What is recovery?
The recovery of a particular substance is defined as the concentration in the dialysate expressed as percent of the concentration in the interstitial fluid. Relative recovery will approach 100% as the flow rate approaches zero, and decrease as the flow rate increases. It is commonly expressed in percent. Absolute recovery is defined as the mass of a substance recovered during a defined time period. It is zero when the flow rate is zero, and will reach a maximum at higher flow rates.
What factors affect recovery?
The absolute recovery (mol/time unit) of a substance from the tissue depends on (1) the “cut off” of the dialysis membrane (usually defined as the molecular weight in Daltons at which 80% of the molecules are prevented from passing the membrane), (2) the length and diameter of the membrane, (3) the flow rate of the perfusion fluid and (4) the diffusion coefficient of the compound through the extracellular fluid. Other factors such as pH of the medium and degradation of the substance may also affect the recovery. The reverse holds true for substances entering the tissue from the probe.
How do the membrane length and flow rate affect recovery?
A longer membrane and a lower flow rate will give a higher recovery.
How should the perfusion fluid be composed?
Ideally the composition of the perfusion fluid should be as close as possible to the composition of the extracellular fluid. However, you may want to change the concentration of sodium, potassium or calcium in order to influence the membrane function in the region you are studying. CMA offers Perfusion Fluid T1 for peripheral tissue and Perfusion Fluid CNS for brain tissue.
What is the pH of the CNS perfusion fluid?
The CNS perfusion fluid is intentionally not buffered to allow it to equilibrate to the same pH as the brain’s interstitial fluid. The pH of a non-buffered solution varies between 5 and 8.
How does the pH of the CNS perfusion fluid impact the tissue?
Since our perfusion fluid is unbuffered it minimally impacts the pH of the tissue. The perfusate will assume the same pH as the surrounding tissue. However, buffering agents within the interstitial fluid may diffuse across the probe membrane at different rates. This can have a small effect on the pH of the tissue.
What perfusion flow rate should be used?
Use a high flow rate if you want to remove or introduce as many molecules as possible per time unit. Use a low flow rate when you want to obtain a more concentrated dialysate (high recovery). Note that a low flow rate gives smaller volume. Consider also the volume needed for the analysis.
What time is needed to obtain steady state conditions?
The introduction of a probe into the tissue will always cause damage and the recovery of cellular function will not be immediate. An hour is often used to reach “baseline conditions”.
When using a probe membrane with a cut-off of 20,000 Daltons, why isn't the recovery for molecules with 20,000 Da molecular weight 100%?
The ability of molecules to pass the membrane decreases logarithmically with increasing molecular weight. By experience we know that most substances with a molecular weight up to 5,000 Da can be dialyzed when using a 20,000 Da membrane. This is of course very dependent on the substance and the sensitivity of the analytical method.
What causes faulty flow?
You may not have calibrated the pump to the syringe you are using. The syringe might be leaking or you are not using the syringe recommended by CMA.
What causes faulty volume of the microdialysis sample?
The probe may be leaking, the tubing may be blocked, something may be wrong with the pump, or the tubing adapters may be leaking.
Why is my recovery so low? Relative Recovery
The concentration recovered will never be similar to what you see in your media. Unless there is total equilibration between your media and the perfusate, the concentration of a given substance in the dialysate will be lower than its actual concentration in your solution. This applies to in vivo and in situ experiments.
The relationship between dialysate and tissue (or solution) concentration is termed "relative recovery" and is defined as the dialysate/interstitial concentration ratio expressed as a percentage:
Relative recovery= Cmd/Cint=1-exp-K0 A/F
where Cmd and Cint are respectively the concentration of your analyte in the microdialysate and interstitial (tissue or solution), K0 is the average mass transfer, A is the membrane surface and F the dialysate flow rate.
As you can see, increasing the membrane surface or decreasing the flow rate will give you a higher relative recovery as well as membrane pore sizes. However, since full equilibration does not occur across the microdialysis membrane, the concentration of the measured substances in the dialysate is never equal to the true interstitial concentration.
If you need to know the interstitial concentration, it can be calculated using other methods, such as the no-net-flux-method.