Volume 11, No.1
| 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.
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.
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.
Avoid and minimise activities that promote evaporation of alcohol and xylene, with subsequent exposure to air,
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
remove slides from xylene one-at-a-time, quickly dry the back and edges, apply mounting medium as needed, and
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
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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