Table 1 Factors affecting recovery of analytes from the skin by microdialysis

Molecular weight, shape and solubility

The size and water solubility of the analyte affect its diffusion through the microdialysis membrane as well as analyte diffusion in the tissue environment. Small molecules are easily recovered whereas high molecular weight molecules are more difficult to sample

In order to recover large molecules high MWCO probes must be used and the microdialysis setup should be carefully optimized (refer to the parameters listed in this table) to obtain the highest relative recovery possible

[86,87,88]

Molecular stability

Analyte stability in dialysates is important to consider for optimal storage and subsequent analyte detection in the samples

A refrigerated fraction collector can be used during microdialysis sampling. Dialysates containing labile analytes should be stored accordingly (e.g. at − 80 °C)

[89]

Other physicochemical properties (e.g. lipophilicity)

The physicochemical properties of an analyte affect its adherence to the tissue environment (e.g. the extracellular matrix) and the probe components. Such adherence will diminish the fraction of soluble analyte and thus analyte recovery

To improve recovery of highly lipophilic analytes a lipid emulsion can be used for perfusion. Non-specific adsorption can be decreased by adding a blocking-protein such as albumin to the perfusate

[86, 88, 90,91,92,93]

Perfusion flow rate

The in vitro recovery is inversely dependent on the flow rate as this affects the extent of equilibrium established across the semi-permeable membrane

The flow rate should be chosen based on the target analyte(s) and the volume requirement of subsequent analyses

For small molecules: 1–5 µl/min

For macromolecules: < 1.0 µl/min

[82, 86, 87, 89, 94]

Sampling intervals

The length and number of sampling intervals affect the temporal resolution and may also affect the molecular stability of the analyte depending on the collection procedure

The sampling interval should be set based on a combination of the volume requirements of subsequent analyses, the temporal resolution required and the analyte stability

[88, 89]

Membrane molecular weight cut-off

The membrane cut-off is defined as the molecular weight at which 80% of the molecules are unable to pass the membrane, therefore it is not an absolute measure. It relates to the membrane pore size and thus has a great impact on analyte recovery, which is (in part) correlated to its size and shape

The optimal molecular cut-off the microdialysis membrane is partly determined by the molecular weight of the analyte but also the requirement for sample purity. Since differences in membrane material will affect the recovery, more probe types should be tested

[94,95,96]

Probe type

Microdialysis probes used in the skin are usually of the linear or the concentric type. The probe construction determines the maximal membrane area available for diffusion. Furthermore, the design affects the outer probe diameter and the number of penetration sites required for insertion, thus the degree of tissue trauma induced by probe insertion. Linear probes penetrate the skin twice and have a smaller outer diameter in contrast to concentric probes, which penetrates the skin once, as the inlet and outlet are placed in parallel, but at the cost of a larger outer diameter

The choice of probe type relates to commercial availability and to the anatomical site to be sampled. The degree of insertion trauma induced must be considered and so must the potential discomfort for human subjects participating in in vivo studies

Linear probes can either be purchased or self-made in the lab. Self-fabrication of probes allows for customization of the membrane length and material

[58, 94, 97]

Probe/membrane material

The probe materials (including the membrane composition) affect potential non-specific adsorption of molecules to probe components as well as analyte interaction with the membrane. This is often an issue for lipophilic molecules

Inert probe materials should preferably be used. Different membrane- and tubing material can be tested with respect to diffusion of molecules across the membrane and the degree of non-specific adsorption

[89, 98, 99]

Membrane length/surface area

The analyte recovery increases with increasing membrane surface area available for diffusion

In general, the membrane length should be maximized (e.g. spanning 2 cm intradermally). However, it must be adjusted to the tissue in which the sampling is carried out

[89, 94, 98]

Perfusate composition

The composition of the perfusion medium affects recovery of molecules and water movement across the probe

A physiological solution is generally used. Additives such as albumin or dextran might improve analyte recovery and stability, while 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

[89, 91, 93, 94, 98, 100,101,102]

Temperature

In theory, diffusion increases with temperature, which can lead to a higher recovery. However, the physicochemical properties of the analyte (especially for proteins) might influence the temperature dependency

The temperature in vivo is determined by the target tissue but can be manipulated in ex vivo and in vitro experiments. In vitro validation studies should reflect the temperature of the end setup (e.g. be adjusted to body temperature)

[89, 98, 103]

Tissue characteristics

The tortuosity of the tissue fluid space will affect analyte diffusion and therefore the recovery. Furthermore, the tissue metabolism, degree of vascularization as well as cell internalization of the analyte will affect its recovery

To obtain valid results from probe calibration studies these should be carried out in a matrix representing the tissue in which the microdialysis sampling will performed ultimately

[88, 89, 97, 104]

Tissue trauma

Transient local tissue trauma is caused by intradermal insertion of microdialysis probes, both in vivo and ex vivo, leading to a release of trauma-associated molecules (e.g. histamine) and changes in blood flow (in vivo). Furthermore, trauma may be induced when processing skin specimens for ex vivo studies

An equilibration period (e.g. 2 h for in vivo studies) can be included to allow wash out of trauma-induced molecules. However, the equilibration period depends on the experimental read-out and proper controls must be included if the molecule of interest is also induced by dermal trauma

[8, 69, 70, 89, 97, 105, 106]

Blood flow

The local blood flow affects wash-out/clearance of solutes and thus recovery of both exogenous or endogenous molecules at the sampling site

The axon reflex-mediated increase in blood flow is of particular importance for studies on penetration or endogenous release of 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

[70, 97, 106]

Application site

Distribution of various cell types (e.g. mast cells) varies across different body sites, as does tissue thickness, which may affect the results obtained if SMD is performed in different body areas

The volar forearm is most frequently used for in vivo studies as it easily accessible, has a low frequency of hairs and presents with a flat surface area. This body site may thus serve as a “standard” when seeking to compare between different experiments

[5, 16, 56, 63,64,65,66, 72, 97, 107, 108]

Anesthetic procedure (in vivo)

Local anesthetics can be used to ease the discomfort related to the probe insertion procedure. However, the use of anesthetics (such as EMLA cream with an occlusive dressing) might affect the skin barrier and the physiological process investigated

It must be considered whether an anesthetic agent applied affects the cutaneous reactions subject to investigation. When EMLA cream is used a 40–60 min application period is recommended to minimize discomfort. Cooling of the insertion area serves as an alternative

[71, 89, 105, 109, 110]

Probe implantation depth

The implantation depth of the microdialysis probe affects which cell types will be in close vicinity of the probe, as cells are not evenly distributed through the skin layers, and may thus affect the response measured. For studies of percutaneous absorption this parameter must be controlled carefully

The probe depth should be fixed and variations must be diminished. Therefore, intradermal insertion of probes should preferably be carried out by the same skilled operator throughout a study. The precise probe depth can be assessed using ultrasound scanning

[77, 89, 97]