The management of haemoglobin interference for the MALDI-MSI proteomics analysis of thyroid fine needle aspiration biopsies
MALDI-MSI represents an ideal tool to explore the spatial distribution of proteins directly in situ, integrating molecular and cytomorphological information, enabling the discovery of potential diagnostic markers in thyroid cytopathology. However, red cells present in the fine needle aspiration biopsy (FNAB) specimens caused ion suppression of other proteins during the MALDI-MSI analysis due to large amount of haemoglobin. Aim of this study was to set up a sample preparation workflow able to manage this haemoglobin interference. Three protocols were compared using ex vivo cytological samples collected from fresh thyroid nodules of 9 patients who underwent thyroidectomy: (A) conventional air-dried smears, (B) cytological smears immediately fixed in ethanol, and (C) ThinPrep liquid-based preparation. Protocols C and A were also evaluated using real FNABs. Results show that protocol C markedly decreased the amount of haemoglobin, with respect to protocols A and B. Protein profiles obtained with protocols A and B were characterised by high inter-patient variability, probably related to the abundance of the haemoglobin, whereas similar spectra were observed for protocol C, where haemoglobin contents were lower. Our findings suggest protocol C as the sample preparation method for MALDI-MSI analysis.
KeywordsFine needle aspiration MALDI mass spectrometry imaging Proteomics Haemoglobin interference Thyroid ThinPrep
Fine needle aspiration biopsies
Haematoxylin and eosin
Indium tin oxide
Matrix-assisted laser desorption/ionisation
Mass spectrometry imaging
Region of interest
Signal to noise
Total ion current
This work was funded by AIRC (Associazione Italiana per la Ricerca sul Cancro) MFAG GRANT 2016 - Id. 18445, and partially supported by the Italian Ministry of University and Research (MIUR) - Department of Excellence project PREMIA (PREcision MedIcine Approach: bringing biomarker research to clinic).
Compliance with ethical standards
The study was approved by the local Ethical Board – San Gerardo Hospital – AIRC-MFAG 18/11/2016. Signed informed consent of all individual participant samples was obtained.
Conflict of interest
The authors declare that they have no conflict of interest.
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