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Histotechnology and Diagnostic Cytology

Fixation of cell suspensions and further analysis

Does it seem reasonable to use a low concentration of formalin (0.25%) to fix cells in a single cell suspension for 18-24 hrs rather than using a higher concentration for a shorter period of time? Do you think the fixation method may affect subsequent attachment to a glass slide? Do you think excess fixative needs to be extensively removed prior to further analysis?

1. Diluting formalin too much may adversely affect its effectiveness. Formalin fixation is a "clockwork" reaction. The two steps involved include the formation of hydroxymethyl compounds followed by the formation of methylene bridges between proteins (Tahan etal, Mod Pathol 8:177-182, 1995; Battifora, Am J Clin Path 96:144,1991). Therefor a short formalin fixation time would result in inadequate fixation. There has been some discussion in the literature concerning the importance of osmolarity of fixative solutions that needs to be considered (Fox & Benton, J Histotech 10:199,1987).
2. Prefixing cells will affect their subsequent attachment to slides. Formalin fixed frozen sections cut for fat stains are notorious for lifting when slides are placed in the staining solution. The use of APTS or polylysine coated slides are definitely helpful in this regard. When unfixed cells are cytospined or directly smeared onto slides, there is some airdrying that occurs prior to fixation in ethanol. This airdrying, similar to burnt egg on a hotplate, allows quite good attachment and therefor retention of cells on slides. Some authors (Atkinson, Diag Cytopath 2:231-232, 1986) have obtained good results after fixing cell suspensions (from FNA rinses) in Saccomano’s solution. After transport in the above solution, the specimen is cytocentrifuged, the slides airdried (10min) and then postfixed in 95% ethanol.
3. I would think it judicious to remove excess fixative prior to further testing whether this is done prior to or after smearing. Hanks or another cell culture fluid can be used. I have in the past washed cells three times with hanks using centrifugation in between each wash.
4. The preferred cell processing technique will depend on the analysis that is to be used:

• Some antigens, if immunohistochemistry is to be used, are quite resistant to fixation in ethanol. Alcohol fixed smears can be used for such antigens as CD45, EMA, CEA, Calcitonin etc (Dabbs etal, Acta Cytol 39:157-163, 1995; Motoyama etal, Acta Cytol 39: 164-170, 1995)
• Air dried smears are preferred for many lymphoid antigens (Aratake etal, Acta Cytol 32:117-122, 1988) and often use acetone fixation post air drying. Most enzyme histochemical procedures require cells that are airdried (ie unfixed).
• McCorriston (J Clin Path, 42:1101-1103, 1989) have described a preservative solution (containing foetal calf serum and DMSO) that allows long term cryogenic storage of cells.

1. Too dilute a formalin solution may not adequately fix cells.
2. Formalin fixation of 18-24 hrs is preferred rather than shorter periods.
3. The osmolarity of the fixative solution needs to be considered.
4. Use coated slides to aid cell attachment.
5. Gentle removal of excess fixative is preferred ( usually 3 rinses).
6. The processing technique will depend on the analysis to be performed.

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