Evaluation and development of reference materials to support mass spectrometry imaging of endogenous metabolites in tissue

Abstract

Mass spectrometry imaging (MSI) provides spatial molecular distribution maps directly from clinical tissue samples, which can give informative results for cancer research. Implementing accurate and precise measurements in MSI is essential. Unfortunately, progress in MSI is often hindered by a lack of reproducible results. As with any other analytical technique, using reference standards and protocols is essential to ensure a robust method. This need is even higher when analysing cancerous tissues characterised by high heterogeneity and biological variability. This PhD thesis aims to develop and evaluate reference materials to support MALDI and DESI MSI analysis of cancerous tissues. Tissue homogenates have previously been used in other MSI studies as reference materials for quantitation purposes and interlaboratory comparisons. This thesis proves the existence of metabolic activity in tissue homogenates, which has not been considered so far in other studies. It proposes the usage of preserved tissue homogenates for the first time. Formalin-fixed and heat-treated tissue homogenates can preserve the metabolic profile in tissue and ensure good-quality sections when embedded. This thesis also proposes the usage of spotted human serum as a quality control standard for MSI. Human serum proved to have similar molecular coverage to cancerous tissue to support the analysis of low molecular weight metabolites and lipids. Human serum spots can precisely be sampled in volumes of a few nanoliters and lack the section-to-section batch effects and the section heterogeneity that characterises cancerous tissue sections. Moreover, instrument variability can be distinguished from the biological variability characterising cancerous tissue sections when analysed simultaneously with human serum spots. In conclusion, this thesis demonstrates that implementing spotted serum as a quality control reference standard and using preserved tissue homogenates is a step further towards achieving reproducible and accurate MSI measurements that are a real representation of the metabolic state of the tissue sample.