Generalities
MFT-F were performed in the same radiopharmaceutical preparation laboratory, in sessions consecutive to daily routine, in the radiopharmacy unit of the Nuclear Medicine Department of Montpellier Cancer Institute, France. All commonly used products (syringes, needles, vials …) were sterile and pyrogen-free. Incubation of samples produced during the development and implementation of the MFT-F was carried out in an internally qualified oven monitored by an external temperature sensor and located in a secured facility.
Choice of fluorescein concentration
The fluorescence yield of fluorescein varies according to concentration, causing quenching at too high concentrations. Maximum fluorescence is reached at 0.002% fluorescein in aqueous solution while fluorescence decreases for higher concentrations (Amalric, 1972). In order to verify this parameter for a fluorescein solution in TSB, successive dilutions were prepared in glass test tubes (10%, 5%, 2.5%, 1%, 0.5%, 0.05%, 0.01% and 0.002%) and were visually examined under ultraviolet light. From each of these solutions, a 2 μL drop was collected, placed on a sterile field to simulate a splashed drop, and visually examined under UV light.
Tryptic soy broth-fluorescein (TSB-F) vials preparation
This step was carried out inside a class II laminar airflow hood placed in a class D controlled area. Vial preparation procedure is shown in Fig. 1. First, 100 μL of fluorescein 10% solution (pharmaceutical grade) were aseptically added to a 100 mL sealed sterile TSB flask (VWR). Then, 6 elution vials (TC-ELU-5®, CIS bio international) were filled with TSB-F. Vials 1 and 6 (10 mL TSB-F each) were incubated for aseptic validation of the manipulation and vials 2 to 5 (16 mL TSB-F each) were stored at 4–8 °C not more than 10 days, before use during MFT-F. Vials 7 and 8 (2 mL TSB-F each) and two 3 mL syringes (1 mL TSB-F each) capped with a luer-lock stopping plug were prepared as positive and negative controls. Positive controls were spoiled with a few microliters of contaminated TSB medium. Detailed TSB-F preparation protocol is available in Supplementary data S1.
Media growth promotion test
To confirm the ability of TSB to support bacterial growth in the presence of 0.01% fluorescein, a verification procedure was undertaken following USP 71 (USP, 2018) and European Pharmacopoea recommendations (Council of Europe, 2018, 2018). The test used a lyophilized Bacillus subtilis spizizenii preparation kit (EZ-ACCU SHOT™ ATCC®, Microbiologics Corporation). Inside a class II laminar airflow hood, 3 vials with 2 mL TSB-F were each seeded with 140 μL of a reconstituted 710 CFU/mL B. subtilis suspension. As a positive control, a 2 mL TSB vial without fluorescein was seeded the same way. A 2 mL TSB vial and a 2 mL TSB-F vial were used as negative controls. These 7 units were incubated during 3 days at 32 °C and visually examined.
Kit preparation
To facilitate the start-up of an MFT, the single-use equipment necessary for an operator evaluation was priorly gathered in a hermetic plastic bag. A complete kit contained four 5 mL syringes, six 3 mL syringes, four sterile 15 mL elution vials labeled A to D, six 23G 25 mm needles, four 22G 30 mm needles, five 23G 60 mm needles, five luer-lok shutters with injection site, sterile non-woven swabs and one sterile disposable drape.
Pre- and post-evaluation bacteriological controls
Control of sterility throughout the whole aseptic handling process had to be validated before the introduction of a MFT. MFT were carried out in a shielded laminar flow shielded cell (ELIZA Series, Comecer), operational and certified class A, placed in the radiopharmaceutical preparation laboratory (class D controlled area). This shielded cell was equipped with an internal 257 nm UV lamp. Before and after each MFT session, surface sampling of two critical planar surfaces (near the handle of the airlock trapdoor and preparation area in front of the operator) was operated by a qualified radiopharmacist, using contact plates. Surface sampling of five nonplanar surfaces (seal of the airlock trapdoor, dipper in the dose calibrator, left hand corner of the working area and interdigital spaces of both gloves) was also carried out using sterile swabs for dry collection incubated in tubes with TSB. Fingerprints from fingertips and thumbs of both gloves were performed by pressing on contact plates for 10 s. After each MFT session, passive air sampling inside the shielded cell was performed by positioning settle plates at 3 predetermined areas (preparation area in front of the operator, left and right hand corner of the working area) during 4 h. Bacteriological samples inside the shielded cell are summarized in Fig. 2. Surface sampling in the class D controlled area was undertaken weekly throughout the MFT campaign at five predetermined surfaces (two workbenches, computer keyboard, external trapdoor of the airlock and above a mobile material cabinet). Passive air sampling in the radiopharmaceutical preparation laboratory was performed by positioning settle plates at two predetermined areas (workbenches).
Operator evaluation form
To qualify the operators, a three-part validation form was established, both for initial and periodic evaluation (Fig. 3). First section, inspired by our internal procedures, gathered evaluation criteria following FGMP and USP 797 garbing and hygiene recommendations (EudraLed n.d.; USP< 797> n.d.) through 30 items. Radiation protection recommendations were also evaluated (Elsinga et al. 2010). A negative answer should be considered as an error, a total score > 90% (≤ 3 errors) being required to validate this section. Second part of this form provided for the counting and characterization of fluorescein-contaminated areas or devices, with a negative scoring system to rate operators according to the number and critical nature of contaminations. Final scores equal to or superior than 27/30 (90%) are expected to pass each part of the evaluation. Third part is reserved for the daily monitoring of incubated units. Operator evaluation form is available in Supplementary data S2.
Operator evaluation protocol
Operators were individually trained and evaluated by one and only qualified radiopharmacist, after explaining the test issues and process. Initial practical training was in accordance with FGMP and USP guidelines for sterile preparations in hospital pharmacies. MFT sessions were scheduled after working days, without cleaning the shielded cell preparation area before the test. Two operators were evaluated successively during each session. Operators were first evaluated on garbing and hygiene common practice before entering the preparation laboratory (see Fig. 3). Once in the laboratory, operators opened an evaluation kit and carefully disinfected the package of each medical device and the septum of each vial with 70% isopropyl alcohol before entering the shielded cell. In the same way, non-disposable equipment was cleaned using a detergent-disinfectant solution. This equipment included five vial shields identified “Eluate” and from A to D, four 5 mL syringe shields identified from A to D, six 3 mL syringe shields identified from 1 to 6 and one 30 cm long forceps usually used to safely handle radioactivity-containing vials. A single vial of TSB-F mixture was used during a test.
Before starting the test, absence of fluorescent traces in the working area and on the gloves was checked using the shielded cell UV lamp. Once all the equipment was in the shielded cell, the operator began the experimental filling operations as illustrated in Fig. 4. A first sequential 2-vials preparation simulation was started, with TSB-F transfers from “Eluate” vial to vial A or vial B and from vial A to vial B. For every transfer, syringe or vial activity measurement was simulated by placing it in the well of the dose calibrator. At the end of the transfer sequence, 3 simulated patient doses were prepared. A second identical preparation sequence was repeated with the same “Eluate” vial and 2 new preparation vials. Detailed MFT-F protocol is available in Supplementary data S3.
Incubation and reading of pre- and post-evaluation bacteriological controls results
Contact plates, settle plates and sterile swabs in TSB tubes were placed for incubation 7 days at room temperature (22 °C ± 2 °C) to promote the development of environmental germs. Then, the same units were incubated 7 more days at 32 °C under 5% CO2 to promote the development of bacterial contaminants of human origin. Samples were monitored daily by counting the total number of discrete colonies on each plate and checking the turbidity of TSB tubes. Positive and negative control agar plates and TSB tubes were prepared and incubated simultaneously.
Incubation and reading of operator evaluation results
At the end of a MFT, all the equipment used for the evaluation was taken out of the glove box and was carefully examined in the dark by the radiopharmacist, under UV light. Absence of fluorescent traces in the working area and on the gloves was checked using the shielded cell UV lamp. A smearing was then carried out on all the surfaces previously observed (inside the shielded cell and on equipment) using a compress slightly soaked in NaCl 0.9%. The compress was carefully examined under UV light to uncover fluorescent traces and improve the detection sensitivity for micro-projections. The limit of detection on the compress was previously assessed as ≤1 μL TSB-F. Fluorescein contaminations are reported on the Operator evaluation form (see Fig. 3). Before incubation, internal surfaces of vial A to D, “Eluate vial” and syringes S1 to S6 were brought into contact with TSB-F mixture by inversion. For syringes, the piston seal was positioned on the 2 mL graduation to allow aerobic conditions. Each unit was incubated 7 days at room temperature then 7 more days at 32 °C under 5% CO2. Positive and negative controls were incubated similarly and simultaneously. All units were visually inspected daily by the unaided eye, with gentle mixing.
Validation of the operator evaluation protocol
MFT-F controls were done by a single trained radiopharmacist. A first series of 3 MFT-F following the previously described protocol was achieved using good aseptic technique. Then, a second series of 3 MFT-F was achieved with intentional aseptic mistakes. These included no prior disinfection of surfaces, equipment or gloves, and intentional finger dab on the septum and on the luer-lok stoppers of vials. Incubation and daily observation were conducted similarly to the methods used during operators assessment.
Cost study
The cost of making and applying one MFT-F was estimated, considering the price of the equipment used and the time spent by the radiopharmacist. This estimated cost was then compared to the commercially available MFT kits.