Stability of liquid Pap specimens for the detection of high-risk

human papillomavirus (HPV) types with the

Roche AMPLICOR® HPV Test*
Dmitriy Kosarikov1, Anona Ahmad1, Robert Current1, Karen Ding1, Sasha Igdari1,
Snehal Joshi1, Rashmi Kotadia1, Won Jae Lee1, Namneet Rudra1, Qian Zhang1, Terri Young1,
Jeffrey Waldman2, Karen Yu1, Rita Sun1
1
Roche Molecular Systems Inc., 4300 Hacienda Drive, Pleasanton, CA 94588, USA;
2
Planned Parenthood Shasta-Diablo, Walnut Creek, CA 94596, USA

Introduction ● All four HPV-positive specimens remained positive up to the Week 12 time-point. All six HPV-negative
specimens remained negative up to the Week 12 time-point. No DNA extraction column blockage
● The Roche AMPLICOR® human papillomavirus (HPV) Test* is a qualitative in vitro test for the was observed
detection of HPV in clinical specimens. The test utilizes amplification of target DNA by PCR,1 and ● Data obtained for the cervical specimens collected in SurePath™ preservative fluid and stored at 30°C
nucleic acid hybridization, for the detection of high-risk (HR) HPV DNA genotypes (16, 18, 31, 33, 35, are summarized in Table 3 (gray background indicates OD values below the assay cut-off)
39, 45, 51, 52, 56, 58, 59 and 68) in cervical cells collected in liquid media. Liquid-based cytology
media – PreservCyt® (ThinPrep® liquid Pap vial) preservative solution from Cytyc Corporation and Table 3. Summary data for cervical specimens collected in SurePath™ preservative fluid and stored
SurePath™ preservative fluid from TriPath Imaging Inc. – have been validated for use with the at 30ºC
AMPLICOR HPV Test, which is based on four major processes: ID Day 0 Day 3 Day 5 Day 7 Day 10 Day 14 Day 21
– Specimen preparation HPV BG HPV BG HPV BG HPV BG HPV BG HPV BG HPV BG
OD OD OD OD OD OD OD OD OD OD OD OD OD OD
– PCR amplification of target DNA using HPV-specific complementary primers
– Hybridization of the amplified products to oligonucleotide probes SP09 3.694 3.783 3.481 3.834 3.712 3.446 3.275 4.000 4.000 3.593 3.307 4.000 1.302 3.533
SP10 3.910 3.863 4.000 4.000 3.814 3.788 3.920 3.631 4.000 3.632 3.810 3.509 3.676 3.336
– Detection of the probe-bound amplified products by colorimetric determination SP11 0.069 3.663 0.060 3.579 0.058 3.390 0.066 1.982 0.071 2.427 0.066 0.737 0.051 0.761
● The AMPLICOR HPV Test permits simultaneous PCR amplification of HPV target DNA and β-globin SP12 4.000 3.575 4.000 3.816 3.795 3.365 3.957 3.270 3.747 3.119 3.782 3.327 3.719 0.580
DNA (cellular control). The β-globin gene is isolated concurrently, and assesses cellular adequacy, SP13 3.996 4.000 3.554 2.520 4.000 1.133 3.865 1.175 3.783 0.760 3.729 0.626 3.781 0.215
extraction and amplification for each individually processed specimen. The master mix reagent SP14 4.000 2.767 3.640 1.304 3.688 0.299 3.425 0.335 3.536 0.089 3.754 0.143
SP15 2.624 3.762 0.050 1.188 0.055 0.283 0.058 0.288 0.045 0.257 0.055 0.247 Not tested
contains primer pairs for DNA from 13 HR HPV genotypes and β-globin DNA. The detection of SP16 2.627 3.668 0.051 2.617 0.048 1.550 0.048 1.310 0.049 0.839 0.057 1.037
amplified DNA (amplicon) is performed using oligonucleotide probes that permit the independent SP17 0.048 4.000 0.056 3.775 0.067 3.606 0.053 3.184 0.060 0.046 0.058 3.428
N/A
identification of HPV amplicon and β-globin amplicon SP18 0.059 3.485 0.059 3.901 0.057 2.767 0.052 3.330 0.055 1.914 0.054 3.380
SP29 0.068 4.000 0.051 3.630 0.050 3.517 0.047 3.408 0.059 3.352 0.052 2.646 0.056 3.411
Objective SP30 3.994 3.708 3.482 3.523 2.030 2.888 3.412 2.164 3.617 1.139 2.012 0.787 3.226 1.959
Data shown in bold font indicate blockage of the DNA extraction column. Week 2 results for specimen PC08 were due to
● To investigate the stability of liquid Pap specimens collected in PreservCyt® and SurePath™ media and contamination. Day 7 results for specimens SP17 and SP18 were not available for operational reasons. Specimens SP14,
stored at different temperatures, for testing with the AMPLICOR HPV Test SP15 and SP16 were not tested on Day 21, since the stability failure for these samples was confirmed by testing at earlier
time-points
Materials and methods ● Specimens SP15 and SP16 lost the HPV signal on Day 3. Specimen SP14 lost the β-globin signal on
● Cervical specimens were collected at a Planned Parenthood Shasta-Diablo clinic, with informed patient Day 10. Since these failures were confirmed by re-testing on the following test days, there was no further
consent, according to a separate collection protocol. Specimens were stored at the collection site at testing for these specimens on Day 21. Note that 4/12 specimens stored at 30°C exhibited DNA
2–8°C and transported to the testing site at ambient temperature within 8 hours of collection. The extraction column blockage at Day 3. The number of column blockage events gradually increased over
stability study for each specimen was initiated (Day 0 testing) within 24 hours after delivery to the time, reaching 7/12 at Day 10. There was no clear correlation between column blockage and loss of
testing site. The following cervical specimens (in PreservCyt® or SurePath™ media) were monitored: signal
– 14 PreservCyt® for 21 days at 30°C; 7/14 tested HPV-negative at Day 0 ● Data obtained for the cervical specimens collected in SurePath™ preservative fluid and stored at 2–8°C
– 10 PreservCyt® for 12 weeks at 2–8°C; 6/10 tested HPV-negative on Day 0 are shown in Table 4 (gray background indicates OD values below the assay cut-off)
– 12 SurePath™ for 21 days at 30°C; 4/12 tested HPV-negative at Day 0
– 10 SurePath™ for 12 weeks at 2–8°C; 3/10 tested HPV-negative on Day 0 Table 4. Summary data for cervical specimens collected in SurePath™ preservative fluid and stored
● All specimens in this study exhibited a positive β-globin signal at Day 0. Loss of either the HPV or the at 2–8ºC
β-globin signal (confirmed by re-test on the following day) was considered a failure for any given ID Day 0 Week 1 Week 2 Week 4 Week 8 Week 12
specimen HPV BG HPV BG HPV BG HPV BG HPV BG HPV BG
OD OD OD OD OD OD OD OD OD OD OD OD
Results SP01 3.477 3.880
SP02 3.861 3.830
3.739 3.494
4.000 3.723
3.557 3.718
3.690 3.845
0.061 3.682
4.000 3.516 3.684 3.860
Not tested
4.000 3.849
● Data obtained for the cervical specimens collected in PreservCyt® preservative solution and stored at SP03 3.733 3.127 3.862 3.020 2.307 0.343 3.545 0.485 3.777 0.050 Not tested
30°C are summarized in Table 1 (gray background indicates OD values below the assay cut-off) SP04 0.066 4.000 0.074 3.707 0.052 3.563 0.054 2.757 0.049 1.929 0.061 0.266
SP05 4.000 3.942 3.865 3.517 3.666 3.916 3.797 3.251 4.000 3.505 3.830 3.289
Table 1. Summary data for cervical specimens collected in PreservCyt® preservative solution and stored SP06 3.832 3.767 4.000 3.709 4.000 3.495 3.563 3.426 4.000 2.874 4.000 3.081
at 30ºC SP07 4.000 3.664 3.809 3.736 3.858 3.576 3.742 3.235 3.920 3.184 3.918 2.665
SP08 4.000 3.747 3.975 3.713 3.911 3.665 3.926 3.418 4.000 3.373 3.982 3.410
ID Day 0 Day 3 Day 5 Day 7 Day 10 Day 14 Day 21 SP19 0.061 3.759 0.051 2.678 0.051 3.573 0.045 2.902 0.058 1.507
HPV BG HPV BG HPV BG HPV BG HPV BG HPV BG HPV BG
N/A
SP20 0.056 3.627 0.052 3.490 0.052 3.663 0.062 4.000 0.048 3.386
OD OD OD OD OD OD OD OD OD OD OD OD OD OD
Data in black represent the results of a confirmatory re-test. Data shown in bold font indicate that blockage of the DNA
PC11 0.062 4.000 0.051 3.379 0.048 3.638 0.053 3.548 0.054 3.540 0.067 3.428 0.045 4.000
PC12 0.057 3.662 0.049 3.499 0.047 3.498 0.049 3.457 0.055 3.437 0.055 3.535 0.050 3.861 extraction column was encountered. Week 1 results for specimens SP19 and SP20 were not available for operational reasons.
PC13 3.751 3.004 3.655 2.544 3.818 3.434 4.000 3.495 3.724 0.636 3.692 2.897 3.782 3.569 Specimens SP01, SP02 and SP03 were not tested on Week 12, since the stability failure for these samples was confirmed by
PC14 0.050 3.695 0.048 3.541 0.046 3.692 0.049 3.453 0.054 3.335 0.052 3.679 0.055 3.637 re-testing at earlier time-points
PC15 0.055 3.676 0.049 3.559 0.083 3.412 0.055 3.360 0.071 3.439 0.060 0.708 0.062 3.503
PC16 3.809 2.972 3.585 3.244 3.587 3.439 3.833 3.489 4.000 2.281 3.657 2.894 3.785 3.627 ● Specimen SP01 lost the HPV signal on Week 4. Specimen SP03 lost the β-globin signal on Week 8. Both
PC17 0.170 3.446 1.690 3.365 0.596 3.428 0.419 3.515 1.344 3.671 0.414 3.488 1.646 3.579
PC18 2.011 3.554 3.710 3.409 3.776 3.635 3.144 3.728 3.550 3.722 3.423 3.688 3.402 3.844 failures were confirmed by subsequent re-testing; therefore, these specimens were not tested on
PC19 0.057 3.478 0.067 3.856 0.051 1.104 0.053 3.679 0.096 3.719 0.041 3.518 Week 12. DNA extraction column blockage was encountered at the Week 8 time-point in
PC20 3.053 3.667 3.584 4.000 3.442 1.950 3.709 3.628 3.565 3.609 4.000 1.837 3/9 specimens. The rate of column blockage events did not change by the Week 12 time-point
PC21 0.058 3.957 N/A 0.055 3.669 0.058 1.990 0.052 3.827 0.052 3.856 0.063 3.653
PC22 3.644 3.439 3.509 3.728 3.728 0.598 3.722 0.986 4.000 3.719 3.636 3.720
PC23 2.713 3.825 4.000 4.000 3.614 2.247 3.569 3.612 3.468 3.871 3.442 2.502
PC24 4.000 3.317 3.606 3.942 3.994 1.865 3.777 3.722 3.757 3.867 3.784 3.761
OD: Optical density; BG: β-globin; Data in black represent the results of a confirmatory re-test. Day 3 results for specimens PC19
Conclusions
to PC24 were not available for operational reasons ● Cervical specimens collected in PreservCyt® preservative solution remained stable
for testing with the AMPLICOR HPV Test for a minimum of 21 days when stored
● Specimen PC17 was HPV-negative on Day 0, but exhibited a low positive signal for HPV upon testing
at subsequent time-points; this may be due to the HPV titer in the sample being around the limit of at 30ºC, and for a minimum of 12 weeks when stored at 2–8ºC. No DNA
analytical sensitivity for the test. The other six HPV-negative specimens remained negative at each extraction column blockage occurs when PreservCyt® preservative solution samples
subsequent time-point. All of the seven HPV-positive specimens remained positive up to Day 21. No are tested
DNA extraction column blockage was observed ● Cervical specimens collected in SurePath™ preservative fluid remained stable for
● Data obtained for the cervical specimens collected in PreservCyt® preservative solution and stored at testing with the AMPLICOR HPV Test when stored for up to 2 weeks at 2–8ºC. These
2–8°C are shown in Table 2 (gray background indicates OD values below the assay cut-off) specimens are not stable for testing with the AMPLICOR HPV Test when stored
Table 2. Summary data for cervical specimens collected in PreservCyt® preservative solution and at 30ºC. SurePath™ preservative fluid specimens exhibit DNA extraction
stored at 2–8ºC column blockage, which is dependent on the storage conditions and time.
ID Day 0 Week 2 Week 4 Week 8 Week 12
Elevated temperature and/or prolonged storage of these specimens increase the
HPV BG HPV BG HPV BG HPV BG HPV BG incidence of column blockage
OD OD OD OD OD OD OD OD OD OD
PC01
PC02
3.950 3.966
0.057 3.682
3.827 3.687
0.050 3.928
3.690 3.343
0.066 3.669
3.879 3.731
0.081 3.608
3.793 3.541
0.048 3.462 Acknowledgements
PC03 0.060 3.912 0.057 3.569 0.068 3.357 0.092 3.614 0.044 2.696
PC04 0.066 3.833 0.059 3.808 0.065 3.699 0.080 4.000 0.053 3.533 The authors are very thankful to the nursing staff of the Planned Parenthood Shasta-Diablo clinic in Walnut Creek, CA, USA,
PC05 3.744 3.758 3.893 3.189 3.893 2.628 3.774 3.789 4.000 3.768 for their efforts in collecting the clinical specimens for this and other studies
PC06 0.051 3.599 0.064 3.630 0.051 3.830 0.052 3.833 0.053 4.000
PC07 4.000 4.000 3.846 3.578 3.892 2.472 3.743 3.610 3.626 3.914
PC08
PC09
0.061 3.868
0.058 4.000
1.012 3.601
0.058 3.648
0.067 3.655
0.057 3.556
0.074 4.000
0.050 0.939
0.048 3.862
0.056 3.961
Reference
PC10 3.297 3.526 3.336 3.651 3.576 3.556 3.638 3.776 3.215 3.786 1. Myers TW, Gelfand DH. Biochemistry 1991;30:7661–6
Data in black represent the results of a confirmatory re-test. Week 2 results for specimens PC08 were due to contamination.
*Under development
Re-testing was not possible for operational reasons