LEVEL IV

1ST SEM
HISTOPATHOLOGY AY: 2022-2023

Histopathology Title

1. Diagnose stage of cancer

OUTLINE 2. Diagnose pre-cancer stage
I Introduction 3. Other inflammatory diseases
A. Terminologies in histopathology
B. Purpose of histopathology
C. Flowchart
D. Sample Collection and Preservation
E. Sample Transport and Accessioning
F. Grossing
G. Tissue Processing Knowledge of about histopathology starts in the operation
H. Automated Tissue Processing room where the samples are collected which include accurate
I. Embedding, Microtomy, Deparaffinization & Staining patient identification, orientation of samples and adequate
II. Papanicolaou Stain
fixation.
III. Principles and Procedures
IV. Frozen Section (Cryostat) Receiving of specimen, includes specimen acceptability and
completeness of request. Next, is entering specimen details and
INTRODUCTION charging of requested procedure placed by medical technologist/
● Histopathology laboratory clerk/ histotechnician.
o branch of biology which deals with study of diseased
tissue under microscope. Accurate processing of specimen, rest in the hence of MT staff
▪ Greek word. Histo means tissue; Patho means or histotechnician who is entrusted in this process. This includes
disease; logy means study. appropriate proper embedding technique, microtomy, staining
and avoiding unacceptable artifacts.
TERMINOLOGIES IN HISTOPATHOLOGY Finally, inspections of controls to determine correctness of
 Biopsy stains and reporting of slides are all in the hands of expert
o removing of bit of tissue from living being. pathologist.

 Autopsy
o removing of large tissue/ organ from dead body. SAMPLE COLLECTION AND PRESERVATION

 Autolysis Tissues are collected and fixed immediately in a 10% neutral

o Destruction of tissue due to its own enzyme. buffered formalin.

Formalin causes:
 Putrefaction
o Destruction of tissue due to environmental bacteria
 Chemical and physical changes
and microorganisms.  Hardens and preserve the tissue
 Protect from degeneration,
PURPOSE OF HISTOPATHOLOGY autolysis and putrefaction

Pathologist understand between the normal and abnormal cell Ideally, 3 times the volume of fixative in ratio the size of the
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 o Consistency
Large radical specimen resection should not be sliced/cut by the
surgeon – should not be sliced or open by the surgeon but sent
it directly to the histopathology department.
 Loafing
o Cutting the specimen into 3 or more parts.
o Fixative can easily penetrate the tissue
o Applies to large radical specimen:
GROSSING
Precise and systematic gross description
o Color
o Size
o Shape of specimen
 Dissection and selection of sections for microscopic study
o Crucial parts of the pathologic examinations
o Done by pathologist, assisted by medical technologist or
histotechnician
 Cartilaginous, bony and hard tissues are placed in the
decalcifying solution for 1-7 days.
 Number of bits received are noted.
o especially in small biopsies e.g., punch biopsies, core
needle biopsies, gastic tissues
 Representative samples of the tissues are placed in the tissue
cassettes using a scalpel and fine pointed forceps.
o Each tissue cassettes are labeled with accession number
 Inking of samples is integrated into the grossing system to
evaluate the margin of resection on microscopy.
 After grossing, all instrument is rinsed with water and wiped
clean
o To prevent any mixed-up floater of tissues

During grossing:
 Perform in well ventilated, well-lit grossing stations.
 Exhaust and proper light are switched on
 Proper gloves, mask and apron should be worn

(Video):
APPENDECTOMY (APPENDIX)
SAMPLE TRANSPORT & ACCESIONING  Color: grayish-brown
 Transported at room temperature  Shape: tubular
o Complete patients name  Consistency: soft to rubbery
o Date and time collected
o Type/s of specimen
 Measurement: L x W x H (4.0 cm x 1.0 cm x 0.5 cm)

 Well-sealed, leak-proof container
 Must NOT be place into any other solution or dry container
EXCEPT for frozen section
o as irreversible deterioration will take place
o making accurate microscopic interpretation IMPOSSIBLE.
 One of the biggest challenges histopathology facing:
o accurately label and track specimens to avoid
misidentification errors
 Patient and specimen identification are critical elements
o Proper identification of specimen by instructing
technicians to use at least two patient identifiers when
receiving samples
 Specimen identification is maintained across steps like:
1. Cutting of specimen on both ends. NOTE: cut specimen at no
more than the thickness of 5mm.
2. One tangential cut (combined longitudinal and cross cut)
3. Get the tissue cassette and place the tissue in the cassette
(=block 3)
4. Cover the tissue cassette
5. Label with corresponding accession number
TISSUE PROCESSING
 Hardens the tissue, enough to enable cutting into 3-5 microns
sections which can then be stained and examined under a
microscope

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 Once the tissue is properly fixed, it goes through a process  1st station: 10% neutral buffered formalin
which evolves the following steps:  2nd station: 95% alcohol
o Dehydration  3rd station: 95% alcohol
o Clearing  4th station: 95% alcohol
o Infiltration/ impregnation  5th station: 100% alcohol
 6th station: 100% alcohol
 Water should be removed from the tissue
 7th station: 100% alcohol
 Progressively replaced by wax, to make a tissue block suitable  8th station: xylene
for sectioning  9th station: xylene
 We use a mechanism to forced drive out the water called  10th station: xylene
dehydration.  11th station: melted paraffin wax
 Dehydration  12th station: melted paraffin wax
o Removal of water from aqueous-fixed tissue.
o Progressively immersed with ascending concentration of o Reduces training time – intuitive function means it’s easy
alcohol to learn to how to use
o Concentration used: o Variable workloads – 100 or 200 cassettes processing at
a time
o High reliability – proven “workhorse” track record
o Mechanical operation – possible mechanical operation
during power outages keeps specimens from drying out.
 Water is driven out. (Alcohol and wax = immiscible (can
not be mixed)) EMBEDDING, MICROTOMY, DEPARAFFINIZATION &
o We need a reagent that can mix both alcohol and STAINING
wax. It is called as Organic solvent xylene  Materials needed:
o Which will act as bridge for alcohol and wax o Paraffin wax
o Gloves
 Immerse the tissue in xylene to drive out the alcohol. This
step is called Clearing. o Metal molds
o Forceps
 Final destination is the wax. We now chased out the xylene
o Tissue cassettes
and immersed the tissue in molten wax at 60°C, this called
Impregnation/infiltration.
EMBEDDING
 Impregnation/infiltration
1. One cassette at a time to maintain specimen integrity.
o Final xylene is replaced with molten wax which
2. Choose the appropriate metal mold – keeping aware of the
infiltrates the tissue
size restriction of the tissue.
3. Orient the tissue correctly – being certain all the tissue in one
Tissue processing is routinely done on an instrument called
plane parallel to the bottom of the mold.
Automated Tissue Processor.
Ex: 1. Tissue to be on edge
2. on end
3. epithelium facing same direction
4. Place your cassette top over the mold and fill with paraffin.
5. Oven for 1 hour at 70°C.
6. Set on to the cold plate neatly and in order for 30 minutes. –
immediate and rapid solidification of the paraffin block is desired. In
order to facilitate sectioning and to prevent the tendency of having
large crystals in the wax.
(Video): 7. Remove the block from the mold.
AUTOMATED TISSUE PROCESSING 8. Clean the block of excess paraffin – without compromising the
accession number on the block and place in a cold ice bath.
 Leica TP1020 tissue processor
o Is a carousel type semi-enclosed tissue processor with 12
MICROTOMY
stations.
o Can be applied to any of the stations both in manual and  Microtome
automatic operation. o Instrument used to cut section from tissue embedded in
o Advantage: substantially improved infiltration of tissue paraffin wax.
in a shorter time. Instruments with the vacuum feature
are equipped with anodized aluminum containers. 1. Use the lever to secure the tissue block.

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2. The hand wheel is use to move the block downward across MOUNTING
the blade. 1. Apply protective cover slip. (This provides permanent
3. Safety knob must be turned downward and handle pulled preservation of each stained tissue sections)
outward. (Make sure to push the handle back in each time 2. Send the slide to the pathologist for diagnostic examination.
you finish using the microtome.
4. The blade should be replaced or adjusted before you cut. Use
new tissue block and make sure to be careful when handling PAPANICOLAOU STAIN
the razor blades.
 Main use of paps stain:
5. When it needs to be cleaned, use only a brush on an upward
o Use to stain paps smear
motion.
o Paps smear is used for cervical cancer screening in
 The knife holder can be adjusted in three planes:
gynecology
o Front Knob – used to move forward and o Most important stain (routine stain) in the practice of
backward. cytopathology.
o Mabhab (left) – used for lateral adjustments. o Use to stain alcohol fixed cytology slides.
o Knife angle knob – changes the angle of knife
 Stains in Papanicolaou stain (pap stain)
holder
o Polychromatic pap stain 5 dyes in three solutions:
6. Cutting works best when the knife holder is set to a fairly
 One nuclear stain (Haematoxylin)
steep angle.
 Two cytoplasmic counter stain (OG- 6 & EA- 50)
7. Remove the outer layer of wax in the block. (TRIMMING- this
step prevents the tissue from shattering when you begin
cutting the sections). NUCLEAR STAIN (HAEMATOXYLIN)
8. Adjust the knife holder as close as possible to the tissue  Stains cell nuclei in BLUE.
block.  Both progressive and regressive
9. Set the thickness indicator to 5 microns.
 Harris Haemaoxylin- commonly used (regressive technique)
10. Once the tissue is exposed, remove and place it on ice bath.
11. Begin to cut and make a ribbon of 6 or soul sections?
12. Place the ribbon on a warm water bath at 45°C.
TWO CYTOPLASMIC STAIN (OG-6 & EA-50)
13. Two of the ribbon strings will go to the slide. 1. Orange G-6
14. Scoop them up using a frosted slide. - First acidic counter stain
15. Label with corresponding accession number using a pencil - Monochrome stain
and place in the staining rack - Stains the protein keratin
(a normal component of the squamous epithelial in ORANGE)
DEPARAFFINIZATION
2. Eosin Azure – 50
o Removing of excess paraffin wax
- Second counter stain
- Different formulation with a number, according to the
1. Incubate the slide for 30 minutes at 70°C.
proportion of the constituent dye
2. Place in two level of xylene (5 minutes each level)
- Commonly use
3. Five times dips of 95% ethyl alcohol.
- Polychromatic stain (mixture of 3 stains)
4. Ten dips in running water.

EOSIN Y
1. MOUNTING
2. Hematoxylin stain for 20 minutes. (Hematoxylin stains cell’s
nucleus)  Stains the cytoplasmic of cells in shades of PINK or GREEN,
3. Ten times dip in running water. depending on the pH.
4. One dip in 1% acid alcohol (this removes excess stain and
define nuclei) PRINCIPLES AND PROCEDURES
5. Ten times dip in running water. 1. Fixation
6. Two dips in ammonia water. (Ammonia serves as bluing agent) - Fixed with 100% alcohol, 95% ethyl alcohol or spray
7. Five dips in running water. alcohol fixative.
8. Ten dips in eosin Y. (Eosin stains the extracellular matrix and - Stand for 20 minutes before staining (this will help
cytoplasm in pink) hydrates the tissue and prepares sample for the uptake
9. Ten times dip in 95% ethyl alcohol. of nuclear dye).
10. Another, ten times dip in 95% ethyl alcohol. (These 2 levels of 2. Nuclear Staining (Haematoxylin)
alcohol aids in rinsing the slide). - Using Harris Haematoxylin for 5 minutes.
11. Two levels of xylene for 2 minutes each level. - Nucleus is deliberately over stained.
12. Blow dry the slides. 3. Differentiation (1% acid alcohol)
- Take of additional haematoxylin stain

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- (Timing in the acid bath is essential for the final  Specimen disc-
appearance of the nucleus). As often results of magnetic stainless-
hypochromasia steel discs for
- If decolorization is inadequate, contrast between mounting.
counter stain and nuclear stain will be lessened.
- Decolorization acid is removed, by rinsing in running
water for 10 times.
4. Bluing (ammonia)
- Two dips in ammonia water
- Serves as bluing agent
5. Dehydration before cytoplasmic staining
- Use of ascending grades of alcohol
- Prepares the cell sample for uptake of the counter stain
6. Cytoplasmic staining
o Orange G-6: 5 dips
- Stains keratinized cell
o Eosin Azure: 30 secs
- Stains rest of the cell
7. Clearing
- Alcohol replaces with xylene
- Refractive index similar to glass slides, cover slip and
mounting medium
8. Blow Dry & Cover Slipping (Mounting)
- Helps to protect from dust
- Preserve the slide

FROZEN SECTION (CRYOSTAT)

 Things should be aware of:
1. Always wear gloves to avoid frostbite.
o Frostbite- injury caused by freezing of the skin and
underlying tissues.
2. Tissue freezing medium- advanced formulation that
designed to support tissue during cryotomy.
o Place around the tissue or in the mold when you’re
preparing to go on the block to be cut.
3. Pick them up with microscope slides.
o Use in room temperature for the frozen sample to
adhere well.
4. Consult your manual for the temperature suitable for the
specimen.

 Thickness indicator- can set to very high or vey low depending

on the specimen to be cut out.
 PE button – extra freeze samples.
 Temperature can be varied, depending on the tissue being cut,
usually from -20°C to -30°C.
 Clock – length of time which cooling may be maintained.
 Light button- to image clearly the samples

(Inside the machine)

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 Mounting area- to 6. Pick up with microscope slide

secure the tissue 7. Drop it off in 95% ethyl alcohol to fix the sample
block
STAINING
1. Immersed with Haematoxylin stain for 1-2 mins.
 Stage- shaving off the 2. Wash well in distilled water for 10 dips.
block 3. One dip in ammonia water
4. Rinse in distilled water for 10 dips.
5. Counterstain in eosin Y for 1-2 dips.
6. Dehydrate in 2 levels of 95% ethyl alcohol
 Anti-roll cover- to 7. Blow dry the slides to remove excess alcohol.
flattened the sliced 8. Apply a protective cover slip.
tissue 9. Slide is now ready for microscopic examination.

 Blade level- to lock

and unlock the blade. REFERENCES
Emilio Aguinaldo College powerpoint presentation: Lesson 01 –
Histopathology Section
 Stage lock-
manipulate the stage
sideways

 Stage locl (bottom) –

forward and
backward

 One arrow button-

moves the mounting
area microscopically
 Two arrow button-
moves the mounting
area at a greater
distance
 Hand wheel – moves
the mounting area on
upward ad downward
movements.

SAMPLE PREPARATION
1. Apply the media direct to the specimen.
2. Specimen should be on the center (parallel to the bottom of
the disc)
3. Use the heat extraction bar to immediately freeze the tissue
sample.
4. Ideal for large tissue like breast mass and thyroid organ

SECTIONING
1. Put the specimen disc in the mounting area (Secure using the
upper left hand corner button)
2. Figure out how far away is the tissue to the blade. (Use the
second panel to move the tissue sample).
3. May or may not initially see the tissue slices.
4. You can always manipulate the thickness of the slice.
5. Using the brush technique.

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