COMMUNICATION The management of haemoglobin interference for the MALDI-MSI proteomics analysis of thyroid fine needle aspiration biopsies Isabella Piga 1,2 & Giulia Capitoli 3 & Vanna Denti 1 & Silvia Tettamanti 1 & Andrew Smith 1 & Martina Stella 1 & Clizia Chinello 1 & Davide Leni 4 & Mattia Garancini 5 & Stefania Galimberti 3 & Fulvio Magni 1 & Fabio Pagni 2 Received: 1 February 2019 /Revised: 19 April 2019 /Accepted: 9 May 2019 /Published online: 30 May 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract 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. Keywords Fine needle aspiration . MALDI mass spectrometry imaging . Proteomics . Haemoglobin interference . Thyroid . ThinPrep Abbreviations FNAB Fine needle aspiration biopsies Hb Haemoglobin H&E Haematoxylin and eosin ITO Indium tin oxide LBP Liquid-based preparation MALDI Matrix-assisted laser desorption/ionisation MSI Mass spectrometry imaging ROI Region of interest S/N Signal to noise TIC Total ion current Introduction Although most thyroid nodules are diagnosed using a morpho- logical approach, a significant challenge is related with the 20– 30% of fine needle aspiration biopsy (FNAB) cases that are deemed to be indeterminate for malignancy (THY3 and THY4 according to the British system for reporting thyroid cytopathology) [1]. So far, patients with a THY3 diagnosis Parts of this work were presented at the 22nd International Mass Spectrometry Conference, Florence (Italy), in August 2018 and have been awarded with an ABC Best Poster Award. Isabella Piga and Giulia Capitoli contributed equally to this work Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-019-01908-w) contains supplementary material, which is available to authorized users. * Fabio Pagni fabio.pagni@unimib.it 1 School of Medicine and Surgery, Clinical Proteomics and Metabolomics Unit, University of Milano – Bicocca, 20854 Vedano al Lambro, Italy 2 School of Medicine and Surgery, Section of Pathology, University of Milano - Bicocca, Via Cadore 48, 20900 Monza, Italy 3 School of Medicine and Surgery, Centre of Biostatistics for Clinical Epidemiology, University of Milano – Bicocca, 20854 Vedano al Lambro, Italy 4 Department of Radiology, San Gerardo Hospital, 20900 Monza, Italy 5 Department of Surgery, San Gerardo Hospital, 20900 Monza, Italy Analytical and Bioanalytical Chemistry (2019) 411:5007–5012 https://doi.org/10.1007/s00216-019-01908-w