Abstract
Real-time on-site histopathology review of biopsy tissues at the point-of-procedure has great potential for significant clinical value and improved patient care. For instance, on-site review can aid in rapid screening of diagnostic biopsies to reduce false-negative results, or in quantitative assessment of biospecimen quality to increase the efficacy of downstream laboratory and histopathology analysis. However, the only currently available rapid pathology method, frozen section analysis (FSA), is too time- and labor-intensive for use in screening large quantities of biopsy tissues and is too destructive for maximum tissue conservation in multiple small needle core biopsies. In this work we demonstrate the spectrally-compatible combination of the nuclear stain DRAQ5 and the anionic counterstain eosin as a dual-component fluorescent staining analog to hematoxylin and eosin intended for use on fresh, unsectioned tissues. Combined with optical sectioning fluorescence microscopy and pseudo-coloring algorithms, DRAQ5 and eosin (“D&E”) enables very fast, non-destructive psuedohistological imaging of tissues at the point-of-acquisition with minimal tissue handling and processing. D&E was validated against H&E on a one-to-one basis on formalin-fixed paraffin-embedded and frozen section tissues of various human organs using standard epi-fluorescence microscopy, demonstrating high fidelity of the staining mechanism as an H&E analog. The method was then applied to fresh, whole 18G renal needle core biopsies and large needle core prostate biospecimen biopsies using fluorescence structured illumination optical sectioning microscopy. We demonstrate the ability to obtain high-resolution histology-like images of unsectioned, fresh tissues similar to subsequent H&E staining of the tissue. The application of D&E does not interfere with subsequent standard-of-care H&E staining and imaging, preserving the integrity of the tissue for thorough downstream analysis. These results indicate that this dual-stain pseudocoloring method could provide a real-time histology-like image at the time of acquisition and valuable objective tissue analysis for the clinician at the time of service.
Highlights
Real-time assessment of freshly removed, intact tissue specimens can help improve clinical procedures, such as diagnostic biopsy, collection of samples for genetic or molecular testing, and/or surgical tumor resection
The laborintensiveness and destructiveness of traditional histopathology preparations, like formalinfixed, paraffin-embedded processing or frozen section analysis, is due to the need to deposit a thin section of hematoxylin and eosin (H&E) stained tissue on a slide to enable it to be evaluated by light-transmission microscopy
An alternative to frozen section analysis (FSA) or touch-prep could be achieved by the application of topical fluorescent stains onto fresh, unprocessed and unsectioned tissues, which are able to mimic the pathological features of traditional hematoxylin and eosin (H&E) staining [15, 16]
Summary
Real-time assessment of freshly removed, intact tissue specimens can help improve clinical procedures, such as diagnostic biopsy, collection of samples for genetic or molecular testing, and/or surgical tumor resection. The current approach of frozen section analysis (FSA) for inprocedure histopathology is relatively time-consuming and damaging to the tissue and is not feasible for the immediate evaluation of small needle core biopsies [1] Touch preparation of these specimens is currently the most effective method, but can frequently misrepresent tumor content and often needs a specialized cytopathologist for proper interpretation [2]. Topical fluorescent staining is quick [14, 17], on the order of seconds, and when paired with a suitably rapid ex vivo microscopy system, could allow virtual H&E images of fresh tissue surfaces to be obtained within minutes of removal from the patient This type of procedure, which conserves the tissue for cryopreservation, could represent an attractive processing alternative to FSA and/or touch preparation for in-procedure pathology and triage of small biopsy samples for downstream analysis
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