Principle of specimen preparation
To harvest as many viable cells from a specimen for an accurate Cytological assessment.
Accurate interpretation of cellular material is dependent on the following factors:
- Methods of specimen collection.
- Fixation and fixatives.
- Preservation of fluid specimens prior to processing.
- Preparation of material for microscopic examination.
- Staining and mounting of the cell sample.
- Interpretation of the material.
Types of non-gynaecological (general) Cytology specimens:
- Body fluids
- Bronchial washings
- Urine
- CSF
- Sputum
- Cell blocks
- FNA’S
Fixation of Cytology Specimens
The main objective of fixation in cytology is to maintain the morphologic characteristics of the cell in as perfect as possible condition.
Cytology specimen should be smeared on the slide in a thin, even layer and immediately fixed in 95% alcohol or spray fixative. Immediate fixation of cellular material is an essential step for accurate cytological interpretation.
Equal volume of 50% alcohol is added to most fluids for fixation purposes and if delay in transportation is expected.
(50% dilution is made up by mixing equal volumes of 95 – 100% alcohol and distilled water)
Macro description
If slides prepared at peripheral labs, describe the specimen type,(precise site of origin), macroscopic appearance, volume of the specimen, colour, clot present and the number of slides received and/or prepared.
Handling of specimens
Large quantity fluid: Let the specimen stand, this will cause the cells to settle at the bottom. Decant the supernatant carefully, not disturbing the sediment and extract the sediment with a pipette and prepare.
Small insignificant quantity (<0.5 ml) of fluid: Add saline and Cytospin. If the sample can’t be processed the clinician must be informed and comment logged onto Meditech.
When more than one sample is received from the same site, slides must be preparedfrom each sample.
When more than one sample is received from different sites, slides must be labelled accordingly.
Centrifugation: 1900 RPM for 10Min
Cytospin: 1000RPM for 10 Min.
Centrifugation
- In some cases, one will find a small clear portion at the top of the spun specimen (supernatant) and then a “buffy layer” on top of the cell button. If there are malignant cells, they have been found to settle there. Carefully just touch the top of the buffy layer with your pipette and gently suck up some
cells, placing them on a labelled slide Go back in again, this time taking up cells from the bottom of the button or fluid that is there.
- Fibrin Clot: cells may be trapped in the clotted material. Remove the clot and place in a petri dish or on a glass slide. By wringing out the clot, these cells may be released. Using forceps and orange sticks, twist the clot on the slides in opposite directions with a wringing action.
- Sputum : Place sputum in petri dish and view over dark surface. Transfer a small portion of discoloured areas/ suspicious specimen, blood or speckled particles onto glass slides with applicator sticks. Carefully crush specimen between marked slides using a rotation movement with pressure until all large lumps disappear. Slides are then separated by pulling them across each other in opposite directions making a uniform smear.
Cell Blocks
Reagents: Serum, thrombin.
- Centrifuge sample for 10 minutes If the sample was prefixed with alcohol, the sediment must be washed several times with saline before proceeding.
- Add approximately 3 – 4 drops of plasma on top of the sediment.
- Mix thoroughly by pipetting, do not vortex
- Add approximately 3 – 4 drops of Thrombin/Thromborel to mixture and mix well.
- Allow 1 – 5 minutes for the clot to form.
- Place clot into a piece of tissue paper and place in cassette.
- Place marked cassette into container with 10% formalin and take to histology for processing.