Cytology 101: How to get the most from your samples (Proceedings)

Cytology is a relatively easy, relatively non-invasive, fast and inexpensive diagnostic technique. Sometimes you actually get the diagnosis. Other times you don't get a specific diagnosis, but the cytologic findings can help you decide which diagnostic technique might be indicated as a next step. Also some potential diagnoses often can be ruled out.

To perform a good cytologic evaluation you must have some knowledge including:

• The normal histological and cytological appearance of tissues in the area being sampled

• The histological and cytological appearance of potential lesions

• The types of pathologic abnormalities that might cause the lesion being sampled.

This knowledge may be relatively simple in some cases and pretty complicated in others. Many cases probably should be evaluated by a specialist with advanced training in pathology. But some cases are straightforward in their cytologic appearance and can be evaluated by interested practitioners. In addition, screening of a sample before submitting it to an outside laboratory is useful in helping to assure its quality as well as being a learning tool if you compare your interpretation with the cytologist's report.

Frequently sampled tissues for cytology include blood, subcutaneous and cutaneous lesions, lymph nodes, prostate, bone marrow, conjunctiva, ear exudates, vaginal and rectal mucosa, internal organs, urine, respiratory tract, and body cavity fluids.

General approach to the cytology slide

Start with a low power objective to see whether there are actually intact cells present, get an idea as to which cell types are present, and find a suitable area to examine at higher magnification. Parasites and other large organisms are also usually best found at low power.

Appropriate areas to examine have a monolayer of intact cells that are well stained. If cells are in a thick area, they are difficult (if not impossible) to identify and morphologically examine. Avoid ruptured cells and areas where the stain is too light or dark. Some highly cellular samples that stain poorly may have well-stained areas at the edge of the slide, or they may require additional staining.

After finding a good area on low power, the slide should be examined with high power, or oil. At this magnification you can positively identify various cell types, examine cellular detail, and search for small organisms.

In most cases, the most important distinction to make is whether a lesion is inflammatory or neoplastic/hyperplastic. Making this determination can lead you to treat with such therapies as antibiotics and anti-inflammatories for inflammatory lesions or to surgically excise or initiate chemotherapy for neoplastic lesions. Once you decide whether a lesion is inflammatory or neoplastic, there are some other types of information that can be gained from a cytology sample that enhance your ability to treat and give a reasonable prognosis.

Classification of inflammation

Inflammation is classified by the type of cells that predominate. Sometimes inflammation is considered to be mixed if there are roughly equivalent numbers of different types of inflammatory cells. The degree of inflammation (mild, moderate or marked) should be noted.

Suppurative (or purulent or neutrophilic) inflammation

• Usually ≥ 90% neutrophils.

• Septic, suppurative inflammation is the term used if bacteria are seen.

• You can't assume that something is non-septic just because bacteria are not seen cytologically. Culture is used to make the determination.

Mixed inflammation

• More than one type of inflammatory cell is present.

• Usually comprised predominantly of neutrophils with 10-50% macrophages and/or lymphocytes.

• Commonly seen with foreign body reaction or fungal infection.

• May be an indication of chronicity.

Mononuclear or granulomatous inflammation

• The majority of cells are macrophages and giant cells.

• Typical inflammatory process associated with mycobacterial agents and steatitis.

Eosinophilic inflammation

• If >15% of the cells are eosinophils an inflammatory reaction is usually considered to have a significant eosinophilic component.

• Commonly associated with allergic reactions or parasites.

Classification of neoplasia-types

Knowing the type of tumor is very useful in being able to predict its behavior, come up with a plan for treatment, and give an accurate prognosis to the owner. Neoplasms are generally classified as epithelial, mesenchymal or round cell tumors. If you can go further with the identification of the specific cell type (e.g. not just carcinoma, but squamous cell carcinoma), that's great, but not always possible. In fact, with some undifferentiated tumors, even figuring out the classification is not possible.

Tumors of epithelial origin

• These include adenomas (benign), carcinomas (malignant) and adenocarcinomas (malignant and of glandular tissue).

• They usually exfoliate cells pretty easily, so samples are often fairly cellular, the cells are usually present in clumps or sheets, with cell-to-cell adhesion although there can be single cells too. Acinar arrangements may be seen with adenomas adenocarcinomas.

• Cells tend to be large, round to polyhedral, with abundant cytoplasm.

• Examples include squamous cell carcinomas, basal cell tumors, mammary adenomas or adenocarcinomas, and thyroid carcinomas.

Tumors of mesenchymal (connective tissue) origin

• These are mostly different types of sarcomas (with a few exceptions). Cells are seen commonly as single cells, although occasional clusters of cells can be seen. Since these are cells that form connective tissue, it's common that they don't exfoliate very well on needle aspiration, so samples are often not very cellular.

• Cytoplasmic borders tend to be indistinct. Cells often have a "spindle" or fusiform shape, with cytoplasmic tails trailing off from the nucleus. In some tumors, cells are round to oval, especially those originating from bone or cartilage and poorly differentiated sarcomas. It's usually difficult to definitively classify the tissue of origin using cytology.

• Examples include fibromas, fibrosarcomas, osteosarcomas, chondrosarcomas, and hemantiopericytomas.

Round cell tumors

• This category is also known as discrete cell tumors. The reason is obvious-the cells generally appear as individual, small to medium sized, round cells. There are usually reasonably good numbers of cells from a needle aspirate.

• Examples include lymphosarcomas, plasmacytomas, mast cell tumors, histiocytomas, and transmissible venereal tumors (TVT). Melanomas are often in this category as they often are discrete round cells.

Inflammation vs neoplasia? confounding factors

In differentiating between a neoplastic and an inflammatory lesion, remember that neoplasms are often associated with some inflammation either because they have necrotic areas, or they impinge on surrounding tissues or they cause ulceration and surface inflammation (e.g. squamous cell carcinoma). Differentiation can also be made difficult by the fact that inflammation causes reactivity in surrounding cells (also known as dysplasia). Reactive epithelial cells can be mistaken for carcinoma and reactive mesenchymal cells can be mistaken for sarcoma.

Classification of neoplasia: benign or malignant

If you determine that a lesion is neoplastic and have an idea as to whether it is epithelial, mesenchymal or discrete, it is important to determine whether it's benign or malignant, if possible. There are several features that are used as criteria of malignancy. These are not absolute, as some non-neoplastic cells within reactive tissue can contain several criteria of malignancy and some neoplastic cells do not, but they serve as a useful guide when used in context with a particular sample.

General or cytoplasmic criteria of malignancy:

• Variation in cell size/anisocytosis

• Cytoplasmic basophilia

Nuclear criteria of malignancy:

These are a reflection of increased nuclear activity or replication thus some of these criteria also seen in cells undergoing benign hyperplasia.

• Macrokaryosis (large nuclei)

• Increased nuclear:cytoplasmic ratio

• Variation in nuclear size/anisokaryosis

• Multinucleation

• Increased mitotic figures

• Abnormal mitosis

• Coarse nuclear chromatin pattern

• Nuclear molding

• Large nucleoli

• Multiple and/or angular nucleoli