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DEPARTMENT OF HEALTH - TONDO MEDICAL CENTER

HISTOPATHOLOGY LECTURE NOTES

ERNY EMMANUEL DC. SARMIENTO, RMT, MLS (ASCPi)CM

→ Includes heating, microwaving, and cryo-preservation.

OUTLINE 2. Chemical Method
I. Histopathology F. Embedding → Achieved by immersing the specimen in the fixative
A. Biopsy G. Sectioning solution.
B. Autopsy H. Staining

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II. Tissue Processing I. Mounting BENEFITS OF FIXATION

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A. Fixation III. Staining Techniques
B. Decalcification A. H&E Staining 1. Allows thin sectioning of tissue by hardening the tissue.
C. Dehydration B. Papanicolauo’s Stain 2. Prevents autolysis and inactivates infectious agents.

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D. Clearing IV. Immunohistochemistry 3. Improves cell avidity for special stains.
E. Impregnation V. Figures

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PRACTICAL CONSIDERATIONS
1. Specimens should be transferred to fixative quickly in less than
LEARNING OBJECTIVES

(A
1 hour.
✔ Understand basic histologic concepts and techniques. 2. Tissues should be fixed in a sufficient volume of solution.
✔ Be familiar with different reagents and equipment used in Fixative to specimen ratio: 20:1 or at least 10:1.

LS
tissue processing. 3. The size of tissue blocks should be small enough to allow
✔ Answer different questions regarding tissue processing. adequate permeation of fixative through the perforations in
cassettes.

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I. HISTOPATHOLOGY MAIN FACTORS INVOLVED IN FIXATION

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● Histopathology is the diagnosis and study of diseases of the ● Volume
tissues, and involves examining tissues and/or cells under a

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→ 10-20 times the volume of the tissue to be fixed.
microscope. ● pH
→ 6-8
A. BIOPSY ● Temperature
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● A procedure to remove a piece of tissue or a sample of cells → Routine: 40℃
from a living body and tested in the laboratory. → Electron Microscopy: 0-4℃
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● Thickness
B. AUTOPSY → Routine: 2 cm2
→ Electron Microscopy: 1-2 mm2
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● Also known as post-mortem examination. ● Duration

● A specialized surgical procedure used to determine the cause → 24-72 hours
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and manner of death. ● Time Interval

● Tissues are examined from a dead body or a cadaver. → <1 hour
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II. TISSUE PROCESSING EFFECTS OF FIXATIVES IN GENERAL

● Describes the steps required to take an animal or human tissue 1. Reduce the risk of infection during handling and actual
.S

from fixation to the state where it is completely infiltrated with a processing of tissues.
suitable histological wax and can be embedded ready for 2. Harden soft and friable tissues and make the handling and
DC

section cutting on the microtome. cutting of sections easier.

3. Make the cells resistant to damage and distortion caused by
STEPS IN TISSUE PROCESSING the hypotonic and hypertonic solution used.
4. Inhibit bacterial decomposition.
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UE

FIXATION CHARACTERISTICS OF A GOOD FIXATIVE

● The first and most critical step in histotechnology. 1. Must be cheap.
● A process that preserves tissues from decay, thereby
AN

2. Must be stable.
preventing autolysis or putrefaction. 3. Must be safe to handle.
→ Autolysis 4. Must inhibit bacterial decomposition and autolysis.
▪ Tissue digestion by intracellular enzymes that are 5. Must permit rapid and even penetration of tissues.
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released when organelle membranes rupture. 6. Must harden tissues thereby making cutting of sections easier.
→ Putrefaction
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7. Must permit subsequent application of many staining

▪ Bacterial decomposition. procedures to facilitate easier and more profitable examination.
● Should be carried out as soon as possible after removal of the
tissues or soon after death. TYPES OF FIXATIVES
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● PRIMARY GOAL:
→ To preserve the morphologic and chemical integrity of the I. According to Composition
cell in as life-like manner as possible. A. Simple Fixatives
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● SECONDARY GOAL: 1. Aldehydes

→ To harden and protect the tissue from the trauma of further a. Formaldehyde
handling. b. Glutaraldehyde
2. Metallic Fixatives
OBJECTIVES OF FIXATION a. Mercuric Chloride
b. Chromate Fixatives
1. To preserve the tissues. 3. Picric Acid
2. To prevent the breakdown of cellular elements. = Imparts yellowish discoloration to tissues
3. To coagulate or precipitate protoplasmic substances. 4. Acetic Acid
5. Acetone
METHODS OF FIXATION 6. Alcohol
1. Physical Method 7. Osmium Tetroxide

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B. Compound Fixatives ● Advantages:

1. Formalin = It permits relatively good cytologic staining.
II. According to Action = It is a moderately rapid decalcifying agent.
A. Microanatomical Fixatives = Does not require washing out before
1. 10% Formol Saline dehydration.
2. 10% Neutral Buffered Formalin = Recommended for teeth and small pieces of
3. Heidenhain’s Susa bones.
4. Formol Sublimate ● Disadvantages:
5. Zenker’s Solution = The extent of decalcification cannot be
6. Zenker-Formol measured by a chemical test.
7. Bouin’s Solution C. Formic Acid
8. Brasil’s Solution 1. Formic Aicid-Sodium Citrate Solution
B. Cytological Fixatives ● Advantages:

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1. Nuclear Fixatives = It permits better nuclear staining than the nitric

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a. Flemming’s Fluid acid method.
b. Carnoy’s Fluid = Recommended for autopsy materials, bone
c. Bouin’s Fluid marrow, cartilage, and tissues studied for

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d. Newcomer’s Fluid research purposes.
e. Heidenhain’s Susa ● Disadvantages:

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2. Cytoplasmic Fixatives = It is relatively slow.
a. Flemming’s Fluid without Acetic Acid = It requires neutralization with 5% sodium sulfate.
b. Kelly’s Fluid D. Trichloroacetic Acid

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c. Formalin with “post-chroming” ● Advantages:
d. Regaud’s Fluid = It permits good nuclear staining.
e. Orth’s Fluid = It does not require washing out.

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3. Histochemical Fixatives ● Disadvantages:
a. 10% Formol Saline = It is a weak decalcifying agent.

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b. 100% Ethyl Alcohol = It is very slow-acting.
c. Acetone E. Chromic Acid (Flemming’s Fluid)
d. Newcomer’s Fluid ● Advantages:

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= Both fixative and decalcifying agents.

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DECALCIFICATION = May be used for decalcifying minute bone
spicules.
● Removal of calcium ions from a bone or calcified tissue
● Disadvantages:
through a histological process that makes them flexible and
= Nuclear staining with hematoxylin is inhibited.
easier to cut.
O,
= It tends to undergo reduction and forms
→ Not routinely carried out in the laboratory due to
precipitates at the bottom of the container, thus
receiving of calcified tissues is low to none.
requiring frequent changes of solution.
NT

DECALCIFYING AGENTS = Insoluble pigments are formed when decalcified

tissue is dehydrated with alcohol.
IE

I. Acid Decalcifying Agents = Degree of decalcification cannot be measured

A. Nitric Acid by the routine chemical test.
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1. Aqueous 10% Nitric Acid Solution F. Citric Acid-Citrate Buffer Solution (pH 4.5)
● Advantages: ● Advantages:
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= Rapid in action. = It permits excellent nuclear and cytoplasmic

= Produces minimum distortion of tissues. staining.
= Produces good nuclear staining. = It does not produce cell or tissue distortion.
.S

● Disadvantages: ● Disadvantages:
= Prolonged use may cause tissue distortion. = Its action is too slow for routine purposes.
= Can seriously damage tissue stainability. II. Chelating Agents
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= Imparts yellow color with nitrous acid. A. Neutral EDTA

2. Formol Nitric Acid ● Maintains bone tissue integrity and histological
● Advantages: features.
= Rapid acting. Recommended for urgent
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biopsies. DEHYDRATION
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= Nuclear staining is relatively good.

● Process of removing water from the specimen and replacing
= Produces less tissue destruction than 10%
it with dehydrating fluid in preparation for impregnation.
aqueous nitric acid.
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→ Removal of water, particularly fixatives, with the use of the

● Disadvantages:
so-called dehydrating agent in increasing strengths.
= The yellow color imparted by nitrous acid
formation will impair the staining reaction of the
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CHARACTERISTICS OF AN IDEAL DEHYDRATING AGENT

cell.
= The solution should be used inside a fume hood. 1. Should dehydrate rapidly without producing considerable
EM

3. Perenyi’s Fluid shrinkage or distortion of tissues.

● Advantages: 2. Should not evaporate quickly.
= Recommended for routine purposes. 3. Should be able to dehydrate even fatty tissues.
= Nuclear and cytoplasmic staining is good.
NY

4. Should not harden tissues excessively.

= Maceration is avoided due to the presence of 5. Should not remove stains.
chromic acid and alcohol. 6. Should not be toxic to the body.
● Disadvantages:
ER

7. Should not be a fire hazard.

= Slow decalcifying agent.
4. Phloroglucin Nitric Acid COMMONLY USED DEHYDRATING AGENTS
● Advantages:
= It is the most rapid decalcifying agent so far. 1. Alcohol
Recommended for urgent cases. ● Ethyl Alcohol (Ethanol)
● Disadvantages: → Recommended for routine dehydration of tissues.
= Nuclear staining is poor. → Most common and best dehydrating agent.
= Prolonged decalcification produces extreme → Boiling point: 78.3℃
tissue distortion. → Starts by placing the fixed specimen in 70% ethyl
B. Hydrochloric Acid alcohol, progressing through 95% ethyl alcohol, to
1. Von Ebner’s Fluid 100% ethyl alcohol.

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→ For delicate tissues (eg. Embryonic tissues), dehydration − Evaporates rapidly.

starting with 30% ethyl alcohol is recommended. − Dyes are not suitable in tetrahydrofuran.
→ Under no circumstance that ethyl alcohol be started
with a higher concentration. CLEARING
▪ Advantages: ● Also known as de-alcoholization.
− Non-toxic ● Process whereby alcohol or a dehydrating agent is removed
− Miscible in all proportions of water from the tissue and replaced with a substance that will
o Mixes with water dissolve the wax with which the tissue is to be impregnated and
− Can be used in eyes and embryos if graded mounted.
alcohols are used. ● Called “clearing” because most, if not all, clearing agents impart
− Fast acting optical clarity or transparency to the tissue due to their relatively
− Inexpensive and easily obtained. high refractive index.
▪ Disadvantages:

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● Most common: Xylene
− Long periods in absolute ethanol will cause

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excessive shrinkage and hardening.
CHARACTERISTICS OF A GOOD CLEARING AGENT
● Methyl Alcohol (Methanol) blood tissue
-

→ Toxic dehydrating agent. 1. Should be miscible with alcohol.

Pi
→ Recommended for blood tissue films and smear 2. Should be miscible with paraffin wax and mounting medium.
preparations. 3. Should not produce excessive shrinkage, hardening, or damage

SC
● Butyl Alcohol (Butanol) plant animal
- &
of tissue.
→ Recommended for plant and animal micro-techniques. 4. Should not evaporate quickly.
2. Acetone = rapid-actingapsies 5. Should make tissues transparent.

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● Rapid-acting dehydrating agent.
● Utilized for most urgent biopsies which dehydrates in ½ to 2 EXAMPLES OF CLEARING AGENTS
hours.

LS
▪ Advantages: 1. Xylene/Xylol commonly used
-most

− Rapid dehydrating agent. ● Colorless clearing agent that is most commonly used in
histology laboratories.

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− Does not extract methylene blue and other dyes
from stained sections. ● Clearing time is usually ½ to 1 hour.
− May cause less shrinkage of the specimen than ● Mostly used as clearing agent during tissue processing and

T,
ethanol. de-waxing agent during staining.
● Also used in cleaning tissue processors, removal of

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− Miscible with most embedding resins.
▪ Disadvantages: immersion oil from the microscope objective, and in recycling
− Volume must be 20 times that of the tissue. of used slides.
− Evaporates rapidly. ● Some toxicities have been reported such as:
→ CNS disorders
O,
− Flammable.
3. Dioxane -dehy & clean is
,
→ Respiratory depression
● Both dehydrating and clearing agents. → Abdominal pain
NT

● Produces less shrinkage of the tissues than ethyl alcohol. → Dryness and redness of skin
● May leave the tissue for a longer period of time. → Dermatitis
→ Liver diseases
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▪ Advantages:
− Universal solvent → Nephrotoxicity
→ Conjunctivitis
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− Miscible with water, alcohol, xylene, and paraffin.

− Faster dehydrant than ethanol. → Teratogenic and feto-toxic effects
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▪ Disadvantages: ● Advantages of Xylene:

− Needs large volume for dehydration. → Most rapid clearing agent, suitable for urgent biopsies.
− Costs about five times more than ethyl alcohol. → Clears within 15-30 minutes.
→ Makes tissues transparent.
.S

− Toxic
− Odorous → Miscible with both absolute alcohol and paraffin.
4. Cellosolve Irapid eas distortion → It is cheap.
DC

● Rapid dehydrating agent. ● Disadvantages of Xylene:

▪ Advantages: → Highly flammable and should be appropriately stored.
− Rapid dehydrating agent. → Makes tissues excessively hard if used >3 hours.
− Tissue may remain in it for months without injury. → Becomes milky when an incompletely dehydrated tissue
L

− Avoids distortion and does not require graded is immersed in it.

UE

solutions. → May irritate eyes, nose, and respiratory tract. It can be

▪ Disadvantages: absorbed through the skin and cause dermatitis.
− Expensive → Toxic and narcotic at higher concentrations
AN

− Rapidly absorbs water from the air. 2. Benzene -APLASTICANEMrecommended

5. Triethyl Phosphate ● Penetrates and clears tissues rapidly.
● Removes water very readily and produces very little ● Used to be a popular and best routine clearing agent until
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distortion and hardening of tissues. recently when its highly carcinogenic properties were
▪ Advantages: recognized.
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− May be used in routine paraffin technique. − Its use for clearing purposes is therefore strongly
− Does not harden tissues excessively. discouraged.
− May be used as a dehydrating solution in the ● Advantages of Benzene:
staining sequence. → Rapid acting, recommended for urgent biopsies.
NY

▪ Disadvantages: → Clears tissues within 15-60 minutes.

− None. → It volatiles rapidly in the paraffin oven and is therefore
easily eliminated from the tissue.
ER

6. Tetrahydrofuran -
dissolves fats

● Both dehydrating and clearing agent → Miscible with absolute alcohol.

● Can dissolve fats and is miscible with alcohol. → Does not make tissues hard and brittle.
▪ Advantages: → Causes minimum shrinkage.
− Miscible in all proportions with water, ether, → Makes tissues transparent.
chloroform, acetone, and the hydrocarbons xylene, ● Disadvantages of Benzene:
toluene, and benzene. → Highly flammable
− Rapid without excessive shrinkage and hardening. → Excessive exposure may cause APLASTIC ANEMIA.
− Not toxic. 3. Chloroform
▪ Disadvantages ● Slower in action than xylene but causes less brittleness.
− Odorous ● Tissues placed in chloroform do not become
o Should be used in a well-ventilated room. translucent.

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● The clearing agent being used at TMC Histopathology → Orientation

Section. ▪ The process by which a tissue is arranged in precise
● Advantages of Chloroform: positions in the mold during embedding, on the microtome
→ Recommended for routine work (6-24H). before cutting, and on the slide before staining.
→ Miscible with absolute alcohol.
→ Recommended for tough tissues. TYPES OF EMBEDDING MOLDS
→ Suited for large tissue specimens.
1. Leuckhart’s Embedding Mold Fig. 4
→ Not flammable.
● Consists of 2 L-shaped strips of heavy brass or metal.
● Disadvantages of Chloroform:
● The mold is adjustable to give a wide variety of sizes.
→ Relatively toxic to the liver after prolonged inhalation
2. Compound Embedding Unit Fig. 5
and may be prevented by adequate and proper room
● Made up of a series of interlocking plates resting on a flat
ventilation.
metal base, forming several compartments.
→ Does not make tissues transparent.

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● Has the advantage of embedding more specimens at a time.
IMPREGNATION/INFILTRATION 3. Plastic Embedding Rings Fig. 6 and Base Molds

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● Most common type of mold used in routine histopath
● Process whereby the clearing agent is completely removed from laboratory.

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the tissue and replaced by a medium that will completely fill all ● Made up of plastic material or oftentimes a stainless metal to
the tissue cavities and give a firm consistency to the specimen. maintain the temperature of melted paraffin.

SC
● Allows easier handling and cutting of suitably thin sections 4. Disposable Embedding Molds
without any damage or distortion to the tissue and its cellular ● Peel-away, disposable, thin plastic molds.
components. i. Plastic Ice Trays

(A
ii. Paper Boats Fig. 7 – Cheap and easy to make.
EMBEDDING
MICROTOMY
● A.k.a casting/blocking.

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● Process by which the impregnated tissue is placed into a ● Also known as cutting/sectioning.
precisely arranged position in a mold containing a medium ● Process by which processed tissue is trimmed and cut into

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which is then allowed to solidify. uniformly thin slices or “sections” to facilitate studies under the
microscope.
CHARACTERISTICS OF A GOOD INFILTRATING AGENT AND ● Microtome is the basic tool used.

T,
EMBEDDING MEDIUM

RM
TYPES OF MICROTOMES
1. Suitable for sectioning and ribboning.
2. Molten between 30℃ and 60℃. 1. Rocking Microtome Fig. 2
3. Translucent or transparent. ● Invented by Paldwell and Trefall.
4. Capable of flattening after ribonning. ● Simplest among other types of microtomes.
O,
5. Non-toxic. ● For cutting serial sections of large blocks of paraffin
6. Odorless. embedded tissues.
NT

7. Easy to handle. 2. Rotary Microtome Fig. 1

8. Inexpensive. ● Invented by Minot in 1885-1886.
● Most common type of microtome used for both routine and
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TYPES OF INFILTRATING AGENT AND EMBEDDING MEDIUM research laboratories.

● Electrically driven rotary microtomes are also now available
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1. Paraffin Wax and can be ideally used to produce ribbons for serial
● Simplest, most common and best infiltrating and sections.
AR

embedding medium. 3. Sliding Microtome celloidin

-

● Solid at room temperature but melts at about 60-70℃. ● Invented by Adams in 1789
→ Melting points differ depending on the manufacturer, but it ● For cutting celloiding sections.
.S

is usually 60℃. → Base-Sledge Sliding Microtome

● Traditionally, it is advised to use paraffin wax above 2℃ − Has two movable pillars holding the adjustable knife
DC

above its melting point. clamps.

● Advantages of Paraffin Wax: → Standard Sliding Microtome
→ Thin, individual serial sections may be cut with ease. − The block remains stationary while the knife is moved
→ The process is rapid. backward and forward during the process of
L

→ Tissue blocks may be stored in paraffin for an sectioning.

UE

indefinite period of time after embedding without tissue − Most dangerous microtome.
destruction. 4. Freezing Microtome
● Disadvantages of Paraffin Wax: ● Invented by Queckett in 1848
AN

→ Overheated paraffin makes the specimen brittle. ● A simple lever operated valve allows the release of rapid,
→ Prolonged infiltration will cause excessive tissue intermittent bursts of CO2 which will freeze the block holder
shrinkage and hardening, making the cutting of tissues and the tissues evenly.
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difficult. 5. Cryostat or Cold Microtome

→ Inadequate impregnation will cause tissues to crumble ● The cryostat is a refrigerated apparatus used for freezing the
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when sectioned and floated-out in a water bath. tissue into the block holder.
2. Celloidin = laraz ,hollowyo eyes
,
● Consists of a microtome, usually a rotary microtome, kept
● Suitable for specimens with large, hollow cavities which tend inside a cold chamber which has been maintained at a
to collapse. temperature between -5℃ to -30℃ (average is -20℃)
NY

● For hard and dense tissues such as bones and teeth and for ● Capable of freezing fresh tissues within 2-3 minutes and
large tissue sections of the whole embryo, eyes, and brain. cutting sections of 4µ with ease.
3. Gelatin Frozentissuea ● Routinely used for urgent/STAT biopsies.
ER

● Rarely used in routine processing except when dehydration 6. Ultrathin Microtome

is to be avoided. ● Equipped with a glass or gem-grade diamond knife.
● Used as an embedding medium for delicate specimens and ● Used to cut very thin sections typically 60 to 100 nano
frozen tissue sections. micron.

EMBEDDING (TECHNIQUE) MICROTOME KNIVES

● The tissue is placed into a mold containing the embedding 1. Plane-Concave Knife
medium and this medium is allowed to solidify. ● 25mm in length.
● Immersed in melted paraffin wax at a temperature between ● One side of the knife is flat while the other is concave.
5℃ and 10℃ above its melting point and then cooled → Less concave celloidin
-

rapidly at -5℃.

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− Recommended for cutting celloidin-embedded tissue

blocks. CHARACTERISTICS OF A GOOD MOUNTING MEDIUM
→ More concave paraffin
-

1. Should be colorless and transparent.

− Recommended for cutting paraffin-embedded tissue 2. Should be freely miscible with xylene and toluene.
blocks. 3. Should not dry to a non-stick consistency and harden relatively
2. Biconcave Knife paraffin
-

quickly.
● 120mm in length. 4. Should protect the section from physical damage and chemical
● With both sides concave. activity.
● Recommended for cutting paraffin-embedded sections on a 5. Should be resistant to contamination.
rotary microtome.
3. Plane-Wedge Knife Frozen sections ; hard tough spx
-
a

COMMONLY USED MOUNTING MEDIA

● 100mm in length.
● Have both sides straight. 1. AQUEOUS MOUNTING MEDIA

M
● Recommended for frozen sections or for cutting extremely ● Water

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hard and tough specimens embedded in paraffin blocks. → Has low refractive index of 1.33
→ Evaporates easily.
TRIMMING → Only good for temporary mounting.

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● Glycerin
● Removal of excess paraffin wax from embedding to facilitate
→ Refractive index: 1.46

SC
cutting and for the blocks to fit the block holder of the
→ Slow to dry.
microtome.
● Farrant’s Medium
▪ Coarse Trimming
→ Refractive index: 1.43

(A
− Heated spatula is held between the tissue block and
● Apathy’s Medium
the block holder until the wax begins to melt.
→ Refractive index: 1.52
▪ Fine Trimming

LS
→ Used for methylene blue-stained nerve preparations.
− May be done by either setting the thickness adjuster at
● Brun’s Fluid
15mm or by advancing the block using the coarse feed
→ Refractive index: 1.52
mechanism.

M
→ Recommended for mounting frozen sections from water.
− The knife is usually tilted at 0-15° angulation on a
2. Resinous Mounting Media
microtome.
● Canada Balsam

T,
→ Refractive index: 1.524
OTHER EQUIPMENTS USED IN MICROTOMY

RM
→ Extracted from a Canadian tree, Abus balsamea.
● Water Bath → Most common in routine histologic sections.
→ Used to stretch the tissue ribbons after sectioning. ● DPX (Dibutyl Pthalate and Xylene)
→ Water should be between 5℃ and 10℃ below the melting → Refractive index: 1.532
O,
point of the paraffin wax. → Recommended for small tissue sections.
● Drying Oven or Hot Plate ● XAM
NT
→ Used to fix the tissues on the glass slide and also used for → Refractive index: 1.52
deparaffinization of wax surrounding the tissue. ● Clarite
● Adhesives → Refractive index: 1.544
IE

→ A substance which can be smeared on to the slides so that

the sections stick well to the slides. III. STAINING TECHNIQUES
M

▪ Meyer’s Egg Albumin

1. Progressive Staining
− Most common adhesive used.
AR

● The process whereby tissue elements are stained in a

− One part egg white, one part glycerin.
definite sequence until the desired intensity of coloring is
o Glycerin is added to the albumin to prevent molds
attained.
during storage.
2. Regressive Staining
.S

● Clean, Frosted Slides

● The process whereby the tissue is first overstained and
→ Slides must be clean.
the excess stain is removed or decolorized from
→ Frosted slides are recommended for all histotechnique/tissue
DC

unwanted parts of the tissue.

processing to facilitate initial labeling.
● Routine Hematoxylin and Eosin staining is the most
common method.
MUST KNOW 3. Metachromatic Staining
L

● Technique which entails the use of specific dyes which

UE

● BEVEL ANGLE differentiate particular substances by staining them with a

→ 0-15° color that is different from that of the stain itself
● THICKNESS OF SECTION FOR ROUTINE PROCESSING (metachromasia).
AN

→ 4-6µ 4. Metallic Impregnation Staining

● WATER BATH TEMPERATURE ● The staining process where specific tissue elements are
→ 5-10℃ below the melting point of the paraffin wax. demonstrated.
M

● DRY OVEN TEMPERATURE 5. Vital Staining

→ Melting point of paraffin wax (usually 60℃) ● A procedure where living cells take up certain dyes which
EM

selectively stain some elements in cells such as

mitochondria, lipid vesicles, and lysosomes.
STAINING
6. Intravital Staining
● Tissue processing step whereby a stain is introduced to the ● The staining process in which live-cell dyes and other
NY

tissue before studying such tissue under the microscope. labels are injected into the bloodstream before fixation of
● It allows observation and differentiation of cells under the the tissues.
microscope. 7. Supravital Staining
ER

● It is widely used in histopathology and diagnosis, as it allows for ● The process in which live-cell dyes and other labels are
the identification of abnormalities in cell count and structure injected into the bloodstream before the fixation of tissues
under the microscope. demonstrating specific parts of the living cell.

MOUNTING HEMATOXYLIN & EOSIN STAINING

● The last step in tissue processing which results in a ● The cornerstone of tissue-based diagnosis.
permanent histological preparation suitable for microscopy. ● The process uses a hematoxylin dye to stain cell nuclei blue
● Application of cover slip/cover glass with the use of a and an eosin dye to stain other structures pink or red such as
mounting medium/mountant for permanent protection of the the cytoplasm.
histological sample.

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→ 2 minutes
H & E STAINING STEPS
1. Xylene IV. IMMUNOHISTOCHEMISTRY
→ 3 minutes ● Method for localizing specific antigens in tissues or cells based
2. Xylene on antigen-antibody recognition; it seeks to exploit the
→ 2 minutes specificity provided by the binding of an antibody with its antigen
▪ The first 2 submersions to xylene are for at a light microscopic level.
deparaffinization.
3. 100% Ethyl Alcohol A. HISTOCHEMISTRY
→ 2 minutes
4. 95% Ethyl Alcohol ● A science that combines the techniques of biochemistry and
→ 2 minutes histology in the study of the chemical constitution of tissues and

M
▪ These 2 concentrations of alcohol are used for cells. Cell identification.
rehydration of the tissues prior to introduction of

)C
stains. B. IMMUNOLOGY
5. Running Water
● A science that deals with the immune system, cell-mediated and

Pi
6. Hematoxylin Stain
→ 15-30 minutes humoral aspects of immunity and immune responses.

SC
▪ The primary stain which stains the nuclei part of
the cell blue. ANTIBODIES AS SPECIFIC STAINING REAGENTS
7. Running Water ● IHC techniques exploit the fact that immunoglobulin

(A
8. Acid Alcohol molecules can serve both as antibodies and as antigens.
→ 15-30 seconds ● Evaluation of an antibody for use in IHC is based on two main
▪ Used for decolorizing the excess hematoxylin stain. points:

LS
▪ It is very important not to over stain the tissue as it → Sensitivity
may affect the appearance of the stained nuclei. ▪ The selective amount of antigen that an IHC technique is
9. Running Water able to detect.

M
10. Ammonia Water → Specificity
→ Usually, a couple of dips until you notice that the tissue on ▪ The characteristic of the antibody to bind selectively to a

T,
the slide turns bluish. single epitope on an antigen.
▪ Acts as a bluing agent

RM
▪ Used to intensify the blue color stained in the nucleus.
HISTOPATHOLOGY AND IMMUNOHISTOCHEMISTRY
11. Running Water
SUBMISSION GUIDELINES
12. Eosin Stain
→ 5 minutes 1. Fixation and Storage
O,
▪ Eosin Y is the type of eosin used. ● 10% Neutral Buffered Formalin in non-breakable plastic
▪ Secondary stain which stains the cytoplasm pink or containers.
NT
red. ● FFPE block with the best representative sample of the
13. Running Water tumor.
14. 95% Ethyl Alcohol 2. Previous biopsy report.
IE

→ 2 minutes 3. Previous H&E-stained histopathologic slides.

15. 100% Ethyl Alcohol 4. Completely filled request form.
M

→ 2 minutes → Imaging
▪ Ascending concentrations of alcohol is used for → Type of fixation used
AR

the dehydration of the stained tissue. → Name of the IHC marker(s) requested
16. Xylene → Name and contact information of the referring physician
→ 2 minutes
.S

▪ The use of xylene in the last part of the staining TISSUE COLLECTION
process is for further deparaffinization of the tissue.
● Tissue samples must be obtained from:
DC

PAPANICOLAOU STAINING → Punch/Core needle biopsy.

→ Excisional/ Incisional biopsy.
● Also known as Pap’s staining/Pap’s smear. ● The sample preparation stage begins as soon as a piece of
● Is a histological and cytopathological staining technique used to tissue is removed from its nutritional source.
L

differentiate cells in a smear preparation.

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● The most common screening method for cervical cancer. ANTIGEN RETRIEVAL
● Specimen: Vaginal discharge.
● Enables the partial reversal of formaldehyde-induced
AN

PAPANICOLAUO’S STAINING STEPS conformational change in antigens.

● Increases the accessibility of the antibody to the antigen.
1. 95% Ethanol ● Carried out with two methods:
M

→ 10-15 minutes → Heat (HIER)

2. Running Water ▪ Critical factors:
EM

3. Hematoxylin Stain − Temperature

→ 1-3 minutes − pH
4. Running Water − Time of incubation
5. 95% Ethanol ▪ Achieved by:
NY

→ 10 dips − Microwave oven

6. OG Stain − Pressure cooker
→ 1.5 minutes − Vegetable steamers
ER

7. 95% Ethanol − Water bath

→ 10 dips − Automated immuno stainers
8. EA-50 Stain → Enzyme digestion (PIER)
→ 2.5 minutes ▪ Enzymatic activity: pronase, pepsin, ficin, trypsin or
9. 95% Ethanol proteinase K unmask antibody epitopes.
→ 10 dips ▪ Efficacy depends upon:
10. 100% Ethanol − Enzyme concentration
→ 1 minute − Incubation time
11. Xylene ▪ Digestion breaks down formalin cross-linking and hence
→ 2 minutes the antigenic sites.
12. Xylene

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− Under-digestion results in too little staining, because ▪ Produces red or blue precipitates.
the antigens are not fully exposed. ● Detection systems:
− Over-digestion can produce “false-positive” staining, → Direct Method
high background levels and tissue damage. ▪ Labeled Ab reacts directly with Ag in tissue sections
● Choice of antigen retrieval depends on the antigen to be ▪ Single step method
demonstrated. ▪ Short and quick
▪ Insensitive due to little signal amplification
BLOCKING NONSPECIFIC BACKGROUND STAINING → Indirect Method
▪ Unlabeled Primary Ab reacts with Ag and the labeled
● Before using specific antibodies to detect antigens by secondary Ab reacts with the primary Ab.
immunohistochemistry (IHC), all potential nonspecific binding ▪ Sensitive due to signal amplification.
sites in the tissue sample must be blocked to prevent ▪ Economical as single secondary Ab can be used against
nonspecific antibody binding. many Abs from same species.

M
● Two aspects of blocking nonspecific background staining: − PAP/APAAP method
→ Non-Specific Antibody Binding

)C
− ABC method
▪ Problem with Polyclonal Antibody − Streptavidin- Peroxidase Method
▪ Antibodies are highly charged molecules and may bind ● Chromogens:

Pi
non-specifically to tissue components. → 3,3 α- diaminobenzidine tetrahydrochloride (DAB)-
▪ May lead to “false-positive” staining. Brown

SC
▪ Reduced by pre-incubation with normal serum. → 3- amino-9-ethylcarbazole – Red
→ Presence of Endogenous Enzymes → 4-chloro-1-naphthol – Blue
▪ Peroxidase and alkaline phosphatase. → Hanker- Yates reagent – Dark Blue

(A
▪ May lead to “false-negative” staining. → Αlpha naphthol pyronin – Red-purple
▪ Done before the addition of enzymes labeled secondary
agents. COUNTER STAINING

LS
▪ 3% Hydrogen peroxide, cyclopropenone hydrate- used to
inhibit peroxidase activity. ● Provides contrast to the primary stain.
● Most commonly used:

M
PRIMARY ANTIBODY REACTION → Hematoxylin and Eosin Staining
▪ Gold standard

T,
● A process whereby the primary antibody binds to the → Hematoxylin
antigen, followed by a second (labeled) antibody binding the ▪ Stains nucleic acids blue

RM
primary. → Eosin
● Two types of antibodies used: ▪ Stains cytoplasmic components red
→ Monoclonal antibodies
▪ Homogenous population of antibodies directed against a CONTROLS
O,
single epitope.
▪ Does not guarantee antigen specificity. ● Positive controls in immunohistochemistry protocols are
NT
→ Polyclonal antibodies specimens containing the target molecule in its known location
▪ Heterogenous mixture of antibodies directed against and whose histomorphology and cytomorphology can be
various epitopes of the same antigen. visualized by a “stain.”
IE

▪ Often give more nonspecific background staining in ● Positive Control

slides. → Antigen (analyte)
M

▪ Non-patient tissue or cells containing antigen to be

Monoclonal Antibodies Polyclonal Antibodies detected and quantified
AR

▪ Known expected result

Mouse or Rabbit Hybridoma Many different species (mostly ▪ Fixed-processed in same way as patient sample
rabbits) ▪ Fixed processed in manner shown to preserve antigen
.S

under analysis
→ Antibody (reagent)
Cleaner Have more non-specific
▪ Antibody reagent constituted in same way as intended for
DC

reactivity
patient sample
→ Purpose
Increased false-negative results More likely to have success in ▪ Control of all steps of the analysis
if target epitope is damaged or an unknown application
L

▪ Training use for appearance of positive reaction;

altered comparison for semi-quantitation of reaction
UE

▪ Validates all steps of analysis, including fixation and

Expensive to produce Inexpensive processing
▪ Validates all steps of analysis, except fixation or
AN

Recognized only ONE epitope Recognized MULTIPLE processing used by individual laboratory
on an antigen epitopes on one antigen ● Negative (specific)
→ Antigen (analyte)
M

Can produce large amount of Produces large amounts of ▪ Tissues or cells expected to be negative of antibody
EM

SPECIFIC antibodies NON-SPECIFIC antibodies ▪ Processed in same way as patient sample

▪ May be portion of patient sample
→ Antibody (reagent)
DETECTION METHODS ▪ Antibody reagent constituted in same way as intended for
NY

patient sample
● Ag-Ab conjugates are visualized by the use of a label. → Purpose
● Enzymes that produce a colored precipitate in the presence of a ▪ Detection of unintended antibody cross-reactivity to cells
ER

substrate are used as labels or cellular components

● Labels: ● Negative (nonspecific)
→ Horseradish Peroxidase → Antigen (analyte)
▪ Most widely used in combination with DAB – ▪ Patient tissue with components that are the same as
produces brown precipitate. tissue to be studied
▪ It is small and does not hinder the binding of antibodies to ▪ Processed in same way as patient sample
adjacent sites. → Antibody (reagent)
▪ Chance of contamination is minimized. ▪ Diluent (as used with antibody) without antibody
▪ Stable enzyme. ▪ Antibody not specific for antigen of interest in same
▪ Endogenous activity is easily quenched. diluents as used with kit antibody
→ Alkaline Phosphatase → Purpose
▪ Most widely used alternative enzyme tracer. ▪ Detection of unintended background staining

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ER, PR, HER2 ALLRED SCORE

● Hormone receptor status markers used to guide treatment ● A semi-quantitative method for evaluating estrogen receptor
options for breast cancer. (ER) status in breast cancer.
● In the case of breast cancer treatment, a crucial step is to test
the tumor tissue to determine if it has estrogen receptors (ER),
progesterone receptors (PR), and/or human epidermal
growth factor receptor 2 (HER2).
● These markers provide key information about how the
cancer may behave.
● Along with tumor grade and cancer stage, tumor marker status
helps determine the best treatment options for breast

M
cancer patients. ER, together with PR, has been recognized as
a “predictive” marker for which women with breast cancer would

)C
respond to hormonal treatment.
● The current clinical practice for the testing uses either Allred

Pi
score or H-score which is based on laborious manual counting
and estimation of the amount and intensity of positively stained

SC
cancer cells in immunohistochemistry (IHC)-stained slides. Both
will be discussed later.
● Can be done on Needle biopsy of the breast and fine needle

(A
aspiration (FNA) cytologic techniques.
● Hormone Receptor Status ; Hormonal Therapy Benefit

LS
HORMONE RECEPTORS
● ERs and PRs bind hormones that exert their effects in the

M
nucleus.
● Nuclear immunostaining can be demonstrated in normal breast

T,
acini Internal controls for the testing procedure.
● PR-positive:

RM
→ <5%; longer disease-free survival.
● IHC assay determination of ER/PR levels has replaced the
dextran-coated charcoal (DCC) method (1990) HER2/neu
● DCC Method Drawbacks:
O,
→ Tumor sampling error ● The ERBB2 (HER2) gene was originally called NEU as it
→ Heavy reliance on obtaining tissue immediately was first derived from rat neuro/glioblastoma cell lines. It
NT
→ Normal tissue contamination was named HER2 because its primary sequence was very
→ Analytic error similar to human epidermal growth factor receptor (EGFR or
● IHC Method Advantages: ERBB or ERBB1)
IE

→ Histologic documentation of the tumor tissue to be assayed ● ERBB2 gene product was precipitated from adenocarcinoma
→ Appreciation of the heterogeneity of ERs and PRs in tumor cells and was demonstrated to be a 185-kD glycoprotein with
M

cell nuclei tyrosine kinase activity.

→ Rapid assessment of tissue for ERs and PRs ● Approximately 15% to 20% of breast cancers demonstrate
AR

→ Rapid turnaround time HER2 gene amplification or protein overexpression.

→ Lower cost ● Trastuzumab is a monoclonal antibody to HER2 that was
→ Ability to use minute quantities of tissue. approved by the FDA in 1998 for use in metastatic breast
.S

● LUMINAL (ER-POSITIVE/HER2-NEGATIVE) cancer. It improves response rates, time to progression, and

→ Nucleus survival when used alone or in combination with chemotherapy
→ Regulated by estrogen in the treatment of metastatic breast cancer.
DC

→ ER-positive/PR-positive ● In the absence of adjuvant systemic therapy, HER2-positive

→ BRCA-2-associated breast cancer patients have a worse prognosis (i.e., a higher
→ Core biopsy Negative repeat HR analysis for resection rate of recurrence and mortality), which clearly demonstrates
L

specimen the prognostic significance of this gene. An even more

UE

important aspect of determining HER2 status is its role as a

H SCORE predictive factor. HER2 positivity is predictive of response to
anthracycline- and taxane- based therapy, whereas the benefits
● Captures both the intensity and the proportion of the biomarker
AN

derived from non-anthracyclines and non-taxane therapy may

of interest from the IHC image and comprises values between 0 be inferior.
and 300, thereby offering a dynamic range to quantify biomarker ● Antibody Used:
abundance. → CB11 and 4D5 antibodies
M
EM

COMMON DISEASES ASSOCIATED WITH ER, PR, HER2

1. ER (Estrogen Receptor)
● Positive
NY

→ Breast, Mucinous Carcinoma

→ Breast, Tubular Adenoma
→ Invasive Ductal Carcinoma
ER

● Negative
→ Breast carcinomas (triple negative)
→ Endocervical adenocarcinoma
→ Ovarian clear cell carcinoma
2. PR (Progenitor Receptor)
● Positive
→ Fibroadenoma
→ Myofibroblastoma
→ Phyllodes tumors
→ Pseudoangiomatous stromal hyperplasia
→ Breast carcinoma (well differentiated tumors: lobular,
mucinous/colloid, sebaceous)

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● Negative Figure 5. Compound Embedding Mold

→ Apocrine metaplasia and carcinomas
→ Microglandular adenosis
→ Myoepithelium and myoepithelial tumors
→ Breast carcinomas (triple negative)
3. HER2 (Human Epidermal Growth Factor Receptor 2)
● Positive
→ Ductal Carcinoma in situ
→ Invasive Ductal Carcinoma
→ Invasive Breast Carcinoma of No Special Type
→ Metastatic Breast cancer
4. Triple Negative (Negative to ER, PR, HER2)
→ Secretory carcinoma

M
→ Medullary carcinoma Figure 6. Plastic Embedding Ring

)C
→ Adenosquamous carcinoma
→ Squamous cell carcinoma
→ Fibromatosis like metaplastic carcinoma

Pi
V. FIGURES

SC
(A
LS
Figure 7. Paper Boat

REFERENCES

M
✔ Histopathologic Techniques by Jocelyn H. Bruce-Gregorios,

T,
MD
Figure 1. Rotary Microtome ✔ www.microbenotes.com

RM
✔ www.ncbi.nml.nih.gov
✔ Dabbs DJ. Diagnostic Immunohistochemistry: Theranostic
and Genomic Applications. 3rd edition. 2010. Elsevier.
Chapter 1.
O,
✔ Immunohistochemistry METHODS. (n.d.).
https://www.slideshare.net/DRKALPAJYOTI/immunohistoche
NT

mistry-methods
✔ Basics of Immunohistochemistry (IHC). (n.d.).
https://www.slideshare.net/vbiogenex/basics-of-immunohistoc
IE

hemistryihc
✔ Immunohistochemistry Guide - Creative Diagnostics. (n.d.).
M

Figure 2. Rocking Microtome https://www.creativediagnostics.com/Immunohistochemistry-g

uide.htm
AR

✔ Case: TMC-23-650, IHC-23-001

.S
DC
L
UE

Figure 3. Autotechnicon
M AN
EM
NY

Figure 4. Leuckhart’s Embedding Mold

ER

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