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Preparation and fixation of samples

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If the sample contains enough material, after smears and cytospins have been made for immediate evaluation, the method of choice is to spin down the rest into a cell button which is fixed in 10% formalin (formal saline or phosphate-buffered formalin) followed by dehydration through graded alcohols and paraffin embedding for sectioning. This has the advantage of allowing many sections for multiple immunocytochemical tests. Sections from these blocks must be subjected to antigen retrieval by heating in buffer and must be picked up on charged slides to avoid being detached during the heating process.

Most samples received in the cytopathology laboratory do not contain enough material for the above procedure and are prepared by making cytospins or using liquid-based cytology (LBC). Cytospin preparations made from suspensions of cells in their original fluid result in well flattened cells in a small area of the slide which can then be air-dried for post-fixation or immediately “wet fixed” in alcohol. The same procedure can be applied to smears. Liquid-based cytology involves pre-fixing the sample in an alcohol-based fixative before making the preparation in a machine which filters the fluid from the cells then pushes the cell deposit in a thin layer onto a prescribed area of a slide. The preparations are excellent but the volume of fixed cells required for a single slide means that rather few can be made from one sample, and the apparatus and slides are expensive, though the fixative itself is not.

Long-term preservation of samples

Because of price constraints, the preparation of cytospins and the “Thinprep™” LBC method mentioned above may be out of reach of laboratories in developing countries, also the immunocytochemical tests which confirm and expand the original diagnosis made on clinical grounds together with Giemsa- or Papanicolaou-stained smears. Many departments therefore send samples abroad for further diagnosis and it is unfortunate that the limited material often arrives in a form unsuitably preserved for the tests needed. Air-dried smears, for example, even if they have been alcohol-fixed before drying, will probably suffer from antigen deterioration, unless they have been kept frozen and shipped in dry-ice. Wet-fixed or dried, formalin-fixed smears can be rehydrated by up to 5 minutes immersion in normal saline before further fixation or immunostaining (Leong et al, 1999) but in all these cases the results may not be reliable.

However, there are two simple methods for preserving morphology and antigenicity in cytological samples for shipment to another laboratory. One is that advocated by the UKNEQAS for Immunocytochemistry and applies to smears, imprints or cytospin preparations. Either before or after fixation in alcohol and without allowing the preparation to dry, the preparation is covered with a solution of 10% polyethylene glycol 1450 (PEG) in 50% methanol, then allowed to dry before storage (at room temperature) or shipment. The PEG forms a thin film over the preparation which protects the antigens and does not affect the morphology (Maxwell et al, 1999; Kirbis et al, 2011). It is removed before the immunostaining procedure in two changes of 95% methanol and further fixation is carried out as required. A commercial variant of the PEG solution, Cytofixx, is no longer available.

The second method has the advantage of providing more material but the disadvantage of requiring transport in an alcoholic solution which can cause problems with couriers (but see below for storage and transport using the PEG solution). Fine needle aspirates or other types of cell preparation are placed directly in the type of alcoholic fixative used for LBC such as PreservCyt™ (70% methanol in a proprietary buffer) and can be transported in this solution or stored at ambient temperature indefinitely. We have immunostained samples prepared like this from an African country after 6 years’ storage in PreservCyt at room temperature and obtained the same results as when they were first received.  If the sample is bloody or mucinous it can be washed first in a 50% buffered alcohol solution (eg Cytolyt ™) or Esposti’s fluid (see Appendix) for a brief period to remove protein and lyse red blood cells. After centrifugation, the supernatant is discarded and replaced with the fixative. Even without the intermediate wash, which may be difficult unless staff and centrifuges are easily available, lymphoma and other FNA samples placed directly in 2 ml of PreservCyt maintain excellent morphology and immunocytochemical reactions. For long storage, the level of the fixative in the vials should be checked regularly for evaporation and topped up if necessary. In addition to immunocytochemistry, the samples are suitable for DNA extraction for genetic studies and in situ hybridisation (eg EBER for EBV and c-myc translocation) (Van Noorden et al 2011).

In order to conserve the material for future work, we use as little of each PreservCyt-fixed sample as possible. Although cytospins would provide flatter cells they would use too much, and we manage with just 1.5 µl of cell suspension dropped onto a charged slide and allowed to dry for about 1 h. After drying, the area of the sample is ringed with a diamond marker on the back of the slide, otherwise it is difficult to locate when the slide is wet. The preparations are then post-fixed in 10% formal saline for 10 mins, washed in saline and examined with morphological stains and the required standard immunocytochemical methods. These may include antigen retrieval by heating the slides in buffer in a microwave oven for 20 minutes. To date, we have not come across an antigen that cannot be immunostained in this material which comprises lymphomas, neural tumours, carcinomas etc., and we have found that the routine antibody dilutions are applicable. The disadvantage is that one is looking at whole cells under the microscope and because of the occasionally overlapping cells it is sometimes difficult to distinguish nuclear from cytoplasmic immunoreactions. However, blue haematoxylin-counterstained nuclei are generally easily distinguishable from the brown (DAB) immunoperoxidase reaction. Another problem is the matrix that sometimes surrounds the cells and is susceptible to staining by a few antibodies, probably because it contains the antigen, but the cells can be distinguished easily from this background. Of course the success of the method is still dependent on the quality of the original sample.

We make our preparations in advance from the PreservCyt-fixed material and before or after the formalin fixation step, cover them with a PEG solution as described above. The dried slides can then be kept for weeks or months and are suitable for sending to another centre if transporting the samples in the alcoholic fixative is difficult. The method is inexpensive (the fixative and the PEG are cheap) and requires no apparatus except for a micropipette and tips and could thus profitably be used in disadvantaged countries. Charged slides are advisable.

Alcohol-based fixatives permeabilize the cell membrane so that antibody solutions can penetrate the cell. If material is primarily fixed in formalin, it may be necessary to insert a permeabilization step with detergent (eg. Triton X-100, 0.2% or Tween 20, 0.05-0.2%) before the immunostaining protocol.