Report 214

Improvement of the Mechanical Stability of Microdialysis

Catheters

A study made by Sven Burman

at CMA Microdialysis AB

KAROLINSKA INSTITUTET

Avdelningen för medicinsk teknik KTH

Enheten för medicinsk teknik Institutionen för medicinsk

laboratorievetenskap & teknik KI

Stockholm 2004

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Sammanfattning Microdialyis är en provtagningsmetod som används för att övervaka förändringar i olika substanser direkt i vävnad. En kateter med ett semipermeabelt membran inplanteras direkt i vävnaden och en pump perfunderar katetern med en perfusionsvätska. Koncentrations skillanden mellan vävnaden och perfusionsvätskan leder till att ämen i vävnaden diffunderar in i katetern och dessa ämnen kan sedan mätas i en analysator. Membranet är dock ganska ömtåligt vilket gör att det kan gå sönder vid införande genom hinnor och seg vävnad. För att öka den mekaniska stabiliteten provades två prototyper; en kateter med en skyddande nätstrumpa och en kateter med en skyddande hylsa. Recoveryt i de två katetrarna provades både in vitro och in vivo. I in vitro testet så kunde ingen skillnad mellan nätkatetrarna och referenskatetrarna. Recoveryn i hylskatetrarna var dock så dålig och ojämn att de ej provades in vivo. In vivo testet visade att nätkatetrarna faktisk hade ett bättre recovery än referenskatetrarna. Serien som användes var dockför liten för att ge säker statistik. Hållbarheten har ej testats men indikationerna tyder på att näkatetern ger det bästa skyddet. Fler tester av både den mekaniska stabiliteten och recoveryn krävs dock för att få en tillförlitlig statistik.

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Abstract Microdialysis is a sampling method for tissue. It is used to monitor the concentration of different substances, e.g. glucose and lactate, directly in the tissue. A catheter with a semi permeable membrane is implanted into the tissue and a small pump perfuses the catheter with an isotopic solution. Due to a concentration gradient between the extracellular fluid of the tissue and the perfusion fluid of the catheter the substances of interest diffuses into the catheter and can be collected in small vials for analysis. The membrane is however quite vulnerable and during implantation through fascias and in rough tissue it sometimes breaks. In order to increase the mechanical stability of the catheters two ideas have been tested; covering the membrane with a protective braid and covering the membrane with a protective cap with cut-out windows. The recovery of the prototype catheters was tested both in vitro and in vivo. The in vitro test showed no big difference in recovery of the braid catheters compared to reference catheters. The recovery of the cap catheters was however quite poor compared to the reference catheters so it was not tested in vivo. The in vivo test showed a better recovery of the braid catheters than the reference catheters. One possible explanation for this is that the braid creates a fluid space around the membrane that enhances diffusion. The small series and a discrepancy between the reference catheters make the statistics a bit insecure though. The mechanical stability of the catheters has not been tested but the braid probably provides best protection for the membrane. More tests of the recovery and the mechanical stability are however required.

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Thanks to….

… my mentors Petra Åhl and Henrik Falkén …Urban Ungerstedt for all generous help in the whole field of microdialysis …Michel Goiny for all help with the pig test …Grzegorz Nowak for all assistance with the hepatic catheter …Gustav Amberg for all assistance with the fluid dynamics ...Hanna Peyronsson for generous help with the in vitro tests …Anette Karlsson and Henrik Jessen for manufacturing the prototypes …Anders Carlssson for explaining the chemistry behind microdialysis …Krister Sjölander and Yngve Hinas ...and the rest of the staff of CMA Microdialysis AB

Sven Burman, Stockholm April 2004

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TABLE OF CONTENTS

1 INTRODUCTION…………………………………………………….6

1.1 BACKGROUND FOR THIS STUDY………………………….…..6 1.2 OBJECTIVES………………….……………………………………6 1.3 CMA MICRODIALYSIS AB………………………………………6

2 LITTERATURE SURVEY…………………………………………..7 2.1 HISTORY OF DEVELOPMENT…………………………………..7 2.2 BASIC PRINCIPLE…………………………………………….…..8 2.3 AREAS OF USE…………………………………………………11

2.3.1 Measurable substances…………………………..….……11 2.3.2 Applications for microdialysis…………………………….11 2.4 MARKET ANALYSIS…………………………………………….13

2.4.1 Competing techniques…………………………………13 2.4.2 Competing companies………………………………...….14 2.5 PATENTS……………………………………………………….…16 2.5.1 Patents of CMA……..………………………………….…16 2.5.2 Other patents……………………………………………..16 2.6 DESIGN OF MICRODIALYSIS CATHETRS…………………...19

2.7 CMA DESIGN OF INTRODUCERS……………………………..21 2.8 CMA DESIGN OF PUMPS AND ANALYZER………………….21 2.9 PROBLEMS ASSOCIATED WITH MICRODIALYSIS………...22 2.9.1 Problems associated with catheters……………………...23

2.10 SPECIFICATION OF REQUIREMENTS…………………….…26 2.11 THE RECOVERY PROBLEM…………………………………..27 2.12 WAYS OF INCREASING THE RECOVERY………………..…31

2.12.1 Changing the flow in the catheter………………………31 2.13.1 NEW CATHETERS……………………………………………35

2.13.1 The Braid………………………………………………..35 2.13.2 The Cap………………...…………………………….…37

3. EXPERIMENTS……………………………………………………39 3.1 INTRODUCTION…………………………………………………….…39 3.2 IN VITRO TESTS…………………………………………………39

3.2.1 Introduction…………………………………………………….39 3.3 IN VIVO TEST…………………………………………………40

3.3.1 Introduction………………………………………………….…40 3.3.2 Materials………………………………………………………..40 3.3.3 Surgical preparation…………………………………………..40 3.3.4 Experimental procedure………………………………………41 4. RESULTS AND DISSCUSION…………………………..42 4.1 IN VITRO RESULTS……………………………………………..42

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4.2 DISSCUSION OF THE IN VITRO TESTS……………………….45 4.3 IN VIVO RESULTS………………………………………………46 4.4 DISSCUSION OF THE IN VIVO TEST…………………………46

5. CONCLUSION……………………………………………51

6. RECOMMENDATION FOR FUTURE WORK……….52

REFERENCES………………………………………………53

DICTIONARY

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1. Introduction 1.1 Background to this study Microdialysis is an in vivo sampling method that makes it possible to analyze the tissue chemistry with high accuracy. The system comprises a pump, a catheter and an analyzer. This study relates to the mechanical stability of the CMA 60 microdialysis catheter. The catheter is intended for use in adipose tissue and in resting skeletal muscle. The membrane of the catheter is quite long, 30 mm, which makes it vulnerable when implanted into tissue. If the membrane breaks the catheter is useless. There are some different reasons for the membrane breakage, the most frequent problem is probably wrong handling. However the catheter should not break, even if the handling is incorrect. Another reason for membrane failure is implantation in muscle. It is hard to keep a muscle resting for the time period of microdialysis and if the muscle is contracted, the membrane is likely to break. If the membrane comes in contact with the muscle fascia it is also likely to break. It would be desirable to have a catheter that is more durable for use in both the applications of today and future applications. 1.2 Objectives The main objective of this study is to increase the mechanical stability of the membrane. In order to do this the reasons for the breakage must be investigated as well as what mechanical stress the membrane is under. To test the ideas prototype catheters shall be built and tested in vitro as well as in vivo. 1.3 CMA Microdialysis AB CMA Microdialysis AB is a Swedish company founded in 1984. It develops and manufactures a full range of microdialysis equipment, from pumps to catheters and analyzers. As the only company it also manufactures catheters for clinical use and has today five different catheters for use in brain, liver, adipose tissue, resting muscle and the abdominal cavity. A new easy-to-use analyzer for the clinic is also under development. CMA is based in Solna, Sweden and has sales offices in the US, UK, France, Germany and Italy. There are also distributors in most parts of the world, e.g. Australia and Japan. There are presently 72 employees around the world.

Fig 1.1. The membrane of the CMA 60 catheter.

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2. Literature Survey 2.1 History of development The first paper regarding microdialysis was published by Bito in 1966 [1]. He implanted semipermeable sacks filled with a dextran-saline solution into the necks of dogs and removed them after ten weeks of implantation. After removal he analyzed the solution for amino acids and electrolytes. Even though this experiment is quite unlike today’s methods the basic idea is still the same. In 1972 J M R Delgado [2] performed an experiment were he for the first time actively perfused a semi permeable membrane in the brain. This was done by implanting a push-pull cannula ending in a semi permeable bag into the brain of ten monkeys. On some of the cannulas electrodes was added to induce and measure electrical activity. The cannula was implanted and after three weeks they were perfused with a Ringer solution and the perfusate was then analyzed. It contained glutamate, amino acids and glycoproteins. The outcome of this experiment was promising but due to problems with the semi permeable bag the method never gained reputation other than among scientists. The next step in the development of microdialysis was when Urban Ungerstedt [3] introduced a hollow fiber for measuring of neurotransmitters in the brain. The first probe he used was a linear probe. It was put trough a rat brain and different substances were measured. This was of course very difficult to do in an awake rat due to the extensive surgery that had to be performed to implant the probe. To avoid this he developed the loop catheter and when the materials got better he developed and patented the first concentric type catheter. Even though there are many types of catheters the concentric catheter is the by far the most used. The first probe was approved in 1987 and the first catheter for human use was approved in 1995. Today microdialysis is an acknowledged research tool in both humans and animals.

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2.2 Basic principle The aim of microdialysis is to very accurately measure different substances in living tissue. A catheter, which has a semi permeable membrane, is inserted into the tissue. The outside of the membrane is in contact with the tissue and the inside of the membrane is constantly perfused with a solution, termed perfusate. This perfusate has different compositions depending on which tissue is to be measured, but usually it is a Ringers solution that has the same composition as the ISF, the interstitial fluid. The solution creates a concentration gradient across the semipermeable membrane since the substances of interest often has a higher concentration in the tissue than in the perfusate. In order to equalize this gradient, diffusion across the membrane starts. Small water-soluble molecules, such as glucose, lactate and pyruvate can pass freely through the membrane in the opposite direction of the concentration gradient. Larger molecules such as proteins cannot pass through the small pores of the membrane. The small molecules that are present in the tissue will diffuse through the membrane and into the catheter where they can be measured. The idea is that the catheter should imitate a blood vessel, which also takes up and releases substances through diffusion (fig 2.1). Fig 2.1 The basic principle of microdialysis with a catheter imitating a blood vessel.

Cell Blood Vessel

Microdialysis Catheter

Cell

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The achieved concentration of the substance in the perfusate relative to the concentration of the same substance in the tissue is termed the relative recovery [%]. The total amount of substances in the sample is termed the absolute recovery [mol/min]. The relative and absolute recovery is dependent on the flow of the perfusate in the catheter. If the flow is low, the concentration gradient has time to equalize giving a higher relative recovery compared with a higher flow (fig 2.2).

100%

Flow rate [µl/min]

Recovery

A high flow will keep the concentration gradient steep, as it will not have time to be equalized. This will lead to a high mass transport across the membrane and a high absolute recovery (fig 2.3) but the concentration will be quite low which gives a low relative recovery.

100%

Flow rate [µl/min]

Recovery

Fig 2.3 The absolute recovery.

Fig 2.2 The relative recovery.

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If the concentration of the substance changes in the tissue, the absolute recovery will also change but the relative recovery will remain constant. In the clinical application it is the relative recovery that is of interest. The recovery is dependent of at least eight different factors; • The flow rate. A high flow rate in the catheter will lead to an increased

diffusion because the concentration gradient is not given time to be equalized, leading to a low relative recovery and a high absolute recovery

• The area of the membrane. A larger area will of course give a higher

diffusion giving a higher recovery. • Temperature. A higher temperature leads to an increased diffusion and a

higher recovery. The diffusion constant is dependent on the temperature because both the viscosity of the ISF and the mobility of the molecules increases with an increased temperature.

• Properties of the extra cellular fluid and the substances measured. The

Stokes-Einstein equation gives that a lower molecular weight gives a higher recovery, due to the fact that the diffusion coefficient is inversely proportional to the radius of the molecules. The charge of the molecules can also affect the recovery since the membrane sometimes can be charged.

• Properties of the membrane. The adherence of the molecules to the

membrane and the cut-off of the membrane affect the recovery. The cut-off is defined as the molecular weight when 80% of the molecules can pass through the pores of the membrane. The most common cut-off in catheters is 20000 Daltons.

• Properties of the tissue. Some tissues have more resistance than others to

diffusion, which makes it harder for the substances to diffuse through the tissue to the catheter. This is the most important factor affecting the diffusion. The blood flow and metabolism of the tissue can to some extent also influence the recovery.

• The volume under the membrane. If the volume under the membrane is small

there is of course a small volume of perfusate that has to be equalized. So by decreasing the volume under the membrane the area of the membrane can also be reduced.

• The depth in the body also influences the recovery. Over weight persons have

a lower blood perfusion in the outer fat layer, which gives a lower recovery compared to measuring deeper in the adipose tissue.

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2.3 Areas of use 2.3.1 Measurable substances There are a number of different substances that can be measured with microdialysis, the most common ones are lactate, pyruvate, glycerol, glutamate and glucose. A high lactate/pyruvate ratio is a marker of ischemia. This is due to the fact that pyruvate is converted to lactate instead of going to the citric acid cycle during hypoxia caused by reduced blood flow to the tissue. By looking at the ratio between lactate and pyruvate the insecurity of the glucose supply can be eliminated, this is further discussed in section 2.11. The glucose level is more complicated to interpret since it is affected by a number of factors, such as the blood glucose level and the blood flow in the capillaries. Usually a decrease in the glucose level means a decrease in the blood perfusion of the brain and in tissue this means a decrease in tissue pO2. Glutamate is released from neurons during ischemia, and the released glutamate can lead to a harmful influx of calcium to the cells, which in turn can lead to cell damage. Hence glutamate is an indirect marker of cell damage but it can be hard to interpret since the released glutamate is mixed with a large pool of glutamate that is already present in the IFS. In the brain, loss of energy in the cells leads to an influx of calcium to the cells. The calcium activates phospholipases that splits away glycerol from the cell membrane. The level of glycerol is thus a marker of how severe the cell damage is. Glycerol in adipose tissue derive from the splitting of triglycerides (fat) into glycerol and free fatty acids. The sympathetic nerves control this process and a high concentration of glycerol in adipose tissue is an indirect marker of sympathetic stress. 2.3.2 Applications for microdialysis Microdialysis is used in both humans and animals. The use in animals is only for research purposes and the main area of use is in the CNS. Measurements can be performed on awake animals and gives good results. Microdialysis can also be used in other tissues in animals such as adipose tissue, intestine, blood and muscles. When used on animals the microdialysis device is called a probe and when used in humans it is called a catheter.

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For humans there are a number of applications. The largest area of use is for brain measurements. Since the brain lies within a confined space a scull trauma that results in a bleeding will build up the intra cranial pressure, ICP. This may result in a lower blood perfusion, which leads to ischemia. A stroke can also cause ischemia in parts of the brain. The nerve cells will then start to produce lactate in the absence of sufficient oxygen and the lactate/pyruvate ratio can be measured. The catheter is usually inserted through a bolt in the cranium but can also be tunnelated through the scalp. Microdialysis is also used for general brain research. The catheters can also be placed subcutaneously. Usually this is done to have a reference to the brain catheter or the hepatic catheter. But it is also done in plastic surgery to monitor the oxygen saturation and survival of free flaps. The substances that can be measured are the same as in the brain. Microdialysis can also be used during transplantations. It is used during liver transplantations to monitor the liver when implanted in the receiver, to see how it recovers. Due to a great lack of livers the technique can also be used to determine the quality of a liver that may be bad but still is considered for use. Microdialysis can be used at most types of transplantations for monitoring of the transplanted organ. The technique is also used in the abdominal cavity. Here it has many applications, one of them is to measure the environment around an anastomosis. If the anatsomosis is not tight it may leak and cause sepsis. Before sepsis the cells often produce lactate thus an increase in the lactate levels can warn that a sepsis may be imminent. The catheter must be placed in the abdominal cavity during open surgery. There are some applications that lie in the near future. One would be to run microdialysis in the heart muscle. This would give a lot of valuable information about the condition of the heart muscle during for example heart surgery. There are still some problems associated with this to be solved, one is the durability of the catheter which today is poor in a working muscle. Another large future application is continuous glucose measurements in diabetes patients. The market for continuous glucose measuring in diabetes patients is large and there are many techniques competing for it. Something that lies closer in the future is glucose measuring in intensive care patients. Some ICU patients can have large variations in their glucose levels and continuous measuring makes it easier to keep the patients glucose level constant. A study done by Greet van de Berghe [3] showed that the mortality was 3,4 percent lower among patients that had their blood glucose levels stabilized compared to those who hadn’t.

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2.4 Market Analysis 2.4.1 Competing techniques There are other techniques doing the same or similar things as microdialysis. Some techniques are competing with microdialysis and others are used as complements. The biggest competition to microdialysis in the field of plastic surgery is the laser Doppler technique. A beam of laser is scattered when hitting tissue and when hitting moving red blood cells the reflected light undergoes a change in wavelength (Doppler change). When hitting a static object, in this case tissue, the light is reflected unchanged. By measuring distribution of the wavelength of the reflected light the blood flow in a tissue can be calculated. The big advantages with this method are that it is non-invasive, easy to use and has a low cost. The drawback however is that it is quite uncertain. It has to be calibrated to give reliable values but even so, it is not very accurate. It only shows the stability of the blood flow and not if it is enough blood, whereas microdialysis shows how the cells actually react to changes in the blood flow. There are a number of companies manufacturing laser Doppler equipment, among them Oxford Optronix Ltd and Perimed AB. ICP (Intra Cranial Pressure) monitoring is a technique that is often used at the same time as microdialysis and the two techniques complement each other. ICP monitoring is used to measure the pressure in the cranium after for example a scull trauma. The measurement is done by first inserting a bolt trough the cranium of the patient and through the bolt the pressure sensor is inserted into the brain. The bolt usually has multiple channels so microdialysis and ICP measuring can be done through the same bolt. The ICP sensor can also be implanted by tunnelation. The largest companies in this field are Codman and Camino. Another way of measuring the brain tissue oxygenation is by pO2 (oxygen partial pressure) measurement. The procedure is quite similar to the ICP measurement but instead of implanting a pressure sensor an oxygen sensor is implanted, usually trough a bolt. The method is sensitive but like laser Doppler it only measures the amount of oxygen in the tissue, not how the cells actually react to it. Transcranial Doppler is another technique using the Doppler phenomena. Instead of using laser, as in the laser Doppler technique, pulses of ultrasound (usually about 2 MHz) are directed towards the area of interest in the scull with a handheld transducer. The pulses pass through the cranium and are reflected in the tissue with a change of frequency when reflected on moving red blood

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cells. This way the blood perfusion in separate regions of the brain can be evaluated. This technique is mainly used for patients in the risk of getting a stroke and to evaluate the cerebral blood flow for other reasons. The big advantage is that the method is noninvasive, but as in the case of laser Doppler, the measurements are not very exact. It also requires a skilled operator. The technique is widespread and frequently used at neuro intensive care units. A method that may compete with the gastric microdialysis is gastric tonometry. The basic principle is somewhat similar to that of microdialysis. A catheter with a balloon attached is inserted into the stomach. The balloon is made of a semipermeable silicon material and it contains CO2. The CO2 of the stomach can diffuse into the balloon to equilibrate the concentration gradient. Samples from the balloon are withdrawn and analysed regularly, thus the carbon dioxide partial pressure, pCO2, of the gastric mucosa can be measured. The gastric mucosa is a part of the walls of the stomach. It is very sensitive to hypoxia and the stomach and the intestines are the first parts of the body to suffer from low perfusion during for example shock, trauma or sepsis when the blood is redistributed. Hence a change in the CO2 is an early warning sign and the technique is used in for example trauma patients, patients with different types of shock or during major surgery.

Clinical signs are something that is looked at together with microdialysis. In for example plastic surgery pale and cool skin is a warning of low perfusion. Blood samples also gives a lot of information about the levels of for example glucose, lactate and haemoglobin. The disadvantage with blood sampling is that it is not a continuous way of measuring and if it is done continuously it creates discomfort for the patient.

2.4.2 Competing companies

There are six companies competing in the microdialysis field, CMA Microdialysis AB, Microbiotech, Harvard Apparatus, BAS, Applied Neurosience and Eicom. They all manufacture equipment for preclinical research, i.e. for the use in animals. CMA is the only company manufacturing equipment for clinical use. Microbiotech is a small Swedish company that was started by former CMA Microdialysis employees. They manufacture concentric probes, much like CMAs but to evade CMAs patent they have put the membrane on the outside of the sleeve (whereas CMA has the membrane on the inside of the sleeve, making the probes less fragile). They have different sizes and membranes with different cut-off. The also have pumps and other equipment for microdialysis. Their annual turnover is 5 M SEK.

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BAS (Bioanalytical Systems Inc) is a medical technology company based in the US. They manufacture instruments and equipment for chromatography, blood sampling, microdialysis, ultrafiltration and electrochemistry. In the microdialysis field they manufacture concentric probes, mainly for use in the brain, and linear probes for use in several different organs, for example adipose tissue and muscles. The linear probe can be folded creating a loop type probe. They also have a special linear probe for implantation in the bile duct. BAS also manufactures a system for ultrafiltration, a technique that is similar to microdialysis. The probe is of loop type but instead of running a perfusate through it a vacuum is applied, which sucks the interstitial fluid out of the tissue. This gives a sample that has the exact same composition as the ISF in the tissue. It is a slow method however, it can only retract fluid in the rate the body can replace it. The affect on the tissue it is implanted in should be considerable. They also manufacture other equipment such as pumps and fraction collectors that is needed for microdialysis and ultrafiltration. BAS has an annual turnover of 186 M SEK [7]. Another large company manufacturing a wide range of medical technology products is Harvard Apparatus, which is a part of the Harvard Bioscience Group. They manufacture products for surgery, cell and molecular research, syringes, peristaltic pumps, ventilators and anesthesia equipment. They have two types of microdialysis probes, side-by-side or loop type. Like the other companies they also manufacture accessories for microdialysis. Their annual turnover is 402 M SEK [8]. Eicom is a Japanese company specialized in making microdialysis equipment and blood- and NOx analyzers. Like CMA they provide the whole system, from the probe to the analyzer (except the pump). They manufacture different types of probes, a concentric probe in different designs, a linear probe and a probe for insertion into a blood vessel. Last year their sales was 35 M SEK. Applied Neuroscience is a small London based company specialized in manufacturing microdialysis probes and accessories. They have three types of probes; concentric, linear and loop-type. Their concentric probe is very similar to CMAs patented probe and their loop-type probe is quite similar to another patented loop-type probe. Some of their probes have integrated electrodes for measuring evoked potentials in the brain and other has microcannulas for giving injections.

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2.5 Patents 2.5.1 Patents of CMA CMA Microdialysis AB has four patents regarding the catheters and its introduction into the body. The first patent [9], 8206863-6, is from 1982 and describes the concentric microdialysis probe. The membrane is fixed inside of the sleeve, making the construction less fragile. The next patent [10], 9400377-9, is similar to the first, but it is here claimed that the membrane should be fixed to the inside of the sleeve and that the distal end of the membrane also should be fixed inside a supporting structure. In patent 9400378-7 [11] the slit-introducer is described. It is a hollow cannula with a slit along the length of it. The catheter is put in the lumen of the cannula and after it has been inserted to the tissue, the cannula is withdrawn and the catheter is slid trough the slit and left in the tissue. A way of protecting the membrane is suggested in pat SE 9902694 [12]. A mesh sleeve is described which encloses the membrane. The mesh should be in the form of a deformable braid. This would protect the membrane and still permit the extra cellular fluid to come in contact with the membrane. This is one of the solutions that are investigated in this thesis. 2.5.2 Other patents Microbiotech has a patent, WO03055540 [13], in which a microdialysis probe is described. The main difference from their original probe is that the inlet is S-shaped whereas the inlet of their original probe is straight. The use of this is unclear, as is the scientific depth of the patent. In patent WO03000129 [14] Markus Mueller and Hartmut Derendorf has focused on the imagebility of the probes position in the body. This is to make it easier to confirm that the probe is at the desired position in the tissue. It is suggested that a guide inside the probe made of a material which can easily be viewed with an appropriate imaging method, for example MRI (Magnetic resonance imaging) or CT. CMA has solved this problem by putting a gold tip on the catheter which makes it easy to view with CT. In patent WO200232494 [15] Panotopoulos Christos proposes that a multipurpose catheter would solve many problems associated with microdialysis. Such a catheter would combine the functions of infusion, aspiration, biopsy, pressure monitoring and microdialysis. It would consist of one or more adjacent lumens and would probably have a quite large diameter. It would also have more functions than needed in a clinical situation. Disetronic has a patent [16] associated with glucose measurements. If the microdialysis technique gets a breakthrough in this field it is considered to be a

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big market. Their catheter for this is a loop type catheter, which is mostly covered with a supporting structure, and the membrane is in contact with the surrounding tissue only at the distal end. The advantages with this construction is that the in- and outlet wont get deformed which may lead to an impeded flow and that the dead space in the catheter is reduced. The recovery is however smaller due to the small membrane area. This is not a big problem due to the fact that Disetronic uses a special technique for glucose monitoring. Concanvalin A, a glucose specific lectin, is added in the perfusate. It can bind four glucose residues and link them into a gel-like structure. So by measuring the viscosity the glucose levels can be determined. The viscosity is measured by comparing the pressure at the inlet with the pressure at the outlet. This method is under development. Another interesting solution is presented in patent WO 200106928 [17] (Jan Liska and Anders Franco-Cereceda). To get a better membrane area/volume ratio many membrane lumens are placed adjacent to one another in a cylindrical manner, either parallel to the cylinder or in a helical manner. It should be noted that this type of catheter is for intravascular use but the same idea should be applicable in other parts of the body, for example in the abdominal cavity. Jan Liska and Anders Franco-Cereceda suggest a different construction in WO 9945982 [18]. This catheter is for intravascular use and consists of two adjacent lumens, which lies within a larger round lumen. One or more windows made of a semi permeable membrane constitutes the active dialyse area. There can also be four lumens with four windows, which gives a higher recovery. The catheter is to be placed in the coronary sinus to detect signs of myocardial infarction earlier. It is mainly intended for patients in the intensive care unit. Another way of doing this is to measure directly in the heart muscle, but that requires more of the catheter, especially regarding its mechanical stability. Two problems associated with microdialysis is the hosing to and from the catheter. The dead volume gives the measurements a delay and it is also suggested that the hosing would make it more likely that the operator would make more errors. To avoid these problems pat no WO9941606 [19] describes a loop type probe that is, at the in- and outlet, connected to a silicon chip. This chip can analyse a number of substances thus giving a faster response than ordinary measurement methods. In an attempt to make the insertion easier pat WO9857693 [20] discusses a new type of introducer. It is a hollow cannula without a slit. After the cannula is in place the cannula is retracted from the tissue but it is not removed from the hosing. The cannula is intended to be fixed to the patients skin while it is still on the hosing. The idea with this is to make the implantation easier and to protect the catheter better. The protection is probably better but the implantation will hardly be any easier. Also the dead space will increase with

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the length of the cannula. The appropriateness of leaving the cannula on the patients skin (even though it to some extent is protected) can also be questioned. An example of another type of probe is the liner probe. BAS has in pat no US 5706806 [21] patented a linear probe with an extra supporting fiber inside the lumen of the membrane. This will prevent the probe from a total break, even if the membrane gets damaged the probe will stay together. A problem with catheters is that when implanted into tissue the membrane portion may be deformed. This is because it is flaccid and it can lead to blocking of the flux, which leads to little or no perfusate in the microvials. Different flux over time is also believed to be related to this. In patent no. WO 9218191 [22] describes a loop type catheter. To keep the loop open at all times they suggest that a high pressure in the membrane portion would help the membrane keep its shape. The relationship between the factors affection the pressure inside an extremely narrow bore is given by equation 1:

were V is the viscosity of the fluid, L is the length of the tubing, Q is the fluid flow rate and R is the radius of the tubing. Thus a variety of variables can be used to create the desired pressure. However, the high hydrostatic pressure should cause leaking of the perfusate by ultrafiltration. A high hydrostatic pressure should also to some extent counteract the osmotic pressure, which would give a lower recovery. These problems are not discussed in the patent.

P = 8ּVּLּQ

ππππּR4 Equation 1

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2.6 Design of microdialysis catheters There are today four different types of probes. The most common catheter is the concentric catheter. It was patented in 1982 by Urban Ungerstedt. It consists of a tubular formed membrane and an outlet of smaller diameter that passes all the way through the lumen of the membrane (fig 2.4). The inlet is attached to the proximal end of the membrane and the perfusate flows under the membrane from the proximal to the distal end of the probe. At the distal end the perfusate flows into the outlet hose and is transported to the vials. This construction gives a fairly robust probe and if the membrane is long enough the recovery is good. This type of catheter can be used for almost all applications and it is the only construction also used for catheters. It can be fitted with an electrode for stimulation and measurements in the brain. A micro cannula can also be combined with the catheter for microinjections.

Membrane portion

Fig 2.4 The concentric catheter. The loop type catheter is also quite common. It consists of a long membrane that is bent to form a loop (fig 2.6). The inlet is connected to one end of the membrane and the outlet is connected to the other end. This makes the membrane twice as long as other types of catheter and the advantage with the loop probe is that the recovery is good and the membrane area/volume ratio is high. But the membrane area of a loop catheter is not larger than the membrane area of a concentric catheter with the same outer diameter:

r r

A loop catheter = rּ π + rּ π = 2ּ rּπ = Aconcentric catheter

Fig 2.5 A cross section of a loop catheter and a concentric catheter.

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The end of the loop is usually made of a stiffer material to help keep the lumen of the probe open at all times. The disadvantages with the loop catheter are that it is more fragile and more likely to be deformed which may lead to blocking of the flow in the catheter. The latter can to some extent be prevented by using a higher pressure inside the catheter. Almost all microdialysis companies make this type of catheter, but this is probably more due to the fact that it is not protected by a patent rather than it is a good design.

Membrane portion

Fig 2.6 The loop type catheter. The linear catheter consists of a long unbent membrane with the inlet connected to one end and the outlet connected to the other (fig 2.7). It is implanted by tunnelation and is usually put subcutaneously. It can be used to measure for example how a drug penetrates the skin. If the membrane breaks, parts of the catheter may be left in the tissue. A way of avoiding this is to put a supporting fiber in the lumen of the catheter.

Membrane portion

Fig 2.7 The linear catheter. The side-by-side catheter is only made by Harvard Apparatus. The inlet and the outlet are placed beside each other and below this the cylindrical membrane is placed (fig 2.8). The advantage is that the probe can be made very small but there are some analytical limitations and the recovery can be inconsistent due to trapped air making this a poor design.

Fig 2.8 The side-by-side probe

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2.7 CMA design of introducers The catheters are implanted to the tissue by the use of an introducer (fig 2.10). The introducer is usually a guide cannula that encloses the catheter or probe. The cannula can either be slited or completely cylindrical and it is sharp at its distal end. The skin is first pierced with a puncturing needle. The catheter is put in the lumen of the introducer which is then inserted into the tissue. The introducer is then retracted leaving the catheters in the tissue. The introducer is removed from the hosing of the catheter through the slit. The procedure is quite easy to perform and causes little pain to the patient. Another type of introducer is the splitable introducer (fig 2.11). It is a plastic cannula that encloses the catheter. After insertion the cannula can be splited along its side and thus removed from the catheter and hosing. It is presently only used for implanting the CMA 62 liver catheter.

Fig 2.11 The splitable introducer, unsplited (left) and partly splited (right). 2.8 CMA design of pump and analyzer To pump the perfusate through the system a small portable pump is used that has a high accuracy at low flow rates. The CMA 107 pump intended for clinical use has a pulsated flow with one pulse every 100 second. When the perfusate has passed through the catheter it is collected in microvials that are put in the analyzer. The analyzer mixes the sample with the reagens for the substance of interest. The mix changes color at a speed that is proportional to the concentration of the substance. The sample is exposed to a light source and a photodiode is put on the other side to measure the absorption in the sample. As the sample changes color the absorption also changes and thus the concentration of the substance of interest can be determined. This technique is called absorption photometry. The next generation analyzers, the Iscus (fig

Fig 2.10 The CMA 60 introducer.

22

2.12), are under development. It is smaller and easier to use than the CMA 600 analyzer that is in use today.

2.9 Problems associated with microdialysis There are some problems associated with microdialysis. The largest problem in the clinical side is that it still is too complicated and hassleful to run microdialysis. The microvials have to be changed regularly and the data retained from microdialysis can be hard for the physicians to interpret. Sometimes the analyzer gives too much information making it difficult to choose what is important. This is the single largest factor to why microdialysis has not yet been accepted at the clinics. The demands on the physicians are too high and their training in microdialysis is too poor. This makes them avoid using microdialysis if it is possible and thus microdialysis is never going to be fully accepted unless this is changed. Another problem with microdialysis is the delay in the system. The sample must pass from the catheter through the hosing into the microvials before it can be analyzed. This gives a total delay of approximately 20 minutes. This is not a problem in many applications but for example when monitoring glucose in diabetes patient it is better to know what happens in the body instantly than knowing what happened twenty minutes ago, since the condition of the patient can change radically in that time. The delay is partly due to the hosing from the catheter, partly due to the low flow rate in the system. The hosing is also a problem in research since some molecules of the sample may adhere to the hosing before it is collected. Some of the problems associated with microdialysis in general could be avoided with an on-line analyzer, i.e. an analyzer that is connected directly with the outlet of the catheter. It would give a more continuous measurement than today and be easier to use for the operator.

Fig 2.12 The new Iscus analyzer.

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2.9.1 Problems associated with the catheters There are two main problems associated with the CMA 60 catheter. The first is that the recovery of the catheters sometimes can be low. There are four reasons for this; • As stated in section 2.6.1, the pores in the membrane are of different size. The innermost layer consists of smaller pores and the outer layer consists of finger-like pores. Air can sometimes be trapped in these pores (fig 2.13) and after insertion into the tissue this air is difficult to get rid of. The diffusion cannot take place through air filled pores and the more pores that are filled with air the smaller the active membrane area gets resulting in a lower recovery. This can be avoided by dipping the catheter in the perfusate before inserting it into the tissue. • If there is an initial hemorrhage when inserting the catheter, caused by for example the sharp introducer, the blood can cover parts of the membrane and after it has coagulated the diffusion cannot take place resulting in a lower recovery. This problem can to some extent be reduced by heparin or silver treatment of the catheter but it is not used today and the effect on the recovery is unknown •The pain the introducer causes can make the vessels contract. This gives a lower blood supply, which in turn gives a lower recovery. With time the recovery normalizes as the vessels dilate. •Compared with other tissues adipose tissue usually has a lower recovery. This is due to the fact that adipose tissue contains fewer blood vessels and is much “dryer”. The other problem is that the membrane sometimes breaks when the catheter is used in adipose tissue and muscles. In order to investigate this all complaints on CMA 60 catheters were reviewed, see appendix X. It is clear that there are two main reasons for membrane damage, either incorrect handling or implantation into muscle. In some cases the membrane has broken when implanted subcutaneous but it is difficult to determine whether it is due to incorrect handling or to fragile membranes. When the membrane breaks due to

Fig 2.13 Cross section of the membrane where some of the pores are filled with air.

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incorrect handling the users are often inexperienced. It is hard to avoid those damages but they would be fewer with a more protected membrane. The fascia surrounding the muscles is quite rough. If the catheter is not inserted long enough in to the tissue the membrane may lie across the fascia. If the muscle is contracted the fascia is most likely to break the membrane. The fascia can also cause the “sock effect” (see below) when the catheter is retracted.

Fig 2.14 Membrane breakage at the fascia. Even if the whole catheter lies within the muscle the mechanical stress induced by contracting muscle is likely to make the membrane break. The intended use for CMA 60 is either adipose tissue or resting muscle. It would be desirable to have a catheter that can be used in working muscle. The catheter can also break in resting muscle just due to the fact that muscle tissue is quite rough. Also during insertion the membrane is in risk of being damaged. When inserting the catheter into the tissue using the slit cannula the tissue can to some extent enter the lumen of the cannula and push the tip of the catheter backwards (fig. 2.15). This will make the membrane rise out of the protecting cannula and it is most likely to be damaged.

Tissue Fig 2.15 The catheter rises from the introducer due to pressure from the tissue and are thus getting damaged. If the membrane is inside the cannula during the whole insertion, it can still be damaged when the cannula is removed. The sharp tip of the cannula can come in contact with the membrane and cut it up. This can happen if the user is inexperienced with inserting catheters. A quite common problem is that the membrane breaks at the proximal end and slides down the outlet hose, making the membrane resemble a sock, see fig 2.16. The catheter can work without problem during sampling but when it is

25

retracted the membrane has slid down. This indicates that the membrane breaks during the retraction. This doesn’t affect the performance of the catheter since it happens when the measurements are finished. It is not a major problem and the only risk is that pieces of the membrane or the whole tip of the catheter is left in the body. Since the outlet hose is glued to the tip the risk of the tip being torn off has to be considered minimal and usually no pieces of the membrane is left in the body. The membrane always breaks at the proximal part and the reason for that is probably that the friction forces from the tissue builds up along the catheter and reaches a maximum there. The glue can also make the membrane more fragile but that has not yet been investigated.

Fig 2.16 The “sock effect” on a CMA 60 (left) and a closer view of the membrane (right). The membrane can also be damaged during production. If small holes are made in the membrane they may not be detected in the manual inspection every catheter goes through. These holes might not show in the initial stage of use but as they get larger the measuring have to be terminated. It is the difficult to derive the damage to the production since the catheter worked at the initial stage of use. Today, the catheters are just manually inspected and flow tested. A test to find leaks should be performed to ensure a higher quality.

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2.10 Specification of requirements There are some different ways of improving the mechanical stability of the membrane. There are some limitations to consider though, and the most important are as follows; • The relative recovery of the new catheter shall be as close to CMA 60 as possible. • The maximal diameter shall not exceed the inner diameter of the existing introducer. • The active membrane area shall be constant. • The membrane shall be protected from mechanical stress. • The catheter shall be possible to sterilize with β-radiation and shall not be magnetic. • The membrane shall be the same as in CMA 60. • The catheter shall be able to be assembled at CMA Microdialysis AB. The foremost important demand in this thesis is of course that the membrane shall be protected from mechanical stress. Considering the problems associated with the catheters mentioned earlier, it is clear that the protection of the membrane has to be better than it is today on the CMA 60. There are a few different ways of solving this problem. The first way is to put a supporting structure around the membrane. This can be done in several ways, but the main problem with this solution is that the active membrane area is reduced leading to a lower recovery. Another way to solve the problem is either to put a supporting structure in the actual membrane. Gluing different patterns in the membrane is one way of doing this. Regardless of which strategy is chosen the active membrane area will get smaller with a protective structure on the membrane. A decreased membrane area will lead to a lower recovery. So there is a balance between protection and recovery.

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2.11 The Recovery Problem How high relative recovery is really needed in a clinical application? There are two ways to look at the data from microdialysis, either to look at the absolute values or at trends. A value can differ quite a lot between different persons, even at a normal state. This makes it hard to say that a level of a certain substance is pathological when it can be normal for some persons and pathological for others. By looking at trends changes in a specific patient can be monitored, and tips and dips in levels can give an indication that something is wrong. This is the way CMA would like to see it, but the physicians still like the absolute values as well to have some sort of reference. If the only interesting thing would be to look at trends a low recovery would theoretically be sufficient as long as it is constant. But there are some problems associated with a low recovery. If the recovery would be 20% a small error would affect absolute value more than the same small error if the recovery were higher, possibly giving false trends. The curve for the relative recovery is steepest between 90% and 10% recovery.

100%

Flow rate [µl/min]

Recovery

90%

10%

Fig 2.17 The relative recovery curve. A small change in the fluid velocity will affect the recovery more in this region than above 90% where the curve is less steep. A change in recovery will give a change in the absolute values and it can be difficult to determine whether the change is due to a real change in the concentration of the substance or just a change in the recovery. The fluid velocity of the perfusate in the catheter is however relatively constant. The fluid velocity of the perfusate on the x-axis in the graph can be changed for the blood flow in the capillaries. To illustrate this one can imagine two adjacent tubes that have an exchange of substances (fig 2.18). The flow rate in the two tubes is of opposite directions and the relative velocity of the flow determines

28

the magnitude of the exchange. It is easy to see that it doesn’t matter which tube has an increase or decrease in the flow, the change in the relative velocity will still be the same. In the body, it does not matter if the change of the flow is in the capillaries or in the catheter, the relative change in velocity is still the same. Even though the tissue between the capillaries will damp the system a change in the blood flow will affect the recovery and it will be more affected in the steep region of low recovery than in the region of high recovery. A change in the blood flow can also cause other problems. If there is an increase in the blood flow more substances will be transported to the investigated tissue (due to the fact that more blood is transported to the tissue) even though the concentration in the blood is constant. This will lead to false increase of the substances in the catheter. When measuring lactate a way of avoiding this false trend is to look at the lactate/pyruvate ratio. All cells degrade glucose in a process termed the glycolysis. It is a chain of reactions in ten steps degrading glucose to pyruvate and ATP. If there is sufficient oxygen in the cell, most of the pyruvate is converted into Acetyl-CoA and enter the citric acid cycle.

Fig. 2.19 The degradation of glucose.

Glucose

Glycolysis

Aerobic reaction Anaerobic reaction

Lactate To the citric acid cycle

Pyruvate

V 1

V 1

V́ 2

V́ 2

Fig 2.18 The mutual exchange between two adjacent pipes.

29

If there is not sufficient oxygen another reaction will take place. The hydrogen donor NADH+ will supply the pyruvate ion with hydrogen and lactate ions are formed. The oxygen sensitive enzyme LDH catalyzes the reaction, and an absence of oxygen will make the reaction proceed to the right. Fig. 2.20 The lactate pyruvate equilibrium. If more blood is transported to the tissue, it will be more saturated and less lactate will be formed. If less blood reaches the tissue, it will be less saturated and hence more lactate will be formed. The problem is however that the level of lactate also is dependent on the supply of glucose. If the oxygen level in the tissue is constantly low and the level of glucose increases, the lactate level will increase since it is formed from glucose. This increase will only be due to the increased glucose level and totally independent of the oxygen level, which is usually of interest. Hence it can be difficult to determine whether a change in lactate is due to a change in the oxygen level or in the glucose level. This insecurity can be evaded by looking at the lactate/pyruvate ratio. Since both lactate and pyruvate are formed from glucose an increase in glucose will lead to an increase in both lactate and pyruvate and the ratio between them will remain unchanged. A decrease in the oxygen supply will make more of the pyruvate form lactate leading to a higher lactate/pyruvate ratio. Consequently the lactate/pyruvate ratio is insensitive to changes in the glucose level. A larger problem arises if only glucose is of interest. There is today no reliable method to determine if a change in the glucose level is due to a higher blood supply or an actual change in the blood glucose level. This is a problem in the diabetes field were the blood glucose level is of interest. Test with urea has as a reference substance has been done.

pyruvate + NADH+ Lactate + NAD + H+

enzyme: LDH

30

A high recovery is also advantageous because it in some respects actually affects the tissue less than a low recovery. Considering fig 2.21 it is easy to se that a catheter with 80% recovery will drain the tissue of substances along its whole length, while a catheter with 100% recovery cease to drain the tissue of substances when 100% recovery is achieved. At the distal end of the catheter with 100% recovery the perfusate has the same concentration as the tissue and thus the diffusion ceases here. Consequently a catheter with 100% recovery will not affect the tissue at its distal end while a catheter with 80% recovery will.

1 M 1 M5 M

2 M

3 M

4 M

5 M

5 M

5 M

2 M

3 M

4 M

5 M

5 M

5 M

5 M

5 M

5 M

Fig 2.21 Catheter with 80% recovery (left) and 100% recovery (right).

Even though the concentration gradient in a catheter with 100% recovery might not get equalized until the distal end of the catheter, the gradient over the whole length of the catheter will be smaller than one in a catheter with less than 100% recovery, thus affecting the tissue less. Considering all this it easy to see that having a high recovery eliminates a lot of errors. As already stated, the problem with putting a supporting structure on the membrane is that the active membrane area will be smaller giving a lower recovery. So there is a balance between a protected membrane and a high recovery.

31

2.12 Ways of increasing the recovery As already stated, a protected membrane will probably lead to a lower recovery due to a smaller active membrane area. But are there other ways of increasing the recovery? The two main ideas are either to change the flow in the catheter or reducing the volume under the membrane. These ideas will be discussed below. 2.12.1 Changing the flow in the catheter The CMA 106 pump previously used for microdialysis gave a constant flow. The later introduced CMA 107 gives a pulsated flow, with a pulse every 100 seconds and adjustable pulse sizes. It was noticed that the catheters had a higher recovery when the new pump was used. One theory regarding this was that the new pump created a turbulent flow in the catheter, whereas the old pump with a constant flow created a laminar flow. But by looking at Reynolds number for the flow in the catheter it is easy to see that a turbulent flow cannot be the casein the catheters. where U is the velocity of the fluid [m/s] νννν is kinematic viscosity [m2/s] l is the diameter of the tubing [m] This will in the case of CMA 60 with a flow of 0,3µl/min give

In order to have a turbulent flow Reynolds number has to be above 2300. So it is obvious that there are no possibilities for the flow to become turbulent. To illustrate this the viscosity of the perfusate in the catheter at these small dimensions can be compared to syrup.

Re = U ּ l

νννν [6]

Re = 0,0505ּ 10-3 m/sּ 0,07ּ 10-3 m

10-3 m2/s = 3,535ּ 10-3

32

Another possible explanation for the higher recovery with a pulsated flow is that the flow profile changes. Considering the profile for a constant laminar flow (fig 2.22) it is easy to see that the flow is very slow in the edges (zero right at the edge).

v

Fig 2.22 The flow profile in the catheter with a constant flow. The theory is that the perfusate at the edges probably equalizes fast with the surrounding tissue. But since the perfusate moves much slower in the edges the gradient between the tissue and the catheter is reduced, resulting in a slower diffusion and a lower recovery.

v

Fig 2.23 The flow profile with a pulsated flow. With a pulsated flow the flow profile changes (fig 2.23). Between the pulses the perfusate is still and during the pulses it moves with a much higher velocity than with a constant flow. This makes the flow profile more rectangular and gives the perfusate at the edges a higher flow. A higher flow at the edges will

33

transport the already equalized perfusate and replace it with new unequalized perfusate. This might give a higher recovery. But this theory is based on the assumption that the diffusion inside the catheter is slow. Considering the perfusate inside the catheter, there is a concentration gradient due to the diffusion speed, with a high concentration of substances at the membrane and a low concentration at the outlet hose. Fig 2.24 shows how the concentration gradient of the diffused substances may look like in the catheter. As the perfusate flows downwards the gradient gets more equalized. But how long time does the gradient require to be equalized. This is given by the equation

were h is the diffusion distance, in this case the distance between the membrane and the outlet hose and D is the diffusion coefficient. The diffusion coefficients for the six substances are [23] as follows. Some of the constants are estimated but since most of substances has the same molecular size and weight the will vary max 20-30% [24]. A measurement can be performed but considering the short diffusion time it is clear that the estimation is sufficient. Dpyruvate= 1ּ 10-5 cm2/s (estimated) Dlactate= 1ּ 10-5 cm2/s (estimated) Dglucose= 6,7ּ 10-6 cm2/s Dglutamate= 1ּ 10-5 cm2/s (estimated) Dglycerol= 1,06ּ10-5 cm2/s

Tdiff = h2

D [6]

Fig 2.24 The concentration gradient in the catheter

34

Equation 3 gives Tdiff = 7,3 s for glucose Tdiff = 4,6 s for glycerol and Tdiff = 4,9 s for the other substances. At the normal flow of 0,3 µl/min it takes ten minutes for the perfusate to move from the proximal to the distal part of the catheter. The diffusion time may seem short but considering the diffusion distance of only 0,07 mm it is a reasonable time. It is obvious that the concentration gradient has enough time to equalize during that time considering the short diffusion time. This shows that the theory with the flow profile probably is incorrect. The diffusion outside the membrane (in the tissue) is probably the largest obstacle for a higher and faster recovery, see the reports on this subject by Gustav Amberg and Urban Ungerstedt, [4] and [5]. The diffusion in the tissue is dependent of supply, clearance and diffusion speed, factors that of course are very difficult both to influence and measure. Another theory that is more probable discusses the shear forces. A pulse is produced every 100 second and during the time between the pulses the perfusate is still in the catheter. The time of the pulses are in the range of 10 ms which means that the fluid is accelerated during a short time. This acceleration leads to a friction between the fluid and the membrane resulting in a shear force. The magnitude of the shear force, ττττ, is given by equation 6; where µµµµ is the friction coefficient and u is the velocity of the fluid [m/s] This gives that the shear force is dependent on the velocity of the pulses and since the velocity is high during the pulses the shear forces will be equally high. It is probable that the large molecules of the perfusate and the ISF to some extent adhere to the inside of the membrane and thus block the pores or at least slow the diffusion down. The shear force from the fluid will “pull” those large molecules away from the membrane and into the perfusate, thus giving a higher recovery. Large molecules from the tissue are also likely to block the fingerstructure of the membrane, and the shear force may to some extent influence those. In fig 2.22, the flow profile shows that a pulsed flow creates a much higher flow at the membrane than a constant flow does. Consequently more pulses and shorter pulse time would lead to a higher recovery.

ττττ = µµµµ ּ δδδδu

δδδδy [6]

35

2.13 New catheters To improve the mechanical stability a number of ideas was investigated. Three of them, “the Cap” and the “the Braid” was chosen for further testing. 2.13.1 The Braid The braid is an idea patented by CMA Microdialysis in 2001. The idea is to put a hollow braid around the membrane that would give it a good protection. It is very unlikely that the membrane should break as it is almost completely covered by the braid. Even if it would break, all pieces of the membrane would stay inside the braid and not be left in the body when the catheter is retracted. The threads the braid is made up of should be very thin, requiring quite little ISF to fill up the gaps between them. By making the threads of nylon the braid will be flexible. In fig. 2.26 it is shown that the round threads of the braid covers a very small area of the membrane making the active area of the membrane relatively large.

The disadvantages with this construction is that “dry” tissues such as adipose tissue requires a longer start up time due to that it takes longer for those tissues to produce enough ISF to fill up the gaps in the braid. Bubbles of air might also be formed in the gaps of the braid affecting the recovery negative. This can maybe be avoided by dipping the catheter in the perfusate before use. The

Fig 2.25 The membrane covered by a braid.

Fig 2.26 Cross section of a catheter covered by a braid.

Membrane

Threads of the braid

36

production of the catheters is also more complicated due to the tendency of the braid to unravel when cut. The braid was delivered by Gudebrod Inc, a FDA approved company specialized in textile manufacturing. The braid is a nylon monofilament braid with an outer diameter of 0,79 mm and a filament diameter of 0,17 mm (Gudebrods specs). Measurements done at CMA indicated that the filament diameter were 0,14 mm. The pic count is 27 pics per inch, making it a rather loose braid. It would be desirable to have a braid with an even lower pic count and a much smaller filament diameter.

Fig 2.27. The Gudebrod braid covering a membrane.

37

2.13.2 The Cap The idea with the cap is to put a protective cap on the parts of the membrane that are most vulnerable i.e. its distal part and along some of its sides. The cap is made of a stiff tube that is cut open to expose the membrane where it is not so vulnerable. The stiffness of the hose makes it less likely to “rise” from the introducer and be damaged that way. It will also make the membrane keep shape and not be deformed by the tissue.

The panels that was cut out was chosen sothe vulnerable parts of the membrane is protected, whilst the parts that seldom break is exposed to the tissue through the panels. This will hopefully give an acceptable recovery and a good protection. A problem with this design is that some of the ISF may leak in between the cap and the membrane. This would make diffusion take place there as well and the recovery would increase. But since it is not possible to control this it would lead to an inconsistent recovery. To avoid this the edges of the panels are glued to the membrane on some of the catheters. Other disadvantages are that the recovery is lower in this type of catheter than in the CMA 60 and the membrane in the panels are unprotected. The prototypes was made of a medical Pebax tubing (7033), which was striped with 30% BaSO4 and delivered by Optinova AB. The panels was first intended to be cut out with a CO2 laser by Lasernova AB but due to difficulties with melting the tube was cut manually. This was done under microscope and gave acceptable accuracy. Three or four panels were cut out, leaving 1-2 mm of materials between them to keep the rigidity of the tube.

Fig 2.28 The Cap.

38

Fig 2.29 The Pebax cap covering the membrane.

Some prototypes were also made with the existing sleeve. It is made of and delivered by Optinova AB. It is softer than the Pebax tubing and the advantage is that the same piece can be used for both the sleeve and the cap, thus reducing one of the glue joints.

39

3. Experiments 3.1 Introduction The prototypes were tested both in vitro and in vivo to determine how the different solutions influenced the recovery. They were first tested in vitro to see if the recovery was acceptable. The in vitro situation is however a quite unlike the situation in the tissue. The in vivo recovery is affected by supply, clearance and diffusivity in the tissue. In vitro there is no continuous supply of substances and the clearance is only done by the catheters itself but most importantly the diffusivity of the substances is much higher in liquid (in vitro) than in tissue. Thus the prototypes were also tested in vivo to get a better view of their performance.

3.2 In vitro tests The in vitro test was performed at the lab of CMA Microdialysis AB. The in vitro test is usually performed to test the consistency of the produced catheters. The test measures the relative recovery of selected substances in vitro. The substance analyzed is glucose which has twice the molecular weight the other substances has making it the hardest substance to recover. The method has some limitations but gives a notion of the performance of the catheters.

40

3.3 In vivo test 3.3.1 Introduction

Since the in vitro test is a quite poor imitation of tissue the catheters were also tested in vivo. The main objective of the test was to investigate how the catheters performed in tissue. Another question was if there is enough ISF in adipose tissue to fill the space between the threads of the braid. The test was conducted at Karolinska Institute in accordance to an experimental protocol approved by the local ethical committee. 3.3.2 Materials The materials used for the microdialysis part of the experiment were as follows: • Three braid prototype catheters • Three reference catheters • Three Arrow Peel-Away Sheath over Needle Introducers no PN-05014 • Six CMA Splittable Introducers SI-2 • Twelve CMA/106/107 Microdialysis pumps • Twelve CMA 106 syringes • Perfusion fluid T1 • Microvials 3.3.3 Surgical preparation For the test a Crossbred (Swedish Landrace) littermate pig of 26 kg was used. The animal was sedated with an intramuscular premedication of 12 mg/kg Ketamine hydrochloride (Ketaminol Vet®. Veterinaria AG, Zurich, Switzerland) and 0,05 mg/kg atropine sulphate (Atropin® NM Pharma AB, Stockholm, Sweden). Before intubation 8 mg/kg of Propofol ( Fresenius Kabi) was given through an ear vein via a 20G venflon cannula. The animal was then intubated (Tracheal cuff tube ID 6,0) and ventilated by a volume controlled respirator (9000, Siemens, Germany) with the following settings; pressure regulated volume control mode, 200 ml/kgּ.min, 20 breaths/min, inspired oxygen fraction 23%. The ventilation vas controlled by SpO2 on the tail. The maintenance of total anesthesia was obtained through an intravenous infusion of Propofol (around 10 mg/kgּh) and 150 µg/h Fentanyl (given through a peripheral vein in the abdominal wall via a 16G venflon cannula). After a midline laparotomy, a urinary catheter was placed in the urinary bladder by a small cystomy. ECG, heart rate and body temperature were

41

measured and recorded during the whole experiment via a patient monitor/ICU Pilot program. The blood pressure was not measured. After the experiment the animal was killed by an injection of KCl in the heart. 3.5.4 Experimental procedure Since the performance of the cap catheters was so bad in vitro they were not tested in vivo. The catheters were connected to pumps and run for fifteen minutes in a beaker with saline solution. After an initial flush, the flowrate was adjusted to 0,3 µl. When normal flow had been verified the catheters were implanted. The braid catheters were implanted in the adipose tissue on the backside of the neck of the pig. An incision was made and the catheters were implanted directly into the adipose tissue. This was done to avoid implanting through the quite rough skin of the pig and also to assure that the catheters were placed in the adipose tissue. Three braid catheters (A1, A2 and A3) and three reference catheters (B1, B2 and B3) were implanted with splitable introducers on the two sides of the incision, see fig 3.2.

B1

A2

B3 A3

B2

A1

Fig 3.2 The insicion (left and middle) and the placement of the individual catheters (right).

After implantation the samples were taken every 20 minutes. The flow rate was 0,3 µl during the sampling. The samples were analyzed in the CMA 600 analyzer continuously during the experiment.

42

4. Results and discussion 4.1 In vitro results Five tests on the prototype catheters were performed; two tests with the braid catheter, two tests with the cap catheter and one with the glued catheter. The braid catheters The results from the first of the two tests with the braid catheters were as follows. The average relative recoveries of the catheters were; Reference cateheter A: 70,8% with an average deviation of 1,0% Braid catheter B: 69,2% with an average deviation of 0,9% Braid catheter C: 72,0% with an average deviation of 1.9% Over time the relative recovery was:

Invitro test; braid cateher

64,0

66,0

68,0

70,0

72,0

74,0

76,0

1 2 3 4 5 6 7 8Measurement no.

Rel

ativ

e re

cove

ry o

f g

luco

se

Reference catheter A

Braid catheter B

Braid catheters C

Fig 4.1 The relative recovery of the first test with the braid catheters. The second test with the braid catheters gave similar good results. The average relative recoveries of the catheters were: Reference catheter A: 71,9% with an average deviation of 1,8% Braid catheter B: 68,3% with an average deviation of 3,0% Braid catheter C: 69,0% with an average deviation 2,5% If the reference catheters had a relative recovery of 100 %, the braid catheters would also have had a relative recovery of 100%.

43

Over time the relative recovery was:

In vitro test; braid catheters II

56,0

58,0

60,0

62,0

64,0

66,0

68,0

70,0

72,0

74,0

76,0

1 2 3 4 5 6 7 8Measurement no.

Rel

ativ

e g

luco

se r

eco

very

[%

]

Reference catheter A

Braid catheter B

Braid catheter C

Fig 4.2 The relative recovery of the second test of the braid catheters. The cap catheters For the cap catheters the results was somewhat different. The relative recovery of the catheters in the first test was: Reference catheter A: 65,7% with an average deviation of 0,7% Cap catheter B: 41,8% with an average deviation of 0,7% Cap catheter C: 19,0% with an average deviation of 1,2% If the relative recovery of the reference catheter had been 100% the cap catheters would have had a recovery of 64% (catheter B) and 29% ( catheter C), which is quite poor. The difference between the cap catheters may be explained by the fact that fluid had come in between the cap and the membrane thus increasing the active membrane area greatly in one of the catheters.

In vitro test, cap catheters

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Fig 4.3 The relative recovery for the cap catheters over time. At the second test with the cap catheters the cap was glued around the cut-out panels to prevent fluid to enter the space between the cap and the membrane. The average recoveries from that test were as follows: Reference catheter A: 66,9% with an average deviation of 0,4% Cap catheter B: 21,6% with an average deviation of 1,0% Cap catheter C: 30,2% with an average deviation of 4,4% If the reference catheter had a recovery of 100% the cap catheters would have had a recovery of 32% (catheter B) and 45% (catheter C). The relative recovery over time was:

In vitro test; cap catheters II

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Fig 4.4 The relative recovery over time for the second test of the cap catheters.

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4.2 Discussion of the in vitro test The results show that the braid catheters obviously perform very well in vitro. The cap catheters have a quite low recovery, most likely due to the small active membrane area. The recovery was very inconsistent and it is a big problem and can to some extent be due to that the panels were cut out by hand. Since the recovery is so low in vitro it was not tested in vivo. As already stated the in vitro situation test is not a very good imitation of living tissue. It gives however a hint about how the catheters perform in vivo. The series tested were too small to give good statistics, only one glued catheter was for example tested. But it still gives a notion about how the catheters perform but the small series should be considered when conclusions are drawn. Since the catheters were prototypes some inconsistencies can be expected, especially for the cap catheter were the panels were cut out by hand. The membrane could also have been affected negatively during manufacturing, for example when putting the braid on the braid catheters. Since the braid catheters are so sensitive in its present form the handling during the test can have affected the catheters. The membrane can vary between the catheters and the outlet hose can also vary in diameter (see section 2.12.2), which is most likely to affect the recovery. The samples from braid test number two was put in the fridge one night before analyse but that should not affect the results too much. During one of the tests the reagents in the Cobas Mira analyser had to be changed which affected the results. The temperature was not measured and could have changed during the tests and a higher temperature leads to a higher recovery. But most of the catheters were rather stable which indicates that the conditions were quite good.

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4.3 In vivo results The braid catheters Three braid catheters and three reference catheters were tested in vivo. They were implanted in adipose tissue through an incision in the neck. Figure 4.5 shows the glucose recovery for the braid catheters. As seen the discrepancy between the references is quite large and that should be considered when analysing the results. But it is still clear that the performance of the braid catheters is good, even better than the references.

Fig 4.5 The glucose recovery for the braid catheters in vivo. There are obviously some disturbances in the beginning of the measurement but after measurement four it is more stable.

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The same phenomena can be seen in the lactate levels (fig 4.6). The braid catheters performs distinctly better here. It should be noted that the lactate ion is half the size of the glucose molecule and hence easier to recover.

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Fig 4.6 The lactate recovery for the braid catheters in vivo. The pyruvate recovery is illustrated in fig 4.7. As the glucose and lactate recovery the measurements are stable after measurement no. four. The reference catheters show good correspondence and the braid catheters perform as good as the reference catheters.

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Fig 4.7 The pyruvate recovery for the braid catheters in vivo.

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For glycerol the result were as follows (fig 4.8). Even though the discrepancy between reference catheters are not the best, its clear that the braid catheters performs as good as the references.

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Fig 4.8 The glycerol recovery in the braid catheters. Considering all the measurements done with the braid catheters the indication is that they actually have better recovery than the reference catheters. But the poor discrepancy of the reference catheters in some of the measurements must be taken in consideration when analyzing the data. The small series makes the statistics a bit uncertain. But the tests still give a positive indication of the braid catheters.

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4.4 Discussion of the in vivo test Considering these results the braid catheters performs surprisingly well. Even though the active membrane area is smaller the recovery is slightly better than the reference catheters, opposite to how it should be. The most likely theory explaining this is that the space around the membrane created by the braid is filled with fluid that enhances the diffusion across the membrane. The membrane of a normal catheter lies against the tissue and the diffusion goes from (solid) tissue to the fluid inside the catheter and the diffusion in the braid catheter goes from the created fluid space to the fluid inside the catheter. Apparently diffusion from fluid to fluid is easier than from tissue to fluid. This side effect of the braid is of course very advantageous. How it influences microdialysis in a more saturated tissue is still to be investigated. And how does this extra fluid space influence the physiology in a long-term perspective? Will cells start to fill the gaps in the braid? The concentration gradient between the tissue and the catheter will probably be stretched out over a longer distance compared to a normal catheter, how does this influence the diffusion and the delay? There are many things yet to be investigated. The braid used for the prototype braid catheter is not optimal. The braid is made of nylon threads making it quite flaccid. Since the braid is so flaccid, a mechanical force applied to the braid will just be propagated to the membrane, but only to the places where the threads of the braid lie against the membrane. The membrane will of course be deformed at these places, see fig 4.13. This effect can be avoided by using more rigid braid.

Fig 4.13 The membrane after the braid has been removed (this catheter has been quite heavily abused though).

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The series are too small to give good statistics, at least five catheters and five references have to be used. However, the tests still give and indication that both the braid catheters and the glued catheters perform well in vivo but more test are required. The physical status of the pig may have influenced the tests. The body temperature varied quite a lot during the experiment, see fig 4.15. An increased body temperature will cause a vasodilation of the peripheral blood vessels leading to a higher blood flow and hence more transport of substances to the tissue. The heart rate is also presented in fig 4.15.

Ta MG Gris111 040402-0001 HR MG Gris111 040402-0001

2004-04-0210:00 11:00 12:00 13:00 14:00

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Fig 4.15. The body temperature and the heart rate of the pig during the experiment. Due to extensive bleeding during implantation of the blood pressure gage the blood pressure was unfortunately not measured.

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5. Conclusion The best way to protect the membrane is to use a braid. It provides good protection and the recovery in vivo is as good or even better compared to a normal CMA 60. If a more rigid braid was used the catheter might even be protected enough for use in working muscle. The sock effect would be totally prevented. If the right type of braid is used manufacturing of the catheters should not be much more complicated than the manufacturing of catheters today. One disadvantage is that the diameter of the catheter increases. With the right type of braid that increase can be acceptable but a new type of introducer is probably still needed. More testing of the braid catheter is however required to investigate the effects of the braid. The in vitro test showed that the recovery of the cap catheter is too poor for use in normal microdialysis applications. The membrane is however probably well protected and this design might be suitable for other applications. Disetronic Medical Systems for example, uses it in their diabetes project. The inconsistent recovery shown in the in vitro tests would probably be better with a more thorough manufacturing.

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6. Recommendations for future work The first question to be answered is which applications the protection is needed for? And what are the actual problems today? In my point of view, the breaking membranes are not a specific CMA 60 problem, but a problem for all catheters except the brain catheters. It is not a major problem for microdialysis today, but with new, more demanding applications, a protected membrane is crucial. Different solutions are probably needed for different applications. More tests should be conducted on both the braid catheter and the glued catheter. Effort should also be made to find a suitable braid, the best way is probably to have Gudebrod Inc. to make a braid after our specific requirements. With a stiff braid, production of the catheters shouldn’t be much more complicated or time consuming than today. A stiff braid would also provide the best protection for the membrane, maybe even so good that the catheter could be used in working muscle. The braid catheter should also be tested more in vivo to investigate how it affects the physiology in a longer time perspective. Unfortunately the actual durability of the braid catheter has not been tested in this thesis. This is of course essential and should also be done before more in vivo tests are performed. Two problems with this type of testing are that the mechanisms making the membranes break and what forces are applied to the membrane in vivo are unknown. Further I strongly recommend a leakage test of all catheters as part of the quality assurance. Today catheters with damaged membranes can pass the manual inspection and be delivered to customers.

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References [1] Bito L, Davson H, Levin E, Murray M and Snider L. “The concentrations of free amino acids and other electrolytes in cerebrospinal fluid, in vivo dialysate of brain, and blood plasma of the dog.” Journal of Neurochemistry,1966 vol 13 pp 1057 to 1067. [2] Delgado J M R, DeFeudis F V, Roth, Ryugo D K and Mitruka B M. “Dialytrode for long term intracerebral perfusionin awake monkeys.” Arch. int. Pharmacodyn. 198 9-21 (1972). [3]Ungerstedt Uand Pycock C “Functional correlates of dopamine neurotransmission.” Bull Schweiz Akad Med Wiss. 1974 Jul;30(1-3):44-45

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[15] Panotopoulos C. “Multi purpose catheter ” Patent no WO0232494, Espacenet [16] Metzger L. No English title available. Patent no. DE 19937099, Espacenet. [17] Liska J, Franco-Cereceda A. “Microdialysis catheter for insertion in a blood vessel” Patent no. WO 200106928, Espacenet. [18] Liska J, Franco-Cereceda A. “A catheter to be inserted into a blood vessel, and a method for detection of substances” Patent no. WO 9945982, Espacenet. [19] Bergveld P, Bohm S and Olthuis W. “Microdialysis probe integrated with a si-chip” Patent no. WO9941606, Espacenet. [20] Haindl H. “Cannular arrangement” Patent no. WO9857693, Espacenet. [21] Kissinger Candice B “Linear microdialysis probe with support fiber” Patent no. US 5706806, Espacenet. [22] Mishra Pravin K. “Dialysis probe” Patent no. WO 9218191, Espacenet [23] Landolt-Börnstein. “Numerical Data and Functional Relationships in Science and Technology” Sixth Edition, Vol II/5a, 1969 [24] Peter Stilbs, Prof Physical Chemistry, The Royal Institute of Technology. [25] Shaldon S, Koch K M. “The Evolution of a Syntetic Membrane for Renal Theraphy”Polyamide- [26] van den Berghe G, Wouters P, Weekers F, Verwaest C, Bruyninckx F, Schetz M, Vlasselaers D, Ferdinande P, Lauwers P, Bouillon R. “Intensive insulin therapy in the critically ill patients.” N Engl J Med. 2001 Nov 8;345(19):1359-67. Campbell M K. “Biochemistry” Second edition Weast, R C “Handbook of Chemistry and Physics” 57th edition. Rosdahl H. “Microdialysis sampling from skeletal muscle and adipose tissue with special reference to the effects of insulin on tissue blood flow and glucose metabolism” Bäckström T. “Intravasal microdialysis as a novel technique to monitor metabolism in myocardial ischemia and critical illness”

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Nowak, G. “Monitoring and prevention of ischamia-reperfusion injury in liver transplantaion –experimental and clinical studies” Andresson T. “Cutaneous microdialysis : a technique for human in vivo sampling” Röjdmark J. “Microdialysis in reconstructive surgery. A clinical and experimental study focusing on monitoring flap metabolism and viability” Benveniste H and Hüttemeier P C. “Microdialysis –Theory and Application” Progress in Neurobiology. Vol. 35, pp 195-215 Ungerstedt U and Hallströn Å ”In vivo microdialysis -A new approach to the analysis of neurotransmitters in the brain ” Life Sciences, Vol. 41, pp 861-864. Jolly D and Vezina P. “In vivo microdialysis in the rat: low cost and low labor construction of a small diameter, removable, concentric-style microdialysis probe system.” Journal of Neuroscience Methods 68 (1996) pp 259-267 Frothingham E P. and Basbaum A I “Construction of a microdialysis probe with attached microinjection catheter” Journal of Neuroscience Methods, 43 (1992) pp 181-188 Medical Dictionary of Medical Related Terminology Online http://medical-dictionary.com (February 2004) Espacenet online; http://se.espacenet.com/ (December 2003) Perimed AB http://www.lisca.se/ (January 2004) Gastric Tonometry Quick Guide http://www.datex-ohmeda.com/clinical/cw_prev_02_article5.htm (January 2003) Bioanalytical Systems Inc. http://www.bioanalytical.com (December 2003) Harvard Apparatus Inc http://www.harvardapparatus.com (December 2003) Eicom Corporation http://www.eicom.co.jp/ (December 2003)

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DICTIONARY Anastomosis If a part of an intestine is removed the union between the

remaining ends is termed anastomosis. Dialysate The perfusion fluid after it has left the catheter.

ECF Extra cellular fluid. The fluid outside cells.

ICU Intensive care unit. Iscemia A local lack of oxygen in a tissue. ISF Interstitial fluid. The fluid filling the small gaps between

cells.

Perfusate The perfusion fluid before it has entered the catheter. Sepsis Toxic poisoning