Stability of human papillomavirus (HPV) DNA in PreservCyt®

at 4ºC and room temperature, as detected by the

Roche AMPLICOR® HPV Test
Heather A Cubie, Kate S Cuschieri, Catherine Moore
Specialist Virology Centre, Royal Infirmary of Edinburgh, Edinburgh, UK

Introduction Results
● Routine screening of cervical cell specimens for human papillomavirus (HPV) is now ● Women recruited to this study had a concurrent report of ≥ CIN2, and all were initially
recommended as an effective management strategy for women with borderline cervical positive for HPV
abnormalities1–3 ● HPV-positivity was maintained in all samples at both temperatures until
● A cost-effective strategy for HPV testing requires a robust, sensitive and specific assay that 28 days after collection (Table 1)
is compatible with Pap smear and liquid-based cytology (LBC) protocols performed at ● At 28 days after collection, some HPV-positivity was lost at both temperatures (Table 1).
ambient temperature1 Some HPV-positivity was also lost after 28 days
● The cellular suspension contained within LBC collection media, such as PreservCyt® ● When consolidating the results (using aliquots stored at both temperatures), 7/10 samples
preservative solution, is convenient for HPV testing following cytological assessment tested positive at 62 days

● Standard LBC collection media were not originally formulated for the preservation of viral Table 1. Stability of HPV DNA in PreservCyt® at two temperaturesa
DNA

Specimen
Time-point (days)
● Accurate detection of viral DNA from LBC specimens stored at room temperature offers

no.
0 2 7 14 21 28 35 62
a significant advantage over strategies requiring the refrigeration of samples prior to HPV
4°C RT 4°C RT 4°C RT 4°C RT 4°C RT 4°C RT 4°C RT 4°C RT
testing
● The Roche AMPLICOR® HPV Test is a rapid and highly sensitive PCR-based assay that 1 + + + + + + + + + + + + + + – +
accurately detects the 13 most common high-risk HPV genotypes (Figure 1) 2 + + + + + + + + + + + + + + + –
3 + + + + + + + + + + + + + + + –

Native HPV
4 + + + + + + + + + + + + + + + +
genome UNG
digestion

Denature and
5 + + + + + + + + + + – + + + + +
hybridize
6 + + + + + + + + + + – + + + + +
Biotinylated PCR
primers
7 + + + + + + + + + + – – – – – –
Wash, add
avidin-HRP
conjugate
8 + + + + + + + + + + + + – – – –
9 + + + + + + + + + + + – – – – +
10 + + + + + + + + + + – – – – – –
HPV
probe
a+/–
represent AMPLICOR HPV-positive or HPV-negative; RT: Room temperature
Immobilized hybrids react Oxidized Oxidized Hybridization
with avidin-HRP TMB TMB

TMB TMB

HRP produces color

upon addition of
Conclusions
TMB substrate
Add stop solution,
read absorbance
● The integrity of HPV DNA can be maintained in PreservCyt® preservative
HPV: Human papillomavirus; UNG: Uracil-N-glycosylase; PCR: Polymerase chain reaction; solution
HRP: Horseradish peroxidase; TMB: 3,3',5,5'-tetramethylbenzidene

● The stability of HPV DNA in PreservCyt®, as determined by a positive result

Figure 1. The AMPLICOR HPV Test
in the AMPLICOR HPV Test, is comparable between storage at room
temperature and 4ºC
Objective ● Detection of HPV DNA in PreservCyt® samples using the AMPLICOR HPV
● To investigate the stability of HPV DNA in PreservCyt® LBC collection media at 4°C Test should be performed within 21 days of sample collection
and at ambient temperature (20–23°C), using the AMPLICOR HPV Test
● The AMPLICOR HPV Test is a robust assay for the routine detection of HPV
in LBC specimens, and is compatible with cytopathological protocols

Materials and methods involving the storage of samples at room temperature

● Cervical cytology specimens from 10 women with known high-grade cervical disease were
each rinsed into 20 mL of PreservCyt®
● Samples were split into two 10-mL volumes, and each was maintained at either 20–23°C References
or 4°C
● Aliquots of both samples from all 10 specimens were taken at 6 hours and 2, 7, 14, 21, 28, 1. Manos MM. BMJ 2001;322:878–9
35 and 62 days after collection 2. No author given. Am J Obstet Gynecol 2003;188:1383–92
● Automated DNA extraction was performed on all aliquots using a QIAGEN BioRobot
3. Solomon D, Schiffman M, Tarone R. J Natl Cancer Inst 2001;93:293–9
9604, with a modified methodology4
● HPV DNA was detected in extracted material using the AMPLICOR HPV Test 4. Cuschieri KS, Seagar AL, Moore C et al. J Virol Methods 2003;107:107–13