Fernanda Dahrouge Chiarot [1]

INTRODUCTION

In the last five decades, cervical cancer (CC) prevention has progressed effectively due to advances in knowledge about its cause, effect, development, diagnosis and prevention1^,2.

TheHuman Papillomavirus ( HPV) is the main sexually transmitted infection (STI) and is responsible for the development of precursor lesions of CC in cases of persistent viral infection of high-risk oncogenic genotypes. The high-risk genotypes 16 and 18 are responsible for 70% of cervical cancers and precancerous lesions. The HPV virus has a tropism for squamous epithelium and mucous membranes, affecting the lower genital tract and the oropharynx3^,4,5,6,7.

In Brazil, the CC screening system includes early detection of the HPV virus and a vaccination program against the main viral types that can cause the neoplasm4^,8.

In Brazil, cervical cancer is the third most common type of cancer among women. For the year 2022, 16,710 new cases have been estimated, which represents a risk of 15.38 cases per 100,000 ^women9. In the regional analysis, cervical cancer is the first most incident in the North (26.24/100,000) and the second in the Northeast (16.10/100,000) and Midwest (12.35/100,000). In the South (12.60/100,000) it ranks fourth and in the Southeast (8.61/100,000) it ranks ^fifth10. In Brazil, the mortality rate from cervical cancer, adjusted for the world population, was 4.60 deaths/100,000 women in 2020. In the historical series of mortality rates in Brazil and its regions, it can be seen that the highest rates in the country have been found in the North, with a clear upward trend over time between 2000 and ²⁰¹⁷¹¹.

For many years in Brazil, the primary methodology used in the recommended screening program was oncotic cytology or the Pap test9^,12. Organized cervical cancer screening programmes in developed countries, which use oncotic cytology, have shown a significant reduction in the incidence of the disease and in mortality compared to developing ^countries13.

Changes in worldwide screening recommendations have been significant in the last decade, due to evidence of regression of abnormal cytological results in the vast majority of cases, since many deaths still occur each year due to CC, even among women who have been followed up, which indicates that the method has failed. Among the reasons are false-negative (FN) results, mostly due to errors in cell sampling, inadequate fixation of the material, insufficient clinical reports and ^analysis errors14.

The World Health Organization (WHO) and the countries of Canada, France and Ireland recommend HPV genotyping assays as the primary method of screening for CC, due to their greater efficacy in reducing incidence and mortality compared to ^cytology15.

Testing for HPV-DNA using molecular methodologies is more sensitive than oncotic cytology and is a non-invasive method that is easy to collect and enables the virus to be identified. Molecular tests associated with morphological tests have been proposed and adapted in several developed countries for the prevention and screening of neoplasia, increasing the effectiveness of screening programs, as they detect high-risk lesions early in women aged 30 or over with NILM cytology, and reducing the need for coloscopies and unnecessary treatments in patients aged 21 or over with ASC-US cytology5^,6,7.

Screening programs for the early detection of CC and precursor lesions are fundamental for the control and prevention of the disease, so recently in Brazil, a new guideline – Ordinance SECTICS/MS No.3 of March 7, 2024, published in the Federal Official Gazette ¹⁶ – established the use of molecular tests for the detection of high-risk oncogenic HPV virus in cervical cancer screening, aiming to improve the system for identifying the virus in the population and its consequences. Thus, within the scope of the Unified Health System – SUS, the use was recommended for molecular tests for the detection of oncogenic HPV, by nucleic acid amplification technique based on PCR, with partial or extended genotyping for cervical cancer screening in a standard risk population and in accordance with the Ministry of Health Guidelines. The pap smear screening guidelines recommend that the test be carried out every three years and, if a lesion is detected, annually, while molecular testing is recommended every five ^years15. This change allows for better adherence, facilitating access to the test for all women.

Liquid Cytology and Molecular Biology

In the 1990s, a number of innovative liquid-based cytology (LBC) methodologies emerged, which improved the conventional methodology of oncotic cytology developed in the 1940s by the doctor Georgia Papanicolaou and which has not been changed for ^years17. This improvement in methodology made it possible to increase the accuracy, sensitivity and specificity of the test. The way the test is collected has changed: instead of the smear taken directly on the slide, in which the spatula is discarded with all the significant representativeness of the cells collected, in liquid cytology, the cervical-vaginal smear is inserted into a liquid medium that preserves all the cells in which it was collected. In the liquid medium, the brush is homogenized and the cells are washed, allowing the preparation of slides containing concentrated cells in an area of 20 mm, with a reduction in cell overlap ¹⁷. CML allows the cells to remain on the slide in a thin layer, without overlap, making it easier to read the slides and providing better visualization by removing obscure elements such as blood, inflammatory exudate, menstrual debris and mucus18^,19,20.

Many studies have shown that the evolution from conventional to liquid methodology has resulted in a reduction in false-negative cases from 5.6% in CC to 2.2% in CML ²¹.

The purpose of the new technologies for preparing the slide and the liquid preservative media on the market is to maintain stability and intact cell morphology, for excellent analysis and a considerable reduction in the presence of interferents, optimizing the increased ability to detect precancerous lesions when compared to conventional cytology. This dilution of the collected cell brush has allowed other analyses to be expanded from the same collection, bringing a scenario of new opportunities and molecular investigations for the benefit of the patient and her health ^19,20,21,22.

The preservatives in liquid cytology allow the cells collected to remain viable for analysis for a longer period of time, compared to the smear on a conventional cytology slide, which must be stained after being prepared. CML allows other slides to be made to confirm a discordant result or a new slide to be made in the event of pre-analytical errors or problems, such as the material breaking or being lost, without the patient having to be called back for a new collection19^,20,21,22.

With the new SECTICS/MS Ordinance No. 3 of March 7, 2024, published in the Federal Official Gazette, the collection of cytology in liquid media, compared to conventional cytology, will help with HPV genotyping as a primary screening test, as well as enabling the use of reflex cytology, preventing women with positive high-risk HPV, during preventive screening, from having to return to the health service for a new collection for cytological screening purposes16^,23,24,25.

The preservative collectors available and used in molecular assays, before the improvement of liquid-based cytology, were not developed for the purpose of preserving cell morphology; they were designed only for the preservation of genetic material, DNA and/or RNA. These preservatives lysed the cell membranes in order to preserve and conserve only the genetic material at room temperature and later even for longer under ^{refrigeration26}.

With the emergence of new preservatives for CML on the market, which preserve cell morphology and consequently the genetic material, their applicability in molecular assays has proved to be a beneficial and important factor in expanding research into diseases related to pathologies of the lower genital tract. This homologation of specific liquid media for detecting the HPV virus and STIs has made it easier to carry out the primary screening test for CC, as well as the molecular testing of the same cell representativeness from a single collection ^18,19.

With the emergence of different types of preservatives in the field of liquid-based oncology cytology, analytical validations have been necessary and have progressed in laboratories according to their needs and use of kits. The main molecular platforms have carried out validations with CML preservative media, with the aim of developing protocols with the same performance as the usual collectors initially approved. The protocols developed initially prioritized three relevant points to maintain the analytical standards of their technologies: the first was to maintain the initial cell concentration, through centrifugation steps with a previous concentration, since both collectors had different initial volumes, those for molecular assays had 1 ml and those for CML 10 to 20ml. The second step was to break the cell membranes that were preserved in the CML preservatives, so the next step was thermal denaturation and/or pH change. The third step was to identify whether the chemical composition of the preservatives might interfere with molecular reactions, causing reaction inhibitions and potentially leading to unsatisfactory or erroneous results23^,24,25.

The concentration and components present in various collection kits are important factors for the quality of the final product when used for molecular purposes. Methanol-based liquid media have been shown to preserve the morphological structure, as well as allowing the molecular structure to be maintained without reaction interferences. Many CML collection media with formaldehyde in their composition have been shown to significantly interfere with the molecular structure of the cell, thus hindering the efficient use of these collections for molecular investigations. Formalin is widely used as a morphological preservative, but it favors cross-linking of HPV DNA between other targets and nuclear proteins, known as crosslinking, which will result in DNA fragmentation and cause interference in PCR amplification, reducing the sensitivity of the assay and the possibility of false-negative results27^,28.

The Kolplast CellPreserv Liquid Cytology System Liquid Cytology System collector has undergone molecular validation in private and public laboratories, including various Molecular Biology platforms. Its methanol-based composition provides the preservative medium with adequate performance for direct loading for real-time PCR tests, without the need for prior treatments to remove components such as formalin. Its composition has demonstrated, in various studies, optimum analytical performance, without causing interference and/or inhibition of its chemical preservative components in real-time PCR reactions, as well as allowing the material to be collected and transported at room temperature, without the need for prior refrigeration ²⁹.

The liquid medium CellPreserv Kolplast demonstrates high stability, preservation of cell morphology and genetic material in the samples, excellent performance for molecular tests and effectively helps to increase capillarity in the performance of preventive tests, facilitating the management and screening measures for cervical cancer in the population at risk, according to the Ministry of Health Guidelines.

The aim of this study was to evaluate the performance of the liquid medium CellPreserv Kolplast liquid medium for molecular assays to detect the HPV virus.

CASUISTRY AND METHOD

50 specimens of cervical-vaginal brushings were tested, collected from the liquid medium CellPreserv Kolplastat the private laboratory IPOGLab – São Paulo/Brazil. All the samples were tested using molecular techniques considered the gold standard for high-risk oncogenic HPV types.

The molecular methodologies tested were Hybrid Capture 2v2 ³⁰, for the high-risk oncogenic HPV group (types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) (^QIAGEN®) with an analytical sensitivity of 1pg/ml or 5.000 viral copies, and the real-time PCR (polymerase chain reaction) assay using the Cobas 4800 system (^ROCHE®)31 with specific HPV genotyping of high oncogenic risk: HPV 16 and HPV 18 and for the group as a whole: 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, with an analytical sensitivity of 50 viral copies. Both tests were carried out at the IPOGLab laboratory in São Paulo.

The loading protocols in both methodologies were followed as developed for PreservCyt liquid medium (^HOLOGIC®).