## 1 Introduction
Annex 1 of the GMP guidelines which focuses on sterile preparations demands written protocols for hand hygiene procedures designed to minimize transfer of contaminants to clear areas [33]. To standardize these, the World Health Organization (WHO) in 2009 published the _WHO guidelines on hand hygiene in health care_[37]. These guidelines recommend a six-step protocol that was originally developed by the British medical biologist [PERSON] in 1978 [38]. A frequent addition to this WHO protocol is the inclusion of the wrists [39], as depicted in Figure 1. Antiseptic solution 'EPG', containing ethanol, hydrogen peroxide, and glycerin, has been recommended by the WHO to be used for both hand hygiene and antisepsis [40]. Training and qualification for compounding practices require operators to demonstrate proficiency in following both hand washing and hand disinfection protocols. Furthermore, according to the _WHO best practices for injections and related procedures toolkit_, hand hygiene should be performed prior to the onsite preparation of injection materials, that is, before entering a cleanroom [41]. Proficiency is typically assessed through direct observation of the actions performed. However, this method solely examines the execution of the hand rubbing motions, while neglecting to validate the quality of those actions. While the effectiveness of the six-step actions in reducing bacterial load has been demonstrated in a systematic review by [PERSON] et al., differences in contact time, technique execution, and applied volume of soap or disinfectant can lead to variations in outcome [42]. Therefore, expanding hand hygiene training with an objective assessment method focusing on the quality of technique may offer potential benefits. The conventional way to assess the quality of hand hygiene is by microbiological sampling, offering a direct measure of microbial contamination of the hands in the form of colony-forming units (CFUs) on an agar Petri dish. However, this method is time-consuming, as it typically requires incubation periods of 48 h or longer before the result can be read out [43]. Because of this, cleanroom operators are not able to get immediate feedback on their performance, limiting the training potential. Furthermore, the sampling as such will pose challenges. Conventional Petri dishes are too small to capture a hand's entire surface area, resulting in indirect measurements through glove imprints [43; 44] or limited representation of the selected fingers only [38; 45; 46]. Additionally, there is considerable variation in initial contamination levels, making it difficult to determine the actual reduction factor achieved as an estimate of the effectiveness of the procedure applied. An ultraviolet (UV)-eye-based assessment method offers a faster and less labor-intensive alternative to microbiological control, as it can be performed easily and the result can be read out within a few minutes. Moreover, it offers operators immediate feedback on their performance. A UV-dye-based assessment is an indirect fluorescence measurement method based on the principle that if the entire hand surface is subjected to washing and disinfection, it should be free from microorganisms and particles. This concept was tested by [PERSON] et al. and validated against the microbiological assay. A UV-dye-based assessment was demonstrated to yield high sensitivity (95%) and specificity (98%) [47]. However, for implementing this method as a routine qualification procedure for hand hygiene, specific criteria must be investigated to obtain a proof-of-concept. In the GMP guidelines [27; 28; 29], requirements are formulated, but no detailed procedures are described with respect to how to assess them. By having a proof-of-concept, a UV-dye-based assessment may become part of in-house training programs for cleanroom operators. The current pilot study aimed to optimize hand hygiene training by objectively assessing the quality of hand hygiene procedures using a UV-dye-based method in a limited-size group of cleanroom operators.