Cytology & Cytodiagnosis

Diagnostic cytologic staining of Pap smear sample

Cytology is the study of the structure and function of cells, while cytodiagnosis is the use of cytology as a diagnostic tool. Cytodiagnosis relies on well-established, highly specific stains and protocols to facilitate the identification of abnormalities in cells with microscopy. In human cytodiagnosis, there are two areas of cytology (gynecological and non-gynecological) in which specimens are examined for the presence of malignant and premalignant cells.

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Cytologic Sampling

In cytodiagnosis, the cells are extracted from the tissue mass, or sample fluid and are transferred to a cytological slide, stained, examined, and evaluated. The architecture of the original tissue becomes unrecognizable, and cannot aid in the evaluation process. Specimen materials such as sputum, urine, body cavity effusions, and lavage material are centrifuged, and the sediment is subsequently smeared onto a microscope slide. Image-assisted or simple fine‑needle aspiration biopsy (FNAB) material from the breast, thyroid, lymph nodes, prostate, cerebrospinal fluid, and other locations are carefully smeared onto slides. Depending on the staining method, the smeared samples are either fixed immediately (Papanicolaou’s stain) or thoroughly dried in air prior to hematological or any other staining. 

Fixation for Cytology

Diagnostic cytologic staining of Pap smear sample

A precondition for exact cytological diagnosis is the perfect fixation of sample material. Specimens must be fixed immediately after collection to prevent cells from drying out and shrinking. Immediate fixing retains the specimen’s structural features for clear staining and differentiation. Staining artifacts can interfere with diagnosis if specimens are fixed too late. The classic method of fixing is to immerse the microscope slide in 96% ethanol for 30 minutes. A more efficient way is to fix cells with a spray fixative. Spray fixatives are aqueous-alcoholic solutions containing polyethylene glycol (PEG). They are suitable for all types of cytological materials stained by the Papanicolaou method.

Cytodiagnosis Staining Methods

The choice of stain largely depends both on specimen origin and the experience and preference of the investigator. For instance, Papanicolaou’s staining technique is not only used for gynecological smears, like PAP smears, but also is routinely used for non-gynecological material, such as sputum, cerebrospinal fluid, synovial fluid, and urine. Giemsa’s stain is widely used for FNAB specimens from lymph nodes. Pappenheim’s stain is used for urinary sediments, effusions, bronchial lavage material, and FNAB material from various locations (e.g., breast, thyroid, cerebrospinal fluid). Wright’s stain is a hematological stain which can be used for differentiating blood cell types. Stained cells are prepared for microscopy using standard alcohol/xylene dehydration, clearing, and mounting. In addition to manual staining methods, mechanical and automated staining systems can be used to prepare large numbers of samples. A fully automated evaluation system can then be used for screening, wherein stained samples are evaluated, abnormal cells or groups of cells are marked, and images are saved in a gallery for retrieval and further investigation.

Cytodiagnostic Interpretation

Microscopic examination of the material can be performed immediately depending on the fixation, preparation, and staining techniques employed. This feature makes cytodiagnosis suitable for large volume screenings, such as cervical screenings. One critical point in cytodiagnosis is that the results of cytological investigations relate directly to the location from which the specimen was collected. For the sake of efficiency, sampling and preparation techniques must be thoroughly mastered and controlled. Suitable controls should be used with each application to avoid an incorrect result.

The success and efficiency of diagnostic cytology are measured by its ability to detect the early changes in disease state (sensitivity) while simultaneously preventing false-positive diagnoses (specificity). The sensitivity and specificity rely on proper sample collection, fixation, staining, and interpretation. Alongside imaging techniques such as x-ray imaging, computed tomography (CT), ultrasound, nuclear spin tomography, and positron emission tomography (PET); cytology is an indispensable part of the diagnosis. 

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