Xylene (C ₆H ₄(CH ₃) ₂) is a polycyclic aromatic hydrocarbon (PAHs) part of the aromatic BTXs (benzene, toluene, and xylenes) obtained from petroleum, from the dry distillation of wood, and from coke gases. Xylene can have one of the three isomers of dimethylbenzene or a combination thereof, which are used as solvents (in fuel formulations, and glue in plastic model kits etc.). ¹ In medical practice, xylene is used in cellular clarification in the final phase of tissue processing. ^{2 , 3}

Since George Papanicolaou’s research began in 1914 at Weill Cornell University, these PAHs have been used as part of the Papanicolaou (Pap) stain to clear cells at the end (clearing) of the process. ⁴ The preliminary Pap protocol as well as the modifications that he made in 1953 and 1959 indicates the use of three xylene baths (~350 ml each) during the process. ⁵ These protocols, and several modifications ^{6 , 7} suggest the use of one or three xylene baths, causing high exposure to workers (as several hospitals do not have adequate protection barriers), bioaccumulation, and environmental pollution (due to the incorrect handling of these wastes). ⁸

Even though there are techniques for the biodegradation of xylene such as the use of iron and manganese from underground aquifers, the biofiltration of xylene by microorganisms adhered to a Nylon support, ^{9 , 10} and modifications of the Pap stain that avoid the use of xylene (and other pollutants), ^{8 , 11 , 12} laboratories in many countries continue using xylene as a cellular clarifier in two or three baths during the process. According to the standardized operating procedures of each cytology laboratory, these baths are replaced every thousand slides or once a week. ¹²

In this study, we aimed to estimate the impact of xylene during the Pap test in terms of number of liters and costs for two baths of xylene, and also estimated the impact with three baths for the next five years (2020–2025) following the 2018 Peruvian Social Security cytology guideline.

We included 131,456 cytology tests in the HNGAI, 254,106 in the HNERM, 110,858 in the HNASS, and 98,478 in the HNEG. The use of xylene during for the HNGAI, HNERM, HNASS and HNEG was 2,369, 2,299, 1,637, and 1,179 liters, respectively. Overall, a total of 7,484 liters of xylene were used for Pap stains in four EsSalud hospitals during a five-year period in Lima, Peru ( Table 1). The mean rate of xylene use was 473.8 ± 88.1 liters (95%CI 396.5 to 551.1) for HNGAI, 459.8 ± 63.5 liters (95%CI 404.1 to 515.5) for HNERM, 327.4 ± 136.3 liters (95%CI 207.9 to 446.9) for the HNASS, and 235.8 ± 111.9 liters (95% CI 137.7 to 333.9) for the HNEG ( Figure 3). Using this amount of xylene in four hospitals resulted in a cost of USD 60,853 (202,068 soles).

When estimating future costs for the five-year period from 2020 to 2025, the biggest assumption is that the use of three xylene baths in four hospitals is estimated at 9,483 liters and 77,110 USD (256040 soles). The lowest assumption for the use of three xylene baths in the next five years is estimated to be 5,485 liters and 44,590 USD (148,080 soles) (p = 0.0025) ( Table 2). For HNGAI, HNERM, HNASS and HNEG, the maximum volume of the three xylene baths used is 561.9, 523.3, 463.7 and 347.7 liters, respectively. For HNGAI, HNERM, HNASS and HNEG, the minimum volume of the three xylene baths used is 385.7, 396.3, 191.1, 123.9 liters, respectively.

These estimates of future costs showed increases and decreases according to the hospitals ( Figure 4). In the four hospitals, the liter and cost of xylene increased by 30.4% and 30.3% USD on average, respectively. The average reduction in the cost and liters of xylene was 25.3% liters and 24.3% USD, respectively. A difference was determined between the increases in xylene use among EsSalud hospitals (p = 0.0001).

We determined that xylene had a high impact during the Pap test with two baths in four EsSalud hospitals, and a dramatic increase in costs and xylene contamination in the next five years (2020–2025) with the use of three xylene baths on the Pap smear according to the recommendations of the 2018 EsSalud cytology guidelines.

In the environment, xylene can cause air pollution due to the production of toxic gases (when it is thermally decomposed), polluting water, soil, and subsoil. ¹⁶ Inhalation of xylene can irritate the mucous membranes of the nose and throat, and high concentrations can cause nausea, vomiting, headache, respiratory failure, cough, heart abnormalities, proteinuria, and hematuria. ¹⁷ Cancer, leukemia, brain tumors and tissue changes (pulmonary long-term exposure and long-term high concentration) are also related to xylene contamination. ^{18 , 19} Other effects include fetal kidney disease, infertility and miscarriage in children whose mothers have been in contact with xylene for a long time. ²⁰

Our estimates of xylene use in hospitals indicate that 594,898 cervical smears analyzed by Pap staining use >7,000 liters of xylene every five years. The same estimation of the use and contamination by xylene can be applied in other realities such as the 243,374 smears of the Hospital Nacional Docente Madre Niño San Bartolomé in Lima during two periods [118,016 for the conventional Pap staining (2011–2013 years) and 125,358 for the prolonged Pap staining (2013–2014 years)], ^{11 , 21} to the 3,276,045 cervical smears of the Instituto Mexicano del Seguro Social in Mexico, ²² and in the 60 million cervical smears performed annually in the United States. ²³ We can infer that in every country, cervical cancer screening will involve a large amount of xylene, which will threaten the health of laboratory workers and global environmental health.

Implementing a third xylene bath to the Pap test would undoubtedly have even more counterproductive results. As our findings indicate, there will be an increase in the overall costs of the tests (77,110 USD), the time to stain each sample, and the daily exposure of workers to higher amounts of xylene (9,483 liters). A previous study demonstrated that 37 national guidelines on prevention measures for the use of xylene in South American cytology laboratories were of little importance. ²⁴ In Peru, xylene is usually stored after use until disposal. It is not reused due to the regulation of controlled reagents, therefore it bioaccumulates until its elimination as a whole. However, there is no global regulation that controls all health centers, so many hospitals eliminate xylene through the drain as well as other hospitals that massively bioaccumulate xylene until they are finally disposed of without environmental protection due to the inefficient waste management system. In this sense, under the actual conditions of cytological screening in low-and-middle-income countries, adding an additional xylene bath to the Pap stain is unsustainable and therefore should not be considered.

Currently, there are xylene substitutes that allow diagnostic activities to be carried out in the pathology laboratory in a less toxic and eco-friendly environment. These substitutes are mainly the solution of Neo-Clear (Merck, Darmstadt, Germany), Pathoclear (Biopack, Buenos Aires, Argentina), Master clear (American MasterTech Scientific, CA, USA), UltraClear™ (Avantor’s JT Baker, Deventer, Netherlands), Shandon™ (Thermo Scientific, Walthman, MA, USA), and Ottix Plus (DiapathS.pA, Martinengo, Italy), which have shown different degrees of performance and health risks. ^{25 – 27} Regardless of the opportunities these surrogates offer, they come with costs for Pap processing that many cytology labs cannot afford. In view of this, Eco-Pap (patent application pending) brings an alternative to ecological staining, which can reduce the number of reagents, staining time and cost without reducing test performance. ^{8 , 12}

On the other hand, our estimated analysis model for three xylene baths shows that the cost of the Pap stain and the liters of xylene solvent are reduced. However, this assumption could not be further from reality, given that exfoliative cytology has a new stage. ²⁸ This fact is partly due to the co-testing in which the Human Papilomavirus test and cytology are used as a strategy with better performance and diagnostic accuracy. ²⁹ It is also because many countries, mainly low- and middle-income countries, have strategies based on exfoliative cytology due to the benefits that this technique offers and for the economic limitations that these countries face. ^{30 , 31}

Another component that distances this assumption of reducing quantities and costs of xylene from reality is undoubtedly the alarming increase in the incidence and mortality from cervical cancer between 2012 and 2018 according to the International Agency for Research on Cancer (IARC) from 527,624 and 569,847 cases for the former, and from 265,672 to 311,365 cases for the second. ³²

In this sense, the use of cytology as a cervical cancer strategy will continue to develop and grow around the plausibility of other techniques such as molecular biology. Therefore, if a xylene bath is added to the Pap test, the amount and cost of xylene in Peru’s EsSalud Hospitals will increase by an average of 16.6% in the next five years.

This study had limitations. 1) The analysis was conducted in four EsSalud hospitals in Lima, however, there are other hospitals that have not been included. Further studies that include other Social Security hospitals are needed; 2) This is the first study in Peru that evaluates the cost of using xylene in cervical cytology. However, due to restricted access and the limited availability of data from each hospital, the cost found has not included the time of use, the need for skilled labor, process automation, xylene bioaccumulation, waste management, and the use of xylene substitutes. These characteristics should be included in future studies; and 3) The analysis evaluated the xylene used in the Pap tests; however, it is necessary to evaluate its impact on histopathology.

We demonstrated a high economic impact of xylene during the Pap test with two xylene baths in four EsSalud hospitals, in Lima, Peru. Our three-bath estimates for 2020–2025 indicate that the economic growth of xylene use is significant, which is why the 2018 EsSalud cytology guidelines are not applicable.

Nowadays, there are some alternative methods that can make Pap smears free of xylene during the clearing process, thereby becoming ecofriendly, which is beneficial to occupational health and global environmental health.