CHAPTER 3

EXAMINATION OF FRESH TISSUE

Histology is the microscopic study of the normal tissues of the body while
histopathology is the microscopic study of tissues affected by disease. The
procedures adopted for the preparation of material for such studies are known as
histologic or histopathologic techniques. The tissues are usually obtained during
surgery, biopsy, or autopsy. They range from very large specimens or whole
organs to tiny fragments of tissue. The following surgical procedures are usually
performed to obtain the specific-types of tissue that are submitted to a histology
laboratory for processing:
Fine needle aspiration is the simplest, least invasive test and uses the
smallest needle to simply remove cells from the area of abnormality. This
is not always adequate to obtain a diagnosis, depending on the area to be
biopsied.
A core needle biopsy removes not only cells, but also a small amount of
the surrounding tissue. This provides additional information to assist in
the examination of the lesion.
An incisional biopsy takes out even more surrounding tissue. It takes out
some of the abnormality, but not all. The doctor will slice into the lesion
and remove only a portion of it. If the lesion is found to be cancerous,
further surgery may be needed to remove or excise the entire lesion.
An excisional biopsy generally removes the entire area in question.
Punch biopsy is considered the primary technique for obtaining
diagnostic full-thickness skin specimens. It requires basic general
surgical and suture-tying skills and is easy to learn. The technique
involves the use of a circular blade that is rotated down through the
epidermis and dermis, and into the subcutaneous fat, yielding a 3- to 4-
mm cylindrical core of tissue sample.
Shave biopsy - where small fragments of tissue are “shaved” from a
surface (usually skin).
Curettings - where tissue is scooped or spooned to remove tissue or
growths from body cavity such as endometrium or cervical canal.

Specimens are usually received in fixative (preservative) but sometimes

they arrive fresh and must be immediately fixed. Tissue specimens received in
the surgical pathology laboratory should have a request form that lists the patient
information and clinical history along with a description of the site of origin. The
specimens are accessioned by giving them a number that will identify each
specimen for each patient. It is important that specimens are properly identified
to minimize the risk of mislabeling.
Once tissues are removed from the body, their proteins and cells are
digested and broken down by their own enzymes, independent of a bacterial
action. This process is known as autolysis, which is retarded by cold and
accelerated at room temperature. It is more severe in tissues that are rich in
enzymes (e.g. liver, brain, and kidney) and less rapid in elastic and collagen
tissues.
Methods of tissue examination may vary according to the structural and
chemical components of the cells to be studied, and depends on the nature and
amount of the tissue to be evaluated. Fresh tissues are usually examined when
there is an immediate need for evaluation. On the other hand, a better and more
effective means of studying tissues, whether normal or abnormal, is by
examination of adequately preserved sections and smears that are stained to
demonstrate specific structures. The glass slides are then mounted with
coverslips for permanent keeping.
Examination may be done on fresh or preserved tissues, depending on
necessity. Fresh tissues have the advantage of being examined in the living
state, thereby allowing protoplasmic activities such as motion, mitosis, a n d
phagocytosis to be observed. Its use is limited, however, because of the fact
that tissues examined in the fresh state are not permanent, and therefore, are
liable to develop the changes that have usually been observed after death.

Methods of Fresh Tissue Examination

1. Teasing or Dissociation – is a process whereby a selected tissue specimen
is immersed in isotonic salt solution such as normal saline or Ringer’s
solution in a petri dish or watch glass, carefully dissected with a needle
an d separated by direct or zigzag spread using an applicator stick. Selected
pieces of the tissue are transferred carefully to a microscope slide and
mounted as a wet preparation underneath a cover glass, care being taken to
avoid forming bubbles. It is either stained with a supravital dye or
examined unstained by Phase Contrast or Bright Field microscopy. It has
the advantage of permitting the cells to be examined in the living state. The
use of the phase contrast microscope greatly increases the structural detail
of the cells examined in the living state, allowing movement and mitotic
division to be observed. The application of certain stains such as methylene
 blue can be also of great value. The preparations, however, are not
permanent.
2. Squash Preparation (Crushing) is a process whereby small pieces of
tissue (not more than one mm. in diameter) are placed in a microscopic slide
and forcibly compressed with another slide or with a cover glass. If
necessary, a supravital stain may be placed at the junction of the slide and
the cover glass, and allowed to be absorbed by the tissue through capillary
attraction.
3. Smear Preparation – The method of preparing the smear differs
depending on the nature of the material to be examined. As a general rule,
smears are made either by spreading the selected portion of the specimen over
the surface of the slide with a platinum loop. Alternatively, an apposition
smear can be made using a second slide to obtain a relatively uniform
distribution of secretion. Too thin or too thick smears have to be avoided,
since they make the tissues less suitable for examination. Smears may be
examined either as fresh preparations similar to that described for teased
preparations, or by using a supravital staining technique. Smear preparations
can be made permanent by fixing them while still wet, staining them to
demonstrate specific structures and inclusions, and mounting the cleared
specimen beneath a cover glass with a suitable mounting medium. This is
useful for preparing smears of thick secretions such as serous fluids,
concentrated sputum, enzymatic lavage samples from the gastrointestinal
tract, and blood smears. This technique is especially useful in cytological
examinations, particularly for cancer diagnosis.
a. Streaking -With an applicator stick or a platinum loop, the material is
rapidly and gently applied in a direct or zigzag line throughout the slide,
attempting to obtain a relatively uniform distribution of secretion. Too
thin or too thick smears have to be avoided, since they make the tissues
unsuitable for examination.
b. Spreading - A selected portion of the material is transferred to a
clean slide and gently spread into a moderately thick film by teasing
the mucous strands apart with an applicator stick. It is a little more
tedious than streaking, but has the advantage of maintaining cellular
interrelationships of the material to be examined. It is especially
recommended for smear preparations of fresh sputum and bronchial
aspirates, and also for thick mucoid secretions.
c. Pull-Apart – This is done by placing a drop of secretion or sediment
 upon one slide and facing it to another clean slide. The material
disperses evenly over the surface of the two slides. Slight movement of
the two slides in opposite directions may be necessary to initiate the
flow of materials. The two slides are then pulled apart with a single
uninterrupted motion, and the specimen is placed under the microscope
for immediate examination, or applied with vital stains.
4. Touch Preparation (Impression Smear) – This is a special method of
smear preparation whereby the surface of a freshly cut piece of tissue is
brought into contact and pressed on to the surface of a clean glass slide,
allowing the cells to be transferred directly to the slide for examination by
Phase Contrast microscopy or staining for light microscopic study. It has an
added advantage in that the cells may be examined without destroying their
intercellular relationship.

Frozen Section
At times during the performance of surgical procedures, it is necessary to
get a rapid diagnosis of a pathologic process. The surgeon may want to know if
the margins of his resection are free from tumor before closing. An unexpected
disease process may be found that requires immediate diagnosis so the surgeon
can decide what to do next, or it may be necessary to determine if the
appropriate tissue has been obtained for further workup of a disease process.
Immediate diagnosis is accomplished through the use of a frozen section,
especially in intra-operative pathology to help the surgeon in choosing his next
plan of action. It is especially recommended when lipids and nervous tissue
elements are to be demonstrated. Frozen sections are usually done on muscle
and nerve biopsies as well as on surgically removed tumors.
A fresh tissue is frozen on a microtome with C02, or on a cryostat, a cold
chamber kept at an atmospheric temperature of -10° to -20° C. The thin frozen
sections are mounted on a glass slide, fixed immediately and briefly in liquid
fixative, and stained using similar staining techniques as in traditional wax
embedded sections.
For histochemistry, cryostat sections give much faster results than paraffin
sections. However, the morphological detail and resolution of frozen sections
are usually inferior compared to the quality of tissue that has been embedded in
paraffin.
The advantage of the frozen section method is rapid processing time with
less equipment requirement, and less need for ventilation in the laboratory. The
disadvantage is the relatively poor quality of the final slide.
 Frozen sections, both fixed and unfixed, have many applications in
histotechnology, and are commonly used for:
1. Rapid pathologic diagnosis during surgery
2. Diagnostic and research enzyme histochemistry
3. Diagnostic and research demonstration of soluble substances such as
lipids and carbohydrates
4. Immunofluorescent and immunohistochemical staining
5 . Some specialized silver stains, particularly in neuropathology

The tissue for freezing should be fresh, and freezing should be done as
quickly as possible. Slow freezing can cause distortion of tissue due to ice
crystal artifacts. The more commonly used methods of freezing include:
1. Liquid nitrogen
2. Isopentane cooled by liquid nitrogen
3. Carbon dioxide gas
4. Aerosol sprays
Liquid nitrogen is generally used in histochemistry and during intra-
operative procedures, and is the most rapid of the commonly available freezing
agents. Its main disadvantage is that soft tissue is liable to crack due to the
rapid expansion of the ice within the tissue, producing ice crystals or freeze
artifacts. It also overcools urgent biopsy blocks, causing damage to both block
and blade if sectioning is done at -70°C or below . The tissue snap-frozen in
liquid nitrogen must therefore be allowed to equilibrate to cryostat chamber
temperature before sectioning is attempted. The majority of non-fatty unfixed
tissues are sectioned well at temperatures between -10oC and -25°C.
One problem with the use of liquid nitrogen is that it causes a vapor phase
to form around the tissue, acting as an insulator that causes uneven cooling of
tissue, particularly of muscle biopsies, and making diagnostic interpretation
difficult. This problem can be overcome by freezing the tissue in Isopentane,
OCT, or Freon 2.2 that has a high thermal conductivity.
Isopentane is liquid at room temperature. A Pyrex glass beaker containing
isopentane is usually suspended in a flask of liquid nitrogen until half-liquid
and half-solid stage is reached. The beaker is removed from the liquid nitrogen
when small crystals start forming on the side of the beaker (approximately
-170°C), and the tissue to be frozen (affixed on a cork disc, aluminum foil or
cryostat chuck) is dropped into the cooled liquid isopentane. This is an
excellent method for freezing muscle tissue.
Tissue blocks can also be frozen by adapting a conventional freezing
microtome gas supply of carbon dioxide gas from a C02 cylinder.
The use of aerosol sprays has become increasingly popular in recent years,
and is adequate for freezing small pieces of tissue except muscle. Quick-
freezing spray cans of fluorinated hydrocarbons (e.g., Cryokwik) have a distinct
advantage of rapidly freezing blocks of any type of tissue.
Fresh, completely unfixed tissues, or tissues that have been briefly treated
with formalin may not require embedding anymore; instead they may be frozen
and cut in a freezing microtome or cryostat. Two methods of preparing frozen
sections may be resorted to: 1. Cold Knife procedure
2. Cryostat procedure (Cold Microtome)

Cold Knife Procedure

Tissue blocks can be frozen by adapting a conventional freezing
microtome gas supply of carbon dioxide gas from a C02 cylinder, or by using a
specially made piece of equipment known as cryostat. Almost any microtome
can be utilized for the purpose, provided means are made available for freezing
and maintaining the specimen and the knife at low temperatures, usually by
utilizing the carbon dioxide technique.
A piece of filter paper soaked in gum syrup is placed on the microtome
stage, and short bursts of C02 are applied, freezing the filter paper to the stage.
The selected block of tissue, approximately 3-5 mm. thick, is then oriented on
the stage, applied with a few drops of gum syrup and frozen solid with several
intermittent bursts of CO2 , each for 1-2 seconds duration, at intervals of around
4 seconds. It should be frozen just to the point where it will be firm enough to
section. The tissue is then lifted up to the knife manually and trimmed until the
surface is flat. The surface is then warmed with the finger until the hard frozen
tissue starts to thaw and becomes visible to the naked eye. This is the DewLine,
the point at which sections may then be cut at 10 µm thickness.
Sections do not form ribbons but rather stick to the knife blade and should,
therefore, be removed with a camel hair brush or finger moistened with water.
They are then transferred to a dish of distilled water to separate, and picked up
individually for mounting and staining. The water dish is usually placed on a
dark or black background, in order to see the sections which are usually
colorless or very light in color.
Tissues that have been frozen too hard will usually chip into fragments
when cut. The surface of the block may then be softened by warming slightly
with the ball of the finger or thumb. Tissues that have not been sufficiently
frozen will cut thick and crumble, and the block may come away from the
stage.
More bursts of C02 gas should then be given to refreeze the block. Whether
used in a cold environment or not, a different temperature between the tissue
and the knife is usually employed, the latter being colder. Using a cold knife in
a controlled cold environment, optimum condition for sectioning shall be
provided for by the following temperatures: Knife -40° to - 60°C
Tissue -5° to - 10°C
Environment 0° to - 10°C

The success of this procedure depends upon ambient temperature and

humidity. It is very hard, if not impossible, to cut sections in a hot or humid
environment. Ease of cutting and quality of sections are always improved if
done in a cold room. Sections thinner than 6 µ generally cannot be obtained
even from tissues that section well, and with ideal conditions for sectioning.

Cryostat Procedure
This method makes use of the Cryostat, an apparatus used in fresh tissue
microtomy. The cryostat consists of an insulated microtome housed in an
electrically driven refrigerated chamber and maintained at temperatures near
-20°C, where microtome, knife, specimen and atmosphere are kept at the same
temperature. The optimum working temperature of cryostat is -18 to -20°C.
Majority of the sections can be cut in isothermic conditions, where the
temperature for sectioning can be accurately established and controlled. The
tissue for freezing should be fresh, and freezing should be done as quickly as
possible. Slow freezing can cause distortion of tissue due to ice crystal artifacts.

Fig. 3-1. Ice crystal artifacts caused by improper freezing

Fresh frozen tissue requires that the tissue be maintained in the frozen solid
state during cutting of section, thereby supporting and protecting the tissue
from damage and distortion by the knife during the process of cutting. The
tissue must be sufficiently cold to prevent compression and displacement of cell
and tissue structures as the knife passes thru it.
The microtome knife needs to be chilled and maintained at low
temperature to prevent complete melting of the tissue, thereby forming a sticky,
distorted mass along the knife edge. When the tissue is too cold, on the other
hand, resistance to cutting is increased, so that the tissue becomes brittle and is
broken down into fragments upon cutting. The cryostat should be left on at all
times even when not in use, since it will require several hours to reach
operating temperature from a room temperature start. It takes at least one hour
for a knife to come down to operating temperature, so that a spare knife should
always be kept inside the cryostat cabinet. To ensure that the sections will cut
smoothly and freely onto the knife surface, the knife as well as the undersurface
and edge of the anti-roll plate must be kept scrupulously clean and dry. Soft
tissue paper, either dry or moistened with absolute alcohol, may be used to
clean the knife and anti-roll plate. The cryostat should be defrosted during the
weekend, including cleaning and oiling of microtome with special low-
temperature oil.
The success of fresh tissue sectioning depends to a large extent on the
temperature, both of the tissue and of the knife. Certain tissues such as fat or
mucin, and hard or dense structures in a soft matrix require much lower
temperatures to impart a suitable consistency for cutting. These are sectioned
on the cryostat by lowering the tissue or knife temperature or both, either by
placing the block holder in a bath of alcohol or acetone containing dry ice, or
by exposing the tissue to carbon dioxide.

Mounting of Tissue Block

Synthetic water-soluble glycols and resins are generally used as mounting
media for tissue blocks that need to be sectioned on a cryostat. The O.C.T.
(Optimal Cutting Temperature) compound, Lab-Tek Products, Division of
Miles Laboratories is especially recommended. It is marketed in convenient 8
oz. plastic dispensers in three temperature ranges, depending on the tissue being
cut: -5 to -15°C for brain, lymph nodes, liver, spleen, uterine curetting, soft
cellular tumors;
-15 to -25°C for non-fatty breast tissue, ovary, prostate, tongue, and
GI tract;
-35°C for fatty breast and omental tissue.
The cryostat is usually set at -18 to -20°C. Preferably, the tissue block
should be 2-4 mm. thick in order to minimize the risk of the knife hitting the
metal tissue block holder. Small fragments of tissue, such as curettings or brain
biopsies, are placed on a thick base of O.C.T. compound. The blocks are then
surrounded and covered with an additional matrix of O.C.T. compound, and
frozen by liquid nitrogen.
The frozen tissue is mounted on the microtome. Both the microtome knife
and the tissue block are left in the cryostat for 15 minutes at -20°C, to ensure
that they are cooled to the correct temperature. Sections between 5-10 µm are
then cut slowly and steadily, removed from the knife with a camel hair brush,
attached directly to slides of cover-glasses at room temperature, air​ dried, and
fixed (optional).
To mount cryostat sections after cutting, one edge of the glass slide is
lowered gently until it is about 1/2 to 1 mm. from the knife face. The section
will automatically transfer from the cold knife to the relatively warm slide. The
slide should never be pressed down on the section, because this will cause a
frost mark to remain where the section rested on the knife. If this happens, the
frost mark should be wiped away with soft tissue paper.
Overall, cryostat sections provide the simplest, quickest and least labor​-
intensive method for producing frozen sections, and are routinely used for
intraoperative and rapid diagnosis of surgical specimen.
It should be noted that cryostats cut only individual sections, and do not
form ribbons, as in paraffin blocks.

Freezing Previously Fixed Tissue

Cryostat sections of fresh, unfixed tissue usually attach easily to the slide,
even without adhesives, and will preserve enzymes and other substances that
may be studied by histochemical techniques.
The cryostat is also recommended for any technique requiring cold
sectioning of fixed material, e.g., for fats and lipids, and for some special
methods for the nervous system. Sections of formalin-fixed tissue, however,
may not adhere to the slide, and will fall off or be detached during staining.
Clean slides should be coated with albumin or chrome-glycerin jelly so that the
fixed tissue will attach to the slide. Another way, albeit cumbersome, is to
immerse the tissue block in boiling 10% buffered formalin for 1 to 2 minutes
before freezing and sectioning for rapid surgical diagnosis. Special fixatives
such as 10% formol calcium at 4°C may be used in histochemistry and for lipid
demonstration.
Tissues that have been fixed or stored in alcohol should be washed in water
for 12-24 hours before sectioning, since alcohol inhibits freezing.

Examination of nerve and muscle

Muscle and nerve biopsies are divided into separate portions that will
allow for formalin fixation and paraffin embedding, unfixed snap-frozen for
cryostat sections, fixation and resin embedding for electron microscopy (EM)
and, in some rare cases, for biochemical immunoblotting studies. Multiple
fixation processes are required because multiple techniques are to be used. The
portion of a specimen intended for frozen section should be transported on top
of wet ice, on saline-dampened gauze, and rapidly frozen within two hours.
Upon receipt in the histology lab, specimen is oriented in O.C.T. (Optimal
Cutting Temperature) compound and snap-frozen in liquid nitrogen/ isopentane
for optimal results. Orientation, size, and expedient flash freezing are critical to
obtaining undamaged sections of unfixed muscle fibers. Do not allow the tissue
to freeze slowly or to soak up excess saline, as these will cause artifacts that
can be seen microscopically and can interfere with diagnostic interpretation.
A portion of the tissue is oriented on a piece of cardboard, fixed with 10%
buffered formalin and processed for staining with routine Hematoxylin and
Eosin (H&E staining). The biopsy portion for electron microscopy is fixed in a
buffered solution of glutaraldehyde and postfixed in osmium tetroxide, usually
by a specialist in electron microscopy. In some muscular degenerative
disorders, biochemical techniques may also be required.

SPECIAL PROCESSING TECHNIQUES

For histochemical evaluation involving enzyme studies, the tissue needs to
be chemically active, and the important chemical constituents should not have
been removed, altered or displaced. In most instances, frozen section is deemed
to be the most ideal and preferred means of preserving tissues in order to avoid
complete or partial loss of enzymes consequent to chemical fixation.
Difficulties, however, arise in obtaining thin and serial sections of uniform
thickness; since cut sections of tissue tend to disintegrate and cannot be easily
handled without prior fixation. These disadvantages will have to be considered
in determining the necessity and advisability of such sections.
In addition to fresh frozen tissue sectioning, there are methods that may be
resorted to, if chemical fixation of tissue blocks is to be avoided, namely:
1. Freeze-drying
2. Freeze substitution

Like fresh frozen sections, these special techniques have the common
principle of rapidly preserving the tissue block by freezing (quenching). The
aim is to produce instant cessation of cellular activity thereby preventing
chemical alteration of tissue and displacement of cellular tissue components.
Freezing must be rapid, accomplished within seconds to prevent the formation
of ice crystal artefacts in tissue blocks and produce optimum tissue
preservation. The freezing agent commonly employed is liquid nitrogen, and
the tissue is sectioned into thin slices using a cryostat machine under very low
temperature. The use of isopentane, pentane and propane and most recently of
dichloro-difluoromethane, which can be cooled to very low temperature in
order to retain the fluidity of the freezing agents, have contributed much in
giving higher conductivity to this liquefied gas.

Freeze-Drying
Freeze-drying is a special way of preserving tissues by rapid freezing
(quenching) of fresh tissue at -160°C and subsequently removing ice water
molecules (dessication) by transferring the still frozen tissue block into a
vacuum chamber at a higher temperature, e.g. -40°C (sublimation) without the
use of any chemical fixative. This technique is generally not used in routine
surgical laboratories, and is restricted to specialized or research laboratories.
A tissue around 2 mm. thick is plunged into isopentane or propane​-
isopentane mixture which has been chilled to -160° to -180°C with liquid
nitrogen. This will effectively solidify the tissue in 2-3 seconds, thus
preventing the formation of large ice crystals, autolysis and putrefaction. The
frozen tissue is then transferred into a high vacuum drying chamber maintained
at a temperature of -30° to -40°C depending upon the size of the tissue. Water
is sublimated and dehydrated from the tissue, thereby completing the
dessication process within 24-48 hours. Once drying is completed, the tissue is
removed, fixed and embedded, either in molten paraffin wax, water soluble
waxes or celloidin. Infiltration and impregnation are usually performed in a
vacuum embedding oven. The tissue is then sectioned in the usual routine
manner and specific staining is applied, depending upon individual necessity.
This technique is generally time-consuming and expensive. Drying is by
far the most time consuming part of the process, as certain tissues contain 70-
80% water by weight that has to be removed without damage to the tissue.
Furthermore, freeze-dried materials are generally more difficult to section than
ordinary paraffin blocks. The tissue is brittle and inadequately supported due to
the relatively short period for wax impregnation; hence, it is not advisable as a
routine procedure. The tissues are usually flattened directly into an albuminous
glass slide with the aid of the finger. Water must be avoided and warm alcohol,
acetone, mercury are preferred.
However, freeze-drying also h a s many outstanding good features. It
produces minimum tissue shrinkage, and allows tissues to be processed in a
fresh state. It causes minimal chemical change on the cells, most especially on
the protein components, and less displacement of tissue and cellular
consti tuents. This method avoids the chemical alteration of cellular
components, t h e denaturation of proteins, destruction of enzymes, and loss of
tissue constituents that usually occur in the usual histological processing.
This method is particularly important as far as enzyme studies are
concerned. In addition to demonstrating hydrolytic enzymes, mucous
substances, glycogen and proteins, freeze-drying may be used for special
studies, including:
1. Immunocytochemistry
2. Fluorescent antibody studies of polypeptide and polypeptide
hormones
3. Autoradiography
4. Microspectrofluorimetry of autofluorescent substances
5. Formaldehyde-induced fluorescence of biogenic amines (to
demonstrate 5-hydroxytryptamine, adrenaline, and other
catecholamines)
6. Scanning electron microscopy

Freeze-Substitution:
Freeze-substitution is a process of dehydration, performed at temperatures
low enough to avoid the formation of ice crystals and to circumvent the
damaging effects observed after ambient-temperature dehydration. It is similar
to freeze-drying in preparing and preserving tissue blocks for subsequent
sectioning because both involve the rapid freezing of tissues and the
subsequent infiltration and embedding of the frozen tissue block in paraffin or
celloidin. The only variation is that the frozen tissue, instead of being subjected
to dehydration in an expensive vacuum drying apparatus, is fixed in Rossman's
formula or in 1% Acetone and dehydrated in absolute alcohol. Infiltration and
embedding is then carried out in the same way as in paraffin section.
Freeze-substitution is based on rapid freezing of tissues followed by
solution ("substitution") of ice at temperatures well below 0°C. A 1 mm to 3
mm specimen is thrown into 3:1 propane-isopentane that is super cooled by
liquid nitrogen to -175°C (with precautions). Cryostat sections are cut 8-10 µm,
and transferred to water-free acetone (substituting fluid), and cooled to -70oC
for 12 hours to 1 week in order to dissolve ice slowly without distorting tissue
structure. The sections are floated onto coverslips or slides and allowed to dry
for subsequent histochemical staining. This technique is relatively more
economical and less time-consuming than freeze-drying. For best
morphological and histochemical preservation, substituting fluids should in
general contain both chemical fixing agent and solvent for ice, e.g., 1%
solutions of osmium tetroxide in acetone, mercuric chloride in ethanol, or
picric acid in ethanol.

REFERENCES
Andrew W. (1966) Microfabric of Man, Yearbook Medical Publishers, Inc., Chicago.
Anderson G, Bancroft J. (2002) Tissue processing and microtomy. In: Bancroft, J .D. Gamble, M.
Theory and Practice of Histological Techniques. 5th ed., Churchill Livingstone, London, 100.
An Introduction to Specimen Preparation: Geoffrey Rolls Leica Biosystems, Wetzlar, Germany, 30.
May 2011
Bancroft JD. (1975) Histochemical Technique. 2nd Ed. London: Butterworths.
Bancroft J.D, Cook HC. (1994) Manual of Histological Techniques and their
Brown CC. (1969) Primer of Histopathologic Technique, Appleton-Century-Crafts, New York.
Brown RW. (2009) Histologic Preparation: Common Problems and their Solutions. CAP Press.
Chicago. Pp. 35-42.
Carson FL, Hladik C. (2009) Histotechnology: A Self-Instructional Text. 3rd ed., American Society of
Clinical Pathology Press.
Pearse AGE. (1980) Histochemistry, Theoretical and Applied, 4th ed., Vol 1, Churchill Livingston,
Edinburgh.
Sheehan DC, Hrapchak B. (1980) Theory and Practice of Histotechnology, 2nd ed., C.V Mosby Co., St
Louis.
Suvarna SK, Layton C, Bancroft JD. (2013) Bancroft’s Theory and Practice of Histological Techniques,
7th ed., Churchill Livingston Elsevier.
CHAPTER 4
CONVENTIONAL TISSUE PROCESSING

In order to enable the pathologist to diagnose the presence or absence of

disease, the histotechnologist needs to produce a tissue section of good quality
that allows for adequate interpretation of microscopic cellular changes. Solid
tissues need to be fixed and processed to preserve their structures, and eventually
impregnated with an appropriate hardening substance to permit making thin
slices suitable for staining and microscopic evaluation. This can be
accomplished by preserving and carefully processing solid structures and tissues
in the following order: 1. Fixation
2. Decalcification (optional)
3. Dehydration
4. Clearing
5. Impregnation (Infiltration)
6. Embedding
7. Trimming
8. Section-Cutting (Microtomy)
9. Staining
10. Mounting
11. Labeling

“Tissue processing” describes the various steps required to take the tissue from
fixation to the state where it is completely infiltrated with a suitable histological
wax and can be embedded ready for section cutting on the microtome.
Once the tissue has been fixed, it must be processed into a form in which it
can be made into thin microscopic sections. Wet fixed tissues (in aqueous
solutions) cannot be directly infiltrated with paraffin. First, the water from the
tissues must be removed by dehydration. This is usually done with increasing
concentrations of alcohol (70% to 95% to 100%). Because water and paraffin are
not miscible, the specimens must be gradually dehydrated to achieve complete
replacement of water with alcohol. Once successfully dehydrated, the next step
is called "clearing" and consists of removing the dehydrating agent (alcohol)
with a substance that will be miscible with the embedding medium (paraffin).
The commonest clearing agent is xylene. Finally, the tissues are infiltrated with
an embedding agent (almost always paraffin).

Overview of the steps in tissue processing for paraffin sections

1. Fixation
It is important that tissues are handled carefully and appropriately fixed as
soon as possible after arriving in the laboratory. The specimen is placed in a
liquid fixing agent (fixative) such as formaldehyde solution (formalin). This will
slowly penetrate the tissue causing chemical and physical changes that will
harden and preserve the tissue and protect it against subsequent processing steps.
Formalin, usually as a phosphate-buffered solution, is the most popular fixative
for preserving tissues that will be processed for paraffin embedding. Ideally,
specimens should remain in fixative long enough for it to penetrate the tissue
and then for an additional period in order to allow the chemical reactions of
fixation to reach equilibrium (fixation time). Fixation is a critical step in the
preparation of histological sections. If it is not carried out under optimal
conditions or if fixation is delayed, a tissue specimen can be irreversibly
damaged. Following fixation, appropriately trimmed specimens are placed in
suitable labelled cassettes (small perforated baskets) to segregate them from
other specimens. Formalin-fixed, paraffin-embedded tissues may be stored
indefinitely at room temperature, and nucleic acids (both DNA and RNA) may
be recovered from them decades after fixation.
2. Dehydration
Because melted paraffin wax is hydrophobic (not miscible with water), most
of the water in a specimen must be removed before it can be infiltrated with wax.
This process is commonly carried out by immersing specimens in a series of
ethanol (alcohol) solutions with increasing concentration to avoid excessive
distortion of tissue until a water-free tissue in alcohol is reached. Water soluble
proteins are removed at lower concentrations of ethanol. When ethanol
concentration is increased to 100%, certain lipids may be dissolved. A typical
dehydration sequence for specimens not more than 4mm thick would be: 70%
ethanol 15 min
90% ethanol 15 min
100% ethanol 15 min
100% ethanol 15 min
100% ethanol 30 min
100% ethanol 45 min
Fatty tissues such as breast or lipoma may be inadequately processed in
what is normally a successful schedule for other tissues. Ethanol is a poor fat
solvent. To ensure complete dehydration, a superior fat solvent such as acetone
or isopropanol should be added before the final absolute ethanol, and chloroform
or trichloroethane used as the transition solvent.
3. Clearing
The dehydrated tissue is transferred to an intermediate solvent that is fully
miscible with both ethanol and paraffin wax. The term “clearing” has been
chosen because many (but not all) clearing agents impart an optical clarity or
transparency to the tissue due to their relatively high refractive index. Another
important role of the clearing agent is to remove a substantial amount of fat from
the tissue which otherwise presents a barrier to wax infiltration. Following the
dehydration, the tissue is immersed in one to three different xylene immersions.
In these stages, the ethanol is gradually replaced with xylene and when the tissue
is embedded, the xylene will then be replaced by the molten paraffin wax. A
typical clearing sequence for specimens not more than 4mm thick would be:
Xylene 20 min
Xylene 20 min
Xylene 45 min
4. Infiltration
The cleared tissue is infiltrated with a suitable histological wax (usually
paraffin) which is liquid at 60°C and then allowed to cool to 20°C in order to
solidify into a consistency that allows sections to be cut. These waxes are
mixtures of purified paraffin wax and various additives that may include resins
such as styrene or polyethylene that allow them to be sectioned thin enough on a
microtome, forming ribbons that can flatten fully when floated on a warm water
bath.
To completely displace the clearing agent, a typical paraffin infiltration
sequence of paraffin for specimens not more than 4mm thick would be:
Paraffin wax 30 min
Paraffin wax 30 min
Paraffin wax 45 min
5. Embedding
Once the tissue has been processed it is ready to be oriented into a paraffin
block and subsequently sectioned. After infiltration with wax, the tissue is
oriented and placed in a mold that is filled with molten wax to form a solid tissue
block that can later be clamped into a microtome for sectioning. The infiltrated
tissue is removed from the cassette and very carefully oriented in a suitably sized
metal mold so that the “plane of section” can be determined. Correct orientation
of tissue in a mold is the most important step in embedding. Incorrect placement
of tissues may result in diagnostically important tissue elements being missed or
damaged during microtomy. Usually tissues are embedded with the surface to be
cut facing down in the mold. After orienting the section, the mold is filled with
molten wax. The main part of the labelled cassette is placed on top of the mold
and topped up with more wax. The whole mold is placed on a cold plate to
solidify. When this is completed the block with its attached cassette can be
removed from the mold and is ready to be sectioned on a microtome. The choice
of mold will depend on the type of chuck in the microtome that will be used to
section the tissue. Stainless steel, ceramic, paper, plastic, and aluminum foil
molds can be used. The basic method is the same for each.
Double embedding is the process by which tissues are first embedded or
fully infiltrated with a supporting medium such as agar or nitrocellulose, then
infiltrated a second time with wax in which they are also embedded. Double
embedding in agar-paraffin is a reliable and convenient method of handling
minute and friable tissue fragments such as curetting and endoscopic biopsies,
which can be lost during tissue processing. It also overcomes the difficulty of
manipulating small tissue fragments during embedding and facilitates correct
orientation and identification of tissues for histochemistry and
immunohistochemistry. The tissues may shrink by the time they are infiltrated
with wax, but if adequately processed, they will still show good morphological
detail and allow for accurate histopathological evaluation.
6. Section-cutting
Once the tissues have been embedded, they must be cut into sections that are
thin enough to be placed on a slide. This is done with a microtome. Good
microtomy techniques will minimize artifacts that can lead to difficult diagnostic
interpretation of special stains. One of the most directly correlated factors is the
thickness in which a specimen is cut. Specimens for routine Hematoxylin and
Eosin (H&E) are cut 3–5 μm in thickness. Tissues to be examined for amyloid
deposits are better sectioned at 8–12 μm, whereas kidney biopsies should be cut
at 2 μm for optimal viewing of the structures of glomeruli. Paraffin wax does not
provide a sufficiently hard matrix for cutting very thin sections for electron
microscopy. Epoxy resins are the most commonly employed embedding media
for semi-thin and ultrathin sections, but acrylic resins are also used, particularly
where immunohistochemistry is required. Thicker sections (0.35μm to 5μm) of
resin-embedded tissue can also be cut for light microscopy. Again, the
immiscibility of most epoxy and acrylic resins with water necessitates the use of
dehydration, usually with ethanol.
The microtome is nothing more than a knife with a mechanism for
advancing a paraffin block across the knife. Knives are either of the standard
thick metal variety or thin disposable variety (like a disposable razor blade).
Usually this distance can be set, for most paraffin embedded tissues at 6 to 8
microns. Plastic blocks (methacrylate, araldite, or epon) are sectioned with glass
or diamond knives that can cut sections down to about 1 micron. Thin sections
for electron microscopy (0.25 micron) are best done with a diamond knife. The
glass slides containing the specimen are then placed in a warm oven for about 15
minutes to help the section adhere to the slide. If this heat might harm such
things as antigens for immunostaining, then this step can be bypassed and glue-
coated slides can be used instead to pick up the sections.
It is important to have a properly fixed and embedded block or much artifact
can be introduced during the sectioning. Common artifacts include tearing,
ripping, creases, holes or folding of sections, etc. Once sections are cut, they are
floated on a warm water bath that helps remove wrinkles. Then they are picked
up on a glass microscopic slide. The tissue slide is drained and may be gently
heated to evaporate the layer of water between the sections and the glass. When
all the water is gone, the slide may be heated enough to melt the wax, a
procedure that may improve adhesion.
7. Mounting of Tissue Sections
Paraffinized ribbons of serial tissue sections can be removed from the
microtome knife as they are cut, by using a wooden tongue depressor blade. In
this process, a slight traction is exerted on the end of the ribbon, stretching it
gradually over the wooden blade while floating in a warm water bath. The
temperature of the warm bath should be kept at 5–10oC below the melting point
of the embedding wax. If it is too hot, desiccated-looking sections will result,
while cool water baths will produce excessive wrinkling of the tissue. Adding a
few drops of ethyl alcohol to the water may facilitate the mounting of tissue
sections. The ribbon must not be left in the bath for more than 1 or 2 minutes, or
over-hydration of the tissue will be produced, simulating the appearance of
edema fluid when examined microscopically. Because tissue sections do not
adhere well to untreated glass slides, a bonding agent also must be a component
of the water bath. Albumin, and poly-L-lysine are all suitable additives of this
type.
One of the most serious issues faced in histotechnology is misidentification
of tissues, which includes mislabeled specimens, block identification problems,
and tissue contaminants. A potentially dangerous mistake that can take place
when mounting sections from flotation baths is the “shedding” of friable small
tissue fragments that float freely on the surface of the water, and which may be
inadvertently picked up when mounting slides from subsequently processed but
unrelated cases. Known as ‘‘floaters’’, these tiny pieces of unrelated tissue
commonly cause problems in interpretation of the biopsies by the pathologist.
For example, a small piece of unrelated cancerous tissue may inadvertently
“float” and be deposited on the slides of a subsequent case that is being
evaluated for the presence of malignancy.
This may lead to an inaccurate diagnosis and result in medicolegal liabilities
on both the pathologist and the histotechnologist. Meticulous cleaning of the
microtome water bath and frequent clearing or changing of the water will
alleviate the danger of rare contaminants being carried over to subsequent
sections being mounted. Also, the technologist must routinely skim, or otherwise
clear, the surface of the water bath between cases. Another source of floater-type
artefact is the ‘‘tongue blade metastasis,’’ wherein tissue adheres to a wooden
applicator stick that is used to float successively prepared ribbons from two
different cases. In cases where the specimen is limited in size (such as skin,
bronchoscopy and gastrointestinal biopsies), it is advisable to save any
unmounted paraffin ribbon (with appropriate identification) from such cases for
at least a week after they are accessioned, so that remounts can be prepared
when additional sections are requested, without the need for further microtomy
of the tissue block.
8. Staining
The tissue sections mounted on the slide are nearly invisible under a light
microscope so they must be stained to create contrast. After drying in a 60oC, the
slide is passed through another series of chemical reagents. Xylene removes the
paraffin and absolute alcohol removes the xylene. The tissue on the slide is then
rehydrated to prepare it for staining. Most staining procedures in the laboratory,
aside from antibody-based immunohistochemistry (IHC), use chemicals or dyes
that will bind or have affinity for certain components of the cells and
extracellular components. The chemical properties of these dyes produce the
visual appearance that is seen under the microscope. Different staining
procedures are done depending on the tissue component that is being studied.
Potential contamination during the staining procedure may be much higher
than having “floaters” when mounting sections from a water bath. The tissue is
deparaffinized during the first steps in preparing the slides for staining. As the
slides are dipped up and down into the staining baths, the deparaffinized tissue
can fragment and small dis-cohesive pieces can break free and be lifted on the
slide. Contamination in the staining solution is dependent on the volume of
slides being stained and the time point during the day that the samples are taken.
Because the stainer baths are a potential reservoir of tissue contaminants,
changing the staining fluids may alleviate some of the potential for carryover
from this source. And, as higher numbers of the contaminating fragments are
localized to the first xylenes and alcohols, frequently changing these baths in
particular may also be useful.

Automatic Tissue Processing

Tissue processing can be performed manually (hand processing), but when
there is a large volume of tissues that need to be processed, it is more convenient
and much more efficient to use an automated tissue processing machine (“tissue
processor”). This machine allows the specimens to be infiltrated with a sequence
of different solvents finishing in molten paraffin wax. The specimens are in an
aqueous environment to start with (water-based) and must be passed through
multiple changes of dehydrating and clearing solvents (typically ethanol and
xylene) before they can be placed in molten wax (which is hydrophobic and
immiscible with water). The duration and step details of the “processing
schedule” chosen for a particular batch of specimens will depend on the nature
and size of the specimens. The older design of an automatic issue processor is a
carousel which contains a cage in which the tissue cassettes are placed. This
carousel has a number of glass beakers containing solvents and solutions which
ensure that the tissue is dehydrated and cleared ready for paraffin wax
embedding. The carousel vertically agitates the cage in each solution before
moving on to the next solution in the dehydration/ clearing method. The modern
processors have a chamber in which the specimens are held and the different
solutions are pumped in and out of the chamber. In general, the whole process
takes around six hours and is usually set up to run overnight. Tissues that come
off the tissue processor are still in the cassettes and must be manually put into
the blocks by a technician who must pick the tissues out of the cassette and pour
molten paraffin over them. This "embedding" process is very important, because
the tissues must be aligned, or oriented, properly in the block of paraffin.

Fig. 4-1. Automatic Tissue Processor

Due to the viscosity of molten paraffin wax, some form of gentle agitation is
highly desirable. If the processor is to be run overnight, it should be programmed
to hold on the first ethanol bath and not finish until the next morning so the
specimens do not sit in hot paraffin longer than the time indicated. If specimens
are fresh they may incubate in formalin in the first stage on the machine. It is
important to not keep the tissues in hot paraffin too long or else they may
become hard and brittle. Processed tissues can be stored in the cassettes at room
temperature indefinitely. Vacuum infiltration is the impregnation of tissues by a
molten medium under reduced pressure. The procedure assists the complete and
rapid impregnation of tissues with wax and reduces the time tissues are subjected
to high temperatures, thereby minimizing heat-induced tissue hardening,
facilitating complete removal of transition solvents, and prolonging the life of
wax by reducing solvent contamination.

Factors that impact the duration of tissue processing and extent of

infiltration
1. Tissue Density and Thickness
Variable tissue density affects infiltration and subsequent sectioning of
tissues. Spongy tissues (like lungs) are usually more rapidly infiltrated
than hard and dense tissues. Thickness of the tissue also influences the
rate of reagent diffusion and hence processing time. Tissue thickness
should be optimized for particular processing schedules, or alternatively,
processing times should be adjusted to accommodate thick, thin or large
tissue blocks.
2. Agitation
Agitation using manual or automated processors increases the flow of
fresh fluids in and around the tissues. Most tissue processing protocols
utilize automated processors with vertical or rotary oscillation
mechanisms to speed fluid exchange. Without agitation, tissues tend to
settle to the bottom of the processing device or become too tightly
packed, therefore reducing surface area available for fluid exchange.
Fluid interchange between processing reagents and tissues is promoted
by exposure of the maximum tissue surface area. Therefore, tissues
should be loosely packed in baskets to facilitate exchange of reagents
and increase diffusion. Ideally, the cassette perforations should be
perpendicular to the fluid flow. If tissues are allowed to settle on the
bottom of a container or are too tightly packed, tissue surface area
available for fluid exchange will be severely restricted.
3. Temperature
Temperatures in the range of 37° to 45°C, for a limited time can speed
up fluid penetration and tissue processing protocols. However, heat must
be carefully monitored. High temperature can cause the tissue to shrink
and to become hard and brittle, while low temperature increases the
viscosity of reagents used in tissue processing, thereby reducing the rate
of diffusion and increasing processing time. This can be avoided by
maintaining embedding waxes 2o to 3°C above their melting points.
Tissue shrinkage during infiltration in paraffin wax results mainly the
effect of heat on collagen.

4. Vacuum and pressure

Reduced pressure can increase the infiltration rate and decrease the time
needed to complete steps in tissue processing protocols. High pressure
facilitates infiltration of dense specimens with the more viscous
embedding media. Vacuum application during tissue infiltration
improves processing quality and can aid in removal of trapped air from
 porous tissue. Using vacuum during tissue processing protocols can
reduce the infiltration time when dealing with dense and fatty tissue
specimens. Vacuum applied during dehydration, clearing and infiltration
improves the quality of processing in tissues such as lung which
becomes de-aerated during the process. However, duration of wax
infiltration is dependent upon viscosity and is not generally reduced by
applying a vacuum.

Technical Considerations
Baskets and metal cassettes should be clean and wax-free.
Tissues should not be packed too tightly in baskets so as to impede fluid
exchange.
Processors must be free of spilled fluids and accumulated wax
accumulations.
Fluid levels must be higher than the specimen containers.
Timing and delay mechanism must be correctly set and checked against
the appropriate processing schedule.
A processor log should be kept in which the number of specimens
processed, processing reagent changes, temperature checks on the wax
baths and the completion of the routine maintenance schedule are
recorded as part of quality assurance program.
Gentle washing and minimal thickness of cell layers will prevent the
cells from detaching during staining. Make sure that there are enough
sections to make a diagnosis, and ensure that the reagents have been
applied evenly to the slides.
Quality of staining can be compromised by inadequate fixation and
similarly by poor tissue processing. A good technician must evaluate and
determine the processing of choice for each purpose, i.e., special stains
on paraffin, frozen or cell smear preparations.
Make sure glass slides are clean and free from debris.
In general, needle biopsies and bloody specimens should be incubated
conservatively, whereas fatty specimens can be processed for longer than
average.

REFERENCES
Brown RW. (2009) Histologic preparations: common problems and their solutions. Northfield, IL:
College of American Pathologists.
Clayden EC. (1971) Practical section cutting and staining. Edinburgh: Churchill Livingstone, 1971.
Carson FL. (2007) Histotechnology. 2nd ed. Chicago: ASCP Press.
Carson FL, Hladik C. (2009) Histotechnology: a self-instructional text. 3rd edition. Chicago: ASCP
press.
Cook DJ. (2006) Introduction to Techniques and Applications, 2nd ed. Oxfordshire, UK: Scion
Publishing Ltd.
Hegazy R, Hegazy A. (2015) Simplified Method of Tissue Processing (Consuming Time and
Chemicals). Ann. of Int. Med. & Den. Res. 1(2):57-61.
Henry JB. (1991) Clinical diagnosis and management by laboratory methods. 18th ed. Philadelphia:
W.B. Saunders.
Histopathology. New York: Churchill Livingstone, 4.2-1 - 4.2-39.
John DB, Anderson G. (2002) Theory and Practice of Histological Techniques.5th ed., Chap 6. Tissue
Processing and Microtomy including Frozen: Elsevier Churchill Livingstone, Edinburg p. 85-108.
Kothar S, Phan JH, Wang MD. (2013) Eliminating tissue-fold artifacts in histopathological whole-slide
images for improved image based prediction of cancer grade. J Pathol Inform., vol. 4, p. 22.
Meschr A. (2012) Junqueira's Basic Histology: Text & Atlas, 12th edition, Chapter 1. Histology & Its
Methods of Study. The McGraw-Hill Companies.
Panja P, Sriram G, Saraswathi TR, Sivapathasundharam B. (2007) Comparison of three different
methods of tissue processing. J Oral Maxillofac Pathol 11(1):15-17.
Smith PJ, Warfield JL. (2005) Tissue processing and embedding in: The science of laboratory
diagnosis: Section 1: Histopathology, 1-specimen handling and preparation of routine diagnostic
histopathology Crocker J and Burnett D (ed.) 2nd edition, Wiley J & Sons Limited (publishers),
England.
Spencer LT, Bancroft JD. (2008) Tissue processing in: Theory and Practice of Histological Techniques,
Gamble M, Bancroft JD (eds.), Chapter (6th ed., Churchill Livingstone Elsevier Ltd.
Suvarna KS, Layton C, Bancroft JD. (2012) Bancroft’s Theory and Practice of Histological Techniques,
7th ed. London, UK: Churchill Livingstone.
Winsor L. (1994) Tissue processing. In Woods A and Ellis R eds., Laboratory
 CHAPTER 5
RAPID TISSUE PROCESSING

In conventional laboratories, routine use of formalin fixation, overnight

dehydration, paraffin infiltration, manual embedding and sectioning have
produced relatively uniform, high-quality tissue sections for histopathologic
examination. Frozen sections have been used when there is an urgent need for
immediate histopathologic evaluation. In recent years efforts have been made
to streamline the processing time by using microwave heating techniques. This
continuous throughput Rapid Tissue Processing (RTP) has introduced dramatic
changes into the practice of modern histotechnology.
As opposed to conventional tissue processors which use a graded series of
alcohols, a clearing agent (usually xylene) and embedding medium (usually
paraffin wax) in an overnight process, microwave assisted processing allows
dehydration to be done in only one step and employs a four step process
involving a single change each in ethyl alcohol and isopropanol followed by
two changes in paraffin. When exposed to these high microwave temperatures,
the alcohols quickly heat and dissipate, while the paraffin remains inert,
allowing the paraffin to fully infiltrate the specimen. This eliminates the use of
xylene from the process. Processing is undertaken in a dedicated microwave
oven which is fitted with precise temperature control and timer, and an
interlocked fume extraction system to preclude accidental ignition of solvent
vapor. A turntable or in-built radiation dispenser facilitates even reagent
heating.
Microwave processing can be 60% to 80% faster than conventional
processing time with an hourly throughput of up to 120 specimens. The
implementation of RTP allows for rapid turnaround of tissue biopsies, is as
effective as slower conventional tissue processing, and provides excellent
morphology, sharp nuclear detail and crisp cellular images. The reagents used
in molecular pathology can preserve DNA, RNA and proteins in the paraffin
block, and eliminate the need to use fresh tissue for molecular studies,
particularly with respect to the identification of gene activation and detection
of gene products such as messenger RNA (mRNA) and proteins. Microwave
technique is now commonly employed to unmask or retrieve antigens, using 10
mm. citrate buffer (pH 6.0).
Microwave irradiation has been used successfully to expedite tissue
fixation and processing. Microwave, a form of electromagnetic wave-induced
heat, when applied in histotechnology, yields histologic material of similar or
superior quality to that provided by conventional processing methods.
Microwave-assisted processing of tissues, from fixation through paraffin, can
save time and produce better results, when the proper microwave technology is
used. The ability to control temperatures by a number of different parameters
(variable wattage, restrictive temperature probe and water recirculation) makes
it possible to achieve results that were not formerly available from
conventional tissue processing systems. The technology makes it possible to
employ any of the routinely used reagents for paraffin processing, and can be
applied to accelerate routine, special, and metallic, as well as
immunofluorescent stains.
Microwave works as a physical agent similar in mechanism to vacuum,
oven (heat) and agitation to expedite the movement of molecules, thereby
accelerating fixation, decalcification, tissue processing and staining for
immunohistochemistry and electron microscopy. Rapid manual microwave-
stimulated paraffin wax processing of small batches of tissues gives excellent
results comparable to tissues processed by longer automated non-microwave
methods. Calibration of domestic ovens is essential for optimum results. The
accuracy of the temperature probe, duration of cycle time, and net power levels
at various settings must be determined before the oven is used to process
tissues.
The equipment used in rapid tissue processing can vary from kitchen type
microwave ovens to sophisticated semi-automated or automated instruments.
These systems all use the same basic principles, with some distinctive
variations that can accommodate differences in laboratory needs and practice
patterns. Microwave excitation of the molecules increases movement in both
solutions and tissues, resulting in improved tissue penetration and fixation,
with preservation of many tissue antigens of diagnostic interest. Lower
temperatures require longer heating times, while higher temperatures require
shorter heating time to achieve strong intensity of staining.
Microwave processing requires very precise maintenance of temperature,
which is not possible with the use of kitchen-type microwave ovens whose
temperature and output power (in watts) are not precisely specified, leading to
inadvertent overheating and miscalculation of the percentage output power.
Because their magnetrons were not precisely controlled, early work with the
kitchen type microwaves resulted in uneven heating (uneven distribution of
microwave energy) and sometimes unpredictable amounts of delivered energy.
Specifically designed microwave units have been developed, which utilize a
removable reaction vessel (to hold cassettes), with manual reagent exchanges
according to a predetermined schedule. These units represent an economical
method for rapid tissue processing, but only on a relatively small scale. They
are also relatively labor intensive.

Fig. 5-1. Microwave tissue processor

[Link]

Unlike domestic microwave ovens, the laboratory microwave oven does

not produce hotspots or uneven heating in tissues due to the presence of
magnetic stirrer kept beneath which provides an even field of irradiation. The
toxic and flammable solvent vapors generated during processing cannot always
be adequately vented from domestic ovens and present an ignition hazard if the
electrical system is unprotected, unlike laboratory ovens wherein adequate
ventilation is created for the escape of fumes. Laboratory microwaves are now
specifically designed and equipped with a thermocouple temperature probe and
controller to maintain temperatures within a very narrow range of +/- 1°C,
thereby producing consistent and reproducible results.
Staining methods take much shorter time in a microwave oven compared
to conventional tissue processing. In general, microwaved slides have better
contrast, more intense staining, and less non-specific staining, compared with
conventional metal-staining methods. Calibrated laboratory microwaves
equipped with temperature probes and air-bubble agitation device have largely
addressed the earlier problems of inconsistent or uneven heating observed
when kitchen-type microwaves were originally used. The optimum temperature
is 75° to 95oC, for metallic stains, and 55° to 60°C for non-metallic stains.
Researchers have found microwave technology to be beneficial in achieving
epitope recovery in formalin-fixed tissues for many markers within 10 min to
15 min.
An enclosed system similar in configuration to modern vacuum assisted
conventional tissue processors has been recently introduced. This instrument,
when combined with microwave fixation, can reduce processing time to about 3
hours and may also be used in a formalin free environment. Like traditional
instruments, these rapid tissue processors run in 'batch' mode; once the cycle has
begun no additional tissues can be added for processing until the cycle is
completed. There are some size constraints, as large tissue section (greater than
1.5 mm. thick) may show inadequate or uneven penetration of reagents, leading
to incomplete clearing and paraffin impregnation. Large tissue blocks will
require special handling to prevent incomplete fixation or tissue clearing. Small
blocks such as endoscopic biopsies can be processed in a shorter period using
tailor-specific protocol.

Fig. 5-2. Sakura Rapid Tissue Processor

([Link]

This system of continuous throughput rapid tissue processing allows for

the addition of new specimens to the processor every 15 minutes, as a reaction
chamber becomes available. This automated system utilizes computer
controlled microwave delivery and temperature controls in a formalin free
environment. Reagent volumes are small, and all reagents are relatively
inexpensive and non-toxic . Xylene has also been eliminated, and vacuum
assisted paraffin impregnation allows for a complete processing cycle of about
60-70 minutes.
Current Rapid Tissue Processors use an innovative, low-wattage
microwave technology, molecular-friendly reagents and traditional vacuum
infiltration techniques to provide consistent, high quality results in record time.
It has a continuous efficient workflow, consistent high quality 1-hour
processing of up to 120 specimens per hour, maximum standardized same day
diagnosis, formalin- and xylene-free processing. Continuous throughput yields
up to 960 cassettes in an 8-hour shift, loads up to 40 cassettes approximately
every 20 minutes, and immerses specimens for 15 minutes in each of the 4
enclosed processing retorts with the standard program.
Several studies have shown that the quality of tissue sections after rapid
tissue processing is comparable, and in some cases, superior to conventional
tissue processing methods, with no detrimental effects on the commonly used
special staining procedures. Immunohistochemical stains can be performed on
sections without antigen retrieval in some cases, and with more highly diluted
primary antibodies in others.
The most dramatic impact of rapid tissue processing has been in the same
day turnaround time from receiving the biopsy until microscopic evaluation and
completion of surgical pathology reports, a significant departure from
conventional surgical pathology practice. Microwave processing can be
achieved 60% faster than conventional processing time so that urgent cases can
be signed out on the same day. In some institutions the entire sequence of
needle biopsy, tissue processing, slide interpretation and immunohistochemistry
for receptors has been carried out within about 4 to 5 hours.
One critical aspect of using microwave techniques is ensuring that samples
have been adequately fixed. While rapid tissue processing by microwave
methods can accept a number of different tissue fixatives, non-formaldehyde
fixatives have been shown to have several advantages. An alcohol-based
fixative has recently been introduced which, when combined with a formalin-
free microwave based rapid tissue processing, has been demonstrated to permit
the recovery of DNA, RNA, and proteins for molecular analyses. This universal
molecular fixative, known as UMFIX (Sakura Finetek USA Inc., Torrance
California), is a mixture of methanol and polyethylene glycol, and is an
excellent and cost-effective alternative to formalin. UMFIX is non-volatile and
active at room temperature. The morphology of UMFIX-exposed tissue is
comparable to that fixed in formalin, and high molecular weight RNA is
preserved in tissue that is immediately fixed and stored up to 8 weeks at room
temperature. The elimination of formalin in both the fixation process and the
processing has improved the quality of antigen preservation in the tissue
sections. An added benefit is the elimination of noxious formalin fumes from
the workplace, as well as expensive ambient air formalin monitoring programs.
Each laboratory should evaluate its fixation control methods to optimize
the use of microwave processing. Converting to the safer chemicals (i.e., less
fumes, non-regulated disposal) may include rotating smaller quantities more
often, causing a net increase in chemical consumption. The safety benefits of
removing undesired regulated waste in addition to calculating net volumes may
offer immediate cost savings.
 Although mechanical or electrical faults occasionally occur in tissue
processors, processing mishaps where tissues are actually compromised, mainly
occur because of human error. Inappropriate processing schedule or making
fundamental mistake (perhaps in replenishing or sequencing of processing
reagents) can result in the production of tissue specimens that cannot be
sectioned and therefore will not provide any useful microscopic information to
the pathologist.

Precautionary Measures:
Sections must be thin enough so fixative and dehydrating solutions can
penetrate completely (about 1.5 mm.). This may increase the time
required in grossing of specimens.
Certain tissues (i.e., brain and large tissue blocks) require additional
steps before placing into the rapid tissue processor.
Continuous flow processing eliminates batching of specimens and
necessitates ongoing attention to the instrument. As samples complete
the processing cycle, they must be removed from the instrument to
accommodate the next basket of cassettes.
Paraffin must be added to the microwave in liquid form, as microwave
energy will not melt paraffin pellets.
Tissue must be brought to the boiling point of the intermedium (82°C for
ethyl alcohol and 78°C for isopropanol) in the last step, to "flash
evaporate" the remaining ethyl alcohol or propanol.

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