Cytospins are an optimal tool in a modern cytology

Cytospins can be regarded as an optimal tool in a modern cytology practice where immunocytochemical (ICC) detection of several different biomarkers is a standard practice. Using optimized parameters of cytocentrifugation and suspension preparation, a required number of adequate cytospins with enough well distributed, well-preserved cells in a monolayer can be prepared from almost all cytology samples, even from low cellular and heavily haemorrhagic samples. Preparation of several adequate cytospins with identical cell population makes them a perfect tool for ICC. With appropriate manipulation of cell suspension, we can monitor and control deposition of cells on the slide and prepare enough adequate cytospins even from low cellular samples which may yield inadequate cell block sections.

The preparation process is simple and quick, only requiring standard equipment and reagents that would be readily available in cytology laboratories. The only things necessary to prepare the optimal cytospins are knowledge, a good cytocentrifuge and some practice.

A review and explanation of several significant factors affecting cells during cytocentrifugation are presented in an article entitled Principles of cytocentrifugation (1).

The first and very important factor in cytospin preparation is obviously the cell suspension. Cytospins can be prepared from primary cell suspensions (body fluids, e.g. urine, cerebrospinal fluid, serous effusions, cysts) or from secondary cell suspensions, where the cells are suspended in a fixative-based or buffer-based cell medium.

Primary cell suspensions can contain extracellular elements (fibrin, mucus, debris etc.) that can affect deposition of cells on the glass slide, it is always better to prepare a secondary cell suspension where the cell sediment or the sample in the needle/syringe is suspended in the cell medium. This is also an effective way to collect all sample leftovers from the needle and syringe and to wash out some extracellular elements.

Using a fixative-based cell medium enables us to postpone preparation of cytospins, however the buffer-based cell media are far more versatile. Fresh non-fixed cells adhere to glass slides far better than cells that have been fixed. Besides, a non-fixed cell suspension can be used to facilitate red blood cell lysis, or for other ancillary diagnostic methods such as flow cytometry immunophenotyping. There are several different buffers or buffer-based media described in the literature for the preparation of secondary cell suspensions from cytology samples. The most basic requirement is that these buffer solutions are both isotonic and sterile. With the addition of other components, cells may be further protected and kept in ‘good’ condition for up to 48 hours. For example we found addition of bovine serum albumin (BSA) and ethylenediaminetetraacetic acid (EDTA) to the phosphate buffer solution very beneficial as BSA protect cells from mechanical forces during the centrifugation, while EDTA helps loosen intercellular contacts.

The cell density and the volume of a cell suspension used for preparing cytospins are other very important factors. Both of these parameters are interdependent and it is crucial to monitor and manipulate them carefully in order to prepare a required number of optimal cytospins.

The cell density of the sample suspension can be determined either by a cell count or by using rapid on site assessment of sample adequacy (ROSE) concept, where a ‘test’ cytospin is prepared and stained using toluidine blue or a similar rapid stain for the evaluation. Using ROSE for the ‘test’ cytospin is actually very helpful and very valuable as it enables a simple, quick and efficient assessment of the sample adequacy, cellularity and any requirements for further suspension manipulation.

If the ‘test’ cytospin is too cellular, the cell suspension can be diluted, but if the ‘test’ cytospin is paucicellular, the cell suspension can be concentrated.

The maximal volume of the cell suspension for one cytospin is determined by the filter card absorbance. For example, the maximal volume of cell suspension when using white filter cards is set to 500 µL, which is according to our experiences, far too much fluid. 100 to 200 µL of the suspension is the best choice. Using a higher volume of the suspension may result in inadequate deposition of the cells, as the filter might not be able to absorb all of the liquid.

In our practice, a ‘test’ cytospin is prepared from each secondary cell suspension using 100 µL of the suspension. Immediate microscopic evaluation of the rapidly stained ‘test’ cytospin then enables us to assess not only the cellularity of the suspension, but also will give an early indication on the presence of diagnostic cells too.

Haemorrhagic cell suspensions are challenging as erythrocytes can sometimes dilute and overlap diagnostic cells. There are several procedures described for haemolysis, however in order to prevent unknown effect of different reagents on a biomarkers, we decided to remove erythrocytes using simple filtration through a nylon mesh with 10 µL or 20 µL pore size.

It is of utmost importance to have a good cytocentrifuge which enable us to prepare as many as possible cytospins at once, with sets of consumables that are easy to assemble and even more importantly, easy to disassemble.

Centrifugal force, time of centrifugation and acceleration are parameters, which need fine adjustment in each laboratory in order to achieve the optimal results.

Centrifugal force should be strong enough to assure good cell deposition on the slide, while mild enough not to damage cell morphology. The optimal centrifugal force actually depends on the type of cells in the samples and the type of the cell medium. In a fixative based cell medium, the cells are already fixed and are more resistant to centrifugal and all other forces during cytocentrifugation, while in a buffer-based media the cells are vital and more susceptible to the different forces. Addition of bovine serum albumin to the buffer-based medium can protect cells from mechanical forces.

According to the experiences from two different independent and dedicated cytology labs, centrifugal force of 41 x g and 55 x g yield consistent and good results.

The time of centrifugation and the rate of acceleration are especially important in case we need to fix cytospins immediately after preparation and we do not want any air-drying before fixation. In both previously mentioned labs, 3 and 4 minutes of centrifugation, respectively, consistently facilitate an adequate concentration of cells, good preservation of cell morphology without any air-drying artefacts.

With some effort and good cooperation of the whole cytology team, cytospin preparation can be optimized relatively quickly. Good cytospins with high cell recovery and good preservation of cell morphology can greatly enhance cytology service for the patient benefit.