**2. Microdialysis**

Present-day microdialysis is the result of several decades of technological advancement. An understanding of the principles underlying the technique is an essential prerequisite to appreciating its potential uses and limitations.

#### **2.1 Principles, uses and limitations**

#### **2.1.1 Principles**

Microdialysis enables sampling of the extracellular fluid (ECF). A microdialysis catheter or probe with a semi-permeable membrane at its tip is placed into the tissue of interest. Perfusate with a similar composition to the ECF is then slowly and continuously infused through the catheter. Substances of interest diffuse across the semi-permeable membrane into the catheter, and the resulting dialysate is collected in microvials, which are changed at regular intervals and subsequently analysed (see Figure 1).

Diffusion of substances from the ECF, across the membrane, and into the flowing perfusate, is often incomplete. Thus, the concentration of a substance within the dialysate represents a fraction of that in the ECF. The *extraction fraction* or *relative recovery* is defined as the ratio of a substance's concentration in the dialysate (*Cdialysate*) compared to the actual concentration in the ECF (*CECF*).

Relative Recovery = Cdialysate / CECF x 100%

A number of variables may influence the relative recovery including the flow rate, the semipermeable membranes length and pore size, and the properties of the substance of interest itself (see Table 1) (de Lange et al. 1997, de Lange, de Boer and Breimer 1999, Hutchinson et al. 2000, Benjamin et al. 2004, Helmy et al. 2009, Chefer et al. 2009, Blakeley et al. 2009).

Clinical Microdialysis in Glioma 147

**Factor Effect**  Microdialysis dependent

Context dependent

In-vitro studies have calculated the relative recovery for specific molecules under different experimental conditions in which the concentration of a substance in the external medium is known or directly measurable. Using such methods the in-vitro recovery for glucose and its metabolites using a LWCO catheter with a 10mm membrane at a flow rate of 0.3microl/min has been estimated at between 70-100% (Hutchinson et al. 2000, Blakeley and Portnow). The in-vitro recoveries of macromolecules such as cytokines using similar methods with a HWCO catheter are variable but usually far lower (Helmy et al. 2009). Although some investigators have used these calculated relative recoveries to correct dialysate concentrations measured, this has proved unreliable because, as mentioned previously, diffusion within aqueous test solutions differs significantly from diffusion within tissue in-

Several methods of determining relative recovery in-vivo have been described in attempt to overcome the shortcomings of in-vitro estimates (see Table 2) (Benjamin et al. 2004, Chefer et al. 2009, Blakeley and Portnow). These methods include the no-net-flux method, the flowrate method, and the use of standards whose concentration is known (both exogenous and endogenous). In the no-net-flux method, perfusate containing several different concentrations of the analyte of interest (both above and below the anticipated concentration in the ECF) is perfused through the microdialysis probe and the amount of this analyte gained or lost from the probe is determined. Using this method the relative recovery may be calculated as the gradient of the linear regression that describes the dialysate concentration of the analyte being studied as a function of experimenter controlled variations in the perfusate concentration. In the flow-rate method, it is assumed that at a flow rate of zero (i.e. stasis) equilibrium between perfusate and the ECF is eventually achieved and that increasing the flow rate leads to a reduced relative recovery in a predictable but non-linear fashion. By infusing at different flow-rates and measuring the concentration of the analyte of interest, it is therefore possible to calculate the relative recovery (Hutchinson et al. 2000). Other methods rely on the use of an internal standard to estimate in-vivo relative recovery. Often, the perfusate contains a known concentration of a radiolabelled molecule similar to the analyte of interest. By determining the loss of this molecule during microdialysis it is possible to calculate its relative recovery. Alternatively, some investigators have made use of urea – which is assumed to have the same concentration throughout all water compartments in the body – as an endogenous standard. By determining the difference

Table 1. Factors affecting relative recovery

vivo.

Perfusate flow rate Decreasing recovery with faster flow rate Membrane length Increasing recovery with larger membrane

Analyte properties Recovery of molecules of similar size may

Solution properties Recovery in-vitro and in-vivo may be very

Temperature Increasing recovery with temperature

increasing pore size

be very different

different

Membrane pore size Larger molecules recovered with

Reducing the perfusate flow rate increases the time available for diffusion of substances across the semi-permeable membrane, and in turn increases the relative recovery of a substance (Tossman and Ungerstedt 1986, Hutchinson et al. 2000). This must be balanced against the reduced dialysate volumes obtained over time, which usually necessitate longer sampling intervals. Increasing the length of the semi-permeable membrane along which diffusion can occur also increases the relative recovery of a substance (Tossman and Ungerstedt 1986, Hutchinson et al. 2000) but the dimensions of the tissue being probed may limit this. Increasing the pore size of the semi-permeable membrane increases the size of molecules that are able to diffuse across it. Most microdialysis catheters used clinically are low molecular weight cut-off (LWCO) with a membrane pore size permitting molecules of approximately 20kDa (such as glucose and its metabolites) to diffuse across them. Recently high molecular weight cut-off (HWCO) catheters have been utilised with a larger membrane pore size permitting molecules of approximately 100kDa (such as cytokines) to diffuse across them. There are a number of methodological difficulties with using such catheters to measure the concentration of macromolecules (Helmy et al. 2009). One concern is that the increased membrane pore size used may lead to net efflux of fluid from the perfusate into the ECF thus influencing the composition of the ECF itself and compromising the validity of data obtained. There have been efforts to counter this net fluid efflux with the addition of a colloid to the perfusate. Various properties of the molecule being measured may also influence its relative recovery such as its shape, charge, hydrophobicity or hydrophilicity, hydrodynamic radius, and interaction with other molecules, such as dimerisation. The effect of these factors is that even molecules of a similar molecular weight may have considerably different relative recoveries in-vivo. Other factors may also alter the relative recovery. The diffusion coefficient has been estimated to increase by 1-2% for every degree Celsius increase in temperature. The diffusion coefficient within an aqueous solution is almost always greater than in tissue due to the increased diffusional path (or "tortousity") of the latter (Blakeley and Portnow).

Fig. 1. Microdialysis components. micropump is seen on the right, microdialysis catheter in the centre, and microvials on the left.

Reducing the perfusate flow rate increases the time available for diffusion of substances across the semi-permeable membrane, and in turn increases the relative recovery of a substance (Tossman and Ungerstedt 1986, Hutchinson et al. 2000). This must be balanced against the reduced dialysate volumes obtained over time, which usually necessitate longer sampling intervals. Increasing the length of the semi-permeable membrane along which diffusion can occur also increases the relative recovery of a substance (Tossman and Ungerstedt 1986, Hutchinson et al. 2000) but the dimensions of the tissue being probed may limit this. Increasing the pore size of the semi-permeable membrane increases the size of molecules that are able to diffuse across it. Most microdialysis catheters used clinically are low molecular weight cut-off (LWCO) with a membrane pore size permitting molecules of approximately 20kDa (such as glucose and its metabolites) to diffuse across them. Recently high molecular weight cut-off (HWCO) catheters have been utilised with a larger membrane pore size permitting molecules of approximately 100kDa (such as cytokines) to diffuse across them. There are a number of methodological difficulties with using such catheters to measure the concentration of macromolecules (Helmy et al. 2009). One concern is that the increased membrane pore size used may lead to net efflux of fluid from the perfusate into the ECF thus influencing the composition of the ECF itself and compromising the validity of data obtained. There have been efforts to counter this net fluid efflux with the addition of a colloid to the perfusate. Various properties of the molecule being measured may also influence its relative recovery such as its shape, charge, hydrophobicity or hydrophilicity, hydrodynamic radius, and interaction with other molecules, such as dimerisation. The effect of these factors is that even molecules of a similar molecular weight may have considerably different relative recoveries in-vivo. Other factors may also alter the relative recovery. The diffusion coefficient has been estimated to increase by 1-2% for every degree Celsius increase in temperature. The diffusion coefficient within an aqueous solution is almost always greater than in tissue due to the increased diffusional path (or "tortousity") of the

Fig. 1. Microdialysis components. micropump is seen on the right, microdialysis catheter in

latter (Blakeley and Portnow).

the centre, and microvials on the left.


Table 1. Factors affecting relative recovery

In-vitro studies have calculated the relative recovery for specific molecules under different experimental conditions in which the concentration of a substance in the external medium is known or directly measurable. Using such methods the in-vitro recovery for glucose and its metabolites using a LWCO catheter with a 10mm membrane at a flow rate of 0.3microl/min has been estimated at between 70-100% (Hutchinson et al. 2000, Blakeley and Portnow). The in-vitro recoveries of macromolecules such as cytokines using similar methods with a HWCO catheter are variable but usually far lower (Helmy et al. 2009). Although some investigators have used these calculated relative recoveries to correct dialysate concentrations measured, this has proved unreliable because, as mentioned previously, diffusion within aqueous test solutions differs significantly from diffusion within tissue invivo.

Several methods of determining relative recovery in-vivo have been described in attempt to overcome the shortcomings of in-vitro estimates (see Table 2) (Benjamin et al. 2004, Chefer et al. 2009, Blakeley and Portnow). These methods include the no-net-flux method, the flowrate method, and the use of standards whose concentration is known (both exogenous and endogenous). In the no-net-flux method, perfusate containing several different concentrations of the analyte of interest (both above and below the anticipated concentration in the ECF) is perfused through the microdialysis probe and the amount of this analyte gained or lost from the probe is determined. Using this method the relative recovery may be calculated as the gradient of the linear regression that describes the dialysate concentration of the analyte being studied as a function of experimenter controlled variations in the perfusate concentration. In the flow-rate method, it is assumed that at a flow rate of zero (i.e. stasis) equilibrium between perfusate and the ECF is eventually achieved and that increasing the flow rate leads to a reduced relative recovery in a predictable but non-linear fashion. By infusing at different flow-rates and measuring the concentration of the analyte of interest, it is therefore possible to calculate the relative recovery (Hutchinson et al. 2000). Other methods rely on the use of an internal standard to estimate in-vivo relative recovery. Often, the perfusate contains a known concentration of a radiolabelled molecule similar to the analyte of interest. By determining the loss of this molecule during microdialysis it is possible to calculate its relative recovery. Alternatively, some investigators have made use of urea – which is assumed to have the same concentration throughout all water compartments in the body – as an endogenous standard. By determining the difference

Clinical Microdialysis in Glioma 149

provides a unique method of continuously measuring brain and tumour chemistry allowing investigation of metabolites and macromolecules involved in tumourogenesis, the dynamic changes in the concentration these molecules over time, and their response to chemo- and radiotherapy. Finally, retrograde microdialysis offers the potential for the direct

Several confounding factors must be considered when performing or interpreting studies that utilise microdialysis to investigate brain tumours. First, although microdialysis is a direct measure of analytes within the ECF, the concentration of a substance within the dialysate still represents only a fraction of that in the ECF. As discussed above, this relative recovery depends upon a large number of variables and estimation by in-vitro and in-vivo techniques has proved unreliable. Second, the invasive nature of microdialysis probe insertion may result in trauma artefact. A recent consensus meeting on microdialysis in neuro-intensive care recognised that data was unreliable for at least one hour after insertion (Bellander et al. 2004). In patients with brain tumour undergoing resection or debulking, the trauma artefact may be considerably longer, particularly if the macromolecules such as growth factors and cytokines are being monitored. Third, the precise location of the catheter tip may greatly influence the data obtained by microdialysis. Studies that have applied microdialysis to patients with brain tumour have demonstrated significantly different metabolic profiles at the tumour centre, tumour periphery or border, and grossly normal

These confounding factors are at least partially mitigated by the use of physiologically meaningful ratios (rather than absolute concentrations), the omission of the first few hours of data obtained post-insertion, and the careful note of catheter locations intra-operatively and using post-operative imaging (see Table 3). The combination of microdialysis with other research methods such as animal studies, in-vitro techniques and imaging provides a

> **Limitation Strategy**  Relative recovery variable Use physiological ratios rather than

The equipment required for microdialysis includes perfusion fluid, microdialysis syringes, microinfusion pumps, microdialysis catheters, and microvials (See Figure 1). Not all commercially available microdialysis equipment is suitable or certified for human use and this must be carefully considered before selecting study apparatus. Perfusion fluid should be as close to the cerebral ECF as possible and CMA CNS perfusion fluid composed of NaCl

Trauma artefact Minimise trauma and wait for data to

Location of probe Note location intra-operatively and image

absolute concentrations

normalise

post-operatively to confirm

administration of chemotherapeutic agents to brain tumours.

peri-tumoural tissue (Roslin et al. 2003, Marcus et al.).

Table 3. Limitations of microdialysis and strategies to avoid

powerful research paradigm.

**2.2 Equipment and technique** 

**2.2.1 Equipment** 

**2.1.3 Limitations** 

between the concentration of urea in plasma, and the concentration in dialysate collected, an estimate of the relative recovery of similar small molecules may be obtained (Brunner et al. 2000, Sorg et al. 2005).


Table 2. In-vivo methods of determining relative recovery

There are a number of methodological difficulties in estimating relative recovery using these described in-vivo techniques, particularly in the context of glioma research. The no-net-flux method requires an accurate estimation of the concentration of analytes in-vivo but the concentration of the cytokines and growth factors involved in gliomagenesis can vary by several orders of magnitude. The flow-rate method requires very slow flow rates to increase the accuracy of the regression analysis, which in turn necessitates long collection periods to obtain sufficient sample volume. The use of an internal standard relies on the assumption that it has a similar relative recovery to the analyte of interest, which, for the reasons mentioned above, may not be valid. These methodological difficulties in estimating relative recovery using in-vivo techniques have led some commentators to the conclusion that the ratio of the concentration of related physiological substances (such as the ratio of lactate/pyruvate, or pro-/anti-inflammatory cytokines) may be a more robust and valuable measurement than attempts to determine the absolute concentration of these molecules in the ECF (Helmy et al. 2009).
