The Efficacy of Liquid-Based Cervical Cytology Using Direct-to-Vial Sample Collection

The Papanicolaou (Pap) test is one of the most common and effective cancer screening tests used by primary care clinicians. However, a recent study conducted through the Agency for Health Care Policy and Research¹ estimated that the true sensitivity of the Pap test is only 51%. Two thirds of the false-negative results occur because of sampling error. The first liquid-based cervical cytology (LBCC) test was approved for clinical use by the United States Food and Drug Administration in 1996.² With LBCC, standard cervical cytology sampling devices are used to obtain a sample from the cervix. The sampling devices are then rinsed in a buffered alcohol transport and preservative solution instead of immediately transferring the cells to a glass slide. Sampling error is reduced, since all the cells are transferred into solution, in contrast with the conventional Pap test (CPT), in which only 20% of the cells are actually transferred to the glass slide.³ The cytologic specimen in solution is then sent to the laboratory where most of the debris, leukocytes, and erythrocytes are filtered from the specimen and a thin layer Pap test is prepared using a special processor. The cells are dispersed evenly with relatively little clumping or overlapping or obscuring debris (Figures 1A and B). The resulting clean smear is circular and contains approximately half the number of representative cells of a CPT. Residual cervical cells in solution that are not initially used for the Pap test may be tested at a later date for lower genital tract pathogens, such as carcinogenic human papillomavirus.^4,5

Liquid-based thin layer cervical cytology has performed favorably in studies using split sample collection techniques.^6-9 With the split sample technique, the cytologic specimen is first transferred to a glass slide for the CPT before transferring the remaining cervical cells to the liquid transport fluid. Evaluation of the LBCC Pap test using this nonrandom study design may be biased, since it is based solely on cells normally discarded. The CPT slides receive the first and perhaps better cellular sample; thus, the quality of that specimen may be vastly different than that available for LBCC. Currently, there are minimal data available in which LBCC was evaluated as it was intended to be used, for a primary cytologic specimen.^10,11 Thus, the full diagnostic potential of this new Pap test technique has not been fully realized. The purpose of our trial was to determine the rates of specimen adequacy and cervical cytologic and histologically confirmed diagnoses obtained with LBCC using a direct-to-vial collection technique and compare these results with those obtained using the CPT.

Methods

Subjects

In our clinical study one group of women was enrolled prospectively, and a second group was considered retrospectively. The first group (LBCC) consisted of 1004 nonpregnant women aged 18 years or older with an intact cervix. The second group consisted of 2110 women with a similar patient profile who had a CPT collected at the same clinics during the 2 years immediately preceding the initiation of the trial.

To ensure an enriched sample of women with cervical neoplasia, approximately 20% of the subjects in both cohorts were included from women presenting consecutively for a colposcopy examination following an abnormal CPT result. The remaining 80% of the subjects represented a general screening population of women presenting for a routine Pap test. All subjects were seen at 1 of 3 clinics: the Family Medicine Center, the Obstetrics and Gynecology Clinic, or the Comprehensive Cancer Center at the Medical College of Georgia in Augusta.

Equipment and Materials

Samples for CPTs and LBCC (ThinPrep Pap Test, Cytyc Corporation, Boxborough, Mass) were collected using a plastic Ayre spatula and cytobrush (Medscand, North Hollywood, Fla). Cervical cells from the second group were processed by CPT methods. Cervical cells from the prospectively enrolled group were preserved and transferred in LBCC solution (PreservCyt, Cytyc Corporation). Cervical samples for the LBCC test were processed using the ThinPrep 2000 Processor (Cytyc Corporation).¹²

Study Design

Eligible prospectively enrolled women presenting consecutively for a routine Pap test or colposcopy examination at any of the 3 designated clinics signed an internal review board-approved informed consent form before participating. After properly visualizing the cervix, a plastic Ayre spatula was used to collect an ectocervical sample. Another sample was collected using a cytobrush rotated 90° to 180° within the endocervical canal. The spatula and cytobrush were then immediately rinsed vigorously in the LBCC solution. The container was sealed, labeled, and sent to the cytology laboratory for processing according to the manufacturer’s instructions.¹² Cytotechnicians or pathologists in the Department of Pathology, Medical College of Georgia (MCG), evaluated all cytology and histology specimens. Cervical cytology was reported using the Bethesda System.

Computerized records from the MCG Cytology Laboratory were used to establish the second group. CPT records with similar patient profiles and clinic affiliation were selected chronologically beginning the day before the initiation of the trial and working backward in time until 2110 records were obtained (approximately 2 years). Cervical specimens were collected previously from this group using the same types of collection devices and technique as that for the LBCC group. However, immediately following sample collection these endocervical and ectocervical cells were plated on a glass slide, fixed, and sent to the cytology laboratory for Pap staining and diagnosis.

Statistical Analysis

The proportion of subjects in each of the Pap test adequacy groups (satisfactory, unsatisfactory, and satisfactory but limited by [SBLB]) were compared between the sample and control groups for the total study group, the colposcopy and general screening cohorts, and the 3 age groups (18-30 years, 31-40 years, 41 years or older). The chi-square statistic was used to compare the proportions unless the tables included cells with expected counts less than 5; for those cases we used the Fisher exact test as extended to tables larger than 2×2. A summary comparison controlling for age group was made using the Cohran-Mantel-Haenszel chi-square statistic. Average age was compared between the groups using the Student t test. At the given sample size the power was calculated to be 80% or greater for detecting a difference in positive rates of 3.5% between the LBCC and the CPT.

Results

Subjects in both groups were comparable by type of clinical visit. Consequently, 1004 LBCC subjects and 2110 CPT subjects were included in the data analysis. The routine Pap test screening group consisted of 796 and 1585 subjects in the LBCC and CPT groups, respectively, representing 79.0% of each population. Approximately 21.0% of the LBCC and CPT subjects (208 and 425, respectively) were represented by women presenting for colposcopy examination following an abnormal Pap test result. Subjects were also stratified into 1 of 3 age ranges for analytic purposes. Thus, 423 and 602 subjects were aged between 18 and 30 years; 287 and 523 subjects were aged 31 to 40 years; and 294 and 885 subjects were aged 41 years or older for the LBCC and CPT populations, respectively. A statistically significant difference between the age distributions was noted (P <.001) with a greater proportion of younger subjects in the LBCC group (mean age=35.0 years) than in the CPT cohort (mean age=39.8 years).

A greater percentage of satisfactory Pap tests were obtained when using LBCC (84.0%) compared with the CPT (60.5%), P ".001 (Figure 1). Also, significantly fewer unsatisfactory and SBLB Pap tests were reported using LBCC (1.2% and 14.8%) compared with the CPT (3.8% and 35.7%). For the colposcopy group, significantly more satisfactory Pap tests were reported for LBCC compared with the CPT (81.7% and 55.3%, respectively; P <.001). Similar results were also noted for the screening group. These significant results were maintained when the data were adjusted for the 3 age groups (Table 1).

The frequencies of cytologic diagnoses for LBCC and the CPT are seen in Table 2. The LBCC method detected a significantly greater percentage of women with low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL) Pap tests results (7.4% and 3.7%, respectively) compared with the CPT (1.7% and 1.7%, respectively). Similar significant results were noted when the LBCC and CPT groups were stratified into the screening and colposcopy populations. When both groups were adjusted for age, a greater percentage of LSIL and HSIL diagnoses were noted for LBCC in all 3 age ranges compared with the CPT (Table 4). The classification of atypical squamous cells of undetermined significance (ASCUS) cases was not modified by LBCC compared with the CPT. Because of the limited number of squamous cell carcinoma diagnoses reported, a comparison between the 2 groups was not possible.

The frequency of histologic diagnoses in the 2 groups closely paralleled Pap test diagnoses in the same populations. The LBCC and CPT groups had 5.4% and 1.0% cervical intraepithelial neoplasia 1 (CIN 1) histologic diagnoses, respectively, while 5.9% and 1.7% of the women had histologic diagnoses of CIN 2 and 3, respectively. These results were also maintained consistently across all 3 age ranges. The predictive value of a positive LBCC test (93.9%) was similar to that for the predictive value of a positive CPT (87.8%) when based on available histology results (Table 5). The sensitivity and specificity were equivalent for each test. Of all women with an LSIL or more severe Pap test result in each group, 1.9% of women in the CPT group and 6.5% of women in the LBCC group had cervical biopsies indicating CIN 1 or more severe disease.

Discussion

Pap test specimen adequacy results reflect whether the most likely site for neoplasia of the cervix—the transformation zone—has been sampled properly. LBCC provided significantly greater rates of satisfactory specimen adequacy than the CPT. Also, compared with the CPT, LBCC effectively reduced the number of SBLB and unsatisfactory specimen adequacy reports. Our findings are consistent with those of studies published previously that used split sample collection techniques.^6,7 The improvement of Pap test adequacy using LBCC may be because a clearer inspection of pertinent normal and abnormal cells is achieved by eliminating or minimizing obscuring inflammatory cells, red blood cells, other debris, and clumped or overlapped cells.¹³ A significant decrease in unsatisfactory Pap tests reduces the extra cost, time, and patient and clinician inconvenience incurred from the necessity of repeating a nonrepresentative Pap test. These advantages may be of particular importance to patients, clinicians, and third-party payers.

Also, in regard to specimen adequacy, LBCC performed better than the CPT for women of all age ranges. This is especially important for older women who typically have a greater frequency of unsatisfactory Pap tests because their active transformation zones are positioned deeply within the endocervical canal and are often inaccessible to comprehensive sampling. In addition, a thin atrophic epithelium (commonly seen in the older age group) is more easily traumatized during sampling, which causes potentially obscuring bleeding. Yet, LBCC reduced the rate of unsatisfactory Pap tests by approximately 50% for women older than 40 years. Other than the advantage of filtering unwanted cells, the difference may be explained by the fact that 80% of cervical cells are discarded with the collection devices and not transferred to a glass slide using the CPT method.³ In contrast, nearly all cells are transferred to solution with LBCC. Thus, the larger specimen obtained with the sampling method used in LBCC increases the availability of a typically limited number of endocervical cells retrieved from post-treatment and postmenopausal women who are routinely more difficult to sample.

The ability of the Pap test to detect cytologic changes consistent with neoplasia, when present, is critically important. In our trial, the LBCC method detected a greater percentage of women with LSIL and HSIL compared with the CPT. Approximately 4 times the number of women with LSIL and twice the number of cases of HSIL were detected by LBCC compared with the CPT. These results replicate or surpass those found in studies that evaluated LBCC using a split sample method.^6-9 Twice the rate of neoplasia detection by LBCC has been reported in studies using the split sample method. Our results, based on a direct-to-vial study design, may portray the true potential for LBCC to detect cervical neoplasia. A cleaner monolayer Pap test with a more comprehensive cellular specimen likely accounts for these remarkable differences. Similarly, the reduced number of unsatisfactory and SBLB Pap tests using LBCC may contribute to the greater yield of SILs. Of note, LBCC did not detect a significantly greater rate of ASCUS Pap test results; this may create problematic management decisions for some clinicians.¹⁴ The ability of LBCC to detect a greater percentage of women with SILs was maintained for the general screening population, the colposcopy cohort, and across the 3 age ranges. LBCC may be better able to detect women with HSIL, a lesion unlikely to resolve spontaneously. Women with HSIL (Figures 2A and B) and a comparable histologic diagnosis deserve prompt therapy for this true cancer precursor. Although many women with LSIL have disease that may regress to normal, as many as 20% to 40% of these women will have histologically confirmed CIN 2 or 3 that deserves treatment as well.¹⁵

Limitations

Our study is subject to several limitations. Most important, the observed differences in Pap test performance may have been because of actual differences of cervical neoplasia prevalences between the 2 groups, rather than a difference between Pap tests. Our subjects were selected from comparable patient populations by a study design that other researchers^10,11,16-20 have used to evaluate the direct-to-vial technique. Other than sampling a population of women with LBCC and then having them return for a CPT 1 month later, which risks compliance failure and spectrum of cervical disease changes or acquisition of new disease, there is no other study design to evaluate a direct-to-vial technique. One may question whether our increased rate of neoplasia detection demonstrated by LBCC is due to a substantial number of LBCC false-positive results.²¹ Yet, because the positive predictive values for the LBCC and CPT were similar and there were 3 times as many women with biopsy results of CIN 1 or greater in the LBCC group compared with the CPT group when only women with LSIL or greater Pap test results were considered, the increased rates of SIL detection using LBCC appear to represent an increased detection of true disease and not false-positive results, further validating our findings.²¹ The equivalent results for LBCC and CPT specificity (99%) indicate that the increased rate of SIL detection by LBCC was not due to a reduced specificity. Also, the favorable test results demonstrated for LBCC are not entirely dissimilar from those of other studies that used a split sample technique, a more biased study design for LBCC.^6-9 Studies by Vassilakos and colleagues¹⁶ and Papillo and coworkers¹⁰ demonstrated that a liquid-based thin layer cytologic report of LSIL or more severe disease correlated better with histologic results of CIN 1 or greater (80.5% and 80.2%, respectively) than CPTs (71.7% and 72.2%, respectively). Finally, because of the limited number of women with cervical caner in this study population, we were unable to determine if there were any differences between LBCC and the CPT in their ability to detect cervical cancer.

Conclusions

Compared with the CPT, LBCC detected a significantly greater percentage of satisfactory Pap tests and significantly reduced the number of unsatisfactory and SBLB tests. These findings demonstrate that LBCC significantly improves the adequacy of Pap tests and may increase the rate of detection of cervical neoplasia compared with CPT. Further study is necessary and warranted, since failure to detect cancer in a timely fashion affects ultimate cure rates, medical costs, quality of life, and perhaps medicolegal expenses. Although liquid-based thin layer cervical cytology is rapidly replacing the glass slide method throughout the United States, additional studies are also necessary to determine whether LBCC reduces the incidence of cervical cancer.

Acknowledgments

We appreciate the assistance of Cytyc Corporation in providing the supplies and grant support necessary for completion of this study.

We would like to thank the faculty and residents in the Family Medicine Center and OB/GYN Clinic who assisted with specimen collection, Jim Best for processing and interpreting cytologic specimens, Aisha Lavin for data management assistance, and April Dean for manuscript preparation.

The Papanicolaou (Pap) test is one of the most common and effective cancer screening tests used by primary care clinicians. However, a recent study conducted through the Agency for Health Care Policy and Research¹ estimated that the true sensitivity of the Pap test is only 51%. Two thirds of the false-negative results occur because of sampling error. The first liquid-based cervical cytology (LBCC) test was approved for clinical use by the United States Food and Drug Administration in 1996.² With LBCC, standard cervical cytology sampling devices are used to obtain a sample from the cervix. The sampling devices are then rinsed in a buffered alcohol transport and preservative solution instead of immediately transferring the cells to a glass slide. Sampling error is reduced, since all the cells are transferred into solution, in contrast with the conventional Pap test (CPT), in which only 20% of the cells are actually transferred to the glass slide.³ The cytologic specimen in solution is then sent to the laboratory where most of the debris, leukocytes, and erythrocytes are filtered from the specimen and a thin layer Pap test is prepared using a special processor. The cells are dispersed evenly with relatively little clumping or overlapping or obscuring debris (Figures 1A and B). The resulting clean smear is circular and contains approximately half the number of representative cells of a CPT. Residual cervical cells in solution that are not initially used for the Pap test may be tested at a later date for lower genital tract pathogens, such as carcinogenic human papillomavirus.^4,5

Liquid-based thin layer cervical cytology has performed favorably in studies using split sample collection techniques.^6-9 With the split sample technique, the cytologic specimen is first transferred to a glass slide for the CPT before transferring the remaining cervical cells to the liquid transport fluid. Evaluation of the LBCC Pap test using this nonrandom study design may be biased, since it is based solely on cells normally discarded. The CPT slides receive the first and perhaps better cellular sample; thus, the quality of that specimen may be vastly different than that available for LBCC. Currently, there are minimal data available in which LBCC was evaluated as it was intended to be used, for a primary cytologic specimen.^10,11 Thus, the full diagnostic potential of this new Pap test technique has not been fully realized. The purpose of our trial was to determine the rates of specimen adequacy and cervical cytologic and histologically confirmed diagnoses obtained with LBCC using a direct-to-vial collection technique and compare these results with those obtained using the CPT.

Methods

Subjects

In our clinical study one group of women was enrolled prospectively, and a second group was considered retrospectively. The first group (LBCC) consisted of 1004 nonpregnant women aged 18 years or older with an intact cervix. The second group consisted of 2110 women with a similar patient profile who had a CPT collected at the same clinics during the 2 years immediately preceding the initiation of the trial.

To ensure an enriched sample of women with cervical neoplasia, approximately 20% of the subjects in both cohorts were included from women presenting consecutively for a colposcopy examination following an abnormal CPT result. The remaining 80% of the subjects represented a general screening population of women presenting for a routine Pap test. All subjects were seen at 1 of 3 clinics: the Family Medicine Center, the Obstetrics and Gynecology Clinic, or the Comprehensive Cancer Center at the Medical College of Georgia in Augusta.

Equipment and Materials

Samples for CPTs and LBCC (ThinPrep Pap Test, Cytyc Corporation, Boxborough, Mass) were collected using a plastic Ayre spatula and cytobrush (Medscand, North Hollywood, Fla). Cervical cells from the second group were processed by CPT methods. Cervical cells from the prospectively enrolled group were preserved and transferred in LBCC solution (PreservCyt, Cytyc Corporation). Cervical samples for the LBCC test were processed using the ThinPrep 2000 Processor (Cytyc Corporation).¹²

Study Design

Eligible prospectively enrolled women presenting consecutively for a routine Pap test or colposcopy examination at any of the 3 designated clinics signed an internal review board-approved informed consent form before participating. After properly visualizing the cervix, a plastic Ayre spatula was used to collect an ectocervical sample. Another sample was collected using a cytobrush rotated 90° to 180° within the endocervical canal. The spatula and cytobrush were then immediately rinsed vigorously in the LBCC solution. The container was sealed, labeled, and sent to the cytology laboratory for processing according to the manufacturer’s instructions.¹² Cytotechnicians or pathologists in the Department of Pathology, Medical College of Georgia (MCG), evaluated all cytology and histology specimens. Cervical cytology was reported using the Bethesda System.

Computerized records from the MCG Cytology Laboratory were used to establish the second group. CPT records with similar patient profiles and clinic affiliation were selected chronologically beginning the day before the initiation of the trial and working backward in time until 2110 records were obtained (approximately 2 years). Cervical specimens were collected previously from this group using the same types of collection devices and technique as that for the LBCC group. However, immediately following sample collection these endocervical and ectocervical cells were plated on a glass slide, fixed, and sent to the cytology laboratory for Pap staining and diagnosis.

Statistical Analysis

The proportion of subjects in each of the Pap test adequacy groups (satisfactory, unsatisfactory, and satisfactory but limited by [SBLB]) were compared between the sample and control groups for the total study group, the colposcopy and general screening cohorts, and the 3 age groups (18-30 years, 31-40 years, 41 years or older). The chi-square statistic was used to compare the proportions unless the tables included cells with expected counts less than 5; for those cases we used the Fisher exact test as extended to tables larger than 2×2. A summary comparison controlling for age group was made using the Cohran-Mantel-Haenszel chi-square statistic. Average age was compared between the groups using the Student t test. At the given sample size the power was calculated to be 80% or greater for detecting a difference in positive rates of 3.5% between the LBCC and the CPT.

Results

Subjects in both groups were comparable by type of clinical visit. Consequently, 1004 LBCC subjects and 2110 CPT subjects were included in the data analysis. The routine Pap test screening group consisted of 796 and 1585 subjects in the LBCC and CPT groups, respectively, representing 79.0% of each population. Approximately 21.0% of the LBCC and CPT subjects (208 and 425, respectively) were represented by women presenting for colposcopy examination following an abnormal Pap test result. Subjects were also stratified into 1 of 3 age ranges for analytic purposes. Thus, 423 and 602 subjects were aged between 18 and 30 years; 287 and 523 subjects were aged 31 to 40 years; and 294 and 885 subjects were aged 41 years or older for the LBCC and CPT populations, respectively. A statistically significant difference between the age distributions was noted (P <.001) with a greater proportion of younger subjects in the LBCC group (mean age=35.0 years) than in the CPT cohort (mean age=39.8 years).

A greater percentage of satisfactory Pap tests were obtained when using LBCC (84.0%) compared with the CPT (60.5%), P ".001 (Figure 1). Also, significantly fewer unsatisfactory and SBLB Pap tests were reported using LBCC (1.2% and 14.8%) compared with the CPT (3.8% and 35.7%). For the colposcopy group, significantly more satisfactory Pap tests were reported for LBCC compared with the CPT (81.7% and 55.3%, respectively; P <.001). Similar results were also noted for the screening group. These significant results were maintained when the data were adjusted for the 3 age groups (Table 1).

The frequencies of cytologic diagnoses for LBCC and the CPT are seen in Table 2. The LBCC method detected a significantly greater percentage of women with low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL) Pap tests results (7.4% and 3.7%, respectively) compared with the CPT (1.7% and 1.7%, respectively). Similar significant results were noted when the LBCC and CPT groups were stratified into the screening and colposcopy populations. When both groups were adjusted for age, a greater percentage of LSIL and HSIL diagnoses were noted for LBCC in all 3 age ranges compared with the CPT (Table 4). The classification of atypical squamous cells of undetermined significance (ASCUS) cases was not modified by LBCC compared with the CPT. Because of the limited number of squamous cell carcinoma diagnoses reported, a comparison between the 2 groups was not possible.

The frequency of histologic diagnoses in the 2 groups closely paralleled Pap test diagnoses in the same populations. The LBCC and CPT groups had 5.4% and 1.0% cervical intraepithelial neoplasia 1 (CIN 1) histologic diagnoses, respectively, while 5.9% and 1.7% of the women had histologic diagnoses of CIN 2 and 3, respectively. These results were also maintained consistently across all 3 age ranges. The predictive value of a positive LBCC test (93.9%) was similar to that for the predictive value of a positive CPT (87.8%) when based on available histology results (Table 5). The sensitivity and specificity were equivalent for each test. Of all women with an LSIL or more severe Pap test result in each group, 1.9% of women in the CPT group and 6.5% of women in the LBCC group had cervical biopsies indicating CIN 1 or more severe disease.

Discussion

Pap test specimen adequacy results reflect whether the most likely site for neoplasia of the cervix—the transformation zone—has been sampled properly. LBCC provided significantly greater rates of satisfactory specimen adequacy than the CPT. Also, compared with the CPT, LBCC effectively reduced the number of SBLB and unsatisfactory specimen adequacy reports. Our findings are consistent with those of studies published previously that used split sample collection techniques.^6,7 The improvement of Pap test adequacy using LBCC may be because a clearer inspection of pertinent normal and abnormal cells is achieved by eliminating or minimizing obscuring inflammatory cells, red blood cells, other debris, and clumped or overlapped cells.¹³ A significant decrease in unsatisfactory Pap tests reduces the extra cost, time, and patient and clinician inconvenience incurred from the necessity of repeating a nonrepresentative Pap test. These advantages may be of particular importance to patients, clinicians, and third-party payers.

Also, in regard to specimen adequacy, LBCC performed better than the CPT for women of all age ranges. This is especially important for older women who typically have a greater frequency of unsatisfactory Pap tests because their active transformation zones are positioned deeply within the endocervical canal and are often inaccessible to comprehensive sampling. In addition, a thin atrophic epithelium (commonly seen in the older age group) is more easily traumatized during sampling, which causes potentially obscuring bleeding. Yet, LBCC reduced the rate of unsatisfactory Pap tests by approximately 50% for women older than 40 years. Other than the advantage of filtering unwanted cells, the difference may be explained by the fact that 80% of cervical cells are discarded with the collection devices and not transferred to a glass slide using the CPT method.³ In contrast, nearly all cells are transferred to solution with LBCC. Thus, the larger specimen obtained with the sampling method used in LBCC increases the availability of a typically limited number of endocervical cells retrieved from post-treatment and postmenopausal women who are routinely more difficult to sample.

The ability of the Pap test to detect cytologic changes consistent with neoplasia, when present, is critically important. In our trial, the LBCC method detected a greater percentage of women with LSIL and HSIL compared with the CPT. Approximately 4 times the number of women with LSIL and twice the number of cases of HSIL were detected by LBCC compared with the CPT. These results replicate or surpass those found in studies that evaluated LBCC using a split sample method.^6-9 Twice the rate of neoplasia detection by LBCC has been reported in studies using the split sample method. Our results, based on a direct-to-vial study design, may portray the true potential for LBCC to detect cervical neoplasia. A cleaner monolayer Pap test with a more comprehensive cellular specimen likely accounts for these remarkable differences. Similarly, the reduced number of unsatisfactory and SBLB Pap tests using LBCC may contribute to the greater yield of SILs. Of note, LBCC did not detect a significantly greater rate of ASCUS Pap test results; this may create problematic management decisions for some clinicians.¹⁴ The ability of LBCC to detect a greater percentage of women with SILs was maintained for the general screening population, the colposcopy cohort, and across the 3 age ranges. LBCC may be better able to detect women with HSIL, a lesion unlikely to resolve spontaneously. Women with HSIL (Figures 2A and B) and a comparable histologic diagnosis deserve prompt therapy for this true cancer precursor. Although many women with LSIL have disease that may regress to normal, as many as 20% to 40% of these women will have histologically confirmed CIN 2 or 3 that deserves treatment as well.¹⁵

Limitations

Our study is subject to several limitations. Most important, the observed differences in Pap test performance may have been because of actual differences of cervical neoplasia prevalences between the 2 groups, rather than a difference between Pap tests. Our subjects were selected from comparable patient populations by a study design that other researchers^10,11,16-20 have used to evaluate the direct-to-vial technique. Other than sampling a population of women with LBCC and then having them return for a CPT 1 month later, which risks compliance failure and spectrum of cervical disease changes or acquisition of new disease, there is no other study design to evaluate a direct-to-vial technique. One may question whether our increased rate of neoplasia detection demonstrated by LBCC is due to a substantial number of LBCC false-positive results.²¹ Yet, because the positive predictive values for the LBCC and CPT were similar and there were 3 times as many women with biopsy results of CIN 1 or greater in the LBCC group compared with the CPT group when only women with LSIL or greater Pap test results were considered, the increased rates of SIL detection using LBCC appear to represent an increased detection of true disease and not false-positive results, further validating our findings.²¹ The equivalent results for LBCC and CPT specificity (99%) indicate that the increased rate of SIL detection by LBCC was not due to a reduced specificity. Also, the favorable test results demonstrated for LBCC are not entirely dissimilar from those of other studies that used a split sample technique, a more biased study design for LBCC.^6-9 Studies by Vassilakos and colleagues¹⁶ and Papillo and coworkers¹⁰ demonstrated that a liquid-based thin layer cytologic report of LSIL or more severe disease correlated better with histologic results of CIN 1 or greater (80.5% and 80.2%, respectively) than CPTs (71.7% and 72.2%, respectively). Finally, because of the limited number of women with cervical caner in this study population, we were unable to determine if there were any differences between LBCC and the CPT in their ability to detect cervical cancer.

Conclusions

Compared with the CPT, LBCC detected a significantly greater percentage of satisfactory Pap tests and significantly reduced the number of unsatisfactory and SBLB tests. These findings demonstrate that LBCC significantly improves the adequacy of Pap tests and may increase the rate of detection of cervical neoplasia compared with CPT. Further study is necessary and warranted, since failure to detect cancer in a timely fashion affects ultimate cure rates, medical costs, quality of life, and perhaps medicolegal expenses. Although liquid-based thin layer cervical cytology is rapidly replacing the glass slide method throughout the United States, additional studies are also necessary to determine whether LBCC reduces the incidence of cervical cancer.

Acknowledgments

We appreciate the assistance of Cytyc Corporation in providing the supplies and grant support necessary for completion of this study.

We would like to thank the faculty and residents in the Family Medicine Center and OB/GYN Clinic who assisted with specimen collection, Jim Best for processing and interpreting cytologic specimens, Aisha Lavin for data management assistance, and April Dean for manuscript preparation.

The Papanicolaou (Pap) test is one of the most common and effective cancer screening tests used by primary care clinicians. However, a recent study conducted through the Agency for Health Care Policy and Research¹ estimated that the true sensitivity of the Pap test is only 51%. Two thirds of the false-negative results occur because of sampling error. The first liquid-based cervical cytology (LBCC) test was approved for clinical use by the United States Food and Drug Administration in 1996.² With LBCC, standard cervical cytology sampling devices are used to obtain a sample from the cervix. The sampling devices are then rinsed in a buffered alcohol transport and preservative solution instead of immediately transferring the cells to a glass slide. Sampling error is reduced, since all the cells are transferred into solution, in contrast with the conventional Pap test (CPT), in which only 20% of the cells are actually transferred to the glass slide.³ The cytologic specimen in solution is then sent to the laboratory where most of the debris, leukocytes, and erythrocytes are filtered from the specimen and a thin layer Pap test is prepared using a special processor. The cells are dispersed evenly with relatively little clumping or overlapping or obscuring debris (Figures 1A and B). The resulting clean smear is circular and contains approximately half the number of representative cells of a CPT. Residual cervical cells in solution that are not initially used for the Pap test may be tested at a later date for lower genital tract pathogens, such as carcinogenic human papillomavirus.^4,5

Liquid-based thin layer cervical cytology has performed favorably in studies using split sample collection techniques.^6-9 With the split sample technique, the cytologic specimen is first transferred to a glass slide for the CPT before transferring the remaining cervical cells to the liquid transport fluid. Evaluation of the LBCC Pap test using this nonrandom study design may be biased, since it is based solely on cells normally discarded. The CPT slides receive the first and perhaps better cellular sample; thus, the quality of that specimen may be vastly different than that available for LBCC. Currently, there are minimal data available in which LBCC was evaluated as it was intended to be used, for a primary cytologic specimen.^10,11 Thus, the full diagnostic potential of this new Pap test technique has not been fully realized. The purpose of our trial was to determine the rates of specimen adequacy and cervical cytologic and histologically confirmed diagnoses obtained with LBCC using a direct-to-vial collection technique and compare these results with those obtained using the CPT.

Methods

Subjects

In our clinical study one group of women was enrolled prospectively, and a second group was considered retrospectively. The first group (LBCC) consisted of 1004 nonpregnant women aged 18 years or older with an intact cervix. The second group consisted of 2110 women with a similar patient profile who had a CPT collected at the same clinics during the 2 years immediately preceding the initiation of the trial.

To ensure an enriched sample of women with cervical neoplasia, approximately 20% of the subjects in both cohorts were included from women presenting consecutively for a colposcopy examination following an abnormal CPT result. The remaining 80% of the subjects represented a general screening population of women presenting for a routine Pap test. All subjects were seen at 1 of 3 clinics: the Family Medicine Center, the Obstetrics and Gynecology Clinic, or the Comprehensive Cancer Center at the Medical College of Georgia in Augusta.

Equipment and Materials

Samples for CPTs and LBCC (ThinPrep Pap Test, Cytyc Corporation, Boxborough, Mass) were collected using a plastic Ayre spatula and cytobrush (Medscand, North Hollywood, Fla). Cervical cells from the second group were processed by CPT methods. Cervical cells from the prospectively enrolled group were preserved and transferred in LBCC solution (PreservCyt, Cytyc Corporation). Cervical samples for the LBCC test were processed using the ThinPrep 2000 Processor (Cytyc Corporation).¹²

Study Design

Eligible prospectively enrolled women presenting consecutively for a routine Pap test or colposcopy examination at any of the 3 designated clinics signed an internal review board-approved informed consent form before participating. After properly visualizing the cervix, a plastic Ayre spatula was used to collect an ectocervical sample. Another sample was collected using a cytobrush rotated 90° to 180° within the endocervical canal. The spatula and cytobrush were then immediately rinsed vigorously in the LBCC solution. The container was sealed, labeled, and sent to the cytology laboratory for processing according to the manufacturer’s instructions.¹² Cytotechnicians or pathologists in the Department of Pathology, Medical College of Georgia (MCG), evaluated all cytology and histology specimens. Cervical cytology was reported using the Bethesda System.

Computerized records from the MCG Cytology Laboratory were used to establish the second group. CPT records with similar patient profiles and clinic affiliation were selected chronologically beginning the day before the initiation of the trial and working backward in time until 2110 records were obtained (approximately 2 years). Cervical specimens were collected previously from this group using the same types of collection devices and technique as that for the LBCC group. However, immediately following sample collection these endocervical and ectocervical cells were plated on a glass slide, fixed, and sent to the cytology laboratory for Pap staining and diagnosis.

Statistical Analysis

The proportion of subjects in each of the Pap test adequacy groups (satisfactory, unsatisfactory, and satisfactory but limited by [SBLB]) were compared between the sample and control groups for the total study group, the colposcopy and general screening cohorts, and the 3 age groups (18-30 years, 31-40 years, 41 years or older). The chi-square statistic was used to compare the proportions unless the tables included cells with expected counts less than 5; for those cases we used the Fisher exact test as extended to tables larger than 2×2. A summary comparison controlling for age group was made using the Cohran-Mantel-Haenszel chi-square statistic. Average age was compared between the groups using the Student t test. At the given sample size the power was calculated to be 80% or greater for detecting a difference in positive rates of 3.5% between the LBCC and the CPT.

Results

Subjects in both groups were comparable by type of clinical visit. Consequently, 1004 LBCC subjects and 2110 CPT subjects were included in the data analysis. The routine Pap test screening group consisted of 796 and 1585 subjects in the LBCC and CPT groups, respectively, representing 79.0% of each population. Approximately 21.0% of the LBCC and CPT subjects (208 and 425, respectively) were represented by women presenting for colposcopy examination following an abnormal Pap test result. Subjects were also stratified into 1 of 3 age ranges for analytic purposes. Thus, 423 and 602 subjects were aged between 18 and 30 years; 287 and 523 subjects were aged 31 to 40 years; and 294 and 885 subjects were aged 41 years or older for the LBCC and CPT populations, respectively. A statistically significant difference between the age distributions was noted (P <.001) with a greater proportion of younger subjects in the LBCC group (mean age=35.0 years) than in the CPT cohort (mean age=39.8 years).

A greater percentage of satisfactory Pap tests were obtained when using LBCC (84.0%) compared with the CPT (60.5%), P ".001 (Figure 1). Also, significantly fewer unsatisfactory and SBLB Pap tests were reported using LBCC (1.2% and 14.8%) compared with the CPT (3.8% and 35.7%). For the colposcopy group, significantly more satisfactory Pap tests were reported for LBCC compared with the CPT (81.7% and 55.3%, respectively; P <.001). Similar results were also noted for the screening group. These significant results were maintained when the data were adjusted for the 3 age groups (Table 1).

The frequencies of cytologic diagnoses for LBCC and the CPT are seen in Table 2. The LBCC method detected a significantly greater percentage of women with low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL) Pap tests results (7.4% and 3.7%, respectively) compared with the CPT (1.7% and 1.7%, respectively). Similar significant results were noted when the LBCC and CPT groups were stratified into the screening and colposcopy populations. When both groups were adjusted for age, a greater percentage of LSIL and HSIL diagnoses were noted for LBCC in all 3 age ranges compared with the CPT (Table 4). The classification of atypical squamous cells of undetermined significance (ASCUS) cases was not modified by LBCC compared with the CPT. Because of the limited number of squamous cell carcinoma diagnoses reported, a comparison between the 2 groups was not possible.

The frequency of histologic diagnoses in the 2 groups closely paralleled Pap test diagnoses in the same populations. The LBCC and CPT groups had 5.4% and 1.0% cervical intraepithelial neoplasia 1 (CIN 1) histologic diagnoses, respectively, while 5.9% and 1.7% of the women had histologic diagnoses of CIN 2 and 3, respectively. These results were also maintained consistently across all 3 age ranges. The predictive value of a positive LBCC test (93.9%) was similar to that for the predictive value of a positive CPT (87.8%) when based on available histology results (Table 5). The sensitivity and specificity were equivalent for each test. Of all women with an LSIL or more severe Pap test result in each group, 1.9% of women in the CPT group and 6.5% of women in the LBCC group had cervical biopsies indicating CIN 1 or more severe disease.

Discussion

Pap test specimen adequacy results reflect whether the most likely site for neoplasia of the cervix—the transformation zone—has been sampled properly. LBCC provided significantly greater rates of satisfactory specimen adequacy than the CPT. Also, compared with the CPT, LBCC effectively reduced the number of SBLB and unsatisfactory specimen adequacy reports. Our findings are consistent with those of studies published previously that used split sample collection techniques.^6,7 The improvement of Pap test adequacy using LBCC may be because a clearer inspection of pertinent normal and abnormal cells is achieved by eliminating or minimizing obscuring inflammatory cells, red blood cells, other debris, and clumped or overlapped cells.¹³ A significant decrease in unsatisfactory Pap tests reduces the extra cost, time, and patient and clinician inconvenience incurred from the necessity of repeating a nonrepresentative Pap test. These advantages may be of particular importance to patients, clinicians, and third-party payers.

Also, in regard to specimen adequacy, LBCC performed better than the CPT for women of all age ranges. This is especially important for older women who typically have a greater frequency of unsatisfactory Pap tests because their active transformation zones are positioned deeply within the endocervical canal and are often inaccessible to comprehensive sampling. In addition, a thin atrophic epithelium (commonly seen in the older age group) is more easily traumatized during sampling, which causes potentially obscuring bleeding. Yet, LBCC reduced the rate of unsatisfactory Pap tests by approximately 50% for women older than 40 years. Other than the advantage of filtering unwanted cells, the difference may be explained by the fact that 80% of cervical cells are discarded with the collection devices and not transferred to a glass slide using the CPT method.³ In contrast, nearly all cells are transferred to solution with LBCC. Thus, the larger specimen obtained with the sampling method used in LBCC increases the availability of a typically limited number of endocervical cells retrieved from post-treatment and postmenopausal women who are routinely more difficult to sample.

The ability of the Pap test to detect cytologic changes consistent with neoplasia, when present, is critically important. In our trial, the LBCC method detected a greater percentage of women with LSIL and HSIL compared with the CPT. Approximately 4 times the number of women with LSIL and twice the number of cases of HSIL were detected by LBCC compared with the CPT. These results replicate or surpass those found in studies that evaluated LBCC using a split sample method.^6-9 Twice the rate of neoplasia detection by LBCC has been reported in studies using the split sample method. Our results, based on a direct-to-vial study design, may portray the true potential for LBCC to detect cervical neoplasia. A cleaner monolayer Pap test with a more comprehensive cellular specimen likely accounts for these remarkable differences. Similarly, the reduced number of unsatisfactory and SBLB Pap tests using LBCC may contribute to the greater yield of SILs. Of note, LBCC did not detect a significantly greater rate of ASCUS Pap test results; this may create problematic management decisions for some clinicians.¹⁴ The ability of LBCC to detect a greater percentage of women with SILs was maintained for the general screening population, the colposcopy cohort, and across the 3 age ranges. LBCC may be better able to detect women with HSIL, a lesion unlikely to resolve spontaneously. Women with HSIL (Figures 2A and B) and a comparable histologic diagnosis deserve prompt therapy for this true cancer precursor. Although many women with LSIL have disease that may regress to normal, as many as 20% to 40% of these women will have histologically confirmed CIN 2 or 3 that deserves treatment as well.¹⁵

Limitations

Our study is subject to several limitations. Most important, the observed differences in Pap test performance may have been because of actual differences of cervical neoplasia prevalences between the 2 groups, rather than a difference between Pap tests. Our subjects were selected from comparable patient populations by a study design that other researchers^10,11,16-20 have used to evaluate the direct-to-vial technique. Other than sampling a population of women with LBCC and then having them return for a CPT 1 month later, which risks compliance failure and spectrum of cervical disease changes or acquisition of new disease, there is no other study design to evaluate a direct-to-vial technique. One may question whether our increased rate of neoplasia detection demonstrated by LBCC is due to a substantial number of LBCC false-positive results.²¹ Yet, because the positive predictive values for the LBCC and CPT were similar and there were 3 times as many women with biopsy results of CIN 1 or greater in the LBCC group compared with the CPT group when only women with LSIL or greater Pap test results were considered, the increased rates of SIL detection using LBCC appear to represent an increased detection of true disease and not false-positive results, further validating our findings.²¹ The equivalent results for LBCC and CPT specificity (99%) indicate that the increased rate of SIL detection by LBCC was not due to a reduced specificity. Also, the favorable test results demonstrated for LBCC are not entirely dissimilar from those of other studies that used a split sample technique, a more biased study design for LBCC.^6-9 Studies by Vassilakos and colleagues¹⁶ and Papillo and coworkers¹⁰ demonstrated that a liquid-based thin layer cytologic report of LSIL or more severe disease correlated better with histologic results of CIN 1 or greater (80.5% and 80.2%, respectively) than CPTs (71.7% and 72.2%, respectively). Finally, because of the limited number of women with cervical caner in this study population, we were unable to determine if there were any differences between LBCC and the CPT in their ability to detect cervical cancer.

Conclusions

Compared with the CPT, LBCC detected a significantly greater percentage of satisfactory Pap tests and significantly reduced the number of unsatisfactory and SBLB tests. These findings demonstrate that LBCC significantly improves the adequacy of Pap tests and may increase the rate of detection of cervical neoplasia compared with CPT. Further study is necessary and warranted, since failure to detect cancer in a timely fashion affects ultimate cure rates, medical costs, quality of life, and perhaps medicolegal expenses. Although liquid-based thin layer cervical cytology is rapidly replacing the glass slide method throughout the United States, additional studies are also necessary to determine whether LBCC reduces the incidence of cervical cancer.

Acknowledgments

We appreciate the assistance of Cytyc Corporation in providing the supplies and grant support necessary for completion of this study.

We would like to thank the faculty and residents in the Family Medicine Center and OB/GYN Clinic who assisted with specimen collection, Jim Best for processing and interpreting cytologic specimens, Aisha Lavin for data management assistance, and April Dean for manuscript preparation.