CytoSpin

However, ICC is not used as frequently in cytology as IHC in surgical pathology due to some technical issues, such as scanty or lack of diagnostic material in cell blocks or cytospins, and questionable reliability of immunos-taining on smears. 

Air drying, usually with subsequent chemical post-fixation, is applicable for cell smears, cytospins and cryosections. Snap freezing (usually in liquid nitrogen) is routinely employed for tissue probes for subsequent cryosectioning. Chemical fixation is commonly carried out in aldehydes (e.g. formaldehyde) for tissue probes and cultured cell monolayers, and in acetone or alcohols (methanol or ethanol) for cryosections and cell preparations. 

In preparing cells for cytospin slides, there are times when immediate fixation is necessary. For these instances, the cells can be fixed before centrifugation. Starting at step 2 for the cytocentrifuged specimen (Subheading 3.3.), continue with ... 

Pipet 100 pL of the cell suspension onto treated slides, spread using the edge of a clean slide, and allow to dry. Alternatively, cells can be attached to the slide by cytospin preparation. Continue with step 2 immediately below (Subheading 3.2.2.). 

Collect and pool the cells by centrifngation count the cells (see Notes 4 and 5). The cell snspension is now ready to introdnce into tissne cnltnre, immnnostain, or deposit onto a microslide with a Cytospin. 

The preparations most often affixed to silane- (9) or poly-L-lysine-coated slides are Carnoy-fixed suspensions of metaphase cells for chromosome studies (see Chapter 47) cytospins of cultured cells followed by methanol fixation sections of formalin-fixed, paraffin-embedded tissues and frozen sections fixed with acetone or ethanol/acetic acid after cutting. Each of these approaches imparts different intracellular effects that may modify cytological detail and/or hybridization. (See Chapters 8 and 9 for a discussion of fixatives and frozen-section preparations.)... 

Tissue culture cells are easy to handle under laboratory conditions. It is necessary to fix these cells immediately after cytospin or before paraffin embedding. Different fixatives are used depending on the goal of the experiment. 

Komminoth, P., Long, A. A., Ray, R., and Wolfe, H. J. (1992) In situ polymerase chain reaction detection of viral DNA, single copy genes and gene rearrangements in cell suspensions and cytospins. Diag. Mol. Pathol. 1, 85-97. 

Excessive apphcation of tissue adhesive, gel-coated slides, or the use of albumin when preparing cytospins may contribute to background stain. The use of charged slides (Fisher Superfrost/Plus, Fisher Scientific, Pittsburgh, PA) can eliminate this problem. 

Increased thickness of specimen Tissue sections should be cut 4-5 jm thick, and cell smears should be spread as thinly as possible. Cytospins should be only a monolayer in thickness. 

Cryostat sections and cytocentrifuge preparations should be air-dried for at least 1 h but preferably overnight before immuno-staining. Before immunolabeling, cryostat sections should be fixed in cold acetone for 10 min and cytospin slides should be fixed for 90 s in a 1 1 mixture of acetone and methanol at room temperature. After fixation, follow the IGSS method for paraffin sections from step 3. Cytospin preparations are usually adequately covered by standard... 

Determine differential cell counts on cytospin smears of lung washes (3 min at 100xjj) stained by the Wright-Giemsa method. 

Can be done on cytospins of cell suspensions or on smears, but more difficult to do and interpert... 

After the Cytospin centrifugation and the total cell count, the remaining BAL is centrifuged for 10 min at 425g. The volume of the supernatant is measured and frozen at -20°C. The BAL fluid can be used to test for the presence of different molecules such as cytokines by ELISA. The cell pellet can also be kept for RNA analysis. 

Cytospin apparatus and microscope slides (Shandon, Runcorn, UK). 

Differential cell counts of the BAL and peritoneal fluids are performed on cytospin-prepared slides. Differential counts of leukocytes in peripheral blood are performed on blood smears. The slides are stained with Diff-Quik (American Scientific Products, McGaw Park, IL) and at least 200 cells are counted per sample to determine the percent of neutrophils, macrophages, lymphocytes, and other cells. 

A sample is taken for cytospin to evaluate the percentage of blasts. 

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