PART THREE
Special Techniques in Cytology
stained. The cells are n o t exposed to a ir at any tim e d uring this
fix a tio n m ethod.
Wet Fixation with Air Drying
W e t fix a tio n w ith subsequent a ir drying, i.e. submerge the cell
sam ple im m e d ia te ly in to the fixative s o lu tio n , rem ove the sam -
ple fro m the fixative s o lu tio n after a specified tim e, a ir d ry the
cell sam ple, and place it in to a container fo r tra nsp o rt to the
laboratory. O n arrival in the laboratory, the slide is placed in
95% ethanol o r its eq uivalent p rio r to staining to rehydrate the
cell sample.
Spray fixation
Spray fixa tio n , i.e. im m ed iate fix a tio n o f the w e t cell sam ple
w ith a c om m ercially available spray fixative. The spray-fixed
cell sam ple is allow ed to a ir dry and then placed in a container
fo r tra nsp o rt to the laboratory. O n arrival in the laboratory, the
polyethylene glycol (carbowax) cell coating m ust be rem oved
p rio r to staining.
Spray fixatives u su a lly consist o f an alcohol base and a w axy
substance (carbow ax)21,22 th a t provides a th in protective coating
fo r the cells. W h e n received in the laboratory, the spray-fixed
slides m ust have the w axy substance rem oved p rio r to staining.
To rem ove the w axy coating, the slides are placed in tw o separate
rinses o f 95% ethanol o r tw o separate rinses o f tap water. Slides
are placed in the first ethanol rinse fo r ap p roxim ately 30 m in .
O ne m ay see opaque, w axy particles flo a t to the surface o f the
ethanol s o lu tio n . T his first 95% e tha no l rinse is discarded after
each use. The slides are then placed in to the second 95% etha-
n o l rinse fo r 10-15 m in . T he use o f these tw o ethanol rinses is
u su ally sufficient to dissolve the w axy substance. In very h o t and
h u m id environm ents, it m ay be necessary to add a th ird ethanol
rinse. It sho uld be noted th a t som e physicians spray fix m ore
h ea vily th a n others, and th is factor m ay add to the d iffic u lty o f
treating all specimens in the same fashion.
Som e m anufacturers o f spray fixatives recom m end rem oval o f
carbowax w ith water. W e have fo u n d 95% ethanol to be m ore
effective and less tim e-consum ing than water rinses. A d d itionally,
after the ethanol rinses, the cells are better prepared fo r the uptake
o f dyes than w ith water rinses alone. However, if availab ility and
cost are factors, w ater rinses w ill suffice. It should be noted th at
i f the carbowax is n o t rem oved com pletely, sm all, w axy particles
w ill be carried in to the staining solutions. N uclei w ill then appear
foggy and lack chrom atinic detail and the cytoplasm m ay exhib it
a pale blue color (Fig. 31.1).
Alcohol-based nonaerosol spray fixatives are preferred. O ne
spray container can fix ap p roxim ately 7 00 -1 0 00 cell samples.
It is suggested th a t one use a c om m ercially prepared fixative
designed specifically fo r cytologic use instead o f alcohol-based
hairsprays, as has been done in the past. O ne o f the reasons fo r
th is rec om m end ation is th a t com panies producing hairsprays
m ay a lter th e ir form ulas, replacing viab le com ponents w ith sub-
stitutes th a t m ay alter the q u a lity o f cell preservation.
W h e n using nonaerosol fixatives the spray nozzle m ay som e-
tim es becom e clogged. It is advisable at the b eg inning o f each
day to test the spray em itted fro m the nozzle to ensure the
spray is evenly distributed. N onaerosol sprays are m ore lik e ly
to produce an uneven spray th an aerosol sprays. N onaerosol
spray fixatives m ay be h eld closer to the specim en th an aerosol
sprays. For som e products, 3 -6 inches is recom m ended, whereas
fo r others, 6 -1 0 inches is the best distance. O ne sho uld try to
o b tain an even spray across the entire slide and avoid spraying
Fig. 31.1 Carbowax not removed prior to staining. Note lack of chromatin
detail and hazy appearance in a cell sample from a patient with a high-grade
squamous intraepithelial lesion. (Papanicolaou x HP.)
the labeled o r frosted p o rtio n o f the slide, w here a heavy coating
o f w axy residue makes it d iffic u lt to n um b er o r label the slide.
I f the frosted area is sprayed it m ay be necessary to scrape o ff
o r otherw ise rem ove the w axy coating o n the frosted end o f the
slide before n um b ering o r applying a label to the slide.
Liquid-based Fixation for Papanicolaou Tests
Liquid-based fix a tio n m ay be m e th a n o l- o r ethanol-based
depending on the m anufacturer. Tw o systems are in current use
w h ile o the r systems are s till undergoing developm ent. B oth o f
the curren tly used systems collect cell samples in to liq u id fixative
so lu tio n s th a t create a m on olaye r cell sam ple and enable sub-
sequent autom ated evaluation. H u m a n p a p illo m a viru s (H P V )
D N A testing, im m unocytochem istry, tu m o r classification, and
o th e r special studies (C h la m yd ia and G onorrhea) can also be
p erform ed o n these preparations.
Liquid-based technolog y fu rth e r resolves the issue o f cells
being obscured due to the presence o f in fla m m a tio n and b lood .
C e llu la r preservation and sam p ling problem s in h e re n t w ith the
conventional Papanicolaou sm ear are effectively e lim ina te d
w ith liquid-based Papanicolaou testing. This technolog y entails
direct transfer o f cells fro m the collection device in to a fixative
s o lu tio n . D iagnostic confidence is enhanced w h e n the liq u id -
based m eth od is u tiliz e d to o b tain a representative sample.
C urrently, tw o liquid-based preparation systems are in use
th a t account fo r a p p roxim ately 80% o f a ll Papanicolaou collec-
tio n m ethod s in the U n ite d States.23,24
T hinP re p and SurePath u tiliz e d iffe re n t processing tech-
niques to achieve the same result: e lim in a te background m ilie u
and provide a w e ll-fixe d concentration o f representative epithe-
lia l cells (Figs 31.2 and 31.3). T hinP re p technolog y u tilize s a fil-
ter process whereas the SurePath system incorporates a gravity
sed im en tatio n process.
A T hinP re p specim en m ay be collected w ith a "b ro o m ,"
spatula, o r endocervical brush and is th en rinsed im m e d ia te ly
in to a via l c on tain in g a m ethanol-based s o lu tio n (PreservCyt).
This elim inates sm earing the sam ple on a slide and ensures
th a t a greater p ro p o rtio n o f cellu la r m aterial w ill be captured
fo r m icroscopic evaluation. T he via l c on tain in g the specim en
is placed in the T hinP re p processor, and a plastic cylinder
c ontaining a polycarbonate filte r m em brane is im m ersed in to
the vial. As processing proceeds, e ith er the cylind er o r the via l
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