Cytology Sample Collection, Processing, Staining
Cytology and Sample Processing
Hematoxylin Staining Process
Hematoxylin (Htx) is a colorant used to be oxidized to Hematein (chromogen = benzene + chromophore groups). This process can occur spontaneously through contact with atmospheric oxygen or through the use of oxidants (e.g., mercuric oxide, potassium dichromate). Oxidation causes the appearance of an aniline molecule which acts as a chromophore and has the property to stain. The oxidation of Hematoxylin is called maturation. To the Hematein is added a potassium aluminum mordant (third step, auxochrome groups) to convert it into Hemalum (colorant).
Bluing is the use of different alkaline solutions (e.g., water, lithium carbonate) which is why Hematoxylin-Eosin (H&E) uses blue for the nucleus. Water wash is used to remove the differentiation solution. Review the solution and the color change. Water is not used for washing during differentiation. Differentiation is necessary because the basic Hematoxylin solution must be removed. An acid solution is used, and alcohol is used to remove excess acid and loaded colorant.
Cytodiagnosis
Cytodiagnosis interprets lesions present in cells obtained by exfoliation through isolated clinical procedures. Its advantage is to perform a much broader and repeated study of the lesion. It is simple, fast, minimally invasive, and low cost. It allows rapid identification of most lesions.
Cytopathology
Cytopathology is the part of Anatomical Pathology that studies various morphological alterations through cells. Types of cytological samples and procedures to obtain cells:
Cytological Sample Collection Methods
- By forced exfoliation: rubbing or scraping with various instruments.
- Through spontaneously emitted liquids containing desquamated elements (Sputum, urine).
- Through liquids extracted via endoscopy or exploration instruments, containing cell suspension from organic areas. These internal liquids can originate spontaneously in the area or result from exudates washed with saline.
Specific Sample Processing
Bronchoscopy Samples
Bronchoscopy (bronchial endoscopy) is performed to study the interior of the bronchi. Sample Types:
- Bronchial Aspirate (BAS): Bronchial cell aspiration in the lab. The aspirated content is centrifuged, and the obtained sediment is spread on a slide and then fixed in 96% alcohol.
- Bronchial Washing (BAL): A fibrobronchoscope is introduced, and a saline wash is injected into a lung segment.
- Bronchial Brushing: A brush is introduced through the bronchoscope, scraping the lesions. The cells remain on the brush bristles. Spread on a slide and fix in 96% alcohol.
Fine Needle Aspiration (FNA) Processing
Fine Needle Aspiration (FNA) of lesions in organs without direct access or spontaneous exfoliation (breast, thyroid, lymph nodes). FNA of solid organs like breast and lymph nodes is air-dried, and the material remains unstained for 2-3 minutes. Air drying can also damage the fixation for Pap staining with 96% alcohol.
Sputum Processing
Sputum: two study techniques:
- Direct Smear: Sent to the laboratory without fixing. First, areas with peculiarities are studied macroscopically. Then, smears are made from these areas and spread in a thin layer on a slide. Fix in 96% alcohol.
- Saccomanno Fixative: Sputum material is mixed with fixative (2% Carbowax in 50% alcohol). The mixture is homogenized and processed like any liquid sample.
Smear Classification: Bethesda System
Classification of Smears: The Bethesda System is considered the most appropriate system, based on:
- Sample correctly identified with the patient’s name.
- Adequate clinical information: should include at least age and relevant clinical data.
- Sample must be technically adequate with appropriate spread, fixation, and staining.
- In Pap smears, include squamous cells as well as cells from the transformation zone.
Satisfactory Rating: Meets all the above criteria.
Satisfactory with Constraints: Limited by:
- Lack of clinical information.
- Presence of blood, inflammatory, or contaminating components (e.g., >50% blood, <75% evaluable cells).
- Absence of cells from the transformation zone.
Unsatisfactory Rating: Lack of proper identification, sample damage, or low cellularity.