HURTADO, ABRAM RITZ C.

23 APRIL 2022
DENT 3C | GENERAL PATHOLOGY LABORATORY

Module:1 Laboratory Techniques in Pathology Lab

Answer the following:

A. How are paraffin sections prepared in the lab? Provide a step by step procedure using images with brief
descriptions.
A. OBTAINING A FRESH SPECIMEN

Fresh tissue samples will be obtained from a variety of sources. It should be noted that they can very easily
be damaged during removal from the patient or experimental animal. It's important that they're handled
with care and mended properly as soon as possible after dissection.

B. FIXATION

A liquid fixative, such as formaldehyde solution, is where the specimen is placed. This will slowly penetrate
the tissue, creating chemical and physical changes that will harden and preserve the tissue, as well as
protect it from further processing steps. There are only a few chemicals that can be used for fixation since
they must have specific qualities.
 C. DEHYDRATION

Because melted paraffin wax is hydrophobic (insoluble in water), the majority of the water in a specimen
must be eliminated before wax can be infiltrated. Immersing specimens in a series of ethanol (alcohol)
solutions of increasing concentration until pure, water-free alcohol is obtained. Ethanol is miscible with
water in all quantities, therefore the alcohol gradually replaces the water in the specimen. To minimize
significant tissue distortion, a series of increasing concentrations is used.

D. CLEARING

Despite the fact that the tissue is now essentially water-free, we are still unable to infiltrate it with wax due
to the fact that wax and ethanol are highly incompatible. As a result, we'll need an intermediate solvent
that's completely miscible with both ethanol and paraffin wax. The ethanol in the tissue will be displaced
by this solvent, which will then be displaced by molten paraffin wax. The cleansing agent also removes a
significant amount of fat from the tissue, which would otherwise act as a barrier to wax entry. Xylene is a
common clearing agent, and it takes several changes to completely replace ethanol.
 E. WAX INFILTRATION

A suitable histological wax can now be infiltrated into the tissue. At 60°C, a common wax is liquid and can
be infiltrated into tissue, then cooled to 20°C, where it solidifies to a consistency that permits pieces to be
cut consistently. These waxes are made out of purified paraffin wax and a variety of additives, such as
styrene or polyethylene resins. These wax formulations have very specific physical properties that allow
tissues infiltrated with the wax to be sectioned at a thickness of at least 2µm, to form ribbons as the sections
are cut on the microtome, and to retain enough elasticity to flatten completely during flotation in a warm
water bath.

F. EMBEDDING OR BLOCKING OUT

The specimen must now be formed into a "block" that can be clamped into a microtome for section cutting
after it has been thoroughly infiltrated with wax. An "embedding centre" is used for this phase, where a
mold is filled with molten wax and the specimen is placed within. The specimen is properly oriented in the
mold because the "plane of section," an important consideration in both diagnostic and research histology,
is determined by its positioning. A cassette is placed on top of the mold, which is then filled with more wax
and set on a cold plate to harden. The block, together with its attached cassette, can now be withdrawn
from the mold and ready for microtomy. It's important to note that, if tissue processing is done correctly, the
wax blocks containing the tissue specimens are very stable and can represent an important source f
archival material

B. Download an image of a tissue sample with the following stains. Provide information on the images
A. H&E

• Skin H&E. Note the balanced coloration in this section of skin. The nuclei are stained purple, while the
cytoplasmic components are pink.

B. CONGO RED

• Congo red stained salivary gland section examined under crossed polarized light from patient with
AL amyloid imaged using clinical microscope and same field examined using Metallurgical
microscope
 C. ZIEHL NEELSEN

• Ziehl-Neelsen stain of the purulent soft tissue specimen revealing pleomorphic bacilli.

REFERENCES:

https://www.leicabiosystems.com/knowledge-pathway/an-introduction-to-specimen-processing/

https://paraffinwaxco.com/paraffin-tissue-block/

https://www.leicabiosystems.com/knowledge-pathway/he-staining-overview-a-guide-to-best-practices/

https://diagnosticpathology.biomedcentral.com/articles/10.1186/s13000-019-0822-4

https://www.researchgate.net/figure/Ziehl-Neelsen-stain-of-the-purulent-soft-tissue-specimen-revealing-
pleomorphic-bacilli_fig2_323954567