Cytology Specimen Processing Flashcards
What does cervical cytology involve?
(3)
Screening for cervical cancer
HPV screening
Cytology triage
What does diagnostic cytology invovle
(3)
Not screening
First line investigation
Sensitive and accurate
- ancillary techniques
What are the two main cervical cytology samples
PreservCyt vial
Buffered methanol
What are the six main diagnostic cytology samples
Joint fluid
CSF
Urine
Body cavity fluids
Respiratory samples
FNA samples
What body cavity fluids can be seen in diagnostic cytology samples?
Pleural
Peritoneal
Acetic fluid
What Respiratory samples can be seen in diagnostic cytology samples?
(4)
Sputum
Bronchial wash
Bronchial brush
Bronchoalveolar lavage
What FNA samples can be seen in the diagnostic cytology lab
Breast
Thyroid
Lymph node
Other organs
Why might a Urine sample be sent to diagnostic cytology lab
Looking for bladder cancer
Why might a CSF sample be sent to diagnostic cytology lab
Looking for lymphocytes, metastatic diseases or lymphomas
Why might joint fluid be sent to diagnostic cytology lab
Looking for ctystals -> goitre and pseudo goitre
How is cervical cytology carried out
(4)
Smear test put in LBC medium
Vial received in lab at specimen reception
HPV testing carried out
If HPV positive needs to be processed, screened and reported
Explain what happens if a smear test comes up HPV positive
(3)
Processed for cytology screening: Slide prep + pap stain
Screening: Medical scientists
Reporting: Pathologist
How do we process our slides in cytology
Smear test
How do we remove blood from smears
Glacial acetic acid
What do we do if we see dyskaryosis on a thin prep
Sent the woman on for colposcopy
How do we screen for HPV
Screen for HPV DNA
Roche Cobas 4800 assay - DNA based
Hologic Aptima HPV test - RNA based
Hologic - Genius (AI digital cytopathology)
What does the Hologic - Genius (AI digital cytopathology) do
This allows us to view cells on a computer screen -> don’t have to set up a microscope
What do we look for on cervical cytology
(4)
Triage test following HPV positive - test of disease
Features of squamous dyskaryosis or carcinoma
Features of glandular abnormality
Refer to colposcopy for any abnormality
What might dyskaryosis indicate
(3)
CIN
- LSIL (low-grade squamous intraepithelial lesion)
- HSIL (high-grade squamous intraepithelial lesion)
Malignancy
What would features of glandular abnormality indicate
CGIN (cervical glandular intraepthelial neoplasm)
Adenocarcinoma
What are the steps in diagnostic cytology
Specimen reception
Fixation
Pre-treatments
Preparations (directs, cytospins, LBC)
Staining
Ancillary requests
Reporting
Give an example of a pre-treatment
(2)
Need to remove mucous from respiratory sample
May need to centrifuge sample
What kind of prep is done on a LBC
Thin prep
What is carried out at specimen reception
Minimum data check
All specimens are allocated a unique lab identifier
Gross description (volume/number of slides, colour, consistency)
Why is inspection of samples so important at reception
Dictates pre-treatment and preparation technique
List some specimen considerations
(8)
Collection method
Time lag
Storage
Volume
Consistency
Contaminants
Microbiological hazards (safety cabinet + PPE)
Pre-treatments
What contaminants can be found in specimens
Blood
Mucous
Clots
What are the three main methods of fixation used
Alcohol based
Combinations
Formal calcium - lipids
What are the two main alcohol based methods of fixation, and when are they used?
IMS/ethanol
- cytospin
Methanol
- thinPrep
- air dried
What is a combination method of fixation
Acetone/methanol
What are the benefits of alcohol fixation
Rapid alcohol fixation:
- retains nuclear morphology
- retains cytoplasmic structures
What is an MGG stain
May Grunwald-Giemsa
Can’t do this type of stain on an alcohol fixed sample
Need fresh sample
Need air dried sample so cells will expand
Then fix cells
Write about fixation in cytology
(3)
Irrespective of preparation method, all samples must be optimally fixed to preserve cells
pre or post-fixation
Relating to staining method
Important to use correct fixative
Only exception is flow cytometry as we need to keep cells viable
How do we treat blood contamination
(6)
Haemolyse the red blood cells
Use cytolyt - it’s haemolytic
Multiple washes with cytolyte will give you a clean cell pellet
Then transfer your cells to slide
Can use commercial lysing agents
Can remove by use of density gradient -> lymphoprep will give a monolayer of cells (buffy coat)
How do we treat mucous contamination
Mucolysis
Use Dithiothreitol (DTT) in alcohol
This can be done for MGG staining
What are the three ways of preparing samples
Cells in suspension -> transfer these to a slide for visualisation
Concentration of cells -> centrifuge to get a cell pellet
Slide preparation methods -> cytospin, ThinPrep, direct spread
What does a pink tinge to your sample mean
Red cell contamination
Need to do another cytolyte wash
How does a cytocentrifuge work?
(4)
Cells are alloquated into the funnel
Cells get splattered onto the slide
Filter paper absorbs extra cells
Might do 2 or 3 cytospins for each sample
What is the papanicolaou stain and what are the benefits?
Alcohol fixation (IMS)
Immediate fixation
Good nuclear morphology
Good cytoplasmic contrast and features
What is the May-Grunwals Giemsa stain and what are the benefits?
(6)
Air dry prior to fixation
Methanol fixation
Poor nuclear morphology
Good architectural features
Good for small cell populations e.g lymphocytes or small cell carcinoma
Good for amplifying large malignant cells e.g. adenocarcinoma
What are the components of the papanicolaou stain and what do they stain?
(3)
Haematolylin - nuclear stain
OG6 (Orange 6) -> cytoplasmic stain for keratin/pre-keratin
EA50 (Eosin and light green) -> glycogen and immature cells stained green (protein stain)
What are some ancillary techniques?
IHC
Cell blocks/clots
Flow cytometry
ISH/FISH
Molecular
Give seven IHC stains/groups of stains used?
Cytokeratins
BerEP4
S100/HMB45/Melan A
ER/PR Receptor/Her 2 Neu
TTF-1/p63/Chromogranin/PD-L1
Calcitonin/Thyroglobulin
Lymphoid markers
What panel of marker is used for melanomas
S-100
HMB-45
Melan A
What is BerEp4 used for?
(2)
Histology stain for diagnosis of basal cell carcinoma
Its an antibody to EpCAM (epithelial cell adhesion molecule)
What stains would be used for breast cancer panel
ER receptor
PR receptor
Her 2 Neu
What is TTF-1
Thyroid transcription factor-1
Regulates transcription of genes specific for thyroid, lung and diencephalon
What are the advantages of tissue blocks
Can take more sections if needed to get a wider IHC panel
Immunocytochemistry can be done -> blocks often used to address QC issues
Long term storage - can go back to sample after years
What are the uses of flow cytometry
Quantification of lymphocytes
Immunophenotyping
- reactive and lymphoma
- classification of lymphoma
Very useful in Hodgkins and Non-Hodkgins lymphoma as there are many different classifications
What is ISH/Molecular
(4)
One of the developments in the lab for tumour typing
Uses new advances in genomics
Used for breast, lung (ALK), lymphomas, bladder tumours (UroVysion)
Helps predict response to therapy and risk of recurrence
What is ALK
Anaplastic lymphoma kinase
What are four main aspects of quality control
Stain check
Specimen preparation check
Specimen/form checks
Sign out
What is IQC
Internal control processes and procedure
How the lab ensures quality
What is EQC
External Quality Control
UKNEQAS e.g. staining and interpretive/reporting schemes
How should fresh or preserved specimens be stored
In a fridge for up to 1 week
How should fixed specimens be stored
Can be stored at room temperature but this is dependent on the medium
Retain until reported
How should cell blocks be stored
Stored long term as tissue blocks
How should slides be stored
Stored indefinitely
But there will be some deterioration e.g. colour fading
What is cell analysis
Techniques for preparation and analysis which spans across into non-hospital laboratory setting and research
Give some methods of cell analysis
(4)
Cell culture work
Morphological analysis of cells
Biochemical analysis
Molecular biology based analysis
- Genomics (DNA/RNA)
- Proteomics
