MEDICAL TECHNOLOGY ASSESSMENT PROGRAM

MEDICAL LABORATORY SCIENCE DEPARTMENT

SAINT LOUIS UNIVERSITY

SPECIAL TECHNIQUES IN HISTOPATHOLOGY

Introduction:
Routine H&E staining and special stains play a critical role in tissue-based diagnosis or research. By
staining transparent tissue sections, these stains allow highly trained pathologists and researchers to view,
under a microscope, tissue morphology (structure) or to look for the presence of particular cell types,
structures or even microorganisms such as bacteria.

In the histopathology laboratory, the term “routine histopathological technique” refers to the
procedures involving hematoxylin and eosin stain (H&E) that is used “routinely” with all tissue specimens
to reveal the underlying tissue structures and conditions. The term “special histopathological technique”
has long been used to refer to a large number of alternative staining techniques and alternative procedures
which allow many innovations such as fast diagnosis during an intraoperative procedure (frozen
techniques), detection of antigen markers found in tissues (IHC), and demonstration of tissue components
when the H&E does not provide all the information the pathologist or researcher needs.

The following special histopathological techniques will be discussed for this lecture:
A. Immunohistochemistry
B. Frozen Section Technique
C. Special Stains for special tissues

I. IMMUNOHISTOCHEMISTRY
A. INTRODUCTION
Immunohistochemistry (IHC) is used in histology to detect the presence of specific protein marker
that can assist with accurate tumor classification and diagnosis. IHC is used as a diagnostic tool to assist in
the diagnosis of solid tumors and cytological specimens.
IHC can be broadly classified into two forms based on the type of tissue processing involved:
a. IHC- formalin-fixed, paraffin-embedded (FFPE)
b. IHC-frozen (Fr)

IHC-FFPE IHC-Fr
Fixation Performed before embedding Can be performed before or after
into paraffin wax. cryo-sectioning.
Common Formaldehyde Formaldehyde or Alcohols
Fixative
Sectioning Microtome, 4-10 μm sections Cryostat/Cryotome, 5-20 μm sections
Storage Ideally fresh sections should be cut after Short-term-
4 weeks due to loss of antigenic epitopes. 1 year at -80oC.
Fresh cut sections should not be used
after 1 month. For long term storage
(several years), coating of slide in
paraffin is recommended.
Major Ease of handling and preserves structural Preserves enzyme & antigen function.
Advantage morphology. Useful for study of post-translationally
Blocks can be stored long term. modified protein, DNA, or RNA.
Major Variable fixation times. Fixation can mask Less optimal for studying structural
Limitations epitopes. morphology. Ice crystals can impact
tissue structure.

Tissue should be cut into 10 mm thick and then immersed in fixative solution, 50-100X the tissue
volume, for effective diffusion of the fixative.

B. PRINCIPLE
Immunohistochemistry (IHC) is a method for detecting antigens or haptens in cells of a tissue
section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.
Immunohistochemistry (IHC) uses antibodies to detect cell and tissue proteins and provide semi-
quantitative data about target protein expression, distribution, and localization. Tissues are sectioned from
fixed embedded (e.g. IHC-Paraffin or plastic) or frozen blocks (e. g. IHC-Frozen), and the sections are then
probed with primary antibodies against the antigens of interest.
Target expression can be evaluated with the corresponding labeled primary antibody (direct detection) or,
more commonly, with the addition of labeled secondary antibodies (indirect detection). The antibody-
antigen binding can be visualized in different manners. Enzymes, such as Horseradish Peroxidase (HRP) or
Alkaline Phosphatase (AP), are commonly used to catalyze a color-producing reaction. The label, either
fluorescent or enzymatic, is used to visualize the antigen-antibody complex.

C. MATERIALS
1. Primary Antibody
The first stage of IHC is the application of a primary antibody that binds specifically to the target antigen.
There are two main types of antibody, polyclonal and monoclonal.
a. Polyclonal antibodies have an affinity with, and bind to, multiple epitopes (or parts) or the target
antigen, and as such are more prone to cross-react to non-target antigens.
b. Monoclonal antibodies have an affinity to only one epitope and tend to produce cleaner, more
specific staining but are less sensitive or intense.

2. Secondary Antibody
Next, secondary antibodies bind to the primary antibody. This is known as indirect IHC.
 3. The Detection System
The detection system builds on the secondary. Modern chromogenic detection utilizes enzymes such as
Horseradish Peroxidase (HRP) that are conjugated (joined) to an antibody. Multiple enzymes attached to
the antibody are known as polymers, and they again produce more intense staining as there are more
molecules for the chromogen to attach.

4. Chromogen
Finally, a substrate forms an insoluble colored precipitate that can be visualized under a microscope. There
are two commonly used chromogens:
a. DAB (brown): DAB is used for most applications as it provides strong and permanent stains.
b. AP (red): AP Red (or another red chromogen) is used mainly for skin sections where the brown
DAB may be masked by brown melanin pigment.

Both DAB and AP Red are sometimes used in the same tissue section to allow the pathologist to visualize
two antigens in one slide. This is a process known as double staining.

5. Counterstain
Hematoxylin is used as a counterstain for IHC staining. The blue background is a hematoxylin
counter-stain that is often applied after the chromogen. The counter-stain provides a contrast to the
chromogen and also helps the pathologist visualize the underlying tissue structure.

D. PROCEDURE

First, perform routine

tissue processing
(fixation, dehydration,
clearing, impregnation,
embedding, and tissue
sectioning using the
rotary microtome

1. Fixation
Selection of Fixing Solution
a. Acetone And Alcohol
These two types of solutions, which are primary fixing solutions, play a role of precipitating sugars and fat
as well as maintain the immunologic competence.
Alcohol is ineffective to maintain low molecular weight protein, polypeptide and cytoplasmic
proteins. However, it can be mixed with glacial acetic acid, ethyl ether, chloroform and formaldehyde.
Acetone is often used for frozen tissue and cytological smears because it has a span penetrability and
dehydration property.
 b. Aldehyde
It is a functional cross-linking agent which is widely used due to its span penetrability, low contractibility
and low background. It helps keep the cross-linking between tissues and maintain antigen.
Formalin (10% neutral buffered) is the most widely used
4% paraformaldehyde is better than formaldehyde
Bouin’s solution (containing picric acid) is the most widely used in histology and pathology
c. Non-Aldehyde
These fixation agents are better mixed with glutaric dialdehyde or paraformaldehyde.

2. Tissue Processing
Five major steps are involved tissue processing: fixation, dehydration, clearing, impregnation, and
embedding. This process makes use of automatic tissue processor.
Fixation: Please refer to the Fixation section described above.
Dehydration: This step removes water completely, creates a condition for the next step and hardens
the tissue of interest. The dehydrating agents must be increasing in concentration. Examples of dehydrating
agents are ethanol and acetone.
Clearing: This is the process of dealcoholization. After dehydration, the tissue of interest requires a
clearing step because the dehydrating agent used in the previous step is immiscible with the paraffin from
one of the subsequent steps. The addition of transparent reagent helps paraffin absorb into the tissue.
Common transparent reagents are xylene, benzene and toluene, chloroform, cedar oil
Infiltration/ Impregnation: The tissue must be immersed in molten paraffin wax so that it adsorbs
the wax-substituting transparent agent. Based upon the melting point of wax, immersion should be
performed at 54-64℃.
Embedding: This is a process of providing a mold for the tissue using paraffin wax. After cooling is
completed, the tissue will be ready for sectioning and suitable for storage.

3. Tissue Sectioning
Tissue Section Types
a. Frozen
The most important feature for this type of tissue section is to keep antigen’s immune-competence
completely, especially for the cell surface antigen. Both fresh and fixed tissues can be processed as frozen
tissues. However, the tissues must be dried (or primary fixed) and stored at low temperature.
b. Paraffin-Embedded
Paraffin-embedded tissue section is normally sliced by a rotary microtome to give a thickness of 2-7 μm.
With proper treatment, the section reveals clear tissue structure and exact antigen location to enable high
medical-value pathology researches and retrospective studies. This section type can be stored at 4℃ for
long term use.

4. Antigen Retrieval
Formaldehyde fixation usually generates methylene bridges which cross-link proteins and
therefore mask the epitope of interest. It is essential to unmask the antigen epitopes in order to allow the
antibodies to bind, either by heat (Heat Induced Epitope Retrieval: HIER) or enzymatic digestion
(Proteolytic Induced Epitope Retrieval: PIER).
 a. HIER
The HIER method can be
implemented by
microwave, high pressure
or water bath. It breaks the
methylene bridges and
exposes the epitope to
allow the antibodies to
bind by continuously
heating. The following
antigen retrieval reagent is
required:
0.01 M citrate buffer solution (pH 6.0)
0.01 M PBS buffer (pH7.0)
0.05 M EDTA (pH 8.0)
0.05 M Tris-EDTA (pH 9.0)
0.05 M Tris-HCl (pH 1~12)
1. Microwave Method
Place the sample section into a microwaveable vessel where antigen retrieval reagent is present
Place the vessel inside a microwave oven
Apply microwave radiation to the sample for 5-20 min

2. High Pressure Method

Place the sample section into an appropriate vessel where antigen retrieval reagent is present
Place the vessel inside a pressure cooker
Turn on the cooker and heat the sample until it boils
Once boiling starts, turn off the cooker after the sample is allowed to reach full pressure for 1-4 min
3. Water Bath Method
Place the sample section into an appropriate vessel where antigen retrieval reagent is present
Place the vessel and thermometer inside a water bath chamber
Heat the sample to 92℃ in the chamber
Remove the sample from the chamber after it is heated at 92℃ for 20-40 min

Note:
The temperature and time should be properly controlled for the antigen retrieval methods.
The higher the temperature, the shorter the heating time (vice versa).

b. PIER
Epitope can be exposed by incubation with proteases which can break the methylene bridges. The choice
for digestion enzymes depends on the antigenic components. Pepsin and bromelin are used for retrieving
antigens in intercellular substance. Other enzymes can be used for intracellular antigen exposure.
5. Inactivation and Blocking
Inactivation
When either the horseradish peroxidase (HRP) or alkaline-phosphatase (AP) system is applied for IHC,
activation of endogenous enzymes should be blocked or inhibited to avoid producing non-specific binding.
a. Endogenous HRP Inactivation
Incubate the paraffin embedded section in 3% H2O2 for 10 min. Incubate the frozen section or cell section
in solution composed of methanol and 3% H2O2 (v/v: 4:1) for 30 min
b. Endogenous AP Inactivation
Incubate the sample section in 0.1 mM Levamisole
Note: Levamisole cannot inhibit the AP activation of endogenous intestine tissue
Blocking
Residual sites on the tissue section may bind to secondary
antibody and produce follow-up false positive results.
Therefore, serum from the same species as the secondary
antibody is commonly used for blocking. Animal’s
autoantibody in the serum can bind to the sites in advance.
Blocking should be done at room temperature for 10-30
min (avoid excessive blocking).

6. Detection
IHC detection methods vary and are based on the nature of
analyze reporting and binding chemistry, among other
factors. Three methods are described here:
immunofluorescence (IF), Enzymatic and Affinity.

a. Immunofluorescence Method
This technique is used for the rapid identification of an antigen by exposing it to known antibodies labeled
with the fluorescent dye (i.e., fluorochrome) which produces light when excited by a laser (e.g. argon-ion
laser). Specific antibody binding can be determined by the production of characteristic visible light and
detected by a fluorescence microscope.
Principle
The indirect staining process involves three steps

 Primary antibody binds specifically to target antigen

 Secondary antibody labeled with fluorophore binds to primary antibody
 Fluorophore is detected via microscopy
b. Enzymatic Method
The enzymatic IHC technique was introduced by Nakane and Pierce in 1967. It identifies antigens of
interest by exploiting the principle of antibodies binding specifically to antigens. An enzyme label is reacted
with a substrate to yield an intensely colored product that can be analyzed. The enzymatic technique was
developed with a similar principle to the IF technique but the two are different as an enzyme is used to
label the antibody for the enzymatic method.
1. Labeled-Enzyme Antibody
For this method, the antibody used for antigen detection has been labeled with the enzyme before the
reaction. After reacting with the targeted antigen, the labeled antigen forms an antigen-antibody complex
where the enzyme catalyzes a substrate to yield an insoluble colored product. Subsequently, the product
can be analyzed by a light microscope or electron microscope. The labeled-enzyme
approach can be done by direct or indirect detections.

Direct Detection
The direct method is a one-step staining method which involves a labeled
antibody (e.g. HRP-conjugated antibody) reacting directly with the antigen of
interest. The antigen-antibody-HRP complex is then allowed to react with a DAB
substrate for staining.
Indirect Detection
The indirect method is a two-step process which involves an unlabeled primary
antibody (first layer) that binds to the target antigen in the
sample and an enzyme-labeled secondary antibody
(second layer) that reacts with the primary antibody. The
secondary antibody must be raised against the IgG of the
animal species in which the primary antibody has been
raised. For instance, if the primary antibody is rabbit anti-
human IgG, the enzyme labeled secondary antibody could
be goat anti-rabbit IgG.

c. Affinity Method
The IHC sensitivity can be improved by employing a higher number of enzyme molecules bound to the
tissue. In this regard, the multiple binding sites between the avidin and biotinylated antibodies have been
exploited for IHC signal amplification. Avidin, an egg white protein, has four binding sites for the low-
molecular-weight vitamin biotin to form a large lattice-like complex. Beside avidin, there are other methods
which involve streptavidin which is a tetrameric biotin-binding protein that is isolated from Streptomyces
avidinii. The avidin and streptavidin methods work almost identically as their structures are very similar
(they have very little amino acid homology). Avidin-Biotin Peroxidase Complex (ABC) and Labeled
Streptavidin Binding (LSB) are the two most widely used affinity methods for amplifying the target antigen
signal.

1. ABC
The method involves four sequential steps.
 Incubation of primary antibody with tissue sample to
allow binding to target antigen
 Incubation of biotinylated secondary antibody (which
has specificity against primary antibody) with tissue
sample to allow binding to primary antibody
 Pre-incubation of biotinylated enzyme (HRP or AP)
with free avidin to form large ABC complexes
(Biotinylated enzyme and avidin are mixed together in
a pre-determined ratio to prevent avidin saturation)
  Incubation of the above pre-incubated solution to tissue sample

2. LSAB
This method uses an enzyme-labeled streptavidin to
detect the bound biotinylated primary antibody on
the tissue section. It can also be applied if the complex
in the ABC method is too big for tissue penetration.
Due to its smaller size, the enzyme-labeled
streptavidin is used to enable tissue penetration. The
LSB method can be employed to replace the ABC
method for the former’s ability to improve sensitivity
and reduce signal further.
The following are the steps:
 Incubation of primary antibody with tissue sample to allow binding to target antigen
 Incubation of biotinylated secondary antibody (which has specificity against primary antibody)
with tissue sample to allow binding to primary antibody
 Incubation of streptavidin-enzyme conjugate to tissue sample

7. Chromogens and Counterstain

The most common chromogen used in IHC is DAB. DAB (3,3’-
Diaminobenzidine) is typically used as a signal enhancer in conjunction
with the HRP-based immunostaining systems. The dark brown end-
product derived from DAB is insoluble in water and alcohol, stable and
suitable for long-term storage. Since DAB may cause skin and bladder
cancers, it is advised that personal protective equipment should be used
and skin/mucosa should be avoided.

Counterstains
After staining the target antigen by IHC, a secondary stain is usually
applied to provide contrast that helps the primary stain more distinct. While many of these stains show
specificity for discrete antigens or cellular compartments, other stains will deliver the staining of a whole
cell. Some of the most common counterstains are described as follows:
a. Hematoxylin
Hematoxylin, a natural dye which is extracted from the heartwood of the logwood tree, is used for cell
nucleus staining. Differentiation refers to the process of using reagents (e.g. 1% hydrochloric acid HCl and
alcohol) to remove the color caused by overstaining or non-specific staining on sample tissues. After
running nuclear staining (in aluminum hematoxylin) and differentiation (in HCl and alcohol), the tissue
section is transferred from an acid solution to an alkaline solution (e.g. ammonia water and disodium
hydrogen phosphate solution). During this process, the section will change from red brown into blue. This
procedure is known as bluing.
b. Methyl Green
Methyl green consists of metallic green microcrystals or bright green powders. It becomes bluish green
when dissolved in water. This basic dye can be easily bounded with highly polymerized DNA and changes
the nucleus to green. Counterstain with methyl green takes 2 to 5 min which should be followed by
washing the sample, dehydration and mounting.
c. Nuclear Fast Red
This counterstain will change the nucleus to red after applying to the tissue section for 2 to 5 min.

E. INTERPRETATION OF RESULT
Results are compared with positive and negative tissue antigen controls.
Positive control: a section from a tissue known to express the protein of interest.
Negative control: a section from a tissue known not to express the target antigen.
II. FROZEN SECTION TECHNIQUE
A. INTRODUCTION
A frozen section is a special type of procedure that is typically requested by a doctor at the time of
surgery. The purpose of a frozen section is to provide your surgeon with information that will help with
decision making during the surgery. A specific type of biopsy procedure called the frozen section was
developed in order to make a rapid diagnosis of a mass during surgery.

Advantages of frozen section biopsy

 If more tissue is needed to make an accurate diagnosis, the surgeon is able to obtain an additional
sample, avoiding a second operation.
 If the tissue is determined to be cancerous and is amenable to surgery, the mass can be removed at
that time.
 If the tissue is determined to be benign (not cancerous), then the mass may not always need to be
removed and the surgery can end.
 The frozen section biopsy can help ensure that the mass being removed is the intended tissue for
removal.
 It can help ensure that the entire mass and its surrounding borders are removed.
 It allows for the collection of proper tissue samples for further scientific research.
 The surgeon and pathologist are able to collaborate to care for the patient.

Frozen sections are performed with cryostats, specialized instruments composed of a tissue
microtome (cryotome) installed within a cooling chamber that allows for rapid freezing of tissues and a
continuous process of cutting tissue at low temperatures (Figure 3-2). Current cryostats use automated
electrical refrigeration systems with accurate thermostats.
The cooling chamber of the cryostat has a “work area” to place chucks when cutting another
section. Tissue is ideally cut when the chamber is between -10° to -20° C, depending on the tissue. To
ensure the cryostat is cool enough for all tissues, it is best to set the chamber temperature to -20° C.
Operation principle of the cryostat:
The cryotome consists of an objective to place the chucks containing frozen tissues, a stage to hold
sharp cutting blades, and an advancing/retracting mechanism. The stage holding blades usually has manual
mechanisms that allow for the installation of disposable metal blades or resharpenable knives in older
models. Cryotome stages also have mechanisms to vary the angle of the cutting blade that would allow for
optimal tissue sectioning. The advancing/retracting mechanism of a cryotome is composed of electrical
motors that are controlled with
electronic controls that rapidly
approximate the objective and metal
chucks holding the tissues to the cutting
blade and an external manual handwheel
that advances the tissue toward the sharp
metal blade in fixed increments, ranging
from 1 μm and greater. Some cryostat
models have optional antiroll guides that
assist with keeping sections flat.
However, using a chilled fine brush to
reduce wrinkling of the section as it is cut is a common practice.
Cryostats perform an automatic defrost cycle to reduce frost in the chamber. The time for setting
the cryostat to defrost automatically should be set during the late night or early morning when the need to
use the cryostat is minimal.

B. PRINCIPLE
During the frozen section procedure, the surgeon removes a portion of the tissue mass. This biopsy
is then given to a pathologist (a doctor who examines tissues and uses laboratory tests to make a
diagnosis). The pathologist freezes the tissue in a cryostat machine, cuts it with a microtome, and then
stains it with various dyes so that it can be examined under the microscope. The procedure usually takes
only minutes.
Freezing mechanism in the cryostat:
Frozen specimens are mounted onto a specimen holder with cryocompound and placed on the freezing
shelf and allowed to stabilize at chamber temperature. Fresh specimens can be frozen directly onto a
specimen holder that is placed onto the freezing shelf. A heat extractor can be used to aid freezing and
ensure good adherence and flat front block surface. A heat extractor is a large piece of stainless steel that is
supported on a hinged arm that is usually mounted above the freezing stage. As the surface of the specimen
starts to freeze the heat extractor is gently lowered onto the top of the specimen and allowed to remain
there for a few minutes.

C. MATERIALS
The main equipment used for frozen sections is the cryostat. The equipment consists of a number of unique
component parts described below.

1. Embedding Well Bars

Embedding is performed in wells machined into the face of one-inch thick stainless steel bars.
When cooled to cryostat temperature, these rather
substantial bars provide a powerful heat sink for rapid
freezing. Wells are machined with beveled walls, rounded
edges, and polished surfaces for ease of release. In its
current form, the author uses square wells with rounded
corners of diameters 18, 24, and 30 mm across the base.
Wells of varying sizes and depths can be made to fit
different situations. Well bars are stored at the deepest
convenient point in the cryostat where temperatures are
lowest and brought to higher or more accessible location
during the embedding process. In many cryostats a small
shelf can be installed for this purpose. An ambient cryostat temperature of -23 to -27 °C functions ideally
for rapid freezing.

2. Chucks
Chucks are designed with a crossing grid pattern of sharply cut channels to maximize the gripping
power re-quired to hold the embedded tissue block. The width and depth of the channels allow for
complete penetration of embedding medium when used cold and can therefore be stored at freezing
temperatures to facilitate rapid freezing. The crossing grid pattern allows for extrusion of excess
embedding medium so that chucks can be pressed flat to the well bar face. The chucks are made of stainless
steel, maximizing their freezing power and durability and are stored in a bin at a convenient location low in
the cryostat. Chucks freeze optimally when used cold but can also be used warm with use of the over-chuck
freezing block described below. Stemmed chucks are most suitable for this process. The stem is the focal
point to apply the necessary sharp tap resulting in an easy release of the block from the well. These chucks
fit many of the major brand cryostats and can be used in most cryostats with use of an adaptor.

3. Over-Chuck Freezing Blocks

The over-chuck freezing blocks constructed of rectangular steel function as a heat extractor. They
are designed to fit over the stem of the chuck. The freezing block also serves as a dislodging tool. A light tap
of the chuck stem cleaves the plane of adhesion, holding the formed block to the well.

4. Embedding Shelf
This removable embedding shelf is designed
to be installed on the front wall below the opening of
the cryostat in the most convenient and
ergonomically comfortable location available. The
shelves are made in various sizes to accommodate
most instruments. The shelf accommodates three
well bars capable of embedding 12 blocks. Well bars
can be used on a convenient flat surface such as a
brush holder, in cryostats that are not compatible
with an embedding shelf.
D. PROCEDURE
The procedure starts when the specimen (unfixed) from intraoperative procedure will be
submitted to the laboratory. The pathologist will perform an immediate gross analysis examination of the
sample. After grossing, the tissue sample will be subjected to cryostat procedure.
The specimen:
Specimens for frozen sectioning can be prepared in a variety of ways. They can be frozen in a cryogen
mixture such as isopentane cooled by dry ice, or placed onto a thin layer of cryocompound on a specimen
holder that is placed on the freezing shelf, Peltier stage, or a large cooled metal block placed inside the
cryochamber. The aim is to freeze the specimen as fast as possible to eliminate ice crystal formation
(freeze artifact). To achieve fast freezing the size of the specimen must be carefully controlled, as a guide a
suitable specimen would be 2 cm × 2cm × 2mm.

The following are procedures involved in the operation of a microtome.

A. Freezing the specimen and preparing a block:
1. The selected portion of tissue to freeze is placed on a prepared metal mounting base called a chuck and
the tissue encased in a squirt of rapidly freezing material. (Figure 3.2 a-b); last image: a-c
2. The tissue can be flattened and the freezing expedited with a steel weight/heat extractor to provide a
smooth flat cutting surface. Freezing the tissue takes about 3 min depending on the size of the tissue. (c-d)
3. The chuck is then screwed into the blade/plate cutting machinery (microtome) and the blade gradually
brought up to the face of the frozen tissue block where sections can be cut and placed on microscope slides.

Screw the chuck in the chuck holder for cutting

B. Cutting the frozen section
1. Before mounting the specimen onto the microtome, ensure that the handwheel is in the locked position.
Never attempt to place or remove a specimen from the microtome without ensuring that the handwheel is
locked. In the unlocked position the object head can move when inserting the chuck, placing the operator at
the risk.
2. The specimen holder is clamped firmly onto the object head mounted on the spindle of the microtome.
3. The handwheel is unlocked and the specimen
is lowered until it is at the same height as the
knife holder. The knife holder can then be
unlocked and moved manually toward the
specimen until it is close to the specimen
surface.
4. Adjust the micrometer setting of the
microtome to “trimming” thickness of 15 µm
and begin to turn the microtome handwheel;
the specimen will advance by this set value and
will make contact with the knife and the surface
of the block will be sectioned. This process is
termed “trimming” or “facing” the block, and
the purpose is to achieve a full face section of
the specimen. As soon as this is achieved stop
sectioning and adjust the micrometer setting to
the desired section value, e.g., 5 µm. Brush all
trimmings from the blade, or knife edge, carefully lower the antiroll plate into place and continue
sectioning.
5. Carefully lift the antiroll guide and with a cold brush arrange or move the sections on the pressure plate
or knife surface. Take a glass slide and holding it at an angle gently lower it onto the section.
6. Sections of fresh frozen tissue will adhere to plain glass slides due to the presence of free protein and
lipid. Sections of fixed frozen tissue will need to be mounted on coated slides e.g., poly-l-lysine.

C. Staining the slide

1. Tissue sections (typically two per specimen) are immediately fixed in methanol for 90 s before being
routinely stained manually with hematoxylin and eosin, dehydrated through graded alcohols, and
transferred to xylene.
2. Add a coverslip and mounting material
3. Any remaining specimen can be stored for future use Frozen remnants are then placed in 10% formalin
and processed for routine histopathological technique (fixation, dehydration, clearing, impregnation,
embedding, H&E staining).

E. RELEASE OF RESULT
Results for frozen techniques are released as soon as diagnosis was made by the pathologist,
usually within minutes to less than an hour from the time the specimen was received. Since the surgeon is
waiting for the result while the patient is in the operating room, it must be released immediately. The result
will determine succeeding actions for the intra-operative procedures done on the patient.
 III. SPECIAL STAINS FOR SPECIAL TISSUES
A. INTRODUCTION
"Special stain" is a term used to refer to many alternative staining techniques that are used when
the traditional H&E does not provide all the information the pathologist or researcher needs from a tissue
slide. "Special stains" are processes that generally employ a dye or chemical that has an affinity for the
particular tissue component that is to be demonstrated. They allow the presence/or absence of certain cell
types, structures and/or microorganisms to be viewed microscopically.
Special staining is performed to visualize selected tissue elements, entities and microorganisms.
Based on classical dye staining methods, special stains technique provide valuable information in the
evaluation of numerous abnormal or disease conditions. The following special stains are of high quality,
and they satisfactorily demonstrate, the tissue components or organisms for which they were designed.

SPECIAL INTENDED USE PRINCIPLE CLINICAL RESULTS

STAIN SIGNIFICANCE
Acid Fast Demonstration of Bacteria of the genus Acid Fast Bacillus Acid fast bacilli: Red
acid-fast mycobacterium have stain is intended for Nocardia Filaments:
Mycobacteria the unique property use as a qualitative Red
of being “acid fast” histologic stain to Leprae bacilli: Red
with certain stain. selectively
The term acid fast demonstrate Background: Blue
refer to the mycobacterium
ability if the Tuberculosis and
bacterium to retain a other acid-fast
dye that washes out organisms or
of all other tissue components in the
element when the tissue section.
section is exposed to
a dilute acid solution.
Alcian Blue Evaluation of Acid Alcian blue is a water It stains basophils, Acid
mucins and – soluble amphoteric cartilage, muco- Mucopolysaccharides:
mucopolysaccharides copper polysaccharides Turquoise Blue
phthalocyanin and
dye that stain by salt glycosaminoglycans
linkage with the
acidic
groups of acid
mucopolysaccharides
Alcian Blue / Differentiation of This is a combination Non-small cell lung Acid Muco-
PAS neutral and acidic of two standard cancer (NSCLC) can polysaccharides: Blue
mucopolysaccharides techniques: Periodic be classified into Neutral
Acid-Schiff and several histological polysaccharides:
Alcian Blue at subtypes, most Magenta
pH 2.5 . commonly Cartilage, ground
adenocarcinoma or substance, epithelial
squamous cell mucins: Shades of
carcinoma. Studies purple to very deep
have shown that blue
histochemical stains Cell bodies of fungi:
Alcian blue-PAS are Red to purple
 useful in lung cancer Mucoid capsule of
diagnosis. Cryptococcus: Blue
Amyloid Demonstration of Congo red stain is a Congo red stain is With normal light
(Congo Red) Amyloid in tissue modification of the used as a qualitative microscopy:
sections Highman's technique. histologic stain to Amyloid Pink to red.
The staining reaction selectively Nuclei Blue
is based on the demonstrate
application of congo AMYLOID in tissue With polarized light
red which stains the sections microscopy: Under
pattern of a typical polarized light to pink
proteins (Amyloid). areas change to green
The B-pleated sheets as the filter is rotated.
of amyloid are The background is
suitable in size and dark.
shape to
accommodate the
congo red molecules,
which are held in the
lattice work of B
pleated
sheets. Birefringence
is an intrinsic
property of
the amyloid fibril
congo red complex.
Without both the red
staining congo red
and the apple green
birefringence under
polarization, a
definite identification
cannot be used, a
mayers Hematoxylin
solution is applied to
provide a contrasting
blue nuclear stain.
Giemsa Demonstration of “Romanowsky" GIEMSA staining kit Nuclei : Red-purple
Parasite and H.Pylori stains differentiates · Leukocytes : Purple
bacteria are formed from a leukocyte in bone · Erythrocyte : Pink
mixture of marrow and other · Mast cells : Blue
polychromed hematopoietic tissue · Pathogenic gram-
methylene blue and (lymph nodes). The negative spiral : Blue
eosin, the resultant stain can also be Bacteria (H. Pylori).
precipitate being used to demonstrate
dissolved in methyl some
alcohol. Such stains microorganisms
are acidic-basic such as H.Pylori.
stains (neutral dyes)
and therefore stain
both acidophilic and
basophilic cellular
 components. Giemsa,
a modification of the
classic Romanowsky
stain, is made from
various azure
compounds (thionine
and its methyl
derivatives) with
eosin and methylene
blue, In GIEMSA
staining kit, a
buffered thiazine
eosinate solution is
used to stain cells
differentially with a
characteristic blue or
pink color.
Iron (Perl’s) Detection of ferric Iron stain is based on Iron stain is used as Iron pigment: Bright
iron in tissue the historic Prussian a qualitative blue
sections and blood or blue reaction. The histologic stain to · Nuclei: Red
bone marrow films iron is separated detect iron in · Cytoplasm: Light
from formalin-fixed pink
protein by paraffin embedded
hydrochloric acid. tissue and bone
The free ferric iron marrow. In the
reacts with the disease states of
potassium hemchromatosis and
ferrocyanide to form hemosiderosis,
an insoluble bright excessive amounts
blue ferric of ferric iron and
ferrocyanide or present in the liver,
Prussian blue. spleen and lymph
nodes.
Trichrome Evaluation of Trichrome III GREEN Trichrome stain is · Basement
(masson’s) collagen and muscle staining kit is a used as a qualitative membranes: Black
fibers modification of histologic stain to · Reticulum: Black
Masson's Trichrome study connective · Nuclei: Blue-Purple
stain. The staining tissue, muscle and ·Cytoplasm, Collagen
reaction is based on collagen fibers in . Connective tissue:
the differential effect formalin-fixed, Pink to orange
of acid dye on muscle paraffin – embedded
and tissue. It is also used
collagen. Bouin's to differentiate
solution is applied collagen from muscle
to tissue sections to tissue. The stains
intensify the final are useful for
coloration. After indicating fibrotic
application of change, that is, an
Trichrome III increase in collagen
mordant, the like that which
collagen is stained occurs in liver
 with Trichrome III cirrhosis and
Green, which pyelopnephretis.
contains fast green.
Mucicarmine Evaluation of mucin, Mucin is a term given Mucicarmine stain is · Mucin: Deep rose to
mucopolysaccharides to a secretion used as a qualitative red
and capsule of produced by a histologic stain in · Nuclei: Deep rose to
cryptococcus variety of epithelial the identification of red
and connective primary tumor sites · Capsule of
tissue cells. The and distinguishing Cryptococcus: Deep
rationale underlying mucin-negative rose to red
the remarkable undifferentiated · Other tissue
specificity of squamous cell element: Yellow
mucicarmine for lesions from mucin –
mucin is not fully positive
understood. adenocarcinomas.
Aluminum salts is The stain displays a
believed to form a specificity towards
chelate complex with mucins of epithelial
mucin, to which origin whereas
carmine attaches by mucins of
dye-lake formation. fibroblastic origin
In this method, iron – stain poorly.
hematoxylin is used
for the nuclear stain
and Tartrazine for
the counterstain.
PAS Diastase digested; The staining reaction PAS staining in · Glycogen: Bright
evaluation of is based on the tissue sections and Magenta
glycogen and oxidation of glycol to digestion with · Nuclei: Light purple
glycogen storage aldehyde followed by diastose is useful as
disease selective staining of an aid in the
the aldehyde groups determination and
by schiff's Reagent. diagnosis of
Diastase digests the glycogen storage
glycogen present in disease in tissue.
the tissue so it is
washed out of the
tissue before PAS
staining occurs.
Diastase digestion is
used to differentially
determine if the PAS
– positive component
is glycogen.
Verheoff – To identify collagen VVG is a two-part Although there are Elastic fibres: black
van gieson and elastic tissue combination stain numerous special Nuclei: black
stain for that enables stains for Collagen: red
collagen differentiation of identification of Other tissues: yellow
some connective elastic fibers, VVG is
tissue components in most commonly used
a tissue which are because it is quick,
 not easily and produces intense
distinguished by staining of elastic
H&E staining: fibers.

Verhoeff stain
component: an iron-
haematoxylin stain
that is specific for
elastic fibers. It
forms strong bonds
with elastin, the main
component of elastic
connective tissue.

van Gieson stain

component: a
counterstain that is
specific for collagen.
It was named after
American
bacteriologist Ira van
Gieson, and
comprises two acid
dyes- picric acid and
acid fuchsin.
Gomori's Stains reticulin fibers Reticulin stains use Stains reticulin Reticulin fibers: black
silver and rely on the fibers. Reticulin Nuclei: red
argyrophilic fibers support the
properties of the body and are
fibers. Argyrophilic common in the liver,
cells can adsorb spleen and kidneys.
silver but cannot Characteristic
reduce it. "Adsorb" is reticulin patterns can
a surface-based help diagnose
process, whereas cirrhosis of the liver,
"absorb" means early fibrosis in bone
there is permeation marrow, tumors
through the tissue. including: hemangio-
Argyrophilic cells sarcomas - tumor of
need a reducer/ cells that line blood
developer to convert vessels, fibroblastic
the invisible silver tumors, and rhabdo-
salts to a visible myosarcomas -
metallic silver. tumor of muscles
that are attached to
bone. They can also
help diagnose
epithelial versus
non-epithelial
tumors.
Orcein The orcein stain is Orcein is composed The Orcein stain is inclusion bodies: dark
 used to identify the of mixture of amino- commonly used to brown-purple colour.
inclusion bodies of and hydroxyl- diagnose hepatitis B, Proteins that are
viruses. phenoxazone which causes associated with
compounds. The inclusion body copper: dark purple.
procedure uses a formation in
solution of Orcein hepatocytes.
combined with 70%
ethanol, and
hydrochloric acid.
Weigert's this type of stain is The solution includes Diagnose diseases Elastic fibers: blue-
resorcin used to stain elastic basic fuchsin, which affecting elastic black
fuchsin fibers produces a complex fibers. Many Nuclei: light blue-
(Weigert’s that attaches to disorders are known black
elastic) elastic fibers, causing to have detrimental Collagen: pink or red,
them to become effects on both other tissues: yellow
stained. The complex visceral and
formed from the cutaneous elastic
basic fuchsin, an iron tissue. Marfan
resorcin lake, binds syndrome, caused by
to the elastic fibers, autosomal dominant
resulting in the blue- mutations in the
black staining. The fibrillin-1 gene
nuclear detail is (FBN1), results in a
stained with an iron defective scaffold for
hematoxylin which elastin deposition.
will not over Stains of aortic and
differentiate in the cutaneous tissue
acidic elastic stain samples from animal
solution. models of Marfan
Weigert's Stain syndrome
solution also demonstrated
comprised of decreased density
resorcin, ferric and increased
chloride, ethanol, fragmentation of
distilled water and elastic lamellae and
hydrochloric acid. fibers, with tangled
Haematoxylin and microfibrils.
van Gieson stain are
also used as
counterstains.

Prepared by: Roselle Joy Padaoan, RMT, MT(ASCPi), MPH

References:
https://www.bosterbio.com/protocol-and-troubleshooting/immunohistochemistry-ihc-principle
https://www.sciencedirect.com/science/article/abs/pii/B9780124200678000155
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