HISTOPATH LAB MIDTERMS

LECTURE / TOPIC 5 TRANSCRIBED BY: IVAJ

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LESSON Aims of Fixation

1. To preserve the tissue nearest to
its living state
TOPIC OUTLINE
1 Fixation 2. To prevent any change in shape
2 Tissue changes in fixation and size of the tissue at the time of
3 Types of fixation processing
4 Description of Nature of Fixation − Tissue removed from body→
5 Essential Precautions for Fixation in General permeable→ cell shrink or swell→morphological
6 Mechanism of Fixation change in appearance of cell (ex. nucleus lyse)
7 Factors that Affect Fixation − Nucleus→ important in diagnosis of CANCER
8 Commonly Used Fixatives in the Laboratory − Any change in nuclear material or nucleus →
9 Fixation Artefact indication that something is wrong w/ the patient
10 Formalin Pigment − Indication something wrong w/ cell itself
11 Troubleshooting in fixation • Karryolysis
• Karryohexis
• Mitotic figure (centrosome & centriole in
Fixation of Histology Samples: Principles, Methods cytoplasm
and Types of Fixatives • Abnormality in cell division
Fixation ▪ Determine if hyperactive nucleus or
− 1st step in tissue processing uncontrolled proliferation of cell
− Important to preserve every structure & organelles of
− the PROCESS by which the cell→ diagnosis of patient depends
cells in the tissue are fixed in a
chemical and physical state, 3. To prevent any autolysis
and all the biochemical and − Autolysis gives nothing (no info→ no diagnosis)
proteolytic activities within − Ex. hemolyzed red cell → smear→ ghost cell
the cells are prevented
4. To make the tissue firm or hard
− Preserve − To prevent trauma/ damage caused by tissue
• morphological appearance of tissue processing
• integrity of tissue itself (cell, chemical structure) − For specimen handling
− Tissue should not be brittle
− 1 of the most crucial step in tissue processing − Infiltration: maintain wax at 56C (di namumuo wax
• any error in this step will affect the rest of habang tissue nakalubog at di maluluto tissue)
procedure • Manual procedure: tissue → oven
• ideally fixation starts the moment organ
remove from body of patient → minimal 5. To prevent any bacterial growth in the tissue
change in tissue − Bacterial growth due to contamination in external
environment
• Presence of large amount of blood could
• Tissue from area w/ normal microbiota (ex.
hinder fixation (slow down or stop)
Intestine or appendix→ many bcteria)
▪ wash tissue prior to fixation
− to avoid bacterial decomposition→ fixation
• Bacterial enzyme (secreted by bacteria that
− AUTOLYSIS: due to failure or improper fixation →
digest tissue itself)→Putrefaction→
cell lysis due to release of several isoenzyme that
degradation tissue
destroy structure)
− Remove tissue from body→ devoid of oxygen & 6. To make it possible to have clear stain
nutrients→putrefaction, decomposition, bacterial
− enhance visuality and clarity of tissue during
growth → autolysis → cell will self-destruct
cutting and staining
• MACROscopically→ tissue might look intact
− preserve enzyme in tissue→enzyme react stain
• MICROscopically→ each tissue/cell is already
• increase affinity of tissue to stain
degrading
▪ No diagnosis → Ghost cell (no cytoplasm,
7. To have better optical quality of the cells
organelles, nucleus)
− makes nucleus condense & hyperchromatic
• hyperchromatic: increase intensity of color of
− every delay in process of tissue preparation→ delay
nucleus
of diagnosis→ delay treatment (ex. cancer→
aggressive)
* PRA: use tissue block as detector
− 1 of the most crucial section in terms of specimen→
Fixative
histopath
• Other section can repeat specimen collection − CHEMICAL used to preserve tissue

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• Ex. acetone, alcohol, osmium tetroxide, 2. Hardening of tissue

glutaraldehyde, formaldehyde
• Formalin: most used (most available & cheap) 3. Interference of staining
− hindrance of staining of enzymes
Ideal Fixative
1. Prevention of autolysis of the cells or tissue 4. Changes of optical density by fixation
− change of optical density of the nuclei
2. Prevention of decomposition of the tissue by
bacteria
Types of Fixation
3. Maintaining the volume and shape of the cell as far A. Nature of fixation
as possible B. Chemical properties
− Osmium tetroxide → increase volume & size of C. Component present
cell D. Action on tissue protein
− Formalin→ binds w/ water makes cell shrink; very Types of Classification
corrosive & toxic to human & environment; when Fixative
diluting use fumehood; possible carcinogenic Nature of 1. Immersion fixation
− Relationship of concentration of FORMALIN with fixation 2. Coating fixation
size of cell is INVERSELY PROPORTIONAL 3. Vapour fixation
• Lower cont = high chance shrink cell 4. Perfusion fixation
• High cont = less chance shrink cell 5. Freeze-drying fixation
6. Microwave fixation
4. Consistently high-quality staining particularly Chemical 1. Aldehyde
routine stain properties • Formaldehyde
a. haematoxylin and eosin stain • Glutaraldehyde
b. Papanicolaou’s stain 2. Oxidising agent
− Makes nucleus cell more condense and • Osmium tetroxide
hyperchromatic 3. Protein denaturing agent
• Ethyl alcohol
5. Rapid action • Methyl alcohol
− To prevent autolysis 4. Cross-linking agents
• carbodiimide
6. Cheap 5. Miscellaneous
• Picric acid
7. Non-toxic Component 1. Simple (only 1 chemical present)
*Lot of factor that will affect fixative regardless of type of present • Formaldehyde
fixative • Ethyl alcohol
*Temperature • Glutaraldehyde
− Most common factor that influences the • Picric acid
• Osmium tetroxide
effectiveness or action of fixative to tissue
2. Compound (more than 1 chemical)
− Ideal temp: RT 20-40C
• Bouin’s fluid
− effect of fixation much faster
• Carnoy’s solution
• Increase temp→ decrease fixation time →
Action on 1. Coagulative
destroy enzyme →enzyme (protein)→
protein • Ethyl alcohol
coagulate at high temp • Picric acid
• Protect integrity of enzyme in tissue→ 2. Non-coagulative
decrease temp (0-4C)→ tissue and enzyme • Formaldehyde
preserve→ fixation time increased • Osmium tetroxide
• Glutaraldehyde
Tissue Changes in Fixation
1 Volume changes Description of Nature of Fixation
2 Hardening of tissue 1 Immersion fixation
3 Interference of staining 2 Coating fixation
4 Changes of optical density by fixation 3 Vapour fixation
1. Volume changes 4 Perfusion fixation
− altered membrane permeability→ increased output 5 Freeze-drying
of fluid into cell 6 Microwave fixation
• Some fixative increase volume of cell (osmium 1. Immersion fixation
tetroxide) or shrinkage of cell (formalin)
− commonest way of fixation in the laboratories
− inhibition of the enzymes responsible for
− usually do after removal of tissue from body of
respiration and;
patient
− change of transport Na+ ions
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− immerse tissue into bottle of fixative • wash tissue w/ NSS

2. Coating fixation (wax + fixative) ▪ to remove excess blood
− commonly used in the cytology samples ▪ counteract effect of blood
− advantage of coating fixative ▪ blood have protein
• impart a protective covering over the smear *formalin → bind to tissue → preserve the
• No need to carry liquid fixative in bottle or jar inside part
− Ex. Papsmear (cervix) → ideal fixative 95% methyl *formalin → bind to blood →slow
or ethyl alcohol penetration in tissue
− Fixative→ spray bottle w/ nozzle (10-12 inches • fixation time: penetration of fixative inside of
distance of fixative from specimen) tissue
• Pressure can directly affect tissue − Tissue should be thinly cut in 3–5 mm thickness.
• ideal fixative: alcohol − The amount of fixative fluid should be 20 times
more than the volume of the tissue (to ensure
− Wax: barrier bw/ any mechanical object &
adequate fixation)
specimen
− The tissue with fixative should be in a tightly screw-
3. Vapour fixation
capped bottle (to prevent leakage)
− vapour converts the soluble material to insoluble
material
• mix vapour w/ specimen w/ any liquid → long Mechanism of Fixation
lasting effect of vapour fixative in tissue 1. Dehydration and
− commonly used vapour coagulation of protein
a. formalin (95% ethyl alcohol)
b. formaldehyde − remove water from
c. glutaraldehyde the tissue and causing
d. osmium tetroxide destabilization of the
e. ethyl alcohol hydrogen
4. Perfusion fixation − not used
− This fixative should flow directly to artery • methyl alcohol →
− mainly used in research purpose (taxidermy in carcinogenic
animal) • 70% ethyl
− common application → morgue alcohol →as
• Ex. to preserve heart →inject fixative in artery sanitizer
5. Freeze-drying − alcohol in protein →destroy hydrogen bond
− tissue is cut into thin sections and then rapidly (tertiary structure) →but preserve secondary
frozen into a very low temperature structure
− don’t need to wait for several hrs→ tissue→ instantly
fixed→ rapid decrease of temperature 2. Cross-linking fixatives
• water in tissue solidify thru high vacuum temp→ a. Formaldehyde: formalin cross link w/ water →
preserve tissue methylene glycol
− ideal for b. Glutaraldehyde: rapidly and irreversibly
a. Excellent for enzyme study & electron cross-links the protein → lysine
microscopy c. Osmium tetroxide: causes oxidation of
b. No change of proteins unsaturated bonds in the biological tissue
c. No shrinkage of tissue particularly lipid → glycol osmate in
d. Preservation of glycogen unsaturated fatty acid
6. Microwave fixation
− “scientific or medical microwave”
− factor to speed up the effect of chemical fixative Factors that Affect Fixation
− electromagnetic field w/ range of 300 megahertz to 1 pH
300 gigahertz 2 Temperature (25-40C)
− effect of microwave: tissue w/ chem fixative → 3 Duration of fixation
hasten/fasten effect of fixation; increase effect of 4 Osmolarity of the fixative solution
chemical in fixation 5 Concentration
− Ex. hollow tissue (heart, liver) → microwave→ burst 6 Agitation
a. In routine surgical pathology laboratory 1. pH of the fixative
b. Electron microscopy after osmium tetroxide − Neutral pH is preferable.
fixation − pH 6-8 is the best range.
c. Urgent processing of biopsy − High acidity or alkalinity interferes fixation.
• Buffer: counteract the acidity of formalin or any
fixative
Essential Precautions for Fixation in General
− The tissue should be free from excessive blood
before putting it into fixative.

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2. Temperature (25-40C) Preparation of Different Formalin Solution

− Room temperature suitable for routine work. A. 10% neutral buffered formalin:
− High temperature facilitates fixation. − Formaldehyde, 40%: 100.0 ml
− Low temperature (0-4 °C) suitable for enzyme − Distilled water: 900.0 ml
histochemistry. − Sodium dihydrogen phosphate: 4.0 g
− Disodium hydrogen phosphate: 6.5 g
3. Duration of fixation
− Depth of penetration of fixative is DIRECTLY B. Preparation of 10% formal saline:
PROPORTIONAL to the square root of time of − Formaldehyde, 40%: 100.0 ml
fixation. − Sodium chloride: 9 g
− Formalin: fixes 1 mm/h. − Distilled water: 900.0 ml
• Preserve 24 hr before initial step
− Small tissue: 6 h in formalin is optimum. C. Formal ethanol fixative:
− Large tissue: 24 h is the optimum time, − Ninety-five percent ethyl alcohol: 20 ml
− Prolonged fixation in aldehyde: inhibition of − Formaldehyde, 40%: 10 ml
enzymatic activity. 2. Glutaraldehyde
− is used as a fixative for electron microscopy
4. Osmolarity of the fixative solution because it fixes and preserves the ultrastructure
− Hypertonic: cell shrinkage − cross linking w/ lysine of amino acid
− Hypotonic: cell swelling
− Best: mild hypertonic (400-450 mOsm) − Advantages
a) Better fixation of ultrastructure.
b) Less cell shrinkage.
5. Concentration c) Preservation of protein is better.
− Mild lower concentration with neutral pH is d) Good cross-linking with collagen.
preferable. e) Less irritating.
− Very low concentration prolongs the time of fixation.
− Higher concentration causes rapid fixation with − Disadvantages
undesirable effect. a) Poor penetration and tissue should be less
than 0.5 mm
6. Agitation b) thick
− Agitation increases rate of penetration. c) Less stable compound
− Rapid agitation: damages delicate tissue. d) No lipid fixation
− Slow gentle agitation preferable. e) Polymerizes above pH 7.5. 5.
f) Costly.
3. Osmium Tetroxide
Commonly Used Fixatives in the Laboratory − used for fixation in electron microscopy
1. Formaldehyde/ formalin − direct effect w/ nucleus in cell →nucleus destroy
− amount of formalin should be 20 times the volume − not used to quantify DNA→ DNA damage
of tissue
− commonly used → availability & cheap − Advantages
− Market: 40% = 100% formalin → dilution → 10% a) This is a very good fixative for lipid.
formalin (ideal) b) It preserves cytoplasmic organelles such as
Golgi bodies and mitochondria,
− Advantages: c) Does not make the tissue hard,
a) The penetration rate of formalin is high.
b) Cell morphology well preserved in formalin. − Disadvantages
c) Cheap. a) It does not fix the proteins and carbohydrates
d) Stable. and therefore it should be used in combination
e) Easy to make the solution. with other fixative.
f) Formalin is effective fixation for routine b) Osmium tetroxide may react with ribose group
laboratory staining of the tissue. and may cause clumping of DNA.
*This can be prevented by pretreatment with
− Disadvantages potassium permanganate or post fixation
a) Slow fixation. with uranyl acetate.
b) Formalin reaction with the tissue is reversible, c) Poor penetration in the tissue.
and it can be removed by washing. d) Tissue swelling may occur.
c) Formalin fails to preserve acid e) Toxic and volatizes at room temperature
mucopolysaccharides. producing harmful vapor.
d) Highly vascular tissue may have dark-brown *This vapor is toxic to the eye and respiratory
e) granules (artefact) tract.
f) Exposure to the skin may cause dermatitis. f) Expensive
g) Chronic inhalation may cause bronchitis
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4. Methyl and Ethyl Alcohol

− fixatives of cytology smears
• Absolute alcohol: 950 ml Choice of fixative BASED ON TECHNIQUE
• Water: 50 ml Technique Fixative choice
• Time of fixation: 15–30 min Routine 10% neutral buffered formalin
− Ideal fixative for romanowsky & wright stain histopathology
5. Acetone Electron microscopy Glutaraldehyde solution or
osmium tetroxide
− for enzyme study and immunocytochemistry
Immunohistochemistry 10% neutral buffered formalin,
− recommended fixative to demonstrate negrei bodies
alcoholic formalin
(brain tissue infected w/ rabies)
Immunofluorescence Unfixed cryostat
6. Bouin’s Fixative
Enzyme Fresh frozen section
− excellent fixative for glycogen
histochemistry
− not suitable for DNA quantitative study→ effect in
nucleus of cell
Fixative of choice ACCORDING TO TISSUE
− Advantages: Tissue Fixative Time
a) It is a good fixative for connective tissue and Day-to-day 10% buffered Small tissue: 6h
glycogen. sample (routine) formalin Large tissue: 12-
b) Rapid penetration rate. 24h
Lymph node B5 solution 18h
− Disadvantages: GI tract 10% buffered 6h
a) It produces yellow stain to the tissue. formalin
• Removal of yellow color: tissue should be Testis 10% buffered
washed thoroughly in 70% ethanol. formalin or
• This yellow color can be removed by Bouin’s fluid
dipping the tissue in lithium carbonate in Bone Marrow Bouin’s fluid 3h
70% alcohol. Spleen Zenker’s fluid 6h
Mercury Salt-Containing Fixatives Eye 10% buffered 48h
− rapidly acting fixative formalin
− mercury: highly toxic
a) Zenker’s Fluid - good fixative for nuclear
chromatin and collagen Fixative of choice for DIFFERENT SUBSTANCES
b) Helly’s Fluid - good cytoplasmic fixative Target substance Fixative of choice
c) B5 Fixatives Protein 10% buffered formalin
Lipid Frozen section or osmium
tetroxide
Comparison of different fixatives
Glycogen Alcohol-based fixative
Mucopolysaccharide Frozen section
Enzyme
DNA & RNA Alcohol-based fixative
Iron

Fixation Artefact
− Artefact: unwanted compound produced due to
fixative
− Ex. formalin pigment→ black precipitate in cell
− unbuffered formalin →accumulation of formic acid
→ react to myoglobin or hemoglobin derivative→
acid hematin→ black precipitate
1 Formalin Pigment
2 Mercury Pigments
3 Fuzzy Staining
4 Prolonged fixation
5 Dichromate deposit
1. Formalin Pigment:

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Removing the pigment: -immediately fix

Steps: the tissue after
1. The section is immersed biopsy
in xylene followed by Insoluble Formalin Use buffered
alcohol to bring in water. brownish-black pigment due to formalin
2. Subsequently the section granular pigment acid
is immersed in 1.8% formaldehyde
picric acid in absolute ethyl alcohol for 15 min. haematein
3. It is then washed thoroughly. formation by
4. Section is re-stained. reaction w/
2. Mercury Pigments blood
− Mercury pigment: ex. buoin Intraepithelial Formalin may Keep formalin in
− Wash with Iodine→ high cleft formation evaporate, & closely capped
specificity w/ mercury calcium bottle
• Convert Mercury→ carbonate is
Mercuric iodide→ precipitated
▪ Wash with: Sodium
thiosulfate
▪ Thiosulfate: remove iodide *read fixative in Gregorio book
3. Fuzzy Staining
− Improper fixation either due to insufficient fixative
or too little time in fixative

4. Prolonged fixation
cause shrinkage of the tissue followed by separation
5. Dichromate deposit
difficult to remove

Formalin Pigment
Colour Brownish black
Nature Granular birefringent refractile
Position Extracellular
Mechanism of Formic acid reacts with haemoglobin
formation derivatives of the blood and produces
acid formaldehyde haematein
How to avoid Use buffered formalin
How to Treat with 1.8% picric acid in absolute
remove ethyl alcohol for 15 min.

Troubleshooting in fixation
Problems Cause Remedies
Nuclear margin is Incomplete -check the
indistinct & nuclei fixation concentration
are fuzzy w/ of formalin
bubbling -keep more time
in formalin for
fixation
-cut thin section
for fixation
-do not put too
many cassettes
together
Tissue shrinkage -Poor fixation -proper fixation
w/ large -Prolonged time
artefactual spaces fixation -check fixative
concentration

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