Microdialysis sampling recovery

Adipose tissue poses additional calibration differences for microdialysis sampling devices. The thickness of the tissue (e.g., lean versus obese individuals) and thus the capillary density will affect interindividual microdialysis sampling recovery values. This has been shown by Lutgers et al., who demonstrated decreases in glucose recovery of up to 50% between human volunteers with a skin fold thickness of 20 versus 45 mm.65 This points to how microdialysis sampling recovery is dependent upon analyte supply since glucose will be supplied better to the microdialysis probe in lean individuals with a higher density of capillaries per unit tissue than obese subjects with a lower density of capillaries and thus an increased mass transfer resistance to the probe. Additional reports have also shown less interindividual differences between microdialysis probes implanted in the forearm versus in the subcutaneous tissue.66... 

Since the first reports on microdialysis in living animals, there have been efforts to estimate true (absolute) extracellular concentrations of recovered substances (ZetterstrOm et al., 1983 Tossman et al., 1986). Microdialysis sampling, however, is a dynamic process, and because of a relatively high liquid flow and small membrane area, it does not lead to the complete equilibration of concentrations in the two compartments. Rather, under steady state conditions, only a fraction of any total concentration is recovered. This recovery is referred to as relative or concentration recovery, as opposed to the diffusion flux expressed as absolute or mass recovery. The dependence of recovery on the perfusion flow rate is illustrated in Figure 6.2. As seen, relative recovery will exponentially decrease with increasing flow as the samples become more... 

In brain research, microdialysis sampling employing a miniaturized dialysis unit (probe) containing a dialysis membrane of a few millimeters length has become popular. The probe is implanted into the tissue or organ of the test animal and is infused with an isotonic solution (typically at 0.5-25 L/min). A steady-state osmotic flux across the membrane removes molecules with a mass below the cutoff of the membrane from the extracellular matrix. Microdialysis yields relatively clean samples of volumes in the range 20-100 jU-L. However, the recovery of neuropeptides can be as low as 0.5-15%, leading to a low neuropeptide concentration in the samples [5,6]. 

In microdialysis, the recovery of analyte from the sample depends on a number of factors, including the chemistry of the analyte, temperature, perfusion rate, membrane surface area, membrane characteristics, and the nature of the sample (including its fluid volume percentage and whether it is in motion). MiCTodialysis is typically done at low perfusion flow rates (0.5 to 2 /d/min). As flow rate increases, relative recovery decreases, but absolute recovery increases. Relative recoveries from small membrane probes are in the 1-20% range. SubstantiaUy higher recoveries in the 50-80% range can be obtained with longer loop-type probes. 

In vivo microdialysis sampling is not as simple. One cannot use the in vitro recovery to calculate an in vivo concentration. In many cases, it is not necessary to know the exact in vivo concentration it is sufficient to observe trends. However, if the exact concentration is desired, there are several methods of obtaining an in vivo concentration. 

Microdialysis can achieve both sample recovery and cleanup before analysis. The pore size of the microdialysis membrane can be tailored for selective exclusion of macromolecules such as proteins and enzymes, thus producing cleaner samples with reduced possibility of enzymatic degradation of the sample. 

If Ciniet has a value of zero, then equation (6.1) reduces to what is commonly called relative recovery and denoted in the literature as RR. The terms EE and RR are frequently interchanged and sometimes the general word, recovery, is used to describe the sampling efficiency of the microdialysis probe. It is important to note that the equivalence of EE versus RR under certain conditions such as with highly regulated concentrations of neurotransmitters has been debated in the literature 46... 

Another means of sampling is push-pull perfusion (79) using a probe with discrete inlet and outlet tubes. With this method, a small amount of cerebrospinal fluid is pulled directly from the brain through the outlet tube and replaced with artificial cerebrospinal fluid administered via the inlet tube. This approach has greater spatial resolution than microdialysis and because cerebrospinal fluid is collected directly, no concern develops about incomplete recovery. 

As mentioned before, microdialysis is a sampling technique controlled by diffusion. Because a fluid continuously perfuses the probe, equilibrium conditions will not be reached, and only a fraction of the actual concentration of the analytes present in the medium surrounding the probe will be collected. The amount of analyte collected is normally defined as recovery . 

The flow rates of the microdialysis experiment are such that samples of 1-10 J,L are typically obtained. At typical perfusion rates, the perfusate is not at equilibrium with the extracellular fluid. As such, the concentration of sample in the dialysate is some fraction of that in the surrounding tissue. This is termed the extraction efficiency and is a function of the delivery or recovery of the probe. Not only are the sample volumes small but also the concentration in the dialysate may be low, typically ranging from 1 pM to 1 lM. Because the recovery is typically less than 100%, the limit of detection of the method should be lower than the lowest concentration expected in the dialysate. This presents a tremendous challenge for the analyst. 

Third, an in vitro calibration, which does not necessarily provide a good estimate of the recovery in vivo, was used for the microdialysis probe. In vitro calibration is only an approximate calibration technique because it does not always accurately represent what is happening in vivo. In addition, the efficiency of the microdialysis probe can change over time as the biological surroundings adjust to the presence of the probe. A final cause for the discrepancy could be due to the method of sampling for the conventional analysis. Because blood samples were taken from the rat, fewer samples could be taken within a given time. 

With in vivo microdialysis (Delgado et al., 1972 Ungerstedt, 1984), the extracellular fluid sample is ftirther qualified by a small sack or loop of semipermeable dialysis membrane (Fig. 51B). Artifidal CSF is slowly circulated into and out of the dialysis r on at flow rates of 1 -5 il/min, so that recovery of small molecules across the membrane from the extracellular space is effective. In essence the probe acts as an artificial blood vessel which may be sampled externally as a function of time during basal behavioral periods, drug administration (via the probe or elsewhere), external stimuli, etc. 

Microdialysis probes come in several sizes and geometries they can be much smaller than ultrafiltration probes because the volume of sample collected does not depend on the membrane surface area (Fig. 3A). Therefore, small microdialysis probes are used when precise spatial resolution is desired. When less spatial resolution is needed and higher recoveries are desirable, longer loop-type probes can be used (Fig. 3B). 

Another factor which may indirectly affect recovery for in vivo sampling is the biocompatibility of the probe. The tissue recognizes the probe as a foreign object and may respond by forming a fibrous layer around it. In short-term studies of hours to 2 days, this is not a significant factor. For microdialysis in longer term studies, this fibrous layer would present an additional diffusion barrier and may decrease recovery. In ultrafiltration, since there is bulk flow across the membrane, recovery may not be affected by this barrier however, the added barrier may affect the volume of fluid which is able to reach the membrane. 

Diameter and length of the microdialysis probe are determined by the site of sampling (see below). In general, the larger the area of the membrane, the higher the recovery of the analyte. 

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