they be upset?
The new fixation solution hasn’t been adjusted to account for the fixative binding sites in the tissue. The uptake of eosin in the tissue has probably been drastically changed.
Many aqueous fixatives penetrate poorly because of:
Fatty cell membranes which will impede the aqueous solution from passing through all the cells.
How has formalin pigment been classified by some authors?
Endogenous hematogenous. This is because formalin may occur intracellularly and is formed by the reaction with heme.
What is the primary purpose of fixation?
Stop enzymatic action. Enzymatic action causes autolysis and autolysis causes postmortem changes.
What solution contains: picric acid, formalin and acetic acid?
Bouin.
How much should the volume of fixative exceed the volume of the tissue?
15x-20x.
What fixative contains: formalin, potassium dichromate and mercuric chloride?
Helly solution.
Precipitate left in tissues have been fixed in solutions containing mercuric chloride may be removed by the immersion in:
Iodine. Ex. Lugol’s during the gram stain.
What percentage is commercial stock formaldehyde?
37-40%.
At what pH does the formalin pigment appear?
Anything less than 6.
How can the formalin pigment be removed from tissue?
Rinsing it in alcoholic picric acid.
What causes the putrefaction of tissue?
Bacterial action.
What tissue change will occur if acetic acid is used alone as a fixative?
Cellular swelling.
What is a good fixative for CNS tissue to be stained with silver or gold techniques?
Formalin ammonium bromide, especially with the Cajal method.
After fixing in Bouin solution, how is the excess picric acid removed?
50-70% alcohol.
Carnoy solution is prepared with acetic acid, alcohol and:
Chloroform.
The first and most important procedure in the preparation of a tissue for microscopic examination is the choice of:
The fixative.
Which fixative is the ideal one for fats?
Osmium tetroxide.
When alcohol is used as the primary fixative, one should expect:
Excessive tissue shrinkage.
Generally, an increase in the temperature of the fixation tissue does:
Increases the speed of fixation, but also increases the rate of autolysis.
Fixations containing chromium salts usually require:
Washing in water before being put in alcohol, or a precipitate with form.
What fixative will usually leave the tissue protein uncoagulated?
Osmium tetroxide, this is commonly done with fats.
To prevent the polymerization of formaldehyde, what is usually added to the stock solution?
Methyl alcohol.
What is another name for polymerized formaldehyde?
Paraformaldehyde.
Which group does the formaldehyde react with in protein chains to denature them?
The amino group.
Hollande solution is a modification of what fixative?
Bouin.
Potassium dichromate increases availability of what group for dinging dyes?
-NH2
What is the minimum recommended time for formalin fixation?
6-8 hours.
Methylene bridges are formed during the reaction of certain tissue groups with:
Formaldehyde. Methylene bridges= crosslinking.
What is the disadvantage of using osmium tetroxide as a fixative?
It penetrates very poorly so sections must be cut very thin.
What is the permissible exposure limit of formaldehyde?
0.75 ppm
Formaldehyde crosslinks proteins by reacting with which group?
Amino (NH2).
What fixative must be prepared under a hood because it will fix your nasal tissue?
Osmium tetroxide. It evaporates VERY easilly.
Good fixation is indicated on an EM of a section of a plasma cell if the:
The nuclear envelope shows regularly spaced envelopes. Mitochondria are very sensitive to the quality of fixation.
One advantage of primary osmium tetroxide fixation is that:
Lipids are rendered insoluble so they show up very clearly.
One advantage of primary aldehyde fixation is that:
There is much better penetration than with osmium tetroxide.
When used as a primary fixative for EM, Millonig formaldehyde:
Both light microscopy and EM will be possible.
When used as a primary fixative for EM, glutaraldehyde will:
Require that the specimen be removed after 24 hours or post mortem changes will start to occur.
The pure polymer of formaldehyde is known as:
Paraforaldehyde.
A solution of 4% paraformaldehyde is cloudy. Why is this?
The paraformaldehyde has not been totally depolymerized.
How does depolymerization of paraformaldehyde occur?
Depolymerization of paraformaldehyde occurs with the addition of an acid (usually NAOH) and heating the solution.
A specimen will not be considered adequately fixed if the nuclei show:
Nuclear bubbling.
How do you remove chromium pigment?
It can partially be removed by the addition of acid alcohol.
How do you remove mercury pigment?
It can be removed by the addition of Iodine and sodium thiosulfate.
How do you remove formalin pigment?
It can be removed by the addition of alcoholic picric acid and alkaline alcohol.
The fixative used in a lab changed from formalin to gloxal. It will be most likely be necessary to now:
Check for the correct staining for H Pylori.
H&E stained sections of liver show marked pyknotic, overstained nuclei. This can be prevented in the future by ensuring that the temperature will not exceed:
55C. The absolute maximum is 65C.
H&E stained sections show a brownish pigment deposit lying on top of the tissue. What happened?
This is formalin pigment, the formalin stock should be higher than a 6pH.
H&E stained sections show a brownish pigment lying on top of the tissue. How can this be removed?
This is formalin pigment, it can be removed with alcoholic picric acid.
Can a formalin pigment form in tissue if the pH is higher than 6?
Yes. It can form in the bloodiest areas of the tissue section even if the pH of the formalin stock is 7.
What will happen to tissue left in fixative for too long?
It can become overhardened.
When is Orth fixative recommended?
For pheochromocytomas when IHC is not to be performed.
Should a good fixative penetrate slowly?
No. It should penetrate quickly to avoid postmortem changes to the tissue.
What is the pigment that is closely related to formalin that occurs in the Plasmodium parasites?
Malarial pigment.
in IHC reactions using horseradish peroxidase, how is a solution of hydrogen peroxide in methanol used?
TO block endogenous peroxidase.
In IHC staining of formalin fixed tissue, how are heat-induced epitopes used?
They enhance primary staining.
Following IHC staining, both the positive control and the patient tissue show weak staining. What happened?
The epitope reterival was done incorrectly.
What is the protozoan that causes outbreaks of severe and relentless diarrhea among patients with AIDS?
Cryptosporidium muris.
Immunoperoxidase stained tissues show a reaction of RBCs and granulocytes with the chromogenic substrate. What happened?
The endogenous peroxidase was not correctly blocked.
“Mycoses” is used to describe what kind of disease?
The term is used to describe diseases caused by fungi.
Myelin contains protein, cholesterol, cerebrosides and what else?
Phospholipids.
How can Alipia felis (cat-scratch disease) be demonstrated?
With the Churukian modification of the Warthin-Starry technique.
Rickettsiae are related to which classification of organisms?
They are bacteria that do not posses the “normal” bacterial attributes.
What procedure would be the most suitable for demonstrating secretory granules in a carcinoid tumor?
The Grimelius procedure.
What would be the best preparation of a cytology specimen to perform multiple special stains?
Make a cell block.
Where in the body are the primary cells involved in immune response located (in the lymphoreticular system)?
The thymus, lymph nodes, spleen and bone marrow.
Fixation in a primary chromate fixative is essential if chromaffin substance is to be demonstrated by what stain?
Chromaffin can be demonstrated by the Schmorl technique.
What are the Chromaffin-cell tumors of the adrenal gland known as?
Pheochromocytomas.
A PAP stained smear only shows blue-green cytoplasm in all cells. No pink cytoplasm is noted. What happened?
The slide was overrinsed in 95% and the Eosin Y came out.
