[M] Week 9 Decalcification, Dehydration, Clearing, Impregnation, and Embedding - Part 1 Flashcards
removal of calcium or lime salts are
removed from tissues (most especially bones and teeth) following fixation.
Decalcification
It enables the histotechnologist to cut soft sections of the bone using the microtome, so that they can be processed like any other soft tissue of the body.
Decalcification
should be done after fixation and before impregnation, to ensure and facilitate the normal cutting of sections and to prevent obscuring the microanatomic detail of such sections by bone dust and other cellular debris.
Decalcification
What are the characteristics of a good decalcifying agent
- Capable of removing calcium salts from tissues completely.
- Without producing considerable destruction or distortion of cells and tissue components.
- Without adversely affecting the staining capacity of the cell, particularly of the nucleus.
What are the agents used in decalcification
- Acids
- Chelating Agents
- Ion exchange resins
- Electrical ionization (electrophoresis)
- the most widely used agents for routine decalcification of large amounts of bony tissues.
- stable, readily available, and relatively inexpensive
Acid Decalcifying Agents
What are the Acid Decalcfying Agens
- Nitric acid
- Hydrochloric acid
- Formic acid
- Tricholoroacetic acid
- Sulfurous acid
- Chromic acid
- Citric acid
ACID DECALCIFYING AGENTS
- the most common and the fastest decalcifying agent.
- It is utilized both as a simple solution or combined with other reagents.
- Representative formulations include:
o Aqueous Nitric Acid Solution 10%
o Formol-Nitric Acid
o Perenyi’s Fluid
o Phloroglucin-Nitric Acid
Nitric acid
Nitric acid
Decalcification Time
- 12-24 Hours
- 2-7 Days
- 12-24 Hours
- 1-3 Days
- Aqueous Nitric Acid Solution 10%
- Perenyi’s Fluid
- Phloroglucin-Nitric Acid
- Formol-Nitric Acid
Nitric acid
Advantage: Rapid action. Recommended for urgent biopsy,and for needle and small biopsy specimen.
Disadvantage: It imparts a yellow
color with nitrous acid, thereby
impairing the staining reaction of the tissue
Aqueous Nitric Acid Solution 10%
Nitric acid
Advantage: Rapid action. Recommended for urgent biopsy.
Disadvantage: It imparts a yellow
color with nitrous acid. This may be prevented by neutralizing the tissue with 5% sodium sulfate and washing in running tap water for at least 12 hours
Formol-Nitric Acid
almost same with Aqueous Nitric
Acid Solution 10%
Nitric acid
Advantage: Recommended for routine purposes.
Disadvantage: Complete decalcification cannot be determined by chemical test.
Perenyi’s Fluid
Nitric acid
Advantage:
- Most rapid decalcifying agent.
* Recommended for urgent works.
Disadvantage:
* It imparts a yellow color (neutralized by 5% Sodium sulfate and water).
* Complete decalcification cannot be
determined by chemical test.
Phloroglucin-Nitric Acid
- inferior compared to nitric acid in its role as a decalcifying agent.
- It has slower action and greater distortion of tissue produced on the section calcified.
Representative formulations include:
o Von Ebner’s Fluid
Hydrochloric acid
Hydrochloric acid
Advantage:
- Moderately rapid decalcifying agent
- Recommended for teeth and small pieces of bone
- Does not require washing out before dehydration
Disadvantage:
- The extent of decalcification cannot be measured by a chemical test.
Decal time. Not mentioned
Von Ebner’s Fluid
- a moderate-acting decalcifying agent which produces better nuclear staining with less tissue distortion.
- It is safer to handle than nitric acid or hydrochloric acid.
- It is recommended for routine decalcification of postmortem research tissues.
Formic acid
Formic acid
Decal Time: 2-7 days
Advantage:
* May be used both as a fixative and decalcifying agent
* Recommended for small pieces of bones and teeth
* Suitable for most routine surgical
specimens, particularly when IHC staining is needed
Disadvantage:
It requires neutralization with 5% Sodium sulfate and washing out to remove the acid from the tissue
Formic acid
Formic acid
Decal Time: **3-14 days **
Advantage: Recommended for autopsy materials, bone marrow, cartilage and tissues studied forresearch purposes
Disadvantage: Requires neutralization with 5% Sodium Sulfate
Formic acid-Sodium
citrate Solution
Decal time: 4-8 days
Advantage
* It permits good nuclear staining.
* It does not require washing out; the excess acid may be removed by several changes of 90% alcohol, thus improving tissue dehydration
Disadvantage
* It is a weak decalcifying agent, not used for dense tissues, and is suitable only for small spicules of bone.
* It is very slow-acting; hence, is not recommended for urgent examinations.
Trichloroacetic acid
Its disadvantage is that it is a very weak decalcifying solution suitable only for minute pieces of bone.
Sulfurous acid
Advantage: May be used both as a fixative and decalcifying agent
Disadvantage:
* Nuclear staining with hematoxylin is inhibited
* The extent of decalcification cannot be measured by a chemical test
* Highly corrosive to skin and mucous membranes
* Carcinogenic
Chromic acid (Flemming’s Fluid)
Decal. time: 6 days
Advantage:
* It permits excellent nuclear and cytoplasmic staining.
- It does** not produce cell or tissue distortion**
Disadvantage:
* Its action is too slow for routine purposes.
Citric Acid-Citrate Buffer Solution (pH 4.5)
substances which combine with calcium ions and other salts, such as iron and magnesium deposits, to form weakly dissociated complexes and faclitate removal of calcium salts.
Chelating agents
The most common chelating agent in the market is
ethylenediaminetetraacetic acid (EDTA) salt
commercial name of VERSENE
This chelating agent is recommended only for detailed microscopic studies
ethylenediaminetetraacetic acid (EDTA) salt, with the commercial name of VERSENE,
Although EDTA is traditionally referred as “acid”, it does not act like inorganic or organic acids, but it binds with metallic ions, notably?
calcium and magnesium
Decal. time:
* 1-3 weeks for small specimens
* It may take 6-8 weeks or longer to totally decalcify dense cortical bone
Advantage:
* An excellent bone decalcifier for IHC or enzyme staining, and for electron microscopy
Disadvantage:
* Very slow decalcifying agent. After nearly 2 months, a diagnosis is still pending as the specimen is not ready to be cut because the tissue
is still hard.
Neutral EDTA
- hastens decalcification by removing calcium ions from formic acid containing decalcifying solutions, thereby increasing solubility from the tissue.
- It is not recommended for fluids containing mineral acids such as nitric acid or hydrochloric acid.
Ion exchange resin (ammonium form of polystyrene resin)
Decal time: 1-14 days
Advantage
* The degree of decalcification may be measured by physical or x-ray method
Disadvantage
* The degree of decalcification cannot be measured by a chemical means
* It is very slow, and is therefore not recommended for urgent and routine purposes
Ion exchange resin (ammonium form of polystyrene resin)
- a process whereby positively charged calcium ions are attracted to a negative electrode and subsequently removed from the decalcifying solution.
- You can facilitate the removal of your calcium by applying electricity in your specimen.
Electrophoresis (Electrical Ionization)
- The time required for decalcification is thereby shortened due to the heat and electrolytic reaction produced in the process.
- The principle is similar to that of chelating agents, with the main difference that this process utilizes electricity and is dependent upon a supply of direct current to remove the calcium deposits
Electrophoresis (Electrical Ionization)
Electrophoresis (Electrical Ionization)
Advantage
* This method is satisfactory for ____ ____ ____, processing only a limited number of specimens at a time.
Disadvantage
* Good cytologic and histologic details are, however, not always preserved in tissues that have been ____ ____
- small bone fragments
- electrically decalcified
What are the factors affecting decalcification procdure
- Concentration and Volume of Decalcifying Agent
- Temperature
- Mechanical Agitation
- Size and Consistency of The Tissue
Concentration and Volume of Decalcifying Agent
The recommended ratio of fluid to tissue volume for decalcification is?
20: 1
Concentration and Volume of Decalcifying Agent
Specimens in the decalcifying solution should be?
fully submerged and overflowing
Concentration and Volume of Decalcifying Agent
TOF
The concentration of active agents will affect the rate at which calcium is removed. In general, more concentrated acid solutions decalcify bone more slowly, but are more
harmful to the tissue.
False (more RAPIDLY)
Concentration and Volume of Decalcifying Agent
High concentrations and greater amounts of fluid will?
increase the speed of the process
Temperature (Decalcifying)
The optimum temperature so far recommended is?
room
temperature range of 18°C -30°C
Temperature (Decalcifying)
TOF
Decalcifying agents are faster at a higher temperature
True
High temperatures are avoided as it may cause tissue distortion by increasing the damaging effects of acids on tissue.
Temperature (Decalcifying)
TOF
Microwave, sonication and electrolytic methods produce heat, and must be carefully monitored to prevent excessive temperatures that damage tissue.
True
Mechanical Agitation (Decalcification)
Gentle agitation may affect the decalcification in what way?
increase the rate of decalcification
Gentle fluid agitation is achieved by low-speed rotation, rocking, stirring or bubbling air into the solution
Mechanical Agitation (Decalcification)
TOF
fucking agitation and moving of the tissue in solution usually influences fluid exchange, accelerates the rate of diffusion and speeds up the decalcificationprocess
False (Mechanical Agitation) not fucking
Mechanical Agitation (Decalcification)
vigorously agitates both specimen and fluid, and may cause disruption of tissue, with formation of cellular debris on the floor of the container.
SONICATION
Size and Consistency of The Tissue (Decalcification)
TOF
smaller tissues are quicker to decalcify while larger and dense bone tissues take more time
True
Size and Consistency of The Tissue (Decalcification)
give the time to decalcifiy tissue and dense bone
- Tissue (24-48 hours)
- Dense bone (14 days)
For dense bone tissue it usually require more In such cases, the solution should be changed daily to ensure better penetration and to test for the degree of decalcification.
What are the ways to measure the extent of Decalcification
- Physical or Mechanical Test
- X-Ray or Radiological Method
- Chemical (Calcium Oxalate Test)
MEASURING THE EXTENT OF DECALCIFICATION
Require manipulation, bending, probing or trimming of the specimen to “feel” for remaining calcified areas
Physical or Mechanical Test
While this method may be successful in experienced hands it is generally considered to be unreliable
MEASURING THE EXTENT OF DECALCIFICATION
Disadvantage of Physical or Mechanical test
subjective (what’s soft for you is not
necessarily soft for everyone else)
MEASURING THE EXTENT OF DECALCIFICATION
during the physical test, what can occure during bending or probing and small deposits of calcium can easily be missed
Mechanical Damage
MEASURING THE EXTENT OF DECALCIFICATION
A method of determining the endpoint by carefully weighing the specimen after rinsing and blotting has also been described, and may be an effective method for large specimens.
Physical or Mechanical Test
MEASURING THE EXTENT OF DECALCIFICATION
An alternate method of evaluating tissues mechanically is by?
pricking the tissue with a fine needle or a probe. This method is apt to produce needle tract artifacts and destroy important cellular details
Pricking, slicing, bending or squeezing tissue can disrupt soft tumors from the bone or cause false positive microfractures of fine trabeculae, leading to a potential misdiagnosis.
MEASURING THE EXTENT OF DECALCIFICATION
Physical or Mechanical Test
usually have diminished consistency and are often softer to touch
Decalcified tissues
MEASURING THE EXTENT OF DECALCIFICATION
The BEST METHOD, particularly with large specimens such as femoral heads
X-RAY OR RADIOLOGICAL METHOD
MEASURING THE EXTENT OF DECALCIFICATION
The most ideal, most sensitive and most reliable method of determining extent of decalcification due to its ability to detect even the smallest focus of calcium which appears
**opaque in an X-ray plate. **
X-RAY OR RADIOLOGICAL METHOD
X-ray films are colored white and when it undergoes Xray, it turns the film black. Bones in X-rays appear white due to calcium blocking off the radiation this is the principle behind using X-ray for checking the extent of
decalcification
MEASURING THE EXTENT OF DECALCIFICATION
What are the disadvantages of X-ray or Radiological Method
- Very expensive
- NOT recommended for mercuric chloride-fixed tissues due to the latter’s characteristic radio-opacity which will interfere with the correct interpretation of the plate.
- Exposure to radiation-
MEASURING THE EXTENT OF DECALCIFICATION
- A simple, reliable and convenient method recommended for routine purposes
- Detection of calcium in acid solutions by precipitation of insoluble calcium hydroxide or calcium oxalate
CHEMICAL METHOD (CALCIUM OXALATE TEST)
MEASURING THE EXTENT OF DECALCIFICATION
what are the solutions needed for chemical method?
- Ammonium hydroxide, concentrated
- Saturated aqueous ammonium oxalate
MEASURING THE EXTENT OF DECALCIFICATION
Give the result of the Chemical method
- cloudiness indicates that there is still calcium found in the solution. Need for further decalcification.
- solution remains clear after neutralization
- INCOMPLETE decalcification
- COMPLETE decalcification
Post-Decalcification
After decalcification is complete, the acid can be removed from tissues or
neutralized chemically
This acid is interfering with the rest of the processing; hence we need to remove it.
Post-Decalcification
To remove the, immerse the tissues in?
- saturated lithium carbonate solution
- 5-10% aqueous bicarbonate solution
Other options include running water, formol saline, PBS
Post-Decalcification
Adequate water rinsing for
- Small specimen
- Larger specimen
give the time
- 30 minutes for small specimens
- 1-4 hours for larger specimens
Post-Decalcification
Acid decalcified tissues for frozen sections must be thoroughly washed in water or stored in?
- formol-saline containing 15% sucrose
- phosphate-buffered saline (PBS) with 15-20% sucrose at 4°C before freezing.
- The process of removing intercellular and extracellular water from the tissue following fixation and prior to wax impregnation
- It involves slow substitution of the water in the tissue with an organic solvent.
dehydration
Most dehydrating agents are strong organic
solvents. which may cause
SHRINKAGE and EXTRACTION of cell components
Dehydration
To minimize the effects of shrinkage and extraction, dehydrating agents are used in a?
graded series for short periods of time, and water is* gradually replaced* so that violent osmotic changes do not produce distortions
Dehydration
TOF
In HISTOPATH, drying is never used in specimens and tissues are never dried. Drying is the removal of water by evaporation from a solid, semi-solid or liquid.
True
CHARACTERISTICS OF AN IDEAL DEHYDRATING AGENT
- It should dehydrate ____ without producing considerable shrinkage or distortion of tissues.
- It should NOT ____ very fast.
- It should be able to dehydrate even ____.
- It should NOT harden tissues ____.
- It should NOT remove ____.
- It should NOT be ____ to the body.
- It should NOT be a ____ hazard.
- Rapidly
- Evaporate
- Fatty tissues
- Stains
- Toxic
- Fire Hazard
CHARACTERISTICS OF AN IDEAL DEHYDRATING AGENT
Dehydrating agents should not be less than ____ the volume of the tissue
10 times
CHARACTERISTICS OF AN IDEAL DEHYDRATING AGENT
- Dehydration times should be as ____ as possible.
- Almost any water miscible, ____ ____ can be used as a dehydrating agent
- Brief
- Anhydrous fluid
What are the commonly used DEHYDRATING AGENS
- Alcohol (most common) - particularly, ETHANOL
- Acetone
- Dioxane
- Cellosolve
- Triethyl phosphate
- Tetrahydrofuran
What are the dehydrating agents under alcohol
- Ethyl alcohol (ethanol)
- Methanol
- Butyl alcohol
- Isopropanol
- Pentanol
Dehydrating Agents
- the alcohol recommended for routine dehydration of tissues.
- It is a clear, colorless, flammable fluid.
- It is considered to be the BEST DEHYDRATING AGENT because it is fast-acting, it mixes with water and many organic solvents, and it penetrates tissues easily.
- It is not poisonous and not very expensive.
Ethanol
Dehydrating Agents
Toxic dehydrating agent, primarily employed for blood and tissue films and for smear preparations.
Methanol
Dehydrating Agents
Utilized in plant and animal microtechniques, is a slow dehydrating agent, producing
less shrinkage and hardening than ethyl alcohol and is recommended for tissues which do not require rapid processing.
Butyl Alcohol
Dehydrating Agents
ADVANTAGES:
- Excellent substitute for ethanol
- Less shrinkage and hardening than ethanol
DISADVANTAGES:
Cannot be used in the celloidin technique since nitrocellulose, which is one of the impregnating
agents, is insoluble in it
Isopropanol
Dehydrating Agents
ADVANTAGES:
- Miscible with some of the organic solvents—90% alcohol, toluene, and xylene
DISADVANTAGES:
- Toxic
- Cannot be used in poorly ventilated rooms
- Not miscible with water
Pentanol
ALCOHOLS IN GENERAL
TOF
- It is advisable to transfer fixed tissues directly from water or aqueous fixative directly into absolute ethanol.
- It is not advisable to remove water gently and allow the tissue to slowly adjust to its removal.
- The more delicate the tissue, the more gently this should be done.
- False (it is not advisable)
- False (It is advisable)
- True
Dehydrating Agent
- is a cheap, rapid-acting dehydrating agent utilized for most urgent biopsies which dehydrates in 1/2 to 2 hours.
- a clear, colorless fluid that mixes with water, ethanol and most organic solvents.
Acetone
Dehydrating Agent
- more miscible with epoxy resins than alcohol, but is highly flammable and requires considerable care in handling.
- It is rapid in action but penetrates tissues poorly and causes brittleness in tissues that are placed in acetone for prolonged periods of time.
Acetone
Dehydrating Agent
Acetone - TOF
- Most lipids are removed from tissues with this dehydrating agent.
- Its use has been limited only to large pieces of tissues due to its extreme volatility and inflammability.
- Because of considerable tissue shrinkage produced, acetone is recommended for routine dehydration purposes
- True
- False (Small pieces)
- False (NOT reco)
- both an excellent dehydrating and clearing agent readily miscible in water, melted paraffin, alcohol and xylol.
- It produces less tissue shrinkage as compared to alcohol dehydration.
- Aside from being expensive, also extremely dangerous, and this is its main disadvantage.
DIOXANE (DIETHYLENE DIOXIDE)
DIOXANE (DIETHYLENE DIOXIDE)
In Gregorio’s, there are 2 methods
what are those
Graupner’s method
and Weiseberger’s method
dioxane processing dehydration
DIOXANE (DIETHYLENE DIOXIDE)
Time schedule for dehydration with dioxane:
- (1st) Pure dioxane solution 1 hour
- (2nd) Pure dioxane solution 1 hour (3rd) Pure dioxane solution 2 hours (1st) Paraffin wax 15 mins.
- (2nd) Paraffin wax 45 mins.
- (3rd) Paraffin wax 2 hours
Embed in mold and cool in water
Graupner’s Method
DIOXANE (DIETHYLENE DIOXIDE)
- Tissue is wrapped in a
gauze bag and suspended in a bottle containing dioxane and a little anhydrous calcium oxide - Eater is displaced from the tissue by dioxane and in turn absorbed by
calcium oxide orquicklime - Dehydration period: 3 - 24 hours
Weiseberger’s Method
Dehydrating Agent
- dehydrates rapidly.
- Ethylene glycol ethers are combustible at 110-120°F and are toxic by inhalation, skin contact and ingestion
CELLOSOLVE (ETHYLENE GLYCOL MONOETHYL ETHER)
Dehydrating Agent
- Rapid dehydrating agent as it removes water very readily.
- Produces limited distortion and hardening of tissue
- It is soluble in alcohol, water, ether, benzene, chloroform, acetone and xylene.
TRIETHYLPHOSPHATE
Dehydrating Agent
- is a reagent that both dehydrates and clears tissues since it is miscible in both water and paraffin.
- It can dissolve many substances including fats and is in itself miscible with lower alcohols, ether, chloroform, acetone, benzene and xylene.
- toxic if ingested or inhaled.
- It causes less shrinkage and easier cutting of sections with fewer artifacts.
TETRAHYDOFURAN (THF)
also called as de-alcoholization, is the process whereby alcohol or a dehydrating agent is removed from the tissue and replaced with a substance that will dissolve the wax with which the tissue is to be impregnated (e.g. paraffin) or used as the medium on which the tissue is to be mounted (e.g. Canada balsam)
Clearing
CLEARING
TOF
After removing water from the tissue through dehydration, it becomes saturated with alcohol. However, for the tissue to be sliced thinly and effectively infiltrated, it needs to be properly prepared. Alcohol and paraffin are not compatible, so a clearing step is necessary.
True
CLEARING
During clearing, the alcohol is replaced with a substance that can blend with both alcohol and paraffin, ensuring the tissue is adequately prepared for further processing. In clearing, we will remove the ____ and substitute it with something else. That ‘something else’ should be miscible with both the alcohol and the paraffin.
Alcohol
CLEARING
When dehydrating agent has been entirely replaced by the solvent, the tissue has a ____ ____; hence, the use of the term “clearing agent”
translucent appearance
CHARACTERISTICS OF GOOD CLEARING AGENT
- It should be ____ to promote rapid removal of the dehydrating agent from the tissue
- It should be miscible with, and easily removed by melted ____ and/or by mounting medium to facilitate impregnation and mounting of sections
- It should not produce ____, hardening and damage to tissue
- It should not dissolve out ____
- It should not evaporate quickly in ____
- It should make tissues ____
- Miscible with alcohol
- Paraffin wax
- shrinkage
- aniline dyes
- water bath
- transparent
COMMONLY USED CLEARING AGENTS
- Xylene (most common)
- Toluene
- Benzene
- Chloroform
- Cedarwood oil
- Aniline oil
- Clove oil
- Carbon tetrachloride
Clearing Agents
- Most commonly used in histology laboratories
- Used for clearing, embedding and mounting procedures
Xylene (Xylol)
Clearing Agents
- May be used as a substitute for xylene or benzene for clearing both during embedding and mounting processes
Toluene
Clearing Agents
Preferred by some as clearing agent in the embedding process of tissues.
Benzene
Clearing Agents
Slower in action than xylene, but causes less brittleness
Chloroform
Clearing Agents
- Not common
- It is used to clear both paraffin and celloidin sections during the embedding process.
- It is especially recommended for central nervous system tissues and cytological studies, particularly of smooth muscles and skin
Cedarwood Oil
Clearing Agents
- This is not normally utilized as a routine clearing agent
- However, it is recommended for clearing embryos, insects and very delicate specimens due to its ability to clear 70% alcohol without excessive tissue shrinkage and hardening
Aniline Oil
Clearing Agents
This reagent causes minimum shrinkage of tissues
Clove Oil
Clearing Agent
Match the Clearing time
- 30- mins to 1 hour
- 2-3 days
- 1-2 hours
- 15-60 mins
A. Xylene (Xylol)
B. Toluene
C. Benzene
D. Cedarwood Oil
.
- A
- D
- B
- C
Clearing Agent
Advantage
* It is generally suitable for most routine histologic processing schedules of less than 24 hours and when tissue block size is less than 5 mm in thickness
Disadvantage
* Highly inflammable and should be
appropriately stored
Xylene (Xylol)
Clearing Agent
Disadvantage:
* More expensive than xylene and more toxic, so less commonly used
Toluene
Clearing Agent
Advantage
* It penetrates and clears tissues rapidly
* It is rapid acting, hence is recommended for urgent biopsies (15-60 minutes) and routine purposes
Disadvantage
* Used to be a popular routine clearing agent until highly carcinogenic properties were recognized
Benzene
Clearing Agent
Advantage
* Thicker tissue blocks (up to I cm) can be processed.
Disadvantage
* Tissues placed in chloroform DO NOT become translucent.
Chloroform
Clearing Agent
Disadvantage
* It requires two changes in clearing solution.
Cedarwood Oil
Clearing Agent
Disadvantage
* Has tendency to become adulterated.
* Wax impregnation after clearing with clove oil is slow and difficult
* Tissues become brittle, aniline dyes are removed, and celloidin is dissolved.
* Expensive
Clove Oil
Clearing Agent
what are the other xylene substitutes
- Tetrahydrofuran
- Dioxane
- Terpenes
- Limonene
- Orange oil based-derived from fruits
- Chlorinated hydrocarbons
- Coconut Oil
- Bleached Palm Oil
- Methyl Benzoate and Methyl Salicylate
Clearing Agent
OTHER XYLENE SUBSTITUTES
Are isoprene polymers found in essential oils originally derived from plants, though some are now synthesized.
Terpenes
Clearing Agent
OTHER XYLENE SUBSTITUTES
- One of the recommended xylene substitutes from the terpene family
- A volatile oil found in citrus peels which goes by several trade names.
