Understanding your materials and methods

Imagine you’d perform a clinical study in healthy volunteers to assess the activity of a new anti-inflammatory drug. How to demonstrate the compound’s anti-inflammatory effect in healthy subjects? These subjects don’t have chronic inflammation; they’re healthy so there’s nothing to treat. A solution is to induce inflammation outside the body. This is done by exposing blood from volunteers that received the drug to a foreign trigger. The theory is simple: if the drug has the intended pharmacological activity, it reduces the trigger-induced inflammatory response.

Obviously, this inflammatory response should be completely controlled in terms of magnitude (what’s the cytokine level?) and nature (what is the pathway producing cytokines?). No unintended inflammation may be present in the test tube. For this reason, only blood collection tubes are used that are endotoxin-free. Endotoxin is a bacterial component that is everywhere, contaminating everything that is handled by human hands. This is certainly not something that you’d want to be in your test tubes!

The clinical trial is executed, cytokine analyses are run, and data are reported. Data analysis reveals that the anti-inflammatory drug did not inhibit inflammation at any dose level tested. Moreover, even the blank conditions (without any inflammatory trigger added) show massive cytokine release! A failed clinical study, who to blame?

This is a hypothetical case, but not inconceivable to happen. We discovered that immune cells collected in heparin tubes may become activated, even though the tubes were tested to be endotoxin-free. This activation differed per manufacturer and even per tube batch. Apparently, heparin tubes may contain an unknown trigger activating the immune system. Luckily, we discovered this before the blood collection tubes were used in a clinical study. Hence, we decided to always test each future tube batch for undesired immune stimulation prior to clinical use. Thereby we avoided a failed clinical trial.

The verification of materials and methods may not be the sexiest topic to blog about. However, above example demonstrates the importance of control and understanding of all experimental details in clinical research. Following the text books, the manufacturer’s specifications, or PubMed is not sufficient. Artifacts may be introduced at different levels: preanalytical, analytical and postanalytical variables play a role. An unsuitable blood collection method may activate platelets and interfere in your readouts, a delay in sample handling may result in reduced responsiveness of the cells in your bioassay, your primary readout measure may be subject to diurnal fluctuations, etcetera. Just imagine that we’d not have tested the blood collection tubes preceding our clinical study. The loss of information, time and money would have been enormous!

Matthijs Moerland

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