Cytopreparatory Techniques
Fig. 31.12 Cells from a patient with a breast carcinoma. The slide was
prepared by the pull-apart slide method and stained by the University of
Rochester nongynecologic Papanicolaou staining procedure (x HP).
D epending on m anufacturer protocols, b rushing specimens
m ay be processed b y rin sin g the tip o f th e brush in the collec-
tio n v ia l o r b y s u b m ittin g the tip o f the b rush d irectly in the
collection via l and s u b m ittin g as part o f the specimen. Liq uid -
based prepared slides are ideal fo r im m u n o lo g ic special staining
techniques, especially i f the specim en is unavailab le fo r a cell
b loc k preparation.
Processing results vary depending on the m anufacturer u ti-
lized, the specim en collection m ethod , and the preparatory p ro-
tocol established by the laboratory. It is advised to have sta ff
m em bers participate in m anufacturer tra in in g sessions and to
fo llo w p rep aration and tro u b le sh o o tin g instructions w h en
processing liquid-based specimens.
Cerebrospinal Fluid Preparation
T he preparation and staining o f CSF is perform ed separately
fro m o th e r flu id samples.
Various cytopreparatory m ethod s are in current use: centrifu-
gation, direct smears, and cytocentrifugation. I f the direct smear
m eth od is used, the a d d itio n o f an adhesive s o lu tio n on the glass
slide, such as polylysine, dextran, o r M ayer's a lb u m in , is sug-
gested so cells adhere m ore read ily to the glass surface. P refixation
w ith alcohol is n o t recom m ended fo r CSF because alcohol m ay
precipitate any p rotein present in the flu id sam ple.54-60
S ta in in g o f CSF is p erform ed separately so th a t cross-con-
ta m in a tio n fro m o th e r specim en types does n o t occur. I f all CSF
fluid s prepared in a single day are m icroscopically m o n ito re d by
the cytopreparatory in d ivid u a l, cross-contam ination w ith in the
samples them selves can be recognized.
Preparation o f C lear CSFs o f Less than 5 mL in Volume
A ll preparatory e q uip m e nt fo r CSF as w e ll as the staining solu -
tio n s is kept separate fro m all o th e r fluids. The eq uip m e nt is
m arked so th a t it is n o t confused w ith o th e r b o d y flu id prepara-
to ry equipm ent.
Precautions fo r preparation o f samples fro m patients w ith
C reutzfeld t-Jakob disease include using disposable eq uip m ent
w henever possible.61 For the staining setup, it is recom m ended to
use disposable containers o r 5 0-m L plastic centrifuge test tubes
th a t are discarded after use. Reusable item s shall be im m ersed fo r
2 h in 5% sod ium h yp o ch lorite (un d ilu te d household bleach)
Fig. 31.13 Cells from the same patient as depicted in Fig. 31.12. This is
a ThinPrep preparation (Papanicolaou x HP).
o r autoclaved at 121°C fo r 1 h before being cleaned o r dis-
carded. Surfaces contam inated w ith b od y fluid s d uring process-
ing o f samples sho uld be disinfected fo r 2 h w ith 5% sod ium
h yp o ch lorite before ro u tin e cleaning. The prepared slides are
discarded after the case has been signed out.
Genitourinary Tract Sam ples and Other
W atery Specim ens
U rin a ry tract sam ples c on tain cells th a t are suspended in an
u n frie n d ly e n v iro n m e n t and m ay already be degenerated
before leaving th e body, according to H a rris and cow orkers.62
C rabtree and M u rp h y w rote, "M o s t o f th e m o rp h o lo g ic changes
recognized as degenerative occur p rio r to v o id in g ."63 C ell
preservation is in fu rth e r jeop ard y once th e specim en has le ft
th e b od y.64 V a ria tio n s in c e llu la rity and cell p reservation m ay
also be reflected in various u ro lo g ic diseases. E a rly m o rn in g and
2 4-h sam ples are n o t recom m ended fo r cytolog ic evaluation.
H o lm q u is t states th a t a u rin e sam ple can s it o u t at ro o m te m -
perature i f prepared the same day th a t it was ob tained ;65 i f le ft
o u t overnig ht o r over a weekend, however, the specim en should
be refrigerated. I f a lon g er delay is anticipated, it is then and
o n ly then th a t 70% ethanol (tw o parts u rin e to one part alco-
h o l) m ay be added to the u rin e sample. A lth o u g h refrig eration
o f the cell sam ple m ay retard cell degeneration, th e a d d itio n o f
an ethanol fixative m ay not.
U rin e sam ple p reparation is d iffic u lt to p erform on cool o r
cold samples. Salts crystallize in cold u rin e samples and m ay
cause the filte r to clog. Salts dissolve in s o lu tio n i f the u rin e is
b rou g h t to ro o m o r b od y tem perature before preparation. It is
advisable to le t the u rin e sam ple reach ro o m tem perature before
processing the sample.
I f the sam ple is collected in an alcohol fixative, it is d iffic u lt
fo r the cells to attach them selves to the surface o f a clean glass
slide. Cells collected in alcohol tend to ro u n d up and harden,
and nuclear pyknosis fre q u e n tly occurs. T he use o f a lb u m in as a
cohesive agent (in the sam ple o r o n the slides) does n o t im p rove
the results. I f the specim en has been fixed in a suspension, it
sho uld be centrifuged, the supernatant poured off, and the sedi-
m e n t resuspended, preferably in balanced salt s o lu tio n , before
fu rth e r processing. A lc o h o l added to u rin e samples also causes
p rotein coagulation in the supernatant.
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