31
Cytopreparatory Techniques
Procedure fo r Samples with a Cell Button
16. P o u r o ff th e s u p e rn a ta n t in to a disp osab le p lastic c o n -
ta in er.
17. Resuspend th e b u tto n in 10 m L o f H a n k 's balanced sa lt
s o lu tio n .
18. E xa m in e a d rop o f resuspended flu id m ix e d w ith to lu i-
d in e b lu e and w ith a m icroscop e d e te rm in e th e a m o u n t
o f flu id to be added to th e cham ber. W ith experience,
o ne m ig h t le a rn i f th e re is adequate m a te ria l fo r a d ire c t
p u ll-a p a rt sm ear.
19. Resuspend th e b u tto n w ith 30 m L o f H a n k's s o lu tio n .
20. Place th e test tu b e o n a v o rte x m ix e r to c om p le te th e
susp e nsion o f th e b u tto n in H a n k's s o lu tio n .
21. A d d o ne d rop o f preservative to th e c ytocentrifug e
cha m b e r i f th e slid e is to be P a p an ic olao u -sta ine d .
22. S p in th e sp ecim en fo r 4 m in a t 6 00 rp m , o r i f CSF, fo r
5 m in a t 5 00 rp m .
23. R em ove slides w ith care.
24. Place disp osab le c y to fu n n e l cham bers d ire c tly in to a
b io h a z a rd o u s bag fo r c o n ta m in a te d m a te ria l.
25. Place cham bers a nd slid e clips in to d isin fe c ta n t.
26. Place slides fo r P a p a n ic o la o u s ta in in to 9 5 % e th a n o l o r
fix w ith spray fixative. T h e la tte r m a y h e lp cells adhere to
th e glass slid e surface.
27. A llo w th e a ir-d rie d sam p le to c o n tin u e to a ir d ry b e fore
s ta in in g w ith D iff-Q u ik .
28. R em ove e q u ip m e n t fro m d is in fe c ta n t a fte r 1 5 -2 0 m in .
29. R inse w e ll a nd d ry th o ro u g h ly .
Procedure fo r Samples without a Cell Button
(See steps 1 -1 5 .)
16. In v e rt th e test tu b e to d ra in o ff sup e rna ta nt.
17. Take th e sam p le fro m th e base o f th e te st tu b e and m ix
w ith H a n k's s o lu tio n .
18. M ix o n a v o rte x m ix e r to resuspend cells.
19. A d d p reservative to each cytoc en trifug e cham ber.
20. A d d a fe w drops o f resuspended specim en to th e cham ber.
21. S p in th e sp ecim en fo r 4 m in a t 6 00 rp m , o r i f CSF, fo r
5 m in a t 5 00 rp m .
22. R em ove slides w ith care.
23. Place disp osab le c y to fu n n e l cham bers d ire c tly in to a red
bag.
24. Place cham bers a nd slid e clips in to d isin fe c ta n t.
25. Place slides fo r P a p a n ic o la o u s ta in in to 9 5 % e th a n o l o r
fix w ith spray fixative. T h e la tte r m a y h e lp cells adhere to
th e glass slid e surface.
26. A llo w th e a ir-d rie d sam p le to c o n tin u e to a ir dry.
27. R em ove e q u ip m e n t fro m d is in fe c ta n t a fte r 1 5 -2 0 m in .
28. R inse w e ll a nd d ry th o ro u g h ly .
Respiratory Tract Specimens and Other
Mucoid Samples
M ethod s o f processing m ucoid specimens such as sp utum , b ro n -
chial washings, b ron c ho alve o la r lavage samples, and tracheal
aspirates m ay vary fro m la b o ra to ry to lab oratory.68-74 In fo rm e r
tim es, som e laboratories used the "p ic k and sm ear" m eth od and
som e used the Saccom anno m eth od .75 T he la tte r m eth od was
designed to hom og enize sp utum samples before spreading the
m aterial on to a glass slide. Today, sp utum samples are a ra rity
in m ost laboratories.
The fo llo w in g is adapted fro m the procedure used at the
N orthw estern M e m o ria l H o sp ital cytop atholog y lab o rato ry fo r
m ucoid specimens (E. Lucas, personal c o m m u nica tion , 2007).
Unfixed Mucoid Respiratory Tract Sam ples
1. Label a cen trifug e tu b e w ith th e p a tie n t id e n tific a tio n
procedure.
2. G e n tly shake th e specim en.
3. P o u r up to 45 cc o f flu id in to th e cen trifug e tube.
4. Secure th e cap and place th e cen trifug e tu b e in to th e
centrifuge.
5. Balance th e tu b e w ith an eq ual v o lu m e tube.
6. C e n trifu g e th e sp ecim en a t 1800 rp m fo r 5 m in .
7. C a re fu lly rem o ve th e tu b e w ith o u t d is tu rb in g th e p e lle t.
8. P o u r o ff th e s u p e rn a ta n t in to th e o rig in a l sp ecim en c on -
ta in er.
9. A d d 30 cc o f C y to L y t to th e specim en.
10. V o rte x th e specim en.
11. Secure th e cap a nd place th e c entrifug e tu b e in to th e
h o ld e r o f th e centrifuge.
12. Balance th e c entrifug e b y p la c in g a n o th e r tu b e o f equal
v o lu m e d ire c tly o p p o site th e sp ecim en tube.
1 3 . C lose th e lid o f th e centrifuge.
14. C e n trifu g e th e sp ecim en a t 1800 rp m fo r 5 m in .
1 5 . W h e n th e c entrifug e stops c o m p le te ly o p e n th e lid and
c a re fu lly rem o ve th e tubes w ith o u t d is tu rb in g th e p e llet.
16. P o u r o ff th e s u p e rn a ta n t in to a lab e le d w aste c on tain er.
I f sam p le is s till m u c o id rep eat Steps 9 -1 5 u n til desired
p e lle t is achieved.
17. Prepare fo u r glass slides b y h a n d w ritin g th e case
n u m b e r and p a tie n t's id e n tific a tio n u sin g a so lve n t-
re sista n t m arker.
1 8 . A tta c h a c y to fu n n e l to each o f th e fo u r lab e le d glass
slides.
1 9 . P ip ette o ne to tw o drop s o f sp ecim en in to each cyto-
fu n n e l w e ll.
20. Cap w ith th e c y to fu n n e l cap.
21. Place in th e cytocentrifug e, b e in g careful to balance w ith
a n o th e r s lid e /c y to fu n n e l setup.
22. S p in fo r 1200 rp m fo r 4 m in .
2 3 . Prepare o ne C o p lin ja r fille d w ith 9 5 % e th a n o l fo r each
case.
24. O nce c yto c e n trig u g a tio n is com p lete, im m e d ia te ly
rem o ve a ll slides and im m e rs e in th e C o p lin ja r o f 9 5%
e th a n o l.
2 5 .S ta in in n o n g yn e c o lo g ic setup.
2 6 .M o u n t and coverslip.
Ferruginous Bodies
S p utum samples processed b y the above m ethod s m ay n o t be
o p tim a l to detect ferrug inous bodies. B ronchial washings m ay
yie ld better results th a n e ith er sp utum samples o r b ronchoal-
veolar lavage. Specimens fro m patients w ith a h is to ry o f asbes-
tos exposure m ig h t be prepared fo r Prussian b lue staining to
im p rove the detection rate o f ferrug inous bodies.2,76-79
Pneum ocystis carinii
D etection o f
P
c a r in ii
in b ron c hial wash samples stained w ith
the D iff-Q u ik m eth od is fa irly reliab le and rapid. The ro u tin e
Papanicolaou stain m ay be less sensitive fo r the washings b u t
is im p roved in b ronchoalveolar lavage specimens. S e nsitivity is
increased w ith the use o f G ro c o tt-G o m o ri m eth en a m ine silver
997
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