Exfoliative cytology

EXFOLIATIVE CYTOLOGY
  • Exfoliative cytology is the microscopic examination of cells that either exfoliate freely from epithelial surfaces or that are removed from tissues by mechanical means such as aspiration, scraping or flushing.

Objectives

  • Cytological assessment during oestrus cycle
  • Early diagnosis of cancer
  • Recognizing certain infection and allergic reaction
  • Cytological assessment in endocrine disorders to assess the hormonal effects
  • Recognition of sex chromatin Types of specimen

Major categories include

  • Fluid specimens- Ascitic fluid, thoracic fluid, pericardial fluid, synovial fluid, CSF and other secretions and inflammatory exudates Needle aspirations
  • Excisional or incisional biopsy
  • Exfoliative cytology including irrigation of hollow organs, direct smears, scrapings, curetings and pressure massage.
  • Slide preparation from fluid specimens
  • Slides should be prepared as quickly as possible following collection in order to minimize cellular degeneration. Care should be taken to prepare a thin film.
  • The smear should be prepared in a fashion similar to that used for preparation of peripheral blood smears. “Feathered edges” has to be ensured as many large cells accumulate in this area.
  • If the fluid is viscid, slow spreading of the material with another slide may be adequate.
  • With extremely viscid material it may be necessary to make a “squash” preparationby placing another slide on the specimen and by gently drawing the two slides apart.
  • Depending upon the type of stain to be used, the slide is either air dried or fixed while wet.
  • Solid tissue touch preparation/ touch imprints
  • Blot the freshly cut surface of the tumours or solid organs with blotting paper or filter paper. Make smears by gently touching the cut surface with clean grease free glass slide. The slides are then fixed in either – alcohol (1:1) mixture.

Fine needle aspiration biopsy smear

  • Fluids or tissue pieces are obtained by needle puncture and aspirations from deep lesions and then direct smears or touch imprints are prepared.

Bovine cervical mucus examination

  • Cervical mucus smear is air dried and examined under microscope for fern pattern with all its primary, secondary and tertiary branches in normal animal. If endometritis is present, the normal fern pattern will be lost.

Vaginal cytology

  • The canine vaginal smears should be stained by either Romanowsky stain or supravital stain. Supravital staining provides more exact preservation of the details of the cells present. Oestrous cycle stage can be assessed by the type of cells that is exfoliated.
  • The characteristic cellular changes in the various stages of the estrous cycle are as follows

STAGE

CHARACTERISTIC CELLS

Proestrous

Large number of erythrocytes

Estrous

Superficial cornified epithelial cells with a pyknotic nuclei or no nucleus predominate

Metestrous

Parabasal cells containing a neutrophil (Metestrual cells) or vacuole (foam cells) in the cytoplasm are often present

Anestrous

Intermediate and parabasal epithelial cells predominate

Fixation

  • The preferred fixative for fluid specimen is an equal part of diethyl ether and 95% ethyl alcohol.
  • An alternative fixative for fluids is the addition of fresh material to an equal amount of 50% ethyl alcohol at the time of collection. Fixation should be for a minimum of 30 minutes, although slides may be left in the fixative for several days. The fixed material is centrifuged at 1500 rpm for half an hour and after decanting the supernatant smears are prepared from sediments.

Staining of cytological smears

  • The technique selected should be simple and rapid, and the stain should provide sufficient cellular details to permit identification.

Wright's stain

Wright’s stain is suitable for cytological studies and has the advantage of being the stain most frequently used for blood films.

  • Stain preparation
    • Place 0.1 g of powdered Wright’s stain in a mortar. Add 60 mL of methylated alcohol, a few milliliters at a time, and grind the stain for several minutes till all the alcohol is added. Transfer the stain to a tightly stoppered brown bottle and store in the dark for 2-4 weeks. Filter prior to use.
  • Phosphate buffer
    • Na2 HPO4 3.8 g
    • KH2 PO4 5.47 g
    • Dissolve in 500 mL distilled water and make up the total volume to 1000ml
    • Commercial Wright's stock solution may be used. The working solution is prepared by mixing the stain solution and distilled water in the ratio of 1:1.
  • Procedure
    • Place slide on a rack and add sufficient quantity of Wright’s stain to cover the slide. Allow the stain to act for two minutes.
    • Add equal quantity of buffer and mix thoroughly by blowing on the slide until a metallic sheen appears on the surface of the buffer-stain mixture. Allow the diluted stain to react for 3-5 minutes.
    • Float off the scum and wash with neutral distilled water using a wash bottle or beaker. This is to be carefully carried out as excessive washing will destain the slide.
    • Air dry and examine.

Schalm's new methylene blue stain

This is a rapid but non-permanent staining procedure.

  • Stain preparation
    • New methylene blue 0.5 g
    • Normal Saline (0.85 %) 99 mL
    • Formaldehyde (40%) 1 mL
    • Filter and store in a brown bottle
  • Procedure
    • Air dry the slide. Place a small drop of stain on the preparation. Place a cover slip. The slide is ready for examination.

Wright - Giemsa staining

  • Commercial Wright's stock solution may be used. The working solution is prepared by mixing the stain solution with distilled water in the ratio of 1:1
  • Commercial Giemsa stock solution is used. The working solution is prepared freshly each time by mixing 1 ml of stock solution with 9 ml of distilled water
  • The air dried slides are placed on the staining rack and flooded with Wright's working stain solutions for 3 min
  • The slides are washed in running water and flooded with the Giemsa working solution and allowed to act for 20 min.
  • The slides are washed in running tap water and air dried.

May - Grunwald giemsa staining

  • Commercial May - Grunwald stock solution may be used. The working solution is prepared by mixing the stock solution with distilled water in the ratio of 1:1
  • The Giemsa working solution is prepared by mixing 1 ml of stock solution with 9 mL of distilled water
  • The air dried slides were placed on the staining rack and flooded with May - Grunwald working solution for 3 min. Then stain with Giemsa working solution for 20 min.
  • The slides are washed in running tap water and air dried.

H & E staining

  • The wet fixed smears are stained with Harri's haematoxylin for 20 min by placing in a coplin jar
  • After 20 min they are taken out and washed in running tap water
  • The slides are dipped in 1 per cent acid alcohol and washed immediately
  • The smears are kept in running water for 5 - 10 min for "blueing"
  • The smears are then stained with one per cent eosin for 1 to 2 min.
  • The smears are washed in running tap water and then air dried
  • The dried smears are dipped in xylol and then mounted by using DPX mountant.

Papanicolaou stain

  • The stains to be used are Harris haematoxylin, Orange G and Eosin Azure-65.
  • Harris haematoxylin working solution
  • To 1000ml of stock Harris haematoxylin add 4 ml of glacial acetic acid
  • Orange G stock solution
  • Dissolve 10 g of orange G in water and bring to a total volume of 100 ml

Working solution of Orange G

  • Orange G stock solution : 25 mL
  • 95% ethyl alcohol : 25 mL
  • Phophotungstic acid : 75 mg
  • EA-65 stock solution

Prepare 10% aqueous solution of

  • Light green S.F. yellow
  • Bismark brown
  • Eosin Y

EA-65 alcoholic solutions from stock aqueous solutions

  • Light green S.F. yellow: 0.05% solution in 95% ethanol
  • Bismark brown: 0.05% solution in 95% ethanol
  • Eosin Y: 0.5% solution in 95% ethanol

EA-65 Staining solution

  • Light green yellowish alcohol solution : 90 mL
  • Bismark brown alcohol solution : 20 mL
  • Eosin Y alcohol solution : 90 mL
  • Phosphotungstic acid : 1.2 g
  • Filter and store in a brown bottle.

Staining Procedure

  • After fixation, transfer the slide directly into 80% alcohol and through 70% and 50% solution to distilled water
  • Stain in working solution of Harris haematoxylin for 45 seconds
  • Using three separate containers, gently but thoroughly rinse the slide in successive containers of water
  • Rinse the slide in 50% alcohol
  • Rinse in 70%, 80% and 95% alcohol solutions
  • Stain in working solution of orange G for one minute, 15 seconds
  • Using 95% alcohol in three separate containers, rinse the slide three times
  • Stain in EA-65 staining solution for 3 minutes
  • Rinse three times in 95% alcohol using three separate containers
  • Dehydrate using absolute alcohol
  • Clear with a mixture of equal parts of absolute alcohol and xylene
  • Rinse in two changes of xylene
  • Mount in a suitable mounting medium

After staining, the slides are scanned with the low power of a microscope to determine specimen cellularity. Giant cells and polymorphonuclear neutrophilic leucocytes can be identified at this power. The presence or absence of sheets of cells and cell clusters should be noted. Areas for high power examination are selected and examined to identify specific cellular characteristics.

Cytomorphologic criteria for malignancy

  • General criteria:
  • Large cell size: Often the first feature observed
  • Aggregates of cells
  • Monomorphism: Belong to the same cell line
  • Pleomorphism: Variability in cell size and shape

Nuclear criteria

  • High nuclear/cytoplasmic ratio: large nuclei
  • Anisokaryosis: Variability in nuclear size
  • Multinucleation of individual cells
  • Normal and abnormal mitotic figures
  • Nuclear molding or deformation by adjacent cells
  • Nucleoli enlarged or varible in size or shape
  • Thickened nuclear membrane (requires Papanicolaou stain)
  • Nuclear chromocentres vary in size or shape (Pap stain)
  • Cytoplasmic criteria
  • Hyperchromasia

Cytoplasmic inclusions: Phagocytosed materials (cells, cell debris) or secretory products (mucin, melanin).

Last modified: Saturday, 24 March 2012, 7:15 AM