Slide-to-Slide Tissue Transfer and Array Assembly From Limited Samples for Comprehensive Molecular Profiling

Abstract

Tissue microarrays (TMA) have become an important tool in high-throughput molecular profiling of tissue samples in the translational research setting. Unfortunately, high-throughput profiling in small biopsy specimens or rare tumor samples (eg, orphan diseases or unusual tumors) is often precluded owing to limited amounts of tissue. To overcome these challenges, we devised a method that allows tissue transfer and construction of TMAs from individual 2- to 5-μm sections for subsequent molecular profiling. We named the technique slide-to-slide (STS) transfer, and it requires a series of chemical exposures (so-called xylene-methacrylate exchange) in combination with rehydrated lifting, microdissection of donor tissues into multiple small tissue fragments (methacrylate-tissue tiles), and subsequent remounting on separate recipient slides (STS array slide). We developed the STS technique by assessing the efficacy and analytical performance using the following key metrics: (a) dropout rate, (b) transfer efficacy, (c) success rates using different antigen-retrieval methods, (d) success rates of immunohistochemical stains, (e) fluorescent in situ hybridization success rates, and (f) DNA and (g) RNA extraction yields from single slides, which all functioned appropriately. The dropout rate ranged from 0.7% to 6.2%; however, we applied the same STS technique successfully to fill these dropouts ("rescue" transfer). Hematoxylin and eosin assessment of donor slides confirmed a transfer efficacy of >93%, depending on the size of the tissue (range, 76%-100%). Fluorescent in situ hybridization success rates and nucleic acid yields were comparable with those of traditional workflows. In this study, we present a quick, reliable, and cost-effective method that offers the key advantages of TMAs and other molecular techniques-even when tissue is sparse. The perspectives of this technology in biomedical sciences and clinical practice are promising, given that it allows laboratories to create more data with less tissue.

Keywords: biomarker; limitations; limited tissue; orphan disease; tissue rescue.

Figure 1.. Schematic of the slide-to-slide (STS) array workflow.

Key workflow steps are: (1) slide selection of a stained or unstained (i.e., blank) section and coverslip lifting, (2) covering donor slide with methacrylate, (3) O/N hydration; (4) lifting tissue that is now fixed to methacrylate, (5) microdissection into tiles, (6) transferring lifted tissue tiles to recipient slide (TMA assembly), (7) removal of methacrylate and downstream processing. Abbreviations: FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; O/N, overnight; STS, slide-to-slide; TMA, tissue microarray.

Figure 2.. Illustrated workflow.

Key steps in the workflow for the assembly of a slide-to-slide (STS) array: (a) Step1: Coverslip lift of H&E-stained slide or matched unstained/blank section; (b) Step 2: Cover in methacrylate, dry, outline region of interest and prepare recipient slide(s) (STS-arrays) and (c) donor slides (now ready for hydrated lift); (d) Step 3: Hydrate overnight to loosen tissue section by immersion of slides in water; (e) Step 4: manual lifting; (f) Step 5: Microdissection of the region of interest and splitting of methacrylate with adherent tissue into tiles; (g) array assembly requires previously prepared recipient slides. (h) Step 6: STS array assembly. (i) Step 7: Dissolve methacrylate in xylene and proceed to downstream assays, for example IHC. Abbreviations: H&E, haematoxylin and eosin staining; STS, slide-to-slide; IHC, immunohistochemistry

Figure 3.. Performance Assessment of the STS- technique.

(a) Drop-out rate; (b) Transfer efficacy; (c) Principal antigen retrieval technique; (d) IHC staining performance; (e) FISH assay performance; (f) average yields of nucleic acid extraction from single stained slides for DNA and RNA. Abbreviations: PBS, phosphate buffered saline; STS, slide-to-slide; IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; Hyb., hybridizations; ng, nanogram; don., donor slide;