Embedding and Microtomy

Embedding

Introduction
After the completion of the processing cycle, the tissues are removed from the tissue processing machines to the blocking room for embedding. Each batch of blocks is tallied against the listing of the blocks for the corresponding batch to ensure that no blocks are missing before embedding.

Principles
The tissue is placed in molten paraffin (56* melting point) such that after the paraffin cools, the tissue and paraffin will form a block of sufficient consistency to cut thin sections

Materials
Embedding machine
Embedding moulds
Forceps

Embedding technique
1. Open the processing cassette carefully and view the tissue.
2. Select a mould that best corresponds to the size of the tissue.
3. Partially fill the mould with paraffin.
4. With warm forceps remove the tissue from the cassette and place it at the bottom of the mould – refer to specimen orientation below.
5. Gently press the surface of the tissue against the solidifying wax to hold it in the desired position.
6.Ensure no tissue is stuck on the lid then discard the lid of the cassette and place the bottom of the cassette firmly on the top of the mould.
7. Fill the combined mould and cassette with paraffin.
8. Place the mould on ice to solidify the wax, thus separating embedding tissue-cassette from the mould.
9. The tissue and cassette forms a paraffin block ready for sectioning.

Specimen orientation
1. Tissue sections are embedded flat to ensure that complete section is obtained.
2. Orientation should be such that the resistance the tissue offers the knife proceeds from the lesser amount towards the greater amount as the block is sectioned. This prevents the harder tissue from compressing the softer tissues and produces a smoother section.
3. There should be an adequate margin of embedding medium surrounding all sides of the tissue for maximum cutting support.
4. Tubular structures such as vas deferens, veins, arteries and fallopian tubes must be embedded such that the knife cuts across the lumen. These should be placed vertically in the mould.
5. Tissues with epithelial surfaces such as skin, intestine, gallbladder, urinary bladder and uterus must be positioned such that the plane of the section is across all tissue layers. The epithelial surface should be placed such that the plane of section is perpendicular.
6. Multiple specimens should be placed side by side close together so that call pieces can be sectioned.
7. Rectangular tissues should be orientated parallel to each other and with their long axis perpendicular to the plane of section.
8. Small bisected cysts should be embedded with the cut surface down and ensure that no air bubbles are trapped in the paraffin.
9. Muscle biopsy in 2 pieces should be embedded with one piece in a longitudinal and the other in a vertical position.

Microtomy

Introduction
Paraffin blocks from the blocking room are brought to the cutting room for sectioning

Principles
Microtomy is the use of a microtome to make thin sections for microscopy. Rotary microtomes are used in the laboratory

Materials
Room Temperature floatation bath
Warm floatation bath – 48 plus-minus 4 degrees Celsius thermostatically controlled
Cryoplate
Disposable microtome blade
Microscope glass slides
Rotatry microtome
Soft pencil

Solution preparation
1. Type II Water 2000ml

Allow the water in the floatation bath to reach 48 plus-minus 4 degrees Celsius on a thermostat before sectioning. Technologist must record this temperature on the temperature chart before sectioning

2. 1% alcohol floatation bath
95% alcohol 5ml
Type II Water 500ml

This is used to float the tissue section prior to transferring on the heated floatation bath above. It is an alternative when folds on the tissue are difficult to get rid, as alcohol having low vapour pressure will increase the surface tension when transferring the tissue onto the heated floatation bath.

Microtomy Technique
1. Rough cutting – Secure the paraffin block in the block holder of the microtome. Adjust it to ensure that it clears the knife. Readjust the block holder screws to place the block parallel to the knife if necessary. During the rough-cut, while being manually advanced, the block is repeatedly sectioned at 20 microns thickness per slice. Sectioning stops when the entire surface of the tissue is exposed. The block is then removed from the holder.
2. All blocks should be rough cut before sectioning because:

a.Dense and hard tissue that may cause nicks and blunt the knife more rapidly can be identified e.g. bone, cervix and thyroid. Such blocks could be separated from the rest and appropriately pre-treated before sectioning. These blocks should be sectioned last as they may still cause nicks and score lines to the blade.
b. Sutures and staples attached to the surgical specimens may not have been removed and can be detected during rough cutting.
c. Hard bone is identified and additional surface decalcification can be done, by placing the block face down in decalcifying agent e.g. RDO for further decalcification prior to sectioning.
d. Fibrous tissue e.g. fibroids can be softened in mollifex or 10% fabric softener for 5 minutes. The paraffin blocks are washed in water after the appropriate treatment porior to sectioning.
3. Chilling – the blocks are then placed face down on the cryoplate to chill the block to facilitate fast sectioning. This renders the block sufficiently hard for thin sections.
4. Sectioning – secure and adjust the block in a similar manner as rough cutting. Using the handwheel, section the block at 3-4 microns, by allowing the block to advance automatically. Some tissue biopsies are sectioned at different thickness.
5. Flotation- gradually lower the section or sections if a ribbon is cut, onto the water bath. If difficulty is encountered in spreading of the tissue, float the section on the alcohol bath first so as to increase the surface tension before transferring onto the water bath. Allow the section to remain on the water bath until it has spread sufficiently. Transfer the section onto a glass slide. The corresponding biopsy number is written on the frosted end of the glass slide.
6. Separation of slides – Slides for routine HE stain, special stains and unstained sections should be separated on different racks.

Serial Sections
First submission
Tissues less than 0.5cm in size
Gastric biopsies
i. 1 HE slide with 6 serial sections
Liver biopsies
i. Slide 1 – 2 consecutive complete sections for HE
ii. Slide 2 – 1 section for MT
iii. Slide 3 – 1 section for Ret
iv. Slide 4 – 2 consecutive complete sections for HE
Bone marrow
i. Section at 2um thick
ii. 1 HE slide with 6 serial sections for small specimen or 3 HE sections on 3 slides labelled VL1, VL2, VL3 for larger specimens and 1 slide for Retic
Renal biopsies
i. Section at 2um thick
ii. 10 slides with 3 sections each as follows:
1. HE on slide 2, 5 and 8
2. Pas on slide 1m 6 and 9
3. PaAg on slide 4
4. PgMT on slide 3, 7 and 10
TBLB – Trans bronchial lung bx
i. Section at 5um thick
ii. 10 slides with 2 sections each as follows:
1. HE on slide 2, 5 and 8
2. TB stain on slide 10 for TB cases only
Endomyocardial bx
i. Section at 5um thick.
ii. 10 slides with 2 sections each as follows:
1. HE on slide 4, 6, 10
2. MT on slide 7
Prostatic needle bx
i. 1 HE slide with 6 serial sections
Tissue biopsies less than 0.5cm in size not specificed above
i. 1 HE slide with 6 serial sections
Larger tissue biopsies
i. 1 HE slide
Request for variable levels
For small tissue approximately less than 0.5cm in size
i. Repeat 6 serial sections on 1 slide labelled VL 1 for HE
For larger tissue approximately more than 0.5cm in size
i. Repeat 3 serial sections on 3 slides labelled VL1, VL2 and VL3 respectively for HE
Request for special stains
Addition sections will be cut and stained as requested
For additional levels of routine biopsy specimens or irregular number of sections required, serial sections will be cut according to the request of the pathologist.
All personnel receiving verbal or phone orders must read back the entire order to verify accuracy and transcription.

Desmond Heng
TG02
0503179D