Institutional Study on Unsatisfactory Pap Smears: Causes and Clinical Impact

Authors: Santosh Tummidi, Varsha Kalambul Vijayalal, Souvik Bhattacharya, Indranil Chakrabarti, Subrat Panda

Abstract

Introduction:

Cervical cancer screening using cervical Papanicolaou (Pap) smears has been well established since the mid-20th century. Specimen adequacy is considered the most relevant quality assurance component since the invention of the Bethesda system for reporting cervical cytology. Cervical smears that are reported as unsatisfactory (US) can significantly delay diagnosis and increase the risk of cancer in follow-up tests. They also lead to unnecessary use of resources.

Materials and Methods:

The cases signed out as US for one and half year duration in the cytology laboratory were retrieved, and demographic details were collected.

Results:

Among the 4,139 Pap tests accepted and reported for the period, 240 (5.8%) were signed out as US. The ratio of conventional to ThinPrep smears was 2.4:1. 70% cases were conventional Pap smears. 66% (159/ 240) cases of the patients were in the fourth and fifth decade age group. Low squamous cellularity was the most common cause (80%) of US smears, obscured by inflammation (12%); other causes were obscuration by blood (0.8%) and mucus (0.4%), drying artifacts (1.6%) and cytolysis (0.4%).

Discussion and Conclusion:

US smears largely depend on various factors, that is, the expertise of the person taking a sample, the date of collection in the menstrual cycle, the method of sampling, sampling devices and staining procedures. The variability in sample collection, including occasional collection by personnel with inadequate training, may also have contributed to the US smears. The result of our study is expected to have a significant impact on proper sampling and the use of adequate quality control measures. In addition, through our follow-up data, we could emphasize the importance of follow-up and human papilloma virus co-testing in patients with US smears.

INTRODUCTION

Cervical cancer ranks 4th amongst the most commonly diagnosed cancers, as well as the 4th most common cancer-causing mortality in women globally. In 2020, there were an estimated 604,127 cases and 341,831 deaths attributed to cervical cancer. Cervical carcinoma attains the status of being the second most (183%) recurrent form of carcinoma in Indian women, as well as the second most recurrent form of carcinoma among women aged 15–44 years.[1] Approximately 5.0% of women in the general populace are estimated to harbor cervical human papilloma virus (HPV)-16/18 infection at any given moment, and 83.2% of invasive cervical carcinomas are ascribed to HPVs 16 or 18.[2]

Cervical cytology screening plays a crucial role in monitoring cancer prognosis. Cervical cancer screening using cervical Pap smears is well-established. It has led to a notable decrease in the incidence of invasive cervical carcinoma among those who are screened. Nevertheless, approximately 33% of the cases where invasive cancer was not prevented can be linked to false negative results from Pap tests.[3]

Specimen adequacy is considered the most relevant quality assurance component since the invention of the Bethesda system (TBS) for reporting cervical cytology. The cervical smears reported as unsatisfactory (US) have a profound impact on the timely diagnosis, increased risk of malignancy on subsequent follow-up, and unnecessary wastage of resources.[3] Liquid-based preparations for cervical cytology have gained popularity as it is expected to reduce US rates.[4] However, it is also important to analyze the clinical determinants leading to US sampling along with the smear characteristics to minimize the US rates.[5]

US cervical cytology smears lead to screening failures and resource wastage. US Papanicolaou (PAP) smear indicates unreliability in detecting cervical epithelial abnormalities. Considering them as negative for malignancy is incorrect and may lack follow-up measures. This study will explore the clinical/pathological parameters associated with US conventional pap smears. Optimal collecting devices or better sampling techniques can be considered to reduce US Pap smears. The causes of US smears and the steps needed to reduce their rate are the main focus of the study.

AIMS AND OBJECTIVES

The primary objectives were to find the US Pap smear causes and the factors associated with them. Their correlation with LMP, method of procedure, type of sample, age and other clinical details.

MATERIALS AND METHODS

Study period

The study was an ambispective study for a period of 1 ½ years. A total of 4,139 Pap smears were recorded. Both conventional Pap tests and liquid-based cytology (LBC) samples were collected by the gynecology department and subsequently sent to the cytopathology lab for analysis.

Sample type

Conventional Pap test samples were obtained using an Ayre spatula and/or an endocervical brush and were immediately transferred to glass slides. These slides were then fixed with 95% ethanol before being dispatched to the cytopathology lab. For the ThinPrep (TP) samples, the collection was performed using a TP vial containing a PreserveCyt transport medium. These samples underwent further processing in the cytology lab via the TP processor. All slides, including both conventional and TP, were subjected to progressive Pap staining utilizing an automated slide stainer to ensure consistent and accurate results.

Sample size

Our sample size included all consecutive patient samples during the study period. Based on available studies and previous experiences, the US rate of routine PAP is very high (approx. 5%) compared to the standard US rate of <1%. Using this data, with a 1% alpha error, 20% loss to follow-up, and 95% power, the sample size was calculated to be 87 cases. However, a total of 240 samples deemed US in the cytology lab were investigated, comprising 169 conventional Pap smears and 71 LBC specimens. Relevant demographic information, clinical history, and menstrual history were extracted from the requisition forms associated with each case.

Inclusion criteria

Pap tests signed out as US for evaluation were analyzed among consecutive cases examined during the study period.

Exclusion criteria

Patients having pelvic irradiation, pelvic malignancy, chemotherapy, and hysterectomy and no proper clinical history were excluded.

Sampling procedure

Patients attending the Gynecology OPD met the inclusion criteria and consented to participate in the study. To obtain a PAP smear, a speculum is inserted into the vagina, and a spatula is inserted into the opening of the cervix and twirled around to collect a sample of cells, which is then smeared on glass slides. Following institutional policy, Pap smears are performed by clinicians who have a minimum of 3 to more than 12 years of experience in sampling. Each patient’s record includes the name of the clinician responsible for sampling, ensuring accountability and maintaining high standards of care. Usually, 2 smeared slides are obtained (1 ectocervix + 1 endocervix) for reporting. The slides with the cervical cytology requisition form will be received by the pathology department for processing. We collected demographic and relevant clinical information, such as age, sex, site of PAP smear, from the cervical cytology requisition form. A record of the number of slides received per patient is kept. Cases with improper labeling and broken slides are not accepted. Our strict adherence to protocols ensures that staining is meticulously monitored, with cytopathologists regularly checking staining quality. Every abnormal case is reviewed by a minimum of two pathologists to ensure accuracy. Additionally, both the Thin Prep processor and slide auto-stainer undergo regular external quality assurance checks to maintain high standards.

**Evaluation ** The primary aim of this study was to re-examine the US slides to elucidate the underlying causes of the inadequate smears, employing the criteria established by the 2014 Bethesda System for Reporting Cervical Cytology (TBS 2014). Additionally, the secondary objective focused on categorizing the age, clinical manifestations, and menstrual history of patients who received US results. All slides underwent a thorough reevaluation, and the reasons for suboptimal results were categorized as 1) Low squamous cellularity [Figure 1]; 2) Obscuration due to inflammation; 3) Obscuration due to hemorrhage; 4) Artifacts from air drying; 5) Presence of lubricant gel; 6) Bacterial contamination; 7) Mucous interference. [Figure 2a–d] The categorization criteria employed were semi-quantitative, based on TBS 2014. TBS states that for reliable diagnosis of epithelial cell abnormalities, a minimum of 5,000 cells and 8,000–12,000 well-visualized and well-preserved cells is essential in LBC and conventional preparations, respectively.

The cellularity of conventional smears was evaluated using reference images from the Bethesda system. For instance, if a 4 × field containing 1,000 cells served as the reference image, a specimen would require a minimum of eight 4 × fields to be considered to have sufficient cellularity. An estimation of the total cellularity can be obtained by conducting representative field cell counts for TP smears. Counting five fields along a horizontal diameter and five fields along a vertical diameter (SKML protocol) at 10 × was used.[6] Apart from the adequacy criteria, smears where over 75% of the squamous cells were obscured, and no abnormal cells were visualized, were classified as US.[1]

Whenever the above criteria were met, the slides were reviewed separately by two cytopathologists with three years of experience. A senior cytopathologist with more than 12 years of experience then reviewed the findings of both residents as part of quality control. The semi-quantitative analysis of the degree of cellularity, severity of inflammation, obscuration by blood, and other factors mentioned above was separately tabulated. Follow-up details were retrieved from the electronic medical records, including repeat cytology and histopathology reports. All data were entered into an Excel spreadsheet. Categorical data were tabulated as numerical values and percentages.

RESULTS

Of 4139 Pap tests, 240 (5.7%) were deemed US by two pathologists. We had both type of sample, that is, conventional and LBC (TP). The ratio of conventional smears to TP smears was 2.4:1. 70% of cases were conventional Pap smears. [Figure 3a] The ages of the women ranged from 15 years to 84 years. 66% (159/240) cases of the patients were in the 4th–5th decade age group with a mean age of 42 years. [Figure 3b]

We conducted an analysis of the clinical symptoms observed among the patients. The most prevalent symptom identified was excessive bleeding, affecting 35% of the patients, followed by lower abdominal pain and white discharge. Notably, one-third of the sampling occurred during the early phase of the menstrual cycle (within the first 14 days). [Figure 3c] Additionally, 18% of the cases had attained menopause. However, menstrual history was unavailable for 23% of the patients.

Low squamous cellularity was observed in all cases (100%) of our study; however, obscuration by inflammation was an additional finding in 24.6% of cases. The inflammation was graded into mild (<25% obscuration), moderate (25%–75% obscuration), and severe (>75% obscuration). 75.4% of the cases had no inflammation, with 10.8% having minimal inflammation, whereas in 13.8% of cases, inflammation was a confounding factor to low squamous cellularity [Figure 4a, 5] [Table 1]. Overall, 12% of cases had blood and mucus, drying artifacts, and cytolysis, in addition to low squamous cellularity [Figure 4b, 6].

Out of the 240 cases reviewed by the residents, both reviewers agreed on the diagnosis of 235 cases. In five cases, their interpretations differed. This resulted in an interobserver variability of 2.08%, showing a high level of agreement. However, the senior pathologist’s report remained US and that was included for the study.

After collecting follow-up details for 4 months, we successfully documented outcomes in 27 cases, which accounted for 11.3% of the cohort. The follow-up findings spanned a range from Negative for Intraepithelial Lesion/Malignancy (NILM) to Keratinizing Squamous Cell Carcinoma. Among the 27 follow-up cases, 20 patients underwent a repeat Pap test within a 6-month period, resulting in 16 tests returning NILM. [Table 2]

One notable case involved a 25-year-old presenting with white discharge and an US initial Pap test, which was later classified as Atypical Squamous Cells of Undetermined Significance upon retesting. [Figure 7a and b] Conversely, a 40-year-old with lower abdominal pain received a diagnosis of Atypical Glandular Cells (not otherwise specified) in her follow-up Pap smear.

Histological examination revealed benign endocervical polyps in 4 cases, while 3 cases were determined to be malignant. A significant finding was a 64-year-old post-menopausal woman, whose histological follow-up diagnosed her with non-keratinizing squamous cell carcinoma, following presentation with lower abdominal pain. Additionally, two middle-aged women were diagnosed with high-grade squamous intraepithelial lesions and keratinizing squamous cell carcinoma upon biopsy, respectively. [Figure 7c and d]

Retesting was performed exclusively for TP cases where low squamous cellularity was presumed to result from a technical artifact, specifically the peripheral collection of cells known as the halo effect. Other causes of retesting include technical errors such as thick smear, poor staining or patchy cellularity. Retesting was possible only for LBC cases, and we had to perform retesting due to the above-mentioned causes in less than 1% of the total LBC smears. For conventional smears, retesting was advised in the report.

DISCUSSION

US Pap smears represent a major challenge for screening cervical cancers. The data from epidemiological studies indicate that properly conducted cervical cancer screening using the Pap smear has significantly affected both the mortality and morbidity rates associated with cervical cancer.[7]

Cervical cytology smears are examined microscopically using TBS, introduced in 1988. The Bethesda reporting system features standardized, clear, and reproducible terminology that emphasizes the latest findings related to cervical neoplasia. This system was updated in 1999, 2001, and most recently in 2014. The reporting framework includes an assessment of the adequacy of the specimen, the presence of cervical abnormalities, and if there are any, the degree of those abnormalities.[3,7]

TBS considers specimen adequacy as the most important component of quality assurance. TBS 2014 has a clear distinction between satisfactory and US samples. US cases are broadly divided into rejected specimens and fully evaluated US specimens. The reasons for US smears can be low squamous cellularity, inflammation, hemorrhage and other obscuring factors.[3]

US smears largely depend on various factors, including the expertise of the person taking a sample, the date of collection in the menstrual cycle, the method of sampling, sampling devices, and staining procedures. The data from the College of American Pathologists (CAP) survey in 2024 show that the median value for US specimens is 1.5–1.4 for conventional and TP, respectively.[8,9] Our study had higher US rates (5.7%), which might be influenced by improper sampling or techniques. This rate is comparable to those reported in a few studies carried out in India, Taiwan, and Italy.[5,9,10] Low squamous cellularity was the most common cause in the present study, in concordance with the previous studies.[6,11,12,13] [Table 3]

Older age is considered an important risk factor for US Pap smears.[4,6,9] Contrary to earlier studies, 66% of the patients belonged to the middle-aged group. Being a government hospital, this could be attributed to screening initiatives implemented across the nation, specifically targeting the middle-aged demographic. In addition, the older age group often presents to the clinic at later stages of the disease, which usually requires a colposcopic biopsy. Gupta et al. [5] studied that age greater than 45 years has a significant correlation with US rates. In our study, the most common clinical symptoms were abdominal uterine bleeding, lower abdominal pain, and white discharge. Vaginal bleeding, lower abdominal pain, postpartum status, and endocervical polyps were associated with US smears in a few of the previous studies.[5,6,14]

The US rate among conventional smears ranges from 4.3%–11.5% as compared to LBC with 0.3%–1.7%.[15] Studies have shown that LBC method had a significantly higher rate of SILs in the US repeat procedures as compared to Pap test with satisfactory and NILM cases.[16] A study done by Kalinicheva et al. has shown that the US rate in LBC is lower compared to conventional methods (1%–5.9%), in LBC by TP (1.1%–3.4%) had higher US rate as compared to SP 0.3%–1.3% method.[17]

Our 1.5-year-old data suggest that low squamous cellularity continues to be the most common cause of US reports, followed by obscuration by inflammation and blood. The least common cause was air drying artifact. This is in concordance with many previous studies.[4,6,18,19] [Tables 4 and 5] The low squamous cellularity can be due to technical in sampling or any significant medical conditions that lead to improper sampling or less cellularity due to atrophy of the cervix.[5,8,20] The guidelines from the American Society for Colposcopy and Cervical Pathology (ASCCP) recommend that women, regardless of their negative history of gynaecological issues, should have repeat Pap tests performed within 2–4 months. If the repeat Pap test is also deemed US, further investigations, such as colposcopy and/or biopsy, may be necessary.[21]

In our research, only 11.3% of patients had available follow-up reports. 6.67% of them underwent repeat Pap tests, which were classified as NILM. Benign conditions, such as endocervical polyps, can also result in US smear results. Four patients were found to have endocervical polyps. The other patients (5 out of 27) received various diagnoses ranging from atypical squamous cells to squamous cell carcinoma within a period of 6 months. One patient also had a US repeat test.

Management of US smears is follow-up after 2–4 months, but patients usually don’t comply to this follow-up. Similar problems have been reported in literature.[13,20,21,22,23,24] According to the 2012 ASCCP guidelines, patients in the US receiving Pap smears should have a follow-up test within 2–4 months. For individuals with inflammatory results, it is advised to retest after addressing the inflammation. Colposcopy is suggested for those with a history of HPV positivity or in cases of recurring US Pap smear results. The clinicians should counsel the patients and convince for follow-up for the US cases with repeat examination as there are high chances of abnormal results in future. At our institute, we give effort to identify the cause of US cases. The percentage of US is an important quality indicator as per CAP protocol. All US cases are reported by two pathologists before releasing. Simultaneously efforts in form of interaction with the gynecologist, technician, nursing officers are done to ensure the check on US cases.[25,26]

Regular training sessions are organized for junior doctors to improve sampling techniques and reduce US rates. The samples were processed as received by the lab and run by cyto-technicians with at least 3–4 years of experience in cytology. The role of technicians was only to load the LBC samples into the TP machine/conventional method slides, followed by staining with an automated stainer, which had the prefixed PAP protocol. Routine training for cyto-technicians and quality control measures are systematically implemented in our laboratory. Nevertheless, factors such as training in sample collection and other pre-analytical variables were not monitored routinely, which might have contributed.

Our limitations included a small study population, retrospective non-randomized nature of the study, and less follow-up cases. A prospective study involving a larger population could facilitate corrective actions, such as repeat testing of remaining samples with inadequate LBC results and patient counseling regarding the importance of timely follow-up. Additionally, it may help minimize observer bias, as the report would not be known in advance. More information about the sampling techniques done by the clinician would have helped in assessing the pre-analytical causes of US smears.

The latest update incorporates the use of HPV co-testing for cervical cancer screening. When sampling is done correctly, using the HPV test as the main screening method can also reduce the likelihood of insufficient cellularity. The reason for HPV testing is that the number of cells needed, regardless of the HPV type, is significantly lower than the 5,000-cell adequacy standard for Pap tests. Moreover, a negative HPV test in the context of a US Pap test may indicate an insufficient sample due to low squamous cellularity and may not be genuinely negative. Therefore, it is advised to use both tests together.[27,28]

CONCLUSION

US smears can undermine the effectiveness of cervical cancer screening by increasing false negative rates. Adjunct HPV testing may help mitigate this issue. Our study was to focus on the various causes of US smears and on preventing the false-negative results. Proper sampling techniques and processing of the sample can decrease the US rates. Clinical symptoms like abnormal uterine bleeding, lower abdominal pain, and other inflammatory conditions might contribute to US sampling. The findings of our study indicate that clinical symptoms such as abnormal uterine bleeding, lower abdominal pain, and benign pathological conditions, including endocervical polyps, may contribute to inadequate sampling outcomes. LBC overcomes most of the causes of US smears in conventional smears, while scant cellularity remains the sole important cause of US smears. To conclude, clinical correlation, patient examination, proper sampling, and processing can reduce the number of US smears.

Consent to publish

Written consent to publish was taken from the patients involved in the study.

Author contributions

TS conducted the literature search, data acquisition, data analysis, clinical study, and manuscript preparation, and will also stand as guarantor. VKV carried out the literature search, data acquisition, and manuscript preparation. SB carried out concepts, design and data analysis. IC & SP participated in the clinical study and manuscript preparation. All the authors have read and approved the final manuscript.

Data availability statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

Ethical policy and Institutional Review Board statement

Written informed consent was obtained from all participants. The study protocol was reviewed and approved by IEC, AIIMS, Kalyani, with approval number IEC/AIIMS/ Kalyani/Certificate/2024/078.

Conflicts of interest

There are no conflicts of interest.

Abbreviations

ASCCP American Society for Colposcopy and Cervical PathologyCAP College of American pathologistHPV Human Papilloma VirusLBC Liquid Based CytologyNILM Negative of Intraepithelial malignancyPAP PapanicolaouSIL Squamous Intraepithelial lesionsSP Sure PathTP ThinPrepTBS The Bethesda SystemUS Unsatisfactory