Setting up cytology in your practice (Proceedings)
Cytolopathology: the laboratory and microscopic evaluation of cells and fluids. Sources of cells include tissue aspirates, imprints, scrapings, body fluids, or lavage fluids.
Pros: Cytology is useful for establishing a diagnosis or can narrow the list of differentials for effusions, masses, lymphadenopathy, or organomegaly. It is rapid, inexpensive, and (usually) only minimally invasive.
Cons: Cytology does not provide information about tissue architecture. Obtaining an appropriate sample is critical for obtaining the correct diagnosis. Secondary processes can confound the diagnosis. Complications are possible and include tumor hemorrhage, infection, and tumor seeding.Equipment and supplies needed to perform cytology in practice are minimal and include a good microscope, 22-25 gauge needles, 3-20 ml syringes, glass microscope slides, a hematologic stain appropriate for cytology, and immersion oil.
There a few key considerations when choosing a microscope. First, it is ideal to have two microscopes in practice – a basic microscope for performing fecals and urinalyses and a higher end microscope for cytology. Microscopes suitable for cytology are called 'compound microscopes' because a combination of lenses are used to visualize the image. The microscope head supports the oculars which may be straight or inclined. An inclined binocular head that can move up and down are the most comfortable and allow adjustment for users of multiple heights. A trinocular head should be purchased if you anticipate photomicrography, addition of a second head, or a video link. While these may seen like a luxury, they can be very useful for training technicians within the clinic, obtaining second opinions from colleagues, and demonstrating abnormalities to clients. With the relatively minor cost investment of a trinocular head, additional attachments can be added at a later time point. The nosepiece is the part of the microscope that holds the objectives. This should rotate smoothly.
Objectives are the most important, and most costly, components of the microscope. There are three basic types of objectives – achromatic, semi-apochromatics (also called fluorite) and apochromatic. Achromatics are the most common, least expensive, and least corrected form of objectives. Fluorite objectives have additional spherical correction. Apochromatics, the most expensive and most corrected optics, are primarily used for research and high-end photography. Plan objectives have additional flat field correction – important for the ability to simultaneously focus on the center and the periphery of the field. Most microscope objectives are either plan achromatic or plan fluorite.
Objectives are often referred to as 10x, 40x, etc. The number is the numerical aperture and is a measure of the resolving power of the objective with higher numbers indicating higher resolution. For cytology, it is critical to, at minimum, have a 10x and 100x oil immersion objective. Additional objectives however also have usefulness. Addition of a 4x objective allows low power scanning for clumps and large organisms. 20x and 40x objectives allow higher magnification screening and comparison of cell morphology between clusters. However, it should be noted that most 40x objectives require that the sample be coverslipped to allow optimal focus. While usually more expensive than a 40x dry objective lens, a 50x oil immersion lens provides excellent clarity and the ability to visualize a larger field area.
Oculars are another critical part of the microscope. The most common type used in cytology are called Huygenian, or negative oculars. To maximize compatibility, it is best to purchase oculars and objectives from the same source. Wide-field oculars allow visualization of a larger area on the slide which can be particularly useful for examination of cytologic samples as it allows simultaneous viewing and comparison of a greater number of cells than does a standard field ocular. High-point eyepieces are designed such that the 'eye point' is above the surface of the lens of the eye. They are especially useful for those who wear glasses but also help to reduce eye fatigue in microscopists who do not wear glasses.
The microscope condenser should have a numerical aperture equal to or greater that than the numerical aperture of the highest power objective intended for use on the microscope. The mechanical stage should operate smoothly. Most microscopes can accommodate left or right handed stages based on the preference of the operator. The microscopes should have both a coarse and fine focus. The light source may be in the base or the back of the microscope but should be adjustable.
Koehler illumination is used to align the light for microscopy so that the sample is uniformly illuminated, bright, and free from glare. First, focus on the specimen at the objective to be used. Close the field diaphragm which is located at the base of the microscope where light comes out. You should see the light reduced to the center of the field. If the area of light is not in the center of the field, use the centering screws (usually silver) attached to the condenser to center the light. Adjust the condenser vertically using the condenser focus knob until there is a sharp demarcation between the light and dark interface. Finally, re-open the field diaphragm to just outside the field of view. Open the condenser to allow the appropriate amount of light to enter.