Histological Techniques Laboratory Report

MLT428 HISTOLOGICAL TECHNIQUES

HISTOLOGICAL TECHNIQUES LAB REPORT

PARAFFIN SECTION | FROZEN SECTION

Prepared by: ELEANOR JOY JOLMIN MUHAMMAD AMIRUL AIMAN BIN ZAINUDIN NUR SOFIYA BINTI SUKANDAR NURUL AIN NASTASHA FAZIERA BINTI PA’E SITTI NUR AISAH BINTI BINTI JALIL

(2020476542) (2020878128) (2020483166) (2020455886) (2020605096)

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.

FLOWCHART

1. Tissue grossing

6. Tissue 'fishing'

7. Tissue staining

2. Tissue fixation

5. Tissue sectioning

8. Tissue mounting

3. Tissue processing

4. Tissue embedding

9. Tissue labelling

1.1 TISSUE PREPARATION REQUIREMENTS MATERIALS Scalpel blades Scalpel holders Forceps Tissue cassettes Dissecting boards Medisheet Gloves Tissue specimen in 10% buffer formalin

PRINCIPLE Tissue grossing is the process in which specimen samples go through a process of cutting or grossing into smaller parts depending on the size and type of specimen. The specimen is either taken entirely or a ‘representative’ section that was placed into a small cassette.

Tissue fixation is a crucial process where the tissue and cellular composition of cells is preserved to allow the cells to withstand processing. Fixation of tissues also prevent breakdown of the tissues as well as the molecular feature by the enzymatic activity for long-term storage.

EQUIPMENT

R E S U L Ducted fume hood Brand: IRYAS Model: ADe - 6BI

T S

Tissue specimens in 10% formalin

TROUBLESHOOTING Struggle to close the cassette lid.

Tissue sample is cut too thick.

Tissue slices should be thin that is around 3-4 mm.

Tissue trauma observed.

Rough dissection and dissection using blunt blade.

Handle specimen with care and always use sharp blade.

Presence of debris or foreign matters.

Chopping board is dirty.

Always starts off with a properly cleaned chopping board.

PROCEDURE

.

1. Personal protective equipment (PPE) was worn.

2. Cassette was labelled according to the type of specimen with student name, student ID, date and specimen type using pencil.

3. The grossing workstation was prepared by turning on the grossing station fume hood and the instruments are ensured ready to be used.

4. Specimen grossing was performed one at a time and placed the specimen into the cassette.

5. The tissue cassette was placed into 10% Neutral Buffered Formalin.

6. Formalin waste was discarded into the formalin waste container.

CONCLUSION In conclusion, skills in examining and choosing the best orientation and part to cut for tissue processing was successfully developed at the third cycle. Choosing which part to be cut is important in order to be able to examine the desired tissue part for disease diagnosis.

1.2 TISSUE PROCESSING REQUIREMENTS MATERIALS

Fixed tissue blocks (10% formalin) Forceps Gloves

REAGENTS

10% Neutral Buffered Formalin Alcohol (Absolute, 95%, 80%, 70%, 50%) Absolute Alcohol and Xylene Mixture (50:50) Paraffin Wax Distilled Water Xylene

EQUIPMENT

Brand: Shandon Citadel

Model: SHA 69800001

PRINCIPLE Tissue processing involves physical and chemical methods. The interchange between the tissue’s internal fluid and its surrounding solution occurs during the tissue immersion will extract the water, replacing it with the support medium.

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 at different concentrations and periods.

PROCEDURE A. Reagents preparation 1. 2L of 50% alcohol was prepared by adding 1000ml of absolute alcohol to 1000ml of distilled water.

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.

4. 2L of 95% alcohol was prepared by adding 1900ml of absolute alcohol to 100ml of distilled water.

5. 2L of alcohol and xylene mixture was prepared by adding 1000ml of absolute alcohol to 1000ml of xylene.

6. All the other reagents were also prepared and put into its respective place in the tissue processor.

B. Tissue processing 1. The cassettes containing the grossed samples were put into the cassettes basket.

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 Tissue section become cracked and folded

Excessive dehydration

Soak the block with a wet gauze before sectioning

During sectioning, tissue is coming out from the block

Dehydration inadequate and there is defective paraffin infiltration in tissue

Change the processing program and give adequate time for dehydration

Poor processing

Improper dehydration

Fluid are changed according to the schedule

The edges the tissue section affected by microchattering

Excessive dehydration

Change the dehydration time. It is better to process the smaller biopsy 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.

REQUIREMENTS

1.3 TISSUE EMBEDDING PRINCIPLE

MATERIALS:

Processed tissue blocks The process of orienting tissue in the correct direction and surround it in a molten liquid such as wax by using a mould. Allow it to solidify and generate a block for cutting. The aims are to provide sufficient external support of the tissue, avoid tissue distortion during the cutting process and preserve the tissue for archival purposes.

APPARATUS

Forceps Scraper Steel block mould Gauze Tissue paper Gloves Aprons Masks

REAGENT

Paraffin wax

Paraffin wax tissue embedding centre Brand: Slee Mainz Model: MPS/C MPS/P MPS/W

EQUIPMENT

PROCEDURE

Paraffin wax tissue embedding centre

1. The temperature of the paraffin tank is examined to ensure that it is higher than the melting point of paraffin wax; within 54oC-60oC. 2. The light and cryo console is switched on.

3. The tissue cassettes are placed into the cassette bath.

4. The cassette is opened to view the tissue sample.

5. Mould chosen must be suitable for the tissue size.

R

6. A small amount of molten paraffin is poured into the mould.

7. By using warm forceps, the tissue sample is transferred from the

E

cassette into the mould. The tissue surface that needs to be

S

observed must be facing downwards in a correct orientation.

U

8. The mould is transferred onto the cold plate.

9. The tissue is gently pressed and held until the thin layer of paraffin

L

solidifies.

T

10. The labeled cassette is placed on top of the mould and the mould is filled with molten paraffin wax until it fully covers the cassette.

S Processed tissue blocks

11. The forceps are wiped to clean the excess wax on it.

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

TROUBLESHOOTING Cracks on Too much Place the wax mould the tissue pressure when on the hot plate when blocks are placing the placing the tissue seen. tissue in the into the wax. wax when it already started to solidify.

Only a The embedded Add just a little fraction of tissue being in pressure when tissue is a different placing tissue to level seen level. the tissue on the wax.

Epithelium Wrong cannot be orientation of seen tissue properly embedded

The right orientation is identified and is embedded facing downwards

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.

CONCLUSION Embedding tissue is important in preventing the tissue distortion during cutting, preserving the tissue for archival use and providing support to the tissue to be 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 on the block. In tissue embedding, a proper tissue orientation is very important to make it easier to be cut by microtome during tissue sectioning.

1.4 TISSUE SECTIONING REQUIREMENTS

PRINCIPLE

MATERIALS: Paraffin blocks APPARATUS Slide racks Glass slides Microtome blades Brush Gauze Newspaper Forceps Thermometer

Sectioning is the process of cutting paraffin embedded tissue blocks into thin slices sections. This thin tissue sections able to absorb dyes during tissue staining, allowing visualization of the tissue structures. Sectioning also necessarily reduces the specimen to a two-dimensional representation during the microscopic examination. On the other hand, thick slices layer will result in over-staining and interferes microscopic observation.

Pasteur pipettes Applicator stick Pencils Biohazard bags

Slee Mainz CUT5062

R E S U L T S

XH-1001 Thermo Scientific

EQUIPMENT Rotary microtome Tissue floatation bath Freezer Drying oven

Tissue sections on microtome.

Floating out (fishing) of selected tissue section.

Glass slide containing tissue section.

PROCEDURE 1.Setting up microtome: (a) The microtome is switched On. (b) The handwheel is turned to its highest position and its stop is activated. The fixing lever is adjusted for the orientation of the specimen to the desired angle and then closed. (c) The finger protection guard is removed to insert the microtome blade at 90C. The right lever is turned counterclockwise to loosen the blade fixation and the blade is inserted from one side. (d) The right lever is turned clockwise to tighten the blade fixation and handwheel lock is released for cutting.

2. Trimming:

(a) The tissue block is placed either vertically or horizontally onto the standard object clamp. (b) For trimming mode, the “Macro” word appeared on the display panel when the “M” button is selected. (c) The trimming thickness is set and the “+” or “–” used to increase or decrease the value.

• For large tissue pieces: 10-20 microns • For small biopsies: 5-10 microns (d) The motorized coarse advance button is pressed to move the specimen block towards the blade. (e) The finger protection guard is removed and trimming started until the complete tissue section appears on the ribbon. The trimmed paraffin blocks are placed in the freezer at -20°C to cool. (f) The used microtome blade is removed and disposed into the sharps container.

3. Sectioning: (a) The finger protection guard is removed to insert the new microtome blade The handwheel lock is released for cutting. (b) The specimen block is inserted after the finger protection was used. (c) The tissue block to be sectioned is taken out of the freezer once it is cooled and placed onto the standard object clamp. (d) For sectioning mode, the “Macro” word disappeared on the display panel when the “M” button is selected. (e) The sectioning thickness is set ranging from 3-5 microns. and the “+” or “–” is used to increase or decrease the value. (f) The motorized coarse advance button is pressed to move the specimen block towards the blade. (g) The finger protection guard is removed. (h) Paraffin tissue block sectioning started to produce ribbon sections. (i) The ribbon sections are pulled from the blade using an applicator stick or forceps and are transferred into the floatation or water bath filled with distilled water (45°C). (j) The ribbons are laid on the floatation bath to prevent wrinkles or bubbles from forming on the tissue and The best section is selected from the ribbon sections. (k) Using a glass slide, floating out (fishing) of selected sections is performed onto the slides. (l) The glass slide containing the tissue section is placed onto the slide rack.

4. The microtome handle is locked after completion while, microtome blade is removed and kept in a designated container while blunt used blades are disposed in a sharp container.

5. Residual tissue, wax is removed from the microtome using a brush and disposed into the biohazard bags. The microtome is cleaned by paraffin repellent.

6. The microtome and floatation bath is switched “Off” and cleaned.

TROUBESHOOTING

CONCLUSION Skills required to properly use a microtome is successfully achieved which perfect ribbon of tissue sections were able to be produced. this manual process require consistent and delicate handwork aas the tissue section is very delicate and easily damaged.

(m) The glass slide is placed into the

drying oven for 37(overnight) or 5860°C (15-30 minutes) after letting the glass slide to stand on the slide rack at room temperature to remove water. (n) The slide(s) is labelled on the frosted end with pencil and placed into the slide rack. The label: student name, student ID number, date and type of specimen.

Sections did not form ribbons.

Dull blade.

Block surface not parallel to the blade.

Use a new blade.

Adjust the block holder angle.

Sections roll up upon cutting.

Dirty or dull blade.

Block too warm.

Clean blade with brush or paraffin repellent.

Cooldown block.

Sections are wrinkled.

Dull or dirty blade.

Slow or uneven rotation.

Use new blade.

Apply consistent wheel rotation.

1.5 TISSUE STAINING REQUIREMENTS Materials

PRINCIPLE Reagents

Unstained tissue slides Hematoxylin 3G (Sakura) Eosin (Sakura) Xylene Apparatus Alcohol (Absolute, 95%) Slide racks Distilled water Forceps Tap water Filter paper Tissue paper Funnel Measuring cylinder

The principle of tissue staining is to enhance the important features or part of the tissue. In tissue staining of histopathology, hematoxylin and eosin the dye is routinely used. Hematoxylin 3G is a basic dye that is usually used to stain the nucleus components of the tissue and give the bluish colour. On the other hand, eosin is used to stain cytoplasmic components of the tissue and give rise to a pinkish color.

PROCEDURE A. Preparation of various concentration of alcohol.

95% Alcohol (400ml) 380ml Absolute Alcohol+20ml Distilled Water

R E S U L T S

Stained slides in the staining basket

B. Tissue staining i. Unstained slides is removed from the oven. ii. Slides are stained according to the procedure below.

1. Xylene (3 minutes) 2. Xylene (3 minutes) 3. Xylene (3 minutes) 4. Absolute alcohol (1 minute) 5. Absolute alcohol (1 minute) 6. 95% alcohol (1 minute)

7. Wash in running tap water (1 minute) 8. Distilled water (1 minute) 9. Haematoxylin 3G (Sakura) (5 minutes) 10. Wash in running tap water (5 minutes) 11. Distilled water (30 seconds) 12. Eosin (2 minutes) 13. Distilled water (10 seconds) 14. 95% alcohol (10 seconds) 15. 95% alcohol (10 seconds) 16. Absolute alcohol (1 minute) 17. Absolute alcohol (1 minute) 18. Absolute alcohol (1 minute) 19. Xylene (1 minute) 20. Xylene (2 minutes)

TROUBESHOOTING The hematoxylin stain is too light. (The nuclei are too pale)

The efficacy of hematoxylin is reduced.

Discard the hematoxylin and replace with new hematoxylin.

The hematoxylin is too dark. (The nuclei are overstained).

The section is too thick.

Recut the section with appropriate thickness.

The eosin is too pale.

The section is too thin.

Recut the section if required after checking the thickness of the section.

Cytoplasm is overstained and has poor differentiation.

The section have been stained in eosin for too long.

The section dehydrated in alcohol too fast.

Decrease the staining time.

Allow more time for appropriate dehydration with alcohol.

CONCLUSION Good tissue staining and reagent preparation skills were able to be achieved in the third and fourth cycles.

Although all the prepared slides were stained according to the given standard operating procedure, some tissues sample has a darker color than the other tissues. This shows that tissue staining are also affected by the thickness of the tissues.

1.6 SLIDE MOUNTING AND LABELLING

REQUIREMENTS

Materials Slide holder Coverslip Applicator stick Self-adhesive label sticker Medisheet

PRINCIPLE

Chemical

To preserve a stained tissue for viewing under the microscope, it must be mounted on a clear slide and covered with a thin clear coverslip. A mounting medium is used to adhere the coverslip onto the slide. Care must be taken to avoid air bubbles under the coverslip.

Mounting medium: Coverseal-X

Equipment

PROCEDURE

Ductless fume hood Brand: ESCO Model: Ascent MAX

R E S U L T S

Slides with coverslip and dried mounting medium.

.

1. Task was performed in a fume hood.

2. Personal protective equipment (PPE) was worn while performing the procedure.

3. Appropriate coverslip was chosen according to the size of the tissue section.

4. An adequate mounting medium was placed on one edge of the coverslip.

5. The slide was lowered gently until it touches the mounting 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 on the coverslip sides.

Putting too many drops of mounting medium .

One to two drops of mounting medium is already sufficient to cover the tissue and cover slip.

Air bubble formed during mounting.

Air bubble can form when the coverslip is closed too rapidly

Air bubble formed can be removed by pressing gently on the air bubble towards the edge of cover slip by using applicator stick.

Cover slip moved after mounting.

This could due to insufficient drying

Allow the mounting medium to dry sufficiently before storing the slide.

The techniques for mounting and covering the stained tissue slides without any air bubbles or artifacts was successfully achieved after the second cycle.

Presence of artifact or air bubbles will interfere during tissue microscopic examination

2.0 FROZEN SECTION REQUIREMENTS

Material Fresh tissue

Apparatus Scalpel and blade Forceps Mould Microtome blade Brush Slide racks Applicator stick Glass slide Coverslip Self-adhesive sticker label Medisheet Gloves

Reagents Tap water Haematoxylin 3G (Sakura) Eosin (Sakura) Distilled water Absolute alcohol 95% alcohol Xylene 10% formalin

PRINCIPLE

Equipments

The frozen section process involves rapid freezing of the tissue sample that will convert water in the tissue into ice. The firm state of the ice within the tissue acts as a support medium during tissue sectioning instead of using paraffin wax.

Using a cryostat, the tissue will undergo freezing and sectioning at the temperature of -20oC. This technique is used to immediately diagnose a tissue when a rapid result is required. It is also used for immunohistochemistry and immunofluorescence study.

Ductless fume hood ( Brand: ESCO, Model: Ascent MAX)

Medium Tissue freezing medium Mounting medium (Coverseal-X)

Cryostat ( Brand: Slee Mainz MEV)

PROCEDURE A. Reagents preparation 1. The staining reagents was prepared like hematoxylin, eosin, absolute alcohol and 95% alcohol which was prepared by adding 380 ml of Absolute alcohol into 20 ml of distilled water.

B. Tissue sectioning 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.

C. Tissue staining and mounting.

1. Rapid H&E staining was performed following the procedure in the Table 2 below.

2. The stained slides were then mounted.

Table 2: Procedure for rapid haematoxylin & eosin staining

Derived from laboratory manual for MLT428 Histological Techniques

2.0 FROZEN SECTION TROUBESHOOTING Artifact that has been frozen

Ice crystals form within the tissue.

Uneven tissue embedding

The tissue surface is uneven and the vital formation may be lost.

Before freezing, make the tissue even at the cutting surface.

Loosen of block during chucking

Tissue may be too cold when placed on the chuck.

Take the tissue out and reattach it on a clean check which is not too cold.

Tissue crumpled

The tissue in the block is either too hot or too cool.

Tissue with a thin stripe

Nicks on the blade caused the perpendicular tear in the tissue.

Make sure the block tissue is in optimum temperature: -15oC to -20oC.

Widely striped and tearing of the tissue

Tissue is sticking with the blade.

The tissue is freezing rapidly.

Do not place the tissue specimen in saline solution before freezing.

Blade should be replace with a sharper one.

The blade must be clean or replace with a new one.

CONCLUSION

Unstained slide

R E S

Credit: Damien Harkin https://www.youtube.com/watch?v=UnCLhowHucU

U L T S

Stained slide

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

Frozen section is mostly important for providing an immediate report of the sample. Diagnostic accuracy of the slides from frozen section technique can be considered high.

However, a small sample size and without the use of special stains can become a limitation to this procedure. This requires close cooperation between the pathologist and the surgeon for a definitive and accurate diagnosis.

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