TIPS AND TRICK FOR GETTING THE MOST FROM CYTOLOGY IN PRACTICEBalázs Szladovits, DVM, FHEA, MRCVS, Diplomate ACVP (Clin Path)Senior lecturer in clinical pathologyDepartment of Pathobiology and Population SciencesRoyal Veterinary College, Hawkshead Lane, North Mymms, AL9 7TAbszladovits@rvc.ac.uk

IntroductionCytology is a great diagnostic tool in clinical practice, given the minimal invasiveness to the patient and relative low cost of the procedure. In order to get the best out of this diagnostic tool, it is useful to avoid common sampling mistakes, and for in house assessment, staining and microscope setup is critically important. Developing key microscopic skill can also greatly help with efficiency, as available time is always a limitation.

Sample qualityCytology greatly relies on sample quality, which is mostly linked to the skill of the collector. People have been taught for many years to use significant amount of suction during sampling, and also commonly use forceful spraying action to make the preparations. Fortunately it is now getting more widely recognized, that this old technique usually harms the sample and results in inferior smears, thus hindering the clinical utility of cytology. One of the main goals during sampling is to collect a representative sample, thus targeting only a single area can lead to e.g. sampling only the necrotic center of a mass (this can be useful clinical information but not the full picture). It is also key to try to avoid blood contamination of the sample, as this makes the microscopic assessment much more difficult and can cause erroneous interpretation (e.g. if there are many neutrophils present in the peripheral blood). Making the smear is another common phase where errors are commonly made by either not spreading the cells well enough, or causing too much damage to the cells. Only intact cells are assessed during the microscopic examination. Excised tissues can also be examined cytologically with the help of an imprint. It is essential to have a fresh cut surface, which needs to be blotted dry, in order not to only “imprint” the tissue juices and blood coming to the surface.

During in house examination, the quality of the staining is another common limitation, especially when Diff-Quik type stains are not being refreshed frequently (about every 2-3 weeks) or the jars that hold the stains are not cleaned properly at the time of the change. The first jar is the alcohol fixative, which tends to evaporate quicker, thus important to keep it closed. Replacement of the entire fluid is important at the time of refreshing the whole set, even if it was recently topped up. The longer the slide is in this jar, the better, thus the number of dips is only important if somebody is in a hurry. The red stain (2nd jar) can not overstain, thus the numbers of dips again only important to avoid understaining. The blue jar is where the exposure time is important, as it is possible to both under- or overstain the slide. If somebody finds a mirrory film on top of the blue stain when it is opened first in the morning, it must not be pushed into the fluid with a slide, but blotted (soaked up) with

a paper towel. The flushing of the slide (4 th jar) meant to be done with distilled water, but tap water appears to work as well.

In case of sending cytology samples to a diagnostic laboratory, sample handling can also introduce problems. Keeping glass slides in the fridge or exposing them to cold during shipping can cause condensation of water on the cold glass when it is processed in a warm environment, which will lyse the cells. Also formalin fumes during collection or shipping will ruin cytology or hematology preparations (e.g. having a formalin jar open in the vicinity). Lastly, breakage of slides during shipping will obviously prevent the chance to reach any conclusions.

Microscopic setupKnowing the correct setup of the microscope makes a huge difference in the quality of the image we are getting, thus the ease of making cytologic diagnoses. Most common mistake is to have the condenser down (as used for urine sediment analysis), which gives too much contrast, and thus hides the cell details, making cytology or hematology smear examination very difficult. Most scopes have a dial or small lever on the actual condenser as well, which has the same effect as lowering the condenser, thus this need to be open, too. Lastly, a commonly missed fact is that the 40x dry objective needs an extra layer of glass (i.e. coverslip) to give a sharp image – this can be just put on top of the slide, no need to glue it down. Using a 50x oil lens (quite expensive) makes the diagnostic use of the microscope much easier.

Key microscopic skillsDeveloping these key skills can greatly increase efficiency, thus making somebody much more likely to use this modality, vs. wasting lots of time tends to kill the desire. A useful tip is to look at a cell, cluster or structure on high magnification, so our brain can develop an image, but then also look at the same thing on one or two magnifications lower – you will be surprised, that one can still see the exact same image, but will appear to be further away. Practicing this will lead to one being able to “see” more on lower magnification. Anything that can be done on one lower magnification increases the efficiency exponentially. Another key skill is to screen the slides on the lowest magnification we have (e.g. 4x or 10x), and being able to recognize/find the area where there are many intact, well spread and well stained cells present. The quicker one can find this “window”, the more one will be able to use the available time to get the most out of the slide. Being able to judge sizes can also greatly help with recognition of cells and/or comparison of cells to atlas images. Erythrocytes are almost always present, thus comparing e.g. the diameter of a nucleus to the diameter of a red blood cell can help to realize whether the cells are small or large (e.g. identifying a cells as a macrophage vs. plasma cell commonly causes difficulty in beginners, but the former is a large cell, while the latter is a small one).

Currently there are several excellent color atlases of veterinary cytology available on the market, the use of which is strongly recommended to develop our knowledge and help with the recognition of lesions in the microscope. Recognition of the most common skin lesions (lipoma, follicular cyst, mast cell tumor) can be achieved relatively easily, while more complex cytology can always be referred to specialists.