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Introduction to Histotechnology and Cytotechnology
Chapter Outline
• Introduction to Histophathology and Exfoliative Cytology
• Basic Terminology
• Histopathology Laboratory Equipment
▪ Microscope
▪ Embedding Equipment
▪ Microtome
▪ Flotation Bath
36
Venk Mani and Surajit Ghosh
▪ Dryer Oven and Slide Warmer
▪ Tissue Processor and Stainer
▪ Incubators
▪ Freezers and Refrigerators
• Laboratory Supplies
▪ Microscope Slides and Coverslips
▪ Containers for Specimens
• Reagents
▪ Mounting Media
▪ Preparation of Reagent Solutions
• Routine and Special Staining: A Review
▪ Introduction
▪ Histology Sample Preparation
▪ Preparing Tissue for Staining
▪ Automating Staining Process
• Review Questions
1127
1128
Medical Laboratory Technology: Volume 3
IntroductIon to HIstopHatHology and ExfolIatIvE cytology
A histopathology laboratory prepares tissue sections for establishing a histopathological
diagnosis. It is the science of studying the changes in a human body brought about by disease,
identifying the disease and its cause, so that appropriate treatment can be given. Because of
its convincing physical evidence, histotechnology has proved to be one of the most eective
tools in diagnosing tissue abnormalities and cancerous conditions. In recent years, with the
advent of freezing microtomy, the teamwork of many surgeons and histopathologists has
greatly contributed to the progress of medical science. Histotechnology is an art by itself. It is
concerned with the processing and preparation of the body tissues in such a manner as to be
able to probe its inside for the sake of diagnosis and treatment.
The specimens submied to a histopathology laboratory could be from the gastroenterologists in the form of small pieces of tissues (biopsies); endoscopic sinus surgery specimen from
an ENT surgeon; appendix, gallbladder for gallstones, enlarged lymph nodes for lymphoma
workup, amputated limb for diabetic gangrene, etc. from general surgeons; and a whole
kidney or liver for malignant tumours, radical mastectomy specimen in breast cancer, etc.
and margins of a skin tumour for frozen sections from surgical oncology.
These specimens are submied either fresh (unxed) or immersed in a xative uid. As
a histotechnician you are neither involved in the collection of the specimens nor in their
laboratory evaluation. You are, however, responsible for the handling and preparation of
the specimens to facilitate their gross and microscopic examinations which are done only
by a histopathologist. With recent advances in histotechnology, a histotechnologist, apart
from routine processing of the tissues, should be aware of complicated procedures involving
electron microscopy (EM), special stains, immunohistochemistry (IHC), immunouorescence
(IF), uorescent in situ hybridization (FISH) techniques and also have a knowledge of using
automated processing and staining equipment. The basic steps of specimen processing
include xation, embedding, microtomy, staining, and mounting. These will be discussed
in the following sections. It is expected that a histotechnologist will be suciently trained
to prepare the specimens according to the specications, recognize satisfactory preparation,
identify and remedy the causes when unsatisfactory results are obtained.
Exfoliative cytology is dierent from histology. Here the specimens are constituted by the
body uids, secretions and excretions collected during physical examination of the patient.
Laboratory techniques involve the preparation of smears, xing, staining, mounting and
microscopic examination. A cytopathologist evaluates the smears for exfoliated cells characteristic of not only cancerous and pre-cancerous conditions, but also of a variety of other alterations produced by inammatory and degenerative processes. Other than routine specimen
processing, a histotechnologist should have knowledge of xation and staining of squash
preparations, imprint/impression smears, ne needle aspiration cytology smears and frozen
sections.
As soon as a specimen for surgical pathology or a uid for cytology is received for analysis,
it is the responsibility of the technologist to accept only if the name on the requisition form
and the details wrien on the specimen container label tallies correctly. Once this is done, the
specimen should be given a surgical number which should appear in all further subsequent
steps till the slides are stained for reporting.
There are some inherent problems faced by the laboratory in preparing specimens for
histological studies. As soon as a tissue is removed from the body for histological examination,
it is cut o from its blood supply and begins to decompose (autolysis) and putrefy. Autolysis
is due to action of the enzymes which are liberated aer cell death and putrefaction is caused
by bacterial invasion and destruction of the tissue. This disintegration of the cell is prevented
by either freezing or by adding certain chemical substances to the tissues/cells which are
called xatives. To preserve as nearly as possible the natural state of the tissue cells, it is
Introduction to Histotechnology and Cytotechnology
essential to check the autolysis with a minimum of delay. This process is known as xing.
Based on the chemical action, xatives are further classied as follows:
• Aldehydes: Formaldehyde (10% formalin used in routine histology) and gluteralde-
hyde used in electron microscopy (EM)
• Oxidising agents: Osmium tetroxide, potassium permanganate and potassium dichro-
mate
• Protein denaturing agents: Methyl alcohol, ethyl alcohol and acetic acid
Large specimens are cut into slices 1–2 cm thick and xed in adequate formalin for xation
before submiing 1–2 cm2 and 3–4 mm thick sections from the specimens for histology. To
enhance the speed of xation and processing, oor model microwave processors are being
used lately.
Tissues are either too so or too hard and calcied, which makes them dicult to cut into
microscopic sections. Thus, the procedures of decalcication, dehydration and embedding
precede microtomy and staining.
The process of embedding involves the inltration of paran wax into the tissues, which
provides the necessary hardness to cut sections. Since the tissues contain water, and paran
wax is insoluble in water, the removal of water (dehydration) is the rst step of tissue
preparation for embedding. This is accomplished by using increasing gradients of ethyl
alcohol until the tissues are nally bathed in 100% (absolute) alcohol. The dehydration is
followed by inltrating a clearing agent such as xylene or chloroform into the tissue. The
clearing agent also acts as a solvent for paran wax. Finally, the paran is impregnated into
the tissues in a molten state while the inltrated xylene diuses out into the paran bath.
When the paran is solidied, the tissue is ready to be made into blocks and cut into thin
slices or sections. The thin sections are then prepared for staining. Most stains are soluble in
water, however, and cannot cross the paran barrier to react with the tissue constituents.
Hence the thin sections are rst rehydrated or ‘taken to water’ prior to staining.
To rehydrate the sections, they are rst oated onto a microscope slide. The sections are
then bonded to the slides by heating them above the melting point of the paran in an oven
or on a warming plate. The sections are then dewaxed by immersing in xylene, followed by
dipping in ethyl alcohol in decreasing gradients. The rehydrated sections are then stained
and again subjected to dehydration before they are permanently mounted. The sections will
fog with time if not dehydrated properly. Staining is usually done with multiple stains in
order to dierentiate between various cells, tissues and cell constituents by their dierential
staining properties.
The study of exfoliated cells in body uids does not involve the cuing of sections, but the
cells may need to be concentrated by centrifugation. A smear is made from the uid or the
sediment on a microscope slide, xed and then stained. The techniques of cytotechnology
closely resemble those of histotechnology except in the preparation of specimens prior to
staining.
1129
BasIc tErmInology
Although a glossary of technical terms used in various clinical laboratories has been presented
at the end of this volume, some of the terms commonly used in histotechnology and cytology
are presented here.
Adhesion: The process of placing the section on the slide so that it is not washed away during
dehydration, staining or other treatments. Egg albumin and gelatin are popular adhesives.
Autolysis: Self-digestion and decomposition of tissues.
Biopsy: A fragment of tissue taken out of an organ from a living person and examined. The
literal meaning of the word is ‘to see for oneself’.
1130
Block: Portion of specimen properly cut and trimmed for processing.
Cryostat: A cold box containing the microtome used to cut frozen sections which are fresh
specimens mostly margins of a malignant tumour. The temperature of the box is kept well
below freezing, in the –20 to –30°C range.
Fixation: A process by which the specimen is preserved in its original condition. Formaldehyde (10% buered formalin) is a commonly used xative.
Clearing agent: A substance that makes tissues more transparent for microscopic examination. Xylene and chloroform are the commonly used clearing agents.
Decalcication: A process to remove calcium from bone and other mineralized hard tissues
in order to facilitate the process of cuing thin sections. Decalcication is done aer xation
and before dehydration and paran inltration. Nitric acid, formic acid and hydrochloric
acid are commonly used to decalcify hard tissues.
Dehydration: The process of removing water from tissues. The common procedure is to treat
the specimen blocks with increasing gradients of alcohol followed by treatment with the
clearing agent.
Exfoliative cytology: Entails the microscopic examination and interpretation of cells which
are shed (exfoliated) spontaneously from epithelial surfaces of the body, or which may be
removed from such surfaces or membranes by physical means. Common specimens include
cervical and vaginal smears, buccal smears, bronchial brushings, bladder washings and
various body uids (pleural eusion, pericardial eusion, ascitic uid, joint eusions and
cerebrospinal uid) and secretions.
Freezing microtomy: Preparations of sections from frozen tissues.
Histology: Study of stained sections of tissues under the microscope.
Inltration: A process by which the clearing agent is eliminated from the tissue making room
for the impregnation of the embedding medium (paran wax).
Impregnation: A process that allows the embedding material to enter the tissue while the
clearing agent is diused out.
Microtome: An instrument used for preparing thin sections (4–5 mm) of tissue. The most
common types are rocking microtome and rotary microtome. Microtomy is the process of
section cuing.
Mounting: The arrangement of specimens on slides for microscopic study.
Rehydration: Commonly referred to as ‘taking the section to water’. The goal is to replace the
water insoluble paran wax with water prior to staining with water soluble stains.
Staining: The process of colouring of tissues in order to facilitate their identication under
the microscope.
Smear: Specimen spread on a slide surface to facilitate microscopic examination. This is the
standard method of specimen preparation in exfoliative cytology.
Medical Laboratory Technology: Volume 3
HIstopatHology laBoratory EquIpmEnt
Most histopathology laboratories will require the following equipment and supplies. Some of
these are shown in Figure 36.1.
• Microscope
• Microtome
Introduction to Histotechnology and Cytotechnology
1131
Figure 36.1 Materials commonly used in histological and cytological studies
• Microtome knife (disposable), or razor
• Timer
• Oven, Bunsen burner, forceps, scalpel, dissecting set
• Constant temperature water-bath, otation bath, paran bath
• Equipment for embedding and vacuum inltration
• Containers (boles) for holding specimens
1132
Medical Laboratory Technology: Volume 3
• General glassware as any other laboratory (pipees, beakers, etc.)
• Microscope slides and coverslips
• Slide carrier
• Slide trays for storage
• Coplin staining dish or staining jar: Vertical and horizontal
• Accessories: Labels, diamond pencil for marking glass slides
• Plastic disposable cassees for embedding
• Automatic tissue processor (for advanced laboratories)
• Automatic stainer (for advanced laboratories)
The microtome and the microscope are the two most important pieces of equipment in a
histology laboratory. Other equipment include a paran oven, vacuum embedding oven,
and tissue oating bath. The technician must know about their proper use and care so that
these equipment can give many years of service. Some of the other equipment used in a
histology laboratory such as the refrigerator, balance, incubator, magnetic stirrer, and others
have been discussed in Chapter 4 of Vol. I.
Microscope
A microscope is used to examine thin sections cut by the microtome. It is an integral part of
most of the clinical laboratories except chemistry. The backbone of histotechnology lies with
the use of various types of microscopes—light microscope, polarizing microscope, dark-eld
microscope and uorescence microscope. The uses of phase-contrast microscope and electron
microscope are rather limited in the histology laboratories of developing countries.
Of the various types of microscopes used in a laboratory, the light microscope is used
routinely. Detailed description of the use and care of a light microscope has been presented
in Chapter 4 of Vol. I. Components of light microscope are given in Figure 36.2 for recall.
Figure 36.2 Components of a light microscope
Introduction to Histotechnology and Cytotechnology
1133
For examining objects under the light microscope, staining is done with dierently coloured
dyes in order to create contrast.
Fluorescence is essentially an optical phenomenon in which light of one wavelength is
absorbed by a substance and almost instantly re-emied as light of longer wavelength. In
uorescence microscopy, the substance is bombarded with short-wavelength light in the
ultraviolet (UV), violet or blue range, and visible light is emied. The source of illumination
is high intensity ultraviolet rays (mercury vapour lamp). The object (antigen) is stained with a
uorescent dye, conjugated to the corresponding antibody, through immunological reaction
(Chapter 24 of Vol. II). The antigen-antibody complex glows with the availability of ultraviolet light and thus the object (antigen) is located. It is used in the identication of spirochetes
through IF techniques and is explained in Chapter 20 of Vol. 2.
A polarizing microscope is nding increased use as a diagnostic tool in histopathology
primarily for the identication of crystals. In case of patients with gout, urate crystals are
sought under polarized microscope. It is also specically used to make the identication
of amyloid stained with Congo red. The use of polarizing microscope in exhibiting double
refraction, an isotropism, birefringence has helped in many diagnostic situations. The light
microscope can be converted easily to a polarizing microscope for its wider use.
Dark eld microscopy excludes the directly transmied light and uses only the scaered
or oblique light during viewing. The light is reected from the microscopic object like stars
with a dark background of the sky at night.
This type of microscopy is used primarily
for the study of unstained microorganisms
and is rarely used in routine histopathology.
Embedding Equipment
The embedding centre provides a supply of
melted paran, warm storage for embedding moulds, small warming and chilling
plates for orientation during embedding,
and a large chilling plate. Some centres
include magnifying glasses to aid in specimen orientation. The paran is kept 2–4°C
above the melting point of the paran used.
If the temperature of the embedding parafn is allowed to go too high, the nature of
the paran and the resultant sectioning
qualities will be aected.
The paran oven (Figure 36.3) maintains
a temperature between 50–60°C which can
be accurately adjusted according to need.
The drier oven described above can be
shared. The oven should be large enough to
have space for melting and storing molten
paran, inltration of paran in sections,
drying of slides and warming of solutions
during the preparation of reagents. Vacuum
embedding saves time and is the method of
choice because it allows for a more thorough
impregnation of paran into the tissue
(Figure 36.3). It is specically recommended
for those tissues which are likely to become
over hardened during the usual 2–3 h of
immersion in hot paran. The temperature
of the bath is kept at 56–58°C and the vacuum
is created by a suction pump.
Figure 36.3 Components of (a) parafn embedding oven,
and (b) vaccum parafn embedding bath
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Medical Laboratory Technology: Volume 3
Microtome
Microtomes are used in section cuing. Three basic types of microtomes are commonly
encountered—rocking, rotary and sliding. Routine histological sections are cut aer
embedding.
Use of a rocking microtome (Cambridge model, Figure 36.4) is common in histology
laboratories of developing countries. It is relatively inexpensive, simple to operate, practically
maintenance free and it can produce sections of high quality. For geing serial sections, the
rotary microtome is preferred (Figure 36.4). It is a delicate machine and designed to cut
extremely thin sections. It is slowly replacing the rocking microtome. Rotary microtome
is found in most laboratories where routine paran and frozen sections are the sole
requirements. Most rotary microtomes operate with a screw feed; the block moves up and
down, and either the knife or the block advances a pre-set number of micrometers with
each revolution of the wheel. This type of microtome is found in most cryostats and is the
most commonly used type for sectioning paran-embedded material. Celloidin-embedded
sections are cut by sliding microtome. Sliding microtome holds the block and the knife is
moved along a horizontal plane past the block face. As the knife is returned to the starting
position, it completes each section cycle and a screw feed causes the block to be raised towards
Figure 36.4 Commonly used microtomes in histology laboratory
Introduction to Histotechnology and Cytotechnology
the knife at a predetermined thickness. This type of microtome is used for sectioning celloidin
and large paran blocks; it is not used in routine histopathology.
Microtomy of frozen sections plays a vital role in modern histology laboratories. Frozen
sections do not need to be embedded. So specimens are cut in frozen conditions which gives
the necessary rigidity for the section cuing. This method is useful for rapid diagnosis during
an operation or to examine the sections for a substance (e.g., fat) or structure that would
otherwise be destroyed by preparing the sections in the routine way.
A clinical freezing microtome (Figure 36.4), used in cuing frozen sections is relatively
portable and can be xed on the table top. A chuck with an aached supply of carbon dioxide
allows for horizontal freezing of the tissue section. The knife is kept cold and the section must
be removed from the knife edge and oated in a dish of distilled water. The clinical freezing
microtome has been replaced to a great degree by the cryostat (Figure 36.4). Cryostat is a
refrigerated chamber containing a microtome, usually of the rotary type. It is cooled by a
mechanical refrigeration unit. Although a cryostat is easy to operate, practice and skills are
needed to obtain good frozen sections. The microtome knife of the cryostat must be very sharp
and the edge must be free of defects. The knife stays warm in the freezing microtome. Aer
sectioning the tissues it should not be stored unprotected. It should be wrapped carefully to
exclude air and then stored in a –70°C freezer. Cut sections are stained in the same manner as
the embedded sections.
All good microtomes are correctly adjusted by the manufacturer. If lubricated and cleaned,
they should stay that way for a long time. The instructions from the manufacturer should be
carefully read and reviewed periodically so that all subsequent adjustments and maintenance
will be exactly as the manufacturer recommends. Since the repairing of instruments is dicult
under the existing conditions of the developing countries, especially when foreign equipment
is imported, the technician must get fully acquainted with the manufacturer’s instructions for
use, maintenance and trouble shooting. Dust is probably the single most important enemy of
most equipment. The basic care of a microtome includes regular removal of dust.
All microtomes have three major parts:
• The block holder in which the tissue is held in position.
• The knife carrier and the knife.
• The adjustment screws and ratchet device that line up the tissue in proper relation to
the knife and feed the proper thickness of tissue for successive sections. The microtome
feeding mechanism is graduated in microns (mm).
With all types of microtomes, the micrometer seing is very important. This seing is
only approximate and is not an exact determinant of section thickness; the actual thickness
is determined by the condition of the microtome and the quality of knife edge as well as the
skill of the technologist.
1135
Care of microtome
Maintenance of the microtome is crucial for its proper functioning. Keep the moving parts
well lubricated and clean. Put a cover on the microtome when not in use to prevent dust
accumulation. Do not permit rust, dust or paran to accumulate between the bearing surfaces
of the knife holder, brackets, etc. The surfaces should be cleaned frequently, and then wiped
with good neutral oil (e.g., coconut oil); this will prevent rust formation.
Aer cuing sections on the microtome, all accumulated paran and tissue should be
removed with a so brush. Metal parts are cleaned with xylene (do not use xylene too
frequently as it may remove the painted nish). All moving parts of the microtome must
be kept lubricated with a light lubrication oil (e.g., sewing machine oil), and kept free from
paran. Xylene (or petroleum ether) helps to remove the paran. The rigidity of the knifeholder and the knife are important but never adjust any screw too tightly as it may cause
binding. The instrument should be tight only to the point of smooth rm operation.