Introduction Fine Needle Aspiration biopsy (FNAB) is the gold standard exam to determine the malignant nature of thyroid nodules[1]. Contamination of FNAB samples with red blood cells is problematic for proteomics analysis, given that large amounts of haemoglobin (Hb) suppress other protein signals[2]. Hence, it is paramount to standardise the sample preparation of ex-vivo and in-vivo thyroid FNABs for proteomic MALDI-MSI analysis, in order to minimise Hb interference. Methods Human FNABs were collected and deposited onto conductive glass slides from both ex-vivo(n=9), surgically removed thyroid specimens, and in-vivo(n=12) thyroid specimens for intact proteins MALDI-MSI analysis. Three protocols were compared using ex-vivo biopsies collected from the same thyroid: a) conventional air dried smear; b) cytological smear immediately fixed in ethanol; c) ThinPrep (TP) cytological preparation using a ThinPrep 2000 system. Results The spectral profiles of both ex-vivo and in-vivo conventional air-dried smears were characterized by high 917 inter-patient variability related to the abundance of the Hb peaks. In particular, the strong vascularization of some thyroid nodules is reflected in FNABs with a high content of Hb. The amount of Hb was markedly decreased in TP preparation with respect to both conventional air-dried and fixed smears. On the other hand, the absolute intensity of other protein signals, suppressed with the other two methods, were significantly increased in TP samples. Furthermore, the management of Hb interference of ex-vivo and in vivo TP samples was comparable, indicating the opportunity to use in-vivo TP specimens for MALDI-MSI proteomic analysis and biomarker discovery. The MALDI-MSI approach combined with virtual microdissection permitted to extract specific protein signatures from different histotypes of both benign and malignant thyroid cell clusters. Conclusions The Thin Prep procedure for thyroid FNABs samples preparation combined with MALDI-MSI proteomic analysis allow us to obtain high-quality spectra, follicular cells specific protein profiles and to manage the haemoglobin interference. The application of this reproducible technique to in-vivo cytological samples can help cytopathologists in the diagnosis of thyroid nodules combining both morphological and proteomics information. Novel Aspect This study represents the first example of MALDI-MSI applied to ex-vivo and in-vivo thyroid FNABs, prepared using the ThinPrep preparation, for proteomic analysis. References 1. Russ G, Bonnema SJ, Erdogan MF, Durante C, Ngu R, Leenhardt L. European Thyroid Journal, 6, 225-237 (2017). 2. Amann JM, Chaurand P, Gonzalez A, Mombley J, Massion PP, Carbone DP, Caprioli RM, Clinical Cancer Research, 12, 5142–5150 (2006). Funding: This work was funded thanks to AIRC (AssociazioneItaliana per la RicercasulCancro) MFAG GRANT 2016 - Id. 18445.