HISTOPATHOLOGY LABORATORY

ROUTINE PROCEDURES
LECTURER: Mr. John Marke Bernardo
DATE | A.Y. 2022 – 2023 | PRELIMS | 2ND SEMESTER

OUTLINE ● NOTE:
I. ROUTINE PROCEDURES ○ S = Biopsy
A. Introduction ○ A = Autopsy
II. RECEIVING ● Serial Numbers are assigned
III. GROSS EXAMINATION ○ Uses Barcodes which contain all of the
IV. FIXATION patient’s information. For Data Privacy.
V. DECALCIFICATION (OPTIONAL) ■ You only scan the barcodes using a
VI. CLEARING barcode scanner once you are
VII. INFILTRATION done with the routine procedures
VIII. EMBEDDING and you need to get the result
IX. TRIMMING ■ Upon receiving the sample, it needs
X. SECTIONING to be pre-soaked with formalin. If
XI. STAINING the sample you received is not
XII. MOUNTING pre-soaked, do not dispose of it;
however, you can reject it.
■ Modern routine procedures do not
ROUTINE PROCEDURES
process the entire organ but instead
In order to enable the pathologist to diagnose the presence
process the representation of the
or absence of disease, the histotechnologist needs to
said organ. We only process the
produce a tissue section of good quality that allows for affected portion of the organ.
adequate interpretation of microscopic cellular changes.
GROSS EXAMINATION
Solid tissues need to be fixed and processed to preserve
their structures, and eventually impregnated with an
appropriate hardening substance to permit making thin
slices suitable for staining and microscopic evaluation

This can be accomplished by preserving and carefully

processing solid structures and tissues in the following
order:
I. Receiving (Accessioning)
● Performed by the Pathologist
II. Gross Examination
● Notes the macroscopic characteristics of the tissue
III. Fixation ● Tissue blocks should be small (e.g. 1-2 mm2 for
IV. Decalcification (optional) electron microscopy and 2 cm2 wide for light
V. Dehydration microscopy) and thin (no more than 0.4 cm for
VI. Clearing light microscopy).
VII. Impregnation (Infiltration) FIXATION
VIII. Embedding ● Preserving biological tissues in a state as close to
IX. Trimming living tissue as possible (both chemically and
X. Section-Cutting (Microtomy) structurally).
XI. Staining ● The specimen is placed in a liquid fixing agent
XII. Mounting (fixative) such as formaldehyde solution (formalin).
XIII. Labeling; ○ Fixatives slowly penetrate the tissue causing
RECEIVING (ACCESSIONING) chemical and physical changes that will
● Checking of Labels harden and preserve the tissue and protect it
a. Patient’s Name against subsequent processing steps.
b. Sex ○ Formalin, usually as a phosphate-buffered
c. Clinical Data solution, is the most popular fixative for
d. Surgical findings preserving tissues that will be processed for
e. Nature of Operation paraffin embedding.
f. Name of tissues submitted

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HISTOPATHOLOGY LABORATORY
ROUTINE PROCEDURES
LECTURER: Mr. John Marke Bernardo
DATE | A.Y. 2022 – 2023 | PRELIMS | 2ND SEMESTER

● Fixation is a critical step in the preparation of dust and other cellular debris. Inadequate
histological sections. If it is not carried out under decalcification may result in poor cutting of hard
optimal conditions or if fixation is delayed, a tissue tissues and damage to the knife edge during
specimen can be irreversibly damaged, leading to sectioning
autolysis and putrefaction. SUBTO
PIC TITLE

○ Autolysis DEHYDRATION
■ the enzymatic digestion of cells by
the action of its own enzymes, and
it mostly occurs in dying or dead
cells.
○ Putrefaction
■ the decomposition of organic
matter. especially : the typically
anaerobic splitting of proteins by
bacteria and fungi with the
formation of foul-smelling
incompletely oxidized products

● This process is commonly carried out by

immersing specimens in a series of ethanol
(alcohol) solutions with increasing concentration
to avoid excessive distortion of tissue until a
water-free tissue in alcohol is reached.
○ It starts with alcohol with lower
concentrations to allow water or any
water-soluble proteins present in the sample
● Following fixation, appropriately trimmed specimens
to interact with it, removing it in the process
are placed in suitable labelled cassettes (small
By the time the sample is immersed in
perforated baskets) to segregate them from other
absolute ethanol (100% ethanol), water and
specimens.
certain lipids are already dissolved.
CLEARING
DECALCIFICATION (OPTIONAL) ● Transition step between dehydration and
● The removal of calcium ions from a bone or
Infiltration
calcified tissue through a histological process that
○ The dehydrated tissue is transferred to an
makes them flexible and easier to cut.
intermediate solvent that is fully miscible with
● The principle of decalcification is fairly simple. Strong
both ethanol and paraffin wax.
mineral acids, such as 10% hydrogen chloride
○ The term “clearing” has been chosen
(HCl), or weak organic acids, such as 5-10%
because many (but not all) clearing agents
formic acid (HCOOH), form soluble calcium salts in
impart an optical clarity or transparency
an ion exchange that moves calcium into the
to the tissue due to their relatively high
decalcifying solution.
refractive index.
● In order to protect the cellular and fibrous elements of
■ High refractive index means that it
bone from damage caused by the acids used as
is almost colorless. Clearing
decalcifying agents, it is particularly important to
agents, which have high refractive
thoroughly fix these specimens prior to decalcification.
index renders anhydrous tissues
● Decalcification should be done after fixation and
transparent or clear for easier
before impregnation, to ensure and facilitate the
cutting of tissue,
normal cutting of sections and to prevent obscuring
● Following the dehydration, the tissue is immersed in
the microanatomic detail of such sections by bone
one to three different xylene immersions. In these

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HISTOPATHOLOGY LABORATORY
ROUTINE PROCEDURES
LECTURER: Mr. John Marke Bernardo
DATE | A.Y. 2022 – 2023 | PRELIMS | 2ND SEMESTER
stages, the ethanol is gradually replaced with
xylene and when the tissue is embedded, the
xylene will then be replaced by the molten paraffin
wax.
INFILTRATION (IMPREGNATION)
● Removal of excess clearing agent from the tissues
and replaces it with a medium that will fill the natural
cavities, spaces, and interstices of the tissues.
● The cleared tissue is infiltrated with a suitable
histological wax (usually paraffin) which is liquid at
60°C and then allowed to cool to 20°C in order to
solidify into a consistency that allows sections to be
cut
○ The need for infiltration is critical since
samples such as spongy bones tend to
pulverize during sectioning due to its
cavities. Paraffin wax infiltrates the natural
cavities or interspaces, providing firm
consistency (So it acts like a foundation like
cement).
● These waxes are mixtures of purified paraffin wax and
various additives that may include resins such as
styrene or polyethylene that allow them to be
sectioned thin enough on a microtome, forming
ribbons that can flatten fully when floated on a warm
water bath.
EMBEDDING
(Wala nag gloves kek)
● aka Casting or Blocking
● is done by placing the infiltrated tissue, in a precisely
arranged position, in a mold containing a medium
which is allowed to solidify
● After infiltration with wax, the tissue is oriented and
placed in a mold that is filled with molten wax to form
a solid tissue block that can later be clamped into a
microtome for sectioning. The infiltrated tissue is
removed from the cassette and very carefully oriented
in a suitably sized metal mold so that the “plane of
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section” can be determined. Correct orientation of
tissue in a mold is the most important step in
embedding.
○ So dito nangyayari yung positioning ng
sample. Kung ang doctor gusto niya
cross-sectional orientation ang tissue block,
then you need to change the position into a
cross-sectional view. Longitudinal is for the
length, lateral is for the width. Usually ang
cross-sectional kay nakahiga while ang
lateral kay nakatayo ang tissue.
● Same medium as the infiltrating agent
SECTIONING
● cutting of the embedded tissues into uniformly thin
slices using the microtome
○ So kung mali ang inyong embedding, mali
ang inyong pagposition sa sample,
automatically mali na ang cutting. Naa man
gud arrangement sa cells that can be best
viewed at a cross-sectional, lateral, or
longitudinal view.
○ Sir what if defective ang microtome? Are we
allowed to cut it with a knife? No. Although
we can cut it intentionally, the product itself is
not uniform. The thickness is not thin,
wherein ang thin sa akoa kay thick pa diay
sa microscope. Kasi ang nagaassess ng
viewing is not our naked eyes but the
microscope. Pag masyadong makapal, hindi
magpenetrate ang light source sa
microscope and pagtingin natin sa eye
piece, black lang kasi nablock siya ng
thickness ng iyong cutting
○ This is your rotary microtome. This is the
knife holder and this is your tissue block.
Ang actual appearance kasi ng tissue block
is parang ice cube, wherein ang tissue nasa
gitna. Ano nasa paligid ng tissue sir?
HISTOPATHOLOGY LABORATORY
ROUTINE PROCEDURES
LECTURER: Mr. John Marke Bernardo
DATE | A.Y. 2022 – 2023 | PRELIMS | 2ND SEMESTER

Paraffin wax. So nakatindog ang orientation stain which follows Romanowsky staining
sa iyahang clip. principle.
● Romanowsky staining principle - basic dye would
adhere/stain to acidic cellular components and your
acidic dye to basic cellular components
○ For example, your hematoxylin which is a
basic dye will stain your nucleus while your
acidic eosin will stain your cytoplasm.
○ Sir why are there varying pigments (naay
dark kaayo na blue naa pud light blue)?
Because magdepende na siya sa amount sa
iyang acidity or alkalinity sa iyang cellular
details, OR maybe because wrong ang
inyong incubation sa staining. For example,
nagstain mo. Dapat ang instruction 1 minute
lang, unya nagsige man mo tabi 1 hour na
○ After cutting into thin slices, meron tayo
hinoon, so mas intense ang iyahang
tinatawag na fish out technique with the help
giprovide na stain.
of your glass slides. In order for the wrinkles
○ Usually kung old age na ang tissue samples,
in your thin sections to be resolved,
nagafade ang stains, kay naa man life span
kailangan natin siya itransfer sa water bath
ang stains.
and then slightly stretch it until such time na
flattened na siya. If naflat na siya, we need MOUNTING
to fish out using the glass slide so that your
tissue will sit on top of your glass slide,
rendering now your almost prepared slide.
Ipaslant natin ang glass slide para magdrip
off yung water na galing sa floatation or
water bath.

STAINING

● protecting tissue sections from physical damage by

coating it with a transparent medium then
covering it with a glass slip
○ The purpose of your mounting is to protect
the tissue samples from physical damage.
We need to place some transparent
adhesives in between your tissue sample
slides and your cover slips. Dapat wala yan
siyang bubbles.
● process of adding colors or dyes to the thin tissue
slices for enhanced visualization and differentiation of
cellular structures
○ In order for us to visually appreciate your
center details, you need to apply certain
stains.
○ We have your most commonly used stains
which is H&E stain and your Papanicolau

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