Human Papillomavirus (HPV) DNA Detection with Genotyping
on Roche cobas-4800, SurePath Specimens
Performance Characteristics
Legacy Laboratory Services performed validation studies of SurePath liquid based cytology
specimens for HPV Genotype testing with the Roche cobas® HPV Test. The Roche method is
FDA approved (IVD) on the cobas-4800 for testing pre- and post-aliquot ThinPrep (TP)
specimens. SurePath (SP) samples were evaluated using an aliquot removed from the original
SP vial after processing for Pap testing.
Method Validation Study Results
Patient Correlation Study
Seventy (70) post-aliquot SurePath patient samples were tested for HPV using both Roche’s
Cobas-4800 HPV Test and Qiagen’s Hybrid Capture-2 High Risk HPV DNA Test (Digene HC2). Of
the 70 samples tested, 40 were Digene negative and 30 were Digene positive. All of the
negative samples correlated. In contrast, 7 of the 30 Digene positive samples were negative on
the Cobas-4800. Details are shown in tables 1, 2, and 3. The overall concordance for all samples
was 90% (63/70), and concordance of positive samples was 76.7% (23/30). See tables 1-3.
Table 1. HPV testing results, Cobas-4800 versus Digene.
Cobas-4800
Digene HC2
Pos
Neg
Pos
23
0
Neg
7
40
Page 1 of 10
Table 2. Concordance of HPV testing by Cobas-4800 versus Digene method
Samples included
Number
Cobas-4800 Concordance with
Digene
Total Samples
70
90%
Digene HPV Negative
40
100%
Digene HPV Positive
30
76.7%
Table 3. Digene Positive Samples, Results on Cobas-4800
Number
Percent
Other
HR+
HPV16+
HPV18+
17
56.7%
1
3.3%
2
6.7%
HPV-16
&
Other+
2
6.7%
HPV-18
&
Other+
1
3.3%
Total
Positive
Negative
23
76.7%
7
23.3%
All 7 samples that had discrepant results were submitted to the Roche Molecular System
Laboratory, in Pleasanton, CA for adjudication testing using a Linear Array HPV Genotyping test
plus repeat testing on the Cobas-4800. Table 4 gives a list of the HPV subtypes that are
detected by these different HPV methods. The Digene and Cobas-4800 methods detect the
same subtypes except for HPV-66 which is not detected by Digene. The Linear Array method
detects all the subtypes detected by Digene and Cobas-4800, plus an additional 23 subtypes,
which are currently considered to be low risk HPV subtypes.
Table 4. Comparison of Digene, Cobas-4800 & Linear Array HPV Genotyping
Method
Digene
Cobas-4800
# Subtypes
Detected
13
14
Linear Array
37
HPV Subtypes detected
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68
6, 11, 16, 18,26, 31, 33, 35, 39, 40, 41, 45, 51, 52, 53, 54, 55, 56, 58,
59,61, 62, 64, 66, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39, CP6108
The adjudication testing confirmed 6 out of 7 of Legacy’s Cobas-4800 results. One sample (#6)
gave an “invalid” result by both Linear Array and repeat Cobas-4800 testing, thus it could not be
included in statistical analysis. Of the 6 other samples, 4 were negative by Linear Array and 2
were positive for low risk HPV subtypes that are not detected by either Digene or Cobas-4800.
These results confirmed Legacy’s Cobas-4800 results. Table 5 summarizes these findings. Based
on these findings, all of Legacy’s Cobas-4800 “discrepant” test results were judged to be true
negatives (excluding sample #6 that was “invalid”).
Page 2 of 10
Table 5. Summary of adjudication testing
Legacy
Digene
Legacy Cobas-4800
Roche Lab
#
HPV High
Risk
Other High
Risk
HPV-16
HPV-18
Roche
Linear Array
Roche
Cobas 4800
1
Pos
Neg
Neg
Neg
Neg
Neg
2
Pos
Neg
Neg
Neg
HPV 70 Pos
Neg
3
4
5
6
Pos
Pos
Pos
Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Invalid
Neg
Neg
Neg
Invalid
7
Pos
Neg
Neg
Neg
HPV 84/53
Pos
Neg
Table 6. Sensitivity & Specificity of Cobas-4800 HPV Genotyping with SurePath samples
True Pos
False Pos
True Neg
False Neg
Total
Sensitivity
Specificity
Accuracy
Cobas-4800
23
0
46
0
69
100%
100%
100%
Digene
23
6
40
0
69
100%
87%
91%
Sensitivity = TP/(TP+FN) *100% ; Specificity = TN/(TN+FP)*100%
Accuracy = (TN+TP)/(TN+TP+FP+FN)*100%
These studies suggest that the discrepancies between the Digene HC2 and Cobas-4800 methods
are related to a combination of causes, including cross-reactivity on Digene with low risk HPV
subtypes (table 5, samples 2 & 7). Other discrepancies might be related to differences in cut-off
sensitivity settings for determining positivity.
Page 3 of 10
Precision Study
For each viral genotype (HPV 16, 18 and 68), a negative SurePath patient pool was spiked with
each positive type and divided into 10 aliquots. Each aliquot was run in duplicate daily for 10
days. Legacy's results showed a positive precision of 98.3% (59/60) and a negative precision
was 100% (20/20) which concurs with Roche’s results for precision studies performed with
ThinPrep samples.
Table 7. Positive Precision Study Results
Run #
Run Date
1
12/2/2013
HPV 16
Neg/Pos
Ct
HPV 18
Neg/Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Neg
Pos
Pos
Ct
HPV 68
Neg/Pos
Ct
37.4
Pos
36.9
Pos
36.1
36.8
Pos
37.9
Pos
36.5
2
12/3/2013
37.3
Pos
36.4
Pos
37.4
36.7
Pos
36.8
Pos
36.3
3
12/4/2013
38.0
Pos
37.0
Pos
37.1
38.0
Pos
36.7
Pos
36.9
4
12/5/2013
37.2
Pos
37.0
Pos
36.8
38.0
Pos
36.9
Pos
37.0
5
12/6/2013
37.6
Pos
36.9
Pos
37.1
37.6
Pos
36.7
Pos
36.4
6
12/9/2013
38.5
Pos
37.5
Pos
38.5
38.7
Pos
37.4
Pos
37.9
7
12/10/2013
37.1
Pos
36.9
Pos
37.2
38.0
Pos
37.3
Pos
37.1
8
12/11/2013
38.1
Pos
36.3
Pos
37.3
39.8
Pos
36.4
Pos
38.2
9
12/12/2013
40.2
Pos
36.7
Pos
37.4
na
Pos
37.0
Pos
37.8
10
12/13/2013
38.6
Pos
37.0
Pos
38.4
38.9
Pos
37.8
Pos
38.5
Average Ct
38.0
37.0
37.3
SD
0.94
0.43
0.73
CV%
2.46
1.16
1.95
*Ct = Crossing Threshold; HPV16 Ct positive cutoff = 40.5; HPV18 Ct positive cutoff = 40.0; HPV68 (High Risk pool)
Ct positive cutoff = 40
Page 4 of 10
Table 8. Negative Precision Study Results
Run #
1
Run Date
12/2/2013
2
12/3/2013
3
12/4/2013
4
12/5/2013
5
12/6/2013
Neg/Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Run #
6
Run Date
12/9/2013
7
12/10/2013
8
12/11/2013
9
12/12/2013
10
12/13/2013
Neg/Pos
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Analytical Sensitivity
Using a purchased 16, 18, and 68 high risk HPV, ThinPrep media and SurePath media were
spiked with the same volume of virus suspension. Various dilutions of each were compared to
see if the end-point (negative result) for SurePath was equal to or higher than the ThinPrep.
This was done to determine if the SurePath specimens are at least as sensitive as the ThinPrep.
The results are displayed below in tables 9-11. Samples prepared in SurePath media had the
same (HPV-68) or better sensitivity (HPV-16 & 18) than samples prepared in ThinPrep media.
Table 12 summarizes these findings.
Table 9. Comparison of ThinPrep (TP) and SurePath (SP) for HPV Genotype 16
Dilution
x16
x16
x16
x32
x32
x32
x64
x64
x64
x128
x128
x128
x256
x256
HPV 16
Crossing Threshold (Ct), Positive cutoff = 40.5
ThinPrep (TP)
SurePath (SP)
Pos/Neg
Ct
Pos/Neg
Ct
Mech Fail*
NA
Pos
33.7
Mech Fail
NA
Pos
33.8
Pos
34.9
Pos
35.1
Pos
35.4
Pos
34.7
Pos
35.0
Pos
34.9
Pos
35.9
Pos
36.0
Pos
36.4
Pos
35.8
Pos
36.7
Pos
35.9
Pos
38.1
Pos
36.6
Pos
37.7
Pos
36.9
Pos
37.4
Pos
36.7
Pos
38.3
Pos
38.0
Pos
39.5
Pos
37.1
Page 5 of 10
x256
Pos
39.1
Pos
37.1
x512
Pos
39.4
Pos
40.1
x512
Pos
39.3
Pos
39.0
x512
Pos
40.2
Pos
38.4
x1024
Neg
NA
Pos
40.1
x1024
Neg
NA
Pos
39.4
x1024
Pos
40
Pos
39.7
*Note: “Mechl Fail” - instrument failure; not a sample failure. Recovery for this error is to repeat testing of the
sample affected.
Table 10. Comparison of ThinPrep (TP) and SurePath (SP) for HPV Genotype 18
HPV 18
Crossing Threshold (Ct), Positive cutoff = 40.0
ThinPrep (TP)
SurePath (SP)
Dilution
Pos/Neg
Ct
Pos/Neg
Ct
x16
Pos
37.1
Pos*
35.7
x16
Pos
37.7
Pos
35.9
x16
Pos
37.0
Pos
36.1
x32
Pos
38.5
Pos
37.0
x32
Pos
38.7
Pos**
36.8
HPV 18 continued
Crossing Threshold (Ct), Positive cutoff = 40.0
ThinPrep (TP)
SurePath (SP)
Dilution
Pos/Neg
Ct
Pos/Neg
Ct
x32
Pos
38.5
Pos***
36.8
x64
Pos
38.9
Pos
37.6
x64
Pos
39.6
Pos
37.2
x64
Neg
NA
Pos
37.6
x128
Neg
NA
Pos
38.1
x128
Neg
NA
Pos
38.0
x128
Neg
NA
Pos
38.0
x256
Neg
NA
Pos
39.1
x256
Neg
NA
Pos
38.6
x256
Neg
NA
Pos
39.1
x512
Neg
NA
Neg
NA
x512
Neg
NA
Pos
39.8
x512
Neg
NA
Pos
39.5
x1024
Neg
NA
Neg
NA
x1024
Neg****
NA
Neg
NA
x1024
Neg
NA
Pos
39.6
SurePath Pos*, Pos **, & Pos *** were also positive for HPV 16 with Ct's of 39.9, 40.3, and 39.9.
ThinPrep Neg**** - This sample was negative for HPV 18, but positive for Other HR HPV Genotype (Ct-38.2).
Page 6 of 10
Table 11. Comparison of ThinPrep (TP) and SurePath (SP) for HPV Genotype 68
X64
X64
X64
x128
x128
x128
x256
x256
x256
x512
x512
x512
x1024
x1024
x1024
HPV 68
Crossing Threshold (Ct), Positive cutoff = 40.0
ThinPrep (TP)
SurePath (SP)
Pos/Neg
Ct
Pos/Neg
Ct
Pos
34.2
Pos
34.8
Pos
34.4
Pos
35.4
Pos
34.2
Pos
34.8
Pos
37.2
Pos
36.1
Pos
35.6
Pos
35.4
Pos
35.2
Pos
34.3
Pos
36.2
Pos
36.1
Pos
36.3
Pos
37.1
Pos
37.8
Pos
36.7
Pos
36.9
Pos
38.4
Pos
38.4
Pos
36.3
Pos
38.4
Pos
37.7
Pos
38.9
Pos
36.7
Pos
38.2
Pos
37.9
Mech Fail
NA
Pos
39.0
Table 12. Summary of Analytic Sensitivity Studies
Media type
Last positive dilution
HPV 16
TP
SP
x512
x1024
HPV 18
TP
SP
x64
x512
HPV 68
TP
x1024
SP
x1024
Analytical Specificity
SurePath material was spiked with a cocktail of mixed vaginal microbial flora to determine if the
flora caused a false positive HPV result. The cocktail contained Lactobacillus acidophilus,
Candida albicans, Streptococcus agalactiae (Group B strep), E. Coli, and Peptostreptococcus
anaerobius. A negative SurePath patient pool was prepared by Cytology. This negative pool
was run on the Cobas-4800 before and after it was spiked with the cocktail. All testing,
including before and after spiking, was negative for HPV-16, HPV-18, and HPV-High Risk Other.
No false positive reactions were observed (table 13).
Table 13. Spiked sample results
Panel ID
1. SP Neg Pool
2. SP Neg Pool
Results
Negative
Negative
Page 7 of 10
3. 200 µL Micro Cocktail +
800 µL SP Neg Pool
Negative
4. 500 µL Micro Cocktail +
500 µL SP Neg Pool
5. SP Neg Pool
6. SP Neg Pool
7. SP Neg Pool
8. SP Neg Pool
Negative
Negative
Negative
Negative
Negative
Carryover
Carryover testing on the Pap processor was checked by performing a run on the Cobas 4800
before and after the Pap processing. A 1:10 dilution using SurePath media and a HeLa cell line
(positive for HPV 18) was used for the positive sample source. The results are listed in table 14.
There was no observable carryover from the positive HPV 18 samples on either the pre or post
Cobas 4800 results.
Table 14. Carryover study results, HPV testing on Cobas-4800 before and after SurePath processing.
Pre SurePath Aliquots - 4800 Results
Sample ID
Result
Ct
Neg
Neg
NA
Neg
Neg
NA
Neg
Neg
NA
1:10 HPV 18
Pos 18
30.1
1:10 HPV 18
Pos 18
30.2
Neg
Neg
NA
1:10 HPV 18
Pos 18
30.3
1:10 HPV 18
Pos 18
30.1
Neg
Neg
NA
Neg
Neg
NA
Neg
Neg
NA
Neg
Neg
NA
1:10 HPV 18
Pos 18
29.5
1:10 HPV 18
Pos 18
29.8
Neg
Neg
NA
1:10 HPV 18
Pos 18
30
1:10 HPV 18
Pos 18
28.9
Neg
Neg
NA
1:10 HPV 18
Pos 18
29.9
1:10 HPV 18
Pos 18
29.6
Neg
Neg
NA
Post SurePath Aliquots - 4800 Results
Sample ID
Result
Ct
Neg
Neg
NA
Neg
Neg
NA
Neg
Neg
NA
1:10 HPV 18
HPV18+
30.0
1:10 HPV 18
HPV18+
29.4
Neg
Neg
NA
1:10 HPV 18
HPV18+
29.6
1:10 HPV 18
HPV18+
30.0
Neg
Neg
NA
Neg
Neg
NA
Neg
Neg
NA
Neg
Neg
NA
1:10 HPV 18
HPV18+
29.5
1:10 HPV 18
HPV18+
29.6
Neg
Neg
NA
1:10 HPV 18
HPV18+
29.9
1:10 HPV 18
HPV18+
29.6
Neg
Neg
NA
1:10 HPV 18
HPV18+
29.0
1:10 HPV 18
HPV18+
29.6
Neg
Neg
NA
Page 8 of 10
Interference Study
A SurePath positive pool was produced by combining positives of Other HR HPV, HPV 16 and
HPV 18. The pool was tested by adding two volumes of the Acrometrix® Inhibition panel using
the Low, Mid and High Hemolytic Vials. Table 15 shows results of the interference study. No
inhibition was noted with any of the positive HPV Genotypes up to a hemolysis concentration of
3.8 µg/dL.
Table 15. Hemolysis interference study results run with SurePath samples on Cobas-4800
Panel ID
Low
Interference
High
Interference
Add 200 µL of
Heme Panel to 800
µL of SP Pool
Add 500 µL of
Heme Panel to 500
µL of SP Pool
Results
Hemolytic Low 1.0 µg/dL
Pos/Neg
Positive
Pos/Neg
Positive
Hemolytic Mid 1.9 µg/dL
Positive
Positive
Hemolytic High 3.8 µg/dL
Positive
Positive
Stability Study
Stability of SurePath media was tested with media spiked with genotypes HPV 16, HPV 18 and
HPV 68. Dilutions were made that approached the lower end of the method sensitivity (two
dilutions before the endpoint as observed in the Analytical Sensitivity study). These prepared
samples were tested for baseline results (Day 0), then separate aliquots were stored at room
temperature (21-23°C) and refrigerated (2-8°C). Duplicates from each storage group were
tested at Day 0, Day 3, Day 5, Day 7, Day 10, Day 14, Day 21, and Day 28. The results are listed
in tables 16 and 17. Results of these studies show that the SurePath specimens are stable for 14
days when refrigerated and for 3 days when stored at room temperature.
Table 16. Stability of SurePath specimens - Refrigerated
Day #
Day 0
Day 3
Day 5
Day 7
HPV 16
HPV 18
HPV 68
Pos/Neg
(Ct Value)
Pos
36.9
Pos
38.2
Pos
39.6
Pos
39
Pos
36.4
Pos
37.8
(Ct Value)
Pos
Pos
Pos
Pos
Pos
Pos
(Ct Value)
37.9
39
39.4
38.1
35.1
37.1
Pos/Neg
Pos
Pos
Pos
Pos
Pos
Pos
(Ct Value)
38.3
37.2
38.4
38.1
37.1
36.8
Pos/Neg
Pos
Pos
Pos
Pos
Pos
Pos
Page 9 of 10
Day 10
Day 14
Day 21
Day 28
(Ct Value)
38
38.4
39.2
38.7
37
37.9
Pos/Neg
Pos
Pos
Pos
Pos
Pos
Pos
(Ct Value)
38.7
37.7
39.4
38.5
37.8
37.4
Pos/Neg
Pos
Pos
Pos
Pos
Pos
Pos
(Ct Value)
37.1
38.1
38.2
38.2
37.6
38.7
Pos/Neg
Neg
Pos
Pos
Pos
Pos
Neg
(Ct Value)
n/a
39
39
39.6
38.1
n/a
Pos/Neg
Pos
Pos
Pos
Pos
Pos
Pos
(Ct Value)
40
39
38.3
38.7
39.6
39.5
Table 17. Stability of SurePath specimens - Room Temperature
Day #
Day 0
Day 3
Day 5
Day 7
Day 10
Day 14
Day 21
Day 28
HPV 16
Pos/Neg
(Ct Value)
Pos/Neg
(Ct Value)
Pos/Neg
(Ct Value)
Pos/Neg
(Ct Value)
Pos/Neg
(Ct Value)
Pos/Neg
(Ct Value)
Pos/Neg
(Ct Value)
Pos/Neg
(Ct Value)
Pos
36.9
Pos
38.8
Neg
n/a
Neg
n/a
Neg
n/a
Neg
n/a
Pos
40.2
Neg
n/a
Pos
38.2
Pos
38.2
Pos
39.2
Neg
n/a
Neg
n/a
Neg
n/a
Pos
40.2
Neg
n/a
HPV 18
Pos
39.6
Pos
38.4
Pos
39
Pos
38.9
Neg
n/a
Neg
n/a
Neg
n/a
Neg
n/a
Pos
39
Pos
38.7
Pos
39.8
Pos
38.7
Pos
39.2
Pos
39.5
Neg
n/a
Neg
n/a
HPV 68
Pos
36.4
Pos
37.4
Pos
39.3
Pos
38.8
Neg
n/a
Neg
n/a
Neg
n/a
Pos
39.9
Pos
37.8
Pos
38.3
Pos
37.7
Neg
n/a
Pos
40
Neg
n/a
Neg
n/a
Neg
n/a
Conclusion:
SurePath, post-quot specimens are acceptable for patient testing to evaluate for HPV
Genotyping with the Roche Cobas® HPV test on the Cobas-4800. Specimens must be run within
3 days after collection if kept at room temperature, or within 14 days if refrigerated.
Page 10 of 10