PART THREE
Special Techniques in Cytology
T he p rinciples o f the H C 2 assay are illu stra te d in Fig. 3 6 .8 .
T he test is p erform ed o n cervical sam ples taken w ith th e Digene
Cervical S am p ler and collected in to tw o o p tio n a l m edia: (1)
U C M (U niversal C o lle cting M e d iu m , Digene) o r (B) PreservCyt
liq uid -b ased cytolog y m edia (Th in P rep liq u id Pap v ia l; Cytyc
C orp., M arlb orou g h , M A ), b o th being valid ated fo r use w ith the
H C 2 assay. Before analysis, specim ens m ay be stored at ro o m
tem p erature fo r up to 21 days o r at 2 -8 ° C fo r up to 8 weeks.
In specim en processing, the first step is the release and dena-
tu ra tio n o f nucleic acids using conventional techniques. In the
next step, H P V target D N A is hyb rid ized w ith a m ix tu re o f fu ll-
leng th H P V type-specific labeled R N A probes in s o lu tio n . The
resultant R N A -D N A hybrids are then captured (step 3) o n to
the surface o f a m ic ro tite r w e ll coated b y specific antibodies.
Im m o b iliz e d hybrids are then reacted w ith a lkaline phos-
phatase-conjugated antibod ies specific fo r R N A -D N A hybrids
and detected w ith a chem ilum inescent substrate (step 4). As the
substrate is cleaved by the b o u n d a lkaline phosphatase, lig h t is
em itted th a t is m easured by a lu m in o m e te r (step 5).
The in te n s ity o f em itted lig h t, expressed as relative lig h t units,
is p ro p o rtio n a l to the a m o u n t o f target D N A in the specimen.
The detection lim it has been reported to be ap p roxim ately 5000
genom e equivalent. U sually, 1 pg/m L o f H P V 16 D N A is used
as the p ositive control, and samples are classified H P V D N A -
p ositive i f th e relative lig h t u n it reading ob tained fro m the lu m i-
n om ete r is equal o r greater th a n the m ean o f p ositive control
values. The H C 2 assay does n o t d isting uish specific H P V geno-
types b u t id entifies m u ltip le types together, m aking the vira l
Automated equipment
Sample collection device
Step 1
Release and
denature nucleic acids
with target DNA
Step 3
Capture RNA:DNA
hybrids onto a solid phase
React captured hybrids
with multiple antibody conjugates
chemiluminescent signal
F ig . 3 6 .8 Principles o f th e H y b r id C a p tu re 2 a ssa y.
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