SCAN
Volume 11, No.1
February 2000
SCAN February 2000
Membership
Aims and Objectives
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Further information about the NAC is available from Mike Rowell, the NAC Secretary.
 THE BROWN ARTIFACT - 'Cornflaking' - Gary W. Gill
The brown artifact is air deposited on superficial squamous cells. It is also known as cornflake cells, or simply cornflaking, because of the crinkled appearance imparted to the surface of these polygonal cells. It is considered to be an extrinsic artifact, meaning it arises from factors outside the cells. Among the first published descriptions include: Danos ML. An artifact on stained sides (Tips on Technique). Cytotechn Bulletin. 1969;6(1): 9.

Significance and occurrence
The brown artifact is observed in all cytology laboratories. When present on a few cells, as is usual, the artifact is annoying at best. Not uncommonly, however, its distribution is sufficiently great to be distracting, and in the worst case, obscuring - literally making it impossible to determine whether abnormal cells are present in affected areas. It occurs most often on Pap smears due to the preponderance of superficial squames, is more likely to occur on thick preparations as gravity accelerates the drainage of xylene off the topmost cells and exposes them to air sooner, and is almost never seen on non-squamous cells.

Origin and timing
The brown artifact occurs because preparations dry sufficiently to allow air to come in contact with the surface grooves of superficial cells. As seen clearly in scanning electron micrographs, these grooves, or channels, course in irregular paths across the entire surface and aid in intercellular adhesion. While submicroscopic, their appearance is suggested by the brown artifact seen in progressive stages of formation on different cells of the same preparation, occupying small areas on some cells and greater areas on others. It is difficult to recreate the brown artifact experimentally.

The artifact arises at, or after, the first alcohol in the latter half of the Pap stain, after which only alcohol and xylene are encountered. The most likely trigger point is the interval between removing a slide from the last xylene bath and applying mounting medium. Other, less plausible, possibilities include the interval between successive baths when a slide rack is drained, and vigorous dipping. Some automated stainers allow slides to drain for 30 seconds between successive baths, which may provide the opportunity for evaporation and consequent air deposition. Vigorous dipping may cause air bubbles to collapse against immersed cells, thus depositing air. The latter possibility seems unlikely. The fact that water removes the brown artifact indicates the air does not occur in the first half of the Pap stain where water is commonly used.

Evidence
Although invisible, air has substance - as is annoyingly evident to anyone who has tried to purge an air bubble from a liquid-filled syringe. The brown colour resembles that of the wall of an air bubble trapped in mounting medium, though the latter is black. The black colour is due to destructive interference of light waves by diffraction, which is the bending of light as it passes through a transparent medium (i.e., mounting medium and air) and encounters an edge (i.e., the interface between air and mounting medium, or perhaps between air and the adjacent mounting medium-infiltrated cytoplasm). Destructive interference occurs when the peaks of one light wave coincide with the troughs of another, and cancel each other out, thus producing darkness. Light is bent into the irregular paths of trapped air by the relatively extreme difference in refractive indices between air and mounting media (or fixed protein) 1.0 vs 1.52 respectively. As microscopic illumination passes through superficial squamous cells, which are stained by eosin (a dye that transmits yellow as well as red light), the entrapped air appears brown. The crinkled appearance is consistent with the submicroscopic topography of superficial squamous cells as described above. The brown artifact is not material deposited by spray fixatives, as this would be removed by water.

Removal
The following procedure removes the brown artifact, and, coincidentally, the counterstain dyes:

1. Remove the cover glass by immersing the preparation in xylene for as long as needed.
2. Remove the mounting medium in xylene, three changes x 10 dips each
3. Remove the xylene in absolute alcohol, three changes x 10 dips each
4. Remove the absolute alcohol in water, three changes x 10 dips each
5. Restain, beginning with OG and continue routinely through the Pap stain,
6. Clear and mount.

Removal appears to be related to water's higher surface tension, relative to that of alcohol; scattered residual pockets sometimes remain. Removal might also be due to the slight swelling of cells that occurs upon re-immersion in water, which might loosen the foothold of air in the grooves. If desired, check the completeness of removal microscopically before restaining. Since this entire procedure is time-consuming, prevention is better. I have not found it necessary to use 50% glycerin instead of water to remove this artifact. Nor have I found any correlation between its occurrence and the quality of the alcohol that is used, as is sometimes noted by others.

Prevention
Avoid and minimise activities that promote evaporation of alcohol and xylene, with subsequent exposure to air, including:

 if staining manually, dip slides at the rate of once per second the distance of the entire slide. Do not submerse a slide rack and rapidly agitate it;

 limit the 'hang' time between baths to no more than 10 seconds, an arbitrary time;

 don't remove slides from xylene at one end of the laboratory and carry the rack to the other end. Keep the slides wet until ready for coverslipping

 avoid draughts (e.g., fans, proximity to open doors, foot traffic) - working under a fume hood promotes the incidence of the brown artifact, as air enters the workspace at the rate of 75-100 linear feet per minute;

 set up a chemical splash shield at the front edge of the fume hood to divert airflow and create a quiet zone behind it in which to coverslip - use a narrow shield, as wide shields can make a technician's shoulder muscles ache; and

 remove slides from xylene one-at-a-time, quickly dry the back and edges, apply mounting medium as needed, and cover.

It appears to make no difference whether the mounting medium is applied to the slide or to the cover glass. Applying it to the slide, however, covers the preparation and reduces the exposure time. Speed is key. Do not drain each slide excessively. Some high volume laboratories lay out many slides at a time, face-up, and go back to apply mounting medium and cover glasses. Such a practice is not recommended, as it promotes the incidence of this artifact.

I am unaware of any published report of an additive that will prevent the formation of the brown artifact. No relevant titles are found when searching on 'brown artifact' and 'cornflaking' in PubMed of the National Library of Medicine on the Internet.
Journal Editor - Brian Nation
Department of Histopathology and Cytology, Hereford Hospitals,
NHS Trust, County Hospital, HR1 2ER Hereford
Tel: 01432 364043 / Fax: 01432 364136

SCAN is published by the National Association of Cytologists in England and
printed by Berrington Press, 20 Berrington Street, Hereford, England, UK
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