Analytical and Bioanalytical Chemistry

, Volume 411, Issue 20, pp 5007–5012 | Cite as

The management of haemoglobin interference for the MALDI-MSI proteomics analysis of thyroid fine needle aspiration biopsies

  • Isabella Piga
  • Giulia Capitoli
  • Vanna Denti
  • Silvia Tettamanti
  • Andrew Smith
  • Martina Stella
  • Clizia Chinello
  • Davide Leni
  • Mattia Garancini
  • Stefania Galimberti
  • Fulvio Magni
  • Fabio PagniEmail author


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.

Graphical abstract


Fine needle aspiration MALDI mass spectrometry imaging Proteomics Haemoglobin interference Thyroid ThinPrep 



Fine needle aspiration biopsies




Haematoxylin and eosin


Indium tin oxide


Liquid-based preparation


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.

Supplementary material

216_2019_1908_MOESM1_ESM.pdf (712 kb)
ESM 1 (PDF 711 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • 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
    Email author
  1. 1.School of Medicine and Surgery, Clinical Proteomics and Metabolomics UnitUniversity of Milano – BicoccaVedano al LambroItaly
  2. 2.School of Medicine and Surgery, Section of PathologyUniversity of Milano - BicoccaMonzaItaly
  3. 3.School of Medicine and Surgery, Centre of Biostatistics for Clinical EpidemiologyUniversity of Milano – BicoccaVedano al LambroItaly
  4. 4.Department of RadiologySan Gerardo HospitalMonzaItaly
  5. 5.Department of SurgerySan Gerardo HospitalMonzaItaly

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