Cervical cytology and pathology Flashcards
Define cytology and examples of techniques used to obtain samples
Cytology
Cytology is the interpretation of cells removed from the human body through clinical procedures or exfoliation
- FNA – Negative pressure to aspirate cells with fine needle (+/- imaging)
- Pap smear – cells ‘swept’ from cervix
- Urine, body cavity fluids, sputum – exfoliation
- Bronchoscopy – directed brushing or washing
List and briefly describe the stains in cytology
Two most widely used stains in cytology:
- Papanicolaou stain, developed by Dr. George N. Papanicolaou in 1942.
- Complex stain using alcohol fixation
- cells tend to contract and appear more round
- *Diff Quik – modified haematological stain.
- Rapid, portable stain using air dried fixation
- cells tend to spread out and flatten
These stains impart a characteristic colour to nuclei and cytoplasmic components.
Describe the Pap stain and procedure
- 22 pots, 4 dyes in 3 stains, 30 mins
- Four main steps in the PAP staining procedure:
- Fixation 95% alcohol
- Nuclear staining - Haematoxylin
- Cytoplasmic staining- Orange G & EA
- Clearing – Xylene
Haematoxylin demonstrates NUCLEAR details of chromatin (crisp blue/purple)
Orange G stains CYTOPLASM if keratin or prekeratin is present – shows squamous differentiation (yellow to orange). Note other cytoplasmic proteins can lead to orange staining.
Eosin Azure stains CYTOPLASM
▪ Light green – metabolically active(ribosome rich) metaplastic and intermediate squamous cells (blue/green)
▪ Eosin Y- superficial squamous cells, nucleoli, cilia, ribosomes (pink)
Describe Diff Quik stain
- 5 pots, 2 dyes, 15 secs – FNA rapid on-site evaluation
- Fixation – 1st air dry -> 2nd methanol
- Stain – eosinophilic (sol’n 1)+ basophilic dye (sol’n 2)
Demonstrates:
- CYTOPLASMIC features - granules, intracellular mucin/fat droplets
- EXTRACELLULAR features - colloid, free mucin, ground substance
- MICROBIOLOGICAL agents – fungi, bacteria
- INFLAMMATION – leukocytes
List reasons for unsatisfactory cervical smears
- Insufficient cells – inadequate sample
- Excessive blood
- Inflammation
- Lubricant
- Formalin vapour (shared bag with histology specimen)
- Too thick
- Poor fixation
- Cells squashed/smashed in smearing process
Note: infections can also hamper the view, may need repeat sampling
Describe the histology of the cervix
The outer portion (ectocervix) is lined by non-keratinising stratified squamous epithelium. The inner portion (endocervix) is lined by simple columnar epithelium (with apical mucin production, not shown in this image). Between them is the transformation zone or zone of dysplasia. Where the squamous and columnar cells meet is termed the squamocolumnar junction.
Other features of endocervix:
The pale vacuoles with mucin in the apical portion of the cells can also be seen.
Glands, stroma and blood vessels are also visible.
The columnar cells of the endocervix can either resemble typical columnar cells, standing tall, like skyscrapers, or they can have more of a honeycomb appearance if taken as a cross-section.
Describe the histology of the endocervix
The layers of the endocervix and their different cellular morphology can be appreciated in histology as well as cytology. Superficial cells are large and have a low N:C ratio, as they are typically well differentiated, while parabasal and basal cells are much smaller and have a high N: C ratio.
Describe the cytology of the endocervix
On cytology staining, superficial cells appear pink or red, while intermediate/basal/parabasal cells appear blue. This is due to the different pH in the cytoplasm of these cells.
Describe changes to cervix with age
At birth, the columnar epithelium is found within the cervical canal. With hormonal changes in the young adult, columnar cells extend out into the ectropion and are exposed. Squamocolumnar junction is formed outside.
By maturity, exocervix is restored, with squamo-columnar junction returns to its original site. A new transformation zone is formed with regrowth of squamous epithelium. Prone to neoplasia.
Define dysplasia
Clonal, neoplastic mutations driving unregulated
growth, with architectural/maturational and
cytologic abnormalities but is limited by the basement membrane.
Describe LSILs
LSIL
LSIL * Low grade squamous intraepithelial lesion
* LSIL includes
– productive HPV infection and
– CIN 1/ mild dysplasia changes (dysplasia limited to bottom
third of epithelium)
* (CIN = cervical intraepithelial neoplasia)
* Includes some possible cases
* Koilocyte is the definitive cell of HPV infection
- raisinoid abnormal border
- halo where viral proteins displace nuclear material
Describe HSILs
- High grade squamous intraepithelial lesion
- Includes
– CIN2/Moderate dysplasia and
– CIN3/Severe dysplasia (same as carcinom-in-situ) - Includes cases of possible HSIL
- Small cells, atypical, high N:C ratio, irregular nuclear membrane, hyperchromasia, irregular chromatin
- cells can resemble plasma cells
Briefly describe HPV infection in glandular epithelium of endocervix
Three stages:
- infection; not visible on cyto/histology
- AIS; visible on cyto/histology
- invasive adenocarcinoma
Cervical screening not as reliable in preventing invasive AC. May detect AIS
Compare and contrast low and high risk HPV
Low risk HPV:
- types 6 and 11
- usually exist in episomal form in infected cells
- usually cause warts which are either flat or papillomatous, +/- LSIL
High risk:
- at least 13 types: 16, 18 and non- account for 70% of cases
- HPV causes almost all cervical cancers
- capable of integrating into host genome with disruption of cell cycle proteins, hence carcinogenic
- can cause LSIL and at risk of causing HSIL +/- invasive carcinomas, depending on other cofactors
Note that both types of infection are usually asymptomatic.
Recall that from initial infection, HPV can persist from one to five years, in which it may be cleared or cause CIN1. This can either be cleared or progress to CIN2/3, which can either stay as such, be cleared, or progress over decades to cervical cancer where it invades the basement membrane, oncogenes have been activated and DNA has been integrated into the genome.
What may also be picked up on cervical screening?
pathologists will often find commensal or pathogenic flora of the cervix. This may hamper reporting and require another sample.
Examples include candida, t. vaginalis, BV and herpes virus.
