A ROUTINE AND ORGANIZED SYSTEM IN THE HISTOPATHOLOGY LABORATORY

MLT428
HISTOLOGICAL TECHNIQUES

A ROUTINE AND
ORGANIZED SYSTEM IN THE
HISTOPATHOLOGY LABORATORY

PREPARED BY:

PREPARED FOR:



MADAM HARTINI YUSOF
DR. NUR AYUNIE BINTI ZULKEPLI

DATE OF SUBMISSION:



6TH JANUARY 2023

WORKFLOW IN THE HISTOPATHOLOGY LAB

INTRODUCTION

Histopathology is a study related to microscopic

examination of a biopsy or surgical specimen that

has been processed and fixed onto glass slides in


order to analyze the signs of disease. So, it will

assist the doctor for better diagnosis and also

know the actual cause for the patient's sickness.

ROUTINE WORKFLOW

Tissue
Grossing

Tissue
Processing

Tissue
Embedding

Slide
Drying

Tissue
Floating

Tissue
Sectioning

Tissue
Staining

Slide
Mounting & Labelling

Image
Analysis

TISSUE GROSSING Objective:
1. To trim the fixed tissue specimens into small blocks
before it is processed using a tissue processor.

MATERIAL APPARATUS Scalpel holder and
blade
Human tissue specimens Forceps Ruler
Gloves Medisheet
REAGENTS EQUIPMENT Masks Apron
Goggles Container
10% Neutral Ducted Fume Tissue cassette
Buffered Hood: Iryas Dissecting board
Formalin

PRINCIPLE

Tissue grossing is a process that tissue specimen need to
be cut with ranging between 3 to 4 mm. Then, placing it
into a small tissue cassette which hold the tissue before
soaking in a 10% formalin buffer solution to fix the tissue

before continue to another process.

PROCEDURE

Wore personal

protective equipment


(PPE).

PROBLEM & SOLUTION Cassette was labelled
according to the type
Problem:
1.Tissue too thick (more than 4mm) so cassette of specimen.
cannot be closed tightly
2.Tissue block surface was not smooth due to Grossing station fume Specimen grossing was
trimming technique hood was turned on and performed once at time
instruments was ensured
Solution: Specimen was
1.Slice the tissue specimen thinner (2-3mm) ready to be used described
using a ruler
2.Avoid cutting the tissue specimen forward Specimen was macroscopically
and backward motion. Make sure to cut the transferred into
tissue in one way motion. Formalin waste was
cassette. discarded into the
RESULT
Tissue cassette was formalin waste
Tissue blocks placed into 10% Neutral container

CONCLUSION Buffered Formalin.

Tissue grossing is very essential to ensure the
best tissue condition to be used for the following
procedures such as embedding and sectioning.

TISSUE PROCESSING Objective:
1.To prepare the reagents used in tissue processing
2.To obtain processed tissue

MATERIAL REAGENTS

Fixed tissue blocks in the Commercially prepared 10% Neutral Buffered Formalin
10% Neutral Buffered Formalin 50% Alcohol, 70% Alcohol, 80% Alcohol and
95% Alcohol
APPARATUS EQUIPMENT Absolute Alcohol
Mixture of Absolute Alcohol and Xylene (50:50)
Forceps Automated
Xylene
Gloves Tissue Processor
Paraffin Wax
Masks (Brand: Shandon
Distilled Water
Goggles (For the preparation of 50%, 70%, 80% and 95% Alcohol)
Citadel 1000)

PRINCIPLE

A procedure involving
Dehydration
removal of water from the
Clearing
tissue and replacing it with
Wax infiltration
a medium which solidifies

allowing thin sections to be

cut on a microtome.

PROCEDURE 1 PROCEDURE 2 The cassettes containing

(A) Reagent Preparation (B) Tissue Processing cutted tissue were
Different concentrations of alcohol (50%, 70%, 80%, All the reagents that were
and 95%) and a mixture of alcohol with xylene were prepared were placed inserted into the tissue
prepared according to the ratio listed below: into the automated tissue
processor of each
respective reagent. processor organizer

baskets by using forceps.

RESULT The basket hanger The organizer baskets
were firmly attached were aligned into the
Processed tissue blocks into the operating head basket hanger.
slot of tissue processor.

PROBLEM & SOLUTION Problem: Tissue processing cycle was Full program time:
Contamination of tissue
started by pressing on the 21 hours and 11 minutes
Folding of tissue due
processing reagents due to
“AUTO-START” button on (allowing one minute of

to over dehydration the usage of processing
the hand held controller. every change of position)
reagents for multiple times
ROUTINE OVERNIGHT TISSUE PROCESSING

Solution: CONCLUSION
Replace with new reagents
Tissue processing is a lengthy

Presence of artifacts process which include a crucial step

Problem: ensuring that tissue block produced

Folding, shattering and cracking sections formed from tissue blocks
is suitable for the next process which

processed through paraffin wax due to over dehydration of tissue blocks include embedding and sectioning.

Thus, it is important for the tissue to

Solution: be well-processed according to the

Decrease dehydration of tissue by adjusting timing of processing cycle
proper program time.

TISSUE EMBEDDING Objective:

To embed the processed tissue blocks with paraffin wax

MATERIAL REAGENTS EQUIPMENT

Processed tissue blocks Paraffin wax Paraffin Wax Tissue

Embedding Centre
APPARATUS (Brand: Slee Mainz

MPS/C, MPS/P, MPS/W)
Forceps
Steel block mould
Gloves
Masks

PRINCIPLE PROCEDURE

Embedding is a process whereby the
A cassette containing processed

infiltrated tissues are enclosed in a mass of
tissue was opened in order to

the embedding medium by using a mould
visualize the tissue sample.
and it is known as casting or blocking.
A small amount of molten paraffin was

The tissue blocks produced that are very thin
poured into the mould and the mould

in thickness require a supporting medium

(embedding medium) before sectioning in
was transferred to a cold plate.
which the tissue blocks are embedded.
The processed tissue was transferred

The importance of embedding is to preserve
immediately to the mould using a forceps

the tissue morphology and giving tissue
after the paraffin wax became semi solid.
support during sectioning.
The tissue was gently pressed to

Microscopically PROBLEM & SOLUTION hold the tissue in a fixed position.

Problem: Molten paraffin was then poured into

Contamination of tissue observed
the mould until it fill up the mould.
microscopically
A labeled cassette along with labeled

Solution: paper was placed on top of the mould.
Ensure the paraffin wax is free from

contamination before embedding The paraffin wax was once again

poured into the mould to fully cover

Problem:
Paraffin wax is too soft producing thin
the face of the cassette.
and thick sections (uneven section)

Solution:
Cool the tissue block on cryo console

with a proper timing

Problem: The mould was then immediately cooled
Tissue block is not firmly held during
down by placing it on the cryo console.
sectioning Once the paraffin wax solidified, the paraffin
tissue block was separated from its mould.

Macroscopically Presence of air bubble Solution: CONCLUSION RESULT
Remove any excess wax on the outside

of the cassette before sectioning Tissue embedding is a crucial
Paraffin
step in preserving tissue
block
Problem:
Presence of air bubble/Tissue
morphology and provide support

separates from its wax causing a crack to the tissue for the next process


Solution: which is sectioning. Hence, it is

important to obtain a good

Melt the paraffin block to obtain the

quality paraffin block without any

tissue and re-embed the sample again crack or presence of air bubble

for further process to obtain a

Cracked tissue block perfect tissue slide in the end

PARAFFIN SECTIONING Objective
To produce a thin layer of tissue section for microscopy
MATERIAL EQUIPMENT
PRINCIPLE
Paraffin Rotary microtome
blocks (Slee Mainz CUT5062) Tissue sectioning is the procedure that involved in

slicing tissue that had been dehydrated and infiltrated

Floatation bath with wax into a thin layer using a microtome
(XH-1001, Thermo Scientific To prevent folds, wrinkles or air bubbles on the tissue

3120058) slide
More stable in maintaining tissue morphology
APPARATUS
PROCEDURE
Applicator stick Pencil
Brush Microtome blade Blade was inserted from one side of the
Forceps Clean frosted glass slide blade holder. The blade holder clamp is

RESULT tightened and blade guard is raised

Tissue section on The paraffin block was inserted at the
labeled frosted glass specimen holder of the microtome and
slides
blade angle was adjusted
PROBLEM AND SOLUTION
The slide were labeled with name,
PROBLEM student ID, class, type of specimen and
Wrinkled and compressed ribbons
staining, date
SOLUTION
Replace blade with a new one The 'M' button was selected until 'Macro' word appeared
on the display panel
PROBLEM
Blade lines appear on tissue block Trimming was started until area of tissue
section appears
SOLUTION
Adjust the angle and tighten the The 'M' button was selected until 'Macro' word
blade disappear on the display panel

PROBLEM Sectioning process was continued until a
Wrinkled tissue section on suitable tissue ribbons was selected
floatation bath
Applicator sticks and forceps were used to transfer
SOLUTION ribbons section into the floatation bath.
Ensure the water bath is in an
ideal temperature (42-45 °C) The best section from the ribbons section was selected
and gently separated using an applicator stick
CONCLUSION
Frosted glass slide was used to fish
Tissue sectioning is important to provide a very thin out the tissue section
layer of specimen for microscopy. A good sectioning
procedure will give the best result of tissue when The glass slide was placed on upright
staining and observe under microscope position on the slide rack to remove
water before transfer into drying oven

TISSUE STAINING Objective:

To stain the tissue section using haematoxylin and eosin

MATERIAL Tap water
Slide rack
Unstained slide Xylene
Hematoxylin 3G (Sakura) Reagent containers
Eosin (Sakura) Absolute Alcohol
95% Alcohol
Distilled water

MATERIAL PROCEDURE

Oven (Memmert) (A) Slide Drying
Ductless Fume Hood
(Brand: Esco)

PRINCIPLE

The chemical attraction between tissue and dye that is used for Drying process is to let water come out from the tissues

staining is to highlight important features of the tissue and after the tissue floating process in sectioning process.
enhance the cell contrast.
The chemical attraction between the tissue and dye is the basis 1)The slide was arranged in the oven
principle of H&E staining. Basophilic structures like chromatin, 2)The slide was dried at 37 degree celcius overnight
ribosomes and cytoplasmic areas are rich in RNA and produce a
blue-purple contrast by the basic dye hematoxylin. (B) H&E Staining
In contrast, basic components such red blood cell, muscle and
collagen are counterstained by an acidic eosin in different 1) The reagents for H&E staining were prepared and placed into

shades of pink, orange and red consecutively the containers before proceeding with the staining
2) The unstained slides then were put into the slide racks
RESULTS 3) The slide racks containing unstained slides were then dipped

into the container in accordance to the H&E staining cycle as

A shown below:
C
Type of sample: Uterus Deparaffinize the tissue section
B with xylene for 3 minutes
A: Cytoplasm (Pink/purplish pink)
B: RBCs (Red) Magnification: 10x Xylene for 3 minutes
C: Nucleus (Purple)
Xylene for 3 minutes
CONCLUSION Rehydrate in absolute

Absolute alcohol
alcohol for 1 minute.
H&E staining is commonly used in pathology to diagnose
for 1 minute
diseases and conditions based on the appearance of tissue
95% alcohol for 1

samples and is also used in research to study tissue and cell
Wash in running tap
minute.
structure and function water in 1 minute
Distilled water

PROBLEM AND SOLUTION Stain with
for 1 minute
Haematoxylin 3G

Solution: Increase time spent in xylene to Wash in running tap

completely remove the tissue's paraffin. for 5 minutes. water for 5 minutes
This artifact can also be noticed when the
Distilled water for 30
Counterstain in eosin

frozen section support media has not seconds for 2 minutes.
been properly removed
Wash using distilled
Dehydrate in 95%

Problem: Uneven staining water for 10 seconds. alcohol for 10 seconds

Solution: Make sure all the water 95% alcohol for Absolute alcohol for

is fully removed during fishing 10 seconds 1 minute.

Problem: Sample lost during staining Absolute alcohol for
Absolute alcohol for

1 minute. 1 minute.

Xylene for
Clear in 200ml of

1 minute xylene for 2 minutes.




END OF CYCLE
TOTAL STAINING TIME = 33 MINUTES

MOUNTING & LABELING Objective:

1.To mount the tissue section with a coverslip using a mounting medium
2.To label the stained slide clearly with specimen and stain details

MATERIAL REAGENTS Xylene

H&E-stained slides Mounting medium

(CoverSeal-X)
APPARATUS
PRINCIPLE
Slide racks
Coverslip (22X40mm/24X60mm) The mounting medium completely fills the area between
Gauze the slide and the cover slide in order to cover permanent
Forceps preparation of biological samples.
Applicator stick
Pencil This protects the sample from the outside and makes it
Self-adhesive label sticker easier to observe the preparation under a microscope
since it has glass-like refractive characteristics.
EQUIPMENT
As the mounting medium dries, it will harden and provide
Ductless stiffness and longevity to the preparation.
Fume Hood:
Esco Scientific During observation under 100x, the oil immersion will not
disrupt the tissues structure on the slide.
PROCEDURE Adequate amount of

mounting medium was
RESULT
An appropriate
placed on the edge of

coverslip was chosen
Mounted H&E-stained slide
according to the size of
the coverslip.

the tissue section. The slide was then

gently put and lowered

*This procedure was performed in a
fume hood to prevent hazardous until it touched the
fume from xylene that is toxic to mounting medium.
human to escape to the environment

The slide was examined

macroscopically to


ensure there are no air

bubbles present.

The slides were labeled with the following details: CONCLUSION

Type of specimen Mounting process is an important step in order to preserve the tissue
Staining method section and enhance visualization of the tissue under the microscope. Thus,
Student name it is very important to ensure that there are no air bubbles present on the
Student ID number mounted slide. A proper labelling is also crucial to produce a reliable result.
Date

PROBLEM & SOLUTION

Few air bubbles present
Coverslip lifted
microscopically from glass slide

IMAGE ANALYSIS Objective:

To observe H&E-stained slide under the microscope (10X and 40X)

MATERIAL RESULT

Mounted Type of sample: Uterus
H&E-stained slides

EQUIPMENT

Microscope
(Brand: ZEISS)

Magnification: 10X Magnification: 40X

PROCEDURE CONCLUSION

A microscope was
Image analysis is the last step of the routine workflow in the
set up by connecting
histopathology laboratory. During this process, the cell
it to the microscope
structures of tissue block can be differentiated with the
help of H&E stain which introduce cell contrast. Thus, we are
to a power source able to observe and differentiate cell structures using a
light microscope with 10X and 40X magnification.

The mounted
PROBLEM & SOLUTION
H&E stained

slide was placed
Problem:
onto the stage Presence of air bubbles on mounted slide during

observation under the microscope
The slide was

observed with various
Solution:
Dip the mounted glass slide into xylene for a few seconds
magnification level
Problem:
(10X and 40X) Blurry image formed during observation

Solution:
Clean the objective lens by using Kimwipes tissue or adjust

the fine adjustment knob to focus on the image

DISCUSSION Type of sample: Uterus (40x) Cross section of uterus

Hematoxylin
Endometrium
principally stains
Myometrium
the nuclei of cells

dark-purple

Eosin principally

stains the


cytoplasm with

pink colour

Eosin also stains the Endometrium
red blood cells in red
Type of sample: Uterus (40x)
Type of sample: Uterus (40x)

FROZEN SECTIONING Objective
To perform a quick microscopic analysis of specimen
MATERIAL EQUIPMENT
PRINCIPLE
Fresh tissue specimens Cryostat
(Brand: Slee
Cryostat quickly cool the tissue sample.
Mainz MEV) It turns water into ice by rapid freezing of the tissue
sample.
REAGENTS The ice acts as embedding medium and enables
sectioning of the sample.
Alcohol (Absolute & 95%) Freezing medium
Xylene Cold ethanol PROCEDURE
Distilled water Hematoxylin 3G (Sakura)
Tap water Eosin (Sakura) A fresh tissue specimen was prepared and
slide is labeled
APPARATUS
Tissue was placed and embedded on
Forceps Chuck chuck with Tissue Freezing Medium
Brush Frosted glass slide
Tissue was placed in freeze shelf and froze
RESULT in a freezing chamber

Sectioned tissue specimen The tissue block was placed on the specimen clamp

PROBLEM AND SOLUTION Trimming was started at 15 micron until an
area of tissue section appears
PROBLEM
Formation of ice crystal or artifacts Tissue was sectioned to about 4-5 micron thick

SOLUTION The tissue section was placed on a frosted

Ensure the temperature of freezing chamber always in glass slide and fixed with cold ethanol
an ideal temperature (-20 °C)
Staining was done using Rapid H&E
PROBLEM staining
Tissue specimen lost during staining

SOLUTION
Fix the tissue slide in cold ethanol

CONCLUSION The stained slide was mounted and observed under a
microscope
Frozen section is very useful for rapid diagnosis or STAT
(Short Turn Around Time) specimen and acts as an
alternative method for paraffin sectioning.

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