Multiplex quantitative imaging of human myocardial infarction by mass spectrometry-immunohistochemistry
Simultaneous assessment of a panel of protein markers is becoming essential in order to enhance biomarker research and improve diagnostics. Specifically, postmortem diagnostics of early myocardial ischemia in sudden cardiac death cases could benefit from a multiplex marker assessment in the same tissue section. Current analytical antibody-based techniques (immunohistochemistry and immunofluorescence) limit multiplex analysis usually to not more than three antibodies. In this study, mass spectrometry-immunohistochemistry (MS-IHC) was performed by combining laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) with rare-metal-isotope-tagged antibodies as a technique for multiplex analysis of human postmortem myocardial tissue samples. Tissue sections with myocardial infarction were simultaneously analyzed for seven primary, rare-metal-isotope-tagged antibodies (troponin T, myoglobin, fibronectin, C5b-9, unphosphorylated connexin 43, VEGF-B, and JunB). Comparison between the MS-IHC approach and chromogenic IHC showed similar patterns in ionic and optical images. In addition, absolute quantification was performed by MS-IHC, providing a proportional relationship between the signal intensity and the local marker concentration in tissue sections. These data demonstrated that LA-ICP-MS combined with rare-metal-isotope-tagged antibodies is an efficient strategy for simultaneous testing of multiple markers and allows not only visualization of molecules within the tissue but also quantification of the signal. Such imaging approach has a great potential in both diagnostics and pathology-related research.
KeywordsMass spectrometry-immunohistochemistry Multiplex tissue imaging Biomarker Myocardial ischemia Forensic pathology
We would like to thank Max Villa and Catia Pomponio for the support and assistance with LA-ICP-MS and immunohistochemistry, respectively.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
For this type of study, formal consent is not required. All cases included in this study were obtained from the autopsy database in our center. In agreement with the local ethics committee and the local general prosecutor, these cases can be included in this type of studies, provided that they are anonymized. In this investigation, no information allowing the identification of a person is given. People, who had previously refused, in a written form, their consent to bequeath their body parts for research use, were excluded from the study.
- 1.Campobasso CP, Dell’Erba AS, Addante A, Zotti F, Marzullo A, Colonna MF (2008) Sudden cardiac death and myocardial ischemia indicators: a comparative study of four immunohistochemical markers. Am J Forensic Med Pathol 29(2):154–161. https://doi.org/10.1097/PAF.0b013e318177eab7 CrossRefPubMedGoogle Scholar
- 4.Stack EC, Wang C, Roman KA, Hoyt CC (2014) Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis. Methods (San Diego, Calif) 70(1):46–58. https://doi.org/10.1016/j.ymeth.2014.08.016 CrossRefGoogle Scholar
- 9.Bandura DR, Baranov VI, Ornatsky OI, Antonov A, Kinach R, Lou X, Pavlov S, Vorobiev S, Dick JE, Tanner SD (2009) Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry. Anal Chem 81(16):6813–6822. https://doi.org/10.1021/ac901049w CrossRefGoogle Scholar
- 10.Bendall SC, Simonds EF, Qiu P, Amir el AD, Krutzik PO, Finck R, Bruggner RV, Melamed R, Trejo A, Ornatsky OI, Balderas RS, Plevritis SK, Sachs K, Pe'er D, Tanner SD, Nolan GP (2011) Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science (New York, NY) 332(6030):687–696. https://doi.org/10.1126/science.1198704 CrossRefGoogle Scholar
- 14.Giesen C, Wang HA, Schapiro D, Zivanovic N, Jacobs A, Hattendorf B, Schuffler PJ, Grolimund D, Buhmann JM, Brandt S, Varga Z, Wild PJ, Gunther D, Bodenmiller B (2014) Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry. Nat Methods 11(4):417–422. https://doi.org/10.1038/nmeth.2869 CrossRefPubMedGoogle Scholar
- 16.Bemis KD, Harry A, Eberlin LS, Ferreira C, van de Ven SM, Mallick P, Stolowitz M, Vitek O (2015) Cardinal: an R package for statistical analysis of mass spectrometry-based imaging experiments. Bioinformatics 31(14):2418–2420. https://doi.org/10.1093/bioinformatics/btv146 CrossRefPubMedPubMedCentralGoogle Scholar
- 17.Matusch A, Depboylu C, Palm C, Wu B, Hoglinger GU, Schafer MK, Becker JS (2010) Cerebral bioimaging of Cu, Fe, Zn, and Mn in the MPTP mouse model of Parkinson’s disease using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). J Am Soc Mass Spectrom 21(1):161–171. https://doi.org/10.1016/j.jasms.2009.09.022 CrossRefPubMedGoogle Scholar
- 18.Becker JS, Zoriy MV, Pickhardt C, Palomero-Gallagher N, Zilles K (2005) Imaging of copper, zinc, and other elements in thin section of human brain samples (hippocampus) by laser ablation inductively coupled plasma mass spectrometry. Anal Chem 77(10):3208–3216. https://doi.org/10.1021/ac040184q CrossRefPubMedGoogle Scholar
- 30.Ogawa H, Suefuji H, Soejima H, Nishiyama K, Misumi K, Takazoe K, Miyamoto S, Kajiwara I, Sumida H, Sakamoto T, Yoshimura M, Kugiyama K, Yasue H, Matsuo K (2000) Increased blood vascular endothelial growth factor levels in patients with acute myocardial infarction. Cardiology 93(1–2):93–99CrossRefGoogle Scholar
- 32.Harpster MH, Bandyopadhyay S, Thomas DP, Ivanov PS, Keele JA, Pineguina N, Gao B, Amarendran V, Gomelsky M, McCormick RJ, Stayton MM (2006) Earliest changes in the left ventricular transcriptome postmyocardial infarction. Mamm Genome 17(7):701–715. https://doi.org/10.1007/s00335-005-0120-1 CrossRefPubMedGoogle Scholar
- 34.Ogrinc Potocnik N, Porta T, Becker M, Heeren RM, Ellis SR (2015) Use of advantageous, volatile matrices enabled by next-generation high-speed matrix-assisted laser desorption/ionization time-of-flight imaging employing a scanning laser beam. Rapid Commun Mass Spectrom 29(23):2195–2203. https://doi.org/10.1002/rcm.7379 CrossRefPubMedGoogle Scholar
- 35.Dobrowolska J, Dehnhardt M, Matusch A, Zoriy M, Palomero-Gallagher N, Koscielniak P, Zilles K, Becker JS (2008) Quantitative imaging of zinc, copper and lead in three distinct regions of the human brain by laser ablation inductively coupled plasma mass spectrometry. Talanta 74(4):717–723. https://doi.org/10.1016/j.talanta.2007.06.051 CrossRefPubMedGoogle Scholar
- 40.Chan W, White DA, Wang XY, Bai RF, Liu Y, Yu HY, Zhang YY, Fan F, Schneider HG, Duffy SJ, Taylor AJ, Du XJ, Gao W, Gao XM, Dart AM (2013) Macrophage migration inhibitory factor for the early prediction of infarct size. J Am Heart Assoc 2(5):e000226. https://doi.org/10.1161/JAHA.113.000226 CrossRefPubMedPubMedCentralGoogle Scholar
- 41.White DA, Fang L, Chan W, Morand EF, Kiriazis H, Duffy SJ, Taylor AJ, Dart AM, Du XJ, Gao XM (2013) Pro-inflammatory action of MIF in acute myocardial infarction via activation of peripheral blood mononuclear cells. PLoS One 8(10):e76206. https://doi.org/10.1371/journal.pone.0076206 CrossRefPubMedPubMedCentralGoogle Scholar