Anatomic Pathology / HPV TESTING: HC II VS CISH
High-Risk Human Papillomavirus Detection
A Split-Sample Comparison of Hybrid Capture and Chromogenic
In Situ Hybridization
Carol L. Schiller, MD,1 Angel G. Nickolov, MA,2 Karen L. Kaul, MD, PhD,1
Elizabeth A. Hahn, MA,2 Janine M. Hy, CT(ASCP),1 Maura T. Escobar, CT(ASCP),1
William G. Watkin, MD,1 and Charles D. Sturgis, MD1
Key Words: Human papilloma virus; HPV; Hybrid Capture; HC II; Chromogenic in situ hybridization; CISH; Atypical squamous cells of
undetermined significance; ASC-US
DOI: 10.1309/13NMAK8J3N1YJXU1
Abstract
The American Society for Colposcopy and Cervical
Pathology (ASCCP) has proposed high-risk human
papillomavirus (HPV) testing as the “preferred” triage
for women with atypical squamous cells of
undetermined significance. We studied 401 atypical
squamous cells of undetermined significance liquidbased cervicovaginal cytology split samples for HPV by
chromogenic in situ hybridization (CISH) and by
Hybrid Capture (HC) II (Digene, Gaithersburg, MD);
202 underwent HC II followed by CISH, and 199
underwent CISH followed by HC II. Of 401 vials, 101
(25.2%) were positive for HPV by 1 or more methods.
HC II labeled 83 of 401 (20.7%) samples as positive,
while 38 of 401 (9.5%) were positive by CISH. Positive
attributes of CISH include the provision of a
cytomorphologic link in assessing HPV positivity and
comparative ease of use in laboratories without trained
molecular diagnosticians. Greater efficacy and
quantitative design are advantages of HC II.
Comparing data by sequence of testing showed a lower
likelihood of positive test results on the second ancillary
test than on the first ancillary test, regardless of age or
testing method (odds ratio, second/first = 0.58; P =
.003). This finding suggests that liquid-based
cervicovaginal cytology samples are not homogeneous
throughout. Correlative studies with histology and
polymerase chain reaction may clarify predictive values
for both methods.
The concept of studying epithelial cells exfoliated from
the uterine cervix as a means of establishing the diagnosis of
and screening for cervical carcinoma is roughly 60 years
old.1,2 Although this is a brief period in the scheme of medical
development, it is a comparatively long span for a modern
laboratory medicine test to exist and be used widely in an
unchanged method. In this sense, some would say that
cytopathologists have lingered behind other laboratory professionals in implementing new technologies and pushing diagnostic limits. Hematopathologists, for example, have become
fluent in the biology and terminology of their diagnoses at the
level of nucleic acids, chromosomes, and proteins.3,4 One of
the reasons that hematopathologists are decades ahead of their
cytopathology colleagues is the disease types studied.
Leukemias and lymphomas provided their investigators with
innumerable discrete islands of sameness, each with cytogenetic, molecular-genetic, and protein-level information neatly
packaged for discovery in natural cell suspensions.
Cytopathologists have only recently begun working in a
“liquid” cervical milieu.5,6 This transition is opening many
avenues for clinical use and scientific investigation.
The application of biotechnology to cervicovaginal
cytology was seen first during the 1980s when morphologic
criteria for the diagnosis of preneoplastic cytologic changes
were compared successfully with human papillomavirus
(HPV) DNA hybridization studies.7 These early cytologicmolecular correlation studies were limited to research
settings and relatively small samples. Such studies were
empowering for the cytodiagnostic community because they
provided a “gold standard” for comparison other than
histology, an equally imprecise science.8 Since then, an
ancillary testing corner has been turned on the course of
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cervicovaginal cytology screening. In many centers, such as
ours at Evanston Northwestern Healthcare, Evanston, IL,
almost all cervical cancer screening tests are collected and
processed using a liquid-based approach that permits increased
detection of preneoplastic lesions.9-16 In addition, liquid-based
exfoliative cytology methods prepare samples for filtration of
excess blood and inflammation, resulting in decreased “unsatisfactory” rates. Liquid-based cervical cytology also provides
monolayer distributions for ease of interpretation and, perhaps
most important, permits 1-visit, split-sample preparations of
cellular material for ancillary studies.
Cervicovaginal cytology screening has changed dramatically during the short time from the late 1980s to today.
Information from the multicenter randomized Atypical Squamous Cells of Undetermined Significance/Low-Grade Squamous Intraepithelial Lesion Triage Study has shown that
testing for the genetic material of high-risk HPV types can
be a valuable colposcopy-determining step for women interpreted to have atypical squamous cells of undetermined
significance (ASC-US) in their cervicovaginal samples.17,18
This triage mechanism has been presented by the American
Society for Colposcopy and Cervical Pathology as the
“preferred” management algorithm for women interpreted to
have ASC-US cells in their cervicovaginal cytology
samples.19 In light of these developments, substantial time
and financial resources have been invested in bringing
manageable, reproducible, and reliable HPV DNA testing to
the routine diagnostic cytology arena.
We report our experience with 2 different HPV testing
methods, Hybrid Capture (HC) II (Digene, Gaithersburg,
MD) and chromogenic in situ hybridization (CISH). A
comparison of high-risk HPV testing by the methods was
performed on split samples from liquid-based cervicovaginal
cytology specimens in a population of women diagnosed
with ASC-US at our center. Approximately half of the vials
underwent CISH followed by HC II (group 1), and the
remaining underwent HC II testing followed by CISH (group
2). The results of these studies are discussed with attention
given to testing outcomes, trends, statistical analysis, and
methodological limitations and advantages.
Materials and Methods
Samples, Preparation, and Interpretation
Cellular residua from 401 nonconsecutive, clinically
ordered, ThinPrep (Cytyc, Boxborough, MA) cervicovaginal
vials diagnosed as ASC-US ❚Image 1❚ by pathologists at
Evanston Northwestern Healthcare Laboratory Services
between January 2002 and August 2002 were retrieved and
submitted for testing by HC II HPV high-risk probe (HPV
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❚Image 1❚ ThinPrep cervicovaginal cytology slide (from a 38year-old group 1 patient [samples underwent chromogenic in
situ hybridization followed by Hybrid Capture II testing]) with
cells interpreted as atypical squamous cells of undetermined
significance. Nuclear enlargement and mild hyperchromasia
were identified cytomorphologically (Papanicolaou, ×600).
For proprietary information, see the text.
types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68)
microplate assay and CISH using the INFORM HPV liquidbased prep high-risk probe (HPV types: 16, 18, 31, 33, 35,
39, 45, 51, 52, 56, 58, 68, and 70) (Ventana, Tucson, AZ).
During the study period, 27,479 gynecologic cytology
specimens were processed at Evanston Northwestern Healthcare Laboratory Services, with an overall rate of ASC-US
diagnoses of 4.24% (1,164/27,479). Some vials were
excluded from the study owing to insufficient quantity of
sample to perform further parallel testing. Approximately
half of 401 samples underwent CISH followed by HC II
testing (group 1; n = 199), and the remaining samples underwent HC II testing followed by CISH (group 2; n = 202). HC
II testing of group 2 specimens was performed within 21
days of initial specimen collection. CISH analysis of group 1
and group 2 specimens and HC II testing of group 1 specimens were performed at intervals ranging from 22 to 219
days after collection. The 202 group 2 samples had been
ordered by clinicians to have HPV testing performed
following cytomorphologic diagnosis. Of these, 123 were to
have HPV testing performed as a “reflex” following a diagnosis of ASC-US, 22 were to have HPV testing performed
regardless of cytomorphologic diagnosis, and 57 had supplementary HPV testing ordered after initial specimen
processing. The HC II test was the assay in clinical use for
all ordered HPV testing by the Molecular Diagnostics Laboratory, Evanston Hospital, at that time. The 199 group 1
© American Society for Clinical Pathology
Anatomic Pathology / ORIGINAL ARTICLE
cases did not have HPV testing ordered by submitting physicians. All specimens used for analysis were the residua of
clinically ordered cervicovaginal cytology samples.
All HC II testing was performed in the Molecular Diagnostics Laboratory, Evanston Hospital (K.L.K.). The details
of the HC II method have been described.20,21 Consistent
with the testing protocol, the results of HC II studies were
reported as positive or negative. The CISH procedure was
performed in the Cytology Preparation and Immunocytochemistry Laboratories, Evanston Hospital (C.L.S.).
Unstained slides were prepared from cellular samples in
PreservCyt fixative solution (Cytyc), using the ThinPrep
2000 Processor (Cytyc). Slides were stored refrigerated until
testing. The CISH procedure was performed on the Benchmark Automated Slide Stainer (Ventana), according to the
manufacturer’s standard protocol.22-25
After initial incubation periods and buffer rinses, an
endopeptidase was applied to and incubated on the slides,
permitting exposure of nuclear DNA to the liquid-based prep
high-risk reagent probe. The probe consisted of a cocktail of
fluorescein-labeled DNA oligonucleotides that had been
demonstrated to hybridize to specific high-risk HPV genotypes. Initially, a mouse antifluorescein primary antibody
bound the probe. Then, a biotinylated secondary horse antibody formulation bound to the primary mouse antibodies.
This step was followed by the addition of a streptavidin
enzyme (alkaline phosphatase) conjugate that bound to the
biotin present on the secondary antibody. The specific antibody–secondary antibody–streptavidin enzyme complex
then was visualized by light microscopy using nitroblue
tetrazolium as a substrate for a precipitating enzyme-generated product, resulting in a deep blue to black staining of
cellular areas where there had been successful probe
hybridization. A nuclear fast red–based counterstain was
used.
Positive control slides consisted of commercially available prepared cytocentrifuged slides using a CaSki cell line
preparation containing approximately 400 copies of HPV
type 16 DNA per cell (Ventana). Negative control slides
were prepared in house from the cellular residua of liquidbased cervicovaginal samples that had been diagnosed cytomorphologically as no intraepithelial lesion or malignancy.
The CISH slides were screened by 2 cytotechnologists
(J.M.H. and M.T.E.), who referred all potentially positive
slides for definitive interpretation. Definitive positive or
negative results were determined by the consensus microscopic interpretation of 2 pathologists (C.L.S. and C.D.S.). A
positive result was defined as any convincing blue to black
nuclear staining, whether focal or global within the given
nucleus ❚Image 2❚. Convincing staining within a single cell
was interpreted as positive per manufacturer guidelines. All
testing was performed in a blinded manner.
❚Image 2❚ Positive chromogenic in situ hybridization slide from
the same patient as in Image 1. This study was interpreted as
positive for high-risk human papillomavirus DNA based on
discrete blue-black nuclear staining from successful probe
hybridization, antibody-enzyme complex formation, and
nitroblue tetrazolium substrate precipitation (×600).
Statistical Analysis
The Wilcoxon rank sum test was used to compare the
patient age distributions in groups 1 and 2 (A.G.N. and
E.A.H.). To account for correlated data owing to the
crossover design, we implemented a logistic regression
model for repeated binary measures.26 The outcome variable
was assigned a value of 1 if the test used was reported as
having a positive result for HPV and 0 if the result was negative. There were 3 explanatory variables: age group (<30
years vs 30-39 years vs ≥40 years), test (CISH vs HC II),
and sequence (first ancillary aliquot tested vs second). The
initial model included all 3 explanatory variables and their
second- and third-order interactions. Nonsignificant interaction terms were dropped one at a time by using a backward
elimination strategy and likelihood ratio tests. A P value of
less than .05 was considered statistically significant. Data
were entered into an Excel worksheet (Microsoft, Redmond,
WA) and exported into SAS software (1999, release 8.2;
Cary, NC) for statistical analyses.
Results
The 401 patients whose samples were included in the
study ranged in age from 14 to 90 years, with an overall
mean age of 37.8 years ❚Figure 1❚. The mean age of group 1
patients was 38.7 years (range, 14-76 years) ❚Figure 2❚ and
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Schiller et al / HPV TESTING: HC II VS CISH
group 40 years or older, the odds ratio for a positive CISH
test was 1.94 for the group younger than 30 years and 1.31
for the group 30 to 39 years old. Despite the observed trend,
these 2 odds ratios were not significantly different from 1.00
(P = .082 and P = .574, respectively). Compared with the
group 40 years or older, the odds ratio for a positive HC II
test was 6.64 for the group younger than 30 years and 2.51
for the group 30 to 39 years old. In contrast with the ratios
for CISH, the 2 odds ratios for HC II were significantly
greater than 1.00 (P < .001 and P = .018, respectively; Table
2).
A significant interaction between age and test was identified (Figure 4; Table 3). For group 1, the odds ratio for a
positive CISH result compared with a positive HC II result
was 0.43 (P = .006) for the group younger than 30 years,
0.77 (P = .525) for the group 30 to 39 years old, and 1.47
(P= .343) for the group 40 years or older. The sequence
effect manifested itself through smaller corresponding odds
ratios for group 2, with an odds ratio for a positive CISH
result compared with a positive HC II result of 0.14 (P <
.001) for the group younger than 30 years, 0.25 (P = .001)
for the group 30 to 39 years old, and 0.49 (P = .095) for the
group 40 years or older. The CISH test generally was less
140
70
120
60
No. of Samples
No. of Samples
for group 2 patients was 36.9 years (range, 19-90 years)
❚Figure 3❚. There were more patients aged 30 to 39 years in
group 2 than in group 1 and more patients aged 40 to 49
years in group 1 than in group 2.
In the final logistic model, the primary explanatory variables (test, sequence, and age group) and 1 second-order
interaction (test-age group) were statistically significant. Of
the 401 samples tested, 101 (25.2%) were positive for the
presence of high-risk HPV DNA by one or both methods; 18
(4.5%) were positive by CISH alone and 63 (15.7%) by HC
II alone; 20 (5.0%) were positive by both methods. A total of
38 (9.5%) were positive by CISH and 83 (20.7%) by HC II.
The probabilities of a positive CISH and/or HC II test result
were significantly dependent on the order in which the tests
were administered; specifically, a positive result was less
likely when either test was performed on the second aliquot
removed for ancillary testing from a given sample. The odds
ratio of obtaining a positive result when performed second in
sequence vs when performed first in sequence was 0.58 (P =
.003) ❚Table 1❚.
With respect to age, the likelihood of a positive test
result was associated negatively with age and varied by test
❚Figure 4❚ , ❚Table 2❚ , and ❚Table 3❚ . Compared with the
100
80
60
40
20
50
40
30
20
10
0
<20
0
20-29 30-39 40-49 50-59 60-69 70-79 79+
< 20
Age (y)
No. of Samples
❚Figure 1❚ Age distribution of all 401 patients whose
cervicovaginal cytology samples were studied.
80
70
60
50
40
30
20
10
0
30-39
40-49 50-59
Age (y)
60-69
70-79
❚Figure 2❚ Age distribution of 199 group 1 patients (samples
underwent chromogenic in situ hybridization followed by Hybrid
Capture II testing) whose cervicovaginal cytology samples
were studied. For proprietary information, see the text.
❚Table 1❚
Odds Ratios for a Positive Test Result by Sequence*
<20
20-29 30-39 40-49 50-59 60-69 70-79 79+
Age (y)
❚Figure 3❚ Age distribution of 202 group 2 patients (samples
underwent Hybrid Capture II testing followed by chromogenic
in situ hybridization) whose cervicovaginal cytology samples
were studied. For proprietary information, see the text.
540
540
20-29
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Sequence
Second (N = 401)
First (N = 401)
*
Odds Ratio (95% Confidence Interval)
P
0.58 (0.40-0.82)
1.00
.003
—
Positive human papillomavirus testing results were found with significantly greater
likelihood when testing was conducted on the first ancillary liquid aliquot out of
the vial than when conducted on the second ancillary liquid aliquot, regardless of
testing method.
© American Society for Clinical Pathology
Anatomic Pathology / ORIGINAL ARTICLE
100
Frequency of Positive
Results (%)
likely to have a positive result than the HC II test in groups 1
and 2, especially in younger patients (with the exception of
the 40 years or older age group in group 1, in which the
second-order action of test-age group interaction prevailed).
A comparison of the charts of predicted and observed
probabilities for a positive test result demonstrated that the
logistic model fit the data well and was able to capture the
reversed trend in the group 40 years or older in group 1
❚Figure 5❚, ❚Figure 6❚, ❚Table 4❚, and ❚Table 5❚.
CISH 1st in Sequence
CISH 2nd in Sequence
HC 1st in Sequence
HC 2nd in Sequence
80
60
40
20
0
<20
20-29 30-39 40-49 50-59 60-69 70-79 79+
Age (y)
❚Figure 4❚ Positive high-risk human papillomavirus results by
age, testing method, and split-sample sequence. CISH,
chromogenic in situ hybridization; HC, Hybrid Capture. For
proprietary information, see the text
Discussion
In the 60 years since the introduction of exfoliative
cervicovaginal cytology as a screening test for precursor
dysplastic and malignant cells, the incidence of invasive
cervical carcinoma in the United States has dropped dramatically. Despite this testament to the value of what arguably
has been the most successful cancer screening test in medical
history (cervicovaginal screening cytology), approximately
6,400 American women still died of cervical carcinoma
during the year 2000, and approximately 233,000 women
died of cervical carcinoma worldwide.27,28 How can the
cytopathology community improve these numbers? The
obvious answer would be to somehow ensure that all women
at risk have access to regular screening (universal health
policy) and to somehow ensure that the best possible testing
methods be used for all women. It is beyond the scope of this
article to address the institution of health care policy
changes; the focus of this work is on preventing patients who
are screened and who have cellular atypia of undetermined
significance from slipping through the diagnostic “cracks.”
Because the vast majority of cervical carcinomas are HPVdriven, ancillary HPV testing to triage patients diagnosed
with ASC-US has been proposed as one management option.
Molecular-genetic HPV testing not only has the capability of clarifying cellular atypia but also has the potential to
limit unnecessary and potentially expensive procedures such
as colposcopies and biopsies.
In addition to cytomorphologic testing, many testing
methods have been used to detect the presence of HPV in
cervicovaginal specimens, including viral load quantification,
Southern blot, polymerase chain reaction (PCR), ViraPap
(Life Technologies, Gaithersburg, MD), Hybrid Capture tube
testing, Hybrid Capture microtiter plate assays, and CISH.
While PCR likely would be considered the gold standard for
the detection of viral DNA, its performance requires a laboratory with a highly trained technical and professional staff.
In addition, commercially available high-risk HPV PCR
cocktail assays are only now coming to market. In our study,
we set out to explore and compare the performance characteristics of 2 methods, a Hybrid Capture microtiter plate
assay and CISH. While our data clearly demonstrate that
both HC II and CISH are capable of detecting the presence
of HPV DNA, they also highlight differences in performance
between the 2 methods.
Perhaps owing to its nature as a signal amplification
technique, HC II detected the presence of HPV DNA more
frequently than CISH. In our series overall, a patient was
more than twice as likely to have a test result positive for the
presence of high-risk HPV DNA by HC II (83/401) than by
CISH (38/401). The greater HC II percentage of positivity
was seen in all age groups regardless of the order in which the
tests were performed, except in the group 40 years or older in
group 1, in which a trend toward a greater percentage of positive CISH results was observed (Figures 4 and 5; Table 3).
The comparatively greater HPV DNA detection rate of HC II
is one of the method’s positive attributes. In addition, the
nature of the numeric cutoff value for determining positivity
❚Table 2❚
Odds Ratios (95% Confidence Intervals) for a Positive Test Result by Age Group and Test*
Test
CISH (N = 401)
Hybrid Capture II (N = 401)
<30 y
1.94 (0.92-4.10)
6.64 (3.49-12.63)
P
30-39 y
P
≥40 y
P
.082
<.001
1.31 (0.51-3.37)
2.51 (1.17-5.38)
.574
.018
1.00
1.00
—
—
CISH, chromogenic in situ hybridization.
* Younger women (<40 years) had positive test results for human papillomavirus nucleic acids more commonly than did older women. These differences were statistically
significant with the Hybrid Capture II method. For proprietary information, see the text.
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❚Table 3❚
Odds Ratios for a Positive Test Result by Test and Age Groups*
Group
Odds Ratio (95% Confidence Interval)
P
<30 (n = 2 × 68)
30-39 (n = 2 × 28)
≥40 (n = 2 × 103)
0.43 (0.23-0.79)
0.77 (0.34-1.74)
1.47 (0.66-3.25)
.006
.525
.343
<30 (n = 2 × 71)
30-39 (n = 2 × 57)
≥40 (n = 2 × 74)
0.14 (0.07-0.28)
0.25 (0.11-0.57)
0.49 (0.21-1.13)
<.001
.001
.095
1
2
In group 1 (n = 199), samples underwent chromogenic in situ hybridization (CISH)
followed by Hybrid Capture (HC) II testing; in group 2 (n = 202), samples
underwent HC II testing followed by CISH. The odds of detecting human
papillomavirus genetic material were significantly higher by HC II than by CISH
in younger women. For proprietary information, see the text.
Positive High-Risk
HPV DNA Test (%)
*
50
42.3%
45
40
35 13.2% 32.4%
26.3%
30
25
20
12.2%
12.7%
15
7.1% 7.1% 9.7%
8.8%
10
4.9%
4.1%
5
0
<30 y 30-39 y ≥40 y
<30 y 30-39 y
≥40 y
Group 1
Group 2
❚Figure 5❚ Observed probabilities for positive high-risk human
papillomavirus (HPV) DNA testing by age, method, and
sequence. More overall positive results are noted with Hybrid
Capture (HC) II (white bars) than with chromogenic in situ
hybridization (CISH; black bars). More overall positive results
are noted in the first test in sequence, regardless of method. In
group 1 (n = 199), samples underwent CISH followed by HC II
testing; in group 2 (n = 202), samples underwent HC II testing
followed by CISH. For proprietary information, see the text.
virtually eliminates interobserver variability, removing
subjectivity from the reporting of HC II results.
Despite labeling fewer samples as HPV DNA positive
than HC II, the CISH method offers the opportunity to view
the cytologic location of HPV infection (ie, nuclear localization in cytomorphologically abnormal cells), which permits a
morphologic link in assessing positivity. The flip side is that
CISH results can be difficult to interpret. Multiple factors,
including bacteria, yeast, improper dehydration in the
staining process, and cellular clumping and degeneration,
can cause staining artifacts that, if overinterpreted, might
lead to false-positive results. In our experience, there is a
substantial learning curve in the interpretation of CISH
results, which might introduce interobserver variability and
subjectivity in reporting. One interesting if unexpected
finding in our series was the number of samples that were
positive by CISH but negative by HC II (18/401 total
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HPV DNA Test (%)
Schiller et al / HPV TESTING: HC II VS CISH
50
43.5%
45
40
35 16.0% 30.7%
30
22.6%
25
20
11.4% 14.4%
15
8.9% 6.3%
9.9%
6.9% 10.4%
10
5.4%
5
0
<30 y 30-39 y ≥40 y
<30 y 30-39 y ≥40 y
Group 1
Group 2
❚Figure 6❚ Predicted probabilities for positive high-risk human
papillomavirus (HPV) DNA testing by age, method, and
sequence. Comparison of this graph to the graph in Figure 5
demonstrates that the predicted and observed probabilities
for positive testing mirror each other, indicating a good fit for
the logistic regression model. The model was able to capture
the reversed trend in the group 1 patients who were older
than 40 years. Black bars, positive results with CISH; white
bars, positive results by Hybrid Capture (HC) II testing. Group
1 samples underwent CISH followed by HC II testing; group
2 samples underwent HC II testing followed by CISH. The
age distributions were as follows: group 1: younger than 30
years, 68; 30-39 years, 28; 40 years or older, 103; group 2:
younger than 30 years, 71; 30-39 years, 57; 40 years or older,
74. For proprietary information, see the text.
samples; 18/38 positive CISH results). A possible explanation for this finding is the increased likelihood of finding a
needle in a haystack when you know where to look, ie,
nuclear localization in cytomorphologically abnormal cells
might lend a practical sensitivity to the technique. The slight
difference in probe cocktails of the 2 methods (CISH
includes HPV type 70 but not 59; HC II includes type 59 but
not 70) also might account for some variance in positivity.
Interpretive overdiagnosis (CISH-only false-positive results)
also might explain some of these results. Alternatively, an
unequal distribution of lesional cells in 1 aliquot of a sample
vs another portion might partly explain this finding.
Given the known epidemiology of cervical preneoplasia, 28 it is not entirely surprising that in our series,
patients in the younger than 30 years and the 30- to 39-yearold age groups had a higher likelihood of a positive test
result by either method than patients in the group 40 years or
older. These trends were statistically significant with respect
to HC II results and not with respect to CISH results (Table
2). These trends fit with the idea that HPV, a sexually transmitted agent, is present at higher frequencies in younger
persons. The absence of statistical significance with the
CISH method might relate to the comparatively smaller
number of positive results in all age distributions. Statistical
significance of these trends might or might not be achieved
with a larger sample number.
© American Society for Clinical Pathology
Anatomic Pathology / ORIGINAL ARTICLE
❚Table 4❚
Observed Probabilities for Positive CISH Results by Age and Sequence*
Sequence
No. of Observations
Age (y)
First
Second
CISH First in Sequence
CISH Second in Sequence
Total (N = 401)
<20
20-29
30-39
40-49
50-59
60-69
70-79
>79
20.0
12.7
7.1
10.9
4.2
16.7
0.0
—
50.0
11.6
8.8
7.9
0.0
0.0
0.0
0.0
5
63
28
64
24
12
3
0
2
69
57
38
23
7
5
1
7
132
85
102
47
19
8
1
*
Positive samples contained high-risk human papillomavirus DNA. Sequence data are given as percentages. In group 1 (n = 199), samples underwent chromogenic in situ hybridization
(CISH) followed by Hybrid Capture (HC) II testing; in group 2 (n = 202), samples underwent HC II testing followed by CISH. For proprietary information, see the text.
❚Table 5❚
Observed Probabilities for Positive HC II Results by Age and Sequence*
Sequence
No. of Observations
Age (y)
First
Second
<20
20-29
30-39
40-49
50-59
60-69
70-79
>79
100.0
40.6
26.3
7.9
21.7
14.3
0.0
0.0
40.0
31.7
7.1
6.3
4.2
0.0
0.0
—
*
HC II First in Sequence
HC II Second in Sequence
Total (N = 401)
5
63
28
64
24
12
3
0
7
132
85
102
47
19
8
1
2
69
57
38
23
7
5
1
Positive samples contained high-risk human papillomavirus DNA. Sequence data are given as percentages. In group 1 (n = 199), samples underwent chromogenic in situ
hybridization (CISH) followed by Hybrid Capture (HC) II testing; in group 2 (n = 202), samples underwent HC II testing followed by CISH. For proprietary information, see
the text.
An intriguing finding in our series was the effect of
sequence on testing outcomes. Regardless of patient age or
testing method, samples were more than 1.5 times as likely to
receive a positive test result from the first aliquot than from the
second aliquot, ie, the first ancillary test in the sequence was
more likely to be positive than the second ancillary test
performed on the same liquid-based sample. This observation
was not merely a trend but a statistically significant difference
with a P value of .003 (Table 1). Of particular interest is that the
effect of sequence was seen more dramatically in HC II results
than in CISH results, ie, the comparative decrease in the
number of positive results between the first and second aliquots
was greater for HC II than for CISH. The noted decrease in
positive results as more liquid sample was removed from the
specimen vials might be explained by nonhomogeneous cell
suspensions or by preferential separation of pertinent cellular
material by the proprietary filtration process. Keeping in mind
that all HC II second aliquots came from samples that already
had 2 ThinPrep slides prepared from them (1 for cytologic
diagnosis and 1 for CISH), the cellularity of the sample might
be a limiting factor in sensitivity if an attempt is made at
performing multiple ancillary tests from 1 vial.
Our study is somewhat limited by design flaws, the
most important of which is the imperfect randomization used
in establishing the crossover groups. The use of data from
the clinically ordered HC II tests (group 2 samples) was
done to minimize reagent cost. This resulted in a slightly
different patient population in group 1 than in group 2, as
evidenced by the slightly differing mean ages in the 2 groups
(group 1, 38.7 years; group 2, 36.9 years). The fact that these
patients had clinically ordered HPV testing might indicate a
higher index of suspicion by their health care providers.
Another limitation of study design is the fact that
both test manufacturers (Digene and Ventana) recommend
that their tests be performed within 21 days of sample
collection. These recommendations are made because of
the concern that cellular degeneration over time might
decrease the sensitivity of both tests. Approximately 75%
of our 802 data points were obtained from specimens
older than 21 days (maximum 219 days). This delay in
processing is an admitted flaw and is not reflective of
“real world” practices. In addition, 2 of the CISH-negative control slides, prepared from cytologically negative
cervicovaginal patient samples, each showed a single
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DOI: 10.1309/13NMAK8J3N1YJXU1
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Schiller et al / HPV TESTING: HC II VS CISH
focus of seemingly convincingly positive nuclear staining.
Commercially dispensed known negative controls were
not available.
Last, our overall ASC-US high-risk HPV positivity rate,
in the low 20th percentile range, is lower than what might be
expected in some centers. This is thought to be secondary to
a comparatively low frequency of disease in our suburban,
relatively affluent patient population and not to represent
potential compromise in the patient samples. Our overall
laboratory atypia (ASC-US and higher) rate falls in the 8%
range, and our ASC-US rate falls in the 4% range, resulting
in an ASC-US/squamous intraepithelial lesion ratio of
approximately 1:1. While this might mean that the overall
percentage of HPV positivity in our specific population
might be lower than in some other centers, we believe that
our data truly reflect our population.
According to the recently released Bethesda System
guidelines and the American Society for Colposcopy and
Cervical Pathology recommendations for management,19
HPV testing is the preferred triage mechanism for liquidbased cervicovaginal cytology samples that have been
labeled cytomorphologically as ASC-US. This confined use
of HPV testing underscores the important “ancillary” nature
of HPV testing and its use as an adjunct to cytomorphologic
examination. The goal of the present study was to compare 2
HPV DNA detection methods; however, these data would be
best interpreted by comparison with the current clinically
available gold standard of cytohistologic correlation and with
ultrasensitive DNA detection techniques such as PCR. It is
our goal, after a window of time for collection of correlative
data, to study sensitivities, specificities, and predictive values
of HC II and CISH for the detection of HPV DNA and
morphologically documented preneoplastic and malignant
changes. The presence of HPV genetic material is not by
itself indicative of cervical disease, and some testing
methods might prove too sensitive for routine clinical use.
From the 1Department of Pathology, Evanston Northwestern
Healthcare, Evanston Hospital, and Northwestern University
Feinberg School of Medicine; and 2Center on Outcomes, Research
and Education (CORE), Evanston Northwestern Healthcare,
Evanston, IL.
Presented in part in poster form (abstract 355) at the
meeting of the United States and Canadian Academy of
Pathology, Washington, DC, March 2003.
Address reprint requests to Dr Sturgis: ENH Director of
Cytopathology, 2650 Ridge Ave, Evanston, IL 60201.
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