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MLT428 HISTOLOGICAL TECHNIQUES

HISTOLOGICAL
TECHNIQUES
LAB REPORT

PARAFFIN SECTION | FROZEN SECTION

Prepared by: (2020476542)
ELEANOR JOY JOLMIN (2020878128)
MUHAMMAD AMIRUL AIMAN BIN ZAINUDIN (2020483166)
NUR SOFIYA BINTI SUKANDAR (2020455886)
NURUL AIN NASTASHA FAZIERA BINTI PA’E (2020605096)
SITTI NUR AISAH BINTI BINTI JALIL

Lecturers:
MADAM HARTINI BINTI YUSOF

DR. NUR AYUNIE BINTI ZULKEPLI

1.0 HISTOPATHOLOGY
LABORATORY WORKFLOW

INTRODUCTION

Histopathology involves microscopic examination of tissue specimens. Since fresh tissue is delicate, all
samples received in the laboratory must be preserved or fixed with chemicals in order to maintain the tissue
structure. Paraffin sectioning is a routinely used method in histopathology in which the specimen will be
infiltrated with paraffin wax. Each of the samples will undergo detailed procedures that begin with tissue
fixation with formalin to maintain the tissue structure. The selected part of the specimen will be cut into a
proper size and immersed in several chemicals: formalin, alcohol, xylene, and paraffin at different
concentrations. Then, the tissue specimen will be embedded into a paraffin block. This prepared block will be
sectioned into thin slices and placed onto the microscopic slide. The slides will be dried to allow the excess
wax to melt away. Once the slide is ready, it will be stained using chosen stains such as hemotoxylin and eosin
(H&E) stain, mounted, and viewed under a microscope for clinical observation. The tissue examination will aid
in the clinical diagnosis.

FLOWC
HART

1. Tissue grossing 2. Tissue fixation 3. Tissue processing

6. Tissue 'fishing' 5. Tissue sectioning 4. Tissue embedding

7. Tissue staining 8. Tissue mounting 9. Tissue labelling

1.1 TISSUE PREPARATION

REQUIREMENTS PRINCIPLE
MATERIALS
Tissue grossing is the process in which specimen samples go through a
Scalpel blades process of cutting or grossing into smaller parts depending on the size and
Scalpel holders type of specimen. The specimen is either taken entirely or a ‘representative’
Forceps section that was placed into a small cassette.
Tissue cassettes Tissue fixation is a crucial process where the tissue and cellular composition
Dissecting boards of cells is preserved to allow the cells to withstand processing. Fixation of
Medisheet tissues also prevent breakdown of the tissues as well as the molecular
Gloves feature by the enzymatic activity for long-term storage.
Tissue specimen in 10% buffer
formalin R
E
EQUIPMENT S
U
Ducted fume hood L
Brand: IRYAS Model: ADe - 6BI T
S Tissue specimens in 10% formalin

TROUBLESHOOTING Tissue sample is cut too Tissue slices should be thin
Struggle to close the cassette thick. that is around 3-4 mm.
lid. Handle specimen with care
Rough dissection and and always use sharp blade.
Tissue trauma observed. dissection using blunt blade. Always starts off with a
Presence of debris or foreign Chopping board is dirty. properly cleaned chopping
matters. board.

PROCEDURE CONCLUSION
1.Personal protective equipment (PPE) was worn. In conclusion, skills in examining and
2.Cassette was labelled according to the type of specimen with student name, choosing the best orientation and
student ID, date and specimen type using pencil. part to cut for tissue processing was
3.The grossing workstation was prepared by turning on the grossing station fume successfully developed at the third
hood and the instruments are ensured ready to be used. cycle. Choosing which part to be cut
4.Specimen grossing was performed one at a time and placed the specimen into is important in order to be able to
the cassette. examine the desired tissue part for
5.The tissue cassette was placed into 10% Neutral Buffered Formalin. disease diagnosis.
6.Formalin waste was discarded into the formalin waste container.
.

1.2 TISSUE PROCESSING

REQUIREMENTS PRINCIPLE

MATERIALS Tissue processing involves physical and chemical methods. The interchange
Fixed tissue blocks (10% formalin) between the tissue’s internal fluid and its surrounding solution occurs during the
Forceps tissue immersion will extract the water, replacing it with the support medium.
Gloves Specimen will undergo fixation, dehydration, clearing, and infiltration during this
process by which the selected part of the tissue will be immersed in each chemical
REAGENTS at different concentrations and periods.
10% Neutral Buffered Formalin
Alcohol (Absolute, 95%, 80%, 70%, PROCEDURE
50%) A. Reagents preparation
Absolute Alcohol and Xylene Mixture
(50:50) 1.2L of 50% alcohol was prepared by adding 1000ml of absolute alcohol to 1000ml of distilled
Paraffin Wax water.
Distilled Water
Xylene 2.2L of 70% alcohol was prepared by adding 1400ml of absolute alcohol to 600ml of distilled water.
3.2L of 80% alcohol was prepared by adding 1600ml of absolute alcohol to 400ml of distilled water.
EQUIPMENT 4.2L of 95% alcohol was prepared by adding 1900ml of absolute alcohol to 100ml of distilled water.
Brand: Shandon 5.2L of alcohol and xylene mixture was prepared by adding 1000ml of absolute alcohol to 1000ml of

Citadel xylene.

6.All the other reagents were also prepared and put into its respective place in the tissue processor.
B. Tissue processing
Model: SHA 1.The cassettes containing the grossed samples were put into the cassettes basket.
69800001 2.The basket was placed in the automated tissue processor and the time was set according to its

appointed time like shown in Table 1.

Table 1: Program for routine overnight tissue processing

R
E
S
U
L
T
S Tissue specimens at

the end of the cycle

Derived from laboratory manual for MLT428 Histological Techniques

TROUBLESHOOTING Excessive dehydration Soak the block with a wet gauze
Tissue section become cracked and Dehydration inadequate and there is before sectioning
folded defective paraffin infiltration in tissue Change the processing program and
During sectioning, tissue is coming Improper dehydration give adequate time for dehydration
out from the block Excessive dehydration Fluid are changed according to the
Poor processing schedule
Change the dehydration time. It is
The edges the tissue section better to process the smaller biopsy
affected by microchattering tissue separately from larger tissue
to have proper dehydration.

CONCLUSION
Tissue processing is important to make sure that water is adequately removed and replaced with support medium to harden the
tissue. During this process, skills to prepare the reagents were developed together with the precautions while handling the
reagents. Other than that, the skill to operate the tissue processor had also developed. The skill of reagent preparation and reagents
loading order and operating the timing in tissue processor are important to ensure that the tissue undergoes proper dehydration
and consequently absorbs right support medium.

1.3 TISSUE EMBEDDING

REQUIREMENTS

MATERIALS: PRINCIPLE
Processed tissue blocks
The process of orienting tissue in the
APPARATUS correct direction and surround it in a
Forceps molten liquid such as wax by using a
Scraper mould. Allow it to solidify and generate a
Steel block mould block for cutting. The aims are to provide
Gauze sufficient external support of the tissue,
Tissue paper avoid tissue distortion during the cutting
Gloves process and preserve the tissue for
Aprons archival purposes.
Masks
Paraffin wax tissue embedding centre
Brand: Slee Mainz
REAGENT Model: MPS/C MPS/P MPS/W
Paraffin wax

EQUIPMENT PROCEDURE
Paraffin wax tissue embedding centre

R 1.The temperature of the paraffin tank is examined to ensure that it is
E higher than the melting point of paraffin wax; within 54oC-60oC.
S
U 2.The light and cryo console is switched on.
L 3.The tissue cassettes are placed into the cassette bath.
T 4.The cassette is opened to view the tissue sample.
S 5.Mould chosen must be suitable for the tissue size.
6.A small amount of molten paraffin is poured into the mould.
Processed tissue blocks 7.By using warm forceps, the tissue sample is transferred from the

TROUBLESHOOTING cassette into the mould. The tissue surface that needs to be
observed must be facing downwards in a correct orientation.
Cracks on Too much Place the wax mould 8.The mould is transferred onto the cold plate.
the tissue pressure when on the hot plate when 9.The tissue is gently pressed and held until the thin layer of paraffin
blocks are placing the placing the tissue solidifies.
seen. tissue in the into the wax. 10.The labeled cassette is placed on top of the mould and the mould is
wax when it filled with molten paraffin wax until it fully covers the cassette.
already started 11.The forceps are wiped to clean the excess wax on it.
to solidify. 12.A small piece of labeled paper is placed on top of the cassette.
13.The mould is immediately cooled down by placing it on cryo console.
14.After the wax solidifies, the paraffin tissue block is removed from its
mould.
15.Excess wax is removed from the paraffin tissue block by using
scalpel.
16.The light and cryo console is switched off when all the tissues are
embedded.

Only a The embedded Add just a little CONCLUSION
fraction of tissue being in pressure when Embedding tissue is important in preventing the tissue distortion during cutting,
tissue is a different placing tissue to level preserving the tissue for archival use and providing support to the tissue to be
seen level. the tissue on the wax. cut on the microtome. During embedding, the skill using tissue embedding
center and dealing with paraffin wax was developed in order to avoid the cracks
Epithelium Wrong The right orientation on the block. In tissue embedding, a proper tissue orientation is very important
cannot be orientation of is identified and is to make it easier to be cut by microtome during tissue sectioning.
seen tissue embedded facing
properly embedded downwards

1.4 TISSUE SECTIONING

REQUIREMENTS PRINCIPLE

MATERIALS: Paraffin blocks Sectioning is the process of cutting paraffin embedded tissue blocks into thin
APPARATUS slices sections. This thin tissue sections able to absorb dyes during tissue
staining, allowing visualization of the tissue structures. Sectioning also
Slide racks Pasteur pipettes necessarily reduces the specimen to a two-dimensional representation during
Glass slides Applicator stick the microscopic examination. On the other hand, thick slices layer will result in
Microtome blades Pencils over-staining and interferes microscopic observation.
Brush Biohazard bags
Gauze R
Newspaper Slee Mainz E
Forceps CUT5062 S
Thermometer U
L
EQUIPMENT XH-1001 T
Rotary microtome Thermo Scientific S
Tissue floatation bath
Freezer Tissue sections on
Drying oven
microtome.
Floating out (fishing) of Glass slide containing
selected tissue section. tissue section.

PROCEDURE

1.Setting up microtome: 2. Trimming: 3. Sectioning: (m) The glass slide is p
laced into the
(a) The microtome is
(a) The finger protection guard is removed to insert the new drying oven for 37(overnight) or 58-
switched On. microtome blade The handwheel lock is released for cutting. 60°C (15-30 minutes) after letting the
(b) The handwheel is (a) The tissue block is placed (b) The specimen block is inserted after the finger protection glass slide to stand on the slide rack
turned to its highest either vertically or horizontally was used. at room temperature to remove
position and its stop is onto the standard object clamp. (c) The tissue block to be sectioned is taken out of the freezer water.
activated. The fixing lever (b) For trimming mode, the once it is cooled and placed onto the standard object clamp. (n) The slide(s) is labelled on the
is adjusted for the “Macro” word appeared on the (d) For sectioning mode, the “Macro” word disappeared on the frosted end with pencil and placed
orientation of the display panel when the “M” button display panel when the “M” button is selected. into the slide rack. The label: student
specimen to the desired is selected. (e) The sectioning thickness is set ranging from 3-5 microns. and name, student ID number, date and
angle and then closed. (c) The trimming thickness is set the “+” or “–” is used to increase or decrease the value. type of specimen.
(c) The finger protection and the “+” or “–” used to increase (f) The motorized coarse advance button is pressed to move the
guard is removed to or decrease the value. specimen block towards the blade. 4. The microtome handle is locked
insert the microtome • For large tissue pieces: 10-20 (g) The finger protection guard is removed. after completion while, microtome
blade at 90C. The right microns (h) Paraffin tissue block sectioning started to produce ribbon blade is removed and kept in a
lever is turned • For small biopsies: 5-10 microns sections. designated container while blunt
counterclockwise to (d) The motorized coarse advance (i) The ribbon sections are pulled from the blade using an used blades are disposed in a sharp
loosen the blade fixation button is pressed to move the applicator stick or forceps and are transferred into the floatation container.
and the blade is inserted specimen block towards the blade. or water bath filled with distilled water (45°C). 5. Residual tissue, wax is removed
from one side. (e) The finger protection guard is (j) The ribbons are laid on the floatation bath to prevent wrinkles from the microtome using a brush
(d) The right lever is removed and trimming started or bubbles from forming on the tissue and The best section is and disposed into the biohazard
turned clockwise to until the complete tissue section selected from the ribbon sections. bags. The microtome is cleaned by
tighten the blade fixation appears on the ribbon. The (k) Using a glass slide, floating out (fishing) of selected sections paraffin repellent.
and handwheel lock is trimmed paraffin blocks are placed is performed onto the slides. 6. The microtome and floatation bath
released for cutting. in the freezer at -20°C to cool. (l) The glass slide containing the tissue section is placed onto is switched “Off” and cleaned.
(f) The used microtome blade is the slide rack.
removed and disposed into the
sharps container.

CONCLUSION TROUBESHOOTING
Skills required to properly use a
microtome is successfully achieved Sections did not Dull blade. Use a new blade.
which perfect ribbon of tissue form ribbons. Block surface not parallel Adjust the block holder angle.
sections were able to be produced. to the blade.

this manual process require Sections roll up Dirty or dull blade. Clean blade with brush or paraffin repellent.
consistent and delicate handwork upon cutting. Block too warm. Cooldown block.
aas the tissue section is very delicate
and easily damaged. Sections are Dull or dirty blade. Use new blade.
wrinkled. Slow or uneven rotation. Apply consistent wheel rotation.

1.5 TISSUE STAINING

REQUIREMENTS PRINCIPLE

Materials Reagents The principle of tissue staining is to enhance the
important features or part of the tissue. In tissue
Unstained tissue slides Hematoxylin 3G (Sakura) staining of histopathology, hematoxylin and eosin
Eosin (Sakura) the dye is routinely used. Hematoxylin 3G is a basic
Xylene dye that is usually used to stain the nucleus
Apparatus Alcohol (Absolute, 95%) components of the tissue and give the bluish
Slide racks Distilled water colour. On the other hand, eosin is used to stain
Forceps Tap water cytoplasmic components of the tissue and give rise
Filter paper to a pinkish color.
Tissue paper
Funnel
Measuring cylinder

PROCEDURE

R A. Preparation of various 7. Wash in running tap water (1 minute)
E concentration of alcohol. 8. Distilled water (1 minute)
S 9. Haematoxylin 3G (Sakura) (5 minutes)
U 95% Alcohol (400ml) 10. Wash in running tap water (5 minutes)
L 380ml Absolute Alcohol+20ml 11. Distilled water (30 seconds)
T Distilled Water 12. Eosin (2 minutes)
S Stained slides in B. Tissue staining 13. Distilled water (10 seconds)
i. Unstained slides is removed from the 14. 95% alcohol (10 seconds)
the staining basket oven. 15. 95% alcohol (10 seconds)
ii. Slides are stained according to the 16. Absolute alcohol (1 minute)
procedure below. 17. Absolute alcohol (1 minute)
1.Xylene (3 minutes) 18. Absolute alcohol (1 minute)
2.Xylene (3 minutes) 19. Xylene (1 minute)
3.Xylene (3 minutes) 20. Xylene (2 minutes)
4.Absolute alcohol (1 minute)
5.Absolute alcohol (1 minute)
6.95% alcohol (1 minute)

TROUBESHOOTING

The hematoxylin stain The efficacy of Discard the hematoxylin and CONCLUSION
is too light. (The nuclei hematoxylin is reduced. replace with new hematoxylin. Good tissue staining and reagent
are too pale) The section is too thick. preparation skills were able to be
Recut the section with achieved in the third and fourth cycles.
The hematoxylin is too The section is too thin. appropriate thickness. Although all the prepared slides were
dark. (The nuclei are stained according to the given standard
overstained). Recut the section if required operating procedure, some tissues
after checking the thickness of sample has a darker color than the other
The eosin is too pale. the section. tissues. This shows that tissue staining
are also affected by the thickness of the
Cytoplasm is The section have been Decrease the staining time. tissues.
overstained and has stained in eosin for too Allow more time for
poor differentiation. long. appropriate dehydration with
The section dehydrated in alcohol.
alcohol too fast.

1.6 SLIDE MOUNTING AND
LABELLING
REQUIREMENTS PRINCIPLE

Materials Chemical To preserve a stained tissue for viewing under the
microscope, it must be mounted on a clear slide and
Slide holder Mounting medium: Coverseal-X covered with a thin clear coverslip. A mounting medium is
Coverslip used to adhere the coverslip onto the slide. Care must be
Applicator taken to avoid air bubbles under the coverslip.
stick
Self-adhesive PROCEDURE
label sticker
Medisheet

Equipment

Ductless fume hood 1.Task was performed in a fume hood.
Brand: ESCO Model: Ascent MAX 2.Personal protective equipment (PPE) was worn while

R performing the procedure.
E 3.Appropriate coverslip was chosen according to the size of the
S
U tissue section.
L 4.An adequate mounting medium was placed on one edge of the
T
S Slides with coverslip and dried coverslip.
5.The slide was lowered gently until it touches the mounting
mounting medium.
medium. Capillary attraction will cause the mounting medium
to flow upwards, carrying the coverslip along with it.
6.The slide was examined macroscopically to ensure there are
no air bubbles. The coverslip was pressed gently with an
applicator stick to remove the air bubbles.
7.Slides was labeled with:

- Type of specimen
- Staining method
- Student name
- Student id name
- Date
8. The sticker label was placed at the frosted end of the slide.
.

TROUBLESHOOTING CONCLUSION

Residues of the mounting medium Putting too many drops of One to two drops of mounting The techniques for
on the coverslip sides. mounting medium . medium is already sufficient to mounting and covering the
cover the tissue and cover slip.
Air bubble formed can be stained tissue slides without
removed by pressing gently on any air bubbles or artifacts
Air bubble formed during Air bubble can form when the the air bubble towards the was successfully achieved
mounting. coverslip is closed too rapidly edge of cover slip by using
applicator stick.
Allow the mounting medium to after the second cycle.
dry sufficiently before storing Presence of artifact or air
the slide. bubbles will interfere during
Cover slip moved after mounting. This could due to insufficient tissue microscopic
drying
examination

2.0 FROZEN SECTION

REQUIREMENTS PRINCIPLE

Material Reagents Equipments The frozen section process involves rapid
freezing of the tissue sample that will
Fresh tissue Tap water Ductless fume hood ( Brand: convert water in the tissue into ice. The
Haematoxylin ESCO, Model: Ascent MAX) firm state of the ice within the tissue acts
Apparatus 3G (Sakura) as a support medium during tissue
Eosin (Sakura) Cryostat ( Brand: Slee Mainz sectioning instead of using paraffin wax.
Scalpel and blade Distilled water MEV) Using a cryostat, the tissue will undergo
Forceps Absolute freezing and sectioning at the temperature
Mould alcohol of -20oC. This technique is used to
Microtome blade 95% alcohol immediately diagnose a tissue when a
Brush Xylene rapid result is required. It is also used for
Slide racks 10% formalin immunohistochemistry and
Applicator stick immunofluorescence study.
Glass slide Medium
Coverslip Tissue freezing
Self-adhesive sticker medium
label Mounting
Medisheet medium
Gloves (Coverseal-X)

PROCEDURE

A. Reagents preparation C. Tissue staining and mounting.
1.The staining reagents was prepared like 1.Rapid H&E staining was performed following the procedure in
hematoxylin, eosin, absolute alcohol and 95% the Table 2 below.
alcohol which was prepared by adding 380 ml 2.The stained slides were then mounted.
of Absolute alcohol into 20 ml of distilled water. Table 2: Procedure for rapid haematoxylin & eosin staining

B. Tissue sectioning Derived from laboratory manual for MLT428 Histological Techniques
1.The temperature of the cryostat was set at -20
degree Celsius.
2.The specimen was grossed and then embedded
on a specimen disc a Tissue Freezing Medium
and was frozen in the designated area in the
cryostat.
3.The embedded specimen disc was placed on
the orientable specimen head.
4.The tissue was trimmed at 10 µm thick until the
specimen tissue was exposed.
5.Sectioning was performed at 5 µm thick until a
ribbon was obtained.
6.The sectioned ribbon of the tissue was placed
onto a labelled slide. The unstained slide was
immediately put into a pre-heated 10% formalin
for about 1 minute.

2.0 FROZEN SECTION

TROUBESHOOTING Ice crystals form within the tissue. The tissue is freezing rapidly.
Artifact that has been frozen Do not place the tissue specimen
The tissue surface is uneven and the in saline solution before freezing.
Uneven tissue embedding vital formation may be lost. Before freezing, make the tissue
Loosen of block during Tissue may be too cold when placed even at the cutting surface.
chucking on the chuck. Take the tissue out and reattach it
Tissue crumpled on a clean check which is not too
Tissue with a thin stripe The tissue in the block is either too cold.
Widely striped and tearing of hot or too cool. Make sure the block tissue is in
the tissue Nicks on the blade caused the optimum temperature: -15oC to
perpendicular tear in the tissue. -20oC.
Tissue is sticking with the blade. Blade should be replace with a
sharper one.
The blade must be clean or replace
with a new one.

CONCLUSION

Frozen section is mostly important
for providing an immediate report of
the sample. Diagnostic accuracy of
Unstained the slides from frozen section
slide technique can be considered high.
However, a small sample size and
R

E without the use of special stains can
become a limitation to this
S Credit: Damien Harkin procedure. This requires close
https://www.youtube.com/watch?v=UnCLhowHucU

U cooperation between the
pathologist and the surgeon for a
L definitive and accurate diagnosis.

T Stained
S slide

Credit: Pierre Gauthier
https://www.researchgate.net/figure/We-selected-one-frozen-section-
histologic-glass-slide-for-each-part-of-the-specimen_fig5_41397630

REFERENCES

An, Y. H., Moreira, P. L., Kang, Q. K., & Gruber, H. E. (2003). Principles of Embedding and Common
Protocols. Handbook of Histology Methods for Bone and Cartilage, 185–197.
https://doi.org/10.1007/978-1-59259-417-7_11

CliniSciences. (n.d.). Mounting media for light microscopy.
https://www.clinisciences.com/en/buy/cat-mounting-media-for-light-microscopy-4120.html

Damien Harkin. (2021). Preparation of frozen tissue sections (Cryotomy).
https://www.youtube.com/watch?v=UnCLhowHucU

Dey, P. (2018). Frozen section: Principle and procedure. Basic and Advanced Laboratory
Techniques in Histopathology and Cytology, 51–55. https://doi.org/10.1007/978-981-10-8252-8_6

Frozen section technique III. Pathology Innovations. (n.d.).
https://www.pathologyinnovations.com/frozen-section-technique-3

Gauthier, P. (2010). Complete frozen section margins.
https://www.researchgate.net/publication/41397630_Complete_Frozen_Section_Margins_with_Me
asurable_1_or_5_mm_Thick_Free_Margin_for_Cancer_of_the_Tongue_Part_2_Clinical_Experience

Histological Techniques. Histology Lab. (n.d.).
https://histologylab.ctl.columbia.edu/histological_techniques/

Histopathology. Lab Tests Online - Explaining Pathology. (n.d.).
https://www.labtestsonline.org.au/inside-the-lab/anatomical-pathology-in-detail/histopathology

Holmes, J. (n.d.). How do you prevent air bubbles in a microscope slide? Cement Answers.
https://cementanswers.com/how-do-you-prevent-air-bubbles-in-a-microscope-slide/

Macleod, C. B., Dow, N., Guo, H., Katz, R., & Martin, E. (2016). Advantages and Disadvantages of
Frozen Section Pathology. CBM Pathology.
https://static1.squarespace.com/static/50f0a19be4b0a42e43ea5e78/t/5a54fdfc652dea585b8f1a26
/1515519485410/Frozen+Section+Pathology+Newsletter.pdf

Rolls, G. (2019). An introduction to specimen processing. Leica Biosystems.
https://www.leicabiosystems.com/knowledge-pathway/an-introduction-to-specimen-processing/

Tissue preparation. (2021). Histology at SIU. https://histology.siu.edu/intro/tissprep.htm

LABORATORY REPORT
HISTOLOGICAL TECHNIQUES

MLT428
OCT2021-FEB2022

THE CYCLE IN HISTOLOGY LABORATORY

NAME 1. MUHAMMAD NABIL MUSAIYAR BIN AHMAD
(2020605034)
GROUP
DATE 2.NURUL AISYAH HANI BINTI JAWAWI
LECTURERS (2020837428)

3.NURUL FATIN MAISARAH BINTI MAT ZIN
(2020455492)

4.NURUL IZZATI BINTI MOHAMED HATTA
(2020837038)

5.SITI NUR ALIAH AZIZAH BINTI MOHD KODORI
(2020614624)

HS241 3A

5 JANUARY 2022

MADAM HARTINI BINTI YUSOF
DR. NUR AYUNIE BINTI ZULKEPLI

INTRODUCTION

What is histology? What is histological

Histology is the study of techniques?
tissue structure.
Histological technique is the technique to produce
Image by General Data https://www.general-data.com/products/lab-consumables tissue specimen that fixed or preserved on the
microscope slide. In order to get this, the tissue need to
go through several process which are grossing,
processing, embedding, sectioning, staining and
mounting and labelling. Then, the structure of the fixed
tissue will observe under microscope.

What is FFPE? What is Frozen Section?

Formalin Fixed Paraffin Embedded (FFPE) is Frozen section is where the tissue sample will
a block preparation to observe the status of rapidly be freeze using cryostat. Then, the
tissue malignancy. This technique use frozen tissue will be sectioning into
formaldehyde for tissue fixation, then microscopic section. This technique usually
continue with paraffin embedding to make a applied in immunofluorescence and enzyme
solid block. immunochemistry studies due it can stain
certain carbohydrate and lipids in tissue.
Advantages
Tissue sample can be stored in room Advantages
temperature. Less time consuming.
Can store for a long period of time for Suitable for molecular genetics analysis.
history perspective. The protein is well preserved in native
The tissue structure can be well state for immunohistochemistry.
preserved.
Disadvantages
Disadvantages Frozen tissue samples will rapidly
Time consuming. degenerate in room temperature.
Limited for certain studies. The sample need to be frozen after the
Tissue sample cannot be use for collection.
molecular analysis. Require an expensive cost for storage.

Precautions during storage and transportation of the specimen.

The specimen need to remove gently to Replace the fixative solution if the colour
avoid mechanical trauma changes or contain blood.

Quickly put the specimen in fixative Use a suitable container and fixation solution
solution to avoid specimen from dry. to maintain the quality of the specimen.

The specimen need to label prior to the The large specimen need to be slice into small
collection and in front of the patient. part so that the fixative can easily penetrate
the tissue.

WORKFLOW CHART

grossing Fume Hood:
Iryas

Processing Tissue Processor :
Citadel 1000

Embedding Paraffin Wax Tissue
Embedding Centre :

Slee Mainz

sectioning Rotary Microtome : Slee
Mainz CUT5062

Staining Manual Slide Staining Set :
Sakura Tissue-Tek

Microscopy Compound Microscope :
Carl Zeiss
Observation H&E Appendix
Result Histology Lab

TISSUE GROSSING MATERIALS &
APPARATUS:
PRINCIPLE EQUIPMENT
Appendix tissue sample
The tissue been cut into desirable Ducted Fume Hood Scalpel holders
size for microscopic examination Brand : IRYAS Scalpel blades
and immersed in 10% neutral Forceps
buffered formalin which act as Tissue cassettes
fixative that helps in maintain the Containers
tissues as life-like state as
possible by inhibit the acidity to Rulers
cause the autolysis of cells. Dissecting boards
Grossing paper / Medisheet
Gloves
Aprons
Masks
Goggles

PROCEDURE

The cassettes was labelled The specimen and the apparatus A sample was choosen and the
with pencil based on the needed for grossing were prepared correct orientation was identified
type of specimen, student and ready to be used. .Specimen grossing was
name and date. performed .

RESULT

TISSUE BLOCK IN 10 % The tissues cassettes was The specimen that
FORMALIN was placed into the 10% have been cut (2-
Neutral Buffered Formalin. 3mm) was sealed in
the cassettes

PROBLEM SOLUTION CONCLUSION

Uneven Cut the cell in 2- Tissue specimen must be cut with correct
trimmed cell 3mm per slice orientation , margin and thickness to
and too thick ensure the cells of the tissue can be
for cassette Use sharp blade when preserved and can be observed to
grossing to avoid the slide diagnose any abnormalities and learn
Specimen been crushed when slicing about the structural level of the tissues.
trauma

TISSUE PROCESSING

PRINCIPLE

PART 1 : DEHYDRATION PART 2 : CLEARING PART 3 : IMPREGNATION

Water, aqueous fixatives, A method of removing The clearing agent from the
and lipid tissue fluids are dehydrating agents tissue was replaced with a
removed from fixed tissues from tissue and medium (wax) that completely
Alcohol was used as replacing them with a fills all of the tissue voids to
dehydrating agents starts wax-soluble solution form a rigid specimen ,
from high to low forming a clear and enabling for easy handling and
concentration. transparent tissue. cutting of suitable thin pieces.

Procedure Material

1. The Tissue block been removed from the 10% Tissue Block
buffered formalin and placed on tissue (appendix ) in 10%
processor basket. buffered formalin

2.The metal lid was placed on top of the tissue Equipment : Tissue Processor
processor basket. Brand : Shandon Citadel 1000

3.The sealed tissue processor was placed inside R E S U
L T Apparatus
the head slot.
Processed Tissue Block
4.The door was closed and the timer was Tissue Block Gloves
started for tissue processing process to start. Lab coat
Forcep
5.The machine will automatically immersed the Tissue processor
tissue cassettes based on the steps and time basket
programmed as the table below. Metal lid

STEPS

PROBLEM SOLUTION

The solution has The solution need
been contaminated to be frequently
from previous test. check and replaced
to make sure the
The alcohol used solution is free
evaporated faster from sediments
at room and have enough
temperature volume for the test.

CONCLUSION

Tissue processing was needed to provide the tissue enough stiffness so it can be
sliced into thin sections for microscopic examination.

TISSUE EMBEDDING

Principle Material & apparatus Equipment

To precisely and perfectly 1.Tissue blocks 4. Steel block mould
orient a specimen into a
that have been 5. Gauze
block of paraffin wax.
processed 6. Tissue paper
Procedures
2. Forceps 7. Gloves

3. Scraper 8.Aprons & Masks Paraffin Wax Tissue Embedding
Centre: Slee Mainz MPS/C / MPS/P /

MPS/W

The tissue was placed into the The mould was placed to a cold
Cassette was taken to A little amount of hot paraffin mould with the sliced surface plate, then flatten the tissue
observe the tissue sample was poured into the mould using warm forceps, gently.
facing down.

The labeled paper was placed on top of the Cassette was placed on top of Then, paraffin wax was added
cassette. The mould then was placed on the mould and wax was added until about a half of the mould.

the cryo console until it fully covered

Problem & Solution Result

Tissue fall out Embed the Conclusion
during sectioning tissue properly
The skill for embedding the tissue
Wrong orientation of Mark the tissue with enough wax and correct
orientation was developed.
tissue with ink.


The tissue is attached to After each embedding, A block of tissue embedded in
paraffin wax was obtained.
the forceps clean the forceps.

TISSUE SECTIONING Rotary
Microtome
PRINCIPLE Slee Mainz
Sectioning refers to the service of cleanly and consistently cutting paraffin embedded CUT5062

tissue into a thin slice. These thin slices are referred as sections.

MATERIALS apparatus EQUIPMENT RESULT
Paraffin-embedded Rotary microtome: Slee
tissue blocks Slide racks Mainz CUT5062 The slide was labelled with
Clean frosted end glass Tissue floatation bath name, student Id,

slide XH-1001 specimen and date.
Tissue Floating Bath High profile microtome Freezer
blade The paraffin-embedded tissue
XH-1001 Clean brush chemicals block was placed onto the

Clean gauze Distilled water standard object clamp.
Paper Paraffin repellent
Tissue paper
Pasteur pipettes
Applicator sticks
Pencil

PROCEDURE

The tissue floatation bath The temperature of The microtome was
was filled with distilled tissue floatation was switched on.
water.
set at 42°C.

SETTING UP

The blade fixation The finger protection guard
was tightened. was removed and the blade
was inserted from one side.

trimming "M" button was selected until appearance The motorized coarse and Trimming was performed
of "Macro" word displayed for trimming fine advance buttons was until the complete
mode. The appropriate trimming thickness
pressed to move the appearance tissue section
was set according to specimen size. specimen towards the on tissue ribbon.
The tissue block was taken out The residual tissue
after 15 minutes to be blade.
sectioned. Trimmed paraffin block ribbons was removed by
was placed in the freezer using brush.

to cool.

The unused area "M" button was The motorize coarse Sectioning paraffin The forceps was
of microtome selected until "Macro" and fine advance button tissue block was used to pull the
blade was used word disappears for was pressed to move performed to ribbon sections
for sectioning. the block towards the produce ribbon from the blade.
sectioning mode. sections.
blade.
sectioning

twTheherefoepodvurperrldinapeecodawestsdeal6.ixdin0et°sCo The slide was let The slide was The residual The best section Twhienatsroitbtrbbhaoaenntshwfs. eeacrttreierodns
to dry on the gently raised wrinkled forming from the ribbon
slightly above
slide rack at room the water tissue was sections was
temperature. removed by using selected.
surface. pasteur pipette.

PROBLEM SOLUTION conclusion

Cutting thick and thin sections within the Ensure the knife is held firmly in place. Sectioning provides the very thin
same section. specimens for the microscopic
examination to study the tissue and to
Sections roll into coil. Reduce section thickness or replace the knife identify the abnormalities or atypical
Sections become wrinkle on the floatation Adjust the temperature of water bath appearance in the tissue.
bath.

TISSUE STAINING

Principle Memmert Oven Sakura Tissue-Tek®
Manual Slide Staining Set
Haematoxylin and Eosin (H&E) is able to create a
chemical attraction between the tissue specimen
and dye. Haematoxylin is a basic dye with positive

charge that will stain the acidic cell structures,
while for eosin is an acidic dye with negative
charge that will stain the basic cell structures.

EosinA,bHsoaleumteatAolxcyohlionland Materials Apparatus
Unstained slide. Slide racks
Measuring cylinder
Reagent Tissue paper
Hematoxylin 3G (Sakura)
Alcohol (Absolute 95% alcohol) Equipment
Eosin (Sakura) Sakura Tissue-Tek® Manual
Xylene Slide Staining Set
Distilled water Memmert Oven
Tap water (Siri No: 830995)

Procedure Steps of staining process 10.Running tap water: 5 minutes
Deparaffinization (55-60℃). 11. Distilled water: 30 seconds
The unstained slide needs to put in 1.Xylene: 3 minutes 12.Eosin: 2 minutes
oven for 30 minutes before starting 2.Xylene: 3 minutes 13.Distilled water: 10 seconds
the staining process. 3.Xylene: 3 minutes 14.95% Alcohol: 10 seconds
Preparation of the 95% alcohol. 4.Absolute Alcohol: 1 minute 15.95% Alcohol: 10seconds
Measure 380ml of absolute alcohol 5.Absolute Alcohol: 1 minute 16.Absolute Alcohol: 1 minute
and add into 20 ml of distilled water. 6.95% Alcohol: 1 minute 17.Absolute Alcohol: 1 minute
Proceed to staining. 7.Running tap water: 1 minute 18.Absolute Alcohol: 1 minute
Remove the unstained slide from 8.Distilled water: 1 minute 19.Xylene: 1 minute
oven and begins staining process by 9.Haematoxylin 3G Sakura: 20.Xylene: 2 minutes
following the steps.
5 minutes Total staining time: 33 minutes

Result Solution Problem

H&E Stained Tissue Stained Slides White spot was spotted Retreated the slide by using
after deparaffinization xylene to remove the white
Nuclei: blue
Cytoplasm: Pink to red process. spot.
Muscle fiber: Pinky red Tissue is wash away Make sure before the
RBCs: Orange/red during staining process. sectioning tissue was in 3
Collagen: Pale pinky red
The reagent is microns.
contaminated with other Replaced the contaminated

reagents. reagent with the new
reagent.

Conclusion

The skill for staining techniques was successfully obtained
by following the correct order of staining procedure.

PRINCIPLE MATERIAL H&E stained slides.
Mounting is the final step of REAGENT
histological techniques. The Mounting medium
mounting medium provide maximum APPARATUS (CoverSeal-X)
degree of transparency to the
stained tissue sections. The major Slide racks
aim of mounting medium is to Coverslip (22 X 40 mm/60 mm)
physically protect the specimen by Applicator stick
connecting the specimen, slide, and Self-adhesive labels sticker
Hair dryer
coverslip together.

PROCEDURES

An appropriate coverslip An adequate amount of The coverslip was gently
according to tissue mounting medium was placed lowered until it touches the
section was chosen.
on the H&E-stained slide. mounting medium.

The mounted H&E stained The slide was labeled with An applicator stick was
slide was dried using a hair specimen type, staining gently pressed against the
dryer before it is stored in coverslip to remove trapped
method, date, student name
a slide box. & student ID and placed at air bubbles.
the frosted end of the slide.

PROBLEM SOLUTION

Too many bubbles trapped Place the slide in xylene to
on the slide. ensure that the coverslip is
removed and repeat the
The coverslip was not placed mounting procedure.
properly on the slide.
Mounting medium too thick. Remove the coverslip and
place a new one on the slide.

Place maximally 2 drops of
mounting medium on the slide.

RESULT : H&E stained slide CONCLUSION

Slide mounting procedure must be done
in order to preserve and support the
H&E stained slide for light microscopy.

FROZEN EQUIPMENT

section

MATERIALS Fresh tissue CHEMICALS / REAGENTS
APPARATUS
Glass slides Tissue freezing medium
Coverslip Hematoxylin 3G (Sakura) Cryostat : Slee Mainz MEV
(22 X 40mm/24 X 60mm) Eosin (Sakura)
Microtome blade 10% Neutral Buffered Formalin
Forcep Alcohol (Absolute, 95%)
Brush Xylene
Applicator stick Distilled water
Coplin jar Tap water

H&E STAINING STEPS

PRINCIPLE
When a tissue sample is rapidly frozen, water is converted to
ice, which serves as an embedding medium and allows the
tissue to be sectioned. If the temperature of the tissue
sample is dropped, the tissue becomes firmer, whereas
increasing the temperature softens the tissue.

PROCEDURES

1) Preparation of reagents (95% alcohol)
400 ml of 95% alcohol was prepared by adding 380 ml
of absolute alcohol into 20 ml of distilled water.

2) Tissue sectioning All apparatus stated above Coplin jar containing 10% After grossing, the specimen
was prepared in the formalin was placed in was embedded on specimen
The temperature of the the oven at 60°C for 1
cryostat was set to appropriate tissue section hour. disc with tissue freezing
-20°C. of the cryostat.
medium & froze in the

designated area of the
The tissue was trimmed
Rapid H&E staining was The section was assigned at 10µ thick until the cryostat.
done & the slide was on the labelled slide & right tissue is exposed &
The specimen disc was
mounted for microscopic away placed in the pre- continued with sectioning placed in the specimen
examination. heated 10% formalin for 1 at 5µ thick.
head.
minute.
CRYOSTAT CLEANING STEPS
The specimen disc was Residual tissue from the The cryostat was Cryostat temperature was reset to
removed from the specimen disc was removed cleaned after being used. -5°C.
The handwheel was locked.
specimen head & left at & placed in a specimen The blade was taken out from blade
room temperature to container containing 10% holder.
Frozen section waste was removed
melt the embedding formalin. with a cold brush.
medium. The section waste tray was emptied.
The storage & shelves were cleaned.
PROBLEM SOLUTION RESULT All specimens were removed from
cryostat.
Tissue cracked. Make sure the cryostat Image by Stephen, R. via The sliding window was closed.
temperature is not too cold. https://www.pathologyinnovations.com/frozen-section-
Streaks line appear technique-3#canvas CONCLUSION
on tissue section. Clean the microtome blade or
Specimen holder replace with a new one. A skill in frozen section method
stucked. was acquired by practicing it
Tissue folded. Clean and unfreeze it by using using the cryostat.
70% ethanol.

Make sure the microtome
blade used is sharp.

REFERENCES

Rolls, G. (2021). Steps to Better Specimen Collection and Transport. Retrieved from
https://www.leicabiosystems.com/knowledge-pathway/steps-to-better-specimen-
collection-transport/
Columbia University. (2016). Histological Techniques. Retrieved from
https://histologylab.ctl.columbia.edu/histological_techniques/

Geneticist. (2018, December 19). What Is A Frozen Section?. Retrieved from
https://www.geneticistinc.com/blog/what-is-a-frozen-section

Geneticist. (2018, January 10). FFPE Tissue Block Uses. Retrieved from
https://www.geneticistinc.com/blog/ffpe-tissue-block-uses

Geneticist. (2018, April 17). The Pros and Cons of FFPE Vs Frozen Tissue Samples. Retrieved
from https://www.geneticistinc.com/blog/the-pros-and-cons-of-ffpe-vs-frozen-tissue-
samples

Batra, S. (2018). Microtomy-The Art of Section Cutting. Histopathology and Cytopathology
Notes. Retrieved from https://paramedicsworld.com/histopathology-cytopathology-
notes/microtomy-art-section-cutting/medical-paramedical-studynotes

Geoffrey Roll (n.d) Steps to Better Grossing . Retrieved from
https://www.leicabiosystems.com/knowledge-pathway/steps-to-better-grossing/

Andrew Lisowski (2019) . Science of Tissue Processing. Retrieved from
https://drp8p5tqcb2p5.cloudfront.net/fileadmin/downloads_lbs/190093_Rev_A_Science_o
f_Tissue_Processing.pdf

Mokobi, F. (2021, May 2021). Hematoxylin and eosin stain (H and E stain or HE stain).
Retrieved from https://microbenotes.com/hematoxylin-and-eosin-stain/