Cytopathology

Cytopathology is a branch of cytology and pathology that studies and diagnoses diseases on the cellular level.

A common application of cytopathology is the Pap smear, used as a screening tool, to detect precancerous cervical lesions and prevent cervical cancer. Cytopathology is also commonly used to investigate thyroid lesions, diseases involving sterile body cavities (peritoneal, pleural, and cerebrospinal), and a wide range of other body sites. It is usually used to aid in the diagnosis of cancer, but also helps in the diagnosis of certain infectious diseases and other inflammatory conditions. Cytopathology is generally used on samples of free cells or tissue fragments, in contrast to histopathology, which studies whole tissues.

Rudolf Ludwig Karl Virchow is considered by many to be one of the fathers of cellular pathology, remembered most for his collection of lectures on the topic, published as Cellular Pathology in 1858.

Testing

Cytopathologic tests are sometimes called smear tests because the samples may be smeared across a glass microscope slide for subsequent staining and microscopic examination. However, cytology samples may be prepared in other ways, including cytocentrifugation. Different types of smear tests may also be used for cancer diagnosis. In this sense, it is termed a cytologic smear.

Collection

This human blood was fractioned by centrifugation. It shows plasma (upper layer), a buffy layer (middle, white colored layer), and erythrocyte layer (bottom).

Two primary methods are used for collecting cells for analysis:

1. Exfoliative cytology: In this method, cells are collected after they have been either spontaneously shed by the body ("spontaneous exfoliation") or manually scraped/brushed off of a surface in the body ("mechanical exfoliation"). An example of spontaneous exfoliation is when cells of the pleural cavity or peritoneal cavity are shed into the pleural or peritoneal fluid. This fluid can be collected via peritoneal washing for examination. Examples of mechanical exfoliation include Pap smears, where cells are scraped from the cervix with a cervical spatula, or bronchial brushings, where a bronchial brush is inserted into the trachea to collect cells from its surface and subject them to cytopathologic analysis.

2. Aspirative cytology: More specifically referred to as fine needle aspiration cytology (FNAC), the aspirative technique tends to be more invasive than exfoliative techniques. In FNAC, a hypodermic needle attached to a syringe is used to collect cells from lesions or masses in various body organs by microcoring, often with the application of negative pressure to increase yield. FNAC can be performed under palpation guidance (i.e., the clinician can feel the lesion) on a mass in superficial regions like the neck, thyroid or breast. FNAC may also be assisted by ultrasound or CAT scan for sampling of deep-seated lesions within the body that cannot be localized via palpation.

FNAC is widely used in many countries, but success rate is dependent on the skill of the practitioner. If performed by a pathologist alone, or as team with pathologist-cytotechnologist, the success rate of proper diagnosis is superior than when performed by a non-pathologist.

Preparation

Numerous methods exist for the initial preparation of collected cells for examination. However, we'll separate them into two categories:

1. Direct smear techniques: While many methods of smearing samples on to a glass slide have been used over the years, two primary uses persist: mucoid and fine needle aspiration smears. Mucoid smears typically focus on sputum and other bronchial specimens, using either a "pick and smear" method or a "mucolysis" method. This method often may be used for exfoliated samples. Fine needle aspiration smears utilize samples from FNAC collection and require a specific, detailed set of protocols to ensure proper diagnostic sensitivity.

2. Cell concentration techniques: These types of techniques tend to be more popular as they provide greater diagnostic sensitivity and this better results. Various types of centrifugation are used "to facilitate the deposition of the suspended cells onto the glass slide." Putting the sample into a centrifuge usually results in layering of the sample, including a vital "buffy" layer containing any white blood and tumor cells present in the sample. A much more concentrated smear can then be made. In some cases, however, the sample may be too small to create a clear buffy layer, requiring a special process called cytocentrifugation, which forces a fluid suspension directly onto a glass slide for analysis. In other cases a density gradient fluid must be introduced before centrifuging in order to better separate thin layers by specific gravity. Other methods include gravity sedimentation, filtration, and cell blocking (as if it were a histological sample).

An additional initial form of preparation exists for the specific case of examining joint aspirates and other samples containing poorly soluble crystals. This "wet preparation" involves placing a drop of the fluid sample onto an unstained slide and then placing a coverslip over the sample, immediately viewing the sample using polarized light microscopy.

Staining

In this example, Burkitt lymphoma is detected from an FNA-retrieved, wet-stained Pap preparation.

While wet preparation is enough in samples containing poorly soluble crystals, the majority of samples initially prepared by direct smear or cell concentration require additional steps to ensure the cells and their constituents can be seen properly under the microscope. Staining is the primary secondary preparation for such cases. Staining typically involves the "washing" of the slide in several solvents to better detect cells that would otherwise remain invisible to the eye when viewed under a microscope.

For wet-stained preparations, two methods of staining are common: the Papanicolaou (Pap) stain and the haematoxylin and eosin (H&E) stain. Pap stains tend to be the most popular for cytopathologic testing due the color range that can be created with it. An H&E stain has the advantage of providing a histological point-of-view for those pathologists accustomed to interpreting histology specimens. For dry-stained preparations, a group of hematologic stains called the Romanowsky stains are used. These types of stains also provide the benefit of a clear, quick analysis for determining specimen adequacy.

Additional techniques are used in special cases where wet- and dry-staining techniques prove inadequate. In the case of serous fluid or FNA diagnosis, one or more antibodies may be applied to the sample (immunocytochemistry) to create a direct visualization via antigen-antibody reactions. Molecular techniques like Southern and Northern blot analysis can also be applied, though molecular cytology is still a new science.

Dehydration, clearing, and mounting

After staining a preparation, it must be finalized for proper viewing under the microscope. This includes the following three closing processes:

• Dehydration: This process involves removing residual water on the slide (using absolute alcohol or a similar substance) in order to ensure clearing properly takes place.
• Clearing: This process renders the preparation optically clear. This is done by replacing the alcohol from dehydration with a solvent that has the same refractive index as the stained material.
• Mounting: A coverslip or appropriate tape is placed over the preparation to ensure optimal flatness for examination.

Examination

The common technique a trained pathologist or clinical technician will use to examine a cytopathologic preparation is by polarized light microscopy. If prepared properly, the components of the sample should appear properly under microscopic view. In rare cases, electron microscopy may be used for tumor sub-classification; however, "various cells in effusion fluids may not be identified and located for proper ultrastructural evaluation."