A Pilot Validation Study Comparing Fluorescence-Imitating Brightfield Imaging, A Slide-Free Imaging Method, With Standard Formalin-Fixed, Paraffin-Embedded Hematoxylin-Eosin-Stained Tissue Section Histology for Primary Surgical Pathology Diagnosis

Abstract

Context.—: Digital pathology using whole slide images has been recently approved to support primary diagnosis in clinical surgical pathology practices. Here we describe a novel imaging method, fluorescence-imitating brightfield imaging, that can capture the surface of fresh tissue without requiring prior fixation, paraffin embedding, tissue sectioning, or staining.

Objective.—: To compare the ability of pathologists to evaluate direct-to-digital images with standard pathology preparations.

Design.—: One hundred surgical pathology samples were obtained. Samples were first digitally imaged, then processed for standard histologic examination on 4-μm hematoxylin-eosin-stained sections and digitally scanned. The resulting digital images from both digital and standard scan sets were viewed by each of 4 reading pathologists. The data set consisted of 100 reference diagnoses and 800 study pathologist reads. Each study read was compared to the reference diagnosis, and also compared to that reader's diagnosis across both modalities.

Results.—: The overall agreement rate, across 800 reads, was 97.9%. This consisted of 400 digital reads at 97.0% versus reference and 400 standard reads versus reference at 98.8%. Minor discordances (defined as alternative diagnoses without clinical treatment or outcome implications) were 6.1% overall, 7.2% for digital, and 5.0% for standard.

Conclusions.—: Pathologists can provide accurate diagnoses from fluorescence-imitating brightfield imaging slide-free images. Concordance and discordance rates are similar to published rates for comparisons of whole slide imaging to standard light microscopy of glass slides for primary diagnosis. It may be possible, therefore, to develop a slide-free, nondestructive approach for primary pathology diagnosis.

Figure 1.

Fluorescence-imitating brightfield imaging image of breast lobules, ducts, stroma, and microvasculature (original magnification ×80).

Figure 2.

Schematic diagram of a fluorescence-imitating brightfield imaging microscope.

Figures 3.

Comparison of fluorescence-imitating brightfield imaging image of cervix (A) with whole slide image scanning (B), the latter following standard hematoxylin-eosin staining after fixation and processing (original magnification×4). Screen shots from PathPresenter are shown here.

Figure 4.

Normal breast. Fluorescence-imitating brightfield imaging (FIBI) image (A). Higher power of boxed areas in A (B and C). Standard hematoxylin-eosin–stained section (D). Higher power of boxed areas in D (E and F). Note lipid and capillaries in adipose tissue visible in the FIBI specimens (original magnifications ×4 [A and D], ×40 [B and E], and ×20 [C and F]).

Figure 5.

Fluorescence-imitating brightfield imaging (A and C) and corresponding standard hematoxylin-eosin–stained images (B and D) of invasive lobular carcinoma of breast. Whole specimen (A and B) and zoomed-in regions (C and D) showing nonneoplastic ducts surrounded by invading lobular carcinoma cells (original magnifications ×4 [A and B] and ×160 [C and D]).

Figure 6.

Fluorescence-imitating brightfield imaging (FIBI) (B and C) and corresponding standard hematoxylin-eosin–stained (H&E) images (A and D) of invasive squamous cell carcinoma of the tongue with necrosis. Whole specimen (A and B) and zoomed-in regions (C and D). Note mitotic figures (circles) visible in both FIBI (C) and standard H&E (D) images (original magnifications ×6 [A and B] and ×300 [C and D]).