PART THREE
Special Techniques in Cytology
the scoring o f at least 100 o r 200 consecutive cells is often
considered as adequate. In the case o f standardized diagnos-
tic assays, the n um b er o f scored cells is u su a lly lower. A m ore
rap id b u t eq ually reliab le scoring m eth od has been developed
fo r diagnostic m u ltita rg e t FISH assays th a t contain fo u r d iffe re n t
probes.4 In th is scanning m ethod , no m ore th a n 25 cells need to
be scored. These cells are selected based on nuclear a b n o rm a li-
ties inc lu d in g enlargem ent, irreg ular borders, and patchy D A PI
staining. In case o f a m p lific a tio n detection (e.g. H ER 2 FISH ),
it m ay be sufficient to score as few as 20 cells. Several im ag-
ing systems have been developed fo r autom atic and unattended
c ounting o f FISH signals in a large n um b er o f interphase nuclei
(Table 3 6 .1 ) .5 These systems use th ree-d im ensional analysis to
capture all signals w ith in the nucleus. The current US Food and
D rug A d m in is tra tio n (FD A )-approved applications in cytology
are scoring o f H ER 2 gene a m p lific a tio n in breast cancer and
aneusom y detection in u rin a ry specimens in c o n ju n c tio n w ith
U ro V ysion m u ltita rg e t FISH testing. The im aging systems gener-
ate image galleries th a t can be edited at the com p uter screen
fo r the fin a l result (Fig. 3 6.2 ). Im p o rta n tly, th ey a llo w fo r per-
m an en t d ocu m e ntation and retrospective reanalysis o f the FISH
results. A sum m ary o f the m ost c om m o n applications o f FISH in
cytop atholog y is show n in Table 3 6.2 .
Polymerase Chain Reaction
Polym erase chain reaction (PCR) was invented in 1983 by
K. M u llis , w h o received the N o b el Prize in C h em istry in 1993
fo r th is groundbreaking discovery. PCR technique opened the
w ay to m od ern m olec ular analysis. It is an in v itro technique
used to a m p lify specific regions o f a D N A strand in an expo-
n e n tia l m anner. T his can be a single gene, ju st a part o f a gene,
o r a noncod ing sequence. M ost PCR m ethod s typ ic a lly a m p lify
DNA double stand
Two split DNA single strands
1. dénaturation
probe in excess
labelled target sequence
\
t
\
3. washing
\
i
\
\
2. hybridization
F ig . 3 6 .1 B a s ic te c h n ic a l s te p s o f flu orescence in situ h ybrid iza tio n .
D N A fragm ents o f up to 10 kilobase pairs. PC R requires the fo l-
lo w in g basic com ponents: a D N A tem p late th a t contains the
region o f the D N A frag m ent to be am p lified , one p a ir o f o r m ore
prim ers th a t are com p lem entary to the D N A regions at the 5'
and 3' ends o f the D N A region th a t is to be am p lified , a D N A
polym erase in order to synthesize a D N A copy o f the region to
be am p lified , and deoxynucleotide triphosphates fro m w h ic h
the D N A polym erase b u ild s the new D N A , in a d d itio n to appro-
priate b u ffe r so lu tio n s and ions. The PCR is carried o u t in sm all
reaction tubes (0.2- to 0.5-m L volum es) con tain in g a reaction
v o lu m e typ ica lly o f 1 5-10 0 m L th a t are inserted in to a th erm al
cycler. T his m achine heats and cools the reaction tubes w ith in it
to the precise tem perature required fo r each step o f the reaction.
The standard PCR u su a lly consists o f a series o f 3 0 -3 5 cycles
(Fig. 3 6 .3 ) .
Microsatellite Analysis/Loss of Heterozygosity
M icrosatellites are noncod ing tandem repeats o f one to six
nucleotides scattered th ro u g h o u t the genome. M icrosatellite
sequences o f an in d ivid u a l are fixed fo r life and are the same in
every tissue. The m icrosatellites o n the tw o alleles are slig h tly
d iffe re n t in m ost ind ivid uals, w h ic h is referred to as heterozy-
gosity. D e le tio n o f one o f the tw o alleles leads to loss o f hetero-
zygosity (L O H ). Therefore L O H analysis b y m icrosatellite-based
assays can detect clonal loss o f one allele at a target locus. The
procedure includes PCR a m p lific a tio n and locus-specific m ic ro -
satellite analysis. N u m e ric genom ic alterations occurring during
tu m o r cell tra n sfo rm a tio n lead to a m easurable s h ift in the n o r-
m a lly balanced ra tio o f the tw o alleles detectable b y capillary
electrophoresis (Fig. 3 6.4). M icrosatellites lin k e d to tu m o r sup-
pressor genes involved in m a lig n a n t tra n sfo rm a tio n are m ost
often analyzed.
There are c urrently few applications fo r L O H analyses in
cytopathology. O th e r th an in FISH, L O H analysis is based on
PCR o f extracted D N A and requires c on trol D N A fro m b lo o d
leukocytes o r fro m n o rm a l tissue o f the same patient. N o rm a l
cells often o u tn u m b e r the tu m o r cells in cytologic specimens.
T his can easily lead to false-negative results o f L O H analysis
due to d ilu tio n o f tu m o r D N A unless the tu m o r cells have been
microdissected. The advantage o f L O H over FISH is the hig her
reso lu tion , since selected D N A stretches o f very sm all size can
be analyzed fo r a llelic im balance. In a d d itio n, m an y d iffe re n t
m icrosatellite m arkers can be included in one assay.
Laser Microdissection
Laser-assisted m icrodissection (L M D ) has becom e an ind is-
pensable to o l fo r m olecular analysis o f extracted nucleic acid
fo r m olec ular applications, inc lu d in g PCR o f tu m o r D N A .6
T a b le 36.1 C om m ercial System s fo r A u to m a te d S canning and Fluorescence in situ H ybridization Spot C ou n tin g
S yste m n a m e
C o m p a n y
C lin ic a l a p p lic a tio n
H o m e p a g e
M e ta fe r/m e ta cyte
M etaSystem s
HER2 a m p lifica tio n , b la d d er cancer (UroVysion)
h ttp ://w w w .m e ta syste m sg ro u p .co m
Ariol
A p p lie d Im a g in g Corp.
HER2 a m p lifica tio n
h ttp ://w w w .a icorp.com
D uet System
B ioview
Bladder cancer (UroVysion), HER2 a m p lifica tio n
h ttp ://w w w .b io v ie w .c o .il
Ikoniscope
Ikonysis
Bladder cancer (UroVysion)
h ttp ://w w w . ikonisys.com
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