HISTOPATHOLOGY

DEHYDRATION

 Process of removing intracellular and extracellular water from the tissue using DEHYDRATING
AGENTS
 Dehydrating agents – are alcohols, used in increasing concentrations
 Most instances, dehydration starts with immersion at 70% ethyl alc. in water, then 90%ethyl alc.
and 100% ethyl alc.
 For delicate tissues (embryonic tissue), start with 30% ethanol.
 GENERAL RULE: dehydrating agents SHOULD NOT be less than 10x the volume of the spx.

Characteristics of an Ideal Dehydrating Solution

1. Dehydrate rapidly
2. Does not evaporate very fast
3. Dehydrate even fatty tissues
4. Does not excessively harden tissues
5. Should not remove stains
6. Should not be toxic to the body
7. Should not be a fire hazard

Excessive dehydration – may cause hard, brittle or shrunken tissues

Incomplete dehydration – most common processing problem that prevents ingress/entrance of
the clearing agents into the tissue making the tissue soft and no-receptive to wax infiltration.

Dehydrating Agents

1. Alcohol
 Direct immersion to high grades of alc.(80-95%) results to shrinkage, hardening and distortion of
tissues
 Concentrated alc.(95% or absolute) hardens ONLY the surface and DOES NOT completely
penetrate the inside
Strength of initial alc. in each conc. depends on the ff:
1. Size of the spx.
2. Nature of the tissue
3. Fixative used

Effects of Alcohol in tissues

1. Concentrated (Above 80%) – hard, brittle and difficult to cut
2. Low concentration (below 70%) – tends to macerate/soften tissues
3. Prolonged storage @ 70-80% - interferes with the staining properties of the spx

Factors that hasten/accelerate dehydration:

1. Temperature @ 37°C – for urgent biopsies (fragmentary biopsies)
2. Anhydrous copper sulfate
 Blue discoloration of copper sulfate crystals indicates full saturation of water
 Placed 1/4 deep the container covered with filter paper
 PURPOSE: To insure complete dehydration, removes water from the dehydrating fluid

 Ethyl Alcohol
 Best dehydrating agent
 For routine dehydration of tissues
 Clear, colorless, flammable
Advantages:
1. Fast acting
2. Mixes with water and organic solvents
3. It is not poisonous and it is not very expensive
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 Methyl Alcohol
 For blood and tissue films and smear preparations
Disadvantage:
1. Toxic

 Butyl Alcohol
 Utilized in plant and animal micro-techniques
Advantages:
1. Produce less shrinkage of cells than ethanol
2. Recommended for tissues that do not require rapid processing
Disadvantage:
1. Slow dehydrating agent

 Isopropyl alcohol – both fixative and dehydrating agent

 Substitute for ethanol

2. Acetone
 Clear, colorless fluid that mixes with water, ethanol and most organic solvents
 NOT recommended for routine dehydration purposes
 Raw material in the preparation of shabu
Advantages:
1. Cheap
2. Rapid acting - used for most urgent biopsies, dehydrates for ½ to 2 hours
3. More miscible with EPOXY STAINS than alc.
Disadvantages:
1. Poor penetration
2. Produce brittleness of tissues
3. Removes lipids
4. Volatile & Inflammable

3. Dioxane / Diethylene dioxide

 Both dehydrating and clearing agent
 Methods;
 Graupner’s – 3 changes of pure dioxane and paraffin wax
 Wiseberger’s – use of calcium oxide or quicklime
Advantages:
1. Readily miscible in water, melted paraffin, alcohol, xylol
2. Tissues can be left in the rgt. for long periods of time
Disadvantages:
1. Tissues treated with dioxane tend to ribbon poorly
2. Expensive
3. Dangerous due to the toxicity of its vapor
4. May create explosive peroxides

4. Cellosolve / Ethylene glycol monoethyl ether

Advantages:
1. Dehydrates rapidly
2. Do not produce hardening or distortion of tissues and can be stored in it for months
Disadvantages:
1. Combustible at 110-120°F (43-49°C)
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2. Toxic when inhaled, ingested and through skin contact

3. Exposure can affect the reproductive, urinary, fetal and blood systems
*REMEDY: use of Propylene-based alcohols

5. Triethyl phosphate
 Readily removes water and produce little distortion and hardening of tissue
 Soluble in alcohol, water, ether benzene, chloroform, acetone, xylene
 Dehydrates stained sections and smears with minimum shrinkage

6. Tetrahydrofuran (THF)
 Both dehydrating and clearing agent
 Dissolves fats
 Miscible with lower alcohols, ether, chloroform, acetone, benzene, xylene
Advantages:
1. For demixing, clearing and dehydrating paraffin sections before and after staining
2. Provide easier cutting with fewer artifacts
3. Does not dissolves out aniline dyes
Disadvantages:
1. Toxic, causes nausea, dizziness, headache and anesthesia
2. Eye and skin irritant and can cause conjuctival irritation
3. with offensive odor, thus requiring a well-ventilated room
4. for protection use Teflon gloves

7. Ethoxyethanol
 Colorless, flammable, strongly hygroscopic
 Miscible with water and most organic sovents

8. Polyethylene glycols
 Clear, viscous, hygroscopic polymers
 Dehydrate and embed tissues that are labile to the solvents and heat of the paraffin wax
method

9. Carboxylol
 Both dehydrating and clearing agent