TITLE: MODULE 3- 4D’S MODEL

Understand the video and make a reflection of the video.

FFPE - Tissue Processing/Embedding/Sectioning for Histology, Immunohistochemistry (IHC),
ISH & FISH

- The first video comprehensively explains the step-by-step tissue processing of a

formalin-fixed paraffin-embedded tissue (FFPE) sample. According to the video and
speaker, the very first step is the receiving of the tissue. Specimens received in the
surgical pathology laboratory contain specimen details and patient information, where it
is received in fresh or in liquid preservatives such as formalin. Then grossing, the
process in which tissue samples are examined and trimmed to proper size. The best part
of the tissue is selected for further microscopic examination to obtain diagnostic
information, placed in small plastic cassettes to hold tissue and allow fluid to pass
through the slits to bathe it. The next step is fixation and tissue processing. The essential
part of all histological and psychological techniques is the preservation of cells and
tissues as they naturally occur. Hence, the objective of tissue processing is to stabilize
tissues or cells and preserve their morphological, anatomical, and biochemical
characteristics for accurate diagnosis. To accomplish this, tissue sections or smears are
usually immersed in fixative solutions like the most commonly used 10% neutral buffered
formalin. Next is the dehydration process, where water present in the tissue is removed.
To minimize tissue distortion or shrinkage from dehydration, delicate specimens are
dehydrated in a concentrated ethanol series from water through 10%, 20%, 50%, 95%,
and 100% ethanol solution. After dehydration, the next step is the clearing process.
Since alcohol and paraffin are not miscible, alcohol must be replaced with an organic
solvent that is visible with both alcohol and paraffin. The tissues are usually put through
three changes of the clearing agents and transferred to paraffin for the next process
which is paraffin embedding. Tissues in the processor are molten paraffin and are still in
the cassettes, were then transferred manually into the embedding molds. Tissues must
be aligned properly at the bottom of the mold, and the bottom part of the cassette
containing the accession number should be placed over the mold. The mold and the
cassette are then filled with more molten wax and the paraffin is then allowed to solidify
by putting the embedding mold on a refrigerated surface. Once the paraffin is solid the
entire block with a cassette pops out of the mold and the block is ready for thin sections
using microtome. This process is called section cutting or sectioning. Once the paraffin
has solidified, the tissue block is removed from the mold and placed in a microtome. Due
to the friction between the knife and the paraffin block, enough heat is generated with
the cutting action, and allows the tissue section to adhere with each new section and
forms a ribbon of sections. The ribbon is then floated on a warm water bath to remove
any compression and wrinkles in the tissue caused by the cutting action, and the desired
section is then picked up using glass slides.
Explain the importance of tissue dehydration
- For tissue processing done in the lab, dehydration is an important step as it is the
process of removing water from the tissue to preserve its quality well. To minimize tissue
distortion or shrinkage from dehydration, delicate specimens are dehydrated in a
concentrated ethanol series from water through 10%, 20%, 50%, 95%, and 100%
ethanol solution. The characteristics of an ideal dehydrator are: it should rapidly
dehydrate tissue w/o producing shrinkage, evaporates very fast , also dehydrates fatty
tissues, should not harden tissue excessively, not remove stains, and shouldn’t be toxic
to the body.

Discuss how clearing of tissue is done.

- After dehydration, the next step on tissue preservation is the clearing process. Since
alcohol and paraffin are not miscible, alcohol or the dehydrating agent must be replaced
with an organic solvent that is visible with both alcohol and paraffin, and this is where the
clearing agent comes in. The tissues are usually put through three changes of the
clearing agents and are transferred to paraffin for the next process which is paraffin
embedding. In the clearing process, tissue becomes translucent. Due to high refractive
indices, embryos and parasites become transparent, and internal structures become
visible to the naked eye.

Compare and contrast impregnation from embedding of tissues