Part 1 (FIXATION) Flashcards
First and most critical step in histotechnology
FIXATION
Stabilization of proteins is the most important reaction for maintaining tissue morphology
FIXATION
Process of preserving the tissue for future examination
FIXATION
have the property of forming cross-links between protein
FIXATION
Primary aim: preserve the morphological and chemical integrity of the cell in life-like manner
FIXATION
Secondary aim: to harden and protect tissue from trauma of further handling
FIXATION
First or Secondary Aim?
preserve the morphological and
chemical integrity of the cell in life-like manner
First Aim
First or Secondary Aim?
to harden and protect tissue from
trauma of further handling
Secondary Aim
In the process, it is possible that the organ or tissue will be exposed to different forces and
pressure.
Secondary Aim of Fixation
Ideal time for fixation
Within 20-30 minutes after
interruption of blood supply.
Once you have already cut off or interrupted the blood supply or remove the organ, it is already the start of death of the cells.
Ideal time for fixation (Within 20-30 minutes)
Usual fixation temperature for
surgical specimen
Room Temperature (22-24 deg)
● Temperature may go higher if
you want to enhance the fixation process.
Fixative-to-Tissue Ratio (F:T Ratio)
F:T Ratio = 20:1
● 20 = Fixative
● 1 = Tissue
Tissue-to-Fixative Ratio (T:F Ratio)
T:F Ratio = 1:20
● 1 = Tissue
● 20 = Fixative
● 20x the tissue volume
Volume
Specimen must be placed in fixative as soon as it is removed from the body (20-30 mins after
interruption of blood supply)
Speed
● Rate of formalin penetration = 1mm per hour
● Formalin was previously a brand name of formaldehyde, but now synonymous with it
Penetration
(4) PRACTICAL CONSIDERATIONS OF FIXATION
1.) Speed
2.) Penetration
3.) Volume
4.) Duration of fixation
Rate of formalin penetration =
= 1mm per hour
is previously the brand name of
acetylsalicylic acid
Aspirin
(2) MECHANISMS INVOLVED IN FIXATION
1) Additive Fixation
2.) Non- Additive Fixation
(3) Examples of Additive Fixation
formalin, mercury, and osmium tetroxide
Example of Non- Additive Fixation
alcoholic fixatives
● Whereby the fixing agent is not taken in, but changes the tissue composition and stabilizes the tissue by removing bound water attached to hydrogen bonds of
certain groups within the protein molecule
● Example: alcoholic fixatives
Non- Additive Fixation
● Whereby the chemical constituent of the fixative is taken in and becomes part of the tissue
● Example: formalin, mercury, and osmium tetroxide
Additive Fixation
Hydrogen Ion Concentration (pH)
Satisfactory fixation:
pH 6-8
(6) MAIN FACTORS INVOLVED IN FIXATION
- Hydrogen Ion
Concentration (pH) - Temperature
- Thickness of
section - Osmolality
- Concentration
- Duration of fixation
Surgical specimen: TEMPERATURE
room temperature (22-24°C)
Electron microscopy and some
histochem: TEMPERATURE
Refrigerator temperature (0-4°C)
Tissue blocks should be either small, thin, or as prescribed by tissue processor manufacturer
Thickness of section
● Formalin aims to harden the
specimen; the longer the specimen stays in the fixative,
the harder it gets
Brain
● Suspended whole in 10%
buffered formalin for 2-3
weeks
Brain
Brain
● Suspended whole in __%
buffered formalin for ___
weeks
Brain
● Suspended whole in 10%
buffered formalin for 2-3
weeks
(2) TYPE OF FIXATIVES ACCORDING TO…
- ACCORDING TO COMPOSITION
- ACCORDING TO ACTION
● One component
Simple Fixatives
● Glacial acetic acid
- Considered as a fixative in itself
- Solidifies at ___°C
- Causes tissues to swell
- Preserve _________
● Glacial acetic acid
- Considered as a fixative in itself
- Solidifies at 17°C
- Causes tissues to swell
- Preserve nucleoprotein
● Glacial acetic acid
- Considered as a fixative in itself
- Solidifies at 17°C
- Causes tissues to swell
- Preserve nucleoprotein
Simple Fixatives
● Made up of 2 or more fixatives
Compound Fixatives
Compound Fixatives example
Zenker’s Fluid
(2) Zenker’s Fluid is made up of:
- Glacial acetic acid
- Mercuric chloride
- Causes the tissues to swell
Glacial acetic acid
- Minimizes the shrinkage effect of the Mercuric chloride
Glacial acetic acid
- Causes the tissues to
shrink
Mercuric chloride
- Minimizes the swelling effect
of the Glacial acetic acid
Mercuric chloride
Glacial acetic acid and Mercuric chloride
● These two _________ each other that minimizes the adverse effect
● These two neutralizes each other that minimizes the adverse effect
For GENERAL microscopic study of tissue structures
MICROANATOMICAL FIXATIVES
(2) According to Action
A. MICROANATOMICAL FIXATIVES
B. CYTOLOGICAL FIXATIVES
Examples
- 10% Formol Saline
- 10% BNF (Buffered neutral formalin)/Neutral buffered
formalin
- Heidenhain’s SuSa
- Bouin’s
- Formol sublimate
- Zenker’s solution
- Zenker-Formol
- Brasil’s
MICROANATOMICAL FIXATIVES
● Preserves SPECIFIC parts and particular microscopic elements of the cell itself
CYTOLOGICAL FIXATIVES
(3) Types of CYTOLOGICAL FIXATIVES
○ Nuclear fixatives
○ Cytoplasmic fixatives
○ Histochemical fixatives
● Preserves the NUCLEAR STRUCTURES of the cells
Nuclear fixatives
● WITH glacial acetic acid
○ Preserves the nucleoproteins
which preserves the nuclear
structures
Nuclear fixatives
Fixatives:
● Flemmings
● Carnoy’s
● Bouin’s
● Newcomer’s
● Heidenhain’s SuSa
Nuclear fixatives
● Preserve the CYTOPLASMIC STRUCTURES
Cytoplasmic fixatives
General pH of Nuclear fixatives
General pH: 4.6 or less (acidic)
Preserve CHEMICAL CONSTITUENTS of cells
Histochemical fixatives
General pH of Cytoplasmic fixatives
General pH: greater than 4.6
● WITHOUT glacial acetic acid
○ Destroys the mitochondria and
golgi bodies (organelles in the
cytoplasm)
Cytoplasmic fixatives
Fixatives:
● Flemming’s fluid w/o acetic acid
● Helly’s fluid
● Formalin with post-chroming
● Regaud’s (Moller’s)
● Orth’s
Cytoplasmic fixatives
Fixatives:
● 10% Formol Saline
● Absolute ETOH (Ethanol)
● Acetone
● Newcomer’s Fluid
Histochemical fixatives
Functions as both a nuclear and
histochemical fixative
Newcomer’s Fluid
All of the following fixatives contain Glacial Acetic Acid except
What is the question looking for? Nuclear or Cytoplasmic fixatives?
Cytoplasmic fixatives
Most commonly used aldehyde fixative
FORMALDEHYDE
Aka Formalin
FORMALDEHYDE
is only a brand name. But due to its popularity, formaldehyde can be called as
“Formalin”
A gas produced from the oxidation of methanol
FORMALDEHYDE
Rate of fixation: FORMALDEHYDE
1mm per hour
Usual fixation Time of FORMALDEHYDE
● 24 hours
2 types of concentration
● 40% Formaldehyde (Stock Solution)
● 10% Formaldehyde (Working
Solution)
- Concentration is too strong resulting to outer part of the tissue tends to overharden
- Not used as routine concentration aldehyde
40% Formaldehyde (Stock
Solution)
- Used to fixed organs and
tissues - An unstable solution and may
produce artifacts that can affect
in the observation of cells in the
microscope
10% Formaldehyde (Working
Solution)
- Needed to be further diluted
with water before using → 10%
formaldehyde
40% Formaldehyde (Stock
Solution)
40% Formaldehyde aka
Stock Solution
10% Formaldehyde aka
Working Solution
Artifacts That May Be Produced By The 10% Formaldehyde (2)
Paraformaldehyde
Acid Formaldehyde Hematin
● White precipitates which may
be found on the solution itself
● Affects microscopic details
Paraformaldehyde
Paraformaldehyde
Removed by: (2)
- Addition of 10% methanol
- Filtration
● Brown or black granules that
may obscure microscopic details
Acid Formaldehyde Hematin
● If present, they can block the
microscopic view of the cells that are underneath them
● Can be a problem because you
won’t be able to see the abnormalities that are on the cells
Acid Formaldehyde Hematin
Acid Formaldehyde Hematin
Removed By: (4)
● Saturated alcoholic picric acid
● Alcoholic Potassium Hydroxide
(KOH)
● Kardasewitsch method
● Lillie’s method
A. ALDEHYDE FIXATIVES (8)
- FORMALDEHYDE
- 10% Formol saline
- 10% Buffered Neutral
Formalin (BNF) - Formol-corrosive
- Glutaraldehyde
- Formol-calcium
- Acrolein
- Karnopsy’s ParaFormaldehyde
Glutaraldehyde Solution (KPG)
● Best fixative for tissue containing iron pigments and for elastic fibers
● Best general tissue fixative
10% Buffered Neutral Formalin (BNF)
2 concentrations:
● 2.5% Solution
● 4% Solution
AKA: Formol-Sublimate
Formol-corrosive
Used for enzyme histochemistry and electron microscopy
Glutaraldehyde
Used for small tissue fragments and needle biopsy
2.5% Solution
Used for the preservation of lipids
Formol-calcium
Used for larger tissue specimens
4% Solution
(2) Used for electron cytochemistry
Acrolein
Karnopsy’s ParaFormaldehyde
Glutaraldehyde Solution (KPG)
40% Formaldehyde can be also called as?
Stock Solution
What is the best fixative for tissue containing pigments and the best general tissue fixative?
10% BNF
What is the next fixative for tissue containing pigments?
10% BNF
What solution can function both as a nuclear and histochemical fixative?
Newcomer’s Fluid
What fixative is used for electron microscopy?
Glutaraldehyde
Most common metallic fixative
MERCURIC CHLORIDE (HgCl2)
● Routine fixative of choice for
tissue photography
● Permits brilliant metachromatic
staining of cells
MERCURIC CHLORIDE (HgCl2)
● Decreases amount of
demonstrable glycogen
● Do not use if you wish to
demonstrate glycogen on tissues
MERCURIC CHLORIDE (HgCl2)
● Causes tissue shrinkage
- MgCl2 is a compound fixative
that causes the tissue to shrink
- To neutralize shrinking, mix with
glacial acetic acid
MERCURIC CHLORIDE (HgCl2)
● Corrodes all metals, except for
the nickel alloy (Monel)
- If you were to fix a tissue in
MgCl2, the material you should
use to get that organ/tissue should be made of Monel nickel
alloy
MERCURIC CHLORIDE (HgCl2)
● Produces black granular deposits
- removed by: De-Zenkerization
Technique
- Reagent: Alcohol Iodine
MERCURIC CHLORIDE (HgCl2)
MERCURIC CHLORIDE (HgCl2)
● Produces black granular deposits
- removed by:
- Reagent:
- removed by: De-Zenkerization
Technique - Reagent: Alcohol Iodine
● Fixation of tumor biopsies (esp. of the skin)
● Came from two German wor
Heidenhain’s SuSa
- Sublimat -
- Säure -
- Sublimat - Mercuric chloride
- Säure - Acid
What is glycogen?
Storage form of glucose in humans
Commonly used for bone marrow biopsies
B-5 fixative
The mercuric chloride deposits on tissues may be removed using?
Alcohol Iodine
Conversion of Glycogen to Glucose =
Glycogenolysis
Conversion of Glucose to Glycogen =
Glycogenesis
What is the meaning of Y in eosin Y?
Y is yellowish
● Chromic acid
● Potassium dichromate
● Regaud’s (Moller’s)
● Orth’s Fluid
CHROMATE FIXATIVES
What is the meaning of LASER?
Light amplified by stimulated emission of radiation
○ For Rickettsia and other bacteria
○ For study of early degenerative process
CHROMATE FIXATIVES
● Generally for acid mucopolysaccharides
LEAD FIXATIVES
● May be used for the fixation of umbilical cord/wharton’s jelly
LEAD FIXATIVES
● Used as:
○ Fixative
○ Decalcifying agent
○ Stain
PICRIC ACID FIXATIVES
● Versatile solution
● Highly explosive when dry
○ When transported it is in a liquid form/solution
PICRIC ACID FIXATIVES
● Produces excessive yellow staining of tissues
○ Can be an advantage/disadvantage
depending on viewpoint
PICRIC ACID FIXATIVES
● Picrates are formed upon protein; precipitates are soluble in water; hence tissues must be first rendered insoluble by direct immersion in 70% ETOH
● Picrate fixatives must never be washed in water before dehydration
PICRIC ACID FIXATIVES
Also an example of picric acid
fixative
Brasil’s alcoholic picro formol
● Recommended for embryos &
pituitary biopsies
Bouin’s solution
What fixative is recommended for embryos?
Bouin’s
What is used for tumor biopsy of skin?
Heidenhain susa
What fixative is used for bone marrow biopsy?
B-5
● Fixes nucleoprotein
○ Able to preserve nucleoprotein
GLACIAL ACETIC ACID
● Destroys mitochondria & golgi bodies
○ Not a component of cytoplasmic fixatives
● Solidifies at _____
GLACIAL ACETIC ACID
17C
● Causes tissues to swell/shrink
○ Can be paired with mercuric chloride because it can shrink which glacial acetic neutralizes the shrinking effect
GLACIAL ACETIC ACID
ALCOHOLIC FIXATIVES
● Generally recommended for:
Glycogen fixation
movement of glycogen granules towards the ends or poles of the
cells
Polarization
One of the disadvantages of these fixatives is
Polarization
What group of fixative which we cannot use for glycogen fixation?
Mercuric chloride
What group of fixative that can decrease the amount of demonstrable glycogen?
Mercuric chloride
● Also known as methyl alcohol
● Used for the fixation of bone marrow smears and blood smears
Methanol
Is bone marrow smear and bone marrow biopsy different?
YES
Used for bone marrow biopsy?
B-5
Used for bone marrow smear?
Methanol
● Used for fixation of cytological smears
(Ex: Pap Smear - 95% Ethanol)
Ethanol
● Most rapid fixative
- Fixation Time:
Carnoy’s Fluid
1-3 hrs
● Uses:
- Preservation of chromosomes**
- Preservation of glycogen
- Fixation of urgent biopsies and
lymph glands
- Preservation of brain tissues for
RABIES diagnosis
- Acetone can also be used
Carnoy’s Fluid
● Uses:
- Preservation of chromosomes**
Carnoy’s Fluid
● Also known as “Gendre’s Fixative”
● Useful in preserving sputum
Alcohol Formalin
● Functions as both nuclear and
histochemical fixative
Newcomer’s Fluid
● Used for the preservation OR fixation of mucopolysaccharides and nuclear proteins
Newcomer’s Fluid
Fixatives for biopsy specimens
Tumor biopsies:
Bone Marrow Biopsies:
Pituitary Biopsies:
Urgent Biopsies & Lymph Glands:
Tumor biopsies: Heidenhain’s SuSa
Bone Marrow Biopsies: B5 Fixative
Pituitary Biopsies: Bouin’s Solution
Urgent Biopsies & Lymph Glands: Carnoy’s Fluid
Precaution: OSMIUM TETROXIDE FIXATIVES
Cause Conjunctivitis or Blindness
● Inhibits hematoxylin and makes counter staining difficult
- Avoid using osmium tetroxide when performing H & E (hematoxylin and eosin) stain.
● Utilized also for electron microscopy
OSMIUM TETROXIDE FIXATIVES
● Kept in a dark-colored bottle to prevent evaporation and reduction by sunlight or organic matter
OSMIUM TETROXIDE FIXATIVES
● It produces black precipitate called
○ It is removed by cold water
OSMIUM TETROXIDE FIXATIVES
2 KINDS OF OSMIUM TETROXIDE
Flemming’s Fluid
Flemming’s Fluid Without Acetic Acid
OSMIUM TETROXIDE
● Example of Nuclear fixative
Flemming’s Fluid
OSMIUM TETROXIDE
● Example of Cytoplasmic fixative
Flemming’s Fluid Without Acetic
Acid
Function as both a Fixative and Decalcifying agent
TRICHLOROACETIC ACID (TCA)
Optimum working temperature for cryostat: -18 to -20
ACETONE
Acetone is used at ice cold temperatures -5 to 4°C
ACETONE
● It is used for the demonstration of water-diffusible enzymes (like
phosphatases and lipases)
ACETONE
● Fixation of brain tissue for rabies diagnosis
○ ** Carnoy’s fixative is also
used for rabies
ACETONE
2 Methods of Fixation
- Direct Method
- Microwave Fixation
It is also applied in bacterial smears
● In gram staining, you put a bacterial colony in the slide then you heat fix it
Direct Method
● Optimum temperature: Microwave Fixation
45 to 55 °C
● It is non-chemical technique useful for preservation of neurochemical substances in the brain (such as acetylcholine)
Microwave Fixation
What is the optimum working temperature for cryostat?
-18 to 20°C
What is the working temperature for acetone?
-4 to 5°C
What is the optimum temperature for microwave
fixation?
45 to 55°C
What is the appropriate temperature for the fixation of
surgical specimens?
Room temperature
What is the temperature utilized for the fixation of electron microscopy and some histochemistry?
0 to 4°C
FIXATIVES FOR ELECTRON MICROSCOPY (5)
● Glutaraldehyde
○ Used in enzyme chemistry but also used in
microscopy
● Platinic Chloride
● Platonic Chloride-formalin (Zamboni’s Gold Chloride)
● Osmium Tetroxide
● 10% BNF (Buffered Neutral Formalin)
FACTORS THAT AFFECT FIXATION
RETARTED BY:
- Size and thickness of the tissue
- Presence of mucus
- Presence of blood
- Presence of fats
- Cold temperature
○ Too large or too thick tissues may not be fixed
right away or preserved properly
○ Rate of penetration of formalin: __________
○ Longer fixation time if tissue is thick or big
Size and thickness of the tissue
1mm/hr
FACTORS THAT AFFECT FIXATION
ENHANCED BY: (3)
- Size and thickness of the tissue
- Agitation
- Moderate heat
○ Agitating the container will help the fixative to penetrate through the tissues
○ The machines in histopath allow continuous agitation of the tissue cassettes while they are
immersed in a formalin fixative during the course of a 24 hour fixation.
Agitation
○ Small or thin tissues will be fixed quickly and properly
Size and thickness of the tissue
○ Specifically 37-56°C
○ Increasing the temperature slightly from room temperature will cause the fixative to better
preserve the tissue.
Moderate heat
