Lec 2: Tissue Processing 1 & 2 Flashcards
The second step in manual tissue processing
Dehydration
The process of removing intracellular and extracellular water from the tissue
Dehydration
Characteristics of an ideal dehydrating solution (7)
- Should dehydrate properly/rapidly without causing shrinkage or distortion
- Should dehydrate even fatty tissues
- Should NOT evaporate quickly
- Should NOT harden tissue excessively
- Should NOT remove stains
- Should NOT be toxic to the body
- Should NOT be a fire hazard
Dehydrating agent should NOT be less than ___ times the volume of the tissue.
10
Commonly used dehydrating agents (6)
- alcohol
- acetone
- cellosolve
- dioxane
- triethyl phosphate
- tetrahydrofuran
- clear, colorless, flammable
- for routine dehydration
- not poisonous
- fast-acting
- penetrates tissue easily
- mixed with water and other organic solvents.
Ethyl Alcohol
• best dehydrating agent
- toxic dehydrating agent
- for blood & tissue film
- for smear preparations
Methyl Alcohol
- slow dehydrating agent
› doesn’t require rapid processing - utilized in plant and animal micro-techniques
- less shrinkage & hardening
Butyl Alcohol
ACETONE
1. boiling point
2. Dehydrates how long?
3. Can tissues be placed in it for a prolonged period of time?
4. How well does it penetrate the tissue?
- 56°C
- 30 minutes to 2 hours
- NO, because it causes brittleness in tissues.
- Penetrates poorly
- clear and colorless
- rapid-acting
- for most urgent biopsies
- extremely volatile & flammable
- mixes with water, ethanol, and most organic solvents
Acetone
- rapid-acting
- combustible (110-120°F) & toxic
- tissue can be stored for months without hardening or distorting.
Cellosolve
CELLOSOLVE
1. boiling point
2. Other name
- 156.4°C
- Ethylene glycol monoethyl ether
The dehydrating agent wherein the reproductive, fetal, urinary, and blood systems are particularly vulnerable to its toxic side effects
Cellosolve
DIOXANE
1. boiling point
2. Refractive index
3. Other name
- 101.5°C
- 1.42
- Diethylene dioxide
- readily miscible with water, melted paraffin, alcohol, and xylol
- tissues can be left for a long time without affecting the staining properties
Dioxane
- miscible in water & paraffin
- can dissolve many substances including fats
- for demixing, clearing, and dehydrating paraffin sections
- odorous
- evaporates rapidly
Tetrahydrofuran
TRIETHYL PHOSPHATE
1. Boiling point
2. Soluble in what substances
- 215°C
- Alcohol, water, ether, benzene, Chloroform, acetone, xylene
- used to dehydrate sections and smears
- minimum shrinkage
- removed water readily
- little distortion and hardening
Triethylphosphate
The killing, penetration, and hardening of tissues
Fixation
- killing: stops metabolic processes
- penetration: fixative penetrates tissue
- hardening: for easier trimming & cutting
First and most critical step involving preserving fresh tissue
Fixation
The alteration of tissues by stabilizing proteins so that the tissues become resistant to further changes
Fixation
Primary Aim of Fixation
To preserve the morphologic and chemical integrity of the cell
Secondary Aim of Fixation
To harden and protect the tissue from trauma
The functions of fixation
- to preserve the tissue
- to prevent breakdown of cellular elements
- to coagulate/precipitate protoplasmic substances
The practical considerations to optimize fixation of tissue (8)
- duration of fixation
- osmolality
- concentration
- penetration
- volume
- speed
- temperature
- thickness
Practical Considerations:
- Specimen should be fixed ___ hour/s from removal.
- Fixative must be ___ times the size of the tissue
- Ratio of fixative to tissue.
- Formalin diffuses into tissue approximately __mm/hr.
- Duration of fixation for LM & EM.
- Specimen should be fixed at least 1 hour/s from removal.
- Fixative must be 20 times the size of the tissue
- 20:1 or 10:1
- Formalin diffuses into tissue approximately 1mm/hr.
- Duration of fixation
a. LM: 2/4–6 hours
b. EM: 3 hours
Practical Considerations:
- Osmolality
a. LM:
b. EM:
c. 10% NBF: - Concentration
a. Formaldehyde:
b. Glutaraldehyde: - Temperature
a. Routine manual:
b. Routine automated:
c. EM:
d. DNA:
e. RNA:
f. microwave: - Thickness of tissue
a. LM:
b. EM:
- Osmolality
a. LM: slightly hypertonic (400-450 mOsm)
b. EM: isotonic (340 mOsm)
c. 10% NBF: 1500 mOsm - Concentration
a. Formaldehyde: 10%
b. Glutaraldehyde: 3%; 0.25% - Temperature
a. Routine manual: 20-22°C
b. Routine automated: 40°C
c. EM: 0-40°C
d. DNA: 65°C
e. RNA: 45°C
f. microwave: 65°C - Thickness of tissue
a. LM: 2cm
b. EM: 1-2mm
Characteristics of a Good Fixative (8)
- cheap & economical
- stable and safe to handle
- fast-acting & even penetration
- inhibits bacterial decomposition & autolysis
- at least isotonic
- hardens tissue
- renders tissue insensitive to subsequent processing
- is compatible with many staining procedures
- What is a simple fixative?
- Give examples.
- Made up of only 1 component substance
- Examples:
a. Aldehyde
b. Metallic fixative
c. Picric Acid
d. Acetic acid
e. Acetone
f. Alcohol
g. Osmium tetroxide
- What is a Compound fixative?
- Give examples
- Made up of 2 or more fixatives added together
- Examples:
a. Zenker’s Fluid
b. Bouin’s fluid
c. Formol saline
Fixatives under Aldehyde Fixative
- formaldehyde (formalin)
- 10% formol saline
- 10% NBF
- Formol-corrosive
- alcoholic formalin
- glutaraldehyde
- formol calcium
- Karnovsky’s Paraformaldehyde Glutaraldehyde
- Acrolein
- buffered at pH 7 to 8
- fixed tissue by forming a cross linkage
Formaldehyde (formalin)
- for fixation of CNS tissues and general post mortem tissue
- made up of 40% formaldehyde + NaCl + distilled water
- fixation time: 12 to 24 hours
10% formol saline
10% Formol Saline
1. Fixation time
2. Composition
- 12 to 24 hours
- 40% formaldehyde + NaCl+ distilled water
- for preservation & storage of surgical, post mortem, and research specimens
- best for tissues containing iron pigments & elastic fibers
10% NBF
10% NBF
Fixation time
4 to 24 hours
- corrosive
- for routine post-mortem tissues
- fixation time: 3 to 24 hours
Formol-corrosive
Formol-corrosive
Fixation time
3 to 24 hours
- denaturing fixative with the additive in the cross links effect of formalin
- for rapid diagnosis
- for preservation of glycogen & micro-incineration
- used to fix sputum and coagulate mucus
Alcoholic Formalin
Alcoholic Formalin
1. Post fixation for ___ hours enhances the immunoperoxidase studies on tissues.
2. Other name.
- 6 hours or more
- Gendre’s Fixative
Glutaraldehyde
1. Composition
2. Fixation time
- 2 formaldehyde linked by 3 carbon chains
- 1½ hour to 2 hours
- for adipose tissue (phospholipid)
Formol Calcium
- surgical biopsy
Acrolein
Examples of the fixatives in Mercuric chloride under Metallic Fixative
- Zenker’s Fluid
- Zenker-formol (Helly’s solution)
- Hedenhain’s Susa
- B-5 Fixative
- for tissue photography: renal tissues, fibrin, CT and muscles
- ADVANTAGE:
» best for hematopoietic & reticuloendothelial tissues - DISADVANTAGE:
» rapidly hardens outer layer of tissue
Mercuric Chloride
• most common metallic fixative
- used to prevent turbidity and formation of dark precipitate
Zenker’s Fluid
Zenker’s Fluid
1. Composition
2. Fixation time
- Mercuric chloride + Glacial acetic acid
- 12 to 24 hours
- excellent for bone marrow, extra medullary hematopoiesis, and intercalated disc of the cardiac muscle.
Zenker-formol (Helly’s)
Zenker-formol
1. Fixation time
2. Other name
- 4 to 24 hours
- Helly’s solution
Hedenhain’s Susa
1. Recommended for ___?
2. Fixation time
- For tumor biopsy especially the skin
- 3 to 12 hours
- for bone marrow biopsy
- enhances nuclear detail to identify normal & abnormal cell types
B-5 Fixative
B-5 Fixative
1. Fixation time
- 4 to 8 hours
Chromate acid preserves ___
Carbohydrates
- For the demonstration of:
» chromatin
» mitochondria
» Golgi bodies
» RBC
Regaud’s Fluid
Regaud’s Fluid
1. Tissue should NOT be thicker than ____
2. Fixation time
3. Other name
- 2-3 mm
- 12 to 48 hours
- Muller’s solution
Potassium Dichromate
1. Preserves ___
2. pH range
- Preserves lipids and mitochondria
- to preserve mitochondria, pH should be 4.5 to 5.2
- recommended for the study of early degenerative processes and tissue necrosis
- for demonstration of rickettsia bacteria and preserves better myelin
- Fixation time: 36 to 72 hours
Orth’s fluid
