Background and Aims.
Post-mortem diagnosis of myocardial ischemia is known to be challenging for forensic and clinical pathologists, especially if death occurred shortly after the onset of ischemia. Many published studies have shown that immunohistochemistry can considerably improve the diagnosis, especially when not a single marker, but a panel of markers, is used. One of the major limits of conventional immunohistochemistry is the inability to detect multiple antigens in a single tissue section, because of spectral overlap of tags, observed using either chromogenic (maximum 3 colors are available) or fluorescent (4-5 colors are available) detection. Moreover, when performing multiplex immunohistochemistry, if indirect detection methods are employed, cross-reactivity between the detection reagents can take place.
Materials and Methods.
Here we present the use of laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS) for the multiplex quantitative imaging of markers in tissue section at cellular resolution (i.e. 1 Oμm). In this feasibility study we tested six primary antibodies, chosen among those we currently use in our routine and research activity in order to detect acute myocardial ischemia. These antibodies (troponin T, myoglobin, fibronectin, C5b-9, connexin 43 and JunB) were labeled each with a rare earth metal isotope prior to their simultaneous incubation on tissue section. We compared the LA-ICP MS approach and conventional immunohistochemistry, using five formalin fixed, paraffin embedded (FFPE) autopsy samples of human ischemic myocardium.
Results and Conclusions.
A good correlation was obtained between ionic and optical images, providing similar detection patterns. Furthermore, we determined the absolute concentration of markers from the LA-ICP MS acquisition, and we were able to show a proportional relationship between signal intensity and local marker concentration in thin tissue sections. The LA-ICP MSbased method is a novel approach for multiplex mapping and imaging of antigen-antibody complexes in tissue, with a relevant diagnostic potential for forensic and clinical pathology.