Microdialysis is not the only sampling method which relies on the use of an implanted semi-permeable membrane. Our In Vivo Ultrafiltration Probes utilize the same method. Microdialysis is an excellent method for monitoring changes in the concentration of a particular analyte, relative to some initial or basal level. However, when you are trying to determine the actual concentration of that analyte in the tissue itself, microdialysis methods require that you perform additional manipulations and extrapolate to determine that figure. In Vivo Ultrafiltration does not dilute the sample. Instead, it extracts the actual extracellular fluid and filters it to exclude high molecular weight compounds such as proteins. The concentration of an analyte in the ultrafiltrate represents the concentration of that analyte in the sampled tissue.
Ultrafiltration Probes are small, flexible tubes which can sterilized and implanted subcutaneously into living animals. The surgery is simple, requiring the implant of the tubing under the skin through a needle-like introducer. A suture secures the probe. Once installed, an Ultrafiltration Probe can be used to painlessly remove fluid from the extracellular space of the surrounding tissue. A common vacutainer provides the motive force which pulls fluid through tiny probes in the probe membrane, up the probe tubing and into the vacutainer. The sampling rate is slow (1 to 3 mL/min) and can not exceed the rate at which the fluid is replaced by the blood vessels within the tissue. The fluid removed is clean, protein free and ready for immediate analysis. Whenever a new sample is needed, a new vacutainer is used. The vacutainer and probe tubing is protected in a lightweight jacket worn by the animal. Small creatures, such as laboratory rodents, are housed in the BAS Raturn system and continuously sampled.
How does In Vivo Ultrafiltration Work?
A small and flexible probe is inserted into the tissue. This probe contains a sealed loop of semi-permeable plastic membrane. The membrane has tiny pores (holes) which allow water, salts and low molecular weight molecules (with molecular weights less than 30,000 daltons) to pass through the membrane and into the probe. Proteins and other large molecules can not pass and are left in the tissue. Tubing connects the membrane loop to the outside of the body. As vacuum is applied to the tubing, often by something as simple as a vacutainer, extracellular fluid is collected as it filters through the membrane, up the tubing and into the vacuum chamber. The concept of in vivo ultrafiltration and the design of probes is covered by USA patents: 4,777,953; 4,854,322; and 5,002,054.
Glucose levels in ultrafiltrates collected from subcutaneous tissue parallel the blood glucose levels. Analysis can be conducted with the glucose strips and meters marketed for home use by human diabetics. The lack of proteins in the sample may, in techniques involving the use of a biosensor electrode, improve the reliability of the measurement. There is about a 30 minute lag time behind blood glucose levels due to the time required for the fluid to move down the probe tubing and into the collecting vessel. Studies have been conducted in dogs for periods of several months.
Alternative Vacuum Source
Ultrafiltration Probes require a vacuum source in order to extract fluid from the tissue which surrounds the probe. There are two options for creating a vacuum: a vacuum tube of the type used for collection of blood, or a peristaltic pump.
Vacuum tubes provide a portable collection method which requires no electrical power, cords or tethering. When the hub device pierces the septum of the tube, the vacuum inside the tube is extended within the probe and fluid flow begins. The tube is replaced with a new vacuum tube whenever another sample is needed. Vacuum tubes might typically be replaced every 30 to 60 minutes. Vacuum tube collection can be used with a tethered small animal, or a freely-roaming large animal. In larger animals (horses, dogs, pigs, sheep) the vacuum tube is usually taped to the skin. The vacuum tube and probe site are protected by a coat or covering as the animal roams freely. To collect ultrafiltrates using vacuum tubes, please order the Ultrafiltration Starter Kit.
Peristaltic pumps provide a more convenient way of sampling from smaller animals housed in a container such as the BAS Raturn system. They can also deliver the sample to an automated, refrigerated fraction collector such as the BAS HoneyComb Fraction Collector. We offer a small peristaltic pump with small ID tubing which can be used for ultrafiltration sampling. To use a peristaltic pump, either order the pump and connectors described below, or determine a way to modify your existing peristaltic pump to create a gas-tight connection to the 0.65 mm OD tubing on the Ultrafiltration Probe.
Probe Sizes
Ultrafiltration Probes uses loops of membrane which are connected to a single collection tube. They are defined by the number of loops of membrane and the length of membrane in each loop. For example, the model UF-3-12 probe has 3 loops of membrane and each membrane loop contains 12 cm of membrane. Therefore, a UF-3-12 probe offers 36 cm of available membrane surface for ultrafiltration. Use a probe like this if you want to get maximum flow and study a uniform tissue such as subcutaneous tissue. Since the membrane is formed into a loop, the loop length is half the membrane length. So in a UF-3-12 (72-6942) probe, the length of the loop is 6 cm. You would use an introducer needle to tunnel under the subcutaneous tissue for a distance of approx. 7 cm in order to insert this probe. The OD of this membrane is 320 micrometers. Other probes models are named in the same manner: UF-1-2 (72-6945) has one loop containing 2 cm of membrane, UF-3-8 (72-6943) has three loops each containing 8 cm of membrane, etc.