Introduction

● Persistent infection with high-risk human papillomavirus (HPV)

genotypes is a necessary
cause of cervical carcinoma; HPV testing offers a way to improve
cervical screening
programs and decrease cancer mortality1–4

● PCR-based HPV DNA assays, such as the Roche AMPLICOR® HPV

Test (Figure 1), have
the advantage of being extremely sensitive and robust, and can be
performed on very small
aliquots of cervical samples in liquid cytology media, and on samples
from archival paraffin-embedded blocks

● The issue of possible cross-contamination with previously amplified

products (amplicon)
must be resolved for PCR-based HPV testing to be clinically reliable

● The AMPLICOR HPV Test prevents cross-contamination, by

incorporating deoxyuridine,
resulting in a product susceptible to degradation by AmpErase® enzyme
(uracil-Nglycosylase),
contained within the master mix

● We performed multiple assays with the AMPLICOR HPV Test, to

assess the level of crosscontamination

Figure 1
 Materials and methods

● Protocols were carried out in three separate areas of the

laboratory (Figure 2)

● A total of five AMPLICOR HPV Test runs, each consisting of 12

replicates of HPV type 16-
positive samples (5 x 105 SiHa cells/mL) and 12 HPV-negative
controls [HPV(-)C], were tested

● HPV-positive cultured cells were spiked into PreservCyt

preservative solution

● DNA extraction was performed using the QIAGEN QIAamp

MinElute Media kit procedure

● The beta-globin (BG) gene is isolated concurrently, and

assesses cellular adequacy, extraction and amplification for each
individually processed specimen

● HPV(+) and HPV(-)C samples were alternated (Figure 3) on the

MinElute vacuum manifold and on the amplification tray, in order to
assess potential carry-over

● PCR was performed on the GeneAmp PCR System 9700, as

shown in Table 1

● Amplicons were then hybridized in parallel in a microwell plate

format against HPV
probes (strips 1–4) and BG probes (strips 5–8)

● The cross-contamination rate was defined as the ratio of the

number of false-positives to the total number of negative samples
tested Figure 2. Partition of work into laboratory areas
Figure 3. AMPLICOR HPV Test amplification tray/microwell layout
Results
● The HPV cross-contamination rate was zero; 0/60 HPV(-)C samples
had HPV
OD450 values ≥0.2

● A low level of human DNA contamination was observed in one

negative sample
(BG OD450 of 0.308 [cut-off, 0.2]), which was declared a false-positive
resulting from a
technical error

● All 60 HPV(+) cell samples were positive for HPV and BG (HPV and
BG
OD450 ≥1.0)

● No HPV(+) or BG OD450 readings of ≤0.2 were observed, indicating

that no significant
sample loss occurred during preparation

Conclusions
● The AMPLICOR HPV Test is highly sensitive and reliable, with no
crosscontamination of HPV DNA observed when tested with high levels
of DNA input

● One sample showed a low level of BG contamination; however, this

may be due to technical error since hybridization of the same sample
against HPV in the multiplex test was negative

References
1. Walboomers JM, Jacobs MV, Manos MM et al. J Pathol 1999

2. Bohmer G, van den Brule AJ, Brummer O et al. Am J Obstet Gynecol

2003

3. Petry KU, Bohmer G, Iftner T et al. Am J Obstet Gynecol 2002

4. Munoz N, Bosch FX, de Sanjose S et al. N Engl J Med 2003