The name PPC or Positive Product Control is dropped regularly when discussing an endotoxin detection method. It is an essential part to demonstrate the validity of an endotoxin test.
The LAL assay is a biological, enzymatical assay. It is based on either a change in turbidity (LAL-turbidimetric), a color difference (LAL chromogenic) or the formation of a clot (gel-clot). The key to these molecular reactions is the proper functioning of the test enzyme from the Limulus Polyphemus Lysate (LAL). Substances present in the test mixture or extract can affect the test enzyme reactivity. If reactivity is decreased, we would expect endotoxin to be underrepresented in the result (inhibition). Similarly, we would see endotoxin overrepresented if enzyme reactivity were increased (enhancement). These effects have the potential to result in either false negative or false positive test results.
For example, let’s assume that a protein denaturing substance is present in a test sample. As the test enzyme is a protein it could potentially be destroyed before the completion of the assay. This would result in a reduction of reactivity, which would subsequently lead to a reduction of clotting, turbidity or colorization ultimately producing a detected endotoxin result that is less than the endotoxin present in the sample. Therefore, we might falsely assume that the sample does not contain endotoxins.
To prevent reporting false negative or false positive results, at Nelson Labs, we test each sample with and without the addition of a known endotoxin spike of normally 0.5 EU/mL. If we subtract the endotoxin concentration from the unspiked sample from the endotoxin concentration of the spiked sample (Positive Product Control (PPC)) we should recover 50-200% of the spike.
If the functioning of the enzyme is as it should, we will measure for an uncontaminated sample an endotoxin content of less than <0.005 EU/mL and for the PPC 0.505 EU/mL, assuming a recovery of 100%.
A recovery of 50-200% might seem a large interval. However, this is due to the statistical phenomenon “A combination of Variances”. Without going into too much detail, this is a case where you subtract a number which is prone to an accepted level of variance from a number which is also prone to a certain level of variance.
PPC’s should be included in every test method. As this is the only mean to demonstrate that the test system is reacting as expected and excludes the occurrence of false negative or positive results. It should be noted however that PPC’s do not exclude false positives caused by Beta-glucans, but this is a topic has been covered in our Beta-glucans post.