HISTOPATHOLOGY

Second Semester, 2022

Instructor: Rosevib Jacobe, RMT
Transes edited by:Ying
April 05, 2022

Examples:
 acetone
 alcohols
3. Coagulant Fixatives
FIXATION o Act by creating a network that allows
solutions to readily penetrate the interior of
the tissue.
Definition:
o killing, penetration, and hardening of tissues Examples:
o alteration of tissues by stabilizing protein so  Zinc salts
that the tissues become resistant to further  Mercuric chloride
changes.  Picric acid
Function of Fixatives:  Ethyl alcohol
o preservation of the tissues by stabilizing from  Methyl alcohol
destruction and post-mortem changes:  Acetone
 necrosis
 autolysis 4. Non-Coagulant Fixatives
 putrefaction (disintegration of tissue w/ o Create a gel that makes it difficult to
formation of foul odor) penetrate by subsequent solutions.
 action of saprophytic bacteria – o Important that sections in non-coagulant
decomposition fixatives be cut thinly for better penetration.
o harden the tissue
 renders the tissue firmer for proper FACTORS AFFECTING FIXATION
grossing and easy of cut thin sections for 1. Temperature
processing o increased fixation temperature accelerates
o act as mordant—preparatory for staining; the fixation of tissues but also increases the
staining is enhanced by fixation rate of autolysis.
o act as germicide/antiseptic  kill and prevents o temperature up to 45degree C has little effect
the growth of microorganism if present. on tissue morphology
o Fixation at room temperature have
Classification of Fixatives: satisfactory results
1. Additive Fixatives 2. Specimen size
o Chemically alter the tissue by bonding with it o specimens should be sent to the surgical
and adding themselves to the tissue. pathology laboratory immediately for proper
o May cause a change in electrical charge as attention by the pathologist
the site of attachment. o Tissue thickness important consideration
Examples: since fixative penetration is limited
 Formaldehyde o Tissues are sectioned at small intervals as
 mercuric chloride formalin penetrates at 3-4 mm/hour
 Chromium trioxide o 3mm sectioning is required
 Picric acid 3. Volume Ratio
 Glutaraldehyde o a critical factor and should be carefully
 Osmium tetroxide observed as much as possible
 Osmium tetroxide o Ideal ration of fixative to specimen is 15-20:1
 Zinc sulfate or chloride 4. Time
2. Non-Additive Fixatives o important on 2 aspects:
a. Cold Ischemia Time
 within 20-30 mins. after interruption
o Act on tissue without chemically combining
of the blood supply
with it.
 must be immersed in the fixative for
o Act by dissociating water from the tissue
no longer than 60 minutes from the
protein groups causing shrinkage and
time of interruption of blood supply.
hardening of the tissue if over fixation occurs
b. Fixation Time
o Predominantly organic compounds

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 HISTOPATHOLOGY
Second Semester, 2022
Instructor: Rosevib Jacobe, RMT
Transes edited by:Ying
April 05, 2022
 for a minimum of 6 hours and a - 1:9 dilution factor
maximum of 48 hours - does not precipitate factor
- does not harden or render albumin insoluble
OSMOLALITY and prevent subsequent hardening by
 Hypertonic gives rise to cell shrinkage alcohols
 Hypotonic/Isotonic causes cell swelling - neither preserves or destroys fat
 Fixative must be slightly hypertonic - good fixative for complex lipids but has no
solution effect on neutral fats
 Formaldehyde: - not the fixative of choice for carbohydrates, it
o 37-40% concentrated formalin preserves proteins so that they hold
o must be dilute in 10% solution glycogen, which is then not easily dissolved
HCHO colorless liquid or gas with a pungent
 Glutaryldehyde:
odor
o 3% normally used; others have
- immediate irritant to the eyes, nose, throat
buffer—effect to the tissue must be
- skin and respiratory system are particularly
balanced out
affected
 pH:
- safety precautions include proper ventilation
o 6-8%
and exhaust, limited or restricted exposure
periods and thorough washing if spilled on
CHOICE OF FIXATIVE
skin.
1. Tissue characteristic - universally accepted as an ideal fixative
a. Size and shape (directly proportional) - effect is considered to be a “soft” fixation
b. consistency of the tissue
2. Fixative factor a. 10% HCHO Routine
a. Ideal for the tissue: - good for CNS and
o Cheaper bone
o available Best for cadavers used
b. formol saline (NaCl
o stable in lab dissection
0.9%)
o isotonic
c. 10% buffered neutral Phosphate – basic buffer
o non-irritating
formalin For frozen section and
o rapid-acting
special staining
o strong penetration
d. Kaisserling’s Utilize 40% HCHO + KOH
+ KNO3
TYPES OF FIXATIVE:
- used in museum
I. According to Composition
fixation for long
II. According to Action
years of fixation
e. Glutaraldehyde Derivative of 40% HCHO
III. According to Composition
- used in electron
 formaldehyde microscopy and
 Glutaraldehyde enzyme
 Glyoxal histochemistry
 Mercuric Chloride
 Osmium tetroxide 2) Mercury – mercuric chloride (corrosive
Simple Aqueous Fixatives  Potassium sublimate, bichloride of mercury)
dichromate - penetrates rapidly and precipitates all
 Zinc Sulfate proteins
 Picric acid - can improve the histologic appearance of
 Acetic acid tissues
 others - has two main disadvantages:
Compound Aqueous 1. mercuric crystals – deposited in the
Fixative tissues, must be removed prior to
Non-aqueous Fixatives staining
2. highly poisonous substance, regulatory
Simple Aqueous Fixatives practices prohibit its disposal into
1) Formaldehyde – 37-40% HCHO sewage systems

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 HISTOPATHOLOGY
Second Semester, 2022
Instructor: Rosevib Jacobe, RMT
Transes edited by:Ying
April 05, 2022
- technique for disposing a fixative with - penetrate well and precipitates all proteins
mercuric chloride depends on precipitation - when used, needs thorough washing as it will
by thioacetamide. continue to react with the tissue structures
and will cause a loss of basophilia
a. Zenker  most popular a. Bouin—picric + 10% formalin + glacial
o mercury + excess glacial acetic acid acetic acid
and salts Na2SO4 and K2Cr207 --used for placenta, umbilical cord and
Uses of Zenker’s: developing organs during pregnancy
1. Connective of tissue fibers – 6) Osmic Acid  very good fixative for small (2-3mm3)
collagen specimens
2. Fixation of bacterial morphology  inorganic acid
3. Special staining – Trichome Staining can be used alone because of 3 ideal
Method characteristics:
b. Helly-Zenker’s formalin  glacial acetic acid is a. highly concentrated – used in electron
removed and replaced with 10% formalin. microscopy
Uses: b. rapid acting – used in frozen selection
1. For biopsy (common) c. strong penetration to the tissue – used for fats
 liver and spleen and lipids
2. For delicate organs
 bone marrow and pituitary  mixtures are not used in routine diagnostic
c. Haidenhain-Susa  Hg + 10% formalin + glacial histotechnology because it penetrates slowly
acetic acid + NaCl and unevenly
Uses:  tissue crumbles if embedded in paraffin
1. for all biopsy specimen except those  cause interference with many staining
used in Helly’s methods
d. Schaudinn  disadvantageous as have Hg + DISADVANTAGES:
absolute alcohol 1. Expensive
Uses: 2. Not easily available
1. temporary fixation of tissues o use only a small amount exception
to the rule (use 2x as much)
7) Chromic Acid (Chromium trioxide)  diluted and
3) Chromic  Potassium dichromate weak acid fixative
- fixes cytoplasm without precipitation -strong oxidizer that is used with other
- never used alone ingredients (can’t be used alone)
- after fixation specimens should be washed - chromic acid + little of osmic acid + K2Cr04
thoroughly to remove an oxide that forms a. Champy- for mitochondria and other
that cannot be removed later in processing cytoplasmic organelles
a. Orth- K2Cr04 + 10% formalin b. Flemming – for fats and lipids
Uses:
1. best for cytoplasmic organelle 8) Glacial Acetic Acid  can be used alone or with
2. for cell mytosis 10% formalin
b. Regaud’s/ Moller  K2Cr04 + glacial acetic - disadvantage: requires the use of concentrated glacial
acid (99.9%)
Uses:
1. for tissues containing yolk (ovary) 9) Acetone used at ice cold temperature from -
and yolk sac (embryo) 5degree C to 4degree C
4) Lead
a. Lillie – Pb+10% formalin + glacial acetic acid ADVANTAGES:
Uses:  Recommended for study of water diffusible
1. very good histochemistry fixative enzymes especially phosphatases and
2. preserve the chemical component of lipases
the cell  Used in fixing brain tissues for diagnosis of
ex: Acid mucopolysaccharide – rabies
connective tissue;
glycoaminoglycans are present
5) Picric leaves tissues soft

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 HISTOPATHOLOGY
Second Semester, 2022
Instructor: Rosevib Jacobe, RMT
Transes edited by:Ying
April 05, 2022
used as a solvent for certain metallic salts
to be used in freeze substitution - Tissue is placed in a primary fixative for
techniques for tissue blocks storage or may require further fixation for
DISADVANTAGES: special staining.
 produces inevitable shrinkage and
distortion Post-Chromatization
 dissolves fats - A form of secondary fixation
- a primarily fixed tissue is placed in aqueous
 Preserves glycogen poorly
of 2.5-3% potassium dichromate for 24hrs to
 evaporates rapidly
act as mordant for better staining effects and
to aid in cytologic preservation of tissues
Fixatives Washing-Out
ALCOHOLS: - Process of removing excess fixative from the
1. Ethyl Alcohol (95%) 75-95% tissue after fixation
a. Carnoy’s –95% alcohol = BEST - improve staining and remove artifacts from
o best fixative to preserve the tissues
lymphatic organs 1. tap water- remove excess chromates
b. Neukomer’s –75% alcohol (isopropyl) from tissues fixed in Kelly’s, Zenker’s ,
o propionic acid + acetone + petroleum and Flemminf’s solutions
ether and dioxane are added o remove excess formalin
o used for fixing nucleoprotein DNA- o excess osmic acid
RNA 2. 50-70% alcohol – used to wash out
c. Gendre’s –absolutue alcohol excess amount of picric acid (Bouin’s
o preserves glycogen but causes solution)
distortation to nuclear detail and 3. Alcoholic iodine – used to remove
shrinks cytoplasm excessive mercuric fixatives
Heat Fixation Microwave Technique
- procedure involves thermal coagulation of - works as a physical agent similar in
tissue proteins for rapid diagnosis mechanism to vacuum, oven (heat) and
- employed for frozen tissue sections and agitation to increase the movement of
preparation of bacteriologic smears molecules and accelerate fixation
- used to accelerate staining, decalcification,
ADVANTAGES: immunochemistry and electron microscopy
 Fixation is better - allows light microscoping techniques used in
 Preserves nuclear and cytoplasmic routine histopathology to be performed
detail adequately
 suitable for frozen tissue preparation
DISADVANTAGES: ADVANTAGES
 produces considerable tissue shrinkage  tissue is heated right through the block
and distortion in a very short time
 Destroys RBC  preserving neurochemical substances in
 Dissolves starch and glycogen brain (acetylcholine)
 for rapid fixation of routine surgical
Secondary Fixation: specimens
1. facilitate and improve the demonstration of  reduces the time taken for
particular substances immunochemistry and in situ
2. make special staining techniques possible (w/ hybridization
secondary fixative acting as mordant) DISADVANTAGES
3. ensure further and complete hardening and  Only penetrate tissue to a thickness of
preservation of tissue. 10-15 mm
- this may be done before dehydration and  no significant cross-linking of protein
on deparaffinized sections before molecules, and subsequent
staining, usually 10% formaline or 10%
 Viable spores and other pathogens may
formol saline as a primary fixative.
remain in tissues processed with
-

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 HISTOPATHOLOGY
Second Semester, 2022
Instructor: Rosevib Jacobe, RMT
Transes edited by:Ying
April 05, 2022
alcohol-based fixative or microwaving - Check the sharpness of scalpel, scissors
alone and quality of other ones like ruler , probes
weighing machines
- every instrument used in the laboratory
should meet electrical safety specifications
and have written instructions regarding its
use
- Flammable materials may only be stored
HISTOTECHNIQUES in approved rooms and only in storage
cabinets that are designed for this purpose
TISSUE PROCESSING:
HAZARDS
 Specimen Accessioning
 Gross Examination  Bouin’s solution is made with picric acid. This
 Fixation acid is only sold in the aqueous state. When it
 Tissue Processing dries out, it becomes explosive
 Sectioning  Many reagent kits have sodium azide as
 H and E staining preservative. You are supposed to flush
 Cover slipping solutions containing sodium azide down the
 Decalcification drain—with lots of water or there is a tendency
for the azide to form metal azides in the
 Artifacts in Histologic Sections
plumbing
 Problems in Tissue Processings
 These are also explosive
 Benzidine, benzene, anthracene and
SAFETY IN THE LAB napthtol containing compounds are
- the lab should be well illuminated and well- carcinogens and should not be used
illuminated  Mercury-containing solution—always be
- Rules and Regulations governing: discarded into proper containers
 formalin and hydrocarbons (such as  Mercury, if poured down a drain, will form
xylene and toluene amalgams with the metal that build up and
- Limits set by the Occupational Safety and cannot be removed.
Health Administration (OSHA) that should  Hazards of usually used formalin
not be exceeded.
- These limits should be revised and revived OBJECTIVE:
to reduce any mishap
- Every chemical compound used in the lab  Tissues from the body taken for diagnosis
should have a materials safety date sheet of disease processes must be processed in
the histopathology laboratory to produce
on file.
 specifies the nature microscopic slides that are viewed under
the microscope by pathologists.
 toxicity
 safety precautions to be taken when  The techniques for processing the tissues,
handling the compound whether biopsies, larger specimens
- The laboratory must have a method for removed at surgery, or tissues from
disposal of hazardous wastes. autopsy
 Health care facilities processing  The persons who do the tissue processing
tissues often contract this to a waste and make the glass microscopic slides are
management company. histotechnologists.
 tissues are collected should be stored
in formalin and may be disposed by Specimen Accessioning
incineration.  Request form
 by putting them through a “tissue  surgical pathology number (identify
grinder” attached to a large sink each specimen for each patient)
(similar to a large garbage disposal  record the patient’s name and number.
unit).

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 HISTOPATHOLOGY
Second Semester, 2022
Instructor: Rosevib Jacobe, RMT
Transes edited by:Ying
April 05, 2022

Penetration
 depends upon the diffusability of each
individual fixative, which is constant
 2-3 mm per hr thinly

Volume
 10:1 ratio

Temperature
 40 degreeC

Concentration of fixative
 formalin is best at 10%
 glutaraldehyde is generally made up at
0.25% to 4%

Time interval
 the faster you can get the tissue and fix it,
the better.

Preparation of 10% buffered neutral formalin

 4% formaldehyde or 10% buffered
formalin is commonly prepared by adding
100 ml 40% formaldehyde to 900ml
distilled water with 4g sodium phosphate,
monobasic and 6.5 sodium phosphate,
dibasic anhydrous.

Tissue Processing:
- the technique of getting fixed tissue into
paraffin.
- can be sectioned at anywhere from 3 to 10
microns, usually 6-8 routinely
- dehydryation
- clearing
- embedding

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