H&E Flashcards
What is the main function of Hematoxylin and Eosin (H & E) staining?
Differentiate acidophilic and basophilic tissue components and provide color contrast between basophilic nucleus and acidophilic cytoplasm.
What is Hematoxylin extracted from?
Logwood (Haematoxylon campechianum).
What happens to freshly cut Hematoxylin when exposed to atmospheric oxidation?
It becomes dark-reddish brown due to oxidation into hematein.
What is the oxidation product of Hematoxylin?
Hematein.
How must Hematoxylin be prepared before staining?
It must be oxidized to hematein.
What is the chemical formula for Hematoxylin?
C16H14O6.
What is the chemical formula for Hematein?
C16H12O6.
What is the process called that converts Hematoxylin to hematein?
Ripening.
What are the common methods of oxidizing Hematoxylin?
- Natural oxidation
- Chemical oxidation (e.g., using sodium iodate, mercuric oxide, potassium permanganate).
What role does the mordant play in Hematoxylin staining?
It enhances the affinity of hematein for tissue components.
What color does Hematoxylin stain basophilic structures?
Blue.
What color does Eosin stain eosinophilic tissues?
Red.
What effect does temperature have on the rate of staining?
Rate of staining increases with an increase in temperature.
What is pyknosis?
Irregular shrinkage or fragmentation of the nucleus.
What pH range is optimal for Eosin staining?
pH 4.6 to pH 5.0.
What are the two types of Hematoxylin staining techniques?
- Regressive staining
- Progressive staining.
What is the purpose of differentiation in staining?
To attack the linkage between tissue and mordant or mordant and dye.
What is the most widely used counterstain in routine tissue staining?
Eosin.
What does an increase in dye concentration do to staining intensity?
It increases the dye binding intensity.
What is the result of overoxidation of Hematoxylin?
Breakdown of the solution and loss of staining capability.
What is the role of aluminum in aluminum-mordanted Hematoxylin solutions?
It acts as a mordant to enhance nuclear staining.
What is the function of glycerol in staining solutions?
To moderate and slow further oxidation of hematein and preserve the stain.
What are the two states in which iron exists for staining?
- Ferrous (++)
- Ferric (+++).
What is the preferred method for preparing iron hematoxylin solutions?
Mixing mordant solution and hematoxylin solutions just prior to use.
What is Eosin Y derived from?
Fluorescein.
What is the significance of the isoelectric point (IEP) of cytoplasmic proteins in Eosin staining?
Staining must occur below pH 6.0 for a net positive charge on the protein.
What color shades can be demonstrated with Eosin staining at the proper pH?
- Three shades of pink
- Deepest red for RBCs.
- Collagen
- Cytoplasm.
What happens to the aluminum-hematein complex at pH less than 8.0?
It remains red and soluble.
What is the effect of using mercury fixatives on Eosin staining?
Tissues fixed with mercury fixative are more receptive to Eosin staining.
What pH range is optimal for staining with eosin?
pH 4.6 to pH 5.0
This range allows for the best demonstration of three shades of pink in staining.
What is the primary staining element that appears as the deepest red?
RBC’s
Red blood cells are the most eosinophilic elements in tissue sections.
What happens to eosin at pH 4.0 or below?
It converts to free acid that can attach nonspecifically, appearing muddy
This affects the staining quality negatively.
What is the role of a mordant in H & E staining?
Links the dye and tissue, forming a dye-lake
Common mordants include iron and aluminum.
List the three ways a mordant may be applied for H & E staining.
- Immediately to the tissue
- Added to the hematoxylin before use
- Incorporated into the stain when prepared
What is the first step in the staining process?
Cutting a ribbon of tissue and placing it on a slide
This is followed by drying the slide in an oven.
What are the two methods for performing H & E staining?
- Progressive
- Regressive
In progressive H & E staining, what is the advantage of using a milder form of hematoxylin?
It avoids over/under differentiation and results in consistent color
The nuclear material turns a deeper blue when rinsed.
What is a critical step in regressive H & E staining?
Differentiating in acid alcohol to remove excess hematoxylin
This step is essential for achieving the desired staining intensity.
What can cause poor H & E staining?
- Over-ripened hematoxylin
- Over-used hematoxylin
- Over-differentiation
- Insufficient differentiation
- Insufficient bluing
- Failure to wash bluing agent
What is the purpose of glacial acetic acid in Harris’ hematoxylin?
It sharpens the stain and suppresses background staining
This increases nuclear selectivity.
What is the oxidizing agent in Harris’ hematoxylin?
Sodium iodate
It has replaced mercuric oxide due to toxicity concerns.
What is the purpose of using chloral hydrate in Mayer’s hematoxylin?
It stabilizes the stain and prevents scum formation
Helps maintain the quality of the staining solution.
What is a unique characteristic of Gill’s hematoxylin?
It can stain mucin found in goblet cells
This is not possible with other hematoxylin solutions.
What is the role of glycerol in Ehrlich’s hematoxylin?
It acts as a stabilizer to prevent scum and precipitate formation
Ensures clarity and quality of staining.
What is Weigert’s hematoxylin primarily used for?
It is a nuclear stain that uses iron as the mordant
It resists decolorization in acid staining solutions.
Fill in the blank: The main mordants in H & E are ______ and ______.
iron, aluminum
What is Weigert’s stain primarily used for?
It is the most widely used nuclear stain that uses iron as the mordant
Weigert’s hematoxylin is favored for its reliability in staining nuclei.
What is the role of ferric chloride in Weigert’s hematoxylin?
It serves as:
* OXIDIZER
* MORDANT
What can be used as an alternative to hematoxylin in the H and E stain?
Celestine blue
A dye-mordant lake is formed when using celestine blue.
List the steps to achieve good H and E staining.
- Microscopically examine a control slide
- Do not allow sections to dry
- Keep solutions covered
- Ensure solutions cover slides completely
- Filter hematoxylin solution
- Follow schedule for changing solutions
- Avoid quick dehydration
- Post-treatment with ammonia water
- Prolonged staining for fixed tissues
- Adjust staining times based on fixative
- Rinse with distilled water if tap water is problematic
- Troubleshoot if staining issues arise
What is a common problem if nuclear staining is not crisp?
Inadequate/incomplete fixation
High temperatures during processing can also contribute to this issue.
What does pale nuclear staining indicate?
It may indicate:
* Inadequate exposure in hematoxylin solution
* Staining with overoxidized hematoxylin
* Exhausted hematoxylin
What might cause dark nuclear staining?
Prolonged exposure to hematoxylin
Tissue sections being cut too thick can also contribute to this issue.
What could lead to pale cytoplasmic staining?
Usually occurs when pH of eosin exceeds 5.0
Carryover from bluing agents may raise pH levels.
How can poorly differentiated eosin be identified?
Only 2 shades of pink are seen, with no contrast between RBCs and connective tissue
Properly differentiated eosin should yield 3 distinct shades of pink.
What should be monitored to avoid poor differentiation in eosin staining?
Ensure:
* Complete fixation
* Proper dehydration and clearing
* Proper pH of eosin
What is indicated by a blue/black precipitate on top of the slide?
Precipitate deposits on top of slides
This can be rectified by filtering hematoxylin daily prior to staining.
What artifact occurs when water and slides turn milky after being placed in water following alcohol during deparaffinization?
This indicates that xylene is present on slides as a result of poor clearing
This can be prevented by backing the slides through the stations and using fresh alcohols.
What should be done if hematoxylin is overoxidized?
Replace with fresh hematoxylin
Ensuring adequate exposure time in hematoxylin can restore nuclear staining.
True or False: The pH of eosin is critical for effective staining.
True
Fill in the blank: If staining problems cannot be identified, cut and stain tissue sections from the previous day with _______.
excellent staining
What is the recommended procedure for automated staining?
Check levels frequently, filter hematoxylin daily, rotate hematoxylin regularly based on volume of slides stained
What causes blue-black precipitate on top of slides?
Precipitate deposits from hematoxylin solutions
Filter hematoxylin daily prior to staining
What does it indicate when water and slides turn milky after alcohol during deparaffinization?
Presence of xylene on slides due to poor clearing
Remedy includes backing slides through fresh alcohols
What problem occurs if white spots are noted post deparaffinization?
Irregular or spotty staining results from improper drying or insufficient time in xylene
Remedy includes treating with absolute alcohol and re-treating with xylene
What causes pale nuclei in tissue sections?
Insufficient time in hematoxylin, over-oxidized hematoxylin, or poor fixation
Remedy includes restaining and possibly increasing time in hematoxylin
What are the causes of over-stained nuclei?
Sections remained in hematoxylin too long or cut too thick
Remedy includes decolourizing and restaining, or re-cutting the tissue
What does red or red-brown coloration in nuclei indicate?
Hematoxylin breakdown or insufficient bluing
Check oxidation status of hematoxylin and increase bluing step
What causes pale staining with eosin?
pH above 5 or excessive dehydration
Adjust pH to 4.6 to 5.0 and ensure bluing reagent is removed
What may cause overstained cytoplasm with poor differentiation?
Eosin too concentrated or overstaining duration too long
Dilute eosin and allow more time in dehydrating solutions
What is the remedy for blue-black precipitate observed on tissue sections?
Filter hematoxylin daily prior to staining slides
This prevents metallic sheen from developing
What causes water bubbles in stained sections?
Inadequate dehydration of tissue sections
Remove coverslip and re-dehydrate with absolute alcohol
What should be done if there is difficulty focusing on tissue areas?
Check for mounting media on top of coverslip
Remove coverslip and remount with a clean one
What causes mounting media to retract from the edge of the coverslip?
Warped coverslip or thinned mounting media
Apply a new coverslip with fresh mounting medium
What indicates that xylene was not completely removed during rehydration?
Milky appearance of water and slides
Change alcohols and dehydrate sections thoroughly
What can cause hazy or milky appearance in the last xylene rinse?
Incomplete removal of water from tissues
Change alcohol solutions and clear in fresh xylene
What causes stained sections to lack transparency and crispness?
Mounting media too thick
Remove coverslip and remount with fresh media
What causes hazy blue nuclei in tissue sections?
Excessive heat during processing or poor fixation
Heat should only be used during paraffin infiltration step
What is the cause of uneven H and E staining?
Water or fixative contamination in paraffin
Substitute toluene for xylene in high humidity areas
What results in poorly defined red-stained nuclei?
Under-oxidized hematoxylin
Use completely oxidized hematoxylin for staining
What causes an overall pink appearance in stained tissue?
Excessive adhesive on the slide
Re-cut and re-stain ensuring minimal adhesive use
What is indicated by dark basophilic staining of nuclei and cytoplasm?
Denaturation of macromolecules by laser or electrocautery
There is no remedy for this artifact
What indicates that a section was allowed to air dry before applying mounting media?
Brown stippling and glossy black nuclei
Rehydrate and remount properly without air drying
What is the Feulgen method used for?
Staining for DNA
It involves hydrolysis of DNA by HCl to generate aldehyde groups
What is a key technical note for the Feulgen reaction?
Hydrolysis time is fixative dependent
Bouin’s solution excessively hydrolyzes nuclei making them unsatisfactory
What can cause unexpected staining results in Schiff reagent?
Contamination or overaged reagent
Prepare fresh batch or decolourize using activated charcoal
What may cause tissues to stain unexpectedly weak in the Feulgen procedure?
Over exposure in acidic fixatives or hydrolysis issues
Avoid acidic decalcification and adjust hydrolysis time
What is the rapid H and E method for frozen sections?
A quick staining technique that takes approximately 4 minutes
It results in a permanent tissue slide
What must be done immediately after cutting frozen sections?
Fix sections immediately
Do not allow air-drying to preserve morphological details
