Podcast Beta
Questions and Answers
Match the fixative with the specimens it is preferred for:
Buffered Formalin = Bone Marrow Zinc Formalin Mixtures = Tooth Specimen Bouin’s Solution = Electron Microscopy
Which fixative is recommended for urgent biopsies?
After fixation, _____ proper will commence.
decalcification
Von Ebner’s fluid permits good nuclear staining.
Signup and view all the answers
What is the purpose of decalcification in histopathology?
Signup and view all the answers
Which tissues must undergo decalcification?
Signup and view all the answers
Decalcification is an optional process in histopathology.
Signup and view all the answers
The basic principle of decalcification is the removal of the substance that makes the bone hard, which is ________.
Signup and view all the answers
What are the criteria of a good decalcifying agent?
Signup and view all the answers
What is the pH of citric acid - citrate buffer solution?
Signup and view all the answers
Why are chelating agents preferred over acids for decalcification?
Signup and view all the answers
Neutral EDTA is very slow for routine purposes.
Signup and view all the answers
Physical tests for decalcification are considered (______/reliable) methods.
Signup and view all the answers
What is the purpose of decalcification in tissue processing?
Signup and view all the answers
What is used to neutralize the decalcifying agent to prevent damage to tissues?
Signup and view all the answers
Electrophoresis is done at room temperature.
Signup and view all the answers
During frozen section, the tissue is fresh and ________.
Signup and view all the answers
Match the ion exchange resin procedure steps:
Signup and view all the answers
What is the desired temperature maintained in the chamber where the microtome, the knife, the specimen, and the atmosphere are kept during frozen section procedures?
Signup and view all the answers
Which of the following freezing agents is generally used and the most rapid of the commonly available ones?
Signup and view all the answers
Freeze drying involves embedding the tissue block with paraffin wax or celloidin.
Signup and view all the answers
__________ is the most commonly used stain for rapid H&E staining in frozen sections.
Signup and view all the answers
Study Notes
Introduction to Decalcification
- Decalcification is a critical process in histopathology when dealing with bones and other calcified tissues.
- It involves the removal of calcium salts to prepare tissues for microscopic examination.
- Tissues that must undergo decalcification include bones, calcified tumors, teeth, and calcified heart valves.
Conventional Tissue Processing
- The conventional tissue processing sequence includes:
- Fixation
- Decalcification (optional)
- Dehydration
- Clearing
- Infiltration
- Embedding
- Trimming
- Sectioning
- Staining
- Mounting
- Labeling
Principle of Decalcification
- The basic principle of decalcification is the removal of calcium from bones and calcified tissues.
- Decalcification can be achieved using strong mineral acids, weak organic acids, or chelating agents.
Factors Affecting the Rate of Decalcification
- Concentration of the decalcifying agent: more concentrated solutions decalcify bone more rapidly.
- Size and consistency of the tissue: larger, denser tissues require longer decalcification periods.
- Temperature: increased temperature increases the rate of decalcification, but also increases the damaging effects of acids on tissue.
- Agitation: gentle agitation can increase the rate of decalcification.
Decalcification Procedure
- Selection of tissue: ideal thickness is 2-5 mm for dense bones and 4-6 mm for soft bones.
- Fixation: essential to fix tissues before decalcification to prevent tissue damage.
- Decalcification: use a decalcifying agent such as nitric acid, hydrochloric acid, or a chelating agent.
- Acid neutralization: necessary to remove excess acid and prevent tissue damage.
- Washing: essential to remove all acid residue from the tissue.
Criteria of a Good Decalcifying Agent
- Complete removal of calcium
- Absence of damage to tissue, cells, or fibers
- Non-impairment of subsequent staining techniques
- Reasonable speed of decalcification### Decalcification Methods
- Nitric acid solution:
- Requires 3 changes of 70%-90% ethanol to remove acid after decalcification
- Permits better nuclear staining than hydrochloric acid method
- Recommended for autopsy materials, bone marrow, cartilage, and tissue studies
- Hydrochloric acid:
- Inferior to nitric acid due to slower action and greater tissue distortion
- 1% solution with 70% alcohol may be used for surface decalcification of tissue blocks
- Trichloroacetic acid:
- Permits good nuclear staining
- Weak decalcifying agent, not suitable for dense tissues
- Suitable for small spicules of bone
- Sulfurous acid:
- Recommended for teeth and small pieces of bone
- Only suitable for minute pieces of bone
- Chromic acid (Flemming's fluid):
- Weak decalcifying agent, not suitable for dense tissues
- May be used as a fixative and decalcifying agent
- Weak organic acids:
- Acetic and formic acid (organic acids)
- Better suited for bone marrow, not as harsh as other acids
- Not used alone as decalcifying agents, but are components of Carnoy's and Bouin's fixatives
- Formic acid:
- Moderate-acting decalcifying agent
- Produces better nuclear staining with less tissue distortion
- Safer to handle than nitric acid or hydrochloric acid
- Citric acid - citrate buffer solution (pH 4.5):
- Permits excellent nuclear and cytoplasmic staining
- Too slow for routine purposes, but suitable for primary decalcification
- Acetic and formic acid (organic acids)
Chelation Method
- Chelating agents:
- Substances that combine with calcium ions and other salts to form weakly dissociated complexes
- Facilitate removal of calcium salt
- Preferred over acid if preservation of nuclear DNA is important or if histochemical methods for nucleic acids and enzyme activities are intended
- EDTA (ethylene diamine tetraacetic acid salt):
- Most common chelating agent in the market
- Combines with calcium, forming an insoluble non-ionized complex
- Works by capturing calcium ions from the surface of the apatite crystal, slowly reducing its size
- Solution should be changed every 3 days
- Neutral EDTA:
- Permits excellent staining results
- Produces minimal cell and tissue distortion
- Inactivate ALP (alkaline phosphatase), actively restored by addition of MgCl
Determining the End Point of Decalcification
- Chemical test procedure 1:
- Insert pipette into the discarded decalcifying solution
- Withdraw approximately 5 ml of the discarded solution and place it in a test tube
- Add approximately 10 ml of the ammonium hydroxide/ammonium oxalate working solution
- Mix well and let it sit overnight
- Turbidity or cloudiness indicates the presence of calcium
- Chemical test procedure 2:
- Take 5 ml of discarded decalcifying fluid
- Add a piece of litmus paper or use a pH meter with magnetic stirrer
- Add ammonium hydroxide drop by drop, shaking after each drop, until litmus paper indicators solution is neutral (pH 7)
- Add 5 ml of saturated ammonium oxalate and shake well
- Allow the solution to stand for 30 minutes
- Formation of white precipitate (calcium hydroxide) indicates a large quantity of calcium
- Over-decalcification:
- Spoils the staining of basophilic elements
- Causes maceration of the softer tissue elements
- Under-decalcification:
- When the tissue is allowed to stay in the decalcifying agent for a very short period of time
- Decalcification may be incomplete, interfering with the normal cutting of sections and staining of specimens
Methods to Check if Decalcification is Complete
- Physical test:
- Unreliable method
- Testing for the pliability of the specimen
- Require manipulation, bending, probing, or trimming of the specimen
- Bubble test:
- Acid reacts with calcium carbonate in bone to produce carbon dioxide seen as layers of bubbles at the surface of the bone during acid decalcification
- If successful, then it is decalcified
- Radiography (X-ray):
- Best method, particularly with large specimens (e.g., femoral heads)
- Not done for EDTA decalcifiers
- Downside: very expensive
Miscellaneous Topics
-
Surface decalcification:
- Method dealing with small unexpected deposits of calcium that may be encountered in paraffin blocks
- Neutralization with 10% HCl for approximately one hour
-
Acid neutralization:
- Chemical used in decalcification should be neutralized to prevent reaction with succeeding pathologic processes
- Accomplished by immersing the decalcified bone in either saturated lithium carbonate solution or 5-10% aqueous sodium bicarbonate solution
-
Tissue softeners:
- Perenyi's fluid
- Acts as both decalcifier and tissue softener
- Softens unduly hard tissues
-
Artifacts:
- Under-decalcification:
- Nuclear detail lost or severely compromised
- Inability to section (tissue is still hard)
- Over-decalcification:
- Disruption of cell membrane and cytologic properties
- Loss of glycogen
- Swelling of tissue, especially collagen### Ion Exchange Resin Procedure
- Under-decalcification:
-
Spread 1/2 inch-thick ion exchange resin over the bottom of a container
-
Place the specimen on top of the resin
-
Add a decalcifying agent 20-30 times the volume of the tissue
-
Allow the tissue to stay in the solution for 1-14 days
-
Measure the end point using physical or X-ray methods
Electrophoresis (Electrical Ionization)
Principle
- A process that attaches positively charged calcium ions to a negative electrode and removes them from the decalcifying solution
- Shortens the time required to 8 hours due to heat and electrolytic reaction
- Not done at room temperature (RT); uses a water bath (30-45°C)
- Decalcifying fluid used: a combination of 88% formic acid and HCl
Disadvantages
- Electrically decalcified tissues generally do not stain well
- Only a limited number of specimens can be processed at one time
Microwave Oven Decalcification
- Place hard tissues in a decalcifying agent in a microwave oven for intermittent periods
- Change the solution regularly until the end-point is reached
- Microwave irradiation speeds up the decalcification process significantly from days to hours
- Temperature restriction: 42-45°C
- Same microwave used in automated tissue processing technique
- Used in immunofluorescent and immunohistochemical staining, and some specialized silver stains
Frozen Section
Definition
- A specific type of biopsy procedure that allows for rapid diagnosis during surgery
Importance
- Necessary for rapid diagnosis of a pathologic process during surgery
- Important in cases where unexpected disease may be found that requires immediate diagnosis
- Allows the surgeon to decide what to do next
Methods
- Freezing microtome or cold knife procedure
- Cryostat/cryocut (cold microtome)
- Cryokwik (quick-freezing spray cans of fluorinated hydrocarbons)
Procedure
- Fresh tissue is frozen on a freezing microtome or cryostat
- Cutting of tissue
- Mounting frozen sections on a glass slide
- Fixed briefly
- Staining
Advantages
- Rapid pathologic diagnosis during surgery
- Diagnostic and research for enzyme histochemistry
- Diagnostic and research demonstration of soluble substances (lipids and carbohydrates)
Special Processing Techniques
Freeze Drying
- Fresh tissue is frozen using liquid nitrogen
- Desiccation (vacuum chamber)
- Infiltration
- Embedding with paraffin wax or celloidin
- End-product: paraffin-embedded block
Freeze Substitution
- Fresh tissue is frozen
- Fixation (Rossman's Formula, 1% Acetone)
- Dehydration (absolute alcohol)
- Embedding
- End-product: paraffin-embedded block
Staining Methods for Frozen Sections
- Rapid H&E Stain (most commonly used)
- Metachromatic Stain (temporary stain)
- Polychrome Stain (temporary stain)
Studying That Suits You
Use AI to generate personalized quizzes and flashcards to suit your learning preferences.
Related Documents
Description
This quiz covers Lesson 6 of Histopathologic & Cytologic Techniques, focusing on Special Processing Techniques Part 1 and 2, taught by Wendell Jeffrey G.Bayron. It deals with the effects of high concentrations and amounts of fluid on tissue processing.
