Clinical benefits of combining H&E and IHC on the same slide include conservation of small specimens, such as biopsies, to maximize the amount of clinical information obtained from the specimen, and the ability to evaluate protein expression within the full H&E context, bringing to bear the trove of information that H&E based staining provides the pathologist. To realize these benefits, we have developed invisible chromogens, which can be multiplexed with conventional stains, and a dual-camera brightfield microscope system that permits the simultaneous viewing and evaluation of each stain. The absorbance spectra of several invisible chromogens plotted in Fig. 1B show clear spectral separation from the individual hematoxylin and eosin absorbance spectra. The invisible chromogens were developed using CDC technology that simplifies selection of chromogen spectral properties13 and allows IHC to be performed ahead of the conventional staining. By performing IHC first, conventional stain is not subjected to IHC reagents that might remove the stain (e.g. eosin removal by aqueous solutions or a variety of non-aqueous solvents), and CDCs are stable to subsequent conventional staining conditions due to the robust covalent attachment.
Simultaneous H&E and IHC is shown in Fig. 3, panels A and B, for the simple case of pancreatic FFPE tissue stained for synaptophysin by IHC using the invisible Cy7 CDC. Using the dual-camera microscope system (Fig. 2), the color camera records the broadband visible light illumination of the H&E stain (Fig. 3A) and the monochrome camera records the NIR illumination of the Cy7 CDC (Fig. 3B). Video of the two camera images, presented side-by-side on the computer monitor, allows the pathologist to visually evaluate both staining patterns on the specimen slide while manually translating the slide. In addition, the H&E stain may be directly viewed through the oculars, with the added precaution of placing bandpass filters within the eyepieces to block transmission of invisible light (see Materials and Methods section and Supplementary Methods document). This permits pathologist examination of the H&E and IHC staining patterns in a manner similar to current practice but on a single slide. To aid cell location, the H&E and IHC images can be combined into a single composite image (Fig. 3C), or a video composite image can be viewed while scanning the specimen using an overlay feature of the software (Fig. 3D–G).
Further multiplexing is possible, as the CDCs in Fig. 1B will support at least duplex IHC. Examples are shown in Fig. 4, panels A-C for tonsil FFPE tissue stained with H&E and CD20/CD8 duplex IHC, and panels D-F for colon tumor FFPE tissue stained with H&E and CD3/CD8 duplex IHC. The two IHC stains are viewed alternately on the monochrome camera by switching between two different invisible illumination channels. A supplementary video may be downloaded that shows the adjacent color and monochrome camera images on the computer monitor while manually scanning a tonsil FFPE specimen and switching between 405 and 770 nm LEDs to alternately present the CD20 DCC CDC and CD8 Cy7 CDC staining patterns, respectively (note video resolution is reduced to limit file size). The image overlay feature is also demonstrated.
The colon tumor example demonstrates important advantages of performing H&E and IHC simultaneously on a single slide. It has been demonstrated in colon carcinoma that the distribution of CD3 and CD8 cells in the core tumor and invasive margin is a strong prognostic marker for disease-free and overall survival19. Typically, three FFPE sections are required for the analysis—one for H&E, and one each for CD3 and CD8. The location of the invasive margin is identified on the H&E slide and then transferred to the CD3- and CD8-stained slides on which the respective cell densities relative to the margin are measured. However, since the CD3 and CD8 cells are enumerated on sequential (serial) FFPE sections, the tumor periphery is somewhat altered on each slide, and boundaries must be adjusted to account for differences in tumor section orientation and tumor alterations with section depth relative to the H&E slide. While this approximation of the tumor and margin boundaries on the IHC slides has been shown to provide clinically significant results, and application of artificial intelligence (AI) simplifies the process, performing H&E and dual IHC on the same slide removes all uncertainty in transferring the boundary locations.
In addition to H&E stain on FFPE tissue, PAP stain can be combined with IHC on cytological preparations, which may have limited specimen due to low cellularity. Historically, morphology of cervical cells, as revealed by PAP staining, served as the primary method for screening patients for cervical cancer and dysplasia potentially leading to cervical cancer. More recently, cervical cells overexpressing both p16 and Ki-67, identified by IHC, have been shown to add additional benefit in identifying abnormal cells20. Combining PAP and Ki-67/p16 duplex IHC, as shown in Fig. 5, panels A-C, enables the evaluation of PAP staining pattern, p16 expression, and Ki-67 expression in every cell, and may provide greater confidence in the final diagnosis.
Mucicarmine special stain is often used to help identify ADC in NSCLC FFPE tissue by the pink coloration due to increased mucin production relative to SCC. TTF-1 and p40 IHC are also employed to distinguish ADC and SCC and duplex IHC for these two markers combined with mucicarmine special stain should re-enforce the carcinoma assignment on a single slide. Agreement between duplex IHC and mucicarmine special stain is evident in Fig. 5, panels D-F (SCC) and panels G-I (ADC) thereby providing greater confidence in their designations of SCC and ADC. Pink mucin staining is minimal in the SCC specimen (Fig. 5D) which also shows p40 positive cells (Fig. 5E), and mucin production is evident in the ADC specimen (Fig. 5G) in agreement with the presence of TTF-1 positive tumor cells (Fig. 5I).
The dual-camera imaging system is compatible with a pathologist’s manual evaluation of specimens at the microscope since the H&E color camera and monochrome CDC biomarker videos can be viewed simultaneously on a computer monitor while scanning the specimen. The highly corrected 20X microscope objective and LED illuminators used on the dual-camera system can provide both camera images in focus at video rates above 30 fps for the visible and LED illumination between 385 and 770 nm, which includes the HCC, DCC, and Cy7 CDCs. With filtered tungsten illumination, simultaneous focus and similarly high video rates are achieved for the visible, 769 nm (Cy7), and 880 nm (ir870) illumination channels (refer to Supplementary Methods document for detail on chromatic aberration correction and exposure times). Multispectral imaging, using only the monochrome camera, can replace the dual-camera system, if interactive manual scanning of whole slide specimens is not needed, or for further documentation, archiving, and quantification of selected microscope fields. In multispectral imaging, narrowband visible light channels are utilized in addition to the invisible light channels to image both the visible conventional stains and CDCs. Image processing can be applied to multispectral images to remove spectral crosstalk and provide images of individual chromogens and stains with reduced or eliminated interference from other chromogens and stains, as shown in Figs. 6 and 7. Even though the unprocessed images of the colon CD3 stain in Figs. 4E and 6D can be visually evaluated, quantification of CD3 staining can be improved by removing the faint nuclear stain, visible in Fig. 6D, to produce the unmixed image in Fig. 6G that is more effectively segmented for cell counting, measuring spatial relationships, and quantifying expression levels. Even the eosin image (Fig. 6A) is improved by removing faint hematoxylin crosstalk as seen in Fig. 6I. While spectral crosstalk between the Cy7 and HCC CDCs is not visually apparent in the processed images, increasing the number of multiplexed IHC targets or confining the CDCs to a smaller spectral range can increase spectral overlaps between dyes. This was seen in the mucicarmine special stain example (Fig. 7, panels A–F) in which the special stain absorbance in the blue and UV wavelengths (Fig. 1A) required duplex IHC to use two NIR absorbing CDCs, resulting at times in noticeable bleeding of the ir870 CDC absorbance into the Cy7 channel. Image processing to unmix the TTF-1 and p40 multispectral images shows complete removal of the TTF-1 staining in the p40 image (Fig. 7, panel E vs panel B) to reveal the expected lack of p40 staining (Fig. 7E) in this ADC NSCLC specimen.
In multispectral imaging, color images of the conventional stains are generated by combining images recorded at two or more visible illumination bands positioned near the peak absorbance of individual dyes comprising the conventional stain. Figures 6C and 7A show H&E color composite images created from images recorded under illumination near the eosin and hematoxylin absorbance peaks for the colon and ADC NSCLC specimens, respectively, and provide good reproductions of images recorded with the color camera. When the eosin, hematoxylin, and chromogen images are unmixed, composite images can be prepared from any combination of these component images, such as in Fig. 7D in which only the hematoxylin and TTF-1 images are combined to provide a representation equivalent to a single TTF-1 IHC with hematoxylin counterstain. Color composite images can also serve to highlight aspects of the IHC staining, such as co-expression, as shown in Fig. 6F in which CD3 and CD8 have been pseudo-colored magenta and cyan, respectively, producing blue in regions of CD3/CD8 co-expression. Note that cyan cells (CD8 only) are not apparent in the figure since any T-cells expressing CD8 should also co-express CD3, thereby producing the blue coloration.
Not all conventional stains are suitable for multiplexing with invisible IHC. These include black and some brown stains that absorb across the UV, visible, and NIR spectrum, such as iron-hematoxylin complex staining of elastin in Verhoeff’s Van Gieson stain. Invisible IHC can still be used with these stains, but the black stains and pigments will be visible in the monochrome UV and NIR images as well as the visible images. Yellow stains may also obscure the UV and blue portions of the spectrum, as seen here for the tartrazine component of the mucicarmine special stain, limiting invisible chromogens to the NIR.
From the above examples, other applications of invisible chromogens may be imagined. One of these applications would be to augment AI evaluation of H&E stained tissues to increase diagnostic sensitivity and specificity. While AI can come close to, or in some cases match or exceed pathologist interpretation21, AI might be improved by enhancing certain cellular, stromal, and overall tissue features with invisible IHC, or identifying tumor-specific markers, providing additional information for diagnosis and correlation with outcomes.
In summary, conventional histological and cytological stains can be combined with IHC on the same slide through the use of invisible chromogens. Clinical benefits include conserving precious specimen material and providing IHC information within the full morphological context and with additional cell specific information that H&E, PAP, and other conventional stains provide. Conventional visible stains and invisible IHC stains can be viewed and evaluated in real-time using a dual camera approach, or multispectral imaging and image processing can be utilized for spectral unmixing, quantitative analysis, and composite image formation.
