ROUTINE PROCEDURES IN HISTOPATHOLOGY LABORATORY

● Histology is the microscopic study of the normal (sometimes), usually

tissues of the body thyroid gland
● Histopathology is the microscopic study of tissues
affected by disease
o These types of tissues are not normal anymore.
We expect to see the abnormality at the cellular ● Surgeon would submit
level and find whatever is causing the said this type of specimen
disease also to have an idea
● The tissues are usually obtained during surgery, whether they need to
biopsy, or autopsy proceed to a bigger
o We can also have simple biopsies wherein the type of surgery or not.
patient is not inside the operation room. Just the They will just have an
minor operation room or sometimes even in the idea whether they’re
doctor’s clinic dealing with benign or
o If the individual is already dead and then the malignant because we
family wants to know the cause of death, we cannot really diagnose
would also be removing organs from the dead it 100% as malignant
body and then send it for histopathologic reading
CORE NEEDLE ● Removes cells and
or screening
BIOPSY small amount of
● The picture shows a
surrounding tissue
tissue already
● The bore of the needle
embedded in paraffin
will be bigger compared
● Paraffin is the same
to the FNAB because
as your wax.
this time around, even
small around of tissues
TYPES OF SURGICAL PROCEDURES
will be removed
FINE NEEDLE ● Simples, least invasive ● This is usually done
ASPIRATION ● This is use when the when the surgeon
specimen or the organ wants toa have an idea
is readily palpable and as to what type of
the patient does not breast tumor he/she is
have to undergo looking at
general anesthesia, just ● Commonly done for
blocking of the nerves breast tumors
Sample of what a fine or even just lidocaine to ● Between FNAB and
needle aspiration or dull or remove the pain Core Needle, you can
FNAB would look like altogether see more abnormalities
under the microscope. ● A wider bore needle will or have a better view of
You will see cellular be inserted directly over what you’re dealing
components as well as the organ and then with if the surgeon will
debris and liquid. You tissue as well as a little submit your core
will not see much tissue bid of liquid will be needle biopsy
because this is only a withdrawn ● Surgeon don’t want to
needle aspirate ● Organs that usually can get a bigger sample
be sample through first so that they will
FNAB would be: Lymph also have an idea as to
nodes, breast what type of surgery

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 ROUTINE PROCEDURES IN HISTOPATHOLOGY LABORATORY
they will perform later
on SHAVE BIOPSY ● Small fragments of
● If it’s frankly malignant tissue are “shaved from
then they would a surface (usually skin)
probably do their MRM
or mastectomy (for
breast)

INCISIONAL BIOPSY ● Only a portion of the

tumor or the desired
area would be
removed. This will be CURRETINGS ● Tissue is scoped or
bigger than your core spooned to remove
needle biopsy tissue or growths from
As you can see in the ● They will directly get it body cavity such as
picture, the surgeon from the mass endometrium or
directly removes a cervical canal
sample from the area of ● Commonly done in
question. It is a good patient who had
representation of that abortion regardless of
specific area compared the cause of the
to the first two abortion
EXCISIONAL BIOPSY ● Removes the entire ● OBGYN would send a
area in question samples of this
● Even the surround will curretings so expect to
be removed as well see a lot of tissues
● This is to ensure that
the margins or lines of
resection are free of the
tumor cells because if
the surgeon is already
having more than 50%
of idea that what he or
she is dealing is
malignancy, the best
option for the patient is ● The picture above is an example of your tongue
to ensure that the area squamous carcinoma
surrounding the tumor ● The area encircled would be the area of interest or
is also free from that the tumor per se. This is where the surgeon will
cancer cells or free remove
from those cancer cells ● From this type it would be excisional biopsy because
you can also see on the right side the epidermis. Not
only the tumor was removed but also the
PUNCH BIOPSY ● Another type of sample
surrounding tissue
which is usually done
● The reason for this is to ensure that the surgeon has
by dermatologist
completely removed the entre tumor itself and the
● Diagnostic
surrounding tissue would not have any more of the
full-thickness skin
cancer cells
specimens
● Why is this important?

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 ROUTINE PROCEDURES IN HISTOPATHOLOGY LABORATORY
o So that recurrence of the cancer or the tumor o Since it’s fluid you don’t have to do anything. It
cells will not happen will just flow across the slide naturally or
METHODS OF FRESH TISSUE EXAMINATION normally – capillary action
1. Teasing or Dissociation
2. Squash Preparation (Crushing)
3. Smear Preparation
4. Touch Preparation (Impression Smear)
TEASING OR DISSOCIATION
● Selected tissue is immersed in isotonic solution
(normal saline or Ringer’s solution – types of
dextrose fluids)
● Carefully dissected with a needle and separated by
direct or zigzag spreading using an applicator stick
o So, you would need to be able to have a good
idea at what you’re dealing with or you’re looking
at so that you can carefully choose the tissues
that you will use
● Avoid forming bubbles!
o It will also affect the quality of the slide
● Can be examined stained (supravital dye) or
unstained (Phase Contrast or Bright Field
microscopy)
● ADVANTAGE
● Compress it between slide and you will have your
o Cells are examined in the living state
squashed specimen
▪ We can see if they’re dividing, or we can
● Disadvantage: cells are quite distorted already
appreciate the parts of the cell when they
(because you forcibly compressed the
are sill in the viable living state. Even the
specimen/tissue)
mitosis can be appreciated
3. SMEAR PREPARATION
● Unfortunately, tissues will not be quite visible for this
● Examination of sediments / Small tissue sections by
one
spreading materials lightly on slide using applicator
stick or a slide.
● Useful in cytological examination as well as in
hematologic examination
● For cytologic purposes, an example would be Pap
smear
o Very good procedure to determine whether the
patient has cervical malignancy or none.
o One of the reasons why cervical cancer has
gone down over the decades because of this
SQUASH PREPARATION (CRUSHING) examination. This has helped in lowering down
● Less than 1 mm tissue are forcibly compressed incidences of cervical cancer
(stained when necessary), and examined under the ● TYPES OF SMEAR PREPARATION
microscope o Streaking
o By the word crushing, you would forcibly press o Spreading
the specimen between two glass slides or o Pull-apart
between a glass slide and a cover slip A. STREAKING
o It has to be small because the specimen will ● Used for preparing mucoid secretions, vaginal
spread out secretions, sputum and gastric content
● Supravital stain may be placed at the junction of the o Very useful when dealing with thick specimen
slide and the cover glass, and allowed to be ● Use a spatula, dissecting needle or applicator stick
absorbed by the tissue through capillary attraction and streak in a zigzag fashion

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 ROUTINE PROCEDURES IN HISTOPATHOLOGY LABORATORY
● The things used in the laboratory are made to cater
for the sizes of the materials as well
● If it’s a liquid culture, transfer one or two loopfuls to
the center of the slide and spread it out thinly.
B. SPREADING
● 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
o Remember, always start with a clean slide.
Always use clean materials so that no dirt, slimy
or oily substances will be transferred to the
specimen. Start with a clean slide and clean
spreaders
● It is a little more tedious than streaking.
o Because you have to tease the mucus trans
apart gently
● Advantage of maintaining cellular interrelationships.
o Cellular interrelationship will be very much
viewable because you did not streak it. Instead,
you just gently spread them apart
● Fresh sputum and bronchial aspirates, and also for
thick mucoid secretions
● Can also be done for some cytologic samples like
pap smear
● Image shows how you do the spreading. Make sure
that you do not go beyond the edges of the slide.
● About 1 inch (1x1) sample would do. So that it will
no flow over the slide
● Make sure that the specimen that you put is not too
much because we don’t want to have a thick smear.
o Because once we stain it, it will become very
dark and will be hard to visualize
C. PULL-APART
● Placing a drop of secretion or sediment upon one
slide and facing it to another clean slide
● Two slides are then pulled apart with a single
uninterrupted motion
● Specimen is placed under the microscope for
immediate examination, or applied with vital stains
● The image above shows how you do the pull-apart
preparation
● Put the specimen on one slide and put another slide
on top of the specimen
● Separate them or pull them apart quickly with one
uninterrupted motion so that the specimen will be
very optimal for viewing under the slide
● Do not have any hesitation strokes
● Put a drop or two of the specimen on one slide
● Put another slide on top of it and then in one
single motion, separate the two slides by sliding
or pulling them apart.
4. TOUCH PREPARATION (IMPRESSION SMEAR)
● Surface of a freshly cut piece of tissue is brought
into contact and pressed on to the surface of a clean
glass slide
● ADVANTAGE: cells may be examined without
destroying their intercellular relationship
o You will be able to visualize the cells clearly
● This is done when we have samples from orthopedic
surgeons that means, BONE. So that, we can see
whether there are tumor cells
● DISADVANTAGE: If the person doing it has heavy
hands, they might be able to destroy the specimen.
So, you need to do this preparation gently. Just
touch. You don’t have to crush the specimen
● Just lightly pat or touch the specimen on the slide
and then preserve the specimen for a different type
of procedure
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 ROUTINE PROCEDURES IN HISTOPATHOLOGY LABORATORY
● Usually done for bone imprints and even lymph node
imprints
FROZEN SECTION
● For rapid diagnosis; when surgeons need to check
their lines of resection.
● Fresh tissue frozen on a microtome with CO2, or on
a cryostat.
o Microtome with CO2 are already old school
o Cryostat – being used in the laboratory today; an
automated machine which will greatly aid the
pathologist when they’re performing the frozen
section biopsies
o Frozen section biopsies are performed by the
pathologists and residents on training
● 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.
o Fixed immediately; you have to put it in an
embedding medium
o Do not put it in formalin because you need the
tissue to be fresh
o The embedding medium is easily frozen
o Still stain the sample but do it rapidly
● Fresh frozen samples; The patient is still on the
operating table that means it still opened. And then
the surgeon wants to find out whether the lines of
resection or margins of resection are clear of the
tumor cells already
o One reason why fresh frozen section biopsy
would be requested
● Why is it rapid?
o Because the specimen will not undergo the
recourse of processing like fixation🡪 gross
cutting🡪 embedding in paraffin🡪sectioning
🡪placing it on the slide 🡪 staining 🡪 cover slip
(mounting)
● Since the patient is still under anesthesia and still on
the table, we need to move very fast.
● The surgeon will submit few samples and then we
will perform the FSB or the Frozen Section Biopsy
and ideally it is done within 30 minutes
● Afterwards, once we have given our impression (not
the final diagnosis), we will then put the specimen in
a fixative and the regular type of processing will then
commence
o This is not the end of the tissue. Just a preview
because frozen section biopsies are not final
diagnosis type of specimens. This is just to
inform the surgeon that:
▪ (1) no need to further resect because there
are no more tumor cells on the surrounding
tissue that they have submitted or
▪ (2) Sorry but this is a malignant case. You
need to perform a bigger or more
comprehensive procedure on the patient; or
▪ (3) the tumor is very big. You might have to
close up and might have to perform
chemoradiotherapy first so that the tumor
will become smaller before you can proceed
again
● There are so many reasons why frozen samples or
frozen biopsies are being performed. Not only for the
liens of resection but also to check the quality of the
specimen whether they already got the tumor or not.
To check whether the ongoing procedure is still
relevant to the patient. To know whether we are
dealing with benign or malignant tumors
● Microtome with CO2 are already old school
● Cryostat – being used in the laboratory today; an
automated machine which will greatly aid the
pathologist when they’re performing the frozen
section biopsies
● Frozen section biopsies are performed by the
pathologists and residents on training
● Advantages
o Give much faster results than paraffin embedded
tissues.
o Rapid processing time.
o Less equipment needed.
▪ You will just need your cryostat machine, a
forceps, and then your coupling jars filled with
your stains
▪ You will not need your embedding medium so
on and so forth
▪ You will not need a separate microtome
anymore
o Less ventilation necessary.
▪ You will not be using fixatives you will not be
using your formalin formulas are very noxious
agents and they can really irritate your eyes
and your nose, it can really make you tear up
and give you very bad allergies if you get
exposed to it
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 ROUTINE PROCEDURES IN HISTOPATHOLOGY LABORATORY
● Disadvantages
o Relatively poor quality of final slide.
▪ Because it is fresh, the tissue is very soft, that
is one of the reasons why fixation is important
so that you will be able to trim and cut the
sample specimens carefully
o Morphological details and resolution of image are
inferior compared to paraffin embedded tissues.
▪ One of the important things when thick when
for fixation is that it will solidify the organs so
you will have a very good view of the tissue or
organ you're dealing with because the
abnormal tissues will stand out from the
normal one
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
MORE COMMONLY USED METHODS OF
FREEZING
1. Liquid nitrogen
2. Isopentane cooled by liquid nitrogen
3. Carbon dioxide gas
4. Aerosol sprays
● As water freezes, the hydrogen bonds push the
water molecules farther apart from each other
increasing the intra or intermolecular space resulting
in expansion
● They become more stable compared to just the
liquid water
1. LIQUID NITROGEN
● Most rapid of the commonly available freezing
agents
● Main disadvantage is that soft tissue is liable to
crack due to the rapid expansion of the ice within the
tissue
● Other disadvantage: act as an insulator that causes
uneven cooling of tissue particularly of muscle
biopsies
o Because muscle muscles are quite hard and
thick so we don't want an uneven pulling,
because that means that the most interior portion
of the tissue will still be soft and this can cause
damage to the tissue
2. ISOPENTANE COOLED BY LIQUID
NITROGEN
● Liquid at room temperature
● Fill a flask with ½ liquid nitrogen
● A metal beaker is filled 2/3 with isopentane and
placed in a liquid nitrogen enough to come up to
about 1/3 of the metal beaker. Prepare at least 10
minutes before freezing sample.
o This has not been this is not being done in more
advanced laboratories anymore because we
have the cryostat
3. AEROSOL SPRAYS
● Is adequate for freezing small pieces of tissue
except muscle
● Disadvantage for this one is melting is very fast
● You have to continuously spray with our aerosol
spray
CRYOSTAT PROCEDURE (COLD
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 ROUTINE PROCEDURES IN HISTOPATHOLOGY LABORATORY
MICROTOME)
● A cryostat is the instrument to freeze the tissue and
also to cut the frozen tissue for microscopic section
● “a microtome inside a freezer”
● The surgical specimen is placed on a cryomold,
frozen and is then secured in a chuck and frozen
rapidly to about -20 to -30 degrees celsius
● The usual histology slice is cut at 5 to 10
micrometers
● The optimum working temperature of cryostat is -18
to -20°c.
● 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.
● The one farthest left would be your compound, that
will be your freezing medium and then you will put it
on the disc or the specimen holder or specimen disc
● Then you put the specimen on top, the oct will serve
as the freezing medium
● The green thing in the middle would be your
specimen
● OCT is short for “optimal cutting temperature.”
o Polyvinyl alcohol
o Polyethylene glycol
o Non-reactive ingredients
● It will serve as the embedding medium
● You put it on the specimen disk the one being held
by the technologist, technician or pathologist
● You put the OCT on top you don't put too much
because you want to perform it quite fast
o The thicker the compound applied the longer the
procedure will be
● And then you freeze it
● And then put the specimen on top
● And then put another layer of the compound just to
seal the specimen inside
● Then you trim it until you get your desired sample
FROZEN SECTION TUTORIAL: EMBEDDING AND
CUTTING SPECIMEN (VIDEO 1)
● Step 1: Gross the Specimen
TIPS FOR GROSSING IN THE FROZEN SECTION
ROOM:
1. Gross one case at a time
2. Keep things minimalistic (e.g., Estimate the size of
small bits of tissue
3. Remove staples, sutures, bone, and fat whenever
possible
4. Keep the tissue as dry as possible
● We are going to cut a piece of tissue. The molds are
kept in the cryostat. Take the mold out at the very
last minute so it doesn’t warm up
● Step 2: Embed the tissue using a mold
o When you put the tissue into the mold make
sure that you control the tissue the whole way.
Anywhere that you touch the tissue, it’s going to
stick so control the tissue and make sure that
you place the tissue in the center of the mold, so
you have border of OCT around the outside and
you’re going to need that in order to tease off the
section as you’re cutting it.
● Avoid unnecessary tissue-block contact before
embedding (tissue will stick to the cold block where it
touches)
● Place the tissue in the center with a 3-5mm border
o Try doing it in one motion. I’m going to touch it
there and flatten it down so now I have space
around here which is 3-5mm border
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 ROUTINE PROCEDURES IN HISTOPATHOLOGY LABORATORY
● Overfill the mold with OCT (chuck won’t stick fi
underfilled)
o Fill this up to overflowing when you put the block
on top of this thing. You want the OCT to ooze
out from between these grooves which is going
to help the block adhere to this

● Tighten this up and I’m going to move this blade so

that we can start on the end right here and as we
continue to cut, we’ll slide this down as this portion
of the blade becomes dull
● Load a new blade and/or move it to an unused area
● Default settings
● Press it down firmly. Bring this over put this into the o Section thickness – 5 microns
fire stick. Hold this down and spray o Blade angle – 5
● Other tips for embedding specimens: ● Remove excess OCT before loading onto cryostat
1. Orientation (ensure the block is loaded flat)
▪ True margins of oriented specimens go face o Make sure that the block is frozen
DOWN
▪ Mucosal margins are oriented so that you
can see the mucosa and submucosa on the
same plan
▪ Ureters should be embedded so you can
see the full cross section
2. Batch specimens from the same case whenever
possible

● Place the block into the holder and position it to cut. I

put it on this angle right here because generally
when you’re cutting tissue you want to cut from soft
to hard to small to large and that will just facilitate
the cutting
● When you spray it, it will freeze a lot more quickly
than just letting it freeze by itself
● Step 3: Prep the cryostat and load the block
o I’m going to put a blade in. In the meantime, it is
(the block) is continuing to freeze

● Step 4: Trim the block

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 ROUTINE PROCEDURES IN HISTOPATHOLOGY LABORATORY
o When the little light is lit up it means that the ● Step 6: Transfer the tissue to the slide
block holder has retracted all the way back to o Hold the slide firmly and have a lot of control
the hold position over it
o When you press the rapid advance button down ● Step 7: Immediately fix the tissue in methanol
for about 10 seconds it is going to bring the thing PREPARATION OF FROZEN TISSUE SECTIONS –
out about 5 mm CRYOTOMY (VIDEO 2)
● Coarse advance until the block almost touches the ● Sectioning of formal and fixed and paraffin
blade (~10s/5mm) embedded tissue is widely recognized as the
● Gently tap the coarse advance while turning the standard for most diagnostic applications
wheel to trim the block MICROTOMY CRYOTOMY
o You can now see that the entire surface is being
cut Sectioning of Sectioning of frozen tissue
formalin-fixed
paraffin-embedded
tissue

Good morphology Supports rapid

investigations i.e.,
intra-operative histology
(e.g., Mohs surgery)

Supports follow-up Avoids use of formalin

investigations
Better for some antigens
● Step 5: Take a section Support enzyme
o The brush should always be cold. If you’re using histochemistry
brush put it down until you’re actually going to
take the section. If the brush gets warm, spray it. Relatively slow Poorer morphology
The reason is when the brush is warm it will diagnosis (i.e., hours)
grab on the OCT, melt, and ruin the section
● Keep the brush cold to prevent it from sticking to the
section
1) Leave it in the cryostat until ready to sue
2) Spray if warm

● So here our senior technician is demonstrating the

inside of the cryostat
● There are a couple of specimens over on the left
hand side there one of which is already prepared in
our plastic embedding medium and then a fresh
piece of tissue there as well
● Now when I’ve taken the section, notice that I don’t
separate it from the block. This is still attacked right
up here and that allows you to be able to manipulate
the section a little bit
● Keep the section attached to the block
● Hold down the section (might jump because of static
electricity)

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 ROUTINE PROCEDURES IN HISTOPATHOLOGY LABORATORY
● Alternatively, you can actually use a mold such as
the one that can be seen on the right hand side here
to completely surround and freeze your piece of
tissue and then you can transfer that once again to
the stub

● The first approach that can be used is to use a

plastic embedding medium such as oct compound
as a way of adhering the fresh frozen tissue to a
stub or disc

● once that's completely frozen such as this one here

prepared a bit earlier we can then transfer that to the
specimen head or chuck

● Then once secured in place the process from here is

remarkably similar to performing regular microtome
paraffin
● The blades that we're using are identical to the ones
that we use for our paraffin microtomes sections

● Then we'll place a bit more of the oct on top in order

to secure in place

● Once the blade has been inserted and clamped in

place we simply move that to an area where it's
convenient to keep a track of how much of the blade
that we've used

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 ROUTINE PROCEDURES IN HISTOPATHOLOGY LABORATORY
● Then the position of the tissue is altered using this
motorized feed, switch up above, as well as the
hand wheel on the right-hand side
● Having positioned the tissue close enough to the
blade you then face the block in a fairly similar
fashion to working with paraffin blocks
● There's quite a lot of excess plastic mounting
medium coming off the surface
● It can sometimes be useful to trim any excess
mounting media from around the edges just to make
it a little bit easier to shape those sections to the
desired size and shape
● Having done that we can then mount the stub back
onto the chuck and then complete the facing until
we're happy that the entire face of the block of tissue
is showing through
● notice at the moment that the tissue coming off the
face of the block is quite crumpled but that's okay
whilst we're just cutting down through the layers to
get a complete face
● Now looking pretty close to what we need having
done that we then insert what's known as an anti-roll
plate and adjust the section thickness down to the
desired level in the order of three to five microns
● The benefit of that anti-roll plate now becomes more
apparent as we continue to turn the hand wheel
● Now makes it a lot easier to visualize those sections
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 ROUTINE PROCEDURES IN HISTOPATHOLOGY LABORATORY
RAPID H&E PROTOCOL
1. Fix for 1 minute in methanol
2. Rinse in water
3. Stain with Mayers’s Hx for 30 secs
4. Rinse in water
5. “blue” by applying 0.1% ammonia for 10 seconds
6. Rinse in water
7. Stain with eosin for 20 seconds
8. Dehydrate, clear and mount

● Remove the anti-roll plate and using a couple of

paint brushes here just very carefully dissecting
those sections within the ribbon

● Then just grab a microscope slide and using this

rolling like action you'll see the sections adhering
onto the glass
FRESH TISSUE RELATIVE OPTIMAL
WATER TEMPERATURE
CONTENT CUTTING

Adrenal glands, High -10 to -15

cartilage, testis, degrees Celsius
uterus

Bone marrow, Intermediate -15 to -25

brain, bladder, degrees Celsius
cervix, heart,
gut, kidney,
liver, lymph
node, muscle,
pancreas,
prostate,
spleen, thyroid

Fatty skin Low -25 to -35

degrees Celsius

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