Cytology is the microscopic examination of cells obtained from the body by aspiration or scraping for diagnostic purposes, such as cancer of the uterine cervix. The main advantage of this test is that the collection of cytological specimens does not require anesthesia, is painless and there is no bleeding neither inflammation.

To carry out a cytology, three steps are required:

  1. Specimen collection.
  2. Use a proper technique: procedure, preparation of smears, fixation, and cytological staining.
  3. Microscopic evaluation of smears.

Performing a cytological staining

To perform a cytology staining, stains reveal the structures of the cells to examine such as the nucleus, the cytoplasm, and cellular granules. Experience is necessary to obtain an optimal smear, a fine balance between too thick and too thin smears.

After preparing the smears, fixation, and staining is essential. Papanicolau (pap) cytological staining is one of the most used procedures and comprises of several synthetic dyes that are either acidic (anionic) or basic (cationic) and with an affinity for the components of the cells. Basic dyes have an attraction for nucleic and ribosomes which have negative charge; whilst the acidic dyes have a propensity for cytoplasm, mitochondria and cilia that have a positive charge.

 

cytological staining

 

Steps for a cytological staining

Pap stain is recommended for routine diagnosis. The general steps for a cytological staining per-se are the following ones:

  • Fixation: the cytology smears are fixed in 95% ethyl alcohol or other substitutes for a minimum of 15 minutes.
  • Staining: the cytological staining differentiates cells structures playing with different dyes.

Nuclear staining: is done by using a hematoxylin stain. Its modified form is used in Papanicolaou staining in regressive method, in which we deliberately over the stain with hematoxylin and remove the excess stain by using a differentiating solution such as acid alcohol (0.05% HCl in 70% ethyl alcohol) or 0.05% aqueous solution of HCl alone.

Cytoplasmic staining: cytoplasmic stains are OG-6 and EA-36. Both are synthetic stains and OG-6 is a monochrome stain while EA-36 is a polychrome stain.

  • Dehydration: dehydration rinses the smears in absolute alcohol for two or three changes for the removal of water. Alternative to 100% ethanol is 100% isopropanol and 100% denatured alcohol.
  • Clearing: cells are not transparent while the smear is in the staining or alcohol solutions. During clearing, Xylene replaces the alcohol , which is also miscible in the mounting medium.
  • Slide mounting: after staining, slides are coverslipped with a mountant that bonds them and protects the sample from air drying, oxidation, or fading of the stain. 

Which are the critical points of a cytological staining?

Rapid fixation in alcohol (wet version) is essential for pap staining, which brings out nuclear details clearly, allowing better identification of malignant cells. However, this is a critical step if the smears are not quickly made and fixed, the cytoplasm takes up more eosin (red color), and nuclear details are less clear. Hence critical steps for evaluation of a smear are its poor preparation, fixation, or staining.

 

The automatization of a cytological staining in a small or medium laboratory: the PolyStainer 

Automating slide staining is a must for labs that wish to increase their yield. IUL’s PolyStainer performs standardized staining procedures that free lab staff from labor-intensive staining procedures. General microbiology and histology labs that seek to reduce sample staining workloads will benefit from its use.

Load up to 20 microscope slides in the PolyStainer basket. Next, the robotic arm will dip and stir these through four exchangeable plastic wells. Dip the slides in the fifth drainable water, which renews after rinsing each slide basket. Last, slides can be fan dried with or without heat. Program the PolyStainer according to your intern protocol.

immunohistochemistry