F Ameloot

H Mezzine

G Khairallah

G Hayek

M Zaidi

L Lhuillier

M Talbi

M Sot

J M Perone
email: jm.perone@chr-metz-thionville.fr

Reconstruction of exenteration socket with Integra® dermal substitute and skin graft

Keywords: exenteration, orbital tumors, dermal substitute, Integra®, epithesis exentération, tumeurs orbitaires, substitut dermique, Integra®, epithèses

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Reconstruction of exenteration socket with Integra® dermal substitute and skin graft

Reconstruction de cavité d'exentération avec substitut dermique Integra® et greffe de peau

Cet article est issu d'une communication orale présentée lors du 120e congrès de la Société

Française d'Ophtalmologie en mai 2014

Auteurs :

François Ameloot 1 , Hicham Mezzine 2 , Grégoire Khairallah 2 , George Hayek 1 , Mohamed Zaidi 1 , Louis Lhuillier 1 , Marouane Talbi 2 , Maxime Sot 1 , Jean Marc Perone 1, * 1-Département d'ophtalmologie, Mercy Hospital, Regional Hospital Center of Metz-Thionville, 57085 Metz CEDEX 3, France 2-Service de chirurgie maxillo-faciale, Mercy Hospital, Regional Hospital Center of Metz-Thionville, 57085 Metz CEDEX 3, France

INTRODUCTION :

Orbital exenteration is a surgical procedure used for the treatment of certain ocular, orbital, and palpebral malignancies that invaded the orbit and cannot be treated conservatively.

It is an extremely mutilating procedure, removing all the orbital elements, and leaving nude the orbital bony walls [START_REF] Pollack | Orbital and periorbital tumors. Indications for exenteration[END_REF][START_REF] Mouriaux | Orbital exenteration][END_REF]. Exenterations can be partial (with eyelid preservation) or total (by extending the procedure to the adjacent tissues or periorbital cavities) when the lesion is extensive [START_REF] Mouriaux | Orbital exenteration][END_REF][START_REF] Kiratli | Orbital exenteration: Institutional review of evolving trends in indications and rehabilitation techniques[END_REF]. The main etiologies requiring exenteration are the palpebral malignancies invading the orbit by contiguity : epithelial tumors (basal cell epithelioma and squamous cell carcinoma), sebaceous carcinoma, conjunctival melanoma and squamous cell carcinoma [START_REF] Soysal | Orbital exenteration: a 10-year experience of a general oncology hospital[END_REF][START_REF] George | Orbital tumors: the ophthalmologist's point of view (biopsies -approachesexenteration)[END_REF].

Total orbital exenteration is the most commonly used technique and involves the bulk removal of eyelids and orbital contents covered by its periosteum. After having incised the skin until the bony rim and circularly around the entire orbital circumference, the periosteum is opened, and the periosteal sac is detached to the apex where the neurovascular pedicule is then carefully coagulated and sectioned [START_REF] Mouriaux | Orbital exenteration][END_REF][START_REF] Kiratli | Orbital exenteration: Institutional review of evolving trends in indications and rehabilitation techniques[END_REF][START_REF] Soysal | Orbital exenteration: a 10-year experience of a general oncology hospital[END_REF][START_REF] George | Orbital tumors: the ophthalmologist's point of view (biopsies -approachesexenteration)[END_REF]. The orbital walls are thus left nude and various methods are available for the cavity repair : spontaneous epithelialization, graft or flap.

Techniques such as locoregional flap (temporal muscle, frontal cutaneous) or free flap (rectus muscle of the abdomen, thigh) have long been the preferred solutions because they allow a correct filling of the orbital hollowness and even of a greater cavity volume if an enlarged exenteration was performed. Locoregional flap are robust and tolerate radiotherapy [START_REF] Hanasono | An algorithmic approach to reconstructive surgery and prosthetic rehabilitation after orbital exenteration[END_REF]. In case of total exenteration without adjuvant treatment, spontaneous epithelialization was the alternative choice versus thin skin grafts [START_REF] Kiratli | Orbital exenteration: Institutional review of evolving trends in indications and rehabilitation techniques[END_REF].

Recently, the appearance of epithesis techniques (made up of silicon), which is magnetically fixed to pillars that are osteo-integrated onto the orbital rim, significantly improved the rehabilitations of the exenteration cavities [START_REF] Larson | The use of osseointegration and rare earth magnetic coupling for oculofacial prosthesis retention in the exenterated orbit[END_REF]. This requires, however, to opt either for a reconstruction solution leaving the orbital cavity open, or for a reoperation on the previously disposed flap, to ensure their correct placement and stability [START_REF] Hanasono | An algorithmic approach to reconstructive surgery and prosthetic rehabilitation after orbital exenteration[END_REF].

Orbital exenteration leaves a significant aesthetic and psychological scar and the rehabilitation of the exenteration cavity is therefore a major issue in the management of these patients. Its goal is to restore at best the aesthetic unity of the face with the possibility of adaptation of an epithesis, while allowing a satisfactory oncological surveillance but also the use of adjuvant treatments if necessary.

We describe here the case of a patient who benefited from a new alternative to the reconstruction of the orbital cavity after exenteration.

CASE DESCRIPTION :

We report the case of a 42-year-old patient, referred to our department for a neglected retractile lesion of the outer half of the lower right eyelid, causing an irritative discomfort that became unbearable. After anamnesis, the patient described a history of chalazion having badly evolved with a stable scar for several months. He had already consulted several specialists for this lesion for the last 3 years, but being very anxious, he had always refused care. The clinical examination was delicate, but there was an ulcerative and retractile lesion, with madarosis on the outer half of the lower right eyelid, retraction of the conjunctival sac and a scar entropion of the lower eyelid (Fig. 1).

Because of the patient's anxiety, a biopsy under general anesthesia was made. While under general anesthesia, an external cantholysis was performed, this helped us to visualize the inferior and lateral conjunctival sac that was infiltrated by lesional tissue. Tissue induration was also noted extending to and below the outer orbital rim, reaching the orbit under the external canthal tendon. A large resection of the outer half of the lower eyelid, the outer canthus, the lateral third of the upper eyelid and the infiltrated conjunctiva was performed.

The extemporaneous examination found a carcinomatous infiltration by an epidermoid carcinoma moderately differentiated with invasion of all the edges including the deep ones.

Given the importance of the lesion and its local invasion, no additional resection was performed, and the decision was made to wait for the final histopathological results. The diagnosis of a moderately differentiated epidermoid carcinoma infiltrating the orbit was confirmed and the case of the patient was presented during a multidisciplinary consultation meeting. Total exenteration was decided with the possibility of performing adjuvant radiotherapy and chemotherapy according to the final pathological findings.

Total exenteration was performed under general anesthesia. After a skin incision to the bony rim, the orbital contents were removed from the periosteal sac with exposure of the bony walls of the orbital cavity (Fig. 2). Extemporaneous examination confirmed healthy margins greater than 5 mm. In addition, resection of the temporal and infero-temporal orbital bone ridges was performed (Fig. 3).

The rehabilitation of the exenteration cavity is carried out by setting up an Integra patch. Developed in 1981 by Yanna and Burke [START_REF] Burke | Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury[END_REF] to provide immediate wound coverage after excision, Integra® allows the regeneration of a functional dermis with a histologically normal architecture after extracellular matrix degradation. The patch consists of a bilayer substitute composed of an extracellular matrix covered with a Silastic® film. The synthetic surface layer is a 0.10 mm thick polysiloxane polymer film for water and gas exchange. The 2 mm thick dermal layer is a combination of bovine collagen Type I fibers and chondroitin-6-sulfate glycosaminoglycan, cross-linked by glutaraldehyde [START_REF] Lamy | The role of skin substitutes in the surgical treatment of extensive burns covering more than 60 % of total body surface area. A review of patients over a 10-year period at the Tours University Hospital[END_REF]. The dermal layer colonized by the cells of the patient is definitive and should be ideally grafted between D14 and D21 after removal of the superficial layer. Definitive healing takes place about one month after the Integra application (Fig. 4).

After hydrating the Integra leaflet for 2 minutes in physiological saline solution, it is placed in the exenteration cavity and sutured directly to the skin margins (Figure 5). The patch is finally plated on the orbital walls by a Jelonet leaflet topped by a compressive pledget (Figure 6). The duration of the intervention was 140 minutes.

An antibiotic prophylaxis with Amoxicillin + clavulanic acid was introduced for 10 days and the patient was able to leave 2 days after the surgery. Cavity monitoring and dressing repair were then performed every 3 days. Healing was satisfactory with the appearance of a budding tissue under the silicone film, 10 days only after the exenteration.

The definitive anatomopathological findings revealed a moderately differentiated epidermoid carcinoma infiltrating the orbit with healthy margins of 0.5 cm along the inferotemporal periosteum. Perineural sheathing and carcinomatous endovascular emboli were also present. Therefore, an adjuvant treatment by radio and chemotherapy was decided during a new multidisciplinary consultation meeting.

Four weeks after the first procedure, the patient underwent another general anesthesia procedure for port-a-cath placement.

Therefore, the decision to perform skin grafting in the exenteration cavity at the same time was taken. After ablation of the silicone film, we found a well vascularized budding of neodermal tissue.

A thin skin graft (0.2 mm), taken from the inside of the right thigh and expanded 1.7 times, was then placed in the cavity and sutured onto the skin margins but also attached directly to the neodermal tissue in the bottom of the cavity. The graft was then covered with Jelonet and a pledget to best conform with the orbital walls. The grafting was complete and rapid. Total re-epithelialization of the cavity was obtained 7 weeks after the exenteration.

Radiotherapy treatment was started the following week, with a dose of 30 grays divided into 15 fractions over the course of 20 days, concomitantly with 3 cures of CISPLATYL (A cure once every 15 days). A low cumulative dose of 30 grays was possible because the adjuvant treatment also included chemotherapy, otherwise this dose would have been too low for isolated radiotherapy. The tolerance of the graft with respect to radiotherapy was excellent, no disunion, no necrosis and no desquamation was noted (Figure 7).

Six months after the exenteration, a stable cavity was obtained, without post radiotherapy integument complications. The cavity was easy to monitor, free of recurrencies, and well opened, allowing us to easily start the process of cosmetic rehabilitation by an epithesis.

Epiplating titanium plates (Medicon, Tuttlingen, Germany) were directly fitted with their magnets on the superior and infero-lateral orbital rims (Fig. 8).

The healing of the skin above the plates and around the magnets was correct. 4 weeks after the establishment of the anchors, molding of the cavity for the creation of the silicone epithesis adapted to the osteo-integrated implants was performed (fig. 9).

In summary, the use of INTEGRA® for reconstruction, allowed us to obtain a rehabilitated cavity, ready to be equipped with an epithesis and easy to monitor. The reconstruction took only 7 months after the orbital exenteration with adjuvant radio and chemotherapy.

DISCUSSION :

In case of orbital invasion, the management of oculo-orbito-palpebral malignancies sometimes requires exenteration. In the literature, after exenteration, the most commonly used reconstruction techniques are spontaneous epithelialization (32 to 80 % of cases), muscle flaps (25 to 77 % of cases) and skin grafts (from 23 to 63 % of cases) [START_REF] Hanasono | An algorithmic approach to reconstructive surgery and prosthetic rehabilitation after orbital exenteration[END_REF][START_REF] Langlois | Evaluation of reconstructive techniques after orbital exenteration in 56 cases][END_REF][START_REF] Nassab | Orbital exenteration for advanced periorbital skin cancers: 20 years experience[END_REF][START_REF] Mohr | Orbital exenteration: surgical and reconstructive strategies[END_REF][START_REF] Rahman | Orbital exenteration: a 13 year Manchester experience[END_REF][START_REF] Nemet | Orbital exenteration: a 15-year study of 38 cases[END_REF]. The recent appearance of epithesis supported by osteo-integrated implants has significantly improved the cosmetic rehabilitation of exenteration cavities, and their use makes it possible to adapt stable epitheses in 20 to 95 % of the cases [START_REF] Hanasono | An algorithmic approach to reconstructive surgery and prosthetic rehabilitation after orbital exenteration[END_REF][START_REF] Langlois | Evaluation of reconstructive techniques after orbital exenteration in 56 cases][END_REF][START_REF] Greig | Reconstruction of the exenterated orbit with osseointegrated implants[END_REF], with a satisfactory result for the patients [START_REF] Larson | The use of osseointegration and rare earth magnetic coupling for oculofacial prosthesis retention in the exenterated orbit[END_REF][START_REF] Moran | Implant-retained prosthetic rehabilitation of orbital defects[END_REF].

The choice of the reconstruction technique should be guided by the size of the deficit to be filled, the use of a possible adjuvant radiotherapy (requiring a rapid making of a cavity that can tolerate it) and finally the patient's will for wearing of an epithesis (requiring the creation of a so-called open cavity) [START_REF] Hanasono | An algorithmic approach to reconstructive surgery and prosthetic rehabilitation after orbital exenteration[END_REF][START_REF] Mohr | Orbital exenteration: surgical and reconstructive strategies[END_REF].

After exenteration, the spontaneous epithelialization of the cavity by granulation is a frequently used reconstruction solution. It makes it possible to obtain cavities that are easier to adapt with epithesis, and also easy to monitor, without the associated difficulties and complications with the removal and the use of a flap or graft. However, it requires frequent care and regular monitoring. The healing average duration can vary between 3 to 10 months, depending on the studies, and is highly dependent on a possible necessary adjuvant radiotherapy [START_REF] Mohr | Orbital exenteration: surgical and reconstructive strategies[END_REF][START_REF] Rahman | Orbital exenteration: a 13 year Manchester experience[END_REF][START_REF] Nemet | Orbital exenteration: a 15-year study of 38 cases[END_REF][START_REF] Putterman | Orbital exenteration with spontaneous granulation[END_REF].

Adaptation of osteo-integrated implants comes later than with the other means of reconstruction and is usually postponed around 11 to 19 months after exenteration in case of granulation [START_REF] Langlois | Evaluation of reconstructive techniques after orbital exenteration in 56 cases][END_REF][START_REF] Rahman | Orbital exenteration: a 13 year Manchester experience[END_REF]. There is also a higher rate of fistula-like complications than with grafts or flaps [START_REF] Putterman | Orbital exenteration with spontaneous granulation[END_REF][START_REF] Bartley | Orbital exenteration at the Mayo Clinic. 1967-1986[END_REF] ranging from 28 % in the series of Rahman et al. [START_REF] Rahman | Orbital exenteration: a 13 year Manchester experience[END_REF] to 68 % in the series of Mohr et al. [START_REF] Mohr | Orbital exenteration: surgical and reconstructive strategies[END_REF]. These fistulas arise because of the secretions create a chronic flow with an inflammatory cavity. All of which is uncomfortable for the patients and therefore hindering the prosthetic adaptation.

Fine or dermal skin grafts accelerate the recovery of the bony walls of the cavity. Healing is usually achieved in 6 to 8 weeks. Although they allow the rapid creation of an ideal open cavity for future prosthetic adaptation, they have a significant failure rate given the difficulty of fixing them directly onto the bone. Moreover, the tissues created being thin, they hardly tolerate radiotherapy and healing problems can also occur during the placement of bone implants [START_REF] Nassab | Orbital exenteration for advanced periorbital skin cancers: 20 years experience[END_REF][START_REF] Rahman | Orbital exenteration: a 13 year Manchester experience[END_REF][START_REF] Nemet | Orbital exenteration: a 15-year study of 38 cases[END_REF]. For Hanasono et al. [START_REF] Hanasono | An algorithmic approach to reconstructive surgery and prosthetic rehabilitation after orbital exenteration[END_REF] their indications are limited to partial or total exenterations, not requiring radiotherapy and where rapid prosthetic rehabilitation is desired.

The immediate reconstruction of the cavities by loco-regional flaps (temporal or frontal) or free flaps can cover large skin deficits and has the advantage of reconstructing the cavity in a single operation. It can be helpful especially for patients with precarious general conditions, but this lengthens the duration of the surgery by several hours as well as the duration of hospitalization. [START_REF] Hanasono | An algorithmic approach to reconstructive surgery and prosthetic rehabilitation after orbital exenteration[END_REF]. Their large volume and rapid healing allow an optimum filling of the cavity thus limiting the risk of fistula. Their robustness gives them good tolerance to adjuvant radiotherapy [START_REF] Benazzou | Orbital exenteration][END_REF]. Their cosmetic results are however less satisfactory than the adaptation of an epithesis, and in the case of desired secondary rehabilitation, as they create so-called full cavities, the flaps will hinder the adaptation of epithesis and revision procedures may be necessary [START_REF] Hanasono | An algorithmic approach to reconstructive surgery and prosthetic rehabilitation after orbital exenteration[END_REF][START_REF] Tyers | Orbital exenteration for invasive skin tumours[END_REF].

In addition, there is a risk of comorbidities and significant aesthetic damage related to the site of sampling. Finally, the flap will make it more difficult to monitor the cavity for early detection of potential recurrences that may occur in up to 24 % of the cases [START_REF] Nemet | Orbital exenteration: a 15-year study of 38 cases[END_REF]. Developed in the 80s by the team of Yanna and Burke [START_REF] Burke | Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury[END_REF], the Integra® has been available in France for about twenty years and used for the management of burn victims where it has shown its effectiveness [START_REF] Lamy | The role of skin substitutes in the surgical treatment of extensive burns covering more than 60 % of total body surface area. A review of patients over a 10-year period at the Tours University Hospital[END_REF][START_REF] Dantzer | Integra, a new surgical alternative for the treatment of massive burns. Clinical evaluation of acute and reconstructive surgery: 39 cases[END_REF]. Currently, because of its ease of use, its ability to bud directly onto the bone, and its satisfactory aesthetic and functional results, it is increasingly used in plastic and reconstructive surgery. It makes it possible to obtain a rapid quality skin coverage (4 to 6 weeks).

Also, quality skin coverage can be obtained in large losses of substance, including the scalp or the face, all of which without using flaps, reduce the operating time, hospitalization time, and comorbidities related to the flap sampling sites [START_REF] Khan | Use of dermal regeneration template (Integra) for reconstruction of full-thickness complex oncologic scalp defects[END_REF][START_REF] Koenen | One-stage reconstruction of deep facial defects with a single layer dermal regeneration template[END_REF][START_REF] Felcht | Two-stage reconstruction of head and neck defects after tumor resection with a dermal regeneration template[END_REF][START_REF] Ozgonul | The Use of Integra® Dermal Regeneration Template for the Orbital Exenteration Socket: A Novel Technique[END_REF].

Finally, tissues reconstructed with the help of Integra® have a good tolerance with respect to radiotherapy [START_REF] Tufaro | The use of artificial dermis in the reconstruction of oncologic surgical defects[END_REF]. In our case, the placement of the dermal substitute, combined with a secondary skin graft, has shown to be effective in reconstructing a complex loss of substance with exposed orbital bone. It allowed a quality healing of the exenterated cavity in just 6 weeks and all while having well tolerated an adjuvant radiotherapy that could have been deleterious to a transplant. A few similar cases of use of Integra® dermal substitute, secondary to exenterations, have been described in the literature in recent years : a series of 7 cases described by Rafailov et al. in 2017 [START_REF] Rafailov | Use of bilayer matrix wound dressing in the exenterated socket[END_REF], a series of 5 cases described by Ozgonul et al. [START_REF] Ozgonul | The Use of Integra® Dermal Regeneration Template for the Orbital Exenteration Socket: A Novel Technique[END_REF] as well as a unique case described by Patel et al. in 2018 [START_REF] Patel | Int Ophthalmol. Two-stage rapid exenteration reconstruction to allow early radiation therapy for an aggressive orbital[END_REF].

For all the described cases, the procedure and the hospitalization duration were shorter than with graft or flap reconstruction techniques [START_REF] Hanasono | An algorithmic approach to reconstructive surgery and prosthetic rehabilitation after orbital exenteration[END_REF]. Postoperative care was simpler, shorter and not very demanding compared to that required with spontaneous epithelialization. However, a second intervention was required in the month following exenteration, to perform thin skin grafting. Some one-step procedures were described and showed satisfactory results as well [START_REF] Koenen | One-stage reconstruction of deep facial defects with a single layer dermal regeneration template[END_REF][START_REF] Demiri | Reconstruction of skin avulsion injuries of the upper extremity with integra(®) dermal regeneration template and skin grafts in a single-stage procedure[END_REF].

In our patient cosmetic rehabilitation was started only 6 months after the exenteration in order to be able to monitor the reaction of the reconstructed cavity in the aftermath of the radiotherapy, but also to detect a possible retraction of the reconstructed cutaneous tissue by dermal substitutes, whose surface area can decrease by 30 % [START_REF] Koenen | One-stage reconstruction of deep facial defects with a single layer dermal regeneration template[END_REF][START_REF] Felcht | Two-stage reconstruction of head and neck defects after tumor resection with a dermal regeneration template[END_REF]. Healing on the bone implants was satisfactory and the well open cavity allowed easy adaptation of the epithesis with stability and comfort. As in the previous publications, we didn't note any complications such as early fistula, dermatitis or necrosis after radiotherapy of the cavity. In addition, the use of the Integra® made it possible to avoid any complications or facial unaesthetic scarring related to the site of flap removal. Finally, in the absence of flap, the monitoring of a possible recurrence in the cavity was easy and since healing was rapid it was not confused with budding tissue.

Legends of the figures

Figure 1 :

 1 Figure 1 : initial aspect of the lesion during management, with an ulcerative and retractile lesion of the outer half of the lower right eyelid.

Figure 2 :

 2 Figure 2 : extirpation of the periosteal sac with the orbital content.

Figure 3 :

 3 Figure 3 : orbital cavity exposed with resection of temporal orbital rim.

Figure 4 :

 4 Figure 4 : from left to right and from bottom to top : Integra® patch ; initial skin deficit ; 6 days after placement of the patch, we note the formation of the neo-dermis under the Silicone sheet ; Removal of the silicone film ; placement of an expanded thin skin graft on the new dermis ; J30 complete healing with reformation of the cutaneous tissue.

Figure 5 :

 5 Figure 5 : Integra® patch set up in the orbital cavity.

Figure 6 :

 6 Figure 6 : Jelonet® tamponade to patch the orbital wall patch.

Figure 7 :

 7 Figure 7 : Cavity 15 weeks after the exenteration and 1 month after the end of the adjuvant radiotherapy.

Figure 8 :

 8 Figure 8 : Cavity equipped with osteo-integrated implant 7 months after the exenteration.

Figure 9 :

 9 Figure 9 : final epithesis.

Figure 1 :

 1 Figure 1: initial aspect of the lesion during management, with an ulcerative and retractile lesion of the outer half of the lower right eyelid.

Figure 2 :

 2 Figure 2: extirpation of the periosteal sac with the orbital content

Figure 3 :

 3 Figure 3 : orbital cavity exposed with resection of temporal orbital rim

Figure 4 :

 4 Figure 4: from left to right and from bottom to top: Integra® patch; initial skin deficit; 6 days after placement of the patch, we note the formation of the neo-dermis under the silicone sheet; Removal of the silicone film; placement of an expanded thin skin graft on the new dermis; J30 complete healing with reformation of the cutaneous tissue

Figure 5 :

 5 Figure 5: Integra® patch set up in the orbital cavity

Figure 6 :

 6 Figure 6: Jelonet® tamponade to patch the orbital wall patch.

Figure 8 :

 8 Figure 8: Cavity equipped with osteo-integrated implant 7 months after the exenteration.

Figure 7 :

 7 Figure 7: Cavity 15 weeks after the exenteration and 1 month after the end of the adjuvant radiotherapy.

CONCLUSION :

The use of Integra® Dermal Substitute combined with a thin skin graft appears to be a simple and satisfactory alternative for the reconstruction of orbital exenterated cavities. It allows rapid healing with a good local tolerance to radiotherapy. It is fast, simple and harmless for the rest of the face. Finally, this combination allows early cosmetic rehabilitation, while ensuring optimal local cancer monitoring. A comparative study would confirm the interest of this treatment in post-exenteration reconstructions.

Conflict of interest :

The authors declare having no conflict of interest related to this article.

BIBLIOGRAPHY :


M.D Nicolas Javaud
email: nicolas.javaud@aphp.fr

M.D Adrien Altar
email: dr.adrien.altar@gmail.com

M.D Olivier Fain
email: olivier.fain@aphp.fr

M.D Paul-Georges Reuter
email: paul-georges.reuter@aphp.fr

M.D Michel Desmaizieres
email: michel.desmaizieres@aphp.fr

M.D Tomislav Petrovic
email: tomislav.petrovic@aphp.fr

M.D Aiham Ghazali
email: aiham.ghazali@aphp.fr

M.D Isabelle Durand-Zaleski
email: isabelle.durand-zaleski@aphp.fr

M.D Laurence Bouillet
email: lbouillet@chu-grenoble.fr

M.D Eric Vicaut
email: eric.vicaut@aphp.fr

M.D David Launay
email: david.launay@chru-lille.fr

M.D Ludovic Martin
email: lumartin@chu-angers.fr

M.D Bernard Floccard
email: bernard.floccard@chu-lyon.fr

M.D Anne Gompel
email: annegompel@gmail.com

M.D Alain Sobel
email: alain.sobel@aphp.fr

M.D Isabelle Boccon-Gibod
email: iboccon-gibod@chu-grenoble.fr

M.D Gisele Kanny
email: gisele.kanny@univ-lorraine.fr

M.D Frederic Lapostolle
email: frederic.lapostolle@aphp.fr

M.D Frederic Adnet
email: frederic.adnet@aphp.fr

Hereditary angioedema, emergency management of attacks by a call center

Keywords: hereditary angioedema, emergency departments, call center

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Hereditary angioedema (HAE) is a rare disorder with an estimated prevalence from 1/50,000 to 1/100,000 [START_REF] Longhurst | Hereditary angio-oedema[END_REF]. HAE type I and II are associated with SERPING1 gene mutations encoding for C1-inhibitor (C1-INH) and with C1-INH deficiency (plasma C1-INH levels below 50% of the normal values). Eighty-five percentage of patients will have low quantitative and functional plasma levels of C1-inh (type I). For patients with normal quantitative plasma levels of C1-Inh, diagnosis requires decreased C1-Inh functional levels (type II) [START_REF] Cicardi | Classification, diagnosis, and approach to treatment for angioedema: consensus report from the Hereditary Angioedema International Working Group[END_REF][START_REF] Zuraw | A focused parameter update: hereditary angioedema, acquired C1 inhibitor deficiency, and angiotensin-converting enzyme inhibitorassociated angioedema[END_REF]. Factor XII-HAE (FXII-HAE) are associated with mutation of the gene encoding human coagulation factor XII and patients have normal antigenic and functional C1-INH levels with family history of angioedema [START_REF] Cicardi | Classification, diagnosis, and approach to treatment for angioedema: consensus report from the Hereditary Angioedema International Working Group[END_REF][START_REF] Zuraw | A focused parameter update: hereditary angioedema, acquired C1 inhibitor deficiency, and angiotensin-converting enzyme inhibitorassociated angioedema[END_REF]. Patients with HAE are characterized by spontaneous, unpredictable and recurrent edema attacks which can be potentially life threatening when the upper airway is involved (larynx and tongue). Other severe manifestations are abdominal attacks that may be painful, mimic abdominal emergencies or be associated with ascites.

Severe attacks can also be edema of the face that may be disfiguring. Attacks can also affect extremities and external genital organs and are debilitating [START_REF] Bork | Clinical studies of sudden upper airway obstruction in patients with hereditary angioedema due to C1 esterase inhibitor deficiency[END_REF][START_REF] Bork | Fatal laryngeal attacks and mortality in hereditary angioedema due to C1-INH deficiency[END_REF][START_REF] Guichon | One hypovolaemic shock...two kinin pathway abnormalities[END_REF]. Treatment options are large including treatments of acute attacks, pre-procedural attack prophylaxis and long-term prophylaxis and are codified in recent guidelines [START_REF] Maurer | The international WAO/EAACI guideline for the management of hereditary angioedema -the 2017 revision and update[END_REF]. However, early access to HAE specific emergency treatments is a challenge for the emergency medicine [START_REF] Javaud | The early treatment of bradykinin angioedema, a challenge for emergency medicine[END_REF]. Hence, although international guidelines for management of HAE attacks exist, current management was suboptimal because emergency medical teams are often unaware of the guidelines and of new specific treatments [START_REF] Otani | Emergency Department Management of Hereditary Angioedema Attacks: Patient Perspectives[END_REF][START_REF] Banerji | Current state of hereditary angioedema management: a patient survey[END_REF][START_REF] Moellman | A consensus parameter for the evaluation and management of angioedema in the emergency department[END_REF][START_REF] Cicardi | Evidence-based recommendations for the therapeutic management of angioedema owing to hereditary C1 inhibitor deficiency: consensus report of an International Working Group[END_REF][START_REF] Ucar | Difficulties encountered in the emergency department by patients with hereditary angioedema experiencing acute attacks[END_REF]. In a recent study, 18 over 39 patients (46%) did not have any specific HAE treatment or received inappropriate treatment in emergency departments (ED), even though ED use is common in HAE [START_REF] Otani | Emergency Department Management of Hereditary Angioedema Attacks: Patient Perspectives[END_REF]. Indeed, in a retrospective study of 193 HAE patients with 8 attacks per patient per year, approximatively 11% of patients visited the ED [START_REF] Bouillet | Hereditary angioedema with C1 inhibitor deficiency: clinical presentation and quality of life of 193 French patients[END_REF]. One strategy successfully described for other chronic disease, is to provide care support 3 to reduce inappropriate treatment and optimize management of HAE attacks [START_REF] Wennberg | A randomized trial of a telephone care-management strategy[END_REF]. This strategy shows that a driven, targeted, population-based program can decrease hospitalizations and thereby reduce total medical costs for the targeted population as a whole [START_REF] Wennberg | A randomized trial of a telephone care-management strategy[END_REF].

The objective of the present study was to determine the management of the acute HAE attacks in the "real life setting" through a call center in France.

METHODS

Study design

This was an ancillary study of the SOS-HAE study (NCT01679912) [START_REF] Javaud | Dedicated call center (SOS-HAE) for hereditary angioedema attacks: study protocol for a randomized controlled trial[END_REF], a multicenter cluster-randomized prospective trial that assessed morbidity (number of hospital admissions) after a telephone care-management used for patients with HAE. HAE patients were prospectively recruited from eight French participating reference centers. The randomized units were the reference centers (clusters). Patients from intervention centers were assigned to receive care management through national centralization of calls made by patients during attacks. Patients from control centers were assigned to usual practice. All patients were followed for 2 years. Informed consent process was based on modified version of Zelen's method with 2-step for randomization and inclusion. Patients in usual practice group were not aware of the availability of the intervention.

Outcome

The primary outcome was the rate of hospitalization

Statistical analysis

The unit of analysis was the attack, data from the intervention group were analyzed.

Descriptive statistics were used to characterize sociodemographic and clinical characteristics.

Data are expressed as mean (SD) for normally distributed variables, median (interquartile range) for non-Gaussian quantitative variables, and as numbers and percentages for categorical variables. The study was approved by local ethics committee (Comité de Protection des Personnes Ile-De-France Paris X, Aulnay-sous-Bois, France).

RESULTS

Characteristics of Study Subjects

Of the 200 patients included in the SOS AOH study, 100 could access the call center in the follow-up period of 2 years and were analyzed (table 1). The median (quartile) age was 38 (29-53) years, and 66 (66%) were female. Eighty (80%) patients had HAE type I, 8 (8%) had HAE type II and 12 (12%) patients had FXII-HAE. Main coexisting conditions were hypertension for 11 (11%) of them, active cancer for 7 (7%), dyslipidemia for 6 (6%) and tobacco use for 5 (5%). Two patients were lost at 22 and 24 months of follow-up. Two patients died at 12 and 14 months of follow-up (due to breast cancer and leukemia, respectively). A large majority of study patients were receiving long-term prophylaxis without plasma-derived C1-INH treatment. All patients had a specific emergency treatment at home and almost all (98%) had a nurse-led formation on self-administration of specific therapy at the beginning of the trial.

Two-years prospective follow-up

Fifty-one (51%) patients had experienced at least one time the call center during the followup. During 2 years, there were 166 calls (table 2) to call center including 129 (78%) severe, 24 (14%) moderate and 13 (8%) mild attacks. Nine over 166 (5%) attacks for 9 different patients resulted in hospital admission to the hospital (in the short-stay unit, ie, <24 hours) during the follow-up period after 17 (10%) EMS dispatch. Call center was directly contacted by the patient in the large majority of cases (96%). Median time from symptom onset to call center call was 3.4 (1.1-7.8) hours. The site of the attacks prompting call to call center were the abdomen (86 of 166, 52%), the limbs (59 of 166, 36%), the cheeks (21 of 166, 13%), the lips (19 of 166, 11%), the upper airways (18 of 166, 11%) (including larynx, tongue and uvula) and the eyelids (4 of 166, 2%). Thirty-nine (23%) attacks were multisite. The trigger was identified for 89 (54%) attacks: stress in 55 (33%) attacks, trauma in 22 (13%) attacks, infection in 8 (5%) attacks and discontinuation of LTP in 4 (3%) attacks. All attacks were treated at home after call center contact with icatibant (30 mg subcutaneously; 93 of 166, 56%), C1-INH concentrate (20 UI/kg intravenously; 14 of 166, 8%) or tranexamic acid (1 gr orally; 59 of 166, 36%). Median time from symptom onset to treatment administration was 3.4 (1.4-6.4) hours. For 17 (10%) attacks, EMS was sent home. The course was favorable for all 166 attacks with a median time from treatment to onset of symptom relief of 1.9 (0.5-11.9) hours. No invasive upper airway management (orotracheal intubation and/or tracheostomy) was required.

DISCUSSION

This first prospective study accurately describes epidemiologic characteristics of patients and the manegement of HAE attacks in France, related to call center. The hospitalization rate was small in our study. The findings suggest that HAE may lead to substantial use of call center, mainly driven by abdominal attacks and to dispatch EMS when edema attacks can be potentially life threatening. The manegement is shortened by the call center.

This cohort presents similar characteristics as those in France or others countries and some differences [START_REF] Bouillet | Hereditary angioedema with C1 inhibitor deficiency: clinical presentation and quality of life of 193 French patients[END_REF][START_REF] Aygören-Pürsün | Socioeconomic burden of hereditary angioedema: results from the hereditary angioedema burden of illness study in Europe[END_REF][START_REF] Wilson | Economic costs associated with acute attacks and long-term management of hereditary angioedema[END_REF]. The predominance of female patients, the median age of patients, the rate of severe attacks and the distribution of attacks sites were similar. The median age in call center group is quite low and this is probably due that younger patients more prone to refer to the call center. It can be seen that 76% of patients in the call center group had a HAE sick parents whereas only 56% of patients in the control group who did have a sick parent. In other words, having a sick parent seemed to make the patient aware of contactoing the call center.

The predominance of emotional distress as triggering factor was similar to previous French cohort [START_REF] Bouillet | Hereditary angioedema with C1 inhibitor deficiency: clinical presentation and quality of life of 193 French patients[END_REF]. Higher rates of abdominal and limb attacks (around 60-70%) were found in previous study. Lower rates (around 5-10%) of laryngeal and face attacks were also consistent with this earlier study [19]. However, the rate of treated attacks with emergency specific drugs (icatibant or C1-INH concentrate) was higher (107/166; 64%) than in the French study (26%) [START_REF] Bouillet | Hereditary angioedema with C1 inhibitor deficiency: clinical presentation and quality of life of 193 French patients[END_REF]. This rate corresponds exactly to the total number of attacks minus the rate of limbs attacks. This was probably due to specialist advice support for management of severe hereditary angioedema attacks and to an education program implemented by reference centers that promoted self-home treatment in accordance with recent recommendations [START_REF] Longhurst | HAE international home therapy consensus document[END_REF].

However, tranexamic acid was used although international guidelines do not advise using antifibrinolytics for on-demand treatment of HAE attacks, as this drug show no or only minimal effects when used for on-demand treatment [START_REF] Maurer | The international WAO/EAACI guideline for the management of hereditary angioedema -the 2017 revision and update[END_REF]. Further, the hospitalizations and emergency visits rate were significantly fewer in our study than those reported by Bouillet et al [START_REF] Bouillet | Hereditary angioedema with C1 inhibitor deficiency: clinical presentation and quality of life of 193 French patients[END_REF] and previous economic study [START_REF] Aygören-Pürsün | Socioeconomic burden of hereditary angioedema: results from the hereditary angioedema burden of illness study in Europe[END_REF][START_REF] Wilson | Economic costs associated with acute attacks and long-term management of hereditary angioedema[END_REF].

The most significant risk factor for EMS dispatch in this study was upper airway involvement, which affects half of HAE patients at least once during their lifetime [START_REF] Bork | Fatal laryngeal attacks and mortality in hereditary angioedema due to C1-INH deficiency[END_REF]. Its incidence was similar to previous described. The relation between laryngeal edema and ED visits is known in two retrospective studies [START_REF] Javaud | Bradykinin-mediated angioedema: factors associated with admission to an intensive care unit, a multicenter study[END_REF][START_REF] Javaud | Factors associated with hospital admission in hereditary angioedema attacks: a multicenter prospective study[END_REF]. In this study, facial edema was never followed by laryngeal edema unlike in the retrospective study by Bork et al [23]. This was probably because all episodes in the present study were treated by C1-INH concentrate or icatibant at home. Median time from attack onset to call center call was fairly short (3.4 hours) but this first medical contact was shorter than reported in previous prospective study [START_REF] Javaud | Factors associated with hospital admission in hereditary angioedema attacks: a multicenter prospective study[END_REF]. Moreover, median time from call center contact to specific treatment was very short. Optimal patient management could further increase the number of immediate self-administration before call.

Implementation by EMS of guideline-recommended systematic treatment of acute attacks, through use of emergency instruction sheets, yielded good results for time from first contact to treatment. Education program might increase the number of immediate self-administration treatment.

Our study has important strengths. Our sample includes a large number of HAE patients with epidemiologic data and confirmed results that have been suggested in the first SOS-HAE analysis. Of note, this study comprises one of the largest samples, one quarter of the total rare HAE population in France, of patients with epidemiologic evaluation. Hence, there is no lack of external validity and generalizations of findings may be valid since a broad population and not a selection of high-risk group was included.

LIMITATIONS

Potential limitations of the current study need to be considered when interpreting the data.

First, as an ancillary study, it was just designed to describe epidemiologic data and no causal link could be demonstrated. Last, we included only patients who have had a follow-up into an expert HAE network, which could lead to a potential source of inclusion bias with "overperforming" HAE patients.

CONCLUSION

HAE is a rare disease whose morbidity is high with a significant impact of daily activity. Our data show that patients have frequent and severe attacks. Use of emergency departments and hospitalizations are reduced by the use of a coordinated national call center in HAE after therapeutic education program that promoted self-administration of specific treatment and use of call to call center.

Table 3. Treatments recommendations for emergency physicians

Recommendations

From

  March 2013 to June 2014, for SOS-HAE study, we prospectively enrolled patients older than 18 years with documented diagnosis of HAE (type I, II or FXII). Pregnant or breastfeeding women, patients with recent history (less than 1 month) of myocardial infarction or ischemic stroke were excluded (given the protective effect of bradykinin in these patients who would therefore receive bradykinin B2 receptor antagonist). Patients with allergy to icatibant or plasma-derived C1-inhibitor (C1-INH) were excluded. A 1:1 ratio randomly assigned centers (not patients) to receive intervention or usual practice. All patients in the intervention arm were given an SOS-HAE card indicating what to do in the case of severe attack. Emergency physicians receiving the call have received special training and can manage the attack. We included for this analysis, which was planned from the start, all patients of the intervention arm of the SOS-HAE study. time (in hours) before call center call (time of symptom onset to time of call), time (in hours) from attack onset to treatment start (time of symptom onset to hour of icatibant or C1-INH concentrate injection), time (in hours) to symptom relief (hour of icatibant or C1-INH concentrate injection to hour of onset of symptom relief).

  Vital distress -Immediately and as soon as possible, administer o Icatibant (Firazyr®): 30 mg subcutaneously or o Plasma-derived C1-INH (Bérinert®): 20 UI/Kg intravenously -Switch the call to the local SAMU to send French EMS -Gain control of upper airway Severe attacks • Laryngeal • Face • Abdominal -Immediate and as soon as possible, administer o Icatibant (Firazyr®): 30 mg subcutaneously or o Plasma-derived C1-INH (Bérinert®): 20 UI/Kg intravenously -If treatments are unavailable at home, switch the call to the local SAMU to send an ambulance headed towards a hospital with specific treatments available, or being able to get them by French EMS -Gain control of upper airway Non-severe attacks (members, genital) -Tranexamic acid : 1 gr / 6h except for patients who are breastfeeding or have thromboembolic pathology Surveillance in all cases -Monitoring by phone in 30 minutes, 1 hour, 4 hours, 12 hours and 24 hours after the beginning of the attack -Advice to call back « SOS HAE » call center in case of secondary worsening

[START_REF] Javaud | Factors associated with hospital admission in hereditary angioedema attacks: a multicenter prospective study[END_REF] No., number of patients; HAE, Hereditary Angioedema; FXII, HAE with mutation of the gene encoding human coagulation factor XII;

Funding: supported by the Programme Hospitalier de Recherche Clinique 2011 of the French Ministry of Health.

Data collection

The complete method details can be found in the original article [START_REF] Javaud | Dedicated call center (SOS-HAE) for hereditary angioedema attacks: study protocol for a randomized controlled trial[END_REF]. We collected the patients' demographic and clinical characteristics at the time of recruitment, and standardized data pertaining to their subsequent 2 years follow-up. We used clinical data in accordance with the recent Hereditary Angioedema International Working Group Guidelines [START_REF] Cicardi | Classification, diagnosis, and approach to treatment for angioedema: consensus report from the Hereditary Angioedema International Working Group[END_REF] and particularly data related to the current attack. For each attack, the emergency physicians prospectively collected standardized clinical data: possible trigger of attack, reason for call, day and time of onset of symptoms, day and time of call to call center, edema site, treatment availability at home, self-administered home therapy, time treatment started and course of attack (onset of symptom relief and time of symptom resolution), prehospital emergency medical service (EMS) dispatch, admission to the ED. The following times were calculated:

AUTHOR CONTRIBUTIONS

• NJ and FA initiated and coordinated the research.

• NJ, FA, OF, EV, IDZ, LB, DL, LM, BF, AG, AS, IBG, BC and GK designed the study. • NJ, FA, IDZ, PGR, AA, FL, EV managed and analyzed data.

• NJ, FA, AA, OF, PGR, MD, TP, AG, EV, LB, IDZ, and FL participated in the data collection and interpretation. • NJ, FA, FL wrote the article. All authors contributed substantially to the study and approved the final version of the article.

Table 1. Baseline characteristics of the study population

Characteristics of patients of the intervention group


P Pellicioli
email: p.pellicioli@esrf.fr

S Bartzsch

M Donzelli

M Krisch

E Bräuer-Krisch

High resolution radiochromic film dosimetry: comparison of a microdensitometer and an optical microscope

Keywords: microbeam radiation therapy, radiochromic film dosimetry, microdensitometer, optical microscope

Purpose: Microbeam radiation therapy is a developing technique that promises superior tumour control and better normal tissue tolerance using spatially fractionated X-ray beams only tens of micrometres wide. Radiochromic film dosimetry at micrometric scale was performed using a microdensitometer, but this instrument presents limitations in accuracy and precision, therefore the use of a microscope is suggested as alternative. The detailed procedures developed to use the two devices are reported allowing a comparison.

Methods: Films were irradiated with single microbeams and with arrays of 50µm wide microbeams spaced by a 400µm pitch, using a polychromatic beam with mean energy of 100keV. The film dose measurements were performed using two independent instruments: a microdensitometer (MDM) and an optical microscope (OM).

Results:

The mean values of the absolute dose measured with the two instruments differ by less than 5% but the OM provides reproducibility with a standard deviation of 1.2% compared to up to 7% for the MDM. The resolution of the OM was determined to be ~1-2µm in both planar directions able to resolve pencil beams irradiation, while the MDM reaches at the best 20µm resolution along scanning direction. The uncertainties related to the data acquisition are 2.5-3% for the OM and 9-15% for the MDM.

Conclusion:

The comparison between the two devices validates that the OM provides equivalent results to the MDM with better precision, reproducibility and resolution. In addition, the possibility to study dose distributions in two-dimensions over wider areas definitely sanctions the OM as substitute of the MDM.

INTRODUCTION

Microbeam Radiation Therapy (MRT) is a recent pre-clinical technique that promises to be more effective than conventional radiotherapy techniques (RT) in tumour treatment [START_REF] Laissue | Neuropathology of ablation of rat gliosarcomas and contiguous brain tissues using a microplanar beam of synchrotronwiggler-generated X rays[END_REF][START_REF] Bräuer-Krisch | Effects of pulsed, spatially fractionated, microscopic synchrotron X-ray beams on normal and tumoral brain tissue[END_REF][START_REF] Uyama | A narrow microbeam is more effective for tumor growth suppression than a wide microbeam: an in vivo study using implanted human glioma cells[END_REF] with fewer biological side effects [START_REF] Laissue | The weanling piglet cerebellum: a surrogate for tolerance to MRT (microbeam radiation therapy) in pediatric neurooncology[END_REF][START_REF] Bouchet | Effects of microbeam radiation therapy on normal and tumoral blood vessels[END_REF]. Pre-clinical studies suggest that MRT may be extremely useful to treat certain pathologies such as brain gliomas in paediatric patients, where the surrounding healthy tissue is particularly sensitive to radiation [START_REF] Laissue | Prospects for microbeam radiation therapy of brain tumours in children to reduce neurological sequelae[END_REF].

The main feature of MRT is the use of arrays of quasi-parallel microbeams, typically 50 μm wide beams spaced by 400 µm pitch, with hundreds of gray (Gy) in the peak and a low dose between the beams further referred to as valley dose. In addition, medium-energy X-ray photons (mean energy around 100 keV) are used instead of MeV photons to reduce the travel range of secondary electrons in the tissues, and the divergence of the beam must be small to preserve the microbeams shape throughout the target. The extremely high dose rates achievable with synchrotron X-rays [START_REF] Blattmann | Applications of synchrotron X-rays to radiotherapy[END_REF][START_REF] Duke | Synchrotron radiation : production and properties[END_REF][START_REF] Fournier | Absorbed dose-towater protocol applied to synchrotron-generated x-rays at very high dose rates[END_REF] allow reducing the exposure time and avoiding the blur of the microbeams traces due to tissue motion that in the human brain has a motion range up to hundreds of micrometres with a maximum motion velocity of 2 mm/s [START_REF] Soellinger | 3D cine displacement-encoded MRI of pulsatile brain motion[END_REF][START_REF] Donzelli | Improving dose calculation and treatment planning techniques for microbeam radiation therapy with computational methods[END_REF][START_REF] De Sola | Impact of cardiosynchronous brain pulsations on Monte Carlo calculated doses for synchrotron micro-and minibeam radiation therapy[END_REF].

MRT is currently best performed at a third generation synchrotron where microbeams are generated by a high brilliance X-ray source [START_REF] Laissue | Neuropathology of ablation of rat gliosarcomas and contiguous brain tissues using a microplanar beam of synchrotronwiggler-generated X rays[END_REF] able to satisfy the beam characteristics described above. All the experiments performed and reported in this paper were performed in Grenoble, France, at the biomedical beamline ID17 of the ESRF -the European Synchrotron -where the X-ray source is placed at around 40 m distance from the target [START_REF] Martínez-Rovira | Development and commissioning of a Monte Carlo photon beam model for the forthcoming clinical trials in microbeam radiation therapy[END_REF] and allows the generation of quasi-parallel microbeams at a dose rate of around 14 kGy/s. Until now, absolute dosimetry at micrometric scale has been a challenge in MRT because the irradiation parameters involved are extreme compared to classical approaches. The high dose rate of the source can cause detector saturation; the resolution on a micrometric scale at the target position is necessary to resolve the dose distribution of the microbeams; the materials used in the realization of the detector should be tissue equivalent around 100 keV photon energy. Potential high resolution detectors and dosimeters such as silicon strip, MOSFET, TLD and others have been designed and tested since the beginning of the development of MRT [START_REF] Bräuer-Krisch | Potential high resolution dosimeters for MRT[END_REF], however a final and definitive solution is not yet established because none of these devices is able to satisfy and achieve all the requirements in MRT. Detector developments are therefore still an active R&D area [START_REF] Bräuer-Krisch | Medical physics aspects of the synchrotron radiation therapies: Microbeam radiation therapy (MRT) and synchrotron stereotactic radiotherapy (SSRT)[END_REF].

Between the most recent works on detectors applied for MRT studies, PRESAGE® dosimetry plastics are able to visualize the delivered dose in a 3D volume [START_REF] Doran | Establishing the suitability of quantitative optical CT microscopy of PRESAGE®radiochromic dosimeters for the verification of synchrotron microbeam therapy[END_REF] but the combination of the spatial resolution of the material with the optical computed tomography system used for the analysis still does not allow to properly resolve the peak dose of the microbeams [START_REF] Mcerlean | Assessment of optical CT as a future QA tool for synchrotron x-ray microbeam therapy[END_REF]. Silicon strip detectors constantly evolved since their first realization and their sensitive volume was recently decreased to a thickness of 5 µm when used in passive mode [START_REF] Fournier | X-Tream quality assurance in synchrotron X-ray microbeam radiation therapy[END_REF]. The PTW microDiamond™ detector (PTW-Freiburg GmbH, Freiburg, Germany) has a nominal circular active volume of 1.1 mm radius and 1 μm thickness able to resolve the microbeam dose distribution if scanned perpendicular to the microbeam extension [START_REF] Livingstone | Characterization of a synthetic single crystal diamond detector for dosimetry in spatially fractionated synchrotron x-ray fields[END_REF]. These electronic dosimeters allow punctual dose measurements in real time, but their geometry requires a precise and time-consuming alignment procedure if a micrometric dose resolution is desired and they are not suitable for delivering dose maps in a reasonable amount of time. In the last years, the use of fibre-optical dosimeters for the dose evaluation in MRT was tested [START_REF] Archer | X-ray microbeam measurements with a high resolution scintillator fibre-optic dosimeter[END_REF] as well, and only recently a 20 µm dose resolution was achieved opening the possibility of microbeams characterization [START_REF] Archer | Synchrotron X-ray microbeam dosimetry with a 20 micrometre resolution scintillator fibre-optic dosimeter[END_REF].

The current study focuses on the use of radiochromic films to evaluate doses on a micrometre scale. Radiochromic films are already used for dose evaluation in conventional RT for relative dosimetry and quality assurance [START_REF] Mack | High precision film dosimetry with GAFCHROMIC® films for quality assurance especially when using small fields[END_REF][START_REF] Devic | Radiochromic film dosimetry: Past, present, and future[END_REF], and they are a promising detector candidate for MRT with the ability to detect 2-dimensional dose maps with less than 5 µm spatial resolution. Films do not allow real-time dosimetry, but they are relatively easy to handle and used for dose measurement in plastic phantoms [START_REF] Devic | Reference radiochromic film dosimetry: Review of technical aspects[END_REF].

Until recently, the read-out of the films irradiated with microbeams was performed using microdensitometers [START_REF] Martínez-Rovira | Development and commissioning of a Monte Carlo photon beam model for the forthcoming clinical trials in microbeam radiation therapy[END_REF][START_REF] Crosbie | A method of dosimetry for synchrotron microbeam radiation therapy using radiochromic films of different sensitivity[END_REF] but a new way involving optical microscopes was suggested for the analysis of the radiochromic film [START_REF] Nariyama | Spectromicroscopic film dosimetry for high-energy microbeam from synchrotron radiation[END_REF][START_REF] Bartzsch | Micrometer-resolved film dosimetry using a microscope in microbeam radiation therapy[END_REF] to overcome the limitations in accuracy, precision and time consumption in the use of the microdensitometers. The nominal specifications of a recent optical microscope easily surpass the specification of a microdensitometer but an actual film read-out comparison between the two instruments and their protocols for the read-out operation was never performed. As first step in the acceptance of the most recent protocol, it is fundamental to verify that the use of the microscope can effectively be considered a valid implementation for film dosimetry without introducing measurement differences.

The aim of this study is to compare a Joyce Loebl 3CS microdensitometer (MDM) and an inverted Zeiss Axio Vert.A1 optical microscope (OM) in terms of accuracy, reproducibility, stability and versatility for the analysis of radiochromic film on a micrometric scale. The comparison of the dose values obtained from the same set of irradiated films using the two devices will be presented to verify the absence of major differences in film dosimetry for MRT. Focus will be placed on the description of the most recent and updated protocols developed for both instruments, explaining how the same read-out approach must be adapted at the devices' limits and characteristics. The OM will be proven to be superior in reliability with a less time-consuming readout procedure. Furthermore, profiting from the much higher spatial resolution of the OM in both spatial directions, we analysed films irradiated with the pencil beam configuration. Pencil beam irradiation is based on a beam cross section of only 50 × 50 µm 2 and dose evaluation is even more challenging compared to microbeams because the spatial fractionation is present in two directions. This level of analysis cannot be performed with other detectors tested in MRT while it can be reached using radiochromic film dosimetry.

MATERIALS

Radiochromic Films

Gafchromic® is a brand of radiochromic films developed by Ashland industries, designed for quantitative measurements of absorbed radiation dose. The Gafchromic® HD-V2 series was used in this study because its dynamic dose range is between 10 and 1000 Gy (Ashland®, 2015) and matches the dose delivered in MRT. The HD-V2 film is composed of only two layers: an active layer nominally 12 μm thick and a polyester substrate nominally 97 µm thick. The active material includes the diacetylene monomer, a radio-sensitive active monomer that polymerises after irradiation and causes a colour/opacity change [START_REF] Cheung | Post-irradiation colouration of Gafchromic EBT radiochromic film[END_REF][START_REF] Williams | Radiochromic film dosimetry and its applications in radiotherapy[END_REF] related to the dose delivered.

One important feature of Gafchromic® films is their near tissue equivalence [START_REF] Williams | Radiochromic film dosimetry and its applications in radiotherapy[END_REF]. Moreover, they do not require post-exposure chemical treatment. However, it was observed that they need about 24 hours after irradiation until the optical density stabilises [START_REF] Butson | Radiochromic film for medical radiation dosimetry[END_REF]. Under these conditions, real-time dose measurements are not possible, and the read-out of the film is done 1-4 days after the exposure. Studies on the granularity of the materials used in the film fabrication demonstrate that the resolution of this HD-V2 detector is about 2 μm to 3 μm [START_REF] Bartzsch | Micrometer-resolved film dosimetry using a microscope in microbeam radiation therapy[END_REF], sufficient to sample the 50 µm wide beams used.

Microdensitometer

Until recently, the analysis of MRT irradiated films was only performed using a microdensitometer (MDM) such as the Joyce Loebl Automation Ltd 3CS available at the ESRF. This instrument was designed for the measurement of optical densities through a true double-beam light system, where two light beams from a halogen source are split into two different paths: one passes through the film while the second is attenuated by an optical wedge of known density. A photomultiplier receives alternately the two beams and the output signal produced by the amplifier is sent to the motor connected to the wedge. If an intensity difference between the two beams is present, the motor moves the wedge and equalises the intensity such that the position of the wedge corresponds to the optical density of the sample.

For the analysis, the film is loaded on a mechanical platform that can be moved along the plane in steps of 1.2 μm. Under standard working conditions, the horizontal resolution (perpendicular to the microbeams and along the scanning direction) is between 20 μm and 25 μm [START_REF] Crosbie | A method of dosimetry for synchrotron microbeam radiation therapy using radiochromic films of different sensitivity[END_REF][START_REF] Morri | Experimental dosimetry of high dose rates X-ray microbeams fields for the MRT[END_REF], calculated from the modulation transfer function (MTF). The resolution parallel to the microbeams is defined by the minimum iris dimension of the MDM optics and it cannot be reduced below 2 mm due to mechanical restrictions.

Optical Microscope

Since 2013, an inverted Zeiss Axio Vert.A1 optical microscope is installed at the ESRF for film read out. It is equipped with a light-emitting diode (LED) source (wavelength range from 400 nm to 700 nm) and a motorised scanning stage that controls the position with a precision of 0.1 μm. A tunable condenser aperture determines the depth of focus which can be adjusted between 2 μm and 45 μm. The aperture dimension influences also the light intensity on the specimen but the image brightness can be regulated by changing the LED voltage or the exposure time of the charge-coupled device (CCD) camera.

A Zeiss AxioCam MRm camera [START_REF] Imaging | Microscopy from Carl Zeiss AxioCam MRm from Carl Zeiss -More Information at Low Light Intensities[END_REF] is mounted to acquire images with 12-bit grey level using a CCD with a basic resolution of 1388 x 1040 and a pixel size of 6.45 μm in both directions. The Zeiss EC Plan-Neofluar objectives are mounted with magnification/numerical aperture of 5x/0.16 to obtain a nominal resolution for each image pixel of 1.29 μm on the film surface. With this setup, the objective provides a field of view of 1.79 × 1.34 mm 2 but a wide film area can be obtained by using the Zeiss mosaiX software that is able to coordinate the motorized scanning stage movement with the camera acquisition and stitch images together to increase the final field of view. The optical resolution of the OM was measured by the MTF normalized to the value at zero frequency and it is 0.1 at ~330 line pairs per millimeter, corresponding to a spatial resolution of 1.52 µm.

METHODS

Film preparation and irradiation

In this work, the largest field used was 3 × 3 cm 2 therefore a single sheet of Gafchromic® film was cut into several pieces avoiding the possible variation in sensitivity response between sheets. To reduce the structural defects due to the cut, a 5 mm wide border of non-irradiated film was left on the margin of the field edges. Due to the manufacturing process, variations in the film sensitivity are higher along the short edge of the film sheet [START_REF] Niroomand-Rad | Comparison of uniformity of dose response of double layer radiochromic films (MD-55-2) measured at 5 institutions[END_REF]. For this reason, the set of film for calibration and measurements was always taken from the same column as defined by the long sheet edge of the same film sheet. Every piece of film was marked in order to keep the orientation constant during irradiation and read-out operations. Films were prepared and handled in dimmed light, wrapped in aluminium foil for storage and exposed to ambient light less than one minute during irradiation preparation. For the irradiation, the film was placed in a solid water phantom consisting of 12 plates of homogeneous water-equivalent RW3 material [35], measuring 30 × 30 × 1 cm 3 each.

Absolute dosimetry was performed for broad beams (BB) of 2 × 2 cm 2 size following as closely as possible the IAEA TRS 398 protocol [START_REF] Musolino | Absorbed Dose Determination in External Beam Radiotherapy: An International Code of Practice for Dosimetry Based on Standards of Absorbed Dose to Water[END_REF] for low energy photons. The polychromatic X-ray irradiation spectrum used had a peak energy of 88 keV and a mean energy of 105 keV. This spectrum configuration of the ID17 biomedical beamline at the ESRF is used in pre-clinical experiments; the x-ray spectrum and a detailed discussion are presented in the work of Crosbie et al. [START_REF] Crosbie | Energy spectra considerations for synchrotron radiotherapy trials on the ID17 bio-medical beamline at the European Synchrotron Radiation Facility[END_REF]. Absolute dose measurement is performed placing an ionization chamber inside the water-equivalent phantom at the same position where films irradiation takes place; in this way, the energy spectrum reaching both detectors is the same and the energy variation of the spectrum along the propagation inside the phantom [START_REF] Tomic | Monte Carlo simulations of different CT X-ray energy spectra within CTDI phantom and the influence of its changes on radiochromic film measurements[END_REF] does not affect the results. A calibrated PTW Pinpoint chamber TW31014 [START_REF] Ptw | Ionizing Radiation[END_REF] in combination with a PTW electrometer (Webline) were used to measure the radiation dose at 2 cm depth from the surface of the RW3 phantom [START_REF] Fournier | Absorbed dose-towater protocol applied to synchrotron-generated x-rays at very high dose rates[END_REF][START_REF] Bräuer-Krisch | Medical physics aspects of the synchrotron radiation therapies: Microbeam radiation therapy (MRT) and synchrotron stereotactic radiotherapy (SSRT)[END_REF]. Following the absolute dosimetry procedure, five Gafchromic® films were irradiated for calibration in a limited dose range with BB doses of 60 Gy, 90 Gy, 100 Gy, 110 Gy and 140 Gy.

The irradiation with microbeams were defined starting from simulated output factor (OF) values. Nowadays, differences between dose simulation for MRT and experimental data vary up to 10% and further investigations are ongoing to reduce the gap. This complex problem is not addressed in this paper and previous simulated data were only used as indication for the microbeams irradiation. To match the delivered peak dose on the film around the centre of the established calibration curve, the irradiations were based on the OF values defined using Monte Carlo (MC) radiation transport simulations [START_REF] Clavel | Microdosimetrie dans le cadre des assais precliniques de la radiotherapie a microfausceaux[END_REF]. For the definition of the single microbeam irradiation, a couple of motorized slits were used to tune the desired beam width. Three films were irradiated with 25 µm, 50 µm and 75 µm wide beams.

Valley dose measurements were previously performed [START_REF] Martínez-Rovira | Development and commissioning of a Monte Carlo photon beam model for the forthcoming clinical trials in microbeam radiation therapy[END_REF][START_REF] Bräuer-Krisch | Medical physics aspects of the synchrotron radiation therapies: Microbeam radiation therapy (MRT) and synchrotron stereotactic radiotherapy (SSRT)[END_REF][START_REF] Bartzsch | Micrometer-resolved film dosimetry using a microscope in microbeam radiation therapy[END_REF] and they present an important investigation since the absolute valley dose can be considered as the limiting factor for dose prescription in MRT [START_REF] Smyth | The normal tissue effects of microbeam radiotherapy: What do we know, and what do we need to know to plan a human clinical trial?[END_REF]. Due to the big difference between peak and valley dose values within the same array of microbeams, the two quantities were evaluated separately, on two different films, because it was not possible to have both values within the defined calibration curve at the same time. For a peak dose value centred within the calibration curve, the valley dose value is too small to be properly measured. The delivered peak dose was therefore increased on the second film to match the valley dose value within the calibration curve. For an array of microbeams, the ratio between peak and valley dose, the so called PVDR value, can be simulated using MC [START_REF] Martínez-Rovira | Development and commissioning of a Monte Carlo photon beam model for the forthcoming clinical trials in microbeam radiation therapy[END_REF] and it provides an estimate of the factor to be applied to increase the peak dose and bring the valley dose within the calibration curve.

For the valley dose evaluation, three films were exposed to an array of 50 μm wide microbeams spaced by a 400 µm pitch, and three field dimensions were used: 10 × 10 mm 2 , 20 × 20 mm 2 and 30 × 30 mm 2 . The overall horizontal extension of the radiation fields was defined by a couple of motorized slits. After the beam passes these slits it is split up by a multislit collimator (MSC) [START_REF] Bräuer-Krisch | Characterization of a tungsten/gas multislit collimator for microbeam radiation therapy at the European Synchrotron Radiation Facility[END_REF], i.e. a tungsten carbide block machined with 50 µm apertures that spatially fractionates the beam coming from the X-ray source. The minimum vertical extension of the microbeams was defined by a fixed aperture 500 µm high while the final height of the irradiated beam was obtained by vertically scanning the phantom in front of the synchrotron source. The combination of a motorized stage with a fast shutter specifically designed for MRT irradiation allows the definition of the beam height with sub-millimetric precision [START_REF] Renier | A white-beam fast-shutter for microbeam radiation therapy at the ESRF[END_REF]. An example of a film irradiated with microbeams is reported in figure 1.

Figure 1. HD-V2 film irradiated with an array of 50 µm wide microbeams spaced by a 400 µm pitch; the irradiated field is 1 × 1 cm 2 . A reference coordinate system is defined.

x y

mm

More recently, pencil beam irradiations have been performed with the purpose to increase healthy tissues tolerance, as compared to planar microbeams at identical peak doses, opening the possibility for whole brain radiotherapy [START_REF] Schültke | Pencilbeam Irradiation Technique for Whole Brain Radiotherapy: Technical and Biological Challenges in a Small Animal Model[END_REF]. Pencil beams are a few micrometre wide squared beamlets arranged into a two-dimensional array, resulting in higher PVDR. The realization of pencil beams is possible combining the use of two MSC to create the beam fractionation in both vertical and horizontal direction. This extreme configuration brings even more challenges in dosimetry at micrometric scale and so far, no experimental dose evaluation is present in literature. With the new dosimetry protocol based on the use of the OM, the dose analysis of pencil beams has become technically possible.

During previous in-vivo pre-clinical experiments [START_REF] Schültke | Pencilbeam Irradiation Technique for Whole Brain Radiotherapy: Technical and Biological Challenges in a Small Animal Model[END_REF], a whole mouse brain was irradiated with pencil beams and for dose verification, film dosimetry was performed. The films were positioned at 3 mm depth in a water equivalent phantom of similar size as a mouse head and two films were irradiated separately adjusting the exposure time to have peak and valley dose values centred in the calibration curve, as done for microbeams irradiation. The same radiation field size used by Schültke et al. [START_REF] Schültke | Pencilbeam Irradiation Technique for Whole Brain Radiotherapy: Technical and Biological Challenges in a Small Animal Model[END_REF] was reproduced: a 20 mm wide and 8 mm high field of pencil beams with peaks of 50 × 50 μm 2 area, spaced by a 400 μm pitch. An example of a Gafchromic® film irradiated with pencil beams is reported in figure 2.

Figure 2. Gafchromic® film irradiated with pencil beams.

Read-out operation and calibration curves

The films were first analysed with the OM and then with the MDM since the microscope read-out is faster and causes less degradations to the films. Two metal masks with a central hole were pressing the film piece flat allowing for a focused image across the camera image. The use of glass slides is not recommended because the reflection of the light between the smooth polyester substrate of the HD-V2 film and the glass creates an interference pattern, known as Newton rings, visible in the acquired images that degrades the image quality.

A picture of the setup is reported in figure 3.

mm

Figure 3. Metal masks used for the film readout. In the bottom right corner, one piece of radiochromic film is held between two metal masks.

The calibration curve was produced fitting the points with the equation suggested by D. Lewis [START_REF] Micke | SU-E-I-63: Multi-Channel Film Dosimetry with Non-Uniformity Correction[END_REF]:

( ) = + / ( -)
where G is the grey value, D(G) the corresponding dose and a, b, c are fitting parameters. This rational function fits properly the calibration points related to the film optical density measured by using transmitted or reflected light [START_REF] Aldelaijan | Comparison of dose response functions for EBT3 model GafChromic TM film dosimetry system[END_REF]. A non-irradiated film was included in the calibration curve because its intrinsic opacity was used to optimise the parameters of the OM avoiding saturation in the acquired images.

The MDM read-out requires a simplified method due to the limited stability of the instrument related to the mechanical drift of the optical components during read-out. It was not possible to identify the technical reason of this behaviour, but it was observed that the focus position of the instruments varies considerably after a few minutes. The value obtained from the same film analysed after 15 minutes without changing any parameter of the device setup can vary by up to 20%. For this reason, using the MDM, the calibration curve was reduced to four homogeneous films (60 Gy, 90 Gy, 100 Gy and 140 Gy) to decrease the time necessary for one read-out.

To discriminate reliable measurements from erroneous ones, one of the BB films was read out twice, at the beginning and at the end of the series and measurements were only considered if the mean optical densities of the two scans show a difference of less than 1%.

Image processing

The read-out procedure of the Gafchromic® films with the OM requires image processing including shading correction, correction of the CCD thermal noise and background light in the room. A white field image F was acquired without any sample loaded and a dark image B was acquired with the light source switched off. For each raw image of the film, the correction was performed in two steps: first, the raw image was corrected pixelby-pixel by the camera noise (dark image B(k,i)):

( , ) = ( , ) -( , )
Where Iraw is the raw image and argument (k,i) denotes the pixel position. Second, the flat field correction (white field image, F(k,i)) was made dividing pixel-by-pixel the image Ib(k,i) by the reference image F(k,i)

and multiplying the result by the average of the pixels in reference image ( , ). It is possible to summarise the corrections of the defects by the formula below:

( , ) = ( , ) ( , ) ( , )
For the films irradiated with BB, the grey value in the centre of the field was correlated with the measured dose of the PTW pinpoint chamber, which was also positioned in the centre of the field during the beam characterization. The dose delivered in the central 3 × 3 mm 2 area of the irradiated field was therefore considered for the creation of the calibration curve.

To align by eye the microbeams on the OM stage is not easy and a mistake of a few degrees affects the dose profile evaluation; therefore, the film image is digitally rotated. The precision in the selection of the microbeams angle from the digital image was checked by the evaluation of the dose profiles steepness for a range of angles around the selected one. Typically, the error made by the user is not more than +/-0.1 degree and this does not affect the dose evaluation.

As reported in the Materials section, the OM spatial resolution is higher compared to the film one and a dose analysis based on a single pixel value is not reasonable because of statistical uncertainties. For this reason, to reduce statistical uncertainties, the average over several pixels was used. For the evaluation of micro-planar beams, the resolution perpendicular to the microbeams (along the x-axis, Fig. 1) needs to be in the order of a few micrometres while parallel to the beam this condition can be relaxed. Consequently, the image was averaged over regions as high as the camera image (1040 pixels ≈ 1.3 mm) and 4 pixels in width equivalent to 5.16 µm. In the case of pencil beam irradiations, an average over a 25 × 25 pixel area was used to evaluate the peak dose and a region ~60 × 60 pixels, centred between four adjacent peaks, was selected for the valley dose evaluation.

The combined use of the motorized stage with the Zeiss software of the OM allows the quick acquisition of images up to 10 cm 2 . However, digital elaboration is required because at the edges of the full image a clear loss of focus was visible with consequent invalidation of the dose analysis. This problem can be caused by a non-perfect alignment of the stage with the optics during the scan or by the presence of film bending when held by the stage. To solve this problem, a "multi-focus approach" was used: the entire area of interest was acquired several times changing each time the focus position. Subsequently, using post image processing, the focused part of each image was selected and reassembled into the final image.

Errors and uncertainties estimation related to the read-out procedure

In the definition of the measurement uncertainties related to the read-out procedure, the major contributions are given by the statistical noise linked to the microscopic inhomogeneities of the film and the intrinsic noise of the CCD camera [START_REF] Bartzsch | Micrometer-resolved film dosimetry using a microscope in microbeam radiation therapy[END_REF]. For the OM read-out, the statistical noise was reduced by averaging over a number of pixels N, as described in the previous section. A smaller N value corresponds to a higher resolution but also to higher uncertainties.

The uncertainty was computed starting from the film images used for the definition of the calibration curve, linking the errors directly to the different dose values. They were divided into blocks Bi containing N pixels each and for every block Bi the mean of its pixels was calculated. In the ideal case, all blocks should have the same pixels mean value, but the defects of the film create differences. For this reason, the uncertainty for a specific dose was determined as the standard deviation of the mean value of the blocks Bi. The same approach was used for the evaluation of the MDM uncertainties where the standard deviation was calculated starting from the dose profile values of the films irradiated with BB. The uncertainties are the same for a defined dose for peak and valley because they are defined for every single point of the dose profile. Averaging over several peaks and valleys, and assuming they have the same dose value, this corresponds to an increase of the sampling area used for the evaluation; therefore, the statistical noise should decrease.

In addition, calibration errors due to the difference between experimental data and the fitted curve were considered. Finally, to quantify the reproducibility and reliability of the data acquired, all the films were analysed three times and the standard deviation of the values obtained from each film was evaluated and used as reproducibility indicator. One single read-out procedure starts with the OM/MDM setup, followed by the calibration curve definition and final evaluation of the film irradiated with microbeams. The reproducibility assessment does not include the uncertainty component relating to individual film variation, and additional components would be required to assess the overall uncertainty of the microbeam dose measured with film.

RESULTS

Single microbeam evaluation

An example of the single microbeam peak dose profile obtained during one single acquisition with the two instruments, the MDM and the OM, is reported in figure 4. Each film is scanned three times with both instruments to obtain better statistics and the mean between the measurements was used for the analysis. Both instruments resulted in similar dose measurements for the peak dose, but the MDM profiles are more irregular than the dose profiles obtained with the OM. The resolution of the MDM is not sufficient to resolve microbeams less than 20 µm wide. In figure 4, the excess dose delivered to the right part of the beam is probably caused by reflection of photons at the inner surface of the collimating slit. Table 1 summarises the results of the measurements: each value corresponds to the average over three measurements and the differences between the measurements for both instruments are less than 3%.

Table 1. Single microbeam dose evaluation

The FWHM is evaluated for the three microbeams and the average value of the three measurements for each film is reported in table 2. The difference between the two instruments' results for FWHM values is around

Dose [Gy] Distance [µm]
2.5% for beams which are more than 50 μm wide, while it increases to around 10% for the thinner beams. The FWHM is systematically lower for the microscope.

FWHM

Valley dose evaluation

In figure 5, an example of a single acquisition for valley profiles obtained with the two instruments is shown. The valley dose is measured in the centre of 1 x 1 cm 2 , 2 x 2 cm 2 and 3 x 3 cm 2 field sizes with the analysis reported in table 3. Even if the profiles measured with the MDM are very noisy as compared to the profile measured with the OM, the results are comparable with dose differences being less than 5.5%. 3. Valley dose measurement for a microbeams array; peak dose used is 6900 Gy for 1×1 cm 2 field, 3800 Gy for 2×2 cm 2 field and 2700 Gy for 3×3 cm 2 field.

Errors and uncertainties

The different components of errors and uncertainties are reported in table 4. The statistical noise was evaluated for each dose value of the calibration curve and regarding the OM, uncertainties were evaluated for the different resolutions used during the analysis while for the MDM, uncertainties were evaluated only for the best achievable resolution. Typically, the films irradiated with low dose present lower percentage errors compared to film irradiated with high dose. Calibration errors due to the difference between experimental data and the fitted curve are in the order of 1-1.5 Gy for OM calibration curves, while for the MDM are higher, 3-5 Gy.

Uncertainties Microscope Microdensitometer

Statistical noise 20.0×2000.0 µm 2 0.5% -2.0% 8% -10% The overall total uncertainty related to the read-out operation is estimated as square root of the sum of the squared contributions of statistical noise, calibration errors, and reproducibility. For the microscope, errors below 3.0% are possible for measurements performed averaging the dose on a 5 × 1300 μm 2 area. In the case of higher resolution, the uncertainty increases to around 6.1% -7.7% for resolutions of 50 × 50 μm 2 . For the MDM, the total error is usually between 8.8% and 14.6% with a resolution of 20 × 2000 μm 2 . Regarding reproducibility and reliability, the variation between the final dose values for the OM is no more than 1.2%, while for the MDM is around 3-7%. For the total read-out uncertainty related to PVDR values, the statistical noise and reproducibility contribution must be considered twice because a separate read-out is necessary to obtain peak and valley dose values, while the calibration error need to be counted only once in case peak and valley dose are chosen to be the same for irradiation on two separate films.

Pencil beams

By using a microscope read-out, the experimental evaluation of pencil beams dose distributions was possible.

In figure 6a and 6b, two digital images are shown, which are used for peak and valley dose evaluation respectively.

Figure 6a. Digital microscope image of pencil beams for peak dose evaluation. Figure 6b. Microscope image of pencil beams irradiated with 65000 Gy in the peak; the mean valley dose between pencil beam is 142 Gy.

The peak dose is evaluated as average between the 12 peaks reported in Figure 6a where areas of ≈35x35 µm 2 in the middle of the square peak fields are used and a dose of 111 ± 7 Gy is determined, with maximum standard deviation between the twelve measured peaks of 3.1%. The film for valley evaluation, reported in figure 6b, displays a pattern of vertical and horizontal lines connecting the individual pencil beams fields.

Using the microscope read-out protocol, all the regions were evaluated separately. Unfortunately, in this case, the regions of the film along the line connecting the pencil beam areas received a too high dose, resulting in saturation of the film. For this reason, these parts of the film where not available for the analysis. Just the areas defined in the centre of four adjacent peaks were considered. In this case, the dose corresponds to the average obtained in a 75x75 µm 2 wide areas and it was determined to be 142 ± 5 Gy, with maximum standard deviation between the evaluated regions of 1.0%. The corresponding experimental PVDR at 3 mm depth for this configuration is 313.

DISCUSSION

Materials discussion

Gafchromic® films are accurate high-resolution radiochromic detectors for MRT and are particularly practicable due to their radiological tissue equivalence. They are widely used for benchmarking and quality assurance checks in pre-clinical research with their capability of visualizing the entire irradiated field at micrometric scale. In this work, HD-V2 Gafchromic® film were selected to be irradiated because they mainly differ from other type of radiochromic films, such as EBT3, in their dynamic dose range.

Irrespective of many drawbacks and technical difficulties, the MDM has been the only instrument available for film analysis in MRT for more than 15 years. However, the OM offers an easier to use read-out and it is simpler to manage compared to the MDM. For example, from past experience, independent observers have difficulties to agree on the focus point at the MDM, while the focus at the OM is easier to define because the film image is displayed live on a monitor.

Another important advantage in the use of the OM is the possibility to define a precise point of the field during the analysis, like the central point of the field, thanks to the combination of the CCD camera, the motorized stage and the Zeiss mosaiX software. With the MDM, the user can just align by eye the film under the optics, with a precision not better than some millimetres and this kind of error affects the measurements.

In addition, the resolution of the MDM along the direction perpendicular to the microbeams is really close to the dimension of the microbeams and in the other direction the best resolution is only 2 mm. Such low resolution is at the limit of usability for the dosimetry of microbeams and does not allow to evaluate the twodimensional dose distribution of pencil beam fields. Here, we present an experimental measurement of a pencil beam dose distributions.

Methods discussion

Regarding the time necessary for the read-out of a series of films, there is a large difference between the two instruments, mainly due to their different mechanical stability. The MDM suffers from a drift of the focus position during the readout and it cannot be adjusted during the procedure, therefore the user is often forced to repeat the measurement. To obtain data from just one film irradiated with microbeams, hours of attempts are necessary. In addition, for each film irradiated with microbeams, the calibration film scans must be repeated because it is practically not possible to read out more than 5 or 6 films before mechanical drifts occur in the instrument. In contrast, using the microscope, all films need to be scanned only once. The image acquisition of the calibration films takes about 20 minutes, and 5 minutes are necessary for each film irradiated with microbeams.

The possibility to read out more films in less time using the OM allows to define a calibration curve with more points in the desired dose range or extend the dose range for the analysis. For the calibration curve of the MDM, no more than four films could be used for a 100 Gy wide dose range while with the OM a calibration curve over a 250 Gy range is possible with steps of 25 Gy. The limits of the microscope calibration curve range are connected to the dynamic range capability of the camera sensor, more specifically to the bright or dark sensitivity limits of the camera. Out of the defined dose range, saturation to the bright sensitivity limit of the camera occurs when trying to measure lower doses while the camera read-out is too noisy when trying to measure higher doses. Adjusting the LED voltage and the camera exposure time it is possible to select and shift the range of the calibration curve and an upper limit of the instrument for films irradiated with around 500 Gy was found.

The use of the OM digital camera provides 2-dimensional images and an image processing protocol has been developed to allow a versatile film analysis. The correction of the image artefacts is performed and the precise vertical alignment of the microbeams is digitally defined to remedy the non-precise manual placement of the films on the mechanical stage. At the end, the absence of a perfectly flat stage and/or sample surface is corrected by rearranging pieces of several image acquisitions with different focal points.

Results discussion

Despite the large technical and specification differences between the MDM and the microscope, the dose values obtained as average of three evaluations for each film differ no more than 4%. Even if the read-out with the two instruments seems to be quite equivalent, the maximum standard deviation between the measurements obtained with the MDM reaches 7% while values between 0.4% and 1.2% were obtained with the OM. The uncertainty estimation reveals that the microscope provides less error prone data compared to the MDM.

Working with the OM, a non-symmetric shape of the single microbeam irradiation was found when the single beam is defined by a pair of motorized slits which allow adjusting the gap according to the desired beam width. The presence of a higher dose at one edge of the beam, as visible in figure 4, is likely caused by a non-perfect parallel alignment of the slit, which leads to a reflection from the slit surface. This problem is not present using the multi slit collimator (MSC) to define the beams and a future project involving the design of a dedicated MSC for OF evaluation with single and fixed apertures of different widths is under development.

The read-out of the film irradiated in pencil beam configuration can be considered successful even if the uncertainties increase because of the high resolution used. Working in the pencil beam scenario the differences between simulated and experimental data increase: the simulation values obtained modelling the experimental set-up used and reported in previous work [START_REF] Schültke | Pencilbeam Irradiation Technique for Whole Brain Radiotherapy: Technical and Biological Challenges in a Small Animal Model[END_REF] present a PVDR value of 530, well above 313 evaluated with the films. Further investigation is required for pencil beams irradiation, starting from the presence of a dose grid visible in figure 6b. This grid is probably related to photon transmission in correspondence of the areas where only one of the two MSCs blocks the photons. Due to this "leakage", the valley dose measured on the film increases, potentially explaining part of the difference in the PVDR values.

CONCLUSIONS

To determine the optical density at micrometric scale of radiochromic films irradiated with microbeams in the order of 50 µm wide, equivalent results with less read-out uncertainties are obtained using an OM instead of a MDM. The microscope allows more precise, accurate and reliable dose evaluation and it is a preferable and more versatile device able to overcome the limitations of the MDM. Using the OM, the 2-dimensional dose distribution of an irradiated film can be evaluated and the study of beams with cross section of 50 × 50 µm 2 can be considered successful. The areas that can be analysed with the OM are considerably larger than with the MDM, allowing future implementation of film dosimetry protocols for the benchmark against dose simulations over the entire radiation field. These fundamental results in dosimetry for MRT clearly validate the transition from the previous protocol defined using the MDM to the one developed using the OM.

Figure 4 .

 4 Figure 4. Peak dose profile of one single microbeam of 75 µm width (FWHM) obtained with the microdensitometer (blue) and the microscope (red)

Figure 5 .

 5 Figure 5. Valley dose distribution profile obtained with the microdensitometer (blue) and the microscope (red)

  Fig. 6aFig.6b

Table 2 .

 2 Single microbeam FWHM evaluation.

		Microdensitometer	Microscope
	Film 75 µm	78.3 µm	77.4 µm
	Film 50 µm	52.4 µm	51.6 µm
	Film 25 µm	28.8 µm	25.8 µm

Table 4 .

 4 Microscope and microdensitometer uncertainties connected to the read-out. Uncertainties data not available for the MDM due to the lack of high-resolution capability have been marked with N/A.

	5.2×1341.6 µm 2 (4×1040 pixels)	0.7% -2.5%	N/A
	51.6×51.6 µm 2 (40×40 pixels)	3.0% -5.3%	N/A
	25.8×25.8 µm 2 (20×20 pixels)	5.5% -7.5%	N/A
	Calibration errors (between 60 and 140 Gy)	0.5% -2.5%	2% -8%
	Reproducibility	0.4% -1.2%	3% -7%
	Combined uncertainties
	20.0×2000.0 µm 2	2.5% -2.6%	8.8% -14.6%
	5.2×1341.6 µm 2 (4×1040 pixels)	2.6% -3.0%	N/A
	51.6×51.6 µm 2 (40×40 pixels)	3.9% -5.5%	N/A
	25.8×25.8 µm 2 (20×20 pixels)	6.1% -7.7%	N/A

Dose [Gy]Distance[µm]

ACKNOWLEDGMENTS

This work was partially funded by the COST action TD1205 Innovative methods in radiotherapy and radiosurgery using synchrotron radiation.

CONFLICT OF INTEREST DISCLOSURE

The authors declare not to have any conflict of interest.