**2.2 Equipment and technique**

#### **2.2.1 Equipment**

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

Clinical Microdialysis in Glioma 151

Imaging should be performed to confirm the catheter position post-operatively. Most commercially available microdialysis catheters are fashioned with a "gold-tip" that is visible on CT to facilitate their identification. The initial microvial (containing flush) is not analysed. Subsequent microvials are numbered and exchanged sequentially at predefined intervals. Microvials are either analysed immediately or stored in -80c for subsequent analysis. Studies have shown that the concentration of glucose and its metabolites within microdialysate is equivalent with measured immediately or after storage in this manner

Glucose and its metabolites are frequently measured using commercial point-of-care analysers such as the ISCUS or CMA 600 (CMA Microdialysis AB, Solna, Sweden), which employ an enzyme-kinetic technique. Several techniques can also be used to analyse the macromolecules present within dialysate. Because the volumes of liquid are small and the concentrations of substances very low, techniques such as Enzyme-Linked Immunosorbent Assay (ELISA), High Performance Liquid Chromatography (HPLC), or Mass Spectroscopy

Although microdialysis is generally a robust technique if difficulties do arise and dialysate is not obtained, a systematic approach is advocated. The micropump should be examined and new batteries placed (if not already done). The catheter insertion site should be examined to check that the catheter remains secure and is not obviously displaced. The microvials can be removed and replaced to ensure that they click into place appropriately. Once confident these components are satisfactory the system may be flushed by re-opening

To date, almost a dozen clinical studies have utilised microdialysis in patients with cerebral tumours; with approximately half of these devoted to investigating gliomagenesis, and the remaining to evaluating treatment with chemo- and radiotherapy. Studies investigating gliomagenesis may be further subdivided into those utilising LWCO or HWCO

In a landmark Swedish study in 2003 Roslin et al studied the baseline concentration of glucose and its metabolites, glycerol and glutamate in patients with high grade glioma

Tumour (T)

Brain Around Tumour (BAT)

(Hutchinson et al. 2000).

(MS) are often employed.

**2.2.3 Troubleshooting** 

microdialysis catheters.

**3.1 Low molecular weight cut off** 

and then closing the lid of the micropump.

**3. In vivo assessment of pathogenesis** 

Table 4. Definitions on the location of microdialysis catheters

**Term Definition** 

Peritumour (PT) Within 5mm-20mm of the tumour or

Within grossly affected tumour tissue, or within 5mm of the resection margin of such tissue

resection margin

Within grossly unaffected brain at least 20mm away from the tumour or resection margin

(147 mM), KCl (2.7 mM), CaCl2 (1.2 mM), and MgCl2 (0.85 mM) in water, is often used. Perfusion fluid is contained in microdialysis syringes with a capacity of approximately 2.5ml. Microinfusion pumps are portable battery driven pumps that compress syringes at a slow predefined rate, which is usually fixed (0.3microl/min) but may be adjustable (0.1microl/min to 5microl/min). Microdialysis catheters vary in their membrane length (10- 30mm) and pore size (LWCO/20kDa or HWCO/100kDa), and by their physical properties (such as shaft size). Conventional clinical microdialysis uses LWCO catheters. Clinical microdialysis studies investigating macromolecules such as cytokines or growth factors require HWCO catheters to maximise recovery of these substances. Fluid is collected in microvials designed to collect micro volume samples and minimise evaporation.

### **2.2.2 Technique**

All patients must be thoroughly counselled beforehand about the potential (but very low) additional risk of haemorrhage and infection, and written informed consent obtained. Preoperatively all the microdialysis equipment should be checked. Particular attention must be paid to the microdialysis catheter, syringe and perfusion fluid to ensure that their packaging remains intact and sterility maintained. Many clinicians advocate priming the catheter so that the system is already functional prior to insertion. This ensures constant fluid flow at the catheter tip and theoretically reduces sedimentation and non-specific binding of proteins to the catheter membrane. The microdialysis syringe is filled with CNS perfusion fluid, and connected to the microdialysis catheter using strict aseptic technique. The microdialysis syringe is placed in the microinfusion pump and a microvial placed at the distal end of the microdialysis catheter to collect the dialysate. Upon closing the lid of the microinfusion pump a 5-minute flush cycle is initiated followed by an automatic decrease to the preset flow rate.

Operative insertion of the microdialysis catheter into cerebral parenchyma may be via a closed or an open technique following tumour biopsy or resection respectively. In the closed technique stereotactic biopsy of brain tumour tissue is performed and then the microdialysis catheter inserted so that the catheter tip lies in the region of interest. Multiple catheters can be placed through a single burr hole using multiple different trajectories. The advantage of this technique is that traumatic artefact is minimised. In the open technique the brain tumour is resected and then the microdialysis catheter placed into the region of interest. Intra-operative real-time three-dimensional ultrasound probes have been used to assist catheter placement (Homapour et al.). Although there is greater traumatic resection artefact associated with open placement the risk of inadvertent complications, such as intracerebral haematoma, is theoretically lower because the catheter is inserted under direct visualisation and blood vessels can be avoided. The precise position of catheters within the brain is critical to interpreting clinical microdialysis studies but a number of terms have been used in the literature with conflicting and overlapping definitions. To avoid confusion during subsequent discussion we will define catheter locations in the following way: Tumour (T) catheters are either within grossly affected tumour tissue, or within 5mm of the resection margin of such tissue; Peritumour (PT) catheters are within 5mm-20mm of the tumour or resection margin; Brain Around Tumour (BAT) catheters are within grossly unaffected brain at least 20mm away from the tumour or resection margin. Once in place the catheter may be secured using a commercial "bolt" or by tunnelling the catheter and stitching it into place, depending on whether a closed or open approach insertion technique is used respectively.

(147 mM), KCl (2.7 mM), CaCl2 (1.2 mM), and MgCl2 (0.85 mM) in water, is often used. Perfusion fluid is contained in microdialysis syringes with a capacity of approximately 2.5ml. Microinfusion pumps are portable battery driven pumps that compress syringes at a slow predefined rate, which is usually fixed (0.3microl/min) but may be adjustable (0.1microl/min to 5microl/min). Microdialysis catheters vary in their membrane length (10- 30mm) and pore size (LWCO/20kDa or HWCO/100kDa), and by their physical properties (such as shaft size). Conventional clinical microdialysis uses LWCO catheters. Clinical microdialysis studies investigating macromolecules such as cytokines or growth factors require HWCO catheters to maximise recovery of these substances. Fluid is collected in

All patients must be thoroughly counselled beforehand about the potential (but very low) additional risk of haemorrhage and infection, and written informed consent obtained. Preoperatively all the microdialysis equipment should be checked. Particular attention must be paid to the microdialysis catheter, syringe and perfusion fluid to ensure that their packaging remains intact and sterility maintained. Many clinicians advocate priming the catheter so that the system is already functional prior to insertion. This ensures constant fluid flow at the catheter tip and theoretically reduces sedimentation and non-specific binding of proteins to the catheter membrane. The microdialysis syringe is filled with CNS perfusion fluid, and connected to the microdialysis catheter using strict aseptic technique. The microdialysis syringe is placed in the microinfusion pump and a microvial placed at the distal end of the microdialysis catheter to collect the dialysate. Upon closing the lid of the microinfusion pump a 5-minute flush cycle is initiated followed by an automatic decrease to the preset

Operative insertion of the microdialysis catheter into cerebral parenchyma may be via a closed or an open technique following tumour biopsy or resection respectively. In the closed technique stereotactic biopsy of brain tumour tissue is performed and then the microdialysis catheter inserted so that the catheter tip lies in the region of interest. Multiple catheters can be placed through a single burr hole using multiple different trajectories. The advantage of this technique is that traumatic artefact is minimised. In the open technique the brain tumour is resected and then the microdialysis catheter placed into the region of interest. Intra-operative real-time three-dimensional ultrasound probes have been used to assist catheter placement (Homapour et al.). Although there is greater traumatic resection artefact associated with open placement the risk of inadvertent complications, such as intracerebral haematoma, is theoretically lower because the catheter is inserted under direct visualisation and blood vessels can be avoided. The precise position of catheters within the brain is critical to interpreting clinical microdialysis studies but a number of terms have been used in the literature with conflicting and overlapping definitions. To avoid confusion during subsequent discussion we will define catheter locations in the following way: Tumour (T) catheters are either within grossly affected tumour tissue, or within 5mm of the resection margin of such tissue; Peritumour (PT) catheters are within 5mm-20mm of the tumour or resection margin; Brain Around Tumour (BAT) catheters are within grossly unaffected brain at least 20mm away from the tumour or resection margin. Once in place the catheter may be secured using a commercial "bolt" or by tunnelling the catheter and stitching it into place, depending on whether a closed or open approach insertion technique is used respectively.

microvials designed to collect micro volume samples and minimise evaporation.

**2.2.2 Technique** 

flow rate.


Table 4. Definitions on the location of microdialysis catheters

Imaging should be performed to confirm the catheter position post-operatively. Most commercially available microdialysis catheters are fashioned with a "gold-tip" that is visible on CT to facilitate their identification. The initial microvial (containing flush) is not analysed. Subsequent microvials are numbered and exchanged sequentially at predefined intervals. Microvials are either analysed immediately or stored in -80c for subsequent analysis. Studies have shown that the concentration of glucose and its metabolites within microdialysate is equivalent with measured immediately or after storage in this manner (Hutchinson et al. 2000).

Glucose and its metabolites are frequently measured using commercial point-of-care analysers such as the ISCUS or CMA 600 (CMA Microdialysis AB, Solna, Sweden), which employ an enzyme-kinetic technique. Several techniques can also be used to analyse the macromolecules present within dialysate. Because the volumes of liquid are small and the concentrations of substances very low, techniques such as Enzyme-Linked Immunosorbent Assay (ELISA), High Performance Liquid Chromatography (HPLC), or Mass Spectroscopy (MS) are often employed.
