HISTOPATHOLOGIC/CYTOLOGIC TECHNIQUES
Extinguishers
LABORATORY SAFETY, INSTRUMENTATION & QUALITY MANAGEMENT SYSTEM
PRELIM: WEEK 1 LAB (01//262023)
HISTOPATHOLOGY
o art of analyzing and interpreting the shapes, sizes and
architectural patterns of cells and tissues within a given specific
clinical background
o Activities:
- pre-analytical, analytical and post-analytical
RISK MANAGEMENT AND SAFETY IN THE LABORATORY
RISK MANAGEMENT
o process of ensuring and maintaining personal as well as
environmental health and safety in the laboratory
o The first step is to identify all electrical, mechanical and biological
hazards that can potentially cause harm in the laboratory.
- An inventory of chemical reagents must be on hand and
obsolete chemicals should be routinely disposed of.
THE FOLLOWING ARE GENERAL SAFETY PRECAUTIONS THAT MUST
BE OBSERVED WHEN WORKING IN THE LABORATORY:
o Protect the hands and forearms by wearing either gloves and a
laboratory coat or suitable long gloves to avoid contact of the
toxic material with the skin. Wash hands frequently throughout
the day and before leaving the lab.
o Procedures involving volatile toxic substances and those
involving solid or liquid toxic substances that may result in the
generation of aerosols should be conducted in a fume hood or
other suitable containment device.
o The laboratory workplace should be well-ventilated, clean and
organized. Smoking, sleeping, eating and drinking are prohibited
in the laboratory. Do not store food and drinks in laboratory
refrigerators. Do not wear shorts, sandals, or open-toed shoes in
laboratory.
o Minors or personal pets are not permitted in the laboratory.
o Secure any dangling jewelry, restrain loose clothing, and tie
back long hair that might get caught in equipment before
starting work.
o Use of cell phones and music headphones should be avoided
while working in the lab. They can be distracting and can
increase the potential for an accident to occur. They can also
become contaminated if handled while working with hazardous
materials.
o Every instrument used in the laboratory should meet electrical
safety specifications and have written instructions regarding its
use.
o Eye wash station, safety shower and first aid kits should be
standard facilities in a laboratory. Fire extinguishers, emergency
shower systems, emergency eye washers, first aid, emergency
blankets, and hoods must be checked monthly.
o To avoid the unnecessary purchase of chemical materials, a
detailed list of chemical materials must be prepared. Only a
minimum amount of volatile chemicals must be kept in the
laboratory.
o Chemical material should be stored and safely secured where
there is sufficient ventilation. Combustible chemical material
must be stored in a heat resistant cabinet. Acids and bases must
be separately stored.
o Every chemical compound used in the laboratory should have a
materials safety data sheet on file that specifies the nature,
toxicity, and safety precautions to be taken when handling the
compound.
o All chemical material must be labeled with the name,
characteristics, danger level, and precautionary measures.
o Laboratories must have available appropriate protective gears
for all individuals: safety devices, goggles, gloves, lab coats, and
face-shields.
o The laboratory must have a method for disposal of hazardous
wastes. Collect and seal absorbed material into labelled
containers for disposal. Tissues that are collected should be
stored in formalin and may be disposed by incineration or by
putting them through a "tissue grinder" attached to a large sink
(similar to a large garbage disposal unit). Used chemicals must
not be released into soil, drains and waterways. Use an
absorbent such as sand, “kitty litter” or a commercial product to
collect spills and contain spread.
o One must always be cautious when handling electrical
appliances and must be aware of the location of safety devices
(fire extinguisher, emergency shower system).
with water, carbon dioxide, dry chemical powder or foam are all
suitable depending on other products involved in a fire. Fire
safety procedures should be posted. There must not be any
obstacle in the vicinity of the laboratory door.
o Avoid handling the sharp ends of instruments. Use forceps or
other tools to remove sharp instruments from baskets and
autoclaves. Workers should use appropriate hand protection
when hands are exposed to hazards such as cuts, lacerations or
thermal burns.
o Laboratory accidents must be documented and investigated
with incident reports and industrial accident reports. Obtain
medical advice (first aid officer, doctor, poisons information
center, ambulance) immediately if major exposure occurs.
TYPES OF HAZARDS
A. CHEMICAL HAZARDS
o Cleaning agents and disinfectants, drugs, anesthetic gases,
solvents, paints, and compressed gases
o Potential exposures to chemical hazards can occur both during
use and with poor storage
o The “lab standard” applies to the laboratory use of chemicals and
mandates written in the Standard Operating Procedures (SOPs)
that address the particular hazards and precautions required for
safe use.
o Permissible Exposure Limits (PELs), Threshold Limit Values
(TLVs), or Occupational Exposure Limits (OELs) are some of the
terms used to define the maximum allowable airborne
concentration of a chemical (vapor, fume or dust) to which a
worker may be exposed
o LABELING
- Every chemical should be labeled with certain basic
information, including: Chemical name and, if a mixture,
names of all ingredients;
- Manufacturer's name and address if purchased
commercially, or name of person making the reagent;
- Date purchased or made;
- Expiration date, if known;
- Hazard warnings and safety procedures.
o DIFFERENT TYPES OF CHEMICALS INCLUDE:
1. Irritants are chemicals that cause reversible inflammatory
effects at the site of contact with living tissue, especially the
skin, eyes and respiratory passages.
2. Corrosive chemicals cause destruction or irreversible
alterations when exposed to living tissue, or destroy certain
inanimate surfaces (generally metal). A chemical may be
corrosive to tissue but not to steel, or vice- versa. Few are
corrosive to both.
3. Sensitizers cause allergic reactions in some exposed
workers, not just in hypersensitive individuals. Sensitization
may occur at work because of the high exposure level.
4. Carcinogens are substances that induce tumors, not only in
experimental animals but also in humans. Examples of
carcinogenic chemicals include chloroform, chromic acid,
formaldehyde, nickel chloride and potassium dichromate.
Carcinogenic dyes include auramine, basic fuchsin, and any
dye derived from benzidine (including Congo red and
diamino- benzidine). 5
5. Toxic materials are capable of causing death by ingestion,
skin contact or inhalation at certain specified
concentrations. These include methanol, chromic acid,
osmium tetroxide and uranyl nitrate.
B. PHYSICAL HAZARDS
o slips and falls from working in wet locations and the ergonomic
hazards of lifting, pushing, pulling, and repetitive tasks. Other
physical hazards often unnoticed are electrical, mechanical,
acoustic, or thermal in nature. Ignoring these can have
potentially serious consequences.
o Many operations in the lab can result in lab workers assuming
sustained or repetitive awkward postures such as looking at
slides on a microscope for extended periods
[SARANILLO, KA.]
C. BIOLOGICAL HAZARDS
o refer to anything that can cause disease in humans, regardless of
their source.
o Biohazards include infectious agents and their toxins as well as
contaminated solutions, specimens or objects.
o Allergens, are one of the most important health hazards, yet they
are frequently overlooked.
o Molds and fungi produce and release millions of spores small enough
to be air, water, or insect- borne which may have negative effects on
human health including allergic reactions, asthma, and other
respiratory problems.
USE AND CARE OF THE MICROSCOPE
o Microscope is one piece of equipment that is used by both the
pathologist and the histotechnologist.
o pathologist examines the slide under the microscope to identify a
disease process or an abnormality that will directly affect the
patient's treatment.
o histotechnologist examines the same slide microscopically for
quality control to determine whether all technical processes are
done properly and if a slide of diagnostic quality has been achieved.
o microscope must accomplish three things:
(1) it must magnify the object
(2) it must resolve the details of the object
(3) it must make these details visible.
COMPOUND MICROSCOPE
o microscope with more than one lens and its own light source. In this
type of microscope, there are ocular lenses in the binocular
eyepieces and objective lenses in a rotating nosepiece closer to the
specimen. Because it contains its own light source at its base, a
compound light microscope is also considered a bright field
microscope, which means that the specimen is lit from below and
viewed from above. Illumination comes from below and contrast in
the sample is caused by absorbance of some of the transmitted light
in dense areas of the sample.
VIEWING HEADS:
o Monocular Heads - only use one eyepiece when viewing the
specimen.
o You are restricted if you want to use an LCD camera because this
would occupy the eyepiece. However, monocular microscopes
are light weight and are inexpensive.
o Binocular heads have two eyepieces and are more convenient
and comfortable to use. It is the most common choice.
o Trinocular Heads - have a third eyepiece tube that can be used
by another person simultaneously or by an LCD camera. The
trinocular option is more expensive than the other two types.
MAIN FRAMEWORK OF COMPOUND MICROSCOPE CONSISTS OF:
o Base -provides support for the microscope. The base should be
large and solid enough to allow the microscope to stand by itself.
o Arm - supports and holds the magnifying and adjustment
system. It can be used as a handle for carrying the microscope.
o Stage - is the flat platform where the slide is placed for
examination.
o Substage - is located directly under the stage and holds the
condenser and diaphragm.
o Mechanical Stage - permits movement of the stage while
holding the • slide in the phase of focus.
THE PARTS OF THE LENS SYSTEM ARE:
o Nosepiece - is located at the end of the body tube for holding the
objectives.
o Objectives - consist of a system of lenses located at the end of
the body tube that is held in place by the nosepiece and is closer
to the slide under examination. The purpose of the objective is
to increase or decrease magnification. The objectives are
mounted on a revolving turret allowing for the change of
objectives. When one objective is focused on the turret, all
lenses will be approximately in focus. If this is true, the
microscope is said to be Par focal.
o Focal length - is the distance between outer lens of objective and
the cover glass of the slide under examination.
Magnification is the process that increases the size of the structure
under examination. It is achieved by the use of the microscope's lens
system. The total magnification of a microscope is the product of the
magnifying power of the objective and eyepiece, with a normal tube
length of 160 mm.
BRIGHT FIELD MICROSCOPY
o generally used in compound microscopes, where light is either
passed through, or reflected off, a specimen.
o Illumination is not altered by devices that alter the properties of light
o "bright field" is derived from the fact that the specimen is dark
and contrasted by the surrounding bright viewing field.
o Simple light microscopes are sometimes referred to as bright
field microscopes where a specimen is placed on the stage of the
microscope and incandescent light from the microscope’s light
source is aimed at a lens beneath the specimen.
o This lens is called a condenser.
o The condenser usually contains an aperture diaphragm to
control and focus light on the specimen; light passes through the
specimen and then is collected by an objective lens situated in a
turret above the stage.
o The objective magnifies the light and transmits it to an oracular
lens or eyepiece and into the user’s eyes.
DARK FIELD ILLUMINATION
o technique used to observe unstained and transparent samples
causing them to be clearly visible and appear brightly lit against
a dark, almost purely black background.
o A dark field microscope blocks this central light with a condenser
so that only oblique rays hit the object. The principal elements
of dark field illumination are the same for both
stereomicroscopes and more conventional compound
microscopes. Illumination of specimens by dark field requires
blocking out of the central light rays along the optical axis of the
microscope, which ordinarily pass through and around
(surrounding) the specimen. Blocking these light rays allows only
those oblique rays originating at large angles to strike the
specimen positioned on the microscope stage.
PHASE CONTRAST MICROSCOPY
o optical microscopy illumination technique in which small phase
shifts in the light passing through a transparent specimen are
converted into amplitude or contrast changes in the image.
o type of light microscopy that enhances contrasts of transparent
and colorless objects by influencing the optical path of light
o phase contrast microscope is able to show components in a cell
or bacteria which would be very difficult to see in an ordinary
light or bright field microscope.
POLARIZED LIGHT MICROSCOPY
o contrast-enhancing technique that improves the quality of the
image obtained with birefringent materials when compared to
other techniques such as bright field microscopy, phase contrast
microscopy and dark field microscopy.
o It is designed to examine specimens that are visible primarily due
to their optically anisotropic character.
o two essential components:
(1) The polarizer situated below the specimen stage usually fixed
in the left-to-right, East-West direction, although this is usually
rotatable through 360 degrees.
(2) The analyzer, usually aligned North-South but again rotatable
on some microscopes, is located above the objectives and can be
moved in and out of the light path as required.
FLUORESCENCE MICROSCOPE
o refers to any microscope that uses fluorescence to generate an
image.
o In fluorescence microscopy, many wavelengths of light, ranging
from the ultraviolet to the visible can be used to cause samples
to fluoresce and allow viewing by eye or with the use of
specifically sensitive cameras.
o When certain compounds are illuminated with high energy light,
they emit light of a lower frequency.
o This effect is known as fluorescence.
ELECTRON MICROSCOPE
o microscope that uses a beam of accelerated electrons as a
source of illumination.
o Because the wavelength of an electron can be up to 100,000
times shorter than that of visible light photons, the electron
microscope has a higher resolving power than a light microscope
and can reveal the structure of smaller objects
[SARANILLO, KA.]
TRANSMISSION ELECTRON MICROSCOPE (TEM)
o works on the same principle as an optical microscope but uses
electrons instead of light and electromagnets instead of glass
lenses.
o Use of electrons instead of light allows a much higher resolution,
giving a two- dimensional view.
o Thin slices of specimen are obtained.
o The electron beams pass through this. It has high magnification
and high resolution.
o A transmission electron microscope can achieve better than 50
nm resolution and magnifications of up to about 10,000,000 x
whereas most light microscopes are limited by diffraction to
about 200 nm resolution and useful magnifications below 2000x.
SCANNING ELECTRON MICROSCOPE (SEM)
o uses electron illumination and looks at the surface of bulk
objects by scanning the surface with a fine electron beam.
o image is seen in 3-D.
o It has high magnification and high resolution.
o The specimen is coated in gold and the electrons bounce off to
give you an exterior view of the specimen.
o The pictures are in black and white.

LABORATORY QUALITY MANAGEMENT SYSTEM

QUALITY
o degree to which healthcare services strive to provide accurate
desired outcomes for patients and are consistent with current
professional knowledge
SAFETY
o “freedom from accidental injury”
o hazards: toxic chemicals, pathogenic microorganisms,
mechanical, electrical and fire
o safety consciousness & safety practices
QUALITY CONTROL
o system of routine technical activities
o provides routine and consistent checks to identify, address
errors and omissions, ensures data integrity, correctness and
completeness and also records all quality control activities
QUALITY ASSURANCE
o planned system of review procedures conducted by personnel
not directly involved in the laboratory process
o data of QC provides the data for QA
- correlation of errors, complaints, failures or other
unexpected results
o Quality assessment programs
- College of American Pathologists (CAP)
- United Kingdom National External Quality Assessment
Service (UK NEQAS)
o Two distinct systems:
- selective system:
o stained preparations from departmental archival
records are used to assess the quality of staining
- distributive system
o participating laboratories are asked to stain
sections that have been submitted by the scheme
organizer
o CONTINUING QUALITY IMPROVEMENT
- system is used to approach, evaluate and identify
opportunities to improve quality before problems occur
through evaluation of all systems/processes in the
laboratory
- goal: improve potential care and safety through recognition
of potential problems/errors before they can occur
PRE-ANALYTICAL PHASE
- sample collection, transport, accession and tissue processing
and submission of the slide for reporting
- can endanger quality of histopathology report
ANALYTICAL PHASE
- component:
➢ slide reading along with relevant data and preparation of
report
POST-ANALYTICAL ASPECTS
- follow the analytical phase
- components:
➢ preparation and delivery of the report
➢ archiving of request form and report
➢ storage of the reported specimen for set retention period
and safe disposal of specimen

[SARANILLO, KA.]
 HISTOPATHOLOGIC/CYTOLOGIC TECHNIQUES
CELL INJURY
PRELIM: WEEK 2 LAB (02/02/2023)

FEATURES OF CELLULAR INJURY

1. CELLULAR SWELLING/LYSIS

2. CYTOPLASMIC LOSS OF GLYCOGEN

3. NUCLEAR PIKNOSIS

4. CELLULAR FATTY CHANGE

[SARANILLO, KA.]
 HISTOPATHOLOGIC/CYTOLOGIC TECHNIQUES ADDITIONAL NOTES
LINKS
CELLULAR ADAPTATION TO INJURY
PRELIM: WEEK 3 LAB (02/09/2023) Virtual Microscope 1 http://histologyguide.com/slidebox/slidebox.html
Virtual Microscope 2 https://vmicro.iusm.iu.edu/virtual_p_nw/nw_path_toc.htm
HYPERTROPHY
o Small spindle-shaped
NORMAL HEART
uterine smooth muscle
cells from a normal (CARDIAC MUSCLE)
uterus.
o Large, plump
hypertrophied smooth
muscle cells from a gravid
uterus

PATHOLOGICAL HYPERTROPHY
o Myocardium in ENLARGED HEART
hypertensive heart disease (CARDIAC MUSCLE)

HYPERPLASIA
o Atypical endometrial
hyperplasia: atypical
glands (majority of
image) juxtaposed NORMAL BREAST
with normal
endometrial glands
(focus in lower right
corner); note the loss
of stratification and slight enlargement of hypochromatic nuclei
in the atypical glands; the normal endometrial glands, in
comparison, are neatly polarized with dark, even chromatin
characteristic of proliferative endometrium (100x)

PATHOLOGICAL HYPERPLASIA LACTATING BREAST

(PHYSIOLOGICAL
HYPERPLASIA)

LUNG WITH OSEOS

METAPLASIA OF
BRONCHIAL
CARTILAGE
ATROPHY
PATHOLOGICAL ATROPHY

GASTRIC ANTRUM
AND FUNDUS WITH
GOBLET CELL
METAPLASIA

METAPLASIA
SEVERE SQUAMOUS
CELL DYSPLASIA,
UTERINE CERVIX

[SARANILLO, KA.]
HISTOPATHOLOGIC/CYTOLOGIC TECHNIQUES LABELLING
o Uses: ELECTRONINC SCANNED LABEL
GENERAL PATHOLOGY AND HISTOPATHOLOGIC TECHNIQUES o For: PROPER IDENTIFICATION
PRELIM: WEEK 4 LAB (02/16/2023)
o Indicates: Specimen code
HISTOPATHOLOGY Specimen number
o “Histos”: tissues Requesting pathologist
o “Pathos”: disease or suffering tissue/organ
o “Logos”: study of Initials of MT
o The study and examination of abnormal tissues in aid of INTRUMENTATION
identification and diagnosis of disease 1. MICROTOME
- Cuts tissues into: THIN SECTIONS/SEGMENTS
HISTOPATHOLOGIC TECHNIQUE - Thickness of a section: 3-5 (um)
o “12-step procedure” - 3 ESSENTIAL PARTS.
o End product: properly mounted stained slide o Knife carrier and knife
o Microtome: cuts tissues o Block holder – aka: CHUCK TISSUE HOLDER
o Base – aka: MICROTOME BODY
- Invented in 1848
- Includes:
- Modifications: Cambridge o Ratchet Feed Wheel
Minot o Pawl
Base-sledge o Adjustment Screws
- Types: Manual and Automatic:
ACCESSIONING MANUAL AUTOMATIC
o aka: “NUMBERING” – most important steps Rocking Vibrating -Lazer microtome
o identification code Rotary (most common) Ultrathin -Computerized
o a combination of numbers and letters Sledge Saw
- S – surgical, A – Autopsy, C – cytological Sliding Hand
Freezing
FIXATION
o FORMALIN: routinely used
- Neutral buffer Formalin – most preferred 2. AUTOMATIC KNIFE SHARPENER
o Preserves: tissues and cellular components - Has: Vibrating Frosted Glass Plate
- Every 4 strokes: the knife turns over and sharpened
DECALCIFICATION
o softening of calcified tissues 3. CRYOSTAT
o fastest decalcification agent PHLOROGLUCIN NITRIC ACID - Aka: CRYOTOME
- Consists of: ROTARY MICROTOME
DEHYDRATION
DEEP FREEZE CABINET
o Removes: excess H2O
- Alternative to: FREEZING MICROTOME
o Preparation for: wax IMPREGNATION
- Temp: -10C to – 30C
o (Ethanol) most routinely/commonly used for dehydrating agent →
- Thickness: 2-16 (um)
increasing concentration
CLEARING 4. AUTOMATIC TISSUE PROCESSOR
o Aka: DEALCHOHOLIZATION - can hold 200 to 300 specimens
o Removes: Alcohol - 2 TYPES
o Replaces with: XYLENE - Rotary/Carousel
o Soluble to: PARAFFIN o Tissue Transfer Processor
o XYLENE: routinely used clearing agent → used to make the tissue clear o Can do fixation up to impregnation process
→ using bright-field microscope - Vacuum infiltration
o Fluid transfer processor
INFILTRATION o (look a box or cabinet)
o Aka: IMPREGNATION
o Fills tissues with: PARAFFIN WAX or PARAPLAST 5. TISSUE EMBEDDING ENTER
- (more effective; used mostly in hospitals) - EG. LEICA EMBEDDING CENTER
o PARAPLAST – to fill the natural spaces, cavities, interstitial spaces, holes - have a paraffin, hotplate, cold plate etc.
of tissues
o Provides: RIGID support for tissues 6. PARAFFIN OVEN
EMBEDDING - Aka: WAX OVEN
o Product: Tissue Block - Dry the fished-out tissue in the paraffin oven
o Uses a: PARAFFIN WAX - After this section can be stain
o Orientation: process of orienting/placing the tissue at the right position. - Temp: 2-5C higher than the melting point of paraffin
- Used to dissolve/minimize the paraffin on the tissue in the process
TRIMMING called DEPARAFFINIZATION before proceeding to staining
o Aka: FACING
o To: Remove excess wax 7. FLOTATION WATER BATH
o Product: TRUNCATED PYRAMID or four-sided prism (routinely used) - Use: to RELAX and SMOOTEN out wrinkled tissue sections prior to
mounting
SECTIONING - Capacity: 2 LITER of distilled water
o Aka: MICROTOMY - Temperature: 5-10 C below Melting Point of Paraffin
o Process of cutting the tissue blocks into thin sections (has a temperature wherein paraffin will not)
o Product: SEGMENTS/SECTION - Dimension: 11 in diameter
o RIBBON: a series of sections (Process: RIBBONING) 4 in height
STAINING - Color (Interior): BLACK → gives contrast to RIBBON to observe
o Uses: DYES better the floating tissue
o Routinely used: HEMATOXYLIN (nucleus) → Color: DARK BLUE - Purpose: The process of transferring of tissue section from the
o Routinely used: EOSIN (cytoplasm) → Color: LIGHT PINK floatation water bath is Fishing out technique
o ACIDOPHILIC: attracts ACIDIC DYES (Nature: BASIC)
o BASOPHILIC: attracts BASIC DYES (Nature: ACIDIC) 8. COPLIN JARS
- Use: containers of STAINS OR REAGENT
MOUNTING
o Secures a: COVER SLIP ON TOP OF A TISSUE SLIDE 9. SLIDE DRYER
o To protect for ease of handling and storage of slide - Use to HASTEN the drying of slides after fishing (FISH OUT)
o Uses: CANADA BALSAM → most common and will permanently fixed - Capacity: 30 Slides
the tissue on the slide
o Routinely used: CANADA BALSAMS 10. AUTOMATED STAINERS
o COLOPHONIUM RESIN: To secure the end of the coverslip - Aka: AUTOSTAINER
- Also know kronig’s cement - Has: ROBOTIC ARM SYSTEM
- Can be: PROGRAMMED

[SARANILLO, KA.]
 HISTOPATHOLOGIC/CYTOLOGIC TECHNIQUES
ACCESSIONING AND FIXATION
MIDTERM: WEEK 8 LAB (03/16/2023)

ACCESIONING
1. Fill the logbook with the needed details:

DATE TIME RECEIVED SPECIMEN ID ORGAN REQ DR. RECEIVED BY

2. Write the number taken from the logbook on the request form.

GROSSING
1. Cut the specimen to fit the tissue cassette (3 x 2 cm; 3-5 mm thick)
2. Place the specimen in the tissue cassette, tie the specimen into the tissue
cassette using a dental floss
3. Close the cassette
4. Using a pencil, write the specimen number of the sides of the tissue
cassette.

FIXATION
1. Prepare 10% FORMALIN from a stock solution (40%)
- Ratio = 1 part tx: 20 parts fixative

FORMULA: C1V1 = C2V2

C1 = initial conc. 40% C2 = final conc. 10%

V1 = X V2 = final volume: 2000ml

40 (X) = (10)(2000ml) = X = 500ml of 40% formalin

40 40
FORMULA: Distilled Water: V2-V1

V2 = 2000 ml
V1 = 500 ml

2000ml – 500ml = 1500ml of DW

2. Place the cassette in 10% formalin (2 changes)

- Adequate fixation (6-12 hours)
- Complete fixation (12-24 hours)

[SARANILLO, KA.]
 HISTOPATHOLOGIC/CYTOLOGIC TECHNIQUES
DEHYDRATION, CLEARING, IMPREGNATION AND EMBEDDING
MIDTERM: WEEK 9 LAB (03/23/2023)
DEHYDRATION
o As soon as the tissue has been fixed, and the bones and teeth
have been decalcified, it is necessary to remove the fixative and
water from the specimen and replace them with dehydrating
fluid in preparation for impregnation. This process of removing
intercellular and extracellular water from the tissue following
fixation and prior to wax impregnation is known as
"dehydration”, and the solutions utilized to make this possible
are called "Dehydrating Agents".
o Most dehydrating agents are strong organic solvents that bring
about some shrinkage and extraction of cell components.
o To minimize these effects, dehydrating agents are used in a
graded series for short periods of time, and water is gradually
replaced so that violent osmotic changes do not produce
distortions.
PROCEDURE
1. Prepare 250mL of 70% and 90% ethyl alcohol from 95% ethyl
alcohol using the following formula:
2. C1V1 = C2V2. Show your computations to your instructor. Label
the containers with the reagent and your group number.
3. Immerse the previously fixed tissue in 70% for 6 hours, 95% ethyl
alcohol for 12 hours, absolute ethanol for 2 hours and another 2
changes of absolute ethanol for 2 hours.
CLEARING
o Clearing (de-alcoholization) is the process whereby alcohol or a
dehydrating agent is removed from the tissue and replaced with
a substance that will dissolve the wax with which the tissue is to
be impregnated (e.g. paraffin) or used as the medium on which
the tissue is to be mounted (e.g. Canada balsam).
o Clearing agent must be fully miscible with both ethanol and
paraffin wax is needed to remove alcohol and other dehydrating
solutions from tissues prior to embedding (usually in paraffin
wax), and from finished slides prior to mounting.
o The most commonly used clearing agent for this purpose is
XYLENE. Glycerin and gum syrup are used when the tissue is to
be cleared directly from water, as in a frozen section.
PROCEDURE
1. Prepare 2 beakers then transfer 250ml of xylene in each beaker.
2. Immerse the previously dehydrated tissue specimen in two
changes of xylene for 1 hour each.
IMPREGNATION
o Impregnation (Infiltration) is the process whereby the clearing
agent is completely removed from the tissue and replaced by a
medium that will completely fill all the tissue cavities and give a
firm consistency to the specimen.
o This allows easier handling and cutting of suitably thin sections
without any damage or distortion to the tissue and its cellular
components.
o Embedding (Casting or Blocking) is the process by which the
impregnated tissue is placed into a precisely arranged position in
a mold containing a medium which is then allowed to solidify.
PROCEDURE
1. Dissolve paraffin wax in five separate beakers. The maximum
temperature should be only 2-5 degrees higher than the melting
point of wax (melting point of wax is 60oC; 55-58oC)
2. Immerse the previously cleared specimen in four changes of wax
having in 15 minutes intervals. Remove the dental floss
EMBEDDING
o (CASTING OR BLOCKING) The process by which the impregnated
tissue is placed into a precisely arranged position in a mold
containing a medium which is then allowed to solidify.
There are generally four types There are three ways by which
of impregnation and paraffin wax impregnation and
embedding medium, namely: embedding of tissues may be
1. Paraffin wax performed:
2. Celloidin (collodion) 1. By manual processing
3. Gelatin 2. By automatic processing
4. Plastic 3. By vacuum embedding
MANUAL PROCESSING
o At least four changes of wax are required at 15 minutes intervals
in order to insure complete removal of the clearing agent from
the tissue.
o The specimen is then immersed in another fresh solution of
melted paraffin for approximately 3 hours to insure complete
embedding or casting of tissue.
AUTOMATIC PROCESSING
o This method makes use of an automatic tissue processing
machine (i.e.,Autotechnicon) which fixes, dehydrates, clears and
infiltrates tissues, thereby decreasing the time and labor needed
during the processing of tissues.
o This results in a more rapid diagnosis with less technicality.
o Usually, only 2- 3 changes of wax are required to remove the
clearing agent and properly impregnate the specimen.
o This is made possible due to constant tissue agitation which
accelerates and improves tissue penetration giving rise to more
consistent results. One example of an automatic tissue
processing machine is the Elliott Bench-Type Processor.
VACUUM EMBEDDING
o Vacuum embedding involves wax impregnation under negative
atmospheric pressure inside an embedding oven. It reduces the
time when tissues are subjected to high temperatures thus
minimizing heat-induced tissue hardening.
o It facilitates complete removal of transition solvents, and
prolongs the life of wax by reducing solvent contamination.
Vacuum hastens the removal of air bubbles and clearing agent
from the tissue block, thereby promoting a more rapid wax
penetration of the tissue.
o This technique is particularly recommended for urgent biopsies,
for delicate tissues such as lung, brain, connective tissues,
decalcified bones, eyes, spleen and central nervous system.
o Recommended for:
- urgent biopsies - decalcified bones
- for delicate tissues - eyes
such as lung and - Spleen
brain - CNS
- connective tissues
SEVERAL TYPES OF BLOCKING-OUT MOLDS MAY BE USED:
1. Leuckhart’s Embedding Mold - consists of two L-shaped strips of
heavy brass or metal arranged on a flat metal plate and which
can be moved to adjust the size of the mold to the size of the
specimen.
2. Compound Embedding Unit is made up of a series of interlocking
plates resting on a flat metal base, forming several
compartments. It has the advantage of embedding more
specimens at a time, thereby reducing the time needed for
blocking
3. Plastic Embedding Rings and Base Mold -consist of a special
stainless steel base mold fitted with a plastic embedding ring,
which later serves as the block holder during cutting.
4. Disposable Embedding Molds a. Peel-Away disposable thin
plastic embedding molds, available in 3 different sizes
PROCEDURE
1. Put a bit of melted wax on the embedding mold.
2. Using a preheated forceps, transfer the impregnated tissue from
the paraffin bath to the mold.
3. Orient the tissue in the perfect central position. Place the filter
paper (containing the specimen number) in such a way that it is
facing outside on the side of the mold.
4. Allow the wax to cool.
[SARANILLO, KA.]
 HISTOPATHOLOGIC/CYTOLOGIC TECHNIQUES ADDITIONAL NOTES:
H&E STAINING ROUTINE H&E STAINING
FINALS: WEEK 15 LAB (05/11/2023) o ROUTINE H&E STAINING in Paraffin Embedded Section
OBJECTIVES o (Regressive Staining)
1. Demonstrate the proper procedure in H & E Staining of tissues o Fixation: Most fixatives can be used except osmic acid
2. Analyze the steps in H & E Staining solutions which inhibit hematoxylin.

PRE-LAB DISCUSSION: PROCEDURE:

HEMATOXYLIN 1. Clear paraffin embedded sections in first xylene bath for 3
o Hematoxylin capechianum / Hematoxylon campechianum minutes.
o Nuclear/basic/1’ stain 2. Transfer to second xylene bath for 2 to 3 minutes.
o WALDEYER: 1st to use hematoxylin 3. Immerse in first bath of absolute ethyl alcohol for 2 minutes.
o Hematoxylin (Ripening) → Hematin (active coloring substance) 4. Transfer to a bath of 95% ethyl alcohol for 1 or 2 minutes.
5. Rinse in running water for 1 minute.
EOSIN Y 6. Stain with Harris alum hematoxylin for 5 minutes (Ehrlich's
o Most commonly used hematoxylin requires 15-30 minutes).
o Cytoplasmic/acidic/2’ stain 7. Wash in running tap water to remove excess stain.
o Counterstain 8. Differentiate in 1% acid-alcohol (1ml concentrated HCl to 99
ml. of 80% ethyl alcohol) for 10-30 sec. monitoring the
DISCUSSION: changes in color microscopically until only the nuclei are
o Hematoxylin and Eosin (H&E) staining is the corner stone of stained.
tissue-based diagnosis. The process stains thin tissue sections so 9. Rinse in tap water.
that pathologists can visualize tissue morphology. The process 10. Blue in ammonia water (average of 5 minutes) or 1% aqueous
uses a hematoxylin dye to stain cell nuclei (and other parts) blue lithium carbonate until the sections appear blue (about 30
and an eosin dye to stain other structures pink or red. seconds).
Hematoxylin binds strongly to acids and consequently binds to 11. Wash in running water for 5 minutes.
nuclear DNA and stains nuclei blue. Properly applied, this 12. Counterstain with 5% aqueous eosin for 5 minutes. If
technique provides exceptional detail of tissue structure and the alcoholic eosin is used, the time can be reduced to 30 seconds
makeup of the cells. This detail is required for tissue-based or 1 minute.
diagnosis, particularly in the detection and classification of 13. If aqueous eosin is used, wash and differentiate in tap water
infection, cancer or metabolic disease. under microscope control until the nuclei appear sharp blue
o The hematoxylin and eosin (H&E) procedure is the 'bread and to blue black and the rest of the tissue appear in shades of
butter' stain in histopathology. In a histology laboratory, almost pink. If alcoholic solution is used, differentiate with 70%
all specimens are initially stained with H&E and additional stains alcohol.
are only ordered if additional information is needed to provide a 14. Dehydrate, clear and mount.
more detailed analysis.
FROZEN SECTION STAINING
MATERIALS/ APPARATUS o Frozen sections mounted on the slides may be stained as in
o Paraffin embedded tissue section paraffin sections although the duration of staining is usually
o Harris Hematoxylin shorter.
o Xylene o H & E staining of Frozen Sections for Rapid Diagnosis
o Ammonia water (Progressive Staining)
o Absolute alcohol
o 1% acid alcohol 1. Orient section in the block and freeze with liquid nitrogen.
o 90% alcohol 2. Cut cryostat sections at 5-10 micron.
o 5% aqueous Eosin 3. Mount sections on to albuminized slides and dip in 10%
o 70% alcohol formalin to fix.
4. Rinse rapidly in water.
PROCEDURE 5. Stain with Harris hematoxylin for 30-45 seconds.
1. Xylene 1 (5 dips) 6. Rinse in tap water.
2. Xylene 2 (5 dips) 7. Blue in ammonia water for 5 seconds.
3. Absolute alcohol (10 dips) 8. Rinse in tap water.
4. 90% alcohol (5 dips) 9. Counterstain with 5% aqueous eosin or 1% alcohol eosin for
5. 70% alcohol (5 dips) one minute.
6. Wash in running water 10. Rinse in tap water.
7. Harris Hematoxylin (5 minutes) 11. Dehydrate in increasing concentrations of alcohol.
8. Wash in running water 12. Clear with xylene.
9. 1% acid-alcohol (10-30 seconds) 13. Mount with cover slide.
10. Wash in running water
11. Ammonia water (5 minutes) o It is somewhat less favored than regressive staining due to the
12. Wash in running water difficulty of producing sufficiently intense progressive
13. 5% aqueous Eosin (5 minutes ) staining of cell structures without staining other parts,
14. 70% alcohol (5 dips) thereby resulting in diffused color and obscured details. For
15. 90% alcohol (5 dips) convenience, reagents for this rapid H&E stain are generally
16. Absolute alcohol (10 dips) arranged in sequence using a series of Coplin jars. This
17. Xylene 1 (5 dips) method takes only 5-10 minutes and produces well-
18. Xylene 2 (5 dips) differentiated sections that are semi-permanent and can be
19. Mount stored. The remaining portion of tissue must be kept for
routine processing and are made for comparison with frozen
sections.

[SARANILLO, KA.]