Lesson 1 Cytology part 1




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 assignment.


Reading Assignment


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)


Writing assignment


There is a writing assignment at the end of part 2 of the lesson. Please turn in your answers by email.


Learning Objectives


Review techniques in sample collection- imprints, scrapings, swabs, aspirates

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 lab


Key Words


Imprint , impression



Aspirate technique

Nonaspirate technique

Spreader slide

Combination slide

Squash prep



Line smear

Romanowsky stain

Eosinophilic, basophilic

New methylene blue

Epithelial cell

Mesenchymal cell


Round cell


Karolysis, karyorrhexis


Anisocytosis, anisokaryosis

Macrocytosis, macrokaryosis

Mitotic figure


Nuclear molding




Collection techniques


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 slide.


Fluids collected from body cavities are the subject of the next lesson.


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 slide


  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 blade.


  The freshly cut tissue surface is scraped with the edge of a surgical blade



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.


Smear techniques


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 smear.


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 "sprayed".


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.


Squash technique


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 rupture cells.


  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 ridges.


Combination slide


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 one end.


  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.


Line smear


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 very cloudy.


  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.


Starfish preparation


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.


Click here to proceed to part 2