HISTOLOGY LABORATORY REPORT (HS2413A GROUP 2)

THE FLOW OF PROCESS IN
HISTOPATHOLOGY LABORATORY

Name & Matrix No
Muhammad Naqiyuddin Izhar (2020483872)
Khairunnisa Irdina binti Rosewan (2020816944)
Muhammad Imran Bin Mohd Azlan (2020879042)
Nurul 'Iffah Hanani Binti Mohd Azri (2020813034)
Wan Nurauni Athirah Binti Wan Hamdan (2020455346)
Nurzaifazatul Farahanum Binti Mohd Shukri (2020449454)

Group: HS2413A

Date: 05/01/2022

Lecturers:
Madam Hartini Yusof
Dr. Nur Ayunie Binti Zulkepli

INTRODUCTION

HISTOLOGICAL TECHNIQUES

Histology is the study of the structure of the tissues of animals and plants. Histological techniques are the processes
that need to be done in order to observe the structure and components of the tissue under the microscope. There is a
standard workflow for histological examinations which starts with gross section, tissue processing, tissue embedding, tissue
sectioning, tissue staining, mounting and labelling and ends with microscopic examination. There are two common methods
used in order to preserve the tissue, which are formalin fixed paraffin embedded (FFPE) and frozen tissue
(Bancroft, J. D., Layton, C., & Suvarna, S. K., 2013)

FORMALIN FIXED EMBEDDED
PARAFFIN (FFPE)

Since the 19th century, FFPE, also known as formalin-fixed paraffin, has been used as a standard procedure for tissue
preservation in clinical samples. The medical community and researchers primarily use formalin-fixed paraffin-embedded
(FFPE) tissue specimens to preserve biopsy samples in formaldehyde, which is subsequently embedded into a paraffin wax
block. The structures of the tissue are preserved during this procedure . This approach has the advantages of being able
to keep the sample at ambient temperature and being cost-effective because it does not require any specific equipment.
Furthermore, because this sample is long-lasting, laboratory professionals can store it for future reference. This is due to
the formalin and wax's ability to preserve cell structures and proteins. However, there are a few drawbacks to FFPE,
including the fact that it takes a long time and is laborious. Apart from that, formalin is employed in this process, which
is very poisonous, volatile, and has an unpleasant odor. (Bancroft, J. D., Layton, C., & Suvarna, S. K. ,2013)

ProcedurHesISTOLOGY WORKFLOW FROZEN SECTION

GROSS SECTION

Figure 1.0 : Grossing the specimen Frozen section, also known as flash
freezing, is a technique in which a

Tissue PROCESSING biopsy is immersed in liquid nitrogen and
stored in an ultra-cold freezer with a

temperature of less than -80 degrees

Figure 1.1: Tissue processor Celsius. This method is mainly used for

intraoperative diagnosis, diagnostic and

TiSSUE EMBEDDING research enzyme histochemistry for
labile enzymes and Immunofluorescence.

Figure 1.2 : Embedding process The benefits of this approach include
the ability to maintain the protein in

tISSUE SECTIONING its original condition, which is ideal for

immunohistochemistry examination.

However, this approach has a number

Figure 1.3 : Tissue block and ribbon of drawbacks, including the fact that

TISSUE STAINING it is expensive because it requires a
special freezer to store the sample,

and hence the sample cannot be

Figure 1.4 : Inserting slides into slides rack Problems andptirsmeoesl.eutrFivouenrdsthfeorrmoaren, extended period of
because a freezer

MOUNTING & LABELLING is required, the frozen sample is
always exposed to mechanical failures

Figure 1.5 : Mounting & labelling the slides and power outages.
(Bancroft, J. D., Layton, C., &

MICROSCOPIC EXAMINATION Suvarna, S. K. ,2013)

Figure 1.6 : Appendix tissue observed under
light microscope (40x)

Flowchart 1: Histology workflow

TISSUE GROSSING

OBJECTIVE

To trim the fixed tissue specimens into small blocks before it is processed using a tissue processor.

PRINCIPLE

Grossing is a term referring to the examination and dissection of surgical specimens together with preparation
of sections from those tissues requiring processing, is the initial step in surgical pathology dissection
(Premalatha & Rao, 2010).

Materials Equipments

Human tissue specimens fixed with comercially Ducted Fume Hood: Iryas

prepared 10% formalin (Provided by MLT) Figure 2.0: Ducted Fume Hood

Apparatus

Scalpel holders Aprons

Scalpel blades Masks

Forceps Goggles

Tissue cassettes

Containers

Rulers
Dissecting boards

Grossing paper/Medisheet
Gloves

Procedures

1.Personal protective equipment was wore.
2.By using a pencil, the cassettes were labeled according to the type of specimen, student name, student

ID number and date.
3.The grossing workstation was prepared. The grossing station fume hood was turned on (lights and suction

vent) and it was ensured ready to be used.
4.Specimen grossing was performed one at a time. Specimen types range from transferring tissue from

specimen containers into cassettes to those requiring sampling.
5.The macroscopic details of the specimens was described.
6.The tissue cassette was placed into 10% Neutral Buffered Formalin.
7.Formalin waste was discarded into the formalin waste container.

Figure 2.1: Labelling Figure 2.2: Prepared Figure 2.3: Cutting Figure 2.4: Loading Figure 2.5: Tissue cassettes
the cassette grossing workstation the specimen the sample into in 10% Neutral Buffered
cassettes
Formalin

Results Problem & Solution

Figure 2.6 Tissue specimen loaded in a cassette with label Avoid overloading cassettes
This will allow ready access to processing reagents and
will prevent distortion of specimens. Overloaded
cassettes will prevent access of processing reagent

Prepare thin slices (3-4 mm thickness)
Prepare a uniform, thin slices so that reagent can
completely penetrate the specimen

(Geoffrey Rolls, 2016)

Conclusion Tissue grossing is important to obtain slides with diagnostic value and to
get an accurate diagnosis since it can directly effect specimen processing.

TISSUE
PROCESSING

SAMPLE OBJECTIVE

sBTwpoueinftcfsheiimlr1e0aedn%ndFtNoharapamttpueafrinlxaidnelidx To provide sufficient rigidity to the tissue so
(NBF) that it can be cut into thin section for
microscopic examination

PRINCIPLE

Tissue processing is designed to remove all extractable
water from the tissue, replacing it with a support medium
that provides sufficient rigidity to enable sectioning of the

tissue without parenchymal damage or distortion (Lena T.

EQUIPMENT USED Spencer and John D. Bancroft, 2017).
Figure 3.0 : tissue cassette CHEMICAL/REAGENT

SMiBrAoirduNanteooldm:: :CSaSHLtH3eAA5d61NT49Di1s80Os0WuN0e0CP0Ir1ToAcDesEsLor

Figure 3.1 : Tissue processor Table 1 : Chemical reagent in tissue processor

1.Place all reagents in the automated PROBLEM
tissue processor and make sure set time
correctly cracked and folded tissue
section
Gross section and fixation
TROUBLESHOOT
M Dehydration- A process removal Figure 3.2 : Tissue processor
E water, adequous fixatives and lipid Change the dehydration time :
process smaller tissue separately
tissue fluid from fixed tissues
from larger tissue
T Clearing- A process removal of
H dehydrating agent from tissue and

replace with a fluid which is wax soluble

O Impregnation- A process whereby Figure 3.3 : Tissue processor
plastic container
clearing agent is completely removed

D from tissue and replaced by medium(wax)

Embedding RESULT Figure 3.4 : Processed tissue
in casette
(Geoffrey Rolls, n.d). The tissue specimen fixed

CONCLUSION into paraffin before

To get sections of diagnostic value, processing of embedding
tissue should be done with appropriate reagents and

very much critical step need to be monitored with
utmost care

TISSUE EMBEDDING

Principle Embedding is the process that provides the specimens with the necessary hardness for

thinner sectioning. Therefore, the specimens or tissue samples were implanted or embedded

in a suitable medium, such as paraffin wax. These processes will also support the specimens

while being cut into much thinner slices during tissue sectioning later. (An et al., 2003)

Material Apparatus Equipment Reagent

Processed tissue Forceps Paraffin Wax Tissue Paraffin Wax
Scraper Embedding Centre
block (Appendix Steel block mould Figure 4.1 : Paraffin Wax
Gauze Brand : SLEE
and Uterus) Tissue paper MAINZ
Gloves Model : MPS/C /
Lab Coat MPS/P / MPS/W
Mask



Figure 4.0 : Paraffin Wax Tissue
Embedding Centre

Procedures Results

The processed tissue cassette was taken A processed tissue block
from the cassette bath and viewed. for the tissue sectioning
Depending on the size of the tissue sample, process is obtained.
the best steel block mould was chosen.
Figure 4.2 : Processed tissue
A small amount of wax was poured block
into the mould.
Problems and Solutions
Warm forceps were used to transfer the
tissue sample into the mould. Improper orientation of the specimen in the paraffin

The sample was positioned in the block
right orientation position, with the
sliced surface facing downward. Make sure the sliced position on the specimen is

The mould was placed on a cooling plate. facing downward and the specimen is placed in the

Using a pair of warm, clean forceps, the correct orientation before placing the mould on the
sample was gently pushed. (In this phase,
the tissue sample was kept in place.) cooling plate ‌(Winsor & Sluys, 2018)


On top of the steel block mould, the
labelled cassette was placed. A crack on the mould's surface

The melted paraffin was poured into the mould When the mould is placed on a cooling plate, use
until it completely covered the cassette's face.
warm forceps to gently push and hold the
When the mould was slightly cool, the marked
paper was placed on top of the cassette. specimen. ‌(Winsor & Sluys, 2018)

The mould was placed on the cryo console The paraffin wax does not infiltrate the tissue sample
to let it cool down. entirely.
The parrafin tissue block was separated from Too much of air bubble on the sample surface.
its mould after the paraffin wax solidified.
A scalpel was used to remove the surplus from Re-embedding the specimens
the paraffin tissue block cassettes.
‌(Biosystems, 2016)
Flowchart 2 : Procedure of embedding tissue
Conclusion

A proper tissue embedding technique gives a
good quality processed tissue sample block,
which is very helpful and can ease the tissue
sectioning procedure.

TISSUE SECTIONING

Principle

Tissue sectioning is a process done to produce thin slice section by using extremely sharp knives or

metals with a rotary microtome (Slee Mainz CUT5062). Once ribbons are produced, it is then placed

into floatation bath (42-45℃) and picked up on slides. The sections thickness range

is from 2 t0 3 µm. (Abdul Hamid, 2020)

Materials Equipments Figure 5.0 :Rotary microtome
used in laboratory
Paraffin blocks
Rotary microtome: Slee Mainz
Apparatus CUT5062
Tissue floatation bath: XH-1001,
Slides rack Thermo Scientific 3120058
Clean frosted end glass slides Freezer
High profile microtome blades Oven
Scraper
Clean brush Figure 5.1 :Equipments
Clean gauze used in laboratory
Paper & pencils
Tissue paper
Pasteur pipettes & applicator sticks

Procedures iii) Cutting sections

i) Setting up the microtome all desired tissues on the slide should be exposed on the

angle of slope of knife at 90° surface
block clamp screw must be firm
and knife screw is tighten a regular cutting rhythm should be maintained

once a ribbon section is produced, fine forceps is used to

hold free end of ribbon (section thickness is set at 3 µm)

ii) Trimming of tissue block last section is brush away with a soft paint from the
Figure 5.4: Trimming and sectioning
any surplus wax is knife process

trimmed to expose iv) Floating out sections

suitable area for tissue action in floating out must be smooth
slightly dragged when ribbon touches the water as it
sectioning (section produce tension in ribbon
folds are removed from section by teasing a part
thickness is set at 10 using forceps

µm) Figure 5.3 :Rotary microtome
used in laboratory

vi) Drying sections Figure 5.5: Floating out tissue
process

drying time is 10 minutes v) Picking up sections Figure 5.6: Picking up section
process
& a hot plate is slide is vertically immersed in the floatation bath &

necessary if staining is position into contact with the section

required urgently remaining water is drained in a vertical position for

cooled sections are several minutes

stored in dust free Flowchart 3: Procedures of sectioning

containers Figure 5.7 :Unstained tissue (Abdul Hamid, 2020)

Results Problems and solutions

Figure 5.8: Unstained Figure 5.9: Ribbon of tissue section 1) Tissue section coiled
tissue on slides Block is cooled down in freezer (10-15 min)

2) Tissue section does not form ribbon & compressed sections
Xylene is used to clean microtome surface
Block is cooled down in freezer (10-15 min)

3) Section in floatation bath appeared wrinkles
Floatation bath is adjusted to suitable temperature
(40-45°C)

Conclusion Sectioning process produces a thin section of block that later can be used
to examine any abnormalities in the tissue cells under microscope

H & E STAINING

Principle

H & E staining is the chemical interaction between the sample tissue and dye. Hematoxylin, a

basic dye present blue-purple contrast on basophilic components, mainly those with nucleic

acid such as chromatin, ribosomes and RNA-rich cytoplasmic regions. An acidic eosin

counterstains the basic elements like RBC, cytoplasm, muscle and collagen in multiple shades

of pink, orange and red. (Giri, D., Says:, D., Says:, G., & Says:, N., 2006)

Materials Figure 6.0 : Unstained tissue Equipment

Unstained Tissue slides Oven
Hotplate & stirrer
Apparatus
Reagents
Slide racks
Filter paper Hematoxylin 3G - Commercial Grade
Forceps Eosin - Commercial Grade
Tissue paper Xylene
Funnel Alcohol (95% & Absolute)
Measuring cylinder Distilled Water
Tripod stand Tap Water

Procedure

1.Place slides into oven at 60°C to allow
removal of wax for around 30 mins.

2.remove slides from oven and allow to
cool for 3-5 mins.

3.Proceed to staining procedure following
the steps and timing as stated below:

Figure 6.1: Staining station

Table 1 : staining sequence Problems & Solutions

Results Presence of artifacts on the stained slide above tissue
Frequently change/filter reagents.

Weak/strong hematoxylin/eosin colour on sample
adjust stain timing based on the stain that is weak
(e.g., hematoxylin: +/-30 secs, eosin: +/- 15 secs).

Figure 6. 3 : H&E stained slide (Cindy Sampias, J., 2018)

Conclusion H & E staining plays an important role in allowing proper evaluation of
sample as well as to ensure findings of sample sectioning is accurate.

MOUNTING & LABELLING
Principle
Labelling is the process of marking the stained slide with the appropriate information required

to identify and archive the slide for study and reference. Mounting is the final step in histology

tissue processing. The function of mounting media is to physically protect the specimen; the

mounting medium bonds specimen, slide and coverslip together with a clear durable film. A

mounting medium with a reflective index close to the fixed tissue will render it transparent,

with only the stained tissue visible. (Giri, D., Says:, D., Says:, G., & Says:, N., 2006)

Materials Apparatus

H&E stained slides Slide racks
Cover slips (22x40
Equipment mm/24x60 mm)
Forceps
Ductless fume hood Gauze
Applicator stick
Reagents Coplin jar
Self-adhesive label
Mounting medium stickers
(CoverSeal-X)
Xylene

Figure 7.0 : H&E stained slide

Procedure

Choose appropriate coverslip
according to size of tissue section.

Place adequate amounts of mounting
medium at the edge of coverslip.

Lower the slide until it touches the mounting medium. Figure 7.1 : Mounting stained slide
Capillary attraction will cause the mounting medium
to flow upwards, carrying the coverslip along with it.

Examine the slide macroscopically to
ensure there are no air bubbles.

Label slides with the following details:
Type of specimen, Staining method,

Student name, Student ID number & Date

Flowchart 4 : Slide mounting procedure Place sticker label at the frosted end
(Ahmad Zaini, n.d.) of the slide and allow slide to dry Figure 7.3 : Mounted slide drying

Results Problems & Solutions
Presence of air bubbles on slide
Figure 7.4 : Mounted slide
gently press coverslip with applicator stick to
remove. If too many are present, place slide in
xylene and repeat the procedure.
Mounting medium excess on top of coverslip
Use scalpel to remove as much as possible without
scratching the area covering tissue. If impossible,
place slide in xylene and repeat the process.

(Ahmad Zaini, n.d.)

Conclusion Mounting procedure is the last but still important step to ensure the tissue sample is
able to be preserved and observed without any artifacts/bubbles to block the sample.

FROZEN SECTION

PRINCIPLE PROCEDURE Figure 8.0: Frozen
section
The frozen section is a quick and rapid tissue section
that uses a device called a cryostat. The cryostat can Personal protective equipment (PPE) need to be wear throughout
perform both freezing and cutting of frozen tissue for procedure
microscopic sectioning.


(Dey, 2018) Cryostat on the scheduled date of frozen section is prepared and
While frozen sections are less structurally durable temperature is set to -20℃. Forceps, low profile microtome blade,
than paraffin-embedded sections, they are far superior
in terms of antigenicity and lipid retention. brush & applicator stick are set aside
(2022 | Histology Research Core | Paraffin & Frozen

Sectioning)
Other than that, this method preserve enzyme and A Coplin jar containing 10% formalin is placed in oven at 60℃ for
antigen expression better than paraffin-embedded 1 hour and is removed before used
Tissue.
Specimen is grossed & each glass slides are labelled (type of
(Frozen vs. Paraffin Sample Preparation, 2022) specimen & date)

MATERIAL, APPARATUS, REAGENT Fresh tissue is embedded on specimen disc with Tissue Freezing
& EQUIPMENT Medium & freeze in designated area


Equipment Reagent
1. Cryostat Tissue-Tek O.C.T Specimen disc is placed in orientable specimen head
Brand : SLEE MAINZ compound

Model : MEV
2. Freezer Apparatus Blades Tissue is trimmed at 10 µm thick until the specimen exposed &
Chuck Forceps sectioning is performed at 5 µm thick
Material Slides

Tissue block Brush
Section is placed onto labelled slide & place in pre-heated 10%
formalin for about 1 minute

Slide is staining with H&E stain

PROBLEM & SOLUTION Slide is mounting and ready to perform microscopic examination


Tissue folding or curling
Use a fine tissue paintbrush to gently flatten the curled The orientable specimen head is removing from the specimen head
tissue or use an anti-roll plate.


Tissue cracking The specimen disc is leaving at room temperature to allow the embedding
This is usually due to over-freezing the embedded sample. medium to melt
Check the cryostat temperature and make sure it is not too

cold by increasing 5 °C until the crack absence.
The residual tissue is removing from specimen disc and placing in specimen
container that containing 10% formalin. The specimen is leaving aside to fix



The cryostat is cleaning after each use



The remaining tissue is processing on the next day for routine processing

Flowchart 5 : Frozen section procedure

Tissue streaks
Frozen debris stuck on the blade causing streak. Clean the
cutting blade or replace the blade.

Stuck chuck Figure 8.4 : Cryostat (front view)
This is usually caused by trapped water inside the specimen (Ahmad Zaini, n.d.)
holder clamp. Run a defrost cycle or use 70% alcohol to clean
it. (Precisionary, 2021)

RESULT

Figure 8.3 : Cryostat

Figure 8. 1 : Invasive mammary Figure 8.2 : Sclerosing adenosis CONCLUSION
carsinoma Reisenbichler, E. S. (2017)
Frozen technique mostly used for rapid diagnosis and has high percentage of actual
expression in cell tissue sample. This method also preserve enzyme and antigen
expression better than paraffin-embedded tissue thus making frozen method is
more reliable to detect cancer cells.

REFERENCES

Abdul Hamid, Z., 2020. MLT428 HISTOLOGICAL TECHNIQUES (PART I) Theory. Faculty of Health
Science Universiti Teknologi MARA.

Ahmad Zaini, M., n.d. Laboratory Manual Histological Techniques. Universiti Teknologi Mara.

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techniques.
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eprocessing.pdf
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Giri, D., Says:, D., Says:, G., & Says:, N. (2018, November 06). Hematoxylin and eosin (H&E) staining :
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Geoffrey Rolls. ( n. d ). An Introduction to Specimen Processing. Advancing Cancer Diagnostics
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REFERENCES

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Paraffin & Frozen Sectioning | Histology Research Core. (2022). Unc.edu.
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