Nasal cytology as a screening tool in formaldehyde-exposed workers

Nasal cytology as a screening tool in formaldehyde-exposed workers Abstract Background Workers in pathology and anatomy laboratories may be exposed to formaldehyde. An evaluation of the early effects of this substance is, therefore, paramount. This preliminary study was conducted to evaluate if nasal cytology could be used as a tool to detect changes in nasal mucosa in workers exposed to formaldehyde. Aims To assess whether nasal cytology was able to reveal any alteration of nasal mucosa in workers exposed to formaldehyde compared to unexposed subjects, and to ascertain whether a specific pattern of alterations correlated with years of exposure in order to evaluate long-term occupational exposure effects. Methods The study included a group of workers exposed to formaldehyde and a group of non-exposed workers. All subjects underwent clinical examination, followed by nasal cytology. Pathological indices from each rhinocytograms were compared between the two groups. Results Nasal cytology revealed a chronic inflammatory non-allergic condition in the exposed group. Qualitative analysis of data distribution of neutrophils and mucous-secreting/ciliated cells ratio showed data clustering with a cut-off set at 15 years of exposure. The mean formaldehyde concentrations ranged from <0.04 to 0.15 parts per million (ppm). The maximum levels of formaldehyde concentrations ranged from 0.2 to 0.67 ppm. Conclusions Our data indicate that nasal cytology may be a promising tool for the health surveillance of workers exposed to formaldehyde and may also represent a useful research tool for the study of the health effects of other chemicals irritants for the upper airways. Formaldehyde, nasal cytology, occupational health, pathology laboratories Introduction Formaldehyde is an important precursor of many materials and chemical compounds. Worldwide levels of formaldehyde production are very high and many people are exposed both occupationally and environmentally. Exposure to formaldehyde can occur from many environmental sources such as combustion processes, building materials and tobacco smoke [1], but the highest level of exposure to formaldehyde occurs in occupational settings, for example pathology laboratories [2] where formaldehyde is commonly used in fixation and preservation of the tissues. Occupational exposure for healthcare professionals, medical laboratories technicians, as well as for teachers and students who handle biological specimens preserved with formaldehyde or formalin (aqueous solution of 40% formaldehyde), is mainly by inhalation because of formaldehyde emission from aqueous solution (embalming fluids). It mainly affects the upper airways mucosa causing nasal mucosal irritation with nasal symptoms and even sinonasal cancers [3]. The International Agency for Research on Cancer (IARC) has classified formaldehyde as a ‘known human carcinogen’ [4] especially for nasal sinus cancer and nasopharyngeal cancer [5], and recent studies have also shown a positive correlation between exposure to formaldehyde and the development of leukaemia, particularly myeloid leukaemia [6]. The nasal and oral mucosa cells, exposed through respiration, are the chief targets of formaldehyde-induced genotoxic effects as previously reported in a group of students exposed to formaldehyde in anatomy class [7] and more recently in a study in anatomy and pathology laboratories and formaldehyde-resins productions [8]. In recent years, nasal cytology has been introduced as a valid method in the differential diagnosis of allergic and non-allergic nasal diseases and it covers an important area of research in sinonasal pathologies [9]. In healthy subjects, the nasal mucosa is characterized by a pseudostratified ciliated columnar epithelium classically composed of four cell types (ciliated, goblet cells, striated and basal cells) and rare neutrophils. Both ciliated and mucous-secreting cells represent the first-line defence located in the airways. From a cellular point of view, nasal pathologies first affect the ciliated cells, determining a rearrangement of the epithelium in favour of mucous-secreting cells (muciparous metaplasia). This process has important physiopathological and clinical consequences, because the increase in goblet cells leads to a reduced efficiency of the mucous-ciliated transport [10]. These events favour the stasis of mucous secretions in the nose—a major risk of bacterial infections [11]. As the turnover of a ciliated cell takes about 3 weeks, frequent inflammation does not allow the re-establishment of a normal ratio between the different cellular subsets [12]. Normal mucous-secreting cells/ciliated cells ratio (MUC/CIL ratio which usually is 1 to 4: one mucous-secreting cell and four ciliated cells) and hyperchromatic supranuclear stria are both markers of anatomical and functional integrity of nasal mucosa [13]. On the other hand, the presence of eosinophils, lymphocytes,macrophages, mast cells, multinucleated ciliated cells (CC), neutrophils and karyorrhexis are markers of cellular modifications of the nasal epithelium caused by exposure to either physical or chemical factors [14]. The simplicity of nasal sampling, the scarce invasiveness of the technique, the cost effectiveness and the reproducibility of the rhinological exams make nasal cytology appealing as a screening tool for workers exposed to chemical irritants to the upper respiratory tract [15]. In this study, we, therefore, assessed whether nasal cytology could be used to identify any alteration of nasal mucosa in workers exposed to formaldehyde. A further aim was to ascertain whether a specific pattern of alterations correlated with years of exposure in order to evaluate long-term occupational exposure effects. Methods The department of otorhinolaryngology, the occupational health department and the department of microbiology of Policlinico Tor Vergata, in Rome (Italy), conducted the study. In 2016, during the annual health surveillance programme (a mandatory task established by the Italian legislation for the healthcare of workers) we recruited, on a voluntary basis, two groups of workers from the same hospital. The first group consisted of laboratory technicians and postgraduate medical students and instructors working in a pathology laboratory, exposed to formaldehyde for at least 1 year. The control group consisted of administrative employees working in the same hospital, but not in the pathology laboratory. For both exposed and control groups, the exclusion criteria were: (i) a positive medical history for any type of rhinopathy, (ii) a positive smoking history, (iii) a positive history of diagnosed allergy and (iv) any currently reported treatment with topical or systemic corticosteroids or antihistamines. All eligible workers provided consent to participate in the research and the study protocol was considered ethically acceptable by the independent ethical committee of the Policlinico Tor Vergata Foundation. An accurate medical history was taken and a predetermined questionnaire was administered to workers to ascertain information about any nasal symptoms, allergy history, smoking habits and any other relevant medical history. Information collected also included years of exposure to formaldehyde, and the use of specific personal protective equipment in the exposed group. An otorhinolaryngological examination was performed by a specialist otolaryngologist, followed by nasal sampling. Nasal cytology for the exposed group of workers was conducted after at least 12 h from the last exposure to formaldehyde, in the morning, just before they started working. The collection technique was performed with a Rhino-Probe® nasal curette by scraping, via anterior rhinoscopy, the middle portion of the inferior turbinate, where there is an optimal ratio between ciliated over mucous-secreting cells and bacteria are absent. This procedure is minimally invasive and does not require local anaesthesia. The sampling was placed on a glass slide, fixed by air drying and then stained using the May-Grünwald-Giemsa method [15]. The slide was then observed through a light microscopy supplied with an object-glass able to magnify up to 100× under oil immersion. Lung function tests and blood tests are included in the standard protocol of the surveillance health programme of workers exposed to chemical agents (in Italy), so these were also included in the study. Spirometric measurements were performed according to American Thoracic Society (ATS)/European Respiratory Society (ERS) guidelines [16] with the same Spirometer (Vmax Spectra 22 Pulmonary Function Analysis Instrument manufactured by SensorMedics). A 12-h fasting venous blood sample was drawn in the morning period, following standardized procedures for samples collection and processing. These tests were performed by an occupational health physician. The pathology laboratory where the sampling took place is 8 m long, 6 m wide and 3 m high. Up to 100 samples were usually processed daily. The laboratory is equipped with combined general and fume hood exhaust system. The general ventilation system consists of two circular anemostats for primary air intake and two rectangular ceiling extractors. Local exhaust consists of three hoods (one Grosslab Senior and two Grosslab Junior) that use a recirculating exhaust system to filter (using formaldehyde neutralizing filters) and recirculate the air. The level of exposure to formaldehyde was evaluated three times during the year: in January, May and September 2016 at the pathology laboratory centre (close to the working area). The air sampling measurements were carried out 1 day per month, during the entire time of a working session (4–6 h), with a Portable Ambient Analyzer (Photoacoustic Field Gas-Monitor— INNOVA 1412) that uses a measurement system based on the photoacoustic infrared detection method. The analytical limit of the instrument for the formaldehyde detection is 0.04 ppm. The percentages of time of exposure to different levels of formaldehyde as presented in Table 3 are the proportion of 4–6 h of measurements. Descriptive statistics, means, medians and SDs were calculated to describe central tendencies in each group. Consequently non-parametric tests of significance (Mann–Whitney U-test for ordinal variables and the binomial test for dichotomous variables) were used to evaluate the difference in the rhinocytogram between the two groups. SPSS ver. 17.0 (SPSS Inc., Chicago, IL, USA) was used to elaborate the data. The means of percentage of the cells (neutrophils, eosinophils, lymphocytes, macrophages and multinucleated CC), as well as the percentage of karyorrhexis, hyperchromatic supranuclear stria and of the mucous-secreting/ciliated cells ratio from each rhinocytogram, were compared between the two groups. Results Among 24 formaldehyde-exposed workers considered for the study, two were excluded because they were exposed for <1 year, two because of a positive history of allergy and five because of a positive smoking history. Among the 14 non-exposed workers, two were excluded because of a positive smoking history, one for a positive history of diagnosed allergy and one for a positive history of chronic rhinitis. The study, therefore, included 15 formaldehyde-exposed workers and 10 non-formaldehyde-exposed controls. Table 1 shows the main characteristics of the subjects included in the study as well as the findings of the comparison of rhinocytograms between the two groups. Table 1. Main characteristics of the subjects and comparison of results from the nasal cytology analysis Formaldehyde exposure  Non-exposed workers, n = 10  Exposed workers, n = 15  P  Sex  Male = 4  Male = 5    Female = 6  Female = 10    (Years)   Mean age ± SD  43.9 ± 11.8  44.7 ± 10.6  NS   Median (min–max)  41 (31–62)  41 (33–64)  (Years of exposure)   Mean ± SD  0 ± 0  16.3 ± 9.7     Median (min–max)  0 (0–0)  15 (3–35)    Neutrophils (%)   Mean ± SD  3.2 ± 1.0  34.0 ± 16.3  <0.001   Median (min–max)  3 (2–5)  30 (15–70)  Eosinophils (%)   Mean ± SD  0 ± 0  5 ± 8  NS   Median (min–max)  0 (0–0)  0 (0–25)  Lymphocytes (%)   Mean ± SD  0 ± 0  4.7 ± 4.3  <0.01   Median (min–max)  0 (0–0)  5 (0–15)  Macrophages (%)   Mean ± SD  0 ± 0  1.2 ± 2.2  NS   Median (min–max)  0 (0–0)  0 (0–6)  Ratio of mucous-secreting cells and ciliated cells   Mean ± SD  0.27 ± 0.02  1.1 ± 0.9  <0.001   Median (min–max)  0 (0.25–0.31)  1(0.29–2.80)  Multinucleated ciliated cells (%)  0  33  <0.001  Karyorrhexis (%)  0  60  <0.001  Hyperchromatic SNS (%)  80  0  <0.001  Formaldehyde exposure  Non-exposed workers, n = 10  Exposed workers, n = 15  P  Sex  Male = 4  Male = 5    Female = 6  Female = 10    (Years)   Mean age ± SD  43.9 ± 11.8  44.7 ± 10.6  NS   Median (min–max)  41 (31–62)  41 (33–64)  (Years of exposure)   Mean ± SD  0 ± 0  16.3 ± 9.7     Median (min–max)  0 (0–0)  15 (3–35)    Neutrophils (%)   Mean ± SD  3.2 ± 1.0  34.0 ± 16.3  <0.001   Median (min–max)  3 (2–5)  30 (15–70)  Eosinophils (%)   Mean ± SD  0 ± 0  5 ± 8  NS   Median (min–max)  0 (0–0)  0 (0–25)  Lymphocytes (%)   Mean ± SD  0 ± 0  4.7 ± 4.3  <0.01   Median (min–max)  0 (0–0)  5 (0–15)  Macrophages (%)   Mean ± SD  0 ± 0  1.2 ± 2.2  NS   Median (min–max)  0 (0–0)  0 (0–6)  Ratio of mucous-secreting cells and ciliated cells   Mean ± SD  0.27 ± 0.02  1.1 ± 0.9  <0.001   Median (min–max)  0 (0.25–0.31)  1(0.29–2.80)  Multinucleated ciliated cells (%)  0  33  <0.001  Karyorrhexis (%)  0  60  <0.001  Hyperchromatic SNS (%)  80  0  <0.001  NS, non-significant; SNS, supranuclear stria. View Large Table 1. Main characteristics of the subjects and comparison of results from the nasal cytology analysis Formaldehyde exposure  Non-exposed workers, n = 10  Exposed workers, n = 15  P  Sex  Male = 4  Male = 5    Female = 6  Female = 10    (Years)   Mean age ± SD  43.9 ± 11.8  44.7 ± 10.6  NS   Median (min–max)  41 (31–62)  41 (33–64)  (Years of exposure)   Mean ± SD  0 ± 0  16.3 ± 9.7     Median (min–max)  0 (0–0)  15 (3–35)    Neutrophils (%)   Mean ± SD  3.2 ± 1.0  34.0 ± 16.3  <0.001   Median (min–max)  3 (2–5)  30 (15–70)  Eosinophils (%)   Mean ± SD  0 ± 0  5 ± 8  NS   Median (min–max)  0 (0–0)  0 (0–25)  Lymphocytes (%)   Mean ± SD  0 ± 0  4.7 ± 4.3  <0.01   Median (min–max)  0 (0–0)  5 (0–15)  Macrophages (%)   Mean ± SD  0 ± 0  1.2 ± 2.2  NS   Median (min–max)  0 (0–0)  0 (0–6)  Ratio of mucous-secreting cells and ciliated cells   Mean ± SD  0.27 ± 0.02  1.1 ± 0.9  <0.001   Median (min–max)  0 (0.25–0.31)  1(0.29–2.80)  Multinucleated ciliated cells (%)  0  33  <0.001  Karyorrhexis (%)  0  60  <0.001  Hyperchromatic SNS (%)  80  0  <0.001  Formaldehyde exposure  Non-exposed workers, n = 10  Exposed workers, n = 15  P  Sex  Male = 4  Male = 5    Female = 6  Female = 10    (Years)   Mean age ± SD  43.9 ± 11.8  44.7 ± 10.6  NS   Median (min–max)  41 (31–62)  41 (33–64)  (Years of exposure)   Mean ± SD  0 ± 0  16.3 ± 9.7     Median (min–max)  0 (0–0)  15 (3–35)    Neutrophils (%)   Mean ± SD  3.2 ± 1.0  34.0 ± 16.3  <0.001   Median (min–max)  3 (2–5)  30 (15–70)  Eosinophils (%)   Mean ± SD  0 ± 0  5 ± 8  NS   Median (min–max)  0 (0–0)  0 (0–25)  Lymphocytes (%)   Mean ± SD  0 ± 0  4.7 ± 4.3  <0.01   Median (min–max)  0 (0–0)  5 (0–15)  Macrophages (%)   Mean ± SD  0 ± 0  1.2 ± 2.2  NS   Median (min–max)  0 (0–0)  0 (0–6)  Ratio of mucous-secreting cells and ciliated cells   Mean ± SD  0.27 ± 0.02  1.1 ± 0.9  <0.001   Median (min–max)  0 (0.25–0.31)  1(0.29–2.80)  Multinucleated ciliated cells (%)  0  33  <0.001  Karyorrhexis (%)  0  60  <0.001  Hyperchromatic SNS (%)  80  0  <0.001  NS, non-significant; SNS, supranuclear stria. View Large The nasal cell examinations showed significant alterations and differences in the exposed group of workers compared to the non-exposed one (Figures 1 and 2). In particular, the largest differences between the two groups were found between neutrophils count, in the exposed group (median: 30; range: 15–70) and control group (median: 3, range: 2–5), and between the mucous-secreting and ciliated cells ratio in the exposed group (median: 1; range: 0.29–2.80) and in the control group (median: 0; range: 0.25–0.31). These results show a damaged nasal mucosa characterized by a significant presence of an inflammatory condition and an ongoing muciparous metaplasia due to an important alteration of muciparous/ciliated cells ratio of subjects exposed to formaldehyde >15 years. So we can deduce that the muciparous metaplasia is highly correlated with the years of exposure to formaldehyde (Figure 2A). Figure 1. View largeDownload slide Normal rhinocytogram: in healthy subjects, the nasal mucosa is composed of numerous ciliated cells with supranuclear stria. May-Grünwald-Giemsa Staining, magnification 1000x. Figure 1. View largeDownload slide Normal rhinocytogram: in healthy subjects, the nasal mucosa is composed of numerous ciliated cells with supranuclear stria. May-Grünwald-Giemsa Staining, magnification 1000x. Figure 2. View largeDownload slide (A) Muciparous metaplasia; (B) minimal persistent flogosis, neutrophil (N) and eosinophil (E), 400×; (C) karyorrhexis (K) of nucleus of ciliated cells; (D) polynucleated cells (P). Staining MGG. (A, C, D) 1000×. Figure 2A and B belong to the same exposed subject, whereas Figure 2B and D were obtained by a second exposed worker. The vast majority of the subjects exposed >15 years presented the concomitant presence of the changes shown in the figure. Figure 2. View largeDownload slide (A) Muciparous metaplasia; (B) minimal persistent flogosis, neutrophil (N) and eosinophil (E), 400×; (C) karyorrhexis (K) of nucleus of ciliated cells; (D) polynucleated cells (P). Staining MGG. (A, C, D) 1000×. Figure 2A and B belong to the same exposed subject, whereas Figure 2B and D were obtained by a second exposed worker. The vast majority of the subjects exposed >15 years presented the concomitant presence of the changes shown in the figure. As expected, since allergic subjects were excluded from the study, no mast cells were found on the rhinocytograms of the two group of workers. In the exposed group of subjects, among the explored variables, data distribution of the percentage of neutrophils and MUC/CIL ratio with years of exposure showed data clustering with a cut-off set at 15 years of exposure (Figure S1, available as Supplementary data at Occupational Medicine Online). Therefore, to better evaluate the presence of long-term effects of formaldehyde exposure, the exposed subjects were grouped as <15 years (six workers) and ≥15 years (nine workers) of exposure. The group with ≥15 years of exposure showed a significant increase of neutrophils (median: 40; range: 15–70, P < 0.05), eosinophils (median: 10, range: 0–25, P < 0.05), multinucleated CC (median: 1; range: 0–1, P < 0.05) and MUC/CIL ratio (median: 2; range: 0.31–2.80, P < 0.01), as compared to the group with <15 years of exposure (23, 15–25; 0, 0–0; 0, 0–0 and 0, 0.29–0.45, respectively). We also noticed that all the exposed workers were affected by the so-called minimal persistent inflammation (MPI), which is a condition classically present in positive mite allergy subjects [17]. MPI is characterized by a persistent infiltration of neutrophils and few eosinophils in the nasal mucosa (Figure 2B) and it was detected in five out of the 15 exposed workers (33%). In particular, all of the exposed workers affected by this MPI were exposed to formaldehyde for >15 years. None of the subjects complained of nasal congestion, noteworthy watering of the eyes or sneezing. All of the subjects exposed to formaldehyde [15] complained of a general discomfort of the olfactory sensory system. Ten (67%) of the exposed workers (six of them exposed from >15 years) complained of dryness of the nose. Four (27%) exposed workers (two of them exposed from >15 years) complained of nose and eyes irritation. All subject reported a clear correlation of the symptoms with their work-shift. No one complained about lower respiratory symptoms. No alterations in lung function or blood tests were detected in exposed subjects. In particular, white blood count as well as other markers of inflammation evaluated (C-reactive protein and erythrocyte sedimentation rate level) were within the normal range. All the exposed workers reported using the recommended respiratory protective equipment correctly, for a short period of time and not on a daily basis, complaining of discomfort on wearing them. The ppm values of formaldehyde concentrations (mean ± SD and median with min–max values) in the laboratory are shown in Table 2. Table 3 shows the percentages of the time of exposure to formaldehyde levels under the detectable limits, into the detectable range and over the threshold limit value (TLV) ceiling according to the American Conference of Governmental Industrial Hygienists (ACGIH). Table 2. Formaldehyde levels (mean ± SD and median with min–max values between parentheses) in air sampling measurements in the pathology laboratory as measured on January, May and September 2016   Formaldehyde concentrations (parts per million)  January 2016   Mean ± SD  0.15 ± 0.21   Median (min–max)  <0.04 (<0.04–0.67)  May 2016   Mean ± SD  <0.04 ± 0.003   Median (min–max)  <0.04 (<0.04–0.23)  September 2016   Mean ± SD  <0.04 ± 0.003   Median (min–max)  <0.04 (<0.04–0.20)    Formaldehyde concentrations (parts per million)  January 2016   Mean ± SD  0.15 ± 0.21   Median (min–max)  <0.04 (<0.04–0.67)  May 2016   Mean ± SD  <0.04 ± 0.003   Median (min–max)  <0.04 (<0.04–0.23)  September 2016   Mean ± SD  <0.04 ± 0.003   Median (min–max)  <0.04 (<0.04–0.20)  View Large Table 2. Formaldehyde levels (mean ± SD and median with min–max values between parentheses) in air sampling measurements in the pathology laboratory as measured on January, May and September 2016   Formaldehyde concentrations (parts per million)  January 2016   Mean ± SD  0.15 ± 0.21   Median (min–max)  <0.04 (<0.04–0.67)  May 2016   Mean ± SD  <0.04 ± 0.003   Median (min–max)  <0.04 (<0.04–0.23)  September 2016   Mean ± SD  <0.04 ± 0.003   Median (min–max)  <0.04 (<0.04–0.20)    Formaldehyde concentrations (parts per million)  January 2016   Mean ± SD  0.15 ± 0.21   Median (min–max)  <0.04 (<0.04–0.67)  May 2016   Mean ± SD  <0.04 ± 0.003   Median (min–max)  <0.04 (<0.04–0.23)  September 2016   Mean ± SD  <0.04 ± 0.003   Median (min–max)  <0.04 (<0.04–0.20)  View Large Table 3. Percentages of time of exposure to different levels of formaldehyde (under the detectable limit (<0.04 ppm)), between the detectable limit and the TLV ceiling (ACGIH) (0.04–0.3 ppm) and over the TLV ceiling as measured during the months of January, May and September 2016 ppm  January  May  September  <0.04  61%  86%  88%  0.04–0.1  13%  14%  12%  0.04–0.1  4%  5%  4%  0.01–0.02  5%  5%  4%  0.01–0.02  4%  4%  4%  >0.3  26%  0  0  ppm  January  May  September  <0.04  61%  86%  88%  0.04–0.1  13%  14%  12%  0.04–0.1  4%  5%  4%  0.01–0.02  5%  5%  4%  0.01–0.02  4%  4%  4%  >0.3  26%  0  0  Numbers and percentages in italics represent a sub fraction of the values included between the detectable limit and the TLV ceiling (0.04–0.3). View Large Table 3. Percentages of time of exposure to different levels of formaldehyde (under the detectable limit (<0.04 ppm)), between the detectable limit and the TLV ceiling (ACGIH) (0.04–0.3 ppm) and over the TLV ceiling as measured during the months of January, May and September 2016 ppm  January  May  September  <0.04  61%  86%  88%  0.04–0.1  13%  14%  12%  0.04–0.1  4%  5%  4%  0.01–0.02  5%  5%  4%  0.01–0.02  4%  4%  4%  >0.3  26%  0  0  ppm  January  May  September  <0.04  61%  86%  88%  0.04–0.1  13%  14%  12%  0.04–0.1  4%  5%  4%  0.01–0.02  5%  5%  4%  0.01–0.02  4%  4%  4%  >0.3  26%  0  0  Numbers and percentages in italics represent a sub fraction of the values included between the detectable limit and the TLV ceiling (0.04–0.3). View Large Discussion The principal finding of this study was that nasal cytology in the exposed group showed significant alterations compared with the control group. The inflammatory changes and the muciparous metaplasia are characteristically observed in nasal mucosa injuries. Of note, they would had been undetected if nasal cytology was not performed, as in traditional health surveillance. The importance of this result is supported by the fact that no changes were found in traditional health assessments (lung function tests and blood exams), performed in the context of the health surveillance programme. This suggests that the nasal cytology test is able to detect early adverse health effects due to formaldehyde that are missed by currently used surveillance programmes. Nasal cytology has recently proven to be useful in revealing chronic inflammatory rhinitis in a group of woodworkers compared with a group of unexposed subjects [18] and it has been used as a screening test for the detection of symptomless patients, particularly in the search of precancerous lesions [19]. Other authors found genotoxic damage in anatomy laboratory workers exposed to formaldehyde, emphasizing the need to develop safety programmes for this group of professional workers [20]. The influence of environmental exposure to formaldehyde has been studied in the nasal mucosa of medical students during cadaveric dissection immediately, before and 6 months after the completion of the course through an olfactory test and the evaluation of nasal mucosal sensitivity to histamine. The authors found temporary abnormalities in the olfactory test. An increased nasal mucosal sensitivity to histamine was only found in subjects with pre-existing allergic rhinitis, and after environmental exposure at high concentrations of formaldehyde [21]. As shown in previous studies, indoor air analyses have constantly shown that the levels of airborne formaldehyde in anatomy laboratories exceed recommended exposure criteria (ranging from 0.30 to 2 ppm) [22]. In our study, the results of measurements showed room averages of formaldehyde concentrations comparable or even lower than most previously reported data. They were, however, higher than the guideline limit of 0.3 short-term exposure TLV set by the ACGIH during one of the three monitoring sessions [23]. Moreover, all the workers who participated to the study stated that during work, they rarely used personal protective equipment for respiratory and eye protection, although they regularly used nitrile gloves for hand protection. We also found that levels of formaldehyde in May and September were substantially lower than in January. Practical advice given by the hygienist about methods of containment and clean up, after reviewing the formaldehyde values of the first monitoring in January, probably contributed to the drop observed in May and September. The health surveillance of workers exposed to formaldehyde in Italy is currently based on a clinical and physical examination, lung function tests and blood tests. In our study, we did not find any significant change in lung function tests or in white cell or other markers of inflammation. This is consistent with what is found in the literature. Most studies in formaldehyde-exposed workers have shown no effects or small non-significant decrease in some of the lung function parameters during the work-shift [24–27]. Similarly blood test results have not identified any major differences with only one report showing small differences in the mean and distributions of values of the white blood cells, lymphocytes, monocytes and eosinophils, in male but not in female subjects exposed to formaldehyde, compared with non-exposed workers [28]. This is the first preliminary study conducted to investigate the cytological characteristics and/or modifications of nasal mucosa in workers exposed to formaldehyde. Moreover, nasal cytology is a reliable, non-invasive, cheap and easy to perform diagnostic tool. Limited numbers of samples and no sequential subjects limited our study, but we found significant data that show the importance of nasal cytology applicability in early diagnosis of nasal mucosa changes in workers exposed to formaldehyde. On the other hand it needs the involvement of a specialist to perform the procedure. In our study, we also found a specific symptomless state of allergic disease characterized by a possible transformation in chronic and persistent inflammation after prolonged exposure to allergenic stimulus (the so-called MPI); so we may conclude that formaldehyde could play a similar role as an allergic agent. In conclusion, further research is required, but our findings suggest that the introduction of this simple method could prove to be useful for the diagnosis, and consequently the prevention, of early changes of nasal mucosa in the workers exposed to formaldehyde. Therefore, we suggest that this test should be routinely used as part of the health surveillance programme for these workers, especially in those exposed for >15 years. Key points Nasal cytology revealed very early inflammatory changes of the nasal mucosa in workers exposed to formaldehyde. Occupational physicians could adopt nasal cytology as a screening tool in workers exposed to formaldehyde. Nasal cytology could represent a useful tool for the study of health effects of several other chemical irritants on the upper airways. Competing interest None declared. References 1. Kim KH, Jahan SA, Lee JT. Exposure to formaldehyde and its potential human health hazards. 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Gelardi M, Luigi Marseglia G, Licari A, et al.   Nasal cytology in children: recent advances. Ital J Pediatr  2012; 38: 51. Google Scholar CrossRef Search ADS PubMed  11. Gelardi M, Fiorella ML, Leo G, Incorvaia C. Cytology in the diagnosis of rhinosinusitis. Pediatr Allergy Immunol  2007; 18 ( Suppl. 18): 50– 52. Google Scholar CrossRef Search ADS PubMed  12. Chapelin C, Coste A, Gilain L, Poron F, Verra F, Escudier E. Modified epithelial cell distribution in chronic airways inflammation. Eur Respir J  1996; 9: 2474– 2478. Google Scholar CrossRef Search ADS PubMed  13. Gelardi M, Cassano P, Cassano M, Fiorella ML. Nasal cytology: description of a hyperchromatic supranuclear stria as a possible marker for the anatomical and functional integrity of the ciliated cell. Am J Rhinol  2003; 17: 263– 268. Google Scholar PubMed  14. Boysen M, Zadig E, Digernes V, Abeler V, Reith A. Nasal mucosa in workers exposed to formaldehyde: a pilot study. Br J Indust Med  1990; 47: 116– 121. 15. Gelardi M, Fiorella ML, Russo C, Fiorella R, Ciprandi G. Role of nasal cytology. Int J Immunopathol Pharmacol  2010; 23: 45– 49. Google Scholar PubMed  16. Miller MR, Hankinson J, Brusasco V, et al.  ; ATS/ERS Task Force. Standardisation of spirometry. Eur Respir J  2005; 26: 319– 338. Google Scholar CrossRef Search ADS PubMed  17. Ciprandi G, Buscaglia S, Pesce G, et al.   Minimal persistent inflammation is present at mucosal level in patients with asymptomatic rhinitis and mite allergy. J Allergy Clin Immunol  1995; 96: 971– 979. Google Scholar CrossRef Search ADS PubMed  18. Lovato A, Staffieri C, Ottaviano G, et al.   Woodworkers and the inflammatory effects of softwood/hardwood dust: evidence from nasal cytology. Eur Arch Otorhinolaryngol  2016; 273: 3195– 3200. Google Scholar CrossRef Search ADS PubMed  19. Maru YK, Munjal S, Gupta Y. Brush cytology and its comparison with histopathological examination in cases of diseases of the nose. J Laryngol Otol  1999; 113: 983– 987. Google Scholar CrossRef Search ADS PubMed  20. Costa S, Coelho P, Costa C, et al.   Genotoxic damage in pathology anatomy laboratory workers exposed to formaldehyde. Toxicology  2008; 252: 40– 48. Google Scholar CrossRef Search ADS PubMed  21. Hisamitsu M, Okamoto Y, Chazono H, et al.   The influence of environmental exposure to formaldehyde in nasal mucosa of medical students during cadaver dissection. Allergol Int  2011; 60: 373– 379. Google Scholar CrossRef Search ADS PubMed  22. Costa S, Carvalho S, Costa C, et al.   Increased levels of chromosomal aberrations and DNA damage in a group of workers exposed to formaldehyde. Mutagenesis  2015; 30: 463– 473. Google Scholar CrossRef Search ADS PubMed  23. Documentation of the threshold limit values and biological exposure indices. 7th edn. Cincinnati, Ohio, 2001. www.acgih.org (20 November 2017, date last accessed). 24. Holness DL, Nethercott JR. Health status of funeral service workers exposed to formaldehyde. 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Published by Oxford University Press on behalf of the Society of Occupational Medicine. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Occupational Medicine Oxford University Press

Nasal cytology as a screening tool in formaldehyde-exposed workers

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Abstract

Abstract Background Workers in pathology and anatomy laboratories may be exposed to formaldehyde. An evaluation of the early effects of this substance is, therefore, paramount. This preliminary study was conducted to evaluate if nasal cytology could be used as a tool to detect changes in nasal mucosa in workers exposed to formaldehyde. Aims To assess whether nasal cytology was able to reveal any alteration of nasal mucosa in workers exposed to formaldehyde compared to unexposed subjects, and to ascertain whether a specific pattern of alterations correlated with years of exposure in order to evaluate long-term occupational exposure effects. Methods The study included a group of workers exposed to formaldehyde and a group of non-exposed workers. All subjects underwent clinical examination, followed by nasal cytology. Pathological indices from each rhinocytograms were compared between the two groups. Results Nasal cytology revealed a chronic inflammatory non-allergic condition in the exposed group. Qualitative analysis of data distribution of neutrophils and mucous-secreting/ciliated cells ratio showed data clustering with a cut-off set at 15 years of exposure. The mean formaldehyde concentrations ranged from <0.04 to 0.15 parts per million (ppm). The maximum levels of formaldehyde concentrations ranged from 0.2 to 0.67 ppm. Conclusions Our data indicate that nasal cytology may be a promising tool for the health surveillance of workers exposed to formaldehyde and may also represent a useful research tool for the study of the health effects of other chemicals irritants for the upper airways. Formaldehyde, nasal cytology, occupational health, pathology laboratories Introduction Formaldehyde is an important precursor of many materials and chemical compounds. Worldwide levels of formaldehyde production are very high and many people are exposed both occupationally and environmentally. Exposure to formaldehyde can occur from many environmental sources such as combustion processes, building materials and tobacco smoke [1], but the highest level of exposure to formaldehyde occurs in occupational settings, for example pathology laboratories [2] where formaldehyde is commonly used in fixation and preservation of the tissues. Occupational exposure for healthcare professionals, medical laboratories technicians, as well as for teachers and students who handle biological specimens preserved with formaldehyde or formalin (aqueous solution of 40% formaldehyde), is mainly by inhalation because of formaldehyde emission from aqueous solution (embalming fluids). It mainly affects the upper airways mucosa causing nasal mucosal irritation with nasal symptoms and even sinonasal cancers [3]. The International Agency for Research on Cancer (IARC) has classified formaldehyde as a ‘known human carcinogen’ [4] especially for nasal sinus cancer and nasopharyngeal cancer [5], and recent studies have also shown a positive correlation between exposure to formaldehyde and the development of leukaemia, particularly myeloid leukaemia [6]. The nasal and oral mucosa cells, exposed through respiration, are the chief targets of formaldehyde-induced genotoxic effects as previously reported in a group of students exposed to formaldehyde in anatomy class [7] and more recently in a study in anatomy and pathology laboratories and formaldehyde-resins productions [8]. In recent years, nasal cytology has been introduced as a valid method in the differential diagnosis of allergic and non-allergic nasal diseases and it covers an important area of research in sinonasal pathologies [9]. In healthy subjects, the nasal mucosa is characterized by a pseudostratified ciliated columnar epithelium classically composed of four cell types (ciliated, goblet cells, striated and basal cells) and rare neutrophils. Both ciliated and mucous-secreting cells represent the first-line defence located in the airways. From a cellular point of view, nasal pathologies first affect the ciliated cells, determining a rearrangement of the epithelium in favour of mucous-secreting cells (muciparous metaplasia). This process has important physiopathological and clinical consequences, because the increase in goblet cells leads to a reduced efficiency of the mucous-ciliated transport [10]. These events favour the stasis of mucous secretions in the nose—a major risk of bacterial infections [11]. As the turnover of a ciliated cell takes about 3 weeks, frequent inflammation does not allow the re-establishment of a normal ratio between the different cellular subsets [12]. Normal mucous-secreting cells/ciliated cells ratio (MUC/CIL ratio which usually is 1 to 4: one mucous-secreting cell and four ciliated cells) and hyperchromatic supranuclear stria are both markers of anatomical and functional integrity of nasal mucosa [13]. On the other hand, the presence of eosinophils, lymphocytes,macrophages, mast cells, multinucleated ciliated cells (CC), neutrophils and karyorrhexis are markers of cellular modifications of the nasal epithelium caused by exposure to either physical or chemical factors [14]. The simplicity of nasal sampling, the scarce invasiveness of the technique, the cost effectiveness and the reproducibility of the rhinological exams make nasal cytology appealing as a screening tool for workers exposed to chemical irritants to the upper respiratory tract [15]. In this study, we, therefore, assessed whether nasal cytology could be used to identify any alteration of nasal mucosa in workers exposed to formaldehyde. A further aim was to ascertain whether a specific pattern of alterations correlated with years of exposure in order to evaluate long-term occupational exposure effects. Methods The department of otorhinolaryngology, the occupational health department and the department of microbiology of Policlinico Tor Vergata, in Rome (Italy), conducted the study. In 2016, during the annual health surveillance programme (a mandatory task established by the Italian legislation for the healthcare of workers) we recruited, on a voluntary basis, two groups of workers from the same hospital. The first group consisted of laboratory technicians and postgraduate medical students and instructors working in a pathology laboratory, exposed to formaldehyde for at least 1 year. The control group consisted of administrative employees working in the same hospital, but not in the pathology laboratory. For both exposed and control groups, the exclusion criteria were: (i) a positive medical history for any type of rhinopathy, (ii) a positive smoking history, (iii) a positive history of diagnosed allergy and (iv) any currently reported treatment with topical or systemic corticosteroids or antihistamines. All eligible workers provided consent to participate in the research and the study protocol was considered ethically acceptable by the independent ethical committee of the Policlinico Tor Vergata Foundation. An accurate medical history was taken and a predetermined questionnaire was administered to workers to ascertain information about any nasal symptoms, allergy history, smoking habits and any other relevant medical history. Information collected also included years of exposure to formaldehyde, and the use of specific personal protective equipment in the exposed group. An otorhinolaryngological examination was performed by a specialist otolaryngologist, followed by nasal sampling. Nasal cytology for the exposed group of workers was conducted after at least 12 h from the last exposure to formaldehyde, in the morning, just before they started working. The collection technique was performed with a Rhino-Probe® nasal curette by scraping, via anterior rhinoscopy, the middle portion of the inferior turbinate, where there is an optimal ratio between ciliated over mucous-secreting cells and bacteria are absent. This procedure is minimally invasive and does not require local anaesthesia. The sampling was placed on a glass slide, fixed by air drying and then stained using the May-Grünwald-Giemsa method [15]. The slide was then observed through a light microscopy supplied with an object-glass able to magnify up to 100× under oil immersion. Lung function tests and blood tests are included in the standard protocol of the surveillance health programme of workers exposed to chemical agents (in Italy), so these were also included in the study. Spirometric measurements were performed according to American Thoracic Society (ATS)/European Respiratory Society (ERS) guidelines [16] with the same Spirometer (Vmax Spectra 22 Pulmonary Function Analysis Instrument manufactured by SensorMedics). A 12-h fasting venous blood sample was drawn in the morning period, following standardized procedures for samples collection and processing. These tests were performed by an occupational health physician. The pathology laboratory where the sampling took place is 8 m long, 6 m wide and 3 m high. Up to 100 samples were usually processed daily. The laboratory is equipped with combined general and fume hood exhaust system. The general ventilation system consists of two circular anemostats for primary air intake and two rectangular ceiling extractors. Local exhaust consists of three hoods (one Grosslab Senior and two Grosslab Junior) that use a recirculating exhaust system to filter (using formaldehyde neutralizing filters) and recirculate the air. The level of exposure to formaldehyde was evaluated three times during the year: in January, May and September 2016 at the pathology laboratory centre (close to the working area). The air sampling measurements were carried out 1 day per month, during the entire time of a working session (4–6 h), with a Portable Ambient Analyzer (Photoacoustic Field Gas-Monitor— INNOVA 1412) that uses a measurement system based on the photoacoustic infrared detection method. The analytical limit of the instrument for the formaldehyde detection is 0.04 ppm. The percentages of time of exposure to different levels of formaldehyde as presented in Table 3 are the proportion of 4–6 h of measurements. Descriptive statistics, means, medians and SDs were calculated to describe central tendencies in each group. Consequently non-parametric tests of significance (Mann–Whitney U-test for ordinal variables and the binomial test for dichotomous variables) were used to evaluate the difference in the rhinocytogram between the two groups. SPSS ver. 17.0 (SPSS Inc., Chicago, IL, USA) was used to elaborate the data. The means of percentage of the cells (neutrophils, eosinophils, lymphocytes, macrophages and multinucleated CC), as well as the percentage of karyorrhexis, hyperchromatic supranuclear stria and of the mucous-secreting/ciliated cells ratio from each rhinocytogram, were compared between the two groups. Results Among 24 formaldehyde-exposed workers considered for the study, two were excluded because they were exposed for <1 year, two because of a positive history of allergy and five because of a positive smoking history. Among the 14 non-exposed workers, two were excluded because of a positive smoking history, one for a positive history of diagnosed allergy and one for a positive history of chronic rhinitis. The study, therefore, included 15 formaldehyde-exposed workers and 10 non-formaldehyde-exposed controls. Table 1 shows the main characteristics of the subjects included in the study as well as the findings of the comparison of rhinocytograms between the two groups. Table 1. Main characteristics of the subjects and comparison of results from the nasal cytology analysis Formaldehyde exposure  Non-exposed workers, n = 10  Exposed workers, n = 15  P  Sex  Male = 4  Male = 5    Female = 6  Female = 10    (Years)   Mean age ± SD  43.9 ± 11.8  44.7 ± 10.6  NS   Median (min–max)  41 (31–62)  41 (33–64)  (Years of exposure)   Mean ± SD  0 ± 0  16.3 ± 9.7     Median (min–max)  0 (0–0)  15 (3–35)    Neutrophils (%)   Mean ± SD  3.2 ± 1.0  34.0 ± 16.3  <0.001   Median (min–max)  3 (2–5)  30 (15–70)  Eosinophils (%)   Mean ± SD  0 ± 0  5 ± 8  NS   Median (min–max)  0 (0–0)  0 (0–25)  Lymphocytes (%)   Mean ± SD  0 ± 0  4.7 ± 4.3  <0.01   Median (min–max)  0 (0–0)  5 (0–15)  Macrophages (%)   Mean ± SD  0 ± 0  1.2 ± 2.2  NS   Median (min–max)  0 (0–0)  0 (0–6)  Ratio of mucous-secreting cells and ciliated cells   Mean ± SD  0.27 ± 0.02  1.1 ± 0.9  <0.001   Median (min–max)  0 (0.25–0.31)  1(0.29–2.80)  Multinucleated ciliated cells (%)  0  33  <0.001  Karyorrhexis (%)  0  60  <0.001  Hyperchromatic SNS (%)  80  0  <0.001  Formaldehyde exposure  Non-exposed workers, n = 10  Exposed workers, n = 15  P  Sex  Male = 4  Male = 5    Female = 6  Female = 10    (Years)   Mean age ± SD  43.9 ± 11.8  44.7 ± 10.6  NS   Median (min–max)  41 (31–62)  41 (33–64)  (Years of exposure)   Mean ± SD  0 ± 0  16.3 ± 9.7     Median (min–max)  0 (0–0)  15 (3–35)    Neutrophils (%)   Mean ± SD  3.2 ± 1.0  34.0 ± 16.3  <0.001   Median (min–max)  3 (2–5)  30 (15–70)  Eosinophils (%)   Mean ± SD  0 ± 0  5 ± 8  NS   Median (min–max)  0 (0–0)  0 (0–25)  Lymphocytes (%)   Mean ± SD  0 ± 0  4.7 ± 4.3  <0.01   Median (min–max)  0 (0–0)  5 (0–15)  Macrophages (%)   Mean ± SD  0 ± 0  1.2 ± 2.2  NS   Median (min–max)  0 (0–0)  0 (0–6)  Ratio of mucous-secreting cells and ciliated cells   Mean ± SD  0.27 ± 0.02  1.1 ± 0.9  <0.001   Median (min–max)  0 (0.25–0.31)  1(0.29–2.80)  Multinucleated ciliated cells (%)  0  33  <0.001  Karyorrhexis (%)  0  60  <0.001  Hyperchromatic SNS (%)  80  0  <0.001  NS, non-significant; SNS, supranuclear stria. View Large Table 1. Main characteristics of the subjects and comparison of results from the nasal cytology analysis Formaldehyde exposure  Non-exposed workers, n = 10  Exposed workers, n = 15  P  Sex  Male = 4  Male = 5    Female = 6  Female = 10    (Years)   Mean age ± SD  43.9 ± 11.8  44.7 ± 10.6  NS   Median (min–max)  41 (31–62)  41 (33–64)  (Years of exposure)   Mean ± SD  0 ± 0  16.3 ± 9.7     Median (min–max)  0 (0–0)  15 (3–35)    Neutrophils (%)   Mean ± SD  3.2 ± 1.0  34.0 ± 16.3  <0.001   Median (min–max)  3 (2–5)  30 (15–70)  Eosinophils (%)   Mean ± SD  0 ± 0  5 ± 8  NS   Median (min–max)  0 (0–0)  0 (0–25)  Lymphocytes (%)   Mean ± SD  0 ± 0  4.7 ± 4.3  <0.01   Median (min–max)  0 (0–0)  5 (0–15)  Macrophages (%)   Mean ± SD  0 ± 0  1.2 ± 2.2  NS   Median (min–max)  0 (0–0)  0 (0–6)  Ratio of mucous-secreting cells and ciliated cells   Mean ± SD  0.27 ± 0.02  1.1 ± 0.9  <0.001   Median (min–max)  0 (0.25–0.31)  1(0.29–2.80)  Multinucleated ciliated cells (%)  0  33  <0.001  Karyorrhexis (%)  0  60  <0.001  Hyperchromatic SNS (%)  80  0  <0.001  Formaldehyde exposure  Non-exposed workers, n = 10  Exposed workers, n = 15  P  Sex  Male = 4  Male = 5    Female = 6  Female = 10    (Years)   Mean age ± SD  43.9 ± 11.8  44.7 ± 10.6  NS   Median (min–max)  41 (31–62)  41 (33–64)  (Years of exposure)   Mean ± SD  0 ± 0  16.3 ± 9.7     Median (min–max)  0 (0–0)  15 (3–35)    Neutrophils (%)   Mean ± SD  3.2 ± 1.0  34.0 ± 16.3  <0.001   Median (min–max)  3 (2–5)  30 (15–70)  Eosinophils (%)   Mean ± SD  0 ± 0  5 ± 8  NS   Median (min–max)  0 (0–0)  0 (0–25)  Lymphocytes (%)   Mean ± SD  0 ± 0  4.7 ± 4.3  <0.01   Median (min–max)  0 (0–0)  5 (0–15)  Macrophages (%)   Mean ± SD  0 ± 0  1.2 ± 2.2  NS   Median (min–max)  0 (0–0)  0 (0–6)  Ratio of mucous-secreting cells and ciliated cells   Mean ± SD  0.27 ± 0.02  1.1 ± 0.9  <0.001   Median (min–max)  0 (0.25–0.31)  1(0.29–2.80)  Multinucleated ciliated cells (%)  0  33  <0.001  Karyorrhexis (%)  0  60  <0.001  Hyperchromatic SNS (%)  80  0  <0.001  NS, non-significant; SNS, supranuclear stria. View Large The nasal cell examinations showed significant alterations and differences in the exposed group of workers compared to the non-exposed one (Figures 1 and 2). In particular, the largest differences between the two groups were found between neutrophils count, in the exposed group (median: 30; range: 15–70) and control group (median: 3, range: 2–5), and between the mucous-secreting and ciliated cells ratio in the exposed group (median: 1; range: 0.29–2.80) and in the control group (median: 0; range: 0.25–0.31). These results show a damaged nasal mucosa characterized by a significant presence of an inflammatory condition and an ongoing muciparous metaplasia due to an important alteration of muciparous/ciliated cells ratio of subjects exposed to formaldehyde >15 years. So we can deduce that the muciparous metaplasia is highly correlated with the years of exposure to formaldehyde (Figure 2A). Figure 1. View largeDownload slide Normal rhinocytogram: in healthy subjects, the nasal mucosa is composed of numerous ciliated cells with supranuclear stria. May-Grünwald-Giemsa Staining, magnification 1000x. Figure 1. View largeDownload slide Normal rhinocytogram: in healthy subjects, the nasal mucosa is composed of numerous ciliated cells with supranuclear stria. May-Grünwald-Giemsa Staining, magnification 1000x. Figure 2. View largeDownload slide (A) Muciparous metaplasia; (B) minimal persistent flogosis, neutrophil (N) and eosinophil (E), 400×; (C) karyorrhexis (K) of nucleus of ciliated cells; (D) polynucleated cells (P). Staining MGG. (A, C, D) 1000×. Figure 2A and B belong to the same exposed subject, whereas Figure 2B and D were obtained by a second exposed worker. The vast majority of the subjects exposed >15 years presented the concomitant presence of the changes shown in the figure. Figure 2. View largeDownload slide (A) Muciparous metaplasia; (B) minimal persistent flogosis, neutrophil (N) and eosinophil (E), 400×; (C) karyorrhexis (K) of nucleus of ciliated cells; (D) polynucleated cells (P). Staining MGG. (A, C, D) 1000×. Figure 2A and B belong to the same exposed subject, whereas Figure 2B and D were obtained by a second exposed worker. The vast majority of the subjects exposed >15 years presented the concomitant presence of the changes shown in the figure. As expected, since allergic subjects were excluded from the study, no mast cells were found on the rhinocytograms of the two group of workers. In the exposed group of subjects, among the explored variables, data distribution of the percentage of neutrophils and MUC/CIL ratio with years of exposure showed data clustering with a cut-off set at 15 years of exposure (Figure S1, available as Supplementary data at Occupational Medicine Online). Therefore, to better evaluate the presence of long-term effects of formaldehyde exposure, the exposed subjects were grouped as <15 years (six workers) and ≥15 years (nine workers) of exposure. The group with ≥15 years of exposure showed a significant increase of neutrophils (median: 40; range: 15–70, P < 0.05), eosinophils (median: 10, range: 0–25, P < 0.05), multinucleated CC (median: 1; range: 0–1, P < 0.05) and MUC/CIL ratio (median: 2; range: 0.31–2.80, P < 0.01), as compared to the group with <15 years of exposure (23, 15–25; 0, 0–0; 0, 0–0 and 0, 0.29–0.45, respectively). We also noticed that all the exposed workers were affected by the so-called minimal persistent inflammation (MPI), which is a condition classically present in positive mite allergy subjects [17]. MPI is characterized by a persistent infiltration of neutrophils and few eosinophils in the nasal mucosa (Figure 2B) and it was detected in five out of the 15 exposed workers (33%). In particular, all of the exposed workers affected by this MPI were exposed to formaldehyde for >15 years. None of the subjects complained of nasal congestion, noteworthy watering of the eyes or sneezing. All of the subjects exposed to formaldehyde [15] complained of a general discomfort of the olfactory sensory system. Ten (67%) of the exposed workers (six of them exposed from >15 years) complained of dryness of the nose. Four (27%) exposed workers (two of them exposed from >15 years) complained of nose and eyes irritation. All subject reported a clear correlation of the symptoms with their work-shift. No one complained about lower respiratory symptoms. No alterations in lung function or blood tests were detected in exposed subjects. In particular, white blood count as well as other markers of inflammation evaluated (C-reactive protein and erythrocyte sedimentation rate level) were within the normal range. All the exposed workers reported using the recommended respiratory protective equipment correctly, for a short period of time and not on a daily basis, complaining of discomfort on wearing them. The ppm values of formaldehyde concentrations (mean ± SD and median with min–max values) in the laboratory are shown in Table 2. Table 3 shows the percentages of the time of exposure to formaldehyde levels under the detectable limits, into the detectable range and over the threshold limit value (TLV) ceiling according to the American Conference of Governmental Industrial Hygienists (ACGIH). Table 2. Formaldehyde levels (mean ± SD and median with min–max values between parentheses) in air sampling measurements in the pathology laboratory as measured on January, May and September 2016   Formaldehyde concentrations (parts per million)  January 2016   Mean ± SD  0.15 ± 0.21   Median (min–max)  <0.04 (<0.04–0.67)  May 2016   Mean ± SD  <0.04 ± 0.003   Median (min–max)  <0.04 (<0.04–0.23)  September 2016   Mean ± SD  <0.04 ± 0.003   Median (min–max)  <0.04 (<0.04–0.20)    Formaldehyde concentrations (parts per million)  January 2016   Mean ± SD  0.15 ± 0.21   Median (min–max)  <0.04 (<0.04–0.67)  May 2016   Mean ± SD  <0.04 ± 0.003   Median (min–max)  <0.04 (<0.04–0.23)  September 2016   Mean ± SD  <0.04 ± 0.003   Median (min–max)  <0.04 (<0.04–0.20)  View Large Table 2. Formaldehyde levels (mean ± SD and median with min–max values between parentheses) in air sampling measurements in the pathology laboratory as measured on January, May and September 2016   Formaldehyde concentrations (parts per million)  January 2016   Mean ± SD  0.15 ± 0.21   Median (min–max)  <0.04 (<0.04–0.67)  May 2016   Mean ± SD  <0.04 ± 0.003   Median (min–max)  <0.04 (<0.04–0.23)  September 2016   Mean ± SD  <0.04 ± 0.003   Median (min–max)  <0.04 (<0.04–0.20)    Formaldehyde concentrations (parts per million)  January 2016   Mean ± SD  0.15 ± 0.21   Median (min–max)  <0.04 (<0.04–0.67)  May 2016   Mean ± SD  <0.04 ± 0.003   Median (min–max)  <0.04 (<0.04–0.23)  September 2016   Mean ± SD  <0.04 ± 0.003   Median (min–max)  <0.04 (<0.04–0.20)  View Large Table 3. Percentages of time of exposure to different levels of formaldehyde (under the detectable limit (<0.04 ppm)), between the detectable limit and the TLV ceiling (ACGIH) (0.04–0.3 ppm) and over the TLV ceiling as measured during the months of January, May and September 2016 ppm  January  May  September  <0.04  61%  86%  88%  0.04–0.1  13%  14%  12%  0.04–0.1  4%  5%  4%  0.01–0.02  5%  5%  4%  0.01–0.02  4%  4%  4%  >0.3  26%  0  0  ppm  January  May  September  <0.04  61%  86%  88%  0.04–0.1  13%  14%  12%  0.04–0.1  4%  5%  4%  0.01–0.02  5%  5%  4%  0.01–0.02  4%  4%  4%  >0.3  26%  0  0  Numbers and percentages in italics represent a sub fraction of the values included between the detectable limit and the TLV ceiling (0.04–0.3). View Large Table 3. Percentages of time of exposure to different levels of formaldehyde (under the detectable limit (<0.04 ppm)), between the detectable limit and the TLV ceiling (ACGIH) (0.04–0.3 ppm) and over the TLV ceiling as measured during the months of January, May and September 2016 ppm  January  May  September  <0.04  61%  86%  88%  0.04–0.1  13%  14%  12%  0.04–0.1  4%  5%  4%  0.01–0.02  5%  5%  4%  0.01–0.02  4%  4%  4%  >0.3  26%  0  0  ppm  January  May  September  <0.04  61%  86%  88%  0.04–0.1  13%  14%  12%  0.04–0.1  4%  5%  4%  0.01–0.02  5%  5%  4%  0.01–0.02  4%  4%  4%  >0.3  26%  0  0  Numbers and percentages in italics represent a sub fraction of the values included between the detectable limit and the TLV ceiling (0.04–0.3). View Large Discussion The principal finding of this study was that nasal cytology in the exposed group showed significant alterations compared with the control group. The inflammatory changes and the muciparous metaplasia are characteristically observed in nasal mucosa injuries. Of note, they would had been undetected if nasal cytology was not performed, as in traditional health surveillance. The importance of this result is supported by the fact that no changes were found in traditional health assessments (lung function tests and blood exams), performed in the context of the health surveillance programme. This suggests that the nasal cytology test is able to detect early adverse health effects due to formaldehyde that are missed by currently used surveillance programmes. Nasal cytology has recently proven to be useful in revealing chronic inflammatory rhinitis in a group of woodworkers compared with a group of unexposed subjects [18] and it has been used as a screening test for the detection of symptomless patients, particularly in the search of precancerous lesions [19]. Other authors found genotoxic damage in anatomy laboratory workers exposed to formaldehyde, emphasizing the need to develop safety programmes for this group of professional workers [20]. The influence of environmental exposure to formaldehyde has been studied in the nasal mucosa of medical students during cadaveric dissection immediately, before and 6 months after the completion of the course through an olfactory test and the evaluation of nasal mucosal sensitivity to histamine. The authors found temporary abnormalities in the olfactory test. An increased nasal mucosal sensitivity to histamine was only found in subjects with pre-existing allergic rhinitis, and after environmental exposure at high concentrations of formaldehyde [21]. As shown in previous studies, indoor air analyses have constantly shown that the levels of airborne formaldehyde in anatomy laboratories exceed recommended exposure criteria (ranging from 0.30 to 2 ppm) [22]. In our study, the results of measurements showed room averages of formaldehyde concentrations comparable or even lower than most previously reported data. They were, however, higher than the guideline limit of 0.3 short-term exposure TLV set by the ACGIH during one of the three monitoring sessions [23]. Moreover, all the workers who participated to the study stated that during work, they rarely used personal protective equipment for respiratory and eye protection, although they regularly used nitrile gloves for hand protection. We also found that levels of formaldehyde in May and September were substantially lower than in January. Practical advice given by the hygienist about methods of containment and clean up, after reviewing the formaldehyde values of the first monitoring in January, probably contributed to the drop observed in May and September. The health surveillance of workers exposed to formaldehyde in Italy is currently based on a clinical and physical examination, lung function tests and blood tests. In our study, we did not find any significant change in lung function tests or in white cell or other markers of inflammation. This is consistent with what is found in the literature. Most studies in formaldehyde-exposed workers have shown no effects or small non-significant decrease in some of the lung function parameters during the work-shift [24–27]. Similarly blood test results have not identified any major differences with only one report showing small differences in the mean and distributions of values of the white blood cells, lymphocytes, monocytes and eosinophils, in male but not in female subjects exposed to formaldehyde, compared with non-exposed workers [28]. This is the first preliminary study conducted to investigate the cytological characteristics and/or modifications of nasal mucosa in workers exposed to formaldehyde. Moreover, nasal cytology is a reliable, non-invasive, cheap and easy to perform diagnostic tool. Limited numbers of samples and no sequential subjects limited our study, but we found significant data that show the importance of nasal cytology applicability in early diagnosis of nasal mucosa changes in workers exposed to formaldehyde. On the other hand it needs the involvement of a specialist to perform the procedure. In our study, we also found a specific symptomless state of allergic disease characterized by a possible transformation in chronic and persistent inflammation after prolonged exposure to allergenic stimulus (the so-called MPI); so we may conclude that formaldehyde could play a similar role as an allergic agent. In conclusion, further research is required, but our findings suggest that the introduction of this simple method could prove to be useful for the diagnosis, and consequently the prevention, of early changes of nasal mucosa in the workers exposed to formaldehyde. Therefore, we suggest that this test should be routinely used as part of the health surveillance programme for these workers, especially in those exposed for >15 years. Key points Nasal cytology revealed very early inflammatory changes of the nasal mucosa in workers exposed to formaldehyde. Occupational physicians could adopt nasal cytology as a screening tool in workers exposed to formaldehyde. Nasal cytology could represent a useful tool for the study of health effects of several other chemical irritants on the upper airways. Competing interest None declared. References 1. Kim KH, Jahan SA, Lee JT. Exposure to formaldehyde and its potential human health hazards. 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Gelardi M, Luigi Marseglia G, Licari A, et al.   Nasal cytology in children: recent advances. Ital J Pediatr  2012; 38: 51. Google Scholar CrossRef Search ADS PubMed  11. Gelardi M, Fiorella ML, Leo G, Incorvaia C. Cytology in the diagnosis of rhinosinusitis. Pediatr Allergy Immunol  2007; 18 ( Suppl. 18): 50– 52. Google Scholar CrossRef Search ADS PubMed  12. Chapelin C, Coste A, Gilain L, Poron F, Verra F, Escudier E. Modified epithelial cell distribution in chronic airways inflammation. Eur Respir J  1996; 9: 2474– 2478. Google Scholar CrossRef Search ADS PubMed  13. Gelardi M, Cassano P, Cassano M, Fiorella ML. Nasal cytology: description of a hyperchromatic supranuclear stria as a possible marker for the anatomical and functional integrity of the ciliated cell. Am J Rhinol  2003; 17: 263– 268. Google Scholar PubMed  14. Boysen M, Zadig E, Digernes V, Abeler V, Reith A. Nasal mucosa in workers exposed to formaldehyde: a pilot study. Br J Indust Med  1990; 47: 116– 121. 15. 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Published by Oxford University Press on behalf of the Society of Occupational Medicine. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Occupational MedicineOxford University Press

Published: Apr 14, 2018

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