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 29
CYTOLOGIC TECHNIQUES

Diagnostic Cytology simply means microscopic examination of cells from
different body sites for diagnostic purposes. Diagnostic cytology includes
exfoliative cytology and fine needle aspiration. For cytologic examination to
be of diagnostic value, the sample must include representative material and the
method used to process the specimen must give rapid results, without
sacrificing cellular detail. Consistency and reliability are most important in
cytological interpretation. Cytologists rely heavily on the quality and
appearance of the stain.
Specimen for cytologic examination may be derived from various sources:
1. Exfoliative cytology
2. Fine needle aspiration
3. Body fluids

Exfoliative Cytology
Exfoliative cytology deals with the microscopic study of cells that have
been desquamated from epithelial surfaces. Exfoliated cells may be found in
smears that have been spontaneously shed or physically removed from
epithelial and mucous membranes. The cytological specimens collected from
female genital tract include cervical smear, vaginal smear, and endometrial
smear. Spontaneous exfoliation is observed in normal cells due to constant
growth and replacement with new cells - more readily observed in malignant
tumor cells. Specimen can be collected from the epithelial surfaces by lightly
scraping the surface, by swabbing, aspirating or washing the surfaces.
Exfoliative cytology is usually recommended for the following:
• Detection of malignant cells in body fluids, mainly used for staging
cancer.
• Detection of precancerous cervical lesions in women (cervicovaginal
smear/Pap smear).
• Assessment of female hormonal status in case of sterility and
endocrine disorders. This is achieved by microscopic evaluation for
determination of maturation index (MI), based on examination of
smears taken from the lateral vaginal walls.
 • For determination of genetic sex -most of the nuclei of females exhibit
conglomeration of chromatin, representing XX chromosomes (Barr
body), which may be demonstrated in the smears from buccal or vaginal
mucosa.
• For detection of infectious agents.

Smear Preparation
Smears should be made from fresh material, prepared in the doctor's office
or radiology units. For direct smear preparation, the material should be
smeared evenly on a clean slide. The patient's identity, including name, age and
date and type of specimen must be legibly indicated on the laboratory
requisition form and the laboratory identifying number should be put on the
slide (preferably on a frosted -end slide with printed label, or with a diamond-
point pen).
Histotechnicians sometimes perform special stains on cytology smears,
blood films and cytopreps from other departments within the laboratory.
Increasingly, the commonly received cytoprep is that of the “thin prep.” These
smears are wet-fixed in 95% ethanol immediately after preparation to preserve
the fine structure of the chromatin and help in the evaluation of nuclear
changes. Air drying is avoided with smears for cytological detection of
neoplasia because it changes the appearances of the cells. Slides bearing blood
or bone marrow smears, on the other hand, are usually air-dried. Marrow
smears are stained in parallel to sections of the bone marrow core biopsy.

Cervical Smear:
Cancer of the uterine cervix is the commonest cancer that can be detected
even at the pre-invasive stage. Cervical cytology screening involves the
microscopic examination of cell samples that have been taken primarily from
the ecto- and endocervix, smeared on glass slides and stained by the
Papanicolaou (Pap) method. The entire procedure is known as Pap smear.
Because cervical cytology is a screening test, abnormal findings must be
confirmed histologically.
The patient should abstain from coitus, not douche the vagina for at least a
day, and not apply intravaginal preparations for at least one week before the
examination. Smears should not be taken during menstrual bleeding, because
of contamination with blood, endometrial component, and tissue debris that
can obscure the cells during smear examination.
Various collecting systems such as cotton swabs, wooden or plastic
spatula, and cervical brushes, so-called cytobrushes, are generally used to
collect gynecology cells. In brush biopsies, more endocervical cells and blood
may be present in the smears, and this may lead to valuation difficulties. The
use of cotton swab for collection of cervical smear is to be discouraged because
of the drying artifacts and loss of cells that are caused by this method. Wooden
spatula is preferable to plastic spatula, because of its mildly rough surface that
can collect more material. Endo-cervical brushing is used strictly for taking
materials from endocervix.
After smear collection, the sample is evenly smeared on to the center of
the non-frosted area of the glass slide and fixed immediately. Excessively thin
or thick smears can result in false-negative reports.

Impression Smear
Impression smears are often used for ulcerated surface lesions to allow an
immediate assessment of the lesion before fixation and processing of the tissue
sample. The preparations should be quickly air dried and then stained. It is also
indicated in the case of tumors especially of lymph nodes. Soon after an
excision biopsy of lymph node, the specimen is cut using a sharp scalpel blade
an imprint of the smear is taken by touching the cut surface with a clean slide
and fixing immediately.

Sputum Smear
• Obtain at least 3 consecutive morning sputum specimens.
• Collect early morning sputum by a deep cough in a wide -mouthed jar
containing Saccomanno fluid (50% ethyl alcohol and 2% carbowax).
If the patient cannot cough up sputum spontaneously, induced sputum
should be collected using inhalation of an aerosol solution for 20 minutes to
produce a deep cough sample. If a more extensive study is to be made, it is
recommended that a minimum of 2-4 slides be prepared, and one is air dried
for Giemsa staining. At least two slides should be stained by Papanicolaou
method.
The collected sputum specimen is poured into a Petri dish and examined
first for blood-flecked or solid particles which are removed, placed on the
slide, crushed slightly with another slide and distributed evenly with a small
amount of the liquid portion. If no solid particles are found, an attempt should
be made to secure representative samples from both thin and thick portions.
With the end of a wooden applicator stick, these samples are evenly spread on
the slides and immediately placed in fixative for a minimum of one hour.
Due to the hardening effect of alcohol, specimen fixed in said solution
should be sent to the laboratory and smears should be made without delay.
 Sputum collected should have been coughed from "down-deep" to ensure
that the specimen is a true representative of the sputum and not just saliva. The
mucus present preserves the cell constituents; however, it is highly
recommended that the specimen be as fresh as possible when examined, if
better cellular detail is to be appreciated.
Alveolar macrophages are found on the sputum smears from a deep cough.
Absence of alveolar macrophages suggests "saliva" rather than sputum and
results an unsatisfactory specimen.


Bronchoscopy specimens:
Bronchial Brushing specimen is directly smeared onto two labeled slides
by pull technique (usually done by a bronchoscopic specialist at the
bronchoscopy laboratory). The slides are then fixed by a spray fixative or
immediately immersed in 95%alcohol. Failure to fix the slides within a few
seconds will produce unsatisfactory cytologic results due to air drying artifact.
Bronchial Washing specimens are freshly collected in the bronchoscopy
collection container and hand delivered to the laboratory.
Bronchial Aspirates: The secretion obtained at bronchoscopy are collected
either by aspiration into a glass suction apparatus or by washing the bronchi
with 1-2 cc. of saline, into a cup, if sufficient in amount, and sent immediately
to the laboratory for preparation and examination. Care should be taken to
collect specimen taken directly from the bronchus, to insure that the smear
contains epithelial cells (ciliated bronchial cells) from the bronchial mucosa,
not just red blood cells and leukocytes.

Smears of Gastric Secretions and Aspirates
Gastric smear preparations are quite difficult to make due to inaccessibility
of the specimen and the presence of gastric juices which have a deleterious effect
on the morphology of exfoliated cells. Smears are usually collected by simple
irrigation and aspiration technique. The collected specimen is then sent to the
laboratory. Specimen should be examined as soon as possible since any delay of
more than I /2 hour, before fixation, will digest the cells and make the specimen
unsatisfactory for evaluation.
The patient should have fasted for at least 8 hours before gastric washing is
performed. Esophageal washings are to be examined immediately.

Smears of Breast Secretion
Cytologic examination of nipple discharge has an extremely low diagnostic
yield for diagnosis of breast carcinoma. Spontaneous nipple discharge is usually
a result of hormonal imbalance in young patients, and when the secretion is
bloody a benign intraductal papilloma should be considered clinically. The
spontaneous nipple discharge should be smeared on a clean glass slide, and
immediately placed in fixative.
In women, except during lactation and the immediate post-lactation period,
any discharge from the nipple is abnormal .The nipple discharge is usually due to
a benign breast lesion such as duct ectasia and papilloma , or due to endocrine
problems. However the major value of cytologic examination of nipple discharge
is potential detection of malignant cells in a patient with clinically undetected
carcinoma.

Collection Technique:
Gently strip the subareolar area and nipple using the thumb and forefinger.
Place the labeled slide upon the nipple and draw it quickly across the nipple. If
more than a drop is collected, use another slide to smear with a pull-up
technique. Immediately drop slide in a bottle of 95% isopropanol or use spray
fixative.
If the secretion is scanty in amount, smears should be restricted to a small
area of the slide to prevent drying. Secretions obtained from both breasts should
be properly identified as left or right. For localized breast lesions producing
discharge upon pressure, the secretion may be smeared directly on the slide after
expressing material from the breast.

Fine Needle Aspiration (FNA)
Fine needle aspiration is the study of cellular samples obtained from organs
that do not shed cells spontaneously, such as breast, thyroid, lymph nodes, liver,
lungs, skin, soft tissues and bones. It is useful in lesions that are easily palpable,
like growth of skin, subcutaneous soft tissue tumors, thyroid, lymph nodes,
salivary glands and breast.
The basic technique uses a 25-gauge needle and a 10-ml syringe. The
needle is inserted into the lesion and repeatedly redirected to sample a number
of areas while applying a small amount of suction on the syringe. Tissues
composed of mesenchymal cells (connective tissue) tightly adhere to each
other and do not exfoliate easily, requiring a larger bore needle and increased
suction may be necessary. The sample should be air dried as quickly as
possible to reduce the effects of shrinkage.
FNA of superficial masses (e.g. breast, thyroid, and peripheral lymph
nodes) is usually done by the clinicians, or in some centers by the pathologists.
Deeply seated lesions such as lung, mediastinum, abdominal organs (liver,
pancreas, etc.) and retroperitoneal organs (kidney, adrenal, lymph nodes) are
performed under laparoscopy computerized tomography (CT scan) or
ultrasound (sonography).

Slide preparation:
When a solid lesion is aspirated, usually a few drops from the tip of the
needle has the most diagnostic material for cytologic evaluation. On the other
hand when the specimen is bloody, the diagnostic cells are diluted and hard to
find on a direct smear. We recommend preparation of maximum 4 slides: using
1-2 drops on each slide and using slide-pull technique (similar to the technique
used for peripheral blood smears). Then rinse the needle in a preservative
solution such as Saccomano fluid, and send it to the laboratory. It will be
processed like any other body effusion specimen at the cytology laboratory.
An ideal aspirate is of creamy consistency with numerous cells suspended
in a small amount of tissue fluid without admixture with blood. In lymph node
aspiration, a cell suspension can be prepared in addition to direct smears.
Smears obtained by FNA are fixed based on the requirements of the stain to be
used.
• Colloid, mucin and smears to be stained with hematological stains
(such as May–Grunwald–Giemsa) should air dried. Smears that are not
correctly made and dried quickly will produce artefacts. Rapid stains
like Diff Quik (2-3 minutes), are particularly useful in preliminary
assessment of adequacy of the sample before the patient is released.
• Smears to be stained by Papanicolaou (Pap) or hematoxylin and eosin
(H&E) should be rapidly fixed in alcohol (wet fixation) to show nuclear
details and allow better identification of malignant cells. If the smears
are allowed to dry and not quickly fixed in alcohol, drying artefact can
occur, the cytoplasm will be more eosinophilic, and nuclear details will
appear fuzzy.
For Transbronchial Fine needle Aspiration (FNA) specimens, a
drop or two should be released onto a slide, smeared by two-slide
pull method and fixed immediately.

Body Fluids:
Cytological investigation of body fluids such as urine samples,
cerebrospinal fluid, pleural or peritoneal effusions, obtained by aspiration, can
cast light on the origin and type of primary tumors as opposed to reactive
processes.
 Serous effusion refers to the fluid accumulated in the three serous cavities
namely pleural, pericardial and peritoneal, which are important sources of
diagnostic material. The presence of malignant cells in serous effusions usually
indicate metastatic involvement and, therefore, a higher stage of cancer.
Collections of specimen are usually processed in the same way as a bronchial
secretion.
Jelly-like clots forming alter removal may be prevented by adding 300
units of Heparin for every 100 ml. of aspirate. This is usually done by
collecting the specimen in heparinized collection containers. They can be
collected in tubes or syringes that may be either plain or pre-heparinized, to
prevent coagulation. Freshly tapped specimens are preferred for cytology. If
immediate processing is not possible, it can be preserved in the refrigerator for
a period of 24-48 hours or pre-fixed in 50% ethanol. Albuminized slides should
be used to prepare smears from prefixed sample.
Cytospin preparation is by far the best method to collect cells from body
fluid (e.g., urine, pleural or peritoneal fluid). For those that have no access to a
cytospin, centrifugation of the preparation and sampling of the centrifuged
sediment is the usual method of cell concentration. For cytological evaluation,
the specimen is centrifuged and one or two drops of sediment are place on a
glass slide, spread out and fixed immediately. A major objection to the use of
cytocentrifuge is the distortion of cellular morphology due to air drying
artifacts, which can be avoided by immediate fixation or by using an equal
volume of polyethylene glycol.

Fig. 29-1. Cytospin of pleural fluid aspirate

Cell Suspensions:
Cell suspensions may be received as the result of direct taps of pleural or
peritoneal effusions, as well as from cerebrospinal fluid (CSF) and synovial
fluid. Most cytologists prefer to receive effusions as rapidly as possible in
sterile containers without fixative or anticoagulant. The optimum amount is 20
to 30 ml. Cells can remain viable for up to 4 days if the specimen is kept
refrigerated at 4°C (do not freeze).
Urine, serous effusions and watery lavages (including bronchoalveolar
lavage) all require concentration of cells prior to transferring them to glass
slides. Centrifugation is the standard technique.

Preparation of cytospin slides:
The specimen is centrifuged as soon as possible at 1000 RPM speed for 1
minute (our suggested method), and the supernatant fluid is removed. A smear
is made of the sediment on a glass slide which has been previously coated with
a thin layer of egg albumin, spreading the smear with another slide as evenly as
possible. When it begins to dry around the edges but is still moist in the center,
the smear is fixed in 95% alcohol.
If smears cannot be prepared immediately, the sediment should be covered
with absolute alcohol and placed in the refrigerator.
Note:
When secretions are small in amount, the smear should be prepared and
fixed in the operating room/bronchoscopy room to prevent specimen drying.

Urinary Tract Specimen
Since the diagnosis is based on the type of specimens under investigation,
it is extremely important to get the information about the type of specimen, and
collection technique when dealing with urinary specimens. Specimens should
be clearly labeled as: • Voided urine (see below)
• Catheterized specimen
• Washings from bladder or renal pelvis
Note: The first voided urine should be discarded, due to the overnight
degeneration of cells. The second urine is preferred. The freshly collected urine
should be sent to the lab immediately. If delay in transportation is anticipated,
refrigeration is recommended. We do not recommend use of any preservative.
Voided urine from males is usually sufficient for cytological evaluation,
but for female patients, a catheterized specimen is recommended to prevent
contamination of specimen with vulvar cells. Early morning specimen yields
the greatest number of cells, but such cells are usually distorted due to
prolonged bladder retention. To obtain a more reliable cytological evaluation,
urine specimen may have to be collected and examined twice -one in the early
morning and another later in the day. At least 50 ml is needed, which must be
centrifuged. Smears of sediments should be prepared and fixed as soon as
possible after collection. Since a great proportion of cells become detached
during fixation, unsatisfactory smears may result in false negative diagnosis. In
general urine cytology alone offers a very low diagnostic yield for detection of
urothelial carcinomas. The collection technique should be always mentioned
on the requisition form. The cytomorphologic features of low grade urothelial
carcinomas may be indistinguishable from those of reactive urothelial
hyperplasia.

Body Cavity Effusions
The fluid specimens should be collected in a clean, non-sterile dry
container, and submitted fresh to the laboratory. If transportation is delayed, the
specimen should be placed in the refrigerator. Use of formalin, alcohol or any
other preservative should be avoided. Heparin, on the other hand, does not
interfere with cytologic preparation and evaluation.
Cells in cerebrospinal fluid (CSF) samples degenerate very quickly and
are usually present in very low numbers. Once the slide has been prepared, it
should be rapidly air dried before staining. The addition of an equal amount of
ethyl alcohol to the cerebrospinal fluid (CSF) is recommended if a delay in
processing is anticipated. For CSF specimens, a minimum amount of 1 cc. is
necessary for cytologic evaluation.

Fixation
In order to secure an optimal specimen, prepared smears must be
immediately fixed by quick immersion in a fixative solution or sprayed (from
about a foot distance) by the fixative. The optimal fixation is done by dropping
the smears (use paper clip on every other slide to avoid slide surface contacts,
and proper exposure to the fixative), in 95% ethyl-alcohol container for 3-5
minutes. The slides may be kept in the fixative for a longer period, when
transportation to the cytology laboratory is required. Exfoliated cells
decompose rapidly, and drying rapidly destroy cellular and nuclear details,
resulting in a speci men that is inadequate for diagnosis. A more efficient and
quicker way is to fix them with a spray fixative or aqueous-alcoholic solutions
containing polyethylene glycol or Carbowax.
If smears cannot be made immediately, the collected material should be
placed in 50% alcohol for all types of effusions . This can be replaced by
Saccomano preservative (50% alcohol and carbowax). Slides that have to be
sent by mail to the laboratory should be air-dried after fixation for I0-1 5
minutes and placed in suitable wooden , cardboard or plastic slide containers to
avoid spillage of the fixative.
When specimen is more than a few drops, the fluid should be centrifuged
first. This is usually done at moderate speed (2000 RPM) for 2 minutes. The
supernatant fluid is decanted and the sediment maybe directly smeared onto
glass slides or directly cytocentrifuged (cytospin) on the coated slides with a
thin film of egg albumin. If sufficient sediment remains (2 cytospin smears are
usually adequate), a cell block can be made (similar to histologic biopsy
material).
Minimum fixation time varies. Some types of smears can be fixed in 10
minutes (with 90% alcohol). A longer fixation is usually not needed. However,
smears may be left in alcohol-based fixative for weeks or months before
staining, provided the container is covered lightly to prevent evaporation of the
solution.

Wet Fixation:
The process of submerging of freshly prepared smears immediately in a
liquid fixative is called wet fixation. This is the ideal method for fixing all
gynecological and non-gynecological smears and any of the following
alcohols can be used.
1. 95% Ethyl Alcohol (Ethanol): This is the ideal fixative recommended
in most laboratories for cytological specimen. As a dehydrating agent, it
causes enough cell shrinkage to yield optimal chromatin detail
characteristics of cytological preparations.
2. Ether alcohol mixture: This fixative was originally recommended by
Papanicolaou. It consists of equal parts of ether and 95% ethyl alcohol. It
is an excellent fixative, but ether is not used in most of the laboratories
because of its safety hazards. , 3. 100% Methanol: is an acceptable
substitute for 95% ethanol. It causes less shrinkage and is more expensive
than ethanol.
4. 80% Propanol and Isopropanol
5. Denatured alcohol
Carbowax (Polyethylene Glycol) fixatives are substitutes for wet fixatives.
They are either aerosols applied by spraying the cellular samples or a liquid
base, which is dropped onto the slide. Prior to staining, the slides should be kept
overnight in 95% alcohol to remove of the coating fixative.
Carnoy’s fixative is a special purpose fixative used to hemolyze the red
blood cells in hemorrhagic samples. It is an excellent nuclear fixative as well as
preservative for glycogen but results in considerable shrinkage of cells and tends
to produce over staining in hematoxylin. Carnoy’s fixative must be prepared
fresh when needed and discarded after each use.
Precautions Observed During Fixation
 1. Identify the slides before preparing smears.
2. Attach a paper clip to the identified end of the slide before
preparing the smear. Paper clip prevents the slide from sticking
together in the fixative 3. Smears should be placed into the fixative
container immediately after preparations.
4. Place each smear in fixative by a single uninterrupted motion to
avoid rippling of smeared material.
5. Avoid striking the bottom of the fixing container forcefully to
prevent dislodging the material from the slide.
Note: To avoid fixation delay, have the fixative bottle open before
preparing the smears. For proper fixation if spray fixative is used, the slide
should be kept in a distance from the slide (approximately 12 inches away).

Staining Methods in Cytology
Papanicolaou staining method is the routine staining procedure used in
cytopathology laboratory. It is a polychrome staining reaction that results in
well stained nuclear chromatin, differential cytoplasmic counterstaining and
cytoplasmic transparency. Cytoplasmic stains are OG-6 and EA-36. Both are
synthetic stains and OG-6 is a monochrome stain while EA-36 is a polychrome
stain. Formalin-fixed cytology preparations must be stained with either H&E or
Papanicolaou stain.
In addition to Pap and H&E stains, many laboratories use May-Grunwald-
Giemsa (MGG) and modified Wright-Giemsa stains for cytological diagnosis
of non –gynecological specimens and Dip Quick for rapid staining. Most
Romanowsky stains used in cytology are aqueous stains as opposed to the
methyl-alcohol based stains used in hematology. Romanowsky stains are
generally used for air-dried fine needle aspirates. Stock solutions of May-
Grunwald reagent and Giemsa stain are now available commercially.
The most frequently used stains for cytochemistry are Periodic Acid Schiff
(PAS) for glycogen, Perl’s stain for hemosiderin, Alcian Blue for mucins,
Grocott Methenamine Silver (GMS) for fungal organisms and Ziehl-Neelsen
stain for acid fast bacilli. Cytology samples may be stained by
immunohistochemical methods similar to those used for histological material.
The use of coated slides will prevent washing off of the cells during processing
and staining. Both peroxidase and alkaline phosphatase may be used for
immunohistochemistry.
To date, Papanicolaou (Pap) smear is considered to be the staining method
of choice for exfoliative cytology. This staining technique, known as the Pap
stain, is still the gold standard today. Sputum or urine specimens containing
squamous epithelial cells also show excellent results when stained according to
the Papanicolaou technique. Based on the number of slides to be prepared, the
staining method selected can be applied either manually or mechanically using
automated staining systems. It has the following advantages: 1. Transparent
blue staining of cytoplasm is obtained due to the action of high alcoholic
content of the cytoplasmic counterstain, allowing overlapping cells to be seen
and identified.
2. Excellent nuclear detail is produced.
3. Color range is predictable and of great value in identification and
classification of cells, producing a good differential coloring of
basophilic and acidophilic cells.
4. It is valuable in comparing cellular appearances in smears with their
counterpart in similarly stained sections.

Papanicolaou stain
Papanicolaou's stain (also known as Pap stain) is a multichromatic staining
cytological technique that is used to differentiate cells in smear preparations of
various bodily secretions, including gynecological smears (Pap smears),
sputum, brushings, washings, urine, cerebrospinal fluid, abdominal fluid,
pleural fluid, synovial fluid, seminal fluid, fine needle aspiration material,
tumor touch samples, or other materials containing cells.
The classic form of Pap stain involves five dyes in three solutions:
• A nuclear stain, hematoxylin, is used to stain the nuclei.
• First OG-6 counterstain (-6 denotes the used concentration of
phosphotungstic acid; other variants are OG-5 and OG-8). Orange G
stains keratin and was originally used to stain the small cells of
keratinizing squamous cell carcinoma present in sputum.
• Second EA (Eosin Azure) counterstain, comprising three dyes; the
number denotes the proportion of the dyes, e.g. EA-36, EA-50, EA-65.
Eosin Y stains the superficial epithelial squamous cells, nucleoli, cilia,
and red blood cells.
• Light Green SF yellowish stains the cytoplasm of other cells, including
non-keratinized squamous cells. Because it is quite expensive and
difficult to obtain. Fast Green FCF can be used as an alternative stain,
but it produces visually different results.
• Bismarck brown Y stains nothing and in contemporary formulations it
is often omitted.
 When performed properly, the stained specimen should display hues from
the entire spectrum: red, orange, yellow, green, blue, and violet. On a well
prepared specimen, the cell nuclei are crisp blue to black. Cells with high
content of keratin and glycogen are yellow. Superficial cells are orange to pink,
and intermediate and parabasal cells are turquoise green to blue. Metaplastic
cells often stain both green and pink at once.
OG and EA stains lose strength more rapidly than hematoxylin and should
be replaced every week or as soon as the staining of cells are no longer sharp
and crisp.
Wet fixation is time consuming, shows drying artifacts and less retention
of material compared to air dried smears. Air dried smears also have
shortcomings such as magnification in the cell size, and often the morphology
of the cells is not crisp. Modifications have been developed in Papanicolaou
stain to improve the staining quality and/or to minimize staining time.
Formula
1. Harris’ hematoxylin
Hematoxylin 5g
Ethanol 50ml
Potassium alum 100g
Distilled water (50°C) 1000ml
Mercuric oxide 2.5g
Glacial acetic acid 40ml
2. Orange G 6
Orange G (10% aqueous) 50ml
Alcohol 950ml
Phosphotungstic acid 0.15g
3. EA 50
0.04 M light green SF 10ml
0.3M eosin Y 20ml
Phosphotungstic acid 2g
Alcohol 750ml
Methanol 250ml
Glacial acetic acid 20ml
Filter all stains before use.

Original Papanicolaou staining method:
1. 96% ethyl alcohol 15 seconds
2. 70% ethyl alcohol 15 seconds
3. 50% ethyl alcohol 15 seconds
4. Distilled water 15 seconds
5. Harris hematoxylin 6 minutes
6. Distilled water 10 dips
7. Hydrochloric acid 0.5% solution, 1-2 quick dips
8. Distilled water 15 seconds
9. Few dips in 0.1% ammoniated water. The smear turns to blue.
10. 50% ethyl alcohol 15 seconds
11. 70% ethyl alcohol 15 seconds
12. 96% ethyl alcohol 15 seconds
13. OG-6 (orange) 2 minutes
14. 96% ethyl alcohol 10 dips
15. 96% ethyl alcohol 10 dips
16. EA 50 eosin yellowish 3 minutes
17. 96% ethyl alcohol (10 dips)
18. 100% ethyl alcohol (10 dips)
19. Xylene (10 dips)
20. Mount: in DPX using coverslip

Fig 29-2. Cervico-vaginal smear showing mature superficial cells with

abundant pink cytoplasm. A few basophilic cells with slightly larger nuclei are
also present.
Results:
Nuclei blue/black
Cytoplasm (non-keratinizing squamous cells) blue/green
Keratinizing cells pink/orange
Precautions:
1. Use stains only after filtering them
2. Change stains frequently
3. Check staining under microscope for good quality control

Pap Staining Procedure for Gynecological Specimen:
Equipment: Shandon Varistain 24-3
Method:
Solutions Time in Seconds
 80% isopropyl alcohol 10
1.
 70% isopropyl alcohol 10
2.
 50% isopropyl alcohol 10
3.
 Distilled water 45
4.
5. Hematoxylin (Harris), Mercury 45
free
 Distilled water 5
6.
 Distilled water 5
7.
 Distilled water 5
8.
 50% isopropyl alcohol 10
9.
 Ammonium hydroxide (NH40H) 60
10.
 70% isopropyl alcohol 5
11.
12. 80% isopropyl alcohol 5
 95% isopropyl alcohol 10
13.
14. OG-6 30
 95% isopropyl alcohol 10
15.
 95% isopropyl alcohol 10
16.
 EA-50 120
17.
 95% isopropyl alcohol 10
18.
 95% isopropyl alcohol 5
19.
 95% isopropyl alcohol 5
20.
 100% isopropyl alcohol 5
21.
 I 00% isopropyl alcohol 5
22.
 I 00% isopropyl alcohol I xylene 10
23.
 Xylene 20
24.

Fig. 29-3. Parabasal cells with smaller cytoplasm and larger nuclei are seen with
a few intermediate cells.

Cells Found in Cervico-vaginal Smears During examination, a wide variety of
vaginal cells may be present, the proportion of which may determine the stage of
sexual period of the patient on the time of ovulation.
Cells found in cervico-vaginal smears are usually divided into: a) Mature
superficial cells - These polygonal squamous cells measure 45-50 µm in
diameter and are usually identified by the presence of pale, pink-staining
cytoplasm and dark pyknotic nuclei, less than 6 µ in diameter. True acidophilia
is characteristic of superficial vaginal cells under estrogen influence (this is not,
however, a reliable index of maturation). Pseudo-acidophilia may be observed
due to the drying of smears especially before fixation, prolapse and drying of
vaginal epithelium, infection and chemicals.
b) Intermediate cells - are medium sized polyhedral or elongated cells
with basophilic vacuolated cytoplasm.
c) Parabasal cells - are round to oval cells with small dense basophilic
cytoplasm, and a total cell diameter of 15-30 µm. They are smaller
than intermediate cells, and have a larger vesicular nucleus. They are
normally found from two weeks of age to puberty, after childbirth, with
abortions and after menopause.
Other cells that may be found in cervico-vaginal smears include: •
Navicular cells are boat-shaped intermediate cells with strong tendency
to fold or curl on edges. Their presence suggests a combined estrogen​-
progesterone effect. They are found in the latter half of the menstrual
cycle, during pregnancy and menopause.
 • Pregnancy cells are round, oval or boat-shaped cells with
translucent basophilic cytoplasm observed greatest at the center of the
cell, due to glycogen accumulation, pushing the nucleus to the side or
towards the cell membrane. This appearance is characteristic due to a
deeper blue stain of the cytoplasm at the periphery .
• Endometrial cells are small cells, slightly cylindrical with less
basophilic cytoplasm, occurring in tightly packed groups of 3 or
more. They are found during and 1-10 days after menstruation, and
are shed in response to ovarian hormones A smear taken during
menstruation will contain endometrial cells and numerous
erythrocytes and leukocytes, frequently rendering the smear
unsatisfactory for a reliable assessment..
• Endocervical glandular cells occur in large groups or small sheets.
The cytoplasm is usually stained pale blue/gray and is finely
vacuolated, often with indistinct cell borders and nuclei with finely
granular chromatin. They may present a honeycomb appearance
when viewed on end.

Vaginal Hormonal Cytology
Vaginal hormonal cytology, aside from being relatively inexpensive, may
be performed regularly without undue risk, even in pregnant women. Vaginal
smears prepared for such purpose are best taken from the upper lateral third of
the vaginal wall, because it is more accessible and less likely to be
contaminated by cellular debris or vaginal discharges.
Smears should be examined first under low magnification to assess the
quality of smear and staining, to detect the presence of RBC and leukocytes
and the type of exfoliated cells, and to give a rough assessment of the
proportion of mature superficial pyknotic acidophilic cells. A quantitative
evaluation of the smear under 40 x objective is then in order.
This test is not commonly used in today's practice of gynecology.


Staining Procedure for Non-Gynecologic Specimens:
We use a modified Papanicolaou technique to provide a standardized
method for staining non-gynecologic specimens. Harris' hematoxylin is the
optimum nuclear stain and the combination of OG- 6 and EA 50 gives the
subtle range of green, blue and pink hues to the cell cytoplasm.
Equipment: Shandon Varistain 24-3



Method:

 Solutions Time
1. 80% Isopropyl alcohol (in
seconds)
8

 70% Isopropyl alcohol 8
2.
 50% Isopropyl alcohol 8
3.
 Distilled water 8
4.
 Hematoxylin (Harris), 35
5. mercury free
 Distilled water 8
6.
 Distilled water 8
7.
 Distilled water 8
8.
 50% Isopropyl alcohol 8
9.
 Ammonium hydroxide 60
10. (NH40H)
 70% Isopropyl alcohol 9
11.
 80% Isopropyl alcohol 8
12.
 95% lsopropyl alcohol 8
13.
 OG-6 15
14.
 95% Isopropyl alcohol 8
15.
 95% lsopropyl alcohol 8
16.
 EA-65 75
17.
 95% Isopropyl alcohol 8
18.
 95% Isopropyl alcohol 8
19.
 95% Isopropyl alcohol 8
20.
 Absolute isopropyl alcohol 5
21.
 Absolute isopropyl alcohol 8
22.
 Absolute Isopropyl alcohol 8
23. and Xylene II
 Xylene 10
24.
Modified Papanicolaou Staining:
This procedure provides optimum nuclear detail information, which is
important for cytologic evaluation of Pap smears, non​gynecological and FNA
cytologic specimens.
1. Stain conventional Pap smears by routine Papanicolaou staining
procedure.
2. For Pap smears prepared with liquid- based technique, an automated
stainer should be used.
3. To avoid contamination:
a. Stain non-gynecologic and FNA specimens separately.
b. Do not stain fluid specimens in close proximity to other cases.
c. Stain suspicious cases separately when necessary.
d. Assess cellularity of fluids and FNA specimens and stain those with
high potential for cross contamination separately.
4. Change all stains weekly or as needed depending on work load.
5. Discard first rinsing alcohol daily, advance subsequent rinses and place
new alcohol as the last rinse. Do the same for xylene.
6. Smears should not be allowed to dry in between any of the staining
steps.
7. Keep a record of changes in staining procedure next to the stainer.
8. All problems with staining should be identified, monitored, corrected,
and recorded.

May-Grunwald Giemsa Stain:
This is one of the common Romanowsky stains used in cytology. It is useful
for studying cell morphology in air-dried smears. It is superior to Papanicolaou
stain when studying the cytoplasm, granules, vacuoles, and basement membrane
material. For nuclear staining Papanicolaou stain is superior.
Notes:
• Do not let the specimens dry at any stage of the staining procedure.
• Wash properly to avoid dye artifacts.
• Staining result is dependent on pH. Alkaline pH increases blue and
acidic pH pink or reddish tinge in the stained specimen.
Take care not to over-stain smears. Under-stained smears can be re-
stained for further periods to increase staining intensity.
 • Staining solutions should be filtered daily in order to remove any loose
cells/cell components.
• When precipitates are formed on the slide, the stains should be mixed
thoroughly or replaced.
It is important to renew alcohol baths (ethanol 96% and 100%)
regularly in order to achieve good differentiation and stain
transparency.
• The last alcohol, xylene or xylene-substitute baths must be absolutely
clean and free of water. Any water remaining can cause the slide to
become decolored as a result of oxidation. Make sure that the smears are
completely dry before staining.
If bacteria and/or fungi are regularly noted within examined smears,
then the staining solutions are likely contaminated and need to be
replaced.
Stains will lose potency over time and need to be replaced.
Rehydration of air-dried smear with normal saline will restore the
transparency of the cell or hemolyzed RBCs, while the use of 4%
formaldehyde/65% ethanol fixative can reduce the time needed for
proper fixation and staining of the smears.

Mounting
Long-term specimens are mounted with mounting media that are first applied
to the slide in dissolved form and then harden as the solvent evaporates. The
refractive index of mounting media is approximately 1.5 and is close to that of
glass. For optimum optical properties, transparency and brilliance of specimens
it is important to ensure that the mounting medium contains the solvent that was
used for clearing. The advantages of mounting agents containing xylene or
toluene include: • Optical brilliance
• Good drying times
• No air bubbles under the coverslip
• Color stability of stained slides

Immunohistochemistry
lmmunohistochemical studies for cell surface antigens on cell smears,
imprints, and cytocentrifuge preparations show sensitivity equal to, or greater
than, that of histologic sections. By contrast, intracytoplasmic and intranuclear
antigens may be more difficult to demonstrate in cytologic material, probably
because cells remain structurally intact and do not allow for easy penetration of
immunologic reagents. False negative immunoperoxidase results for
intracellular antigens are more common in cytologic samples than in histologic
specimens. Cell suspensions are seldom used for routine
immunohistochemistry. Conventional filter preparations produce an undesirable
background staining, probably due to nonspecific absorption of reagents by the
filter. The methods for processing and handling cellblock preparations are
similar to handling and processing histologic samples.
Samples stored for several months, either at room temperature, 4oC or
-20oC can be processed for DNA and protein analysis. Polymerase chain
reaction (PCR) and subsequent direct sequencing can be used to analyze
mutations of p53 and other genes from relatively few suspicious or malignant
cells. Fluorescence In Situ Hybridization (FISH) is the most objective
technique for assessment of Her2/neu of fine needle aspirates for assessment of
breast cancer.

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