Baumann et al. Clin Transl Allergy (2019) 9:24 https://doi.org/10.1186/s13601-019-0262-y Clinical and Translational Allergy REVIEW Open Access Skin microdialysis: methods, applications and future opportunities—an EAACI position paper Katrine Y. Baumann1,2, Martin K. Church3, Geraldine F. Clough4, Sven Roy Quist5,6, Martin Schmelz7, Per Stahl Skov1,8, Chris D. Anderson9, Line Kring Tannert8, Ana Maria Giménez‑Arnau10, Stefan Frischbutter3, Jörg Scheffel3 and Marcus Maurer3* Abstract Skin microdialysis (SMD) is a versatile sampling technique that can be used to recover soluble endogenous and exog‑ enous molecules from the extracellular compartment of human skin. Due to its minimally invasive character, SMD can be applied in both clinical and preclinical settings. Despite being available since the 1990s, the technique has still not reached its full potential use as a tool to explore pathophysiological mechanisms of allergic and inflammatory reac‑ tions in the skin. Therefore, an EAACI Task Force on SMD was formed to disseminate knowledge about the technique and its many applications. This position paper from the task force provides an overview of the current use of SMD in the investigation of the pathogenesis of chronic inflammatory skin diseases, such as atopic dermatitis, chronic urticaria, psoriasis, and in studies of cutaneous events during type 1 hypersensitivity reactions. Furthermore, this paper covers drug hypersensitivity, UVB-induced- and neurogenic inflammation, and drug penetration investigated by SMD. The aim of this paper is to encourage the use of SMD and to make the technique easily accessible by providing an overview of methodology and applications, supported by standardized operating procedures for SMD in vivo and ex vivo. Keywords: Microdialysis, Cutaneous, Inflammation, Interstitial, Dermal What is skin microdialysis? reflecting target tissue status as an alternative to repeated To perform skin microdialysis (SMD) thin tubular dialy- blood sampling. This monitoring situation usually, sis membranes are inserted into the dermis or the sub- because of the insertion technique used, involved place- cutis and perfused at a low speed with a physiological ment of probes in the subcutaneous layer of the skin. It solution (the perfusate) (Fig. 1). Endogenous or exog- allowed early detection of a metabolic deterioration in, enous molecules soluble in the extracellular fluid diffuse for instance, an intensive care patient with sepsis. With into the tubular microdialysis membrane and are col- time, the technique began to be used for specific stud- lected in small vials for analysis. The duration and timing ies elucidating the role of the actual subcutaneous tis- of the collected dialysate samples allows kinetic evalua- sue [1]. Specific placement of probes into the dermal tion of the events occurring in the tissue. layer opened the way for studies of inflammatory events In broad terms, microdialysis has been applied in most prominently driven by that part of the skin. SMD two scenarios. The first and in fact the original use of has also been applied in drug discovery and pharmacoki- the technique aimed to gain continual, real-time data netic/pharmacodynamic (PK/PD) studies (for reviews see [2–4]) and in the study of percutaneous penetration of potentially harmful exogenous agents from the envi- *Correspondence: marcus.maurer@charite.de 3 ronment [5]. SMD has the advantage over other tissue Department of Dermatology and Allergy, Charité – Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany sampling techniques of being minimally invasive, and it Full list of author information is available at the end of the article is well tolerated by human volunteers. As a consequence © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Baumann et al. Clin Transl Allergy (2019) 9:24 Page 2 of 13 Syringe filled with perfusate Microdialysis probe Inlet tubing Epidermis Syringe pump Dermis Sample Fig. 1 Schematic representation of SMD (here with a linear microdialysis probe). The membrane is inserted into the tissue from which it allows recovery of soluble molecules (in red) when perfused using a microperfusion pump. © Niels Peter Hell Knudsen it has been widely used to study normal, diseased and The use of scanning laser Doppler imaging in combi- experimentally perturbed skin function [6–8]. In the nation with SMD has allowed the investigation of quan- 25 years since the introduction of the technique, over 800 titative real-time temporal and spatial changes in blood papers have been published on SMD. flow in response to other potential inflammatory media- The purpose of this paper is to review how the use tors in the skin. For example, the H­ 1-antihistamines ceti- of SMD has helped to improve our understanding of rizine and loratadine were shown to inhibit wheal and chronic inflammatory skin conditions and skin inflam- flare responses to bradykinin as well as histamine [14]. mation in general. We also hope to encourage the use of The obvious conclusion from this study was that brady- SMD for investigating the many remaining unanswered kinin induces histamine release, particularly as cetirizine questions on the mechanisms of cutaneous inflammation was shown not to interact with either the ­B1 or B­ 2 brady- especially in the field of skin allergy and skin hypersensi- kinin receptors [15]. However, microdialysis showed that tivity reactions. this was not the case in most individuals [14]. Instead, the results of the use of SMD suggest that there is co-opera- How SMD has helped us to understand skin tivity between bradykinin and histamine ­H1-receptors in inflammation and skin inflammatory disorders the dermis. A similar scenario has been found with plate- What SMD taught us about cutaneous type 1 let activating factor [16]. hypersensitivity reactions In a further study [7], the cytokine response to dermal The wheal and flare response to dermal provocation with allergen provocation was studied in 11 allergic individu- allergen is a well-established model of type 1 hypersen- als over a period of 6 h using two linear SMD probes sitivity. SMD is an ideal technique with which to inves- inserted 1 cm apart in the volar skin of the forearm. tigate the mechanisms of this response by identification Allergen injection caused a significant rise in interleukin of the inflammatory mediators generated in vivo in real- (IL)-6 within 30 min. However, increased tumor necrosis time [9–11]. factor (TNF)-α levels were found in only 3 individuals at The mechanism of the early phase response has been this time. At both 3 and 6 h, significantly elevated levels investigated by insertion of microdialysis probes into of IL-1α, IL-1β, IL-6 and IL-8 were found. Interestingly, different areas of the wheal and flare [12]. The results IL-6 and IL-8 were also raised at the site 1 cm from the showed that histamine was released in the wheal but not allergen injection. In contrast, adhesion molecule expres- the flare. Further studies [13] showed that the neurogenic sion and leukocyte infiltration were elevated only at the flare was mediated primarily by calcitonin gene related allergen injection site, suggesting a complex relationship peptide (CGRP). between cytokine generation and cellular events in aller- gic inflammation. A further fascinating outcome of this Baumann et al. Clin Transl Allergy (2019) 9:24 Page 3 of 13 study was that, when looked at individually, the cytokine methotrexate. Methotrexate bioavailability was higher profile of every person was different illustrating the need in psoriasis plaques than in non-lesional skin but highly for further human SMD studies to unravel the complexi- individual [29]. Several SMD studies analyzed histamine ties of immunological skin responses. release examined by high-performance liquid chroma- tography (HPLC) in psoriatic skin and showed a ten- How SMD has helped our understanding of atopic fold increase in lesional compared to non-lesional skin dermatitis [30]. Ranitidine was able to reduce histamine release in The particular strength of SMD in studies of atopic lesional skin [31]. dermatitis (AD) is combining analysis of local media- tor concentrations with the assessment of sensory Chronic urticaria: what did we learn from SMD studies? perception. Intra-probe delivery of mast cell-degran- SMD is ideally suited for the investigation of inducible ulating codeine provokes local wheal and C-fiber urticaria, because its signs and symptoms (itchy wheals activation resulting in an axon reflex erythema and and angioedema) can be induced by skin provocation histamine-independent itch in patients with AD [17]. with relevant triggers. Most SMD studies have investi- This response is mediated probably via increased mast gated cold urticaria, first in 1995 when histamine release cell tryptase activating nociceptors via proteinase-acti- was demonstrated in wheals elicited by an ice cube test vated receptors [17, 18]. Higher iron and ascorbic acid in cold urticaria patients [32]. Nuutinen et al. reported as wells as prostanoid levels were found in the skin of similar results [33] but failed to demonstrate leukotriene AD patients [19, 20] whereas no significant increase ­C4 ­(LTC4) release. They concluded that the absence of in nerve growth factor was detected [21]. Intra-probe ­LTC4 could be due to the activation of skin mast cells by delivery of prostaglandin (PG)E2 [22] and low pH per- an IgE-independent mechanism. Taskila et al. also failed fusate [23] were successfully used to show the sensi- to detect L ­TC4 by SMD in volunteers challenged with tized neuronal itch response to normally painful stimuli stinging nettles [34]. In contrast, Horsmanheimo et al., in patients with AD. also using SMD, measured increase of ­LTC4 in volunteers In pain research, SMD has been used to assess the after controlled challenge with mosquito bites [35]. link between local mediator release and neuronal sen- SMD has also been used to monitor the therapeutic sitization in more detail [24, 25]. Thus, using improved effect of desensitization or antihistamines in cold urti- analytical methods, SMD will successfully identify caria patients by measuring histamine or cytokine release clinically relevant local mediator concentrations in AD in response to cold provocation. For example, Tannert such as large signaling peptides, local hormones and et al. investigated cold desensitization in cold urticaria lipids. patients [36] and found, before desensitization, histamine release by SMD in wheals elicited by cold challenge but Insights from SMD studies on psoriasis no histamine release upon a subsequent codeine skin Cytokine profiles of SMD-derived samples analyzed by test. After successful desensitization, no histamine was bead-based multiplex immunoassays have been used released at cold-exposed skin sites while codeine chal- to monitor changes in the micromilieu of the skin of lenge resulted in histamine release indicating that the patients with psoriasis for up to 24 h. Post-equilibration mechanism of cold desensitization is unlikely to be due levels at 17–24 h showed that granulocyte-macrophage to depletion of histamine in skin mast cells. In a study by colony-stimulating factor (GM-CSF) and TNF-α levels Krause et al., the beneficial effect of using increased doses were elevated in psoriasis compared with healthy sub- of the non-sedating antihistamine bilastine in patients jects [26]. In another study, levels for IL-2, IL-6, IL-18 with cold urticaria was shown [37]. SMD in cold chal- and IL-23 were elevated in dialysates of lesional versus lenged patients with cold urticaria treated with increased non-lesional skin prior to therapy. Clinical improvement doses of bilastine showed significantly reduced late phase under 12 weeks of continuous oral therapy with fumaric histamine and proinflammatory cytokine (IL-6 and IL-8) acid esters paralleled the reduced concentrations of these release. cytokines in dialysates [27]. The same group reported that IL-1β was elevated in dialysates from psoriasis The use of SMD in studies of drug hypersensitivity plaques compared with non-lesional skin, and levels were and ultraviolet B (UVB)‑induced skin responses reduced under successful anti-psoriatic fumaric acid Skin provocation testing with drugs or UVB radiation esters therapy [28]. A pharmacokinetic profile was elab- allows for assessing skin responses by SMD, for exam- orated in patients with psoriasis using SMD comparing ple to sample the real-time release of biomarkers. For lesional and non-lesional skin with intravenous micro- drug hypersensitivity studies, the skin can be challenged dialysis after administration of oral or subcutaneous directly by performing skin tests with the drug close to Baumann et al. Clin Transl Allergy (2019) 9:24 Page 4 of 13 the probe to elicit a wheal that develops across the probe. in neurogenic vasodilation in human skin [43]. While Experimentally, the impact of treatment on mediator histamine release following nociceptor activation has release can be studied and compared to placebo treat- been shown in rodent skin [44], this is not the case in ment. While SMD is well suited for assessing drugs humans within the axon reflex flare area [45]. SP-release that elicit immediate reactions in the skin, delayed drug in humans is less pronounced as compared with rodents reactions mediated by T cell activation are more chal- and there is no neurogenic protein extravasation in lenging to study by SMD. Nevertheless, SMD is promis- healthy volunteers [46, 47]. However, there are chronic ing, because mediators of different sizes can be sampled pain conditions in which SP-upregulation might enable by use of probes with varying molecular weight cut-off neurogenic protein extravasation also in humans [48] (MWCO). So far, SMD has been used to a limited extent even in the non-affected limb [49, 50]. More tardy neu- in the investigation of drug hypersensitivity. In one study, ropeptide degradation increases neurogenic vasodilation SMD in penicillin allergic patients demonstrated that [51] and might be of clinical importance in chronic pain histamine was only released in the minority of positive conditions. intracutaneous tests with penicillin [38]. SMD has been used in several studies of the release of How SMD is used to study drug penetration prostanoids and cytokines following UVB radiation [24, and distribution 39–42]. In one study, SMD was performed 24 h before Following on the use of SMD to investigate metabolic and 24 h after UVB challenge, and dialysates were sam- events in the human body, the study of percutaneous pled at 8-h intervals [39]. Probe placement 24 h prior to penetration of exogenous substances has been argu- radiation induced an unspecific proinflammatory, trau- ably the first dermatological area studied by microdi- matic response driven by IL-6, IL-8, TNF-α and IL-1β, alysis [52]. Several ways of delivery to the skin of drugs whereas UVB radiation showed a mixed ­TH1/TH2-related and other agents of interest have been studied [5, 53, cytokine profile, with a late IL-4 and IL-10 dominant 54], and SMD has also been used in animal models and ­TH2-driven shift. A more recent SMD study elaborated a ex vivo models. Topics for discussion and development kinetic profile for inflammation markers 16 h prior and have clustered around membrane permeability and 48 h post radiation [41]. Dialysates were collected at 4-h the “stickiness” of molecules, the analytical sensitivity intervals and analyzed for 5- and 8-iso-PGF2α, 9α,11α- required and issues of lipophilicity and tissue binding of PGF2α and P ­ GE2 using gas-chromatography/mass-spec- individual target molecules. Several useful reviews are trometry and for IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, available illustrating important, generic methodological IL-10, TNF-α, Fas ligand (FasL), interferon-γ-induced issues (e.g. [2, 55–59]). Attempts to fulfill the develop- protein 10 (IP-10), monocyte chemoattractant protein 1 mental and regulatory needs concerning bioavailability (MCP-1), regulated on activation normal T cell expressed and bioequivalence of topical pharmaceuticals have, over and secreted (RANTES), eotaxin, and GM-CSF using the last two decades, involved the use of either tape a multiplex cytometric-bead-array. As a result, 3 peaks stripping (so called DPK—dermato-pharmaco-kinetic with synchronic release of ­TH1-directed inflammatory modelling) or SMD, with the current emphasis on the cytokines and prostanoids could be detected post-UVB latter. In vivo protocols involving SMD have been sug- radiation: an early phase (4–12 h), an intermediate phase gested with numbers of subjects (and thus costs) that (16–24 h) and a late phase (32–40 h). A T ­ H2-directed are far lower than the traditional clinical trial methodol- cytokine response was detectable during intermedi- ogy, which has previously been necessary to demonstrate ate and late phases. This study indicated that the release bioequivalence of a new topical pharmaceutical product. of cytokines and prostanoids is synchronized and that More recently, the open flow variant of SMD involving a slow T ­ H1-to-TH2 shift occurs up to 40 h after UVB outer membranes that are fenestrated rather than being radiation. reliant on pores, has been the subject of intensive devel- opment of technique, application and data interpretation The use of SMD to study neurogenic inflammation [60, 61]. The developments have been necessary in order The activation of peptidergic nociceptors in the skin to standardize potential sources of variability in data such causes the release of neuropeptides that dilate precapil- as blood flow and other interindividual variability. Since lary arterioles (calcitonin-gene related peptide, CGRP) both standard SMD and open flow microperfusion have and increase leakiness of post-capillary venules (sub- been used to demonstrate the chronology of expression stance P, SP), i.e. neurogenic inflammation. SMD has of inflammatory and other tissue indicators, the integra- been used to apply neuropeptides and assess dose– tion of pharmacokinetic and pharmacological data seems response functions for neurogenic inflammation and entirely possible and logical. itch including studies that suggest a role of nitric oxide Baumann et al. Clin Transl Allergy (2019) 9:24 Page 5 of 13 The study of penetration of harmful agents into the Selection of perfusion medium (usually isotonic skin is also possible. The microdosing nature of micro- saline without or with additives to aid analyte recov- dialysis (low concentrations and small areas of skin for ery) and rate of perfusion are driven by the nature of provocation rather than larger areas of skin or systemic the solute to be recovered and by the study aims (see administration) is an important ethical advantage for Table 1). Volume requirements of the assay platform studies on e.g. percutaneous penetration of pesticides or are also highly influential in determining probe perfu- similar potentially toxic agents. In extension, SMD may sion rates and dialysate collection protocols. The recent have uses in studies of dose (dermal delivery) of allergens development of microfluidic platforms for the continu- and even of their fate (by metabolism) in living skin. ous on-line sampling of dialysate may go some way towards addressing this in future [72, 73]. SMD techniques and methodology The members of the EAACI Task Force on SMD have In vivo SMD developed a standard operating procedure (SOP) for There is an extensive and wide ranging technical litera- performing in vivo SMD studies, which is provided in ture on SMD that focusses on key methodological con- the online supplement of this report (see In vivo SMD siderations. The most important of these relate to choice SOP, Additional file 1). of skin site, probe selection and insertion, and to probe perfusate and perfusion rate [62]. Ex vivo SMD The most frequently used site for in vivo SMD is the The application of SMD in studies of human ex vivo volar forearm [5, 16, 63–65] (Fig. 2), although other skin was first described in 1996 by Petersen et al. sites have been used to study regional variations in skin using the technique to measure release of histamine responses [66], pruritic responses (e.g. in the scalp [67]), from skin-resident mast cells in response to intrader- and in the assessment of skin graft and flap viability mal injection of chemokines [74]. Since then, excised [68]. When using the forearm, usually only one arm is human skin has been studied by microdialysis to meas- used at a time to allow the participant some freedom of ure other endogenous molecules [75] and for investiga- movement. tions of cutaneous drug penetration [76–80]. Dermal Probe selection is driven by the physicochemical inflammatory reactions have been studied by SMD in properties of the analyte recovered; its size, charge and animal ex vivo skin [81], but this application has not hydrophilicity determining the MWCO of the dialysis yet been described for human skin specimens. Hence, membrane as well as the construction of the probe itself. human ex vivo skin has an unused potential in trans- Linear probe membranes have a smaller diameter than lational studies, as it facilitates investigations of pre- the larger concentric probes, which are typically used for clinical compounds with respect to their cutaneous systemic drug delivery studies. As a result, narrow inser- effects and metabolism, while reflecting the natural tion needles are used for linear probes, which cause less biological variation in contrast to studies relying on insertion trauma. Insertion of concentric probes, on the cell lines or skin substitutes. However, it is important other hand, requires only one penetration of the skin. to acknowledge that the lack of blood flow and inner- It is important to acknowledge that most, but not all, vation hampers studies of systemic influence on cuta- in vivo human SMD studies use local anesthetic. This neous responses. Furthermore, clearance of molecules has the advantage of reducing the pain of probe insertion from the tissue is also altered ex vivo, and the duration (and encouraging study participation) and limiting the of experiments is limited by the viability of skin speci- wounding response. However, its long action may con- mens. Similar to SMD performed in vivo, an ex vivo found studies in which changes in local blood flow and/or setup must be carefully optimized based on the target neurogenic responses are of interest or where they may analyte(s) (see Table 1). A consensus protocol for per- influence the interpretation of pharmacological studies. forming ex vivo SMD studies, developed by the EAACI There are very few reports about the time necessary for Task Force on SMD members, is provided in the online recovery from trauma associated with probe insertion supplement of this report (see Ex vivo SMD SOP, Addi- or about the specific endogenous compounds generated tional file 2). as a result of this trauma. A 2 h recovery period is usu- ally adopted to allow local blood flow to return to normal The strengths and limitations of SMD (indicating the resolution of the immediate erythematous SMD is a well validated and safe technique that has been response to trauma) [69, 70] or a normal flare response extensively used to sample intrinsic dermal chemicals, to histamine to be re-established (indicating the recovery such as mediators of inflammation, from the skin, and from the local anesthetic) [71]. to deliver extrinsic substances, such as drugs, to the skin. Microdialysis has made major contributions to our Baumann et al. Clin Transl Allergy (2019) 9:24 Page 6 of 13 A B C D E F G H I J Fig. 2 An example of the SMD procedure. a Priming of microdialysis probes prior to insertion, b topical application of local anesthesia, c intradermal insertion of guide cannulas, d introduction of probes through the guide cannulas, e SMD setting and basal sampling, f skin challenge, g skin site before challenge, h wheal and flare reaction in response to intradermal challenge, i collection of dialysates—here in microtubes, j alternative collection of dialysates—here directly into sampling wells. Please refer to the SOPs (in vivo SMD SOP and ex vivo SMD SOP, provided as Additional files 1 and 2, respectively) for a detailed description of the SMD procedure. Pictures are reproduced with kind permission from Line Kring Tannert and Marcus Maurer Table 1 Factors affecting recovery of analytes from the skin by microdialysis Factors influencing analyte Effect Recommendations/considerations References recovery Analyte-related Molecular weight, shape and solubility The size and water solubility of the analyte affect its diffusion In order to recover large molecules high MWCO probes must be [86–88] through the microdialysis membrane as well as analyte diffusion used and the microdialysis setup should be carefully optimized in the tissue environment. Small molecules are easily recovered (refer to the parameters listed in this table) to obtain the highest whereas high molecular weight molecules are more difficult to relative recovery possible sample Baumann et al. Clin Transl Allergy Molecular stability Analyte stability in dialysates is important to consider for optimal A refrigerated fraction collector can be used during microdialysis [89] storage and subsequent analyte detection in the samples sampling. Dialysates containing labile analytes should be stored accordingly (e.g. at − 80 °C) Other physicochemical properties (e.g. The physicochemical properties of an analyte affect its adherence To improve recovery of highly lipophilic analytes a lipid emul‑ [86, 88, 90–93] lipophilicity) to the tissue environment (e.g. the extracellular matrix) and the sion can be used for perfusion. Non-specific adsorption can be (2019) 9:24 probe components. Such adherence will diminish the fraction of decreased by adding a blocking-protein such as albumin to the soluble analyte and thus analyte recovery perfusate Technical Perfusion flow rate The in vitro recovery is inversely dependent on the flow rate as this The flow rate should be chosen based on the target analyte(s) and [82, 86, 87, 89, 94] affects the extent of equilibrium established across the semi-perme‑ the volume requirement of subsequent analyses able membrane For small molecules: 1–5 µl/min For macromolecules: < 1.0 µl/min Sampling intervals The length and number of sampling intervals affect the temporal The sampling interval should be set based on a combination of [88, 89] resolution and may also affect the molecular stability of the analyte the volume requirements of subsequent analyses, the temporal depending on the collection procedure resolution required and the analyte stability Membrane molecular weight cut-off The membrane cut-off is defined as the molecular weight at which The optimal molecular cut-off the microdialysis membrane is partly [94–96] 80% of the molecules are unable to pass the membrane, therefore determined by the molecular weight of the analyte but also the it is not an absolute measure. It relates to the membrane pore size requirement for sample purity. Since differences in membrane and thus has a great impact on analyte recovery, which is (in part) material will affect the recovery, more probe types should be correlated to its size and shape tested Probe type Microdialysis probes used in the skin are usually of the linear or the The choice of probe type relates to commercial availability and to [58, 94, 97] concentric type. The probe construction determines the maximal the anatomical site to be sampled. The degree of insertion trauma membrane area available for diffusion. Furthermore, the design induced must be considered and so must the potential discom‑ affects the outer probe diameter and the number of penetration fort for human subjects participating in in vivo studies sites required for insertion, thus the degree of tissue trauma induced Linear probes can either be purchased or self-made in the lab. Self- by probe insertion. Linear probes penetrate the skin twice and have fabrication of probes allows for customization of the membrane a smaller outer diameter in contrast to concentric probes, which length and material penetrates the skin once, as the inlet and outlet are placed in parallel, but at the cost of a larger outer diameter Probe/membrane material The probe materials (including the membrane composition) affect Inert probe materials should preferably be used. Different mem‑ [89, 98, 99] potential non-specific adsorption of molecules to probe components brane- and tubing material can be tested with respect to diffusion as well as analyte interaction with the membrane. This is often an of molecules across the membrane and the degree of non-spe‑ issue for lipophilic molecules cific adsorption Membrane length/surface area The analyte recovery increases with increasing membrane surface area In general, the membrane length should be maximized (e.g. span‑ [89, 94, 98] available for diffusion ning 2 cm intradermally). However, it must be adjusted to the tissue in which the sampling is carried out Page 7 of 13 Table 1 (continued) Technical Perfusate composition The composition of the perfusion medium affects recovery of mol‑ A physiological solution is generally used. Additives such as albumin [89, 91, 93, 94, 98, ecules and water movement across the probe or dextran might improve analyte recovery and stability, while 100–102] ­preventing fluid leakage from the probe (a frequent issue for probes with a high molecular weight cut-off ) and decrease ­non-specific adsorption to probe components Temperature In theory, diffusion increases with temperature, which can lead to a The temperature in vivo is determined by the target tissue but [89, 98, 103] higher recovery. However, the physicochemical properties of the can be manipulated in ex vivo and in vitro experiments. In vitro Baumann et al. Clin Transl Allergy analyte (especially for proteins) might influence the temperature ­validation studies should reflect the temperature of the end setup dependency (e.g. be adjusted to body temperature) Biological Tissue characteristics The tortuosity of the tissue fluid space will affect analyte diffusion and To obtain valid results from probe calibration studies these should [88, 89, 97, 104] therefore the recovery. Furthermore, the tissue metabolism, degree be carried out in a matrix representing the tissue in which the (2019) 9:24 of vascularization as well as cell internalization of the analyte will microdialysis sampling will performed ultimately affect its recovery Tissue trauma Transient local tissue trauma is caused by intradermal insertion of An equilibration period (e.g. 2 h for in vivo studies) can be included [8, 69, 70, 89, 97, microdialysis probes, both in vivo and ex vivo, leading to a release of to allow wash out of trauma-induced molecules. However, the 105, 106] trauma-associated molecules (e.g. histamine) and changes in blood equilibration period depends on the experimental read-out and flow (in vivo). Furthermore, trauma may be induced when processing proper controls must be included if the molecule of interest is also skin specimens for ex vivo studies induced by dermal trauma Blood flow The local blood flow affects wash-out/clearance of solutes and thus The axon reflex-mediated increase in blood flow is of particular [70, 97, 106] recovery of both exogenous or endogenous molecules at the importance for studies on penetration or endogenous release of sampling site small molecules as the magnitude of clearance is directly related to blood flow; consider control of flow by laser Doppler imaging Ex vivo studies may be affected by the absence of blood flow Application site Distribution of various cell types (e.g. mast cells) varies across different The volar forearm is most frequently used for in vivo studies as it [5, 16, 56, 63–66, body sites, as does tissue thickness, which may affect the results easily accessible, has a low frequency of hairs and presents with a 72, 97, 107, 108] obtained if SMD is performed in different body areas flat surface area. This body site may thus serve as a “standard” when seeking to compare between different experiments Anesthetic procedure (in vivo) Local anesthetics can be used to ease the discomfort related to the It must be considered whether an anesthetic agent applied affects [71, 89, 105, 109, probe insertion procedure. However, the use of anesthetics (such as the cutaneous reactions subject to investigation. When EMLA 110] EMLA cream with an occlusive dressing) might affect the skin barrier cream is used a 40–60 min application period is recommended and the physiological process investigated to minimize discomfort. Cooling of the insertion area serves as an alternative Probe implantation depth The implantation depth of the microdialysis probe affects which cell The probe depth should be fixed and variations must be diminished. [77, 89, 97] types will be in close vicinity of the probe, as cells are not evenly Therefore, intradermal insertion of probes should preferably be distributed through the skin layers, and may thus affect the response carried out by the same skilled operator throughout a study. The measured. For studies of percutaneous absorption this parameter precise probe depth can be assessed using ultrasound scanning must be controlled carefully Page 8 of 13 Baumann et al. Clin Transl Allergy (2019) 9:24 Page 9 of 13 Table 2 Strengths and limitations of SMD for the study of inflammation and allergy in the skin (see text for further information and references) Strengths Limitations • Can be used with equal efficacy in both healthy and diseased skin • Introduction of a microdialysis probe into the skin is a (minimally) invasive • Allows dynamic, real-time assessment of intercellular messengers procedure necessitating appropriate controls in order to assess whether • Provides objective information on signaling pathways between resident particular molecules are truly related to the disease state under investiga‑ inflammatory cells, sensory nerves and the vasculature tion or have been generated as part of the tissue response to probe • Used to explore the temporal and spatial variations in mediator or implantation metabolic profiles • Despite application of local anesthetic, the insertion of microdialysis • Probes with different MWCO allow the recovery of small molecules (e.g. probes may be associated with mild pain histamine) away from metabolic enzymes and the recovery of larger • Diffusion of chemicals in the skin, particularly large molecules, is very molecules (e.g. cytokines and neuropeptides) limited. Consequently, maximum probe perfusion rates need to be low • Use of low perfusion rates and/or the addition of colloid or lipid (0.1–5 µl/min) emulsions to the probe perfusate enhances solute recovery and limit • Small recovery volumes and low concentrations of recovered chemicals hydrostatic fluid loss make the use of assays with an appropriate sensitivity an absolute neces‑ • Can be used in conjunction with other techniques, such as laser Doppler sity blood flux imaging and/or tissue histology in studies of dermal inflam‑ • Microdialysis recovery of high-molecular-mass substances, such as matory and allergic reactions cytokines and neuropeptides, has proved particularly problematic • Probe insertion is easy for the physician and relatively pain free, particu‑ • Reduced recovery due to reduced solute bioavailability within the tissue larly when inserted under local anesthetic space or to the adherence of bioactive molecules onto the material of the • Probes may be left in place for up to several days implanted probe • Probes leave no scarring • Long-term studies require the use of portable pumps, which may affect • Analysis platforms are continually improving e.g. development of micro‑ the ability of study participants to move freely depending on the dura‑ fluidic platforms for continuous on-line assay of dialysates tion and the anatomical site • Experienced personnel are required for optimal results (e.g. to insert probes at a consistent depth) understanding of dermal inflammatory disease and has It is advisable to contact local ethical authorities to driven innovative thinking in PK/PD drug studies. Still, clarify the need for ethical approval of ex vivo SMD stud- there are limitations related to the technique that must ies and to obtain informed consent from skin donors. be acknowledged and considered before using SMD to study inflammation and allergy. Table 2 summarizes Outlook: future applications of SMD some of the technique’s strengths and limitations. SMD has great potential to become a standard and routinely used technique not only in experimental der- Ethical considerations in SMD studies matology and allergology but also in the pharmaceu- The use of SMD in humans has been permitted through tical and cosmetic industry. It provides quantifiable the approval of microdialysis probes by the US Food and data of the mediators involved in the inflammatory Drug Administration (FDA) and the European Union response in situ. SMD has already been successfully Conformite Europeene (CE) [82]. applied in studies of inflammatory skin conditions A significant benefit of SMD is its minimally invasive including immediate hypersensitivity, urticaria, atopic nature compared to alternative tissue sampling tech- dermatitis and drug hypersensitivity. Other skin dis- niques. Still, whenever research is carried out on humans eases for which SMD can help to better characterize or human tissue, potential ethical issues must be consid- pathogenic mechanisms include bullous diseases, mas- ered. The ethical requirements related to the use of SMD tocytosis, autoinflammatory disorders, and allergic depend on the setting in which the technique is applied. contact dermatitis. In vivo studies are always subject to ethical approval from As SMD can be performed in vivo as well as ex vivo, local Ethics Committees (in accordance with the Declara- it can help to replace artificial skin models and animal tion of Helsinki [83]). Whether the sourcing and use of studies to perform skin penetration studies in drug human ex vivo skin for research purposes should also be development. Although SMD is minimally invasive it approved by an ethical committee might be a question must always be performed following ethical require- of the anonymity status of the donor. Acquisition of fully ments in human research. The combination of micro- anonymized tissue may in some countries be exempt dialysis with advanced imaging techniques such as from ethical approval, however, with the entry into confocal microscopy or life imaging of the skin in 3D force of the European General Data Protection Regula- [84] may offer new perspectives. Clinical studies may tion (GDPR) the true anonymity of the donor might be benefit from SMD as it allows for in situ monitoring brought into question. of molecules with a short in vivo half live (for example Baumann et al. Clin Transl Allergy (2019) 9:24 Page 10 of 13 bradykinin) or mediators that are produced only locally Author details 1 RefLab ApS, Copenhagen, Denmark. 2 Department of Immunology and/or in low amounts meaning that changes may not and Microbiology, University of Copenhagen, Copenhagen, Denmark. result in noticeable alterations in plasma/serum levels. 3 Department of Dermatology and Allergy, Charité – Universitätsmedizin SMD offers the possibility to extract these mediators Berlin, Charitéplatz 1, 10117 Berlin, Germany. 4 Faculty of Medicine, Univer‑ sity of Southampton, Southampton, UK. 5 Clinic of Dermatology, Otto- from the site where they are produced. von-Guericke University, Magdeburg, Germany. 6 Skin Center MDZ, Mainz, In addition to the recovery of mediators from Germany. 7 Department of Experimental Pain Research, CBTM, Medical Faculty the skin, SMD probes can be used to administer Mannheim, Heidelberg University, Heidelberg, Germany. 8 Odense Research Center for Anaphylaxis (ORCA), Department of Dermatology and Allergy drugs locally and monitor cutaneous responses [6]. Center, Odense University Hospital, Odense, Denmark. 9 Department of Clini‑ SMD could be applied in studies that involve spe- cal and Experimental Medicine, Linköping University, Linköping, Sweden. cial excipients to deliver active molecules into dif- 10 Department of Dermatology, Hospital del Mar, Institut Mar d’Investigacions Mèdiques, Universitat Autònoma, Barcelona, Spain. ferent layers of the skin such as transdermal delivery systems (laser-assisted drug delivery or micro needle Acknowledgements patches), nanoparticles or the bicosome technology We are grateful to the support of EAACI. This is the work of an EAACI Task Force on Skin Microdialysis. [78, 85]. Microdialysis is not restricted to the skin. Other tissues such as the heart, liver, embryonic tis- Competing interests sue, brain or muscles have been successfully studied by KYB is employed as Industrial Ph.D. student by RefLab ApS and PSS is acting as research consultant for RefLab ApS and EP Medical. The other authors have no microdialysis. competing interests to declare. Current efforts to improve SMD are focused on making this technique more precise and easier to use Availability of data and materials Not applicable. and more sensitive. There is a need for a broad spec- trum of probes and for portable syringe pumps that Consent for publication allow for long-term studies over several days without Not applicable. hospitalization. Advances in miniaturization of pumps Ethics approval and consent to participate and in microfluidics-based collection and analysis will Not applicable. make it even more convenient for the tested subject, Funding particularly in extended sampling studies. Technologi- We are grateful to support by EAACI and the study was funded, in part, by cal advances will help to improve detection thresholds financial support from EAACI. and thus allow for the detection of trace amounts in even lower volumes [72, 73]. Publisher’s Note SMD is a valuable technology for research in derma- Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. tological allergology and beyond, and awareness of and further improvements in SMD will increase its use and Received: 16 March 2019 Accepted: 25 March 2019 utility in experimental and clinical studies. Additional files References 1. Coelho M, Oliveira T, Fernandes R. Biochemistry of adipose tissue: an endocrine organ. Arch Med Sci. 2013;9:191–200. Additional file 1. In vivo SMD SOP. A standard operating procedure 2. Kreilgaard M. Assessment of cutaneous drug delivery using microdialy‑ (SOP) for sampling of soluble molecules from human skin in vivo using sis. Adv Drug Deliv Rev. 2002;54(Suppl.):S99–121. microdialysis—a protocol from the EAACI Task Force on Skin Microdialysis. 3. Brunner M, Langer O. Microdialysis versus other techniques for Additional file 2. Ex vivo SMD SOP. 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