HS2413B_HISTOLOGICAL LAB REPORT GROUP 2

Laboratory Report
Histological Techniques

MLT428
OCT2021-FEB2022

ROUTINE
Practice

in HISTOPATHOLOGY LABORATORY

name student id

Muhamad Zul Azmeer Bin Mohd Nasir 2020473736
Muhammad Syaddad Bin Zainuar 2020846902
Farah Adriana Binti Mokhtar 2020473348
Hajar Hanani Binti Hishamudin 2020492628
Sabrina Valeriana Senai Binti Macdonald 2020828002

group

hs241 3b

LECTURERS

Madam Hartini Yusof
Dr. Nur Ayunie Binti Zulkepli

DATE

5 january 2022

INTRODUCTION

HISTOPATHOLOGY Histopathology is a study related to microscopic examination of a
LABORATORY biopsy or surgical specimen that has been processed and fixed onto

glass slides in order to analyze the signs of disease.

Using pathobiology understanding to evaluate and interpret the
shapes, sizes, and results of the cells and tissues architecture patterns

within a particular clinical context to get an accurate diagnosis.

Histological techniques are a Histological Techniques 1) Formalin-Fixed Paraffin-
series of crucial and important Embedded
Techniques that should be Once the sections are prepared, they
procedures to produce mastered: are usually stained to help distinguish
stained tissue samples on the components of the tissue.
glass slides for microscopic Tissue grossing & fixation 2) Frozen Section
Tissue processing Tissues are frozen rapidly in liquid
examination. Tissue embedding nitrogen, and then cut in a cryostat
Tissue sectioning with a cold knife, then stained and
Tissue staining observed in the microscope.
Tissue mounting

Equipment in Histopathology Laboratory

Tissue Rotating Arm
transfer head
basket Block
attachment Adjustments holder

slot Wax Stage Blade
Reagent container holder
containers Base

Keypad
control

Wax reservoir Mold bin
Wax dispenser Forceps warmer

Cold plate Hot plate Tissue
warmer

ROUTINE WORKFLOW

Specimen fixed in 10% formalin tissue grossing tissue processing

Fixation status of a Specimen is trimmed into 2-3mm Total tissue processing time
specimen is checked. sizes and kept in a cassette. takes about 21 hours.

floating out tissue section tissue sectioning tissue embedding

Tissue section is floated out Tissue block is trimmed first, frozen and Tissue embedded in a block
in water bath at 43°C-45°C. sectioned into thin ribbon of tissues. of paraffin wax.

fishing out tissue slide drying dewaxing using hot plate at 60°C

The best tissue section is selected Slides were allowed to Slides were dewaxed for
and separated by using an air-dried on the slide rack. approximately 4 minutes when
applicator stick.
drying slide using a hot plate.
tissue mounting and labelling
tissue staining

Stained-tissue slides were Stained-slides were allowed Using hematoxylin and eosin
mounted and labelled completely. to dry before mounting. (H&E) stain.

TISSUE PREPARATION

pPrRinIcNipCleIPLE

Tissue samples are immersed in an adequate volume of fixative solution (10% neutral buffered formalin) to preserve them
from decaying or damage. The formalin has a property of forming cross links between proteins, thus stabilizing the tissue
structures. The neutral pH inhibits formation of formalin pigment. 1:10 to 1:20 volume ratio of tissue to fixative is necessary
for optimal fixation.
Grossing includes general inspection of tissue specimen and identification of normal and abnormal components. For
example, specimen from category C such as appendix requires simple dissection with sampling needing a low level of
diagnostic assessment/ preparation.

materials procedure

Human tissue specimens (liver, By using a pencil, cassettes were labelled clearly with the
tonsil, appendix, uterus) student's name/ ID number, type of specimen and date.
10% neutral buffered formalin
(commercially prepared)


equipment Fixation status of the specimen was checked. Margin and
orientation of the specimen were also identified.



Ducted fume Relevant portions of the tissue specimen were selected
hood. before grossing.
Brand: IRYAS


By using a scalpel, specimen grossing was performed one

at a time.

apparatus result

Small tissue specimen (2-3 mm thick) was placed

Scalpel holder Trimmed securely inside a plastic cassette.
Scalpel blade tissue

Forceps
Dissecting board Tissue Tissue cassette was then transferred into a container
Tissue cassette specimen containing 10% Neutral Buffered Formalin (NBF).
Containers in cassette

MediSheet
Kimwipes All the remaining specimens were transferred back into
Gloves the specimen container for future references.
Masks
Tissue grossing and fixation were performed in

the fume hood (grossing station) with appropriate

problem and solution PPE.
Small tissue samples are usually fixed at room

Uneven specimen temperature.

Choose a proper orientation of the
Time of fixation was at least 6 hours but not more
than 24-72 hours. This includes time in a processor

specimen before cutting.
and documentation time.
Improve technique of handling the
scalpel.
Larger specimens may require longer time as
Ensure that the thickness of a specimen formalin slowly penetrates the tissues.
is no more than 3mm.


Overloading cassette
conclusion


Only place required volume of tissue in Correct technique of tissue preparation (labelling cassette, grossing,
a cassette.
fixation) is very important to ensure good performance of subsequent

Second cassette is used if the volume
histology procedures such as embedding and sectioning.
of tissue is too great.

TISSUE PROCESSING

PRINCIPLE EQUIPMENT

Tissue processing is designed to remove all extractable water 3 Stages:
from the tissue, replacing it with a support medium that -Dehydration
provides sufficient rigidity to enable sectioning of the tissue -Clearing
without parenchymal damage or distortion. -Infiltration

APPARATUS MATERIALS Automated Tissue Processor
Brand: SHANDON CITADEL 1000
Forceps Tonsil, appendix and
Tissue processor organiser baskets uterus tissue specimen Model: SHA 69800001
Tissue processor basket lid fixed in 10% formalin.
Tissue processor reagent containers CHEMICAL/REAGENT
Measuring cylinder (1L & 2L)
Beaker (1L & 2L)
Gloves
Aprons
Masks
Goggles

PROCEDURE

Tissue blocks in cassettes that were fixed in 10% buffered formalin
were transferred and stacked onto tissue processor organiser baskets

by using forceps.

Tissue processor baskets filled with cassettes then were inserted into RESULT
the basket hanger with tissue processor basket lid positioned on top
Tissue block in
of it. cassette after
The tissue processor operating head door were open for the complete cycle of
attachment of the basket hangers by securing it into the operating processing.

head slot. PROBLEM
The processing time was confirmed as programmed as on hand-held
controller display & “AUTO START” key were pressed to commenced After a few cycles of tissue processing, the
reagent in the container starts to decrease in
the tissue processing cycle. volume due to evaporation & turns murky as
the tissue block is being socked in the
Dehydration Clearing Infiltration reagents.

Step 2-7 Step 8-10 Step 11 & 12 SOLUTION/TROUBLESHOOTING
Water & aqueous Dehydrating The clearing agent
fixatives are extracted compounds are is entirely removed The chemical reagents were replaced after
from fixed tissues in removed from from the tissue and the fourth cycle with freshly prepared
this procedure. the tissue and substituted with a reagents to ensure the next cycle of
To avoid distortion to replaced with a medium (wax) that specimen blocks were fully covered and
fragile tissue, wax-soluble completely fills all optimized the tissue processing process.
specimens are treated solution. of the tissue voids.
using a graded system The tissue then It provides a
of reagents with have translucent matrix and avoids
increasing appearance. tissue structural
concentrations. damage during
microtomy.

CONCLUSION

Although tissue processing is a very time consuming procedure, it is a very crucial step to emphasize as it
would determine the quality of tissue block specimens whether it is good enough to be carried for the
next steps (embedding, sectioning & staining) without any further problems.

TISSUE EMBEDDING

EQUIPMENT MATERIALS CHEMICAL/REAGENT

Paraffin Wax Tissue Embedding Centre Processed tissue blocks Paraffin wax
brand : Slee Mainz MPS/C/MPS/P/MPS/W (liver, tonsil, appendix, uterus) Brand: SAKURA Tissue-Tek

Paraffin Wax

principle

APPARATUS Embedding is a process which will hold
the processed tissue in place by
Forceps Steel block Plastic cassette Gloves embedding the tissue into a block of
Scraper mould Tissue paper Masks paraffin wax. The tissue specimen was
enclosed in an embedding medium
Processed tissue procedure Tissue sample in using a mould. The embedding medium
cassette was placed the cassette was that being used is paraffin wax. The
Cryo console being viewed first medium will be able to support the
in the and the light embedded tissue blocks during
cassette bath were turned on. The best mould microtomy in order to get a thin ribbon
that corresponds of tissue sections.
Processed tissue then A small amount of to the tissue’s size
transferred from the molten paraffin results
was poured into was chosen
cassette into the the mould Paraffin-
mould by using warm embedded

forceps tissue
blocks
The mould was The tissue was pressed More molten wax
transferred to gently and firmed into the was poured into problems and solution/troubleshooting
the cold plate. wax using a pair of warm the mould until
forceps until the paraffin Wrong orientation of the processed tissue
almost full.
solidified in a thin layer Check for the flat surface of the tissue
beforehand proceed transferred into
The mould was Molten wax was Labelled cassette and the paraffin wax-mould
immediately being poured into the paper were placed on
cooled down by mould until it fully unstable cassette
placing it on the cold covered the face top of the mould.
of the cassette Re-embedded the
plate tissue in the
paraffin wax by
The paraffin tissue block was separated from dewax the
its mould after the paraffin wax solidified previous wax on
the hot surface of
Excess wax from paraffin tissue block cassette was embedding centre
removed by using scalpel.
Air bubbles are entrapped around
conclusion the tissue

Embedding step need to be done very carefully as the blocks' result can Embedded the tissue properly in the
also affecting tissue sectioning process. So, during embedding the molten wax by slowly pouring the
tissue specimen, sturdy hands needed and filled up the molten paraffin molten wax into the mould.
wax slowly to prevent from creating any bubbles, plus placing the
cassette correctly.

TISSUE SECTIONING
prpirnincicpipllee equipment

Rotary microtomes play an important role as they are capable of cutting sections Rotary microtome Slee Mainz CUT 5062
from paraffin blocks as thin as 1μm. As the block goes through the blade, a

segment with such a thickness will be created. When the sections are placed in a

water bath, the wax expands due to surface tension and heat, which aids in the

removal of creases and folds. Sharp and blemish-free blades are required for

satisfactory cutting. A good blade may section poorly prepared paraffin blocks,

but a bad blade can fail to cut even the best material.


apparatus material Floatation water bath XH-1001

Slide racks Paraffin block Hot plate or drying oven HPS-7C
Frosted slides (tonsil, appendix and uterus) Freezer
Brush

Gauze procedure
Pasteur pipettes

Applicator sticks 1.Setting up microtome 2. Trimming of tissue block
Pencil Blade angle was adjusted to Paraffin block was placed vertically or horizontally onto the cassette
Forceps 90 degree. Microtome bladder holder. Pressed forward the paraffin block until it touches the edge of the
Thermometer was placed which is a different knife.
site for trimming and Section thickness was set for 10-20 microns for large tissue pieces; 5-10
result sectioning. microns for small tissue pieces.
Blade fixations was tightened Stopped trimming once complete tissue sections appeared on the
by the right lever clockwise. section ribbon. Trimmed block was replaced in the freezer at -20 degree
and moved to an unused area bladder or placed a new bladder

Ribbon section

Floating out section 4. Floating out sections 3. Cutting sections
Ribbon sections was gently pressed Took out the paraffin block from the freezer. Placed paraffin
by using an applicator stick to block onto the cassette holder.
prevent wrinkles or bubbles. The Changed the “Mode” and thickness of 5-10 microns was
best section from the ribbon was selected.
selected and separated it by using A series of paraffin sections was cut until produce a ribbon of
an applicator stick. serial sections. By using a brush, ribbon section from the knife
Make sure not to leave sections of edge was separated. Ribbon sections was transferred into
ribbon too long to prevent tissues flotation water bath that filled with distilled water (42-45)
expanding and distorted.

5. Picking up sections 6. Drying sections
Sections was collected using a clean slide by using The slides were allowed to dry on the
fishing techniques. The slide was immersed into the slides rack at room temperature. Any
water bath to get the best result. water was removed from the slides
The slide was held vertically beneath the section and before proceeding to the drying oven.
lifted up gently to make sure tissue adherence to the
slide

Tissuperisneccitpiolneingcios naclturuseioanrt that requires a problem & solution/troubleshoot

great deal of talent and experience. Good Ribbon will not form
sectioning will give the best result in visualizing
tissue under the microscope. However, it is Paraffin block too hard; blade might be dirty
important to have properly fixed and embedded
blocks or artefacts including tearing, ripping, Rewarm paraffin block; replacperinwcitihplcelean
holes or folding will appear. blade

Large holes formed in sections
Tissue is not embedded flat

Depending on issue/problems, Re-embed

tissue as required

TISSUE STAINING

PRINCIPLE

In general, tissue staining is used to emphasize the tissue components and improve tissue contrast. In histological studies,
the H&E staining is used to distinguish cell components, as H&E dyes contrasting colour for cell’s nucleus and cytoplasm
components.

Hematoxylin is a basic dye, which stains the cell’s nucleus, gives it bluish colour.
Eosin is an acidic dye, which stains the cytoplasm components of cell, gives it a pink-reddish colour.

CHEMICALS/REAGENTS APPARATUS EQUIPMENTS

Hematoxylin 3G (Sakura) - Commercially Slide racks Oven
prepared Reagent containers Brand: Memmert
Eosin (Sakura) - Commercially prepared Forceps Hotplate & Stirrer
Xylene Filter paper
Alcohol (Absolute, 95%) Tissue paper Brand: Pro
Distilled water Funnel Model: HP-7 Lab
Tap water Measuring cylinder
Tripod stand Plus Series
MATERIALS

Unstained tissue slides

PROCEDURE

Staining Protocol

The tissue sections were Deparaffinization Clearing
dewaxed using the oven for 30 (Step 1-3) (Step 19-20)
minutes or hotplate & stirrer for Completely removed
4 minutes each slides at 60℃. Totally removed the the alcohol from tissue
paraffin from the tissue
After the dewaxing section
protocol was completed, section
the slides were arranged
Hydration Dehydration
in the slide rack. (Step 4-8) (Step 16-18)
The staining procedures began Removed the xylene. Removed water from
Prepared the tissue tissue section
by following the staining section for aqueous Control eosin intensity
protocol reagents exposure. (Step 13-15)
Removed excess stain
After the staining protocol was (Step 9) (Step 12)
completed, the slides were left Haematoxylin stained Eosin stained cytoplasmic
on the slide holder to be air- nucleus of the cell components of the cell

dried. Bluing reagent (Step 11)
(Step 10) Removed
excess stain
Changed reddish -
purple haematoxylin to

blue colour.

RESULTS PROBLEM CONCLUSION

Stained Irregular staining of the tissue section Tissue staining is one of the vital
tissue due to the incompletely paraffin removal procedure in histology laboratory
slides that need to be emphasized as it
Stained by xylene is used to differentiate the
tissue under features in cell. Therefore, it is a
microscope SOLUTION/TROUBLESHOOTING must to follow in detail, every step
in the staining protocol to prevent
Ensure to follow the time for xylene, OR any further problem that can
Replace the xylene with the new one, as the affect the tissue section.
previous one may have been contaminated

with the unfixed tissue section.

TISSUE MOUNTING & LABELLING

PRINCIPLE

Tissue sections that need to be examined at any length of time or to be stored for a specific duration of time
must be mounted using appropriate mounting media. Mounting media are used to protect and preserve tissue
sections and to enhance the microscopic evaluation of the tissue. A mounting medium with an refractive index
close to that of the fixed tissue will provide a transparency, with only the stained tissue elements visible. The
mounting medium used in the laboratory will harden to hold the coverslip firmly in place.

MATERIALS CHEMICAL/REAGENT EQUIPMENT

H&E stained slides Mounting medium Ductless fume hood
Product's name: CoverSeal-X Brand: ESCO

APPARATUS Model: Ascent Max

Slide holders Image retrieved from
Coverslips (22 x 40 mm / 24 x 60 mm) Youtube MedLab Channel,
Applicator sticks
MediSheets Mounting & Labelling
Self-adhesive label stickers

PROCEDURE result

A self-adhesive sticker was labelled with the following Mounted H&E
details: Student’s name, Student’s ID number, Type of stained slide
with a label
specimen, Type of stain and Date.

PROBLEM & SOLUTON

A coverslip was chosen appropriately according to the Incomplete information on the
size of the tissue section. sticker label.


Prepare a complete label before mounting a
A mounting medium was placed adequately on one slide. Ensure that important information such
edge of the coverslip. as name, type of specimen, date and type of

stain used are written clearly on the label.

The slide was gently lowered until it touched the Excess mounting medium
mounting medium. Capillary attraction will cause the flowing out of the slide.
mounting medium to flow upwards, carrying the coverslip
Let the mounted slide dry and
along with it. then use a scalpel to remove

the excess mounting medium.

The slide was examined macroscopically to make sure CONCLUSION
there were no air bubbles.

Mounting should be done properly with
less error concerning air bubbles.
If air bubbles were present, the coverslip was pressed Complete labelling is a must to provide
gently using an applicator stick to remove the air bubbles. reliable result when evaluating the
tissue section.


The written sticker label was pasted at the frosted

end of the slide.



Finally, the slide was placed on the slide holder to be air-
dried.

FROZEN SECTION

principle CHEMICAL/REAGENT

The tissue specimen is rapidly frozen, converting water to ice, which serves Tissue Freezing Medium /
as an embedding medium and allows the tissue to be sectioned (cryotomy). Frozen Section Compound
If the temperature of the tissue sample is decreased, the tissue gets stiffer, 10% Neutral Buffered
Formalin
whereas increasing temperature softens the tissue. Hematoxylin 3G (Sakura)
Eosin (Sakura)
EQUIPMENT APPARATUS Alcohol (95% & Absolute)
Xylene
Cryostat (brand: Slee Mainz MEV) Glass slide Tap water
Oven (brand: Memmert) Coverslip Distilled water
Microtome
Blade MATERIALS
Forceps
Brush Fresh tissue
Applicator stick
Coplin jar
Gloves

procedure results

Two slides were labelled with the surgical pathology "FS" and the medical
record number.

A cryo cassette was constructed. A tiny amount of O.C.T. cryomatrix should Ribbon sectioned Stained tissue slide
be added. The cryocassette was placed on the cryobar and the cryospray was
used on it to freeze. Image retrieved from:
https://www.pathologyinnovations.com/frozen-section-
The block was trimmed carefully at 15 micron intervals until the entire surface of
the tissue is revealed. The knife was wiped gently by using a brush. Then, a technique
portion was cut about 4-5 microns thick..
problems and
The section was extracted from the knife by using a coated glass slide at solutions/troubleshooting
room temperature. Then, two (2) frozen section slides each block were
stained with H&E and immediately transferred to hematoxylin. Ice crystal artifacts

Stained in hematoxylin for 1 minute. Rinsed in tap water. Increase the freezing time of the
1 % Acid alcohol 1-2 dipped. Rinsed in tap water tissue, as it can reduce the size
Stained in 1% Alcoholic Eosin for 30 seconds. Rinsed in tap water of ice crystal artifacts. Therefore,
Dehydrated in 3 different Alcohol the tissue damage can be
Cleared in 3 changes of Xylene minimized.
Mounted with DPX mountant and coverslip.
Tissue tears or streaks
conclusion
Check the entire blade as it may
Frozen section is an alternative method of paraffin section in tissue due to the presence of frozen
diagnosis. It is very useful in dealing with STAT (Short Turn Around debris stuck on it, OR
Time) specimen as it consists of rapid procedures compared to the
paraffin section. However, it has a strict regulation likes a Replace the blade with the new
temperature sensitivity of the tissue sample. Therefore, it is needed one.
to follow each step of the procedures to reduce a possibility of
tissue damage.

REFERENCES

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REFERENCES

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Special Appreciation To
Dr Nur Ayunie Binti Zulkepli

Madam Hartini Yusof
Sir Mohd Nornizam Bin Ahmad Zaini

Miss Salina Binti Shafie
Sir Mohd Nazzihan Md Ajis