PART THREE
Special Techniques in Cytology
to produce consistent staining o f nuclei and cytoplasm th a t can
easily be interpreted b y the im aging system. I f staining reagents
and protocols are substituted o r changed the stain q u a lity can
be com prom ised and slide im aging w ill be unsuccessful. Slides
prepared fo r im aging devices m ust be coverslipped evenly and
fiee o f a ir bubbles. A utom ated coverslipping instrum ents should
be considered fo r use w ith im aging systems to provide consist-
ent coverslipping results th a t reduce slide rejections and im aging
'events'.
T he N orthw estern M e m o ria l H o sp ital cytop atholog y labora-
to ry uses the Sakura Tissue-Tek DRS A u to m a tic Slide Stainer.48
T he progressive sta in in g m eth od is used to stain all cell
samples.
T he T hinP re p Papanicolaou stain has a sto ic hiom etric con-
te n t very near the D N A content o f the nucleus, thus a llo w in g
im aging o f the slide based on th e optical d ensity o f the nucleus.
C o nve ntio na l and ThinP re p slides processed o n the T2000
require a d iffe re n t staining p rotocol th an cervical slides proc-
essed o n the T hinP re p 3000 (E. Lucas, personal com m unica-
tio n , 2007). Graded alcohols are used fo r h yd ra tio n o f the cells.
Surgipath h em a to xylin , O G -6, and EA 50 are used fo r staining
c onventional slides. T hinP re p stains are used fo r liquid-based
samples. Separate staining lines are u tiliz e d fo r nongynecologic
and fine-needle aspirates.
Environmentally Friendly Staining Procedure
In 2006, G ill introd uced and described a m o d ific a tio n o f the
Pap anicolaou sta in in g procedure. T his procedure reduces the
expense o f costly chem icals and xylene disposal and, at the
sam e tim e yield s an effective stain (Fig. 31.5).49 It w o u ld seem
to be a m eth od deserving o f fu rth e r stud y and im p le m e n ta tio n ,
especially fo r in d ivid u a ls setting up new lab oratories in coun-
tries w here th e a va ila b ility o f chem icals and fu n d in g are o f
critical consideration. W a te r is used to rem ove carbowax fro m
spray-fixed slides. There are n o graded alcohols, and p la in tap
w a te r is used to b lue th e h e m a to x y lin and p rio r to OG. Listed
b e lo w is th e procedure fro m the above-cited article b y G ill
as used in th e Tho m a s Jefferson U n iv e rsity H o sp ita l cytology
laboratory.
1. Tap w a te r: 10 dips
2. Tap w a te r: 10 dips
3. G ill's h e m a to x y lin : 1 -2 m in
4 . Tap w a te r: 10 dips
5 . Tap w a te r: 10 dips
6 . Tap w a te r: 2 m in
7 . Tap w a te r: 10 dips
8. O G -6 : 5 s
9 . A b s o lu te e th a n o l: 10 dips
10. 0 .5 % acetic acid (H O A c ): 10 dips
11. 0 .5 % acetic acid (H O A c ): 10 dips
12. EA: 6 -8 m in
1 3 .0 . 5 % acetic acid (H O A c ): 10 dips
14. 0 .5 % acetic acid (H O A c ): 10 dips
1 5 .0 . 5 % acetic acid (H O A c ): 10 dips
16. A b s o lu te e th a n o l: 10 dips
17. A b s o lu te e th a n o l: 10 dips
18. A b s o lu te e th a n o l: 10 dips
19. X yle ne : 10 dips
20. X yle ne : 10 dips
21. X yle n e u n til cover slip p e d
Fig. 31.5 Enviro-pap stain (Thomas Jefferson University Hospital cytology
laboratory) (x LP).
On-Site Quick Staining Procedure
This procedure is used b y the U n ive rsity o f Rochester cyto-
p atholog y lab o rato ry (Figs 31.6 and 31.7).
1. 9 5 % e th a n o l (o n -s ite fix a tio n )
2. 5 0 % e th a n o l: 10 dips
3. Tap w ater: 1 0 -2 0 dips
4. H e m a to x y lin # 2 (R ic ha rd A lla n ): 2 0 -3 0 s
5. Tap w ater: 1 0 -2 0 dips
6. C la rifie r # 2 (R ic ha rd A lla n ): 2 q u ic k dips
7. Tap w ater: 1 0 -2 0 dips
8. B lu e in g reagent (R ic ha rd A lla n ): 20 s
9. Tap w ater: 1 0 -2 0 dips
10. 5 0 % e th a n o l: 10 dips
11. 9 5 % e th a n o l: 10 dips
1 2 .O G (H a rle c o ): 1 5 -2 0 dips
1 3 .9 5 % e th a n o l: 10 dips
1 4 .9 5 % e th a n o l: 10 dips
1 5 .E A 65 (e o s in ) (H a rle c o ): 30 s
1 6 .9 5 % e th a n o l: 10 dips
1 7 .9 5 % e th a n o l: 10 dips
1 8 .9 9 % e th a n o l: 10 dips
1 9 .9 9 % e th a n o l: 10 dips
20. 9 9 % e th a n o l: 10 dips
21. X yle ne : 10 dips
2 2 . X yle ne : 10 dips
2 3 . X yle ne : 10 dips
Helpful Hints When Staining
with the Papanicolaou Method
• W h e n slides are tra n sfe rre d fro m w a te r to h e m a to x y lin ,
th e glassy d rop le ts o f w a te r o n th e slid e m u s t disappear
b e fore p la c e m e n t o f slides in h e m a to x y lin . T h is step m a y
take as lo n g as 30 m in a nd depends o n th e thickness o f
th e cell sam ple. I f th e glassy appearance does n o t disap-
pear fro m th e slide, th e h e m a to x y lin dye does n o t p en-
etrate th e n uc le us and th e cells m a y appear hazy. D iscard
w a te r rin se a fte r each use.
988
previous page 974 ComprehensiveCytopathology 1104p 2008 read online next page 976 ComprehensiveCytopathology 1104p 2008 read online Home Toggle text on/off