**2.1.3 Limitations**

148 Advances in the Biology, Imaging and Therapies for Glioblastoma

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.

**Method Principle** 

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

Until relatively recently few studies had applied microdialysis to patients undergoing surgical biopsy or resection of their brain tumours. To this end, clinical studies using microdialysis in patients with brain tumours offer a number of potential advantages over other methodological approaches. First, in contrast to traditional in-vitro studies, clinical microdialysis studies permit the assessment of brain tumours in-vivo, recognising the complex interactions between tumour- and host-related factors, and the role these interactions play in tumourogenesis. Second, by applying microdialysis to patients with brain tumours, rather than animal models of such tumours, clinical microdialysis eliminates the possibility of erroneous interpretation of interspecies differences or of limitations of the brain tumour model itself. Third, clinical microdialysis provides a direct measure of analytes within the ECF when compared with imaging techniques. Fourth, microdialysis easily allows repeated evaluation over an extended time course. Microdialysis therefore

When analyte concentrations within perfusate and ECF are equal, there is nonet-flux

When flow rate is zero (i.e. stasis) equilibrium occurs between perfusate and ECF

Fraction of exogenous standard lost from perfusate is equal to fraction of analyte extracted from ECF

Fraction of endogenous standard and analyte extracted from ECF is equal

2000, Sorg et al. 2005).

No-net-flux

Variable flow rate

Internal standard (Exogenous e.g. radiolabelled)

> Internal standard (Endogenous e.g. urea)

the ECF (Helmy et al. 2009).

**2.1.2 Uses** 

Table 2. In-vivo methods of determining relative recovery

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

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 powerful research paradigm.


Table 3. Limitations of microdialysis and strategies to avoid
