chapter
31
Cytopreparatory Techniques
Catherine M Keebler and Michael Facik
Contents
In tro d u c tio n
C r o s s - C o n t a m in a tio n M e t h o d t o A v o id F lo a te rs
F ix a tio n
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F ix a tio n M e t h o d s
A ir D ry in g f o r S e le c te d C e ll S a m p le s
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O t h e r C o n s id e r a tio n s
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C o u n te r s ta in s : O r a n g e G a n d E A
A u t o m a t e d V e rs u s M a n u a l S ta in in g p ro c e d u re s
S ta in in g B o d y F lu id s
E n v ir o n m e n ta lly F r ie n d ly S ta in in g p ro c e d u re
I n f e c tio n C o n t r o l
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O n - S it e Q u ic k S t a in in g p ro c e d u re
H e lp fu l H in ts W h e n S ta in in g w it h t h e p a p a n ic o la o u M e t h o d
C o n c lu d in g R e m a rk s
T r o u b le s h o o tin g t h e p a p a n ic o la o u S ta in in g M e t h o d s
Introduction
D iagnostic cytologic criteria are based o n h o w cell samples are
prepared. M ethod s o f preparation, fix a tio n , and staining m ay
vary fro m lab o rato ry to laboratory. M o st m ethod ologies, h o w -
ever, are s im ila r in th e ir basic concepts and goals: to reduce cell-
u la r artifacts and o b tain an o p tim a l diagnostic cell sam ple.1-6
N e w technologies b o th in cell fix a tio n and in preparation
m ay y ie ld p ossib ilities fo r a d d itio na l diagnostic tests to be made
available b y the cytop atholog y laboratory. W h ile these new
technologies m ay require a d iffe re n t approach to sam ple prepa-
ra tio n , the basic principles o f fixa tio n , preparation, and staining
m ethod s rem a in the same, i.e. to o b tain an o p tim a l diagnostic
cell sam ple.7-17
Fixation
General Comments on Fixation
T he purpose o f cytologic fixatives is to m a in ta in , as closely as
possible, the cytom orp holog ic characteristics and diagnostically
essential cytochem ical elem ents o f the cell. A n appropriate fixa-
tive fo r cytodiagnostic purposes sho uld p erform the fo llo w in g
fu n c tio n s:18
• P enetrate cells ra p id ly;
• M in im iz e cell shrinkag e;
• M a in ta in m o rp h o lo g ic in te g rity;
• D eactivate a u to ly tic enzym es;
• Replace c e llu la r w ater;
• F ac ilitate d iffu s io n o f dyes across cell b o un d aries;
• H e lp cells adhere to a glass surface;
• P ro vid e c o n siste n t results o ver tim e ;
• Prod uce a p e rm a n e n t cell record;
• S top c e llu la r a nd m ic ro b ia l g ro w th (a n ti-m ic ro b ia l).
H is to ric a lly , P a p an ic olao u ,19 lik e Reider b efore h im ,20 used
ether and 9 5% e th a n o l (1:1) as th e cytolog ic fixative o f choice.
C u rren tly, 9 5% e th a n o l, 8 0% iso p ro p a n o l, 100% m e th a n o l,
9 5% d enatured a lc oh ol, and variou s c o m m e rc ia lly available
spray fixatives have replaced th e e th e r/e th a n o l m ix tu re as the
fixa tive o f choice fo r safety reasons. F or lab o rato rie s using
liq u id -b a se d techniques, th e type o f fixa tive depends o n the
m e th o d selected. C u rre n t liq u id -b a se d p rep arations enhance
c e llu la r preservation b y u tiliz in g fixa tive s o lu tio n s th a t are
e ith e r e th a n o l- o r m ethanol-b ased . C e ll a lte ra tio n s m ay be
m in im a l a m ong th e variou s fixatives in use and m ay fa ll in to
one o f five categories: w e t fix a tio n , w e t fix a tio n w ith subse-
q u e n t a ir d rying , spray fix a tio n , liq u id -b a se d fix a tio n , and lys-
ing fix a tio n fo r b lo o d y samples.
Fixation Methods
Wet Fixation
W e t fix a tio n , i.e. subm erge the cell sam ple im m e d ia te ly in to the
fixative s o lu tio n . The cell sam ple rem ains in the fixative solu -
tio n u n til it arrives in the laboratory, w here it is accessioned and
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