Background
Application of acetic acid to the cervix followed by colposcopic assessment with or without colposcopically directed biopsy is the standard test used to detect dysplastic lesions of the cervix. However, there is no evidence-based common standard defining how exactly to perform this test.
Objective
To prospectively define the optimal timing for the colposcopic assessment of acetowhite lesions.
Materials and Methods
Consecutive women referred to our colposcopy unit were recruited. Using a standardized colposcopy protocol, we recorded the most severe colposcopic lesion 1, 3, and 5 minutes after application of acetic acid (primary study end point). The time to first appearance of the most severe colposcopic lesion, highest staining intensity, and fading of the most severe colposcopic lesion were video documented (secondary study end points, assessed independently by 3 raters). Results were compared using parametric and nonparametric tests.
Results
A total of 300 women were included. After 1 minute, 290 of 300 patients (96.7%) were diagnosed with the most severe colposcopic lesion. This proportion did not improve after 3 minutes (290/300 [96.7%]) or after 5 minutes (233/264 [88.3%]). The proportion of minor and major changes continuously declined over time from 142 in 300 (47.3%; 1 minute) to 107 in 264 (40.5%; 5 minutes) and from 110 in 300 (36.7%) to 91 in 264 (34.5%), respectively. The median time until the first appearance of the most severe colposcopic lesion was 13.5 (interquartile range, 3−27.25) seconds and was significantly lower in high-grade squamous intraepithelial lesion (7 [interquartile range, 1−20] seconds) compared to low-grade squamous intraepithelial lesion (19 [interquartile range, 9−39.5] seconds; P < .001). We observed fading of acetowhite lesions in 78% of cases, occurring at a median of 191 (interquartile range, 120−295) seconds after application of acetic acid. Fading started earlier in high-grade squamous intraepithelial lesion compared to low-grade squamous intraepithelial lesion (179.5 [interquartile range, 110− 253.25] versus 212.5 [interquartile range, 146.5−300]; P = .044). Overall, the net difference between colposcopic assessments at 3 minutes versus at 1 minute was 1 more high-grade squamous intraepithelial lesion and 1 less low-grade squamous intraepithelial lesion.
Conclusion
It is reasonable to conclude that the best time to identify lesions is 1 minute after the application of acetic acid. Continued evaluation for up to 3 minutes may be considered reasonable for an optimal high-grade squamous intraepithelial lesion yield. However, fading of acetowhite lesions is common, especially in high-grade squamous intraepithelial lesions, and supports a recommendation of not prolonging colposcopy beyond 3 minutes.
Applying acetic acid (AA) to the cervix followed by colposcopic assessment with or without colposcopically directed biopsy is the standard test used to detect dysplastic lesions of the cervix. The AA test is used worldwide and is an integral part of the management of cervical intraepithelial neoplasia. This test is recommended as standard of care by the International Federation for Cervical Pathology and Colposcopy, as well as national societies such as the American Society for Colposcopy and Cervical Pathology (ASCCP) and the German Society of Colposcopy and Cervical Pathology (AG-CPC).
Why was this study conducted?
Although application of acetic acid to the cervix followed by colposcopic assessment is the standard test used to detect dysplastic lesions of the cervix, there is no common standard for performing this test.
Key findings
The optimal timing for the colposcopic assessment of acetowhite lesions is 1 minute after the application of acetic acid. Fading of acetowhite lesions is common, starts after 1 minute, and is more pronounced in high-grade than in low-grade squamous intraepithelial lesions.
What does this add to what is known?
Using a prospective design, this study clearly defines the optimal time frames for colposcopic assessment after application of acetic acid, allowing colposcopists to minimize the examination’s duration without compromising reliability.
Surprisingly, recommendations for how exactly to perform AA testing in order to obtain optimal results vary considerably. For example, historical textbooks such as the original description of colposcopy and the AA test by Hinselmann in 1925 recommended applying AA for up to 5 minutes. Recent recommendations include application of 5% AA followed by a delay of 1−2 minutes before colposcopic assessment, 3% AA for 30−60 seconds before assessment, 5% AA for 1 minute followed by immediate assessment, or application of 5% AA for 1 minute followed by another minute of waiting. In a large randomized screening trial with >130,000 participants, 4% AA with assessment 1 minute after AA application was used as a standard, based on the World Health Organization (WHO) International Agency for Research on Cancer’s handbook on cervical cancer screening and their own previous trial. In many studies, the specifics of AA testing are not defined at all. It is noteworthy that the ASCCP and other national societies do not delineate the details of the AA test in their colposcopy standards.
Therefore, based on the data in the literature and based on existing practice guidelines, there is no common standard for AA testing during colposcopy. Current practice of this important and commonly used diagnostic tool is not evidence based. This statement is supported by a PubMed literature search (search terms: colposcopy, acetic acid test, acetowhite; search date September 27, 2019), through which we identified no controlled trial defining the optimal point of time for AA testing during colposcopy. Because the AA test is an integral part of the management of women with cervical dysplasia, there is a medical need to properly define the specifics of this test in a prospective study.
To address this issue, we designed a prospective study to investigate the AA test in detail in a large patient population. The aim of our study was to properly define the optimal point of time for the colposcopic assessment of acetowhite lesions after AA application. In addition, we wanted to quantify the increases and decreases in acetowhite lesion severity over time, and to specify the proportion and timing of fading of acetowhite staining.
Materials and Methods
We recruited consecutive women referred to the colposcopy outpatient unit of the Department of Obstetrics and Gynecology of the Ruhr-Universität Bochum, Germany, or the doctor’s office of the Institute of Cytology and Immune Cytochemistry, Dortmund, Germany, for further assessment of a pathologic Papanicoloaou (Pap) smear result. After giving informed consent, a detailed personal history was taken and documented. We used a standardized protocol for colposcopy and AA testing and in all women as follows: using a binocular colposcope (Model 1D LED, Leisegang, Berlin, Germany), the native impression of the cervix was recorded with ×7.5 and ×15 magnification. Then, acetic acid 5% was applied to the cervix with 3 pushes using a commercially available household spray can (Wilpeg, Großenlüder, Germany), corresponding to a total volume of 3 mL. Next, the colposcopist assessed and classified the most severe colposcopic lesion (MSCL) 1, 3, and 5 minutes after the application of AA. The primary end point of this study was the proportion of MSCL after 1, 3, and 5 minutes using the International Federation for Cervical Pathology and Colposcopy Rio classification criteria. Secondary study endpoints included the time from AA application to the first appearance of the MSCL, the time from AA application to the highest staining intensity of the MSCL, and the time from AA application to the start of fading of acetowhite staining of the MSCL. These were recorded and video documented for every patient. Of these, 85 videos were selected (with an allocation of 1:2:2 to histological outcome: negative for dysplasia, low-grade squamous intraepithelial lesion [LSIL], high-grade squamous intraepithelial lesion [HSIL]/carcinoma) for detailed assessment by 3 colposcopists to further specify the dynamics of acetowhitening. This sample size was calculated based on the experience that acetowhitening of HSIL is faster than that of LSIL lesions (assumed on average to be most intense after 60 seconds) and assuming a fixed standard deviation of ±15 seconds, so that a difference in time needed to reach maximum intensity of 20% could be detected at an α of 0.05 with a power of at least 90%, and allowing for a dropout/error rate of 15% (video not of suitable quality, wrong group assignment due to clerical errors). G*Power 3.1.9.2 was used for this calculation. All 3 colposcopists, who were blinded to patient information and histological outcomes, assessed each video with regard to the exact time (in seconds) until the first and secondary endpoints occurred. Endpoints not occurring before the end of the observation period (300 seconds after application of AA) were censored with a value of 300 seconds. Waterfall plots were used to visualize the time points of fading in low-grade and high-grade lesions as determined by the 3 raters; each bar represents 1 observation. To determine proportions of nonfaded acetowhite lesions, the time points of fading onset determined by the 3 raters were averaged per video.
All colposcopists were certified specialists with more than 300 annual colposcopies performed. The study was approved by the Ethics Committee of the Medical Faculty of Ruhr-Universität Bochum, Bochum, Germany (registration number 18-6370, dated August 24, 2018).
Assuming a continuous outcome measure such as the time until the appearance of the MSCL, a sample size calculation with the confidence level set at 95% and the confidence interval set at 5% resulted in a sample size of 278 patients for a population size of 1000 women (roughly corresponding to the number of women seen by a colposcopists with 300 cases per year over 3 years). Therefore, we aimed to recruit at least 300 patients, allowing for approximately 10% of dropouts/protocol violations.
Study data were collected and managed using REDCap (Research Electronic Data Capture), a secure, Web-based application designed to support data capture for research studies. After data collection had been concluded, exported data were further processed in Microsoft Excel (Microsoft Inc, Redmond, WA) and prepared for statistical analyses using SigmaPlot 14 (Systat Software Inc, San Jose, CA). Descriptive statistics are reported using means and standard deviations for normally distributed data, and medians and interquartile ranges (IQR) for data not meeting this assumption. Accordingly, statistical analysis was performed using parametric ( t test) or nonparametric tests (Mann−Whitney U test for pairwise comparison and analysis of variance on ranks for multiple comparisons). All P values are 2-tailed, and P < .05 was considered statistically significant.
Results
From September 2018 to May 2019, we prospectively recruited 305 consecutive women from our colposcopy units into this study. Of these, 4 women were excluded because of protocol violation. In 1 case, study documentation was missing. Thus, data from 300 colposcopies were evaluated. Figure 1 gives a detailed account of the patients’ flow through the study and the workflow. Patient characteristics are shown in Table 1 . All patients underwent a standardized colposcopy and AA test protocol as delineated in the Materials and Methods section. Table 2 lists the Pap smear results, the colposcopic findings before and after AA testing, and the corresponding histopathological results. Specifically, type I, type II, and type III transformation zones were identified in 195 (65.0%), 58 (19.3%), and 47 (15.7%) of cases. At the end of colposcopy, that is, 5 minutes after AA application, colposcopic assessment found a normal cervix in 61 cases (23.1%), minor changes in 107 cases (40.5%), major changes in 91 cases (34.5%), and major changes suspicious for invasion in 1 case (0.4%), respectively. The colposcopic findings changed over time. When comparing the colposcopic assessments 1 minute, 3 minutes, and 5 minutes after AA application, the proportion of minor changes continuously declined from 142 (47.3%) after 1 minute to 137 (45.7%) after 3 minutes and 107 (40.5%) after 5 minutes. The proportion of major change lesions also declined over time, from 110 (36.7%) to 114 (38.0%) and to 91 (34.5%), respectively. Of note, this effect was restricted to minor and major changes and was not observed in the categories “nonspecific findings,” “miscellaneous findings,” and “other findings” (as defined by the Rio classification system ).
| Characteristic | Value |
|---|---|
| No. of patients | 300 |
| Age, y | 35.2 (29.4–44.3); range 19.8–78.4 |
| Body mass index, kg/m 2 | 23.7 (21.3–27.3) [44] |
| Parity | 1 (0–2) [44] |
| Allergy, yes/no | 94 (37.2%)/159 [47] |
| Smoking, yes/no | 98 (37.8%)/161 [41] |
| Alcohol abuse, yes/no | 7 (2.4%)/288 [5] |
| Drug abuse, yes/no | 10 (3.5%)/274 [16] |
| Concomitant disease, yes/no | 103 (41.2%)/147 [50] |
| Prescription drug use, yes/no | 118 (48.8%)/124 [58] |
| Immunosuppressive conditions, yes/no | 4 (1.6%)/247 [49] |
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