This lesson reviews concepts in cytology including collection
techniques, slide preparations, and cell evaluation.
This lesson is in 2 parts- you will need to follow another link at
the end of part 1 to finish the lesson and get to the writing
Read chapter 9 pp.287-336 in Laboratory
Procedures for Veterinary Technicians 5th
edition (by Hendrix and Sirois)
Read chapter 11 pp 453-474 in Laboratory Procedures for Veterinary
Technicians 4th edition (by Hendrix)
There is a writing assignment at the end of part 2
of the lesson. Please turn in your answers by email.
Review techniques in sample collection- imprints, scrapings, swabs,
Be able to prepare cytologic smears using a variety of techniques-
combination, squash, feather, starfish, line smear
Know which cytologic smear technique to use for a particular sample
Know 2 basic staining techniques
Be able to identify basic cell types
Be aware of the cellular criteria of neoplasia
Know proper protocol for submission of cytologic samples to an outside
Imprint , impression
New methylene blue
Collection of cytologic samples can be accomplished through imprints,
impression smears, scrapings, aspirates, and swabs.
Imprint technique involves pressing a microscopic slide against a
tissue surface of the animal so that cells are released and stick onto
the slide. In most cases, blood and fluid should be blotted from the
surface first before taking the imprint. Multiple imprints are usually
taken so that there is the option of a variety of stains for
diagnostic purposes. Impression smears involve taking a piece of
tissue separate from the animal and pressing it against a slide
surface to harvest some cells.
Scrapings are prepared from tissue surfaces using a sterile surgical
blade. The edge of the blade is dragged across the surface of the
tissue. Cells will collect on the edge of the blade without cutting
the tissue. The cells on the blade are then transferred to the slide.
Swabs are collected from tissue surfaces or to collect sparse fluid or
exudate accumulations. Moisten a sterile cotton swab and move the swab
across the tissue surface. Cells on the swab are transferred to the
slide by rolling it over the slide surface, not rubbing it.
Cells from subcutaneous tissue masses can be collected into a needle
via aspiration or nonaspiration ("stab") techniques. Aspiration is accomplished using a
needle and syringe. If the mass is large the needle may be redirected
while still in the mass with constant aspiration. If the mass is
small, position the needle, aspirate, then release the plunger before
redirection of the needle. Do not aspirate whenever removing the
needle from the mass as this will contaminate the sample with cells
from surrounding tissue. Usually a small amount of fluid and some
cells are collected into the hub of the needle with aspiration
technique. If material is not aspirated, alternatively you can use the
stab technique. A needle is directed into the mass back and forth
several times to collect cells into the needle lumen. More solid
material is acquired with the stab technique. After either aspiration
or stab technique, the material is expelled from the needle onto a
slide by attaching a syringe loaded with air. The plunger is pushed
quickly to force the material from the needle onto a slide.
Remember--Do not fill the syringe with air while it is attached to
the needle containing the sample as this will tend the damage the
cells to a greater degree.
The syringe is loaded with air and attached to the needle containing
the cells. Pressing the plunger rapidly forces the sample onto the
Fluids collected from body cavities are the subject of the next
Slide preparation techniques
Impression smear technique
Solid tissues harvested via biopsy are often studied via the
impression smear technique. The cut surface of a fresh tissue sample
is lightly pressed against the slide to collect cells onto the slide.
Many technicians develop expertise in cytology by making impression
smears of all excision biopsies. A cytology slide is prepared from the
tissue before it is put into formalin and sent for histopathology. The
cytology slide is evaluated, and once histopathology results are
available, the cytology results can be compared to the histopathology
results for learning purposes.
A freshly cut surface of the tissue is prepared.
Blot the tissue surface to remove excess blood and fluids.
The tissue is lightly pressed in several areas against the
The finished slide. There are several areas on the slide that
will stain and yield cells to view.
If no cells are released via the impression smear technique, then
scraping the freshly cut surface will collect some cells onto the
The freshly cut tissue surface is scraped with the edge of a
The blade is applied against the microscopic slide to transfer
the cells from the blade onto the slide.
The finished slide is ready for drying and staining.
Alternatively, the stab technique could be used.
If the cells collected are in a droplet, the material must be
distributed across the surface of the slide for staining and
evaluation. There are several techniques used to spread the material
over the slide: feather, squash, combination, starfish, and line
If the material expelled onto the slide consists of very small
droplets that do not have many cells, then no further smear technique
is used. Some technicians call this the spray technique. The slide is
used as is, that is, as the droplets landed on the slide, or were
Feather technique (blood smear technique)
The feather smear is the same technique used for blood smears. This
technique is used when the sample contains a lot of cells and there is
a need to spread the cells out.
The droplet is placed near the end of the slide.
A spreader slide is placed at an angle against the drop. The
fluid will spread across the end of the spreader slide.
The spreader slide is moved across the specimen slide until all
of the fluid adhered to the end of the spreader slide is dispersed.
A feather edge is formed at one end of the smear. The feather
edge is generally the best place to examine because the cells are
usually spaced out evenly in this region of the slide where the smear
is not too thick.
The squash technique is used to disperse cells over the slide evenly.
This technique is used for samples that may have cellular clumps that
need to be separated and otherwise would be too thick for
identification. This technique will also tend to rupture delicate
cells if performed in a rough manner. This is the
same technique used for buffy coat examination for microfilaria.
A drop is placed on the slide
Another slide is placed on top of the drop so that the slide
is at a 90 degree angle.
The slides are pulled apart without pressing so that the
material is spread evenly across the slide.
The finished slides are air dried and ready for staining.
Modified squash technique
This technique is a squash technique that supposedly is less likely to
A drop is placed on the slide
Another slide is placed on top of the drop so that the slide is
at a 90 degree angle
the top slide is rotated to a 45 degree angle
The top slide is lifted from the specimen slide.
The finished slide. The material on the slide will have some
The combination technique uses three different preparations for one
sample on a single slide. This technique is often used first if you
have only one drop of sample and you are not sure if it has a lot of
cells in it or not, or what technique would work best.
A droplet is placed on a slide in the middle
A spreader slide is placed on top of one third of the drop on
A squash prep is prepared of that one third of the drop
On the other end of the drop a spreader slide is
backed into the drop and placed on edge
against the drop. A feather edge is formed by moving the spreader slide across
the slide away from the drop.
The finished slide contains a middle portion of the drop that is
undisturbed, a feather preparation on one end and a squash preparation
on the other end. The slide is dried and stained.
For samples with very little cells in them, a line smear is used to
concentrate the cells along a visible line on the slide.
You can guess that a sample does not have very many cells if it is not
A drop is placed on the slide
A spreader slide is placed against the drop on edge
The spreader slide is moved across the slide to spread the
droplet material on the surface. Since the material
is not very cellular, it is not easy to see, so it will seem like you
are spreading nothing.
The spreader slide is stopped and lifted up.
The finished slide shows that there is an edge or line formed.
The line would be the area on the slide to view for cells.
The starfish preparation is used to gently spread very scant samples
on a slide.
A droplet is placed on a slide
A needle is used to spread the material off into small
projections from the drop
The finished slide contains a preparation that forms a flower or
star pattern on the slide.
Whatever preparation technique is used, the slide is air dried and
then stained. Air drying is best accomplished by gently waving the
slide in the air. Using hair dryers or open flames to warm the slide
and hasten drying may distort the cells. Proper air drying also helps
to fix the cells to the slide so they donít fall off during staining.
Now proceed to part 2 by using the link below.
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