Chapter 13: Enzyme Histochemistry Flashcards
What are enzymes?
Enzymes are proteins that catalyze reactions occurring in biological systems.
How does biological oxidation occur?
Biological oxidation occurs with the addition of oxygen, the loss of hydrogen or the loss of electrons.
A compound is reduced when what is removed?
A compound is reduced when there is a loss of oxygen, a gain of hydrogen or a gain of electrons.
The compound or chemical group in which a specific enzyme works is known as the:
Substrate.
Tissue for some enzyme demonstration should be fixed if possible because:
Considerable diffusion artefact may be seen on unfixed frozen sections for some enzymes, particularly those that are water soluble.
Tissue for enzyme studies has been fixed in cold calcium formalin. What would the best storage medium be?
After fixation, tissue may be stored in gum sucrose solutions at 4C for several weeks.
Enzymes that act on substrates be adding water belong to the group known as:
Hydrolases.
The optimum procedure for freezing muscle biopsies is:
Isopentane at -150C.
The supply of isopentane has been depleted, although liquid nitrogen is available, and a muscle biopsy needs to be frozen. In this case, what would be the best freezing procedure?
The muscle needs to be dusted with talc, then frozen with liquid nitrogen.
The most frequently used methods of demonstrating hydrolytic enzymes involve:
Simultaneous coupling.
When used on muscle biopsies, the alpha-napthyl acetate esterase stain demonstrates:
Motor end plates on muscle.
Paraffin sections may be used for what stain?
The napthol AS-D chloroacetate esterase stain is a specific esterase stain that can be used on paraffin sections.
The napthol AS-D chloroacetate esterase stain demonstrates:
Granulocytes.
The ATPase reaction involves:
Simultaneous coupling and metallic substitution.
The final colored product in the ATPase reaction is:
Cobalt sulfide.
With the ATPase stain, pH 9.4, on a normal muscle biopsy, the results would show:
Dark type IIA and IIB fibers and light type I fibers.
What does the acid phosphatase reaction demonstrate?
The alcohol residue of napthol AS-BI phosphate.
What does the acid phosphatase reaction on muscle demonstrate?
Lysosomes.
The end product of the alph-napthol acetate esterase, acid phosphatase and alkaline phosphatase is:
An azo dye.
The NADH diaphorase or reductase technique on muscle demonstrates:
Mitochondria, Z-band material and sarcoplasmic reticulum.
Microscopic evaluation of NADH diaphorase stains show marked precipitate on the section. This is most likely the result of:
The incubating solution was buffered to pH8.5, anything above 8.0 will cause this precipitate.
Succinic dehydrogenase procedures demonstrate:
Mitochondria only.
The absence of phosphorylase activity in muscle indicates:
McArdle disease.
The phosphorylase stained sections were not read for several days. What should we do?
They should be restained in dilute iodine.
Nitro blue tetrazolium functions as a:
A hydrogen acceptor.
The endomysium is a network of connective tissue surrounding:
Each individual muscle fiber.
Skeletal muscle can be identified by the presence of:
Peripheral nuclei.
Fibers that are high in oxidative enzymes are classified as:
Type I fibers.
Type II fibers would be expected to contain an abundance of:
Phosphorylase.
The stain most commonly used for the diagnosis of a glycogen storage disease affecting muscle is:
The PAS.
What is the most important property of enzymes?
They bind to the substrate.
What are four types of histochemical reactions for the demonstration of hydrolytic enzymes:
Simultaneous capture or coupling, Postincubation coupling, Self colored substrate and Intramolecular rearrangement.
What are the four factors that influence enzyme demonstration:
Nonoptimal substrate, Nonoptimal temperature, Inhibitors and Nonoptimal pH.
Microscopic evaluation of an acid phosphatase stain reveals too much pale methyl green counterstain. What happened?
It was mounted with synthetic resin.
Macroscopic evaluation of an NADH diaphorase stain reveals aggregates and crystals of diformazan or a diformazan-protein complex. What happened?
It was mounted with a synthetic resin.
By the time the pathologist was able to read the phosphorylase stained section, the stain had faded. What can you do to fix it?
It needs to be restained in the Gram iodine solution to restore the color.
Sections stained with the napthol AS-D chloroacetate esterase stain shows a brown, refractile, pigmentlike artefact scattered throughout the section. What happened?
The air dried slides were mounted without first being dipped in xylene.
ATPase stained sections are retrieved after storage for several years. Microscopic evaluation shows marked fading of the stain. This indicates that:
Normal fading with time has occurred.
What method stains elastic tissue brown?
Acid Orcein.
The pathologist has requested a Feulgen procedure on a lymph node. What will the staining sequence be fore this specimen?
HCl, Schiff, sulfurous acid and light green.
Fresh silver solutions were properly prepared and used in a Warthin-Starry stain. After completion, the positive control does NOT demonstrate spirochetes. What happened?
Improperly prepared developing solution.
The function of Bouin fluid in trichrome stains is that of a:
It is used as a mordant.
In order to protect the leprosy organism from extraction of the acid-fast component, sections are deparaffinized in xylene and:
Peanut oil.
What is the black pigment that is often found in lungs and hilar lymph nodes, cannot be bleached and cannot be identified by typical chemical reactions?
Anthracotic or carbon pigments.
What tissue component can be stained by the Weigert resourcin-fuchsin, Hard resourcin-fuchsin or orcein?
Elastic fibers.
What is the oxidizer in the Wilder procedure for Retic?
Phosphomolybdic acid.
What is the oxidizer used in the Gomori procedure for Retic?
Potassium permanganate.
What is the purpose of using nonmetallic forceps and chemically-cleaned glassware in silver techniques?
To prevent contamination of the silver solution.
What is an adipocyte?
A fat cell.
What stain does elastic fibers have an affinity for?
Orcein.
What is the most common basic auxochrome encountered in dye chemistry?
-NH2.
What is the acidic auxochrome of eosin?
-COOH.
What is a rhabdomysarcoma?
A malignant neoplasm of skeletal muscle.
If the mucoid capsule of the fungus is intact, which fungi can be differentially stained by using Muci?
Cryptococcus neoformans.
What substance can be demonstrated with hematoxylin without a mordant?
Copper.
ATPase stained sections are retrieved from storage after several years. Microscopic evaluation shows marked fading of the tissue. What happened?
ATPase stains normally fade over time.
How is protease used in IHC?
Protease is used for some enzyme retrieval methods.
What is the method of choice for demonstrating calcium phosphate?
The Von Kossa technique.
What is the Avidin-biotin complex (ABC) immunohistochemical procedure?
The ABC complex binds to biotin-labeled secondary Ab.
When no immunostaining of both the control and the patient tissue is seen, what happened?
The incorrect primary Ab was applied.
What is the main advantage of using DAB rather than AEC?
The DAB chromogen provides a permanent preparation.
What procedure can be used to demonstrate Giardia duodenalis (lablia)?
The Giemsa stain.
What procedure will demonstrate Borrelia burgdorferi?
The Steiner, because it is a spirochete.
An undifferentiated malignant neoplasm stains positive for leukocyte common Ag and negative for carcinoembryonic Ag and cytokeratin. Where is the origin of these malignant cells?
Lymphatic.
What is the major reason for performing a PAP over a direct or indirect conjugate method?
The PAP staining technique offers increased sensitivity of Ag detection.
There is very weak Alcian Blue staining in structures that are expected to be positive. What happened?
There was poor hydration during the deparrafinization process.
What is the tumor that is thought to be associated with Asbestos exposure?
Mesothelioma.
What IHC technique is mainly confined to the demonstration of immunoglobulin and complement in frozen sections of skin and renal biopsies?
Traditional direct technique.
What are the three methods used for heat-induced Ag retrieval?
A microwave oven, a pressure cooker or a 95-98C waterbath.
What are the proteins that form the thick and thin filaments of skeletal muscle fibers?
Actin and myosin.
A birefringent reaction product is produced following staining of calcium deposits with what stain?
The Alizarin Red S.
Neuritic plaques of Alzheimer’s disease consists of abnormal cell processed, often in close proximity to:
Amyloid.
Does the validation of a new Ab require the use of multiple chromogens?
No, it only requires one.
A trainee is having trouble counterstaining a GMS with light green. The counterstain is just not taking. What happened?
The light green must not be acidified.
The PAP counterstain smears look dirty and not optimal. What happened?
The alcohol used to rinse after the counterstains is dirty.
When the ABC complex detection system is used in IHC, the possible false positive staining is common in what kind of tissue?
Liver.
The diagnosis of Alzheimer’s disease is pased on the presence of neurofibrillary tangles and neuritic plaques in what neural tissue?
Cerebellum.
Silver nitrate is not available to perform a requested basement membrane stain. What is another stain that could be done instead?
The PAS could be done instead.
A trainee about to perform a Feulgen reaction reports that no Feulgen reagent can be found in the lab. What should they look for instead?
Schiff reagent.
What does the HMB-45 Ab demonstrate?
Melanin.
What should microscopic evaluation of a brain section stained with the Holzer technique show?
Blue to purple glial fibers.
Microscopic evaluation of an auramine-rhodamine stained control section shows minute, shiny orange structures against a dark background. What is this control positive for?
Mycobacterium tuberculosis.
What will the histochemical procedure for nonspecific esterase demonstrate?
The enzyme’s activity.
What should the collagen stain in a routine H&E?
Pink, with NO gray or brown.
In the coupling method for alkaline phosphatase, what will the napthol be coupled with?
Diazonium.
What stain best demonstrates pneumocystis jiroveci?
GMS>
A control section used in the GMS stain is known to contain Aspergillus. Microscopic evaluation reveals black, dense walls of the hyphae with pale gray internal structures and a green background. How should this result be considered?
Excellent.
If the PAS is negative, the pH 2.5 Alcian blue is positive and the colloidal iron is positive, what does that mean?
The patient tissue most likely contains acid mucopolysaccharides.
Why would acetic acid be added to hematoxylin right before routine H&E staining?
To improve the hematoxylin’s selectivity to chromatin.
What does the Schmorl technique depend on?
The reduction of ferric iron to ferrous iron.
What is the best tissue control type for the Carmine technique?
Liver, because the carmine technique is for glycogen.
A section stained with Fouchet reagent shows discreet emerald green globules. What does this indicate the presence of?
Bilirubin.
The microscopic QC of an H&E reveals lightly stained nuclei lacking sharp chromatin detail. What happened?
Poor tissue fixation.
DNA is NOT demonstrated on a microscopic section of a lymph node treated for 1 hour with 1 N HCl at 60C followed by staining with Schiff reagent for 1 hour. What happened?
There was excessive hydrolysis of the nuclei.
In an immunoperoxidase procedure, the primary Ab used is a mouse monoclonal anti-Leu-4. Which secondary Ab would be appropriate?
Goat anti-mouse.
In a Verhoeff van-Gieson stain, the tunica adventitia of large arteries stains similarly to what kind of tissue?
Since the tunica adventitia is the toughest part of the artery, it would stain similarly to collagen.
What is the difference between rotary and linear automatic stainers?
Rotary stainers permit different times in each compartment.
If you purchase an unlabeled monoclonal Ab to a specific Ag, what is the secondary Ab that is most commonly applied?
Rabbit anti-mouse.
Pyroninophilia indicates the presence of:
Ribonucleic acid.
What is an auxochrome?
The group present in dyes that is responsible for forming ionic bonds.
How is the simultaneous coupling technique used?
It is used in enzyme histochemistry.
What is the color index printed on the side of a stain bottle refer to?
Its standard identification number.
What term is defined as the staining reaction that depends upon ionization of both the dye and the material in which the dye is precipitated?
ADsorption.
The napthol AS-D chloroacetate esterase technique is useful in identifying what type of cells?
Granulocytes.
Microscopic evaluation of a Carnoy fixed control section of kidney known to contain amyloid fails to show green birefringence following staining with Congo red. What happened?
There was a staining problem because Carnoy fixative is the ideal fixative for Congo red staining.
Pure methyl green used at a slightly acidic pH is considered a specific stain for what?
DNA>
The Weil stain will demonstrate which structure of the eye?
The optic nerve.
What is the type of staining in which tissue soaks up dye and is completely penetrated by it?
ABsorption.
Microscopic evaluation of an H&E shows no pink staining. How would you go about isolating the problem?
The pH of the eosin should be checked. Non-acidic eosin will not stain tissue.
The alcian blue, pH 2.5 stained slides show consistent blue staining of the nuclei. How can this problem be fixed in the future?
The time in the Alcian blue solution needs to be decreased.
An insulinoma is a neoplasm that most commonly arises in what organ?
The beta cells of the pancreas because they make insulin.
A pathologist is complaining about the number of floater tissues on the H&E stained sections. What is the most likely cause of the contamination?
The automated “dip and dunk” stainer.
A pathologist notes several air bubbles trapped under the section. What should the microtomist be reminded of?
The flotation bath water should be a allowed to sit overnight before use.
What causes nuclear bubbling?
Incomplete fixation before processing, microwave drying of the slides or insufficient draining of slides before drying.
A pathologist is complaining that the Congo red stained sections are showing faint red instead of apple green birefringence. What happened?
The sections aren’t being cut at 10 microns.
A ribbon of paraffin embedded brain tissue is floated on the water bath. Small holes are noted in the first section and decrease in size in subsequent sections. What is the most appropriate action?
To cut and discard the sections until the holes disappear is the tissue isn’t too exhausted.
Successive cryostat sections catch on one area in the middle of the anti-roll plate. What is happening?
The anti-roll plate has been damaged.
What kind of knife should be used to section glycol methacrylate tissue?
Glass knives.
A microscopic section of liver tissue shows numerous irregular holes throughout the tissue. Why?
Poor sectioning technique, or too aggressive sectioning.
Periodic acid methenamine silver (PAMS) stained kidney sections reveal well impregnated basement membrane. However, within the individual glomeruli the membranes overlap and cannot be evaluated as separate structures. How can this be corrected?
By cutting future sections at 2 to 3 microns.
A tech is having trouble getting all paraffin embedded blocks to ribbon. Examination reveals that the tissue was embedded at 62C melting point paraffin and the room temp is 25C. What should correct the problem in the future?
Immediately ordering some lower melting point paraffin.
Undercalcified bone should be embedded in what medium?
Plastic.
During the sectioning of plastic blocks on an ultramicrotome, alternating thick and thin sections are observed. What’s wrong?
The blade is loose.
A research project requires extra large sections to be cut. What kind of microtome should be used?
A sliding microtome.
Glass knives are being made for cutting thin resin-embedded sections. How often should they be broken?
They should bebroken as needed because glass knives rapidly lose their edge.
