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Liquid Cytology: What is it and how does it compare to conventional cytology.
Major advancements in cervical cancer screening have dramatically lowered the mortality rate worldwide. Cervical cancer is a potentially preventable disease, with the correct screening programme, along with reliable and accurate laboratory techniques. The first major advancement in cervical cancer screening was the Pap test, followed by the use of liquid-based cytology. Since the introduction of liquid-based cytology, there have been debates surrounding its reliability and accuracy in terms of sensitivity in comparison to the use of the conventional Pap test when detecting precancerous lesions. Many trials and studies have been undertaken to compare both techniques with many questions still unanswered, but it is certain that in the future there will be a need for the introduction of molecular aspects when analysing cervical cytology.
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Globally cervical cancer is the second most common form of cancer in women. (Bosch, et al., 1995). It is preventable when the precursor lesions are detected and treated early. Cervical cytology has been implemented for over 50 years, and results have shown that this is the main weapon of defence against this disease. (Koss, 1989). Due to the success in the advancement of screening within cervical cytology, the number of incidences of cervical cancer has decreased by more than 50% in the past 30 years (Karjane, 2018). One of the major changes in the preparation method for screening cervical cancer cells has come in the form of liquid-based cytology. Cells are no longer being smeared onto a glass slide but are now washed into a vial of liquid and filtered, which results in a random sample being presented in a thin layer on a glass slide. (Denton, 2007). Although some countries have changed to liquid-based cytology for cervical screening, controversy remains over the accuracy and reliability of liquid-based cytology in comparison to conventional cytology.
Major advancements in screening techniques have dramatically lowered the death rate from cervical cancer worldwide. The first major advancement in cervical cancer screening was the Papanicolaou (Pap) test in 1928 (Tan & Tatsumura, 2015). Pap testing was responsible for reducing the incidence of cervical cancer between 1955 and the mid-1980s (Institute, 2007). While the second major advancement in cervical cancer screening came in the form of liquid-based cytology (LBC), and is now implemented on over 90% of cervical cytology samples today. Since the introduction of liquid-based cytology, there has been debate surrounding its reliability in comparison to the use of the conventional Pap test when detecting precancerous lesions. (Sawaya, 2010).
Although the conventional Pap test was responsible for the initial advancement in reducing the incidence of cervical cancer, the clinical performance of this technique is not without limitations. When using the Pap test a broad range of sensitivity (30%-87%) has been reported when detecting high-grade lesions (Gynecologists, 2008). It was also found that the conventional Pap test has a false positive rate of about 14% to 33% (Hartmann, 2002). It is known that the Pap test has been successful in detecting and diagnosing squamous cell carcinomas, although it is less useful in the detection of adenocarcinomas which now contribute to 10% of all cervical cancers. There is a possibility that this could be linked to the difficulty in obtaining enough sample to entirely represent the endocervical canal, which is where most glandular lesions occur. When employing the pap technique, the equipment used to obtain a sample during a conventional Pap smear means that only a small portion of the sample taken from the patient is transferred on the slide. Most of the sample is discarded, along with the sampling equipment, which may lead to inaccuracies when testing the slide. The detection of adenocarcinoma is between 45% and 76% due to the varying degree of sensitivity of the Pap smear. The problems faced when using the Pap smear technique results from obscuring by blood or inflammation, bad cell fixation and inhomogeneous distribution of cells on the slides. Worldwide it is accepted that there are major limitations to conventional cytology.
It is the limitations of the conventional Pap test which have encouraged the scientific community to rethink the diagnostic technique used to detect cervical cancer cells. The introduction of liquid-based cytology has decreased the number of inadequate smears resulting in the improved detection of abnormal cells significantly. (Astbury, et al., 2006). In liquid-based cytology, the cervical cells are obtained in an identical manner to the conventional Pap test, but instead, a cervical broom, a spatula and an endocervical brush or an extended spatula are used to collect the cells (Arbryn, et al., 2007). The difference is that the cells are then rinsed into a vial with preservation solution instead of being smeared onto a slide. This technique eliminates blood, mucus, inflammatory cells and mucus which allows for a more representative sample to be used for testing. The residual material in the vial can also be used for further testing such as reflex human papillomavirus (HPV) testing (Siebers, 2009).
In 1996 the ThinPrep Pap test became the first liquid-based cytology technique to be approved by the FDA. The processing of a ThinPrep Pap test sample involves the rotation of a filter within the sample vial which creates currents in the fluid that separates debris and disperses mucus while preserving the cells morphology. Within the filter, a gentle vacuum is created which collects cells on the exterior surface of the membrane. Once the cells are collected on the membrane, the filter is inverted which is then pressed against the slide. The slight positive air pressure causes the cells to adhere to the slide, which results in the even distribution of cells, as can be seen in Fig 1 (Gibb & Martens, 2011)
Figure 1: ThinPrep Sample processing and analysis. (Gibb & Martens, 2011)
The significant trial for the ThinPrep Pap test revealed that the test provides a 65% increase (P< .001) in the detection of low grade squamous intraepithelial lesions (LSIL) or greater cytology and showed improvement in specimen quality in comparison to the conventional Pap test (P<.001) (Lee, et al., 1997). Later studies supported these results. A study carried out in New England by Diaz-Rosario and Kabawat found that there was a 74% increase in the detection of low grade squamous intraepithelial lesions and the detection of high-grade squamous intraepithelial lesions increased by 102.54% (from 0.26% – 0.52%) as a result of using the ThinPrep Pap test in comparison to the conventional Pap test. Other significant results included a decrease in the specimens categorised as “satisfactory but limited” which is due to the absence of obscuring inflammation, blood and poor fixation. This is only achievable when using a liquid-based cytology technique.
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The second liquid-based cytology technique approved by the FDA was the SurePath Pap test. Similar to the ThinPrep Pap it was designed to screen for the presence of atypical cells, cervical cancer or its precursor lesions (US Food and Drug Administration, 1999). When a SurePath Pap test is carried out, cervical cytology samples are collected in a similar manner to that of a ThinPrep test, but the sample is then transferred into a vial with a collection medium (SurePath Preservative Fluid), which allows the entire sample to be captured. In comparison to the ThinPrep technique, SurePath uses a sedimentation process where samples are enriched to remove debris, which is then followed by centrifugation to generate a pellet, which is refined further to apply it onto a slide for analysis. A thin layer cell sample is then produced using the BD Prep Stain Processor. It was found in studies that the SurePath Pap test increased the detection rate of low-grade squamous intraepithelial lesions and high-grade intraepithelial lesions in cytology samples by 47% (P<.0011) and 116% (P<.002) respectively in comparison with the conventional Pap test (Marino & Fremont-Smith, 2001). A similar study found that the SurePath Pap test increased the detection rate of atypical squamous cells of undetermined significance, LSIL, and HSIL, but the major statistic in this study also revealed a significant reduction in unsatisfactory analysis for the SurePath test in comparison to the conventional Pap test (Fremont-Smith, et al., 2004). It is evident that both liquid-based cytology techniques provide specific benefits in comparison to the conventional Pap test, but when deciding which technique is better it is crucial to remember that the two LBC technologies differ in their clinical performance.
It is evident that Pap tests have been extremely effective in aiding clinicians in the detection and treatment of cervical cancer, but that has not completely eradicated the problem. As discussed above, Pap testing can produce false negatives, which is a result of sampling or detection errors (Mandelblatt, et al., 2002). Roughly one-third of errors are connected to slide interpretation, which highlights the subjectivity of cytological diagnosis. In contrast, molecular-based HPV DNA testing can detect patients at risk for developing preinvasive cervical lesions which removes the subjectivity of cervical cytology analysis (Boulet, et al., 2008). There are large amounts of evidence indicating that HPV is found in more than 99% of cases of cervical cancer, with the virus known to be implicated in the development of cervical cancer, both squamous cell carcinomas and adenocarcinomas (Walboomers, et al., 1999). HPV DNA testing has also demonstrated a higher sensitivity in comparison to Pap testing when detecting preinvasive high-grade cervical lesions (Mayrand, et al., 2007). It is evident that in the coming years as screening programmes develop it will be essential to include HPV molecular-based detection along with genotyping technologies. Clinically this will offer additional benefits when screening for cervical cancer.
In the past 60 years, there have been major advancements in cervical cytology screening due to the introduction of Pap testing, which has led to the development of liquid-based cytology. These advancements in screening techniques have dramatically lowered the death rate from cervical cancer worldwide. But both conventional and liquid-based cytology face their limitations, which is why there is so much controversy surrounding the use of one or the other in a laboratory. As discussed, the pivotal trials for ThinPrep and SurePath tests demonstrate the benefits of each individual technique, but these tests will not be sufficient enough to eradicate cervical cancer which is possible. For this to happen it is vital that cervical screening protocols stay in line with technological advances.
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