PART THREE
Special Techniques in Cytology
H o lm q u is t states th a t all u rine samples should be centrifuged
and rinsed once w ith a salt s o lu tio n .65 I f the specimen is b lo o d y or
contains am ple sedim ent, then m ore than one rinse m ay be neces-
sary to help uptake o f dyes in the staining process. C entrifug ation
o f g e nitourinary tract samples is the usual procedure. O ne m ay
also a llo w the u rin e to sedim ent at ro o m temperature. In the latter
case, cells obtained fro m the b o tto m o f the container are usually
adequate in num b er to ob tain a diagnostic cell sample, provided
it is placed in to a conic container fo r C ytospin preparation.
C e n trifu g a tio n and direct smears were the m ost c om m o n
m ethod s fo r preparation. Because u rin e samples have a ten-
dency to becom e detached fro m glass slides d uring fix a tio n
and staining, a coating agent such as a lb u m in 0.1% p olylysine
h yd ro b rom id e o r frosted D a k in slides were used. In 1981, Bales
described a sem iautom atic system fo r h an d ling a h ig h volum e
o f u rin e sam ples.66 The technique is sem iautom ated, and rapid
p rep aration o f u rin e samples can be perform ed b y personnel
w ith lim ite d experience. Samples are coated w ith 2% carbowax
in 70% ethanol. T his m eth od also proved to be excellent in pre-
serving casts, crystals, and b lo o d cells.
T he first m o rn in g void ed u rin e is th o u g h t b y som e to have
p oorer cellular preservation because o f prolonged exposure o f
cells to urine. However, Pearson and colleagues showed th a t if
the p H is low , exposure o f cells to unfixed u rin e fo r as long as 72
h causes little loss o f preservation.67
T he a d d itio n o f a fixative to specimens th a t have a hig h er pH
o r th a t m ust undergo p rolonged storage before preparation can
im p ro ve the preservation o f cellular detail.
N o re la tio n sh ip could be distinguished between the n um b er
o f cells in the o rig ina l sam ple and the v a ria tio n in cell counts in
the m aterial prepared w ith various techniques: a ir d rying and
w e t fixatio n.
S ta in in g o f u rin e samples is th e same as staining o f b od y
fluids, w ith the exception o f the tim e the slides are exposed
to h em ato xylin. A t the U n ive rsity o f Rochester, the tim e in the
h e m a to xylin is reduced to 15 s.
Preparatory Techniques for Direct Smears
and Cytospin Sam ples
Procedure 1 fo r D irect Smears
1. T u rn o n th e b io h a z a rd o u s h o o d h a lf an h o u r b e fore w o rk
is to begin.
2. C heck th e pressure gauge o n th e va c u u m p u m p .
3. P u t o n g o w n a nd gloves.
4. Specim ens are easier to prepare w h e n at ro o m tem perature.
5 . O rg a n ize th e w o rk area.
6. W ip e th e o u tsid e o f a ll sp ecim en c on tain ers w ith 7 0%
is o p ro p a n o l o r 10% h o u s e h o ld b leach (i.e. C lo ro x )
s o lu tio n .
7 . W ip e a ll re q u is itio n fo rm s w ith d e c o n ta m in a tio n s o lu tio n .
8. M a tc h specim ens to re q u is itio n fo rm s . I f th ere are any
discrepancies, contact th e p h ysic ia n w h o requested th e
test.
9 . A ssign a la b o ra to ry n u m b e r to each case.
10. L is t cases o n a lo g sheet.
11. Label th e sp ecim en c on tain er, re q u is itio n fo rm , slides,
a nd test tubes w ith th e la b o ra to ry n u m b e r, p a tie n t
id e n tific a tio n , a nd typ e o f sam ple.
12. Place th e sp ecim en c on tain ers in th e h o o d .
13. M ix specim ens th o ro u g h ly in th e c o lle c tio n c o n ta in e r so
th a t a liq u o ts c o n ta in e q u iv a le n t c e llu la r contents.
14. Place th e w e ll-m ix e d flu id sam p le in to tw o separate
capped and lab e le d disp osab le p lastic cen trifug e test
tubes (a m o u n t in each test tu b e depends o n to ta l flu id
received). T w o 5 0 -m L a liq u o ts are u s u a lly adequate i f
th e sp ecim en has been w e ll m ixed . T w o con ic 1 5 -m L
test tubes m a y be used fo r s m a lle r sam ples.
1 5 . C e n trifu g e th e sam p le in 5 0 -m L p lastic test tubes fo r
10 m in a t 2 5 0 0 rp m .
16. C a re fu lly decant o n e test tube. Save a nd refrig erate th e
second test tube.
17. Place o ne d ro p o f to lu id in e b lu e o n to th e glass slid e
w ith o ne d rop o f s e d im e n t w ith a disp osab le p ip ette
(please check sec tion o n s u p ra v ita l s ta in in g ). W ith expe-
rience, o ne can assess i f th ere is adequate m a te ria l fo r a
p u ll-a p a rt sm ear. I f th e cell b u tto n is lim ite d o r w atery, a
C y to s p in is p e rfo rm e d .
1 8 . M ix drop s to g e th e r w ith a clean a p p lic a to r stick.
1 9 . C o ver w ith a 2 4 x 2 4 m m th in n e s s n o . 1 coverslip.
20. W a it 1 m in .
21. O bserve u n d e r a m icroscop e w ith x 10 objective.
22. E stim a te th e n u m b e r o f cells. C e llu la rity is d e te rm in e d
b y th e n u m b e r o f cells p er m ic rosco p ic fie ld .
N ote: the m icroscope used to exam ine to lu id in e b lue sam -
ples sho uld be in the cytopreparatory laboratory. T he m ic ro -
scope requires d e co nta m in atio n s im ila r to th a t used fo r o the r
e q uip m e nt used in cell preparation.
Decide w h a t type o f preparation sho uld be m ade based on
the c e llu la rity o f the sam ple, type o f cells observed, and clinical
history. D irect smears m ay be m ade at th is tim e. M ake one air-
dried sm ear and three 95% ethanol-fixed slides— th e air-dried
slide is fo r the D iff-Q u ik stain and the w et-fixed slides fo r the
Papanicolaou stain.
Procedure 2 fo r Cytospin Cell Preparation
1. T u rn o n th e b io h a z a rd o u s h o o d h a lf an h o u r b e fore w o rk
is to begin.
2. C heck th e pressure gauge o n th e v a c u u m p u m p .
3. P u t o n g o w n and gloves.
4. R em ove specim ens fro m th e re frig e ra to r and place in
th e h o o d . N o te : specim ens are easier to prepare w h e n at
ro o m te m p erature.
5. O rg an ize th e w o rk area.
6. W ip e th e o u tsid e o f a ll sp ecim en c on tain ers w ith 7 0%
is o p ro p a n o l o r 10% h o u s e h o ld b leach s o lu tio n .
7. W ip e a ll re q u is itio n fo rm s w ith d e c o n ta m in a tio n s o lu tio n .
8. M a tc h specim ens to re q u is itio n fo rm s . I f th ere are any
discrepancies, con tac t th e p h ysic ia n req u e stin g th e test.
9. A ssign a la b o ra to ry n u m b e r to each case.
10. L is t cases o n a lo g sheet.
11. N u m b e r slides.
12. Place th e lab e le d slide, filte r card, sam p le cham ber, and
slid e c lip in to th e C y to s p in head.
1 3 . Place th e w e ll-m ix e d flu id sam p le in to tw o separate,
capped and lab e le d disp osab le p lastic cen trifug e test
tubes (a m o u n t in each test tu b e depends o n to ta l flu id
received). T w o 5 0 -m L a liq u o ts are u s u a lly adequate i f
th e sp ecim en has been w e ll m ixed . T w o con ic 1 5 -m L
test tubes m a y be used fo r s m a lle r sam ples.
14. C e n trifu g e th e sam p le in 5 0 -m L p lastic cen trifug e test
tubes fo r 10 m in a t 2 5 0 0 rp m .
1 5 . U rin e sam ples cen trifug e fo r 10 m in a t 2 0 0 0 rp m .
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