DISCOVERIES 2015, Apr-Jun, 3(2): e46

DOI: 10.15190/d.2015.38
Emerging technologies for diagnostic pathology

PERSPECTIVE Article

Emerging technologies for diagnostic pathology

Octavian Bucur*

Department of Pathology, Harvard Medical School and Beth Israel Deaconess Medical Center,
Boston, MA, USA

*Corresponding author: Octavian Bucur, MD, PhD,, Department of Pathology, Harvard Medical School and
Beth Israel Deaconess Medical Center, 330 Brookline Ave, Boston, MA, 02215, USA. E-mail:
obucur@bidmc.harvard.edu

Submitted: June 27, 2015; Accepted: June 30, 2015; Published: June 30, 2015;
Citation: Bucur O. Emerging technologies in diagnostic pathology. Discoveries 2015, April-June; 3(2): e46. DOI:
10.15190/d.2015.38

ABSTRACT Abbreviations:
Many technological advances have been made in Fluorescence in situ sequencing (FISSEQ); 2
the recent years, several of them with a great dimension (2D); 3 dimension (3D); expansion
potential of significantly improving the diagnostic microscopy (ExpM);
pathology field. This article discusses three of the
most promising technologies, which emerged in the 1. Introduction
last one year. Fluorescent in situ sequencing can
lead to the simultaneous identification of the Several technologies with great potential in
transcriptome-wide RNA in individual cells across significantly advancing the diagnostic pathologies
a tissue sections. 3D microscopy together with have emerged in the recent years. This article
advanced image analysis can be used in diagnostic focuses on three revolutionary technologies that
pathology and will especially be useful in hard to emerged in the last one year. These methods
diagnose cases where the spatial relationship of the promise to advance the diagnostic in pathology by
tissue components is important. Expansion adding another dimension (3D diagnosis),
microscopy physically expands the biological sequencing in situ (fluorescent in situ sequencing)
specimen, and is of great interest for diagnostic and tissue expansion (expansion microscopy).
pathology since the cheap conventional These technologies combined with automated
microscopes can be used to image a symmetrically pathology have the potential of being widely
expanded tissue. In addition, digital analysis and employed by the pathologists in the near future.
computational pathology are an integral part of each
of these three emerging technologies, which 2. Fluorescent in situ sequencing
underline their importance for the future
developments in diagnostic pathology. Many approaches using high-throughput
methodologies have resulted in an extraordinary
Keywords: development of the “omics” fields: genomics,
emerging technologies, methods, diagnosis, transcriptomics, proteomics, metabolomics,
FISSEQ, expansion microscopy, 3D microscopy, interactomics, immunomics, and others1,2 etc.
3D diagnosis, image analysis, computational Simultaneous determination and analysis of
pathology; hundreds/thousands of genes (genomics),
transcripts (transcriptomics), proteins (proteomics)
can provide an unprecedented insight into the

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Emerging technologies for diagnostic pathology

organization, regulation and function of biological diagnosis and post-diagnosis follow up of many
systems1. pathologies, including cancers.
Researchers and pathologists understand
now more than ever the importance of personalized 3. 3D diagnosis in pathology
medicine. The potential impact of next-generation
sequencing and whole-genome analysis in medicine Since the early 20th century, pathological
and, specifically, in clinical laboratory practice is classification of many types of malignancies has
well recognized3,4. However, the current sequencing been primarily based on the visual analysis of
techniques, although high-throughput, do not hematoxylin and eosin stained image slides using
provide much information on the exact location of conventional two dimensional (2D) microscopy8.
its target. Moreover, DNA/RNA FISH identifies the With the recent development of new state-of-the-art
exact location of the target, however, it is not a microscopy platforms, such as the fluorescent
high-throughput technique5, since it is limited in the lightsheet microscopy (the Nature Methods’ method
number of probes that can be simultaneously or of the year 2014)9-11 and multiphoton microscopy
consecutively employed. Identifying the exact (2015 Brain Prize given for the development of 2
location of the sequenced or identified RNA/DNA photon microscopy)12, the rapid acquisition of three
of interest on the tissue section would bring an dimensional (3D) images directly from tissue
enormous benefit not only to the research samples up to several millimeters in thickness is
community, but also to the diagnostic pathology now possible11.
field. Fluorescence in situ sequencing (FISSEQ) Recent results presented a successful
is a highly multiplexed subcellular RNA sequencing pipeline of formalin fixed paraffin embedded tissue
in situ, which permits massively parallel detection sample preparation, imaging with a 3D advanced
of genetic elements, including gene transcripts, microscopy and extraction of quantitative image
important in analyzing cellular phenotypes, gene measurements from the 3D images to build
regulation, and cellular environment in situ6,7. This classification models in breast tissue. This
transcriptome-wide RNA sequencing in situ was classification models help in differentiating normal
validated on multiple specimen types and spatial tissue from non invasive breast cancer and different
scales. Fisseq is compatible with fresh frozen brain grades of invasive breast cancer13,14.
tissue sections and whole-mount embryos, although Thus, 3D microscopy together with
the authors only present the complete sequencing of advanced image analysis can be used in diagnostic
cell lines6. pathology and will be especially useful in hard to
Briefly, the cells/tissue are fixed and the diagnose cases where the spatial relationship of the
RNA is revers transcribed into cDNA using random tissue components is important.
hexamer primers. cDNA is then in situ amplified
and cross-linked, forming the single-stranded DNA 4. Expansion microscopy
nanoballs (also named rolonies or cDNA
amplicons). Fluorescent sequencing, which consists Researchers have successfully created state of the
in using a fluorescent probe hybridized to the art microscopes, which are under continuous
adaptor sequence and imaged by confocal development, in order to provide high resolution
microscopy, is next performed on the DNA images and to see in details the structures of the
nanoballs6,7. different types of cells and tissue9-12. However, a
If successful on tissue sections, this method revolutionary idea recently came to life when a 4-5
would enable the simultaneous identification of the fold expansion of the human brain tissue was
transcriptome-wide RNA in individual cells across reported with minimal distortion15. The authors
a tissue sections. This will not only help scientists named this method, of physical magnification of the
to study in situ cellular interactions (such as the specimen itself, expansion microscopy (ExpM)15.
ones from epithelium and stroma in several types of ExpM would be a great asset in diagnostic
malignancies (breast, gastro-intestinal tumors etc)), pathology, since one could use a cheap
immune cells and cancer associated conventional microscope to image a symmetrically
fibrobalsts/tumor cells etc) but will also help in expanded tissue.

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Emerging technologies for diagnostic pathology

ExpM uses a “swellable” polymer network 4. Haspel RL, Saffitz JE. Genomic oncology
in a biological sample that can expand the sample education: an urgent need, a new approach. Cancer J.
by 4-5 fold15,16. The method uses the well known 2014 Jan-Feb;20(1):91-5.
properties of the polyelectrolyte gels, which after 5. Ginart P, Raj A. RNA sequencing in situ. Nat
water addition they expand. The tissue is placed in a Biotechnol. 2014; 32(6):543-4.
polyelectrolyte gel, followed by its treatment with a 6. Lee JH, Daugharthy ER, Scheiman J, Kalhor R,
protease to homogenize its mechanical Yang JL, Ferrante TC et al. Highly multiplexed
characteristics and the placement of the specimen in subcellular RNA sequencing in situ. Science. 2014;
water for expansion15. 343(6177):1360-3.
7. Lee JH, Daugharthy ER, Scheiman J, Kalhor R,
Expanded tissues (such as the brain) have
Ferrante TC, Terry R. Fluorescent in situ sequencing
the great advantage that the process makes them
(FISSEQ) of RNA for gene expression profiling in
completely or partially transparent. Thus, these intact cells and tissues. Nat Protoc. 2015; 10(3):442-
tissues are well fit for lightsheet microscopy, which 58.
requires transparent specimens, so there is no need 8. Beck AH, Sangoi AR, Leung S, et al. Systematic
for a preclearing step17. analysis of breast cancer morphology uncovers
stromal features associated with survival. Sci Transl
In conclusion, these are only few of the several Med 2011; 3:108ra13.
important technological advances that could help 9. Keller PJ, Schmidt AD, Wittbrodt J, Stelzer EH.
the diagnostic pathology field in the near future. Reconstruction of zebrafish early embryonic
Although these methods are still under development by scanned light sheet microscopy.
development, they have an extraordinary potential Science 2008; 322:1065-9.
in transforming diagnosis of different pathologies. 10. Keller PJ. Imaging morphogenesis: technological
Moreover, these three techniques can be combined advances and biological insights. Science 2013; 340:
together and employed for analysis of the same 1234168
samples, leading to valuable results for both 11. http://www.nature.com/nmeth/journal/v12/n1/f
research and diagnosis. ull/nmeth.3251.html
12. http://www.thebrainprize.org/flx/prize_winners/
Acknowledgements 13. http://www.aacr.org/Documents/15AM_SIT
This work was supported by the Lady TATA awardeesforProgram.pdf
Memorial Trust Fellowship (London, UK). 14. Bucur O, Irshad H, Montaser-Kouhsari L,
Knoblauch NW, Oh Eun-Yeong, Nowak J, Beck AH.
Abstract 3477: 3D morphological hallmarks of breast
Conflict of Interest
carcinogenesis: diagnosis of non-invasive and
The author does not declare any conflict of interest. invasive breast cancer with Lightsheet Microscopy.
Cancer Res. 2014 (Proceedings: AACR 106th Annual
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