Sample Loss: Why Measure It and How to Interpret It

In the realm of assays, precision is paramount. Variations, whether too high or too low input material, have the potential to inadvertently alter experiment outcomes.This is particularly critical in biochemical and biophysical assays, where sample concentration stands as a vital parameter.

Sample loss can wreak havoc during both sample storage and assay preparation, often escaping notice. Sample loss stems from various sources, among which we can list: inherent sample instability, improper storage conditions (e.g.repeated freeze-thaw cycles) or contamination, errors in dilutions and manual handling. Even attractive forces between the sample and assay apparatus, colloquially known as "stickiness," contribute to unnoticeable sample reductions.

Regardless of its source, sample loss is deceptively easy to overlook, and it can profoundly impact assay development and result accuracy.

Methods such as UV absorbance, fluorescent dyes, or western blots offer limited insight into sample concentration, leaving the critical aspect of the amount variations in input material unnoticed throughout the experiment.

In this case, how to measure sample loss?

With Fida 1, sample loss is ’’just’’ one of eight integrated quality control parameters, making sample-loss monitoring a hassle-free task. During each run, sample loss is exposed through a reduction in peak area.  No extra steps needed. This readout is extracted for every measurement performed, meaning that you will always be informed if sample loss occurs during a measurement. Armed with this knowledge, you can troubleshoot efficiently and prevent sample loss strategically.

What can be the reasons behind sample loss?

Causes of sample loss can be different. Most typical ones are as follows:

Stickiness:

‍Understood as the stickiness  of protein or complexes to the glassware like vials,  capillary, etc. In Fida software, sample loss measurement is shown as on the graph below,  where the exact same sample was measured in presence (blue) and absence (red) of a mild detergent. Clear difference of peak area/fluorescence  indicates sample loss due to stickiness.

Errors in sample preparation:

This can simply be a handling error with manual pipetting. It can be easily fixed by repeating the procedure. It seen as loss of  fluorescence or peak area.

Aggregation  or agglutination:    

Sometimes sample loss can occur in a process like aggregation or  agglutination. Here, the soluble protein starts to form precipitates or     aggregates, leading to lowering of overall fluorescence signal. For more  on the topic see our app note ''Characterising Multivalent Complex Formation (Agglutination of multivalent antibodies)''