Cytologic examination is a minimal invasive and cost-effective diagnostic screening tool.
With some systematic preparation the sampling procedure is easily performed everywhere and anytime.
In contrast to histopathology a cytological diagnosis may be established in-house with some training or within a shorter period of time by a specialist in the laboratory. The aim of cytology, however, is not to replace a histopathologic examination!
In many cases a definitive diagnosis can be established immediately, in other cases infor-mation on the general nature of the disease (inflammatory, degenerative, neoplastic) or at least guidance for further diagnostic procedures is achieved.
The diagnostic validity of cytologic examinations depends on various factors. With some planning and practice these factors can be optimised in order to manage samples successfully to achieve a most conclusive diagnostic interpretation.
Few tools are necessary for cytologic sampling:
- Needles (20 G to 22 G)
- Syringe (2 or 5 ml)
- Glass slides
- Pencil or permanent marker for glass slide labelling
- ev. EDTA-tubes
Fig. 1: Sampling tools
Key-Point: Prior to sampling, all necessary materials should be set up in such a way that the cytologic slides can be prepared quickly. Take care not to let the sample dry out or coagulate prior to slide preparation.
When lesions in different locations are sampled or different sampling or preparation techniques are used, it is extremely important to label the slides accordingly (best: in advance) in order to avoid confusion later.
The preferred method of slide preparation depends on the type of lesion to examine.
In superficial lesions touch imprints, scrapings (for native examination) and cytobrush/swab imprints are performed.
Solid masses are sampled by fine needle aspiration biopsy (FNAB) or fine needle biopsy (FNB).
In case of surgical removal of a nodule or obtaining a core biopsy it is also possible to prepare touch imprints. The cut surface of the specimen is blotted first on a paper towel to remove excess fluid and afterwards multiple times to the surface of a glass slide prior to formalin fixation.
Key-Point: Cytologic specimens must never get in contact with formalin. Even formalin fumes will alter morphology and staining properties of the obtained cells completely. Always prepare, store and send cytologic preparations separately from formalin containers!
Touch imprints are best made before and after cleaning of a superficial lesion (slide labelling!). A slide is touched softly onto the ulcerated skin. Care must be taken not to wipe over the lesion, because cells are easily destroyed in that way and there are only chromatin strands visible on the cytologic preparation.
If big crusts are present, their lower surface should also be imprinted, because some micro-organisms e.g. Dermatophilus congolensis are detected frequently in this location.
Scrapings are performed predominantly in order to detect ectoparasites, sometimes fungal hyphae or spores can be found, too.
The affected part of the skin is clipped, if necessary, preferably without removing scales or crusts. A scalpel blade is prepared with few drops of paraffin oil, and with this instrument the skin is scraped several times, depending on the desired depth of the scraping.
The sampled material is applied to a glass slide, maybe with another drop of liquid paraffin, a coverslip is applied. This preparation is scanned unstained in the microscope at 4x or 10x magnification.
Indirect smears are made by gently rolling a swab or a cytobrush over the surface of the lesion and afterwards transferring the gathered material onto a glass slide, likewise rolling it gently over the surface. Prior to sampling, very humid surfaces are blotted dry with lint-less cloth (e.g. watery eyes) in order to avoid drying artefacts on the smears. If the surface of the lesion is very dry the swab or cytobrush can be moistened with some drops of saline to avoid cell rupture during the sampling procedure.
FNAB and FNB
For performing a FNAB the tip of a needle attached to a syringe is inserted into the tissue of interest, that is well fixated by the other hand. The plunger of the syringe is retracted in order to create negative pressure.
Now the needle is advanced repeatedly in different directions of the lesion in order to sample cells of various regions.
The needle must not be drawn out of the lesion as long as negative pressure is applied! Otherwise the aspirated material would be drawn into the syringe, from where it can hardly be retrieved again.
After releasing the vacuum the needle is withdrawn, the syringe is disconnected and filled with air. Now the needle is again attached to the syringe and the sampled material blown out of the needle gently onto to a glass slide, preferably near to the frosted end. In doing so the tip of needle should touch the glass slide in order to avoid spraying of the sample over the slide, because in that way the material dries before it can be spread. The dried droplets are usually to thick for cytologic examination.
Fig. 2: FNAB and squash preparation
A second glass slide is placed over the specimen without applying a lot of pressure. Then the top slide is glided along the surface of the slide that contains the sampled material or both slides are drawn apart in opposite directions. (Fig. 2).
In case too much pressure is applied the cells will rupture during preparation and the cytologic examination will reveal nothing but chromatin strands and nuclear remnants, which’s origin is usually not identifiable anymore.
If much material was sampled, it is best divided on several slides so that the cells may be smeared in a satisfactorily thin manner.
The aim is to prepare the sampled cells in a monolayer. In case several cell layers are present one over another, it is not possible to examine the morphology of the cells, especially the nuclear criteria of malignancy, adequately (cf. Figure 4).
In highly vascular tissue, or if good exfoliation of cells is expected (e.g. in lymphnodes) FNB (fine needle biopsy) is the preferred method of sampling.
In contrast to FNAB technique FNB is performed by placing a needle without syringe into the lesion. While inserting and retracting the needle in different directions it is rotated in order to punch out enough cells.
The further preparation is performed like described for FNAB.
If more than one lesion is present, each of them has to be sampled. In this case special care has to be taken: correct slide labelling is necessary and localisation and sampling sites / labelling should also correlate with the anamnesis.
Masses should be aspirated in more than one location, whenever size allows. Rapidly growing neoplasia sometimes yields only necrotic debris centrally, which is not diagnostic. On the other hand, inflammatory lesions can be surrounded by highly active mesenchymal proliferation that can even mimic sarcoma.
In generalised lymphadenopathy, mandibular nodes should not be chosen as sole location for biopsy. Frequently there will be inflammatory changes in nose or throat, contributing reactive changes to the lymph node populations that can make diagnosis more complicated. If possible, other lymph nodes should be sampled preferentially (please note down the locations).
If aspiration yields fluid, the sample should be placed into an EDTA-tube immediately to avoid clotting. Clotting alters the total protein content and cell count. If a mass was aspirated, a second sampling attempt, aiming for firm tissue should be made, both samples can then be spread out and examined.
Key-Point: From liquid samples, smears should always be prepared at the time of sampling and sent air-dried, together with the fluid.
Stability of cells in fluid is variable and can cause morphologic artefacts: especially fragile cells, like most tumor cells can lyse, making diagnosis impossible or erroneous. Macro-phages or mesothelial cells can be activated in vitro, and start phagocytosis or even displaying criteria of malignancy. And, finally, fresh-prepared smears are important to discriminate septic inflammation from contamination with in vitro growth of microorganisms.
Slides can be prepared by different methods, according to the likely cell content and the viscosity of the liquid.
A direct smear is always important, to estimate the cell count. The sample is spread like a blood smear: place a small drop of sample close to one end of the slide, then a second slide is placed on top in an angle of app. 45° and brought towards the droplet. Allow the fluid to spread out along the edge of the glass and then slide the upper glass in a smooth movement, spreading out all of the material. (Fig. 3).
Fig. 3: Blood smear technique
Samples with low cellularity should additionally be spread with a „stop“. To achieve this, the sample is spread like described above, but instead of spreading all material, the spreading stops after two thirds of the slide, leaving a small amount of fluid in a line. In this line cells will be concentrated – especially high-volume cells like neoplastic cells or macrophages.
Another method of concentration is a sediment smear. An aliquot of the sample is centrifuged at 200-300 G (app. 1500 rpm in a standard small centrifuge) and the supernatant is carefully decanted. The pellet is then resuspended with the remaining fluid and slides are prepared.
Viscous material will be more difficult to spread, the upper glass slide can be held less steeply, and pushed forward slower, making the smear longer and thinner.
Very viscous samples can also be spread by squash preparation.
Key Point: Smears need to be dried at room temperature, prior to staining and/or shipping. If samples are packed while still moist, cells will suffer a wide variety of artefacts, often resulting in destruction of cells and non-diagnostic results.
Staining the specimen
A variety of Romanowsky-type stains are available that are well suited for cytologic specimens. (e.g. Diff-Quik®, Hema-Quick®).
The staining procedure will usually follow the manufacturer’s instructions, but a few adaptations can be helpful:
Dense, highly cellular smears may need longer staining time, also weakened staining after long use of the solutions can sometimes be compensated by longer, or more frequent dipping.
Fig. 4: Lymph node; dense multilayer, understained slide. Cellular detail
can not be evaluated.
Good quality of staining is prerequisite for good diagnostics! If the microscopic evaluation shows that colours are too pale, the staining procedure can simply be repeated.
A convenient way for maintenance of staining solutions is to use aliquots and store them in airtight containers (e.g. urine sampling beakers, plastic cups with snap on lid).
However, if staining solutions are contaminated with bacteria they need to be replaced, contamination happens easily, especially with frequent use or higher ambient temperatures. If samples are sent away for diagnosis, at least one of the slides should be sent unstained, to allow the pathologist to use the stain of their own choice.
Fig. 5: Lymph node; cells are prepared in a monolayer, staining quality is
good, lymphoma can be diagnosed.
Shipment of Samples
Slides should be sent properly packed, suitable break-proof containers are available free of charge from us. Liquid samples should be sent quickly, cool if possible, and accompanied by direct smears.
Points to remember:
• Good preparation is the key to success
• Avoid formalin fumes
• Impression smear: touch, don’t wipe!
• Prepare thin smears (monolayer)
• Don’t apply pressure when preparing smears.
• Put liquid samples into EDTA-tubes ASAP
• Always send freshly prepared smears with liquid samples
• Air-dry slides well before shipping or staining
• Include an anamnesis when sending cytologic samples
Fig.2 from: Cowell & Tyler: Diagnostic Cytology of the Dog and Cat
Fig.3 from: Mischke: Zytologisches Praktikum für die Veterinärmedizin