PART THREE
Special Techniques in Cytology
used. The antibod ies th a t were investigated w ith th is technique
are listed in Table 3 5 .2 .
Cell Blocks
C ell blocks are a lu x u ry to have w h e n studying a cytologic speci-
m en due to th e ir versatility. C ell blocks are all-p urpose cellular
m aterial th a t can be used fo r special stains as w e ll as ICC. The
cell blocks w ith sta n d the s im ila rly processed paraffin-em bedded
surgical tissue specimen. In a d d itio n to the obvious advantage
fo r studying the tissue architecture, 1 0-12 a d d itio na l tissue sec-
tio n s can be cut fo r IC C (Fig. 3 5.2 ). Ten percent neutral buffered
fo rm a lin is used fo r fix a tio n o f tissue fragments. In te rp re ta tio n
o f the results is easier, and storage o f the specim en is easier w ith
u n lim ite d antigen preservation. For these reasons, cell blocks
are the sup erior m eth od fo r IC C fo r any cytologic specimen.
Suspensions o r b lo o d y specimens m ay be fixed in fo rm o l-s a lin e
to lyse red cells, o r the specim en m ay be collected in R P M I salt
s o lu tio n , treated w ith a com m ercial th ro m b in -p la s m a agent to
organize a clot, and th e n fixed in 10% fo rm a lin and processed
lik e a surgical specim en. T he m a in disadvantage o f th is m ethod
is the a va ila b ility o f enough m aterial.
A fu lly autom ated rap id cell b lo c k (RCB) system introduced
recently b y Cytyc (C e llie n t) increases the overall c e llu la rity in
the resulting sections w ith decreased tim e and reagents to m ake
a cell b loc k.28 A p rop rietary tissue cassette and filte r assembly
designed to capture tissue fragm ents also perm its them to be
p o sition ed in a plane fo r m ic ro to m y (Fig. 3 5 .3 ). S m all aliquots
o f xylene, alcohol, and p araffin are ra p id ly draw n th rou g h the
sam ple to produce a broad, u n ifo rm layer o f cells embedded in
paraffin. T he RCB produces a cell b loc k in 15 m in fro m residual
T hinP re p vials o r o th e r specimens and can be used fo r a variety
o f gynecologic samples, FN A biopsies, resp iratory samples, b od y
fluids, and samples fro m o th e r b od y sites. I f fo rm a lin is n o t used
as the p rim a ry fixative, the alternative fixative has to be validated
against the fo rm a lin -fix e d specim en and controls m ust be used
th a t have been fixed in the alternative fixative.
Controls
P ositive and negative controls m ust be p erform ed w ith each test
sample. Tissue controls are typ ic a lly used in m ost laboratories
fo r convenience, b u t th e ideal c on trol sho uld be a com parably
fixed cytology sample. I f tissue controls are used, caution m ust
be exercised in in te rp reta tion , because a d iffe re n t set o f artifacts
is present in tissue com pared w ith cytology samples. C y to l-
ogy controls can be ob tained o n a d a ily basis fro m the surgical
p atholog y bench w ith the use o f aspirates o r direct im p rints.
These preparations sho uld be fixed in the same w ay as the test
sample. It is m ore practical to use cells o n the cytology slide as
a p ositive-neg ative in te rn a l control, depending o n the a ntib od y
and cells th a t are present.
Specim ens of Lim ited Quality
Im m u n o h is to c h e m istry
can
be
ham pered
b y
the
lim ite d
q u a n tity o f cytology specimens. There is very lim ite d in fo r-
m a tio n in the cytology lite rature to address th is issue. Dabbs
et al. described a "d o ub le -lab e lin g m e th o d " to address the
p rob lem o f lim ite d m aterial w h en m ore th an one a ntib od y
is required to m ake a diagnosis. In b rief, im m u n o s ta in in g
was p erform ed on these slides w ith and w ith o u t a preced-
ing d e co lo riza tio n step. The results w ith b o th types o f stains,
w ith o r w ith o u t p rio r d ecolorization, were sim ilar. Background
staining was m ore o f a p rob lem o n the air-dried unfixed cases.9
Table 35.2 Antibodies Investigated with the Thin-layer Technique
A n tib o d y
D ilu tio n
S o u rce
Monoclonal carcinoembryonic
pd
Biomeda (Foster City,
antigen
ca)
polyclonal carcinoembryonic
antigen
pd
Biomeda
CEAD-14
pd
Enteric products
(StonyBrook, NY)
CD43
40
Dako (Carpinteria, CA)
CD68
150
Dako
Chromogranin
pd
Biomeda
Collagen IV
25
Dako
Desmin
75
Dako
Epithelial membrane antigen
100
Dako
Estrogen receptor
50
Dako
progesterone receptor
20
BioGenex (San
Ramon, CA)
Gross cystic disease fluid
protein-15
25
Signet (Dedham, MA)
Ham -56
6000
enzo (Farmingdale, NY)
Human chorionic gonadotropin
1000
Dako
Insulin
1000
Dako
AE1/AE3
200
Boehringer-Mannheim
(Indianapolis, IN)
K903
50
enzo
CAM5.2
pd
Becton Dickinson
(Franklin Lakes, NJ)
CD30
100
Dako
CD20
40
Dako
Laminin
5
Dako
MAC387
75
Dako
HHF-35
pd
Biomeda
Neuron-specific enolase
400
Dako
placenta-like alkaline
phosphatase
20
Dako
prostate-specific antigen
10
Dako
prostatic acid phosphatase
pd
Biomeda
CD34
800
BioGenex
Synaptophysin
30
BioGenex
Thyroglobulin
pd
Dako
UCHL1
200
Dako
Vimentin
pd
Dako
HMB-45
pd
enzo
O13
800
Signet
Lambda
40 000
Dako
Alpha-fetoprotein
pd
Immunstain
Thus recently stained o r archived cytology slides can be used
fo r im m unop eroxid ase studies, w h ic h include c o m m o n ly used
antibod ies such as C A M 5.2, A E 1/A E 3, K903, carcinoem bry-
onic antigen (C EA), desm in, H H F-35 (m uscle-specific actin),
1046
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