How improvements in monitoring and safety practices lowered airborne formaldehyde concentrations at an Italian university hospital: a summary of 20 years of experience

This study was carried out at the Careggi University Hospital, one of Europe’s largest polyclinics. For over 50 years, the Careggi laboratory of Anatomic Pathology had been located in a four-storey historical building, that is, until 2019, when it moved to a new building under a new name: Pathology Laboratory.

Air indoor and personal FA exposure was investigated at three locations: i) operating theatres, where freshly removed surgical samples are put into containers, ii) the Pathology Lab specimen reception, where all pathology samples are received, identified, and prioritised, and iii) the Pathology Lab gross room, where residents, pathologists, and trained technicians examine and dissect tissue specimens. Indoor exposure was measured in vapour and particulate matter samples taken monthly by positioning measuring instruments on the floor at four points, with an inlet at a height of 1.5 meters (27, 28).

Between 1999 and 2007, the gross room received disposable containers from the operating theatres at two benches with aspiration hoods. Fume hoods were not installed in the operating theatres, where small (≤2 cm) and large biopsies (≥2 cm) were immersed in containers with 4 % formalin from a 40 % FA solution prepared in-house by lab staff.

Between 2008 and 2016, the Pathology Laboratory and the operating theatres had fume hoods with a foot pedal for formalin control, which served for anatomic pathology (Diapath, Martinengo and Aquaria, Lacchiarella, Italy, respectively). Pre-filled formalin containers (Diapath) were also introduced into the process.

Between 2017 and 2019, small biopsies were immersed in pre-filled containers with 4 % FA encapsulated in the lid [Securbiop^® (Traces, Carmagnola, Italy), Zero (Meccanica GM, Loreto, Italy), and BiopSafe (Axlab Innovation, BiopSafe ApS, Vedbæk, Denmark)]. The operating theatres used Tissue-SAFE vacuum sealing (VS) of surgical specimens (Milestone, Sorisole, Italy). Fresh specimens were stored in vacuum-sealed plastic bags and refrigerated at 4° C until transfer to the Pathology Laboratory for fixation under controlled conditions. In the Pathology Laboratory, automated and closed operations ensured that the right amount of formalin was dispensed to obtain standard formalin/specimen ratios. When the lab was relocated to the new building, it got new Trimming Tech 130 fume hoods (Bio-Optica, Milan, Italy) and a computer-based system to monitor and control heating, ventilation, air conditioning, and extraction from fume hoods. All fume hoods were used and maintained in strict accordance with operating and safety technical standards.

Figure 1 shows the timeline of deployment of various devices to monitor FA at workplace. Between 1999 and 2012, the hospital used personal active air samplers with Lp-2,4-dinitrophenylhydrazine (DNPH)-S10 coated cartridges on a silica sorbent (Cat. No. 21024-U, Supelco, Bellefonte, PA, USA) (29) attached to a GilAir-3 pump (Gilian, San Diego, CA, USA) at 500 mL/min (for 8 h) and 1300 mL/min (for 15 min) and an eight-position automatic manifold pump area sampler (SkyPost Gas/Bravo M Plus, TCR Tecora, Cogliate, Italy). Excess DNPH reagent from the LpDNPH-S10 cartridge after elution with 3 mL of acetonitrile was removed with Dowex 5OW-X8 (Cat. No. 217492, Merck, Darmstadt, Germany) resin (30). The resin was then washed three times with water and twice activated with 2 mol/L of sulphuric acid for 5 min, before being washed another three times with water and twice with ethanol. A 2-mL volume of diethyl ether was eluted through a freshly activated cation-exchange cartridge before sample elution. The eluate was then evaporated to dryness and the residue was dissolved in 1 mL of toluene containing the isobutyl chloroformate derivative of di-n-butylamine (Giotto Biotech, Sesto Fiorentino, Italy), as internal standard (IS).

Figure 1

Solid phase microextraction (SPME) was used for on-fibre derivatisation before air sampling (31, 32). A Fast-Fit Assembly (FFA, Chromline, Prato, Italy) with 65-μm polydimethylsiloxane/divinylbenzene SPME fibres (PDMS/DVB, Cat. No. FFA-57293-U, Supelco) was doped for 30 s in the headspace of a 4 mL vial, previously equilibrated by magnetic stirring at room temperature for 20 min with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) purchased from Merck (Cat. No. 76735), Fisher Scientific (Cat. No. NC0674130, Waltham, MA, USA), and Alfa Aesar (Cat. No. ALFAA18368.06, Haverhill, MA, USA), solubilised in 1 mL of water (17 mg/mL). For both personal and area samplings we used rapid-SPME (1 min, at the sampling rate of 18.3±0.8 mL/min) and eight-hour TWA-SPME [at the sampling rate for 3-mm Z distance (distance from the needle opening to the sorbent surface) of 0.03±0.0025 mL/min].

For FA-2,4-dinitrophenyl (DNP)-hydrazone and FA-2,3,4,5,6-pentafluorobenzyl (PFB)-oxime analysis we used two 5 % phenyl-95 % PDMS stationary phase columns of 30 m (Cat. No. CP9013, 0.25 mm × 0.25 μm film thickness, Agilent J&W GC Columns, Santa Clara, CA, USA) and 60 m (Cat. No. CP8949, 0.25x 1.0 film thickness, Agilent J&W GC Columns), respectively, installed in a Varian CP-3800 gas chromatograph (GC) connected to a Varian Saturn 2200 electron ionisation mass spectrometer (MS) with a Varian switching valve (Valco, Vici, Houston, TX, USA). The GC automation of the analytical process was done with a Gerstel Multi Purpose Sampler MPS 2 XL dual head (Gerstel GmbH & Co.KG, Mülheim an der Ruhr, Germany) equipped with an Anatune 300 Automated DNPH unit (Anatune, Cambridge, UK) and a Multi Fiber eXchange (MFX) 25-position tray for FFA-SPME fiber injection (Chromline) (33).

A direct reading instrument, Formaldemeter™ htV-M (PPM Technology, Norfolk, UK), was used. It is an active sampler (a 10-mL snatch-sample of air is taken in by an internal pump) with an electrochemical sensor with a resolution of 10 μg/m³ and a mean response time of 60 s. All devices for area sampling were used on a tripod set at the breathing height.

In 2013–2015, FFA-Sep-Pak XpoSure Sampler Plus Short DNPH-coated cartridges on a silica sorbent (Cat. No. WAT047205, Waters, Milford, MA, USA) with magnetic adaptors were introduced together with GilAir Plus pumps (Sensidyne, St. Petersburg, FL, USA) for personal sampling and SkyPost Gas (TCR Tecora) with the GSM module for area sampling.

FA concentrations higher than 370 μg/m³ measured by Formaldemeter™ htV-M were confirmed with a Chromline automatic fibre sampler with Wi-Fi connection. All devices for area sampling were used on a tripod at the breathing height. For personal SPME air sampling we used a Supelco diffusive sampling fibre holder (Cat. No. 57584-U).

To fully automate gas chromatography, we introduced Flex GC xyz autosampler (EST Analytical, Fairfield, CT, USA) assemblies with: i) Multi Tool Exchanges (MTX) to desorb FA-2,4-DNP-hydrazone from the FFA-Sep-Pak XpoSure Sampler Plus Short DNPH-coated cartridges, remove the excess DNPH derivatizing agent with an FFA-polymeric Oasis Plus mixed-mode cation-exchange sorbent (MCX, Cat. No. 186003516, Waters), dispense the diphenylamine solution as IS, and inject the liquid sample, and ii) an MFX 45-position tray for FFA-SPME fibre injection (Chromline) (25).

In 2016–2018, we used FFA-Sep-Pak XpoSure cartridges for a six-position GasCheck Basic automatic collector box (AMS Analitica, Pesaro, Italy) equipped with a GSM module set to 0.3 and 1.2 L/min flow rate for eight-hour and 15-minute sampling, respectively. The GC analysis of FA-2,4-DNP-hydrazone was performed as described above, save for the following changes: large-volume injection (LVI) used a programmed temperature vaporisation (PTV) injector (34), a 35 % phenyl-65 % PDMS stationary phase column (Cat. No. 122-3832UI, DB-35MS UI, Agilent J&W GC Column,), and a nitrogen-phosphorus thermionic specific detector (TSD). We also introduced a Next Environmental Monitoring (NEMo) XT (Ethera, Crolles, France) passive sampler with a nanoporous FA sensor (Cat. No. NE-FOR01x), which uses a sol-gel process based on colour variation with readings taken with an optical reader every two hours. All devices for area sampling (GasCheck Basic automatic collector box, NEMo XT, Formaldemeter™ htV-M, and FFA-SPME Fiber Automatic Sampler) were placed on a tripod at the breathing height and connected wirelessly to the Chromline FA Data Storing System. Personal air sampling continued to use SPME equipped with a diffusive sampling fibre holder and FFA-Sep-Pak XpoSure Sampler Plus Short DNPH-coated cartridges connected to GilAir Plus pumps.

In 2019, the tripod was replaced with an innovative ergonomic armchair with a headrest housing remotely controlled instruments for continuous measurement: a six-position GasCheck Basic automatic collector box with Sep-Pak XpoSure Sampler Plus Short DNPH-coated cartridges and a new, battery powered, NEMo XT with a larger measuring range (up to 2,450 μg/m³). For short-term sampling we used a Gasera Multipoint Sampler with multichannel monitoring via 12 sample inlets, connected with a new direct reading sampler (Gasera One Formaldehyde, Gasera, Turku, Finland). It combines cantilever enhanced photoacoustic detection technology and a quantum cascade laser source operating at mid-infrared fundamental absorption spectra (detection limit of 1.2 μg/m³, user configurable response time starting from 10 s, and a dynamic range over five orders of magnitude beyond the detection limit) and replaced the Formaldemeter™ htV-M and SPME passive sampling. Personal air sampling still relied on FFA-Sep-Pak XpoSure Sampler Plus Short DNPH-coated cartridges connected to GilAir Plus pumps and on GasCheck and NEMO XT on the ergonomic chair’s headrest. This allowed us to compare the methods.

Calibration was verified with a dynamic calibration system with exposure sensors and then with spectrophotometric analysis using an optical reading module. FA dynamic pressures were calibrated with a Harvard Plus 11 syringe-pump (Harvard Apparatus, Holliston, MA, USA) set to 2 μL/min and connected to an adsorbent tube injector system (Supelco). The active-sampling DNPH cartridge, considered to be the gold standard, and the direct reading Gasera One were tested using the following FA concentrations: 20, 40, 80, 160, and 320 μg/μL. For each FA air concentration, five determinations were performed. FA air concentration (C_{FA air}) was calculated according to the following formula:

where, C_FAair is the concentration of the analyte in the air (μg/L), C_Sol is the concentration of the solution (μg/L), F_syringe is the syringe pump flow (μL/min), and F_air is the air flow (L/min). The concentration of water vapour produced by the impinger was determined by measuring the dew point temperature with a photoacoustic infrared Innova type 1312 Multigas Monitor (LumaSense Technologies, Santa Clara, CA, USA). Atmospheric pressure was determined with a GE Druck DPI 705 digital pressure indicator (General Electric, Boston, MA, USA).

We used the Wilcoxon signed-rank sum test to compare DNPH cartridge active sampling (considered the reference method), DNPH cartridge measurements from the GasCheck Basic automatic collector box set in the back of the armchair, and direct readings taken by the NEMo XT in the headrest. We also compared Gasera One direct readings and DNPH cartridge measurements from the GasCheck Basic automatic collector. All outliers were included in the analysis due to our limited sample size.

To assess calibration, that is to compare the theoretical and measured values, we used simple regression models and evaluated the significance of regression coefficients α and β by testing the hypothesis of perfect calibration (H0: β=1, α=0) using F statistics and coefficient restriction. We also calculated the R-squared value to verify goodness of fit for the estimated regression model for each method.

All statistical analyses were run on Stata Statistical Software, release 11 (StataCorp LP, College Station, TX, USA).

Until 2007, FA concentrations would soar up to 1,286 μg/m³ in short-term exposure measurements during the most critical activities and up to 813 μg/m³ in TWA measurements. The likely reasons were on site preparation of containers filled with a 4 % formalin solution (prepared by dissolving 40 % FA) and the lack of fume cupboards with an exhaust air system.

By 2016, these FA readings dropped thanks to the introduction of pre-loaded containers, but continued to be high, with peaks in the gross room reaching 824 μg/m³ and 399 μg/m³ for short-term and TWA measurements, respectively. One of the concerns was poor sealing of the containers before and after they were opened for the insertion of biopsy. Another was dispersion of formalin fumes during container filling.

Since 2017, readings dropped even further, thanks to the use of the closed-circuit system for pre-loaded containers and vacuum sealing, which proved robust and practical. Moreover, the adoption of regulation UNI/TS 11710:2018 (35) (specifying the acceptance limits and robustness of containment, face velocity, and air exchange efficiency required for fume cupboards, as well as the methods and procedures for testing them) led to a more careful planning and arrangement of fume cupboard ventilation. This drop in airborne FA observed in 2017–2019 can also be attributed to extensive training of the staff (e.g., regarding safety equipment and maintenance, scientific updates, devices for safe handling, use of personal protective equipment, and emergency care) and the development of functional waste-container management and disposal.

The decrease in FA exposure is shown in Table 1. From 1999 to 2019, the gross room saw a 93 % decrease in TWA and short-term exposure (Figure 2). In the specimen reception this decrease was 87 % (Figure 3). In the operating theatres only short-term exposure was measured during immersion of biopsies, and it dropped 87 % (Figure 4).

Figure 2

Figure 3

Figure 4

Having an accurate reading of background pollution is important for correct estimation of FA exposure, so we simultaneously monitored FA concentrations in nearby outdoor sites. Indoor FA concentrations before working hours were higher than outdoor concentrations. From 1999 to 2018, outdoor concentrations dropped from 8.3 to 2.1 μg/m³. The decrease recorded in 2019 (average 0.7±0.3 μg/m³) coincided with the end of two major construction projects in the immediate vicinity – municipal tramway and renovation of the Careggi buildings – both of which involved heavy (diesel-fuelled) vehicle operation and traffic. Moreover, in 2019, the Pathology Laboratory moved to a new building located in a less polluted area on a hill near the hospital campus.

The most important improvements in anatomic pathology laboratory workflow were the introduction of closed-circuit systems for small biopsies and of modified atmosphere packaging (MAP) and under-vacuum storage (UVS) for large ones. Closed-circuit systems prevent contact with FA. We have been using several brands of pre-filled formalin containers with lids as well as a container with a floating shield of fluid to confine the FA vapours. This dramatically reduced the use of formalin in the operating theatres at our hospital and provided safer handling in the specimen reception and gross room. A word of caution, though: we noticed that formalin is agitated during transport and that FA vapours can pass into the container’s headspace. It is therefore necessary to let these containers settle after samples were delivered to the specimen reception.

Another innovation came with containers for large biopsies that employ MAP or UVS systems immediately after tissue insertion. T-Filler (Combifill, Bergamo, Italy) dispenses 4 % FA solution into rigid 600 to 5700 mL containers. Tissue Vacuum Plus and Tissue Filling System (Kaltek) utilise MAP technology and dispense formalin into rigid containers of between 250 and 5,000 mL. The Biopreserve (Patholab, Selargius, Italy) system offers 600 to 5,000 mL containers filled with formalin in UVS. The latter two systems use bags only for transporting fresh biopsy or for storing it after fixation in formalin inside a rigid container. The Careggi hospital opted for SealSafe by Milestone, which uses a non-rigid, double polyamide layer polyethylene bags. As an added bonus, these bags greatly reduced storage room occupied by the specimens.

Transporting formalin, with or without specimen, is clearly a critical phase in the workflow, as it increases the risk of spills and exposure to Immediately Dangerous to Life and Health (IDLH) levels. In fact, the Higher Health Council of Italy (36) and the Italian Group of Mammary Pathology (GIPaM) of the Society of Pathology (SIAPEC) (37) have called for improvements in all phases of biopsy handling, including transportation, in order to prevent harm to employees. We therefore adopted specimen transportation chests, which were well sealed and resistant to shocks and vibration. Moreover, the use of UVS and/or MAP systems restricted the use of FA to dedicated areas in the pathology laboratories, since large boxes of formalin fixative no longer had to be transported throughout the hospital. Last but not least, twenty years of continuous staff training in safe handling and management of formalin also minimised errors in the workflow and accidental formalin spills.

The observed drop in airborne FA at our hospital is owed to new best practices adopted. These were identified and implemented expressly thanks to continuous monitoring of workplace activities.

Since FA occupational exposure limit values (OEL) have yet to be universally accepted, in Italy we recommend the following reference guides, given in order of priority: i) the OELs published in the EU Directives not yet implemented by Italian legislation and ii) the TLVs published by ACGIH.

All measuring procedures and sampling strategies aim to compare occupational exposure measurements against the OELs in order to keep workers safe. Italian laws regulate these procedures and strategies to some degree. EN 689:2018 (38) encourages personal sampling devices to be used within the breathing zone, which corresponds to a 30-cm radius around the face.

Only a few studies have compared FA concentrations in personal and area devices in the healthcare sector. Most involve personal and area monitoring in autopsy rooms, where the operations require continuous movement around the dissection table. These found that personal readings were higher than area readings, as they ranged from 0.04 to 8.91 mg/m³ compared to 0.05 to 3.01 mg/m³ (ratio 1–3) (39, 40, 41). However, in one of the few studies conducted in a hospital pathology lab, Vimercati et al. (4) observed that personal and area concentrations were comparable, with an approximate ratio of 1. Our 2019 data (Table 2) are in line with these results.

In 2019, since detected FA concentration values had plummeted by one order of magnitude, we decided to no longer use the SPME technique for eight-hour TWA. The device’s 4 μg/m³ limit of quantification (LOQ) was not always distinguishable from FA in blank PFBHA samples. Instead, combining FFA-SPME’s one-minute sampling and Formaldemeter’s htV-M IH monitoring enabled us to identify peaks in emissions. Unfortunately, the Formaldemeter htV-M has low specificity in certain conditions, especially in open space settings, as is the case in our new Pathology Lab. In these environments, alcohols and alkanes interfere with device readings. At the same time, possible exposure to FA is no longer limited to gross rooms and vacuum-sealing rooms but includes all of the pathology lab. Luckily, rapid FFA-SPME makes up for Formaldemeter’s shortcomings, even though it requires a large number of fibres for sampling. Gasera One Formaldehyde, with a dynamic range 100,000 times above FA detection limit, is a valid alternative. Besides, it is highly selective against carbonyl compounds and other volatile organic compounds (VOCs), while its response time and detection limits are lower by one order of magnitude compared to Formaldemeter htV-M. The same is true for the NEMo IAQ monitor, which employs nanoporous materials, as it separately measures VOCs, carbon dioxide, humidity, and temperature.

Table 3 shows the results of our tests of theoretical FA atmospheric concentrations and corresponding measurements for DNPH-cartridge monitoring method and Gasera One. Given our p level of significance of 0.05, we can infer that our direct-reading Gasera One complies with “perfect calibration” in the lab settings.

We introduced an innovative ergonomic armchair, with a piezoresistive pressure sensor to detect the presence of the operator, a barcode reader for personnel identification, and a headrest equipped with remotely-managed continuous measuring instruments within the breathing zone. It could replace conventional personal sampling in order to eliminate human error but also to reduce personal monitoring cost. The NEMo XT’s small size (175x95x75 mm), and its low weight (450 grams), allow it to be inserted into the headrest, while the shape of the GasCheck Basic automatic collector box allows it to be applied on the back of the armchair. Thanks to remote control and cloud storage of data, this new chair/workstation allows to monitor various locations simultaneously from a central control location and makes monitoring simpler and cheaper at the same time. In addition, continuous monitoring allows to identify the most error-prone steps in the workflow and to overcome them by training staff better or by introducing technical or system refinements.

Our remotely controlled area monitoring approach combined with the ergonomic armchair with continuous measuring instruments provide a complete picture of workplace exposure, eliminating at the same time human error and reducing personnel costs for sampling. A future improvement to this approach would be to use portable GC instruments to evaluate DNPH cartridge samples in the field. In addition to its many advantages, this would provide immediate, real-time results, allowing us to pinpoint which specific task involves the greatest risk, make and in-depth analysis, and suggest a solution.