PART THREE
Special Techniques in Cytology
Labeling
1. Label each slid e w ith th e slid e la b e l generated fo r th e
p a tie n t.
2. Be sure to m a tc h th e n a m e o n th e la b e l w ith th e a p p ro -
p ria te slides.
3. S u b m it th e case a lo n g w ith th e acc om p an yin g re q u is itio n
to a c yto te c h n o lo g is t fo r m ic rosco p ic review .
Please note: the techniques described above assume the operator
is right-handed; a m od ification is made fo r a left-handed operator.
Protective attire sho uld be w o rn at all tim es d uring the
m o u n tin g and cover slip p ing o f slides.
N ote: in som e laboratories, the slides are already labeled
d irectly on the frosted end w ith pencil o r perm anent in k , w h ile
in o th e r laboratories slide labels are com puter-generated w ith
p a tie nt id entifiers (nam e o f p atient and accession n um b er and
in som e cases a specific p a tie nt u n it n um b er given to p atient
w h e n he o r she registers w ith in a h osp ital system).
Automated Cover Slipping Procedure
There are cover slipping instrum ents th a t a utom atically cover-
slipglass slides. O ne such m achine is the Sakura Tissue-Tek Cover-
slipper.52 It can be used fo r conventional and liquid-based cervical
cytology slides m ounted on standard 25 x 75 m m microscope
slides. The use o f special resin-coated film elim inates the need fo r
tra d itio n a l coverslips and m o u n tin g m edia. The fo llo w in g is the
procedure used at the N orthw estern M e m o ria l H osp ital cytopa-
th olog y lab oratory (E. Lucas, personal com m unication, 2007).
1. T u rn th e p o w e r s w itc h to "O n ."
2. O p e n th e glass d o ors o n th e fr o n t o f th e in s tru m e n t b y
pressing th e m e ta l p late o n th e u p p er p a rt o f each d o o r.
3. T h e "D o o r " in d ic a to r lig h t w ill illu m in a te w h e n th e d o o r
is o p en .
4. C heck th e flu id level in th e xy le n e b o ttle .
5. R e fill i f b e lo w 2 50 m L. Be sure th ere is s u ffic ie n t x yle n e
fo r th e e n tire ru n .
6. Be sure th e cap a nd gaskets are p ro p e rly seated o n th e
b o ttle and a ll a ir b ub b le s have b een b led fro m th e lin e
b e fore s ta rtin g th e ru n .
7. Load an e m p ty slid e rack in to th e rec eivin g c h a n n e l w ith
th e "u p s id e " fa cing u p and th e b asket g uid e p in inserted
in to th e g roove o f th e lo a d in g channel.
8. C u t th e film le n g th b y p ressing d o w n o n th e tip o f th e
b lad e h o ld e r.
9. C lose firs t th e rig h t side d o o r th e n th e le ft side d oor.
10. T h e film le n g th d e fau lts to "L o n g ." T h is film le n g th is
used to cover s lip c o n v e n tio n a l P a p a n ic o la o u sm ears.
11. C hange th e film le n g th to "S h o rt" b y pressing th e "S h o rt"
keypad to cover slip liq u id -b a se d P a p a n ic o la o u slides.
12. C o n firm th e xy le n e d rip v o lu m e b y p ressing th e "X y le n e
check" o n th e keypad.
13. A d ju s t v o lu m e , i f necessary, b y tu rn in g th e xy le n e a d ju st-
m e n t k n o b c o u nte rcloc kw ise to increase th e v o lu m e and
clockw ise to decrease th e v o lu m e .
14. C heck th a t a ll lig h ts are o ff w ith th e exc ep tio n o f th e
film le n g th in d ic a to r. T h e in s tru m e n t w ill n o t sta rt
p rocessing a nd an e rro r c o n d itio n m u s t be corrected i f
a n y o th e r lig h t is on.
15. Press th e "S ta rt" keypad. T h e in s tru m e n t w ill b e g in to
process th e slides.
1 6 . W h e n th e cover s lip p in g is com p lete, th e slides are
d ep osited in th e rec eivin g basket.
17. Press "S to p " to end th e m ec ha nica l m o v e m e n t o f th e
in s tru m e n t.
18. R em ove th e e m p ty slid e rack fro m th e rec eivin g basket
and th e c om p le te d slid e rack fro m th e slid e d ry in g area.
1 9 . T u rn th e p o w e r o ff.
20. A t th e end o f th e day, u n th re a d th e film b y p u llin g th e
film back th ro u g h th e th re a d in g m e c h a n is m a nd re w in d
o n to th e ro ll.
N ote: i f the film is le ft threaded on the instru m e nt, it w ill
"re m e m b e r" the shape o f the ro lle r m echanism and w ill n o t lie
fla t w h en placed o n the slide.
Liquid Mounting Media
L iq u id substitute m o u n tin g so lu tio n s are lik e ly to e m it p oten-
tia lly toxic vapors. The y produce an irreg ular layer o f m o u n tin g
m ed ium over the cells. This w avy layer o f m o u n tin g m edia is
d iffic u lt to screen. These so lu tio n s have a lo w e r refractive index
th a n th a t o f cells. They m ay increase fading and, w h en dry, a llo w
the surface layer to scratch and crack w ith age.
Preventing Fading
Som e laboratories add an a n tio x id a n t to the m o u n tin g m ed ium
to prevent eosin fro m fad ing over tim e . B utylated hydroxy-
to lu en e is such a substance.
A 1% concentration, 2,6-di-tert-butyl-p-cresol, helps preserve
R om anow sky-type b lood stains b u t does n o t affect the preser-
va tio n o f h em a to xylin , O G , eosin Y, o r lig h t green SF yello w ish.
Cover slip p ing in h o t and h u m id geographic areas m ay a llo w
m oisture to collect w ith in the cell sam ple. T his m ay lead to
fad ing in the stored specim en.
Destaining and Restaining Slides
General Comments
D estaining and restaining a slide w ith the Papanicolaou m ethod
can be p erform ed on m ost samples b u t produces o p tim a l results
o n ly w h e n the o rig ina l cell sam ple was p rop e rly fixed.
There are several m ethod s fo r rem oving the coverslip. Once
th is is achieved, the next step is rem oval o f the m o u n tin g
m ed ium , w h ic h m ay take several hours' im m e rsio n in xylene.
The old er the slide, the m ore tim e th is step takes. T he tim e varies
depending on the a m o u n t and type o f m o u n tin g m ed ium used.
The next step is rem oval o f the nuclear dye. It is u su ally nec-
essary to place the slide in a d ilu te hyd ro ch lo ric acid, either an
aqueous o r an alcohol-based s o lu tio n . Rem oval o f h em a to xylin
m ay take fro m 5 to 20 m in o r longer, depending on the th ic k -
ness o f the cell sample. The slide is then rinsed g ently in ru n n in g
tap w ater fo r 10-15 m in .
Manual Removal of Coverslip
Procedure 1 fo r R ecent Slides
1. Place th e slid e in a C o p lin ja r c o n ta in in g xyle ne u n til th e
coverslip fa lls aw ay fro m th e slide. Pressure is n o t used to
rem ove th e coverslip. (T h is step m a y take h o u rs o r even
days, d ep end ing o n th e age o f th e slid e and th e m o u n tin g
m e d iu m used.) Keep th e C o p lin ja r tig h tly capped w h ile
w a itin g fo r th e coverslip to be dislodged. O nce th e cover
slip has fa lle n aw ay fro m th e slide, proceed to step 2.
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