Multiplex immunohistochemistry as a novel tool for the topographic assessment of the bone marrow stem cell niche.

Immunohistochemistry (IHC) using specific antibodies is a well-established method for the visualization of distinct cell populations. With increasing availability of suitable methods for complex tissue analyses, new demands have arisen to provide next to complex quantitative data information on protein expression, spatial distribution and cell-cell interactions in tissue sections. During the last decade, tissue preparation, fluorescent dyes, hardware imaging and software analysis were improved to solve problems concerning quantitative preciseness and tissue autofluorescence of multicolor staining. Automated cell segmentation as well as subcellular multiparameter analysis of fluorescence-based multiplexed IHC techniques, such as multispectral imaging (MSI), allows the quantification and localization of multiple proteins in the same tissue section. This technique gives us the opportunity to visualize and record the spatial relationship between different cells and is currently employed for formalin-fixed, paraffin-embedded (FFPE) samples, but has not yet been developed for calcified bone marrow (BM) biopsies. This chapter summarizes a novel protocol developed for decalcified FFPE BM samples. In addition, it discusses the technical aspects and pitfalls using this material thereby extending the use of MSI for analysis of BM malignancies. It provides an overview on the characterization and distribution of cell populations and protein expression patterns regarding their prognostic and predictive value, and their use for guidance of therapeutic decisions.