2004 Summer Meeting - Amsterdam - The Pathological Society of ...

209
In-Situ Hybridization Identification of the Y-Chromosome as
a Tool to Characterize a Graft Versus Host (GvH) Model in
the Rat
GM Elliott , E Holness , KC Hickling , RV Bundick , PJ Kerry
AstraZeneca R&D Charnwood, Loughborough, United Kingdom
The GvH model of injecting DA rat lymphocytes into the footpad of DA/Lewis
F1 rats is used to investigate efficacy of immunomodulating xenobiotics, often
using popliteal lymph node weight as the experimental endpoint. The relative
contribution of donor/host cells is poorly understood and the Y-chromosome is
one way of investigating the contribution of (male) donor cells to (female)
recipient lymph node expansion. Techniques to demonstrate Y-chromosomes
in the mouse are available but problematical in rat.
In situ hybridization on formalin-fixed sections of lymph node from male and
female control rats and from female rats 7 days post-injection with male donor
cells was carried out to identify Y-chromosomes using a Cy3 labelled probe
(Cambio CA-1631).
Cells containing Y-chromosomes were evenly distributed throughout control
male lymph nodes and were absent in female nodes. Male donor cells
(lymphocytes and blastoid) were found evenly distributed or in clusters
throughout female recipient lymph nodes. Alleles of another chromosome
(Chromosome 12) were present in cells from all nodes but were clearly
distinguishable from Y-chromosomes. This method shows promise in aiding
mechanistic understanding of the mode of action of novel immunomodulating
agents. Additional work is required to investigate the time course and
phenotype of donor cell participation.
210
TmaDB: A Tissue Microarray Database
A Sharma-Oates 1 , P Quirke 1 , D R Westhead 2
1 Academic Unit of Pathology, University of Leeds, Leeds, United
Kingdom, 2 School of Biochemistry and Microbiology, University of Leeds,
Leeds, United Kingdom
A single tissue microarray (TMA) experiment can generate a vast quantity of
data, as a consequence a systematic approach is required for the storage and
analysis of such data.
To facilitate such analyses a relational database (known as TmaDB) has been
developed to archive numerous aspects of TMA data including experimental
design, experimental protocol, results from the various immunocytological and
histochemical staining experiments including the scanned images for each of
the TMA cores. Other aspects of the data stored in the database include
pathology reports associated with each specimen on the TMA slide, location of
the various TMAs and the individual specimen blocks (from which cores were
taken) in the laboratory and their current status i.e. if they can be sectioned into
further slides or if they are exhausted. The database that has been designed
incorporates most of the published common data elements for TMA
experiments. TmaDB is therefore compatible with the TMA data exchange
specification developed by the Association of Pathology Informatics
community.
Furthermore, TMA experiments from several types of cancer can be stored in a
single database. TmaDB will provide a comprehensive repository for TMA data
such that a large number of results from the numerous immunostaining
experiments can be compared for each of the TMA cores in a simple way by
tiling each of the images with a description of the result. This will allow a
systematic, large-scale comparison of tumour samples to facilitate the
identification of gene products of clinical importance such as therapeutic or
prognostic markers. Finally, the ultimate goal of this work to establish a
standard for reporting TMA data that is equivalent to MIAME for microarray
data.
211
Software Development for Processing and Analysis of
Histopathologic Images
PM Pavlopoulos 2 , N Kavantzas 1 , P Korkolopoulou 1 , S
Bouzoukas 2 , E Patsouris 1
1 Department of Pathology School of Medicine National and Kapodistrian
University of Athens, Athens, Greece, 2 Urologic Clinic 1st Social Security
Hospital of Athens, Athens, Greece
Aim: Development of an easy to use and adaptable image processing and
analysis PC software program for Windows operating system.
Material and Methods: The application was developed in Visual Basic 6.0
(SP5) (Microsoft Corp.) environment. For user friendliness the program was
designed with multiple document interface and Windows XP compatibility.
Results: The application can open and edit the most common file types (*.bmp,
*.jpg, *.gif). It has basic functions such as reversal or posterization of the
images and it can apply mathematical operations as well as brightness, contrast
and color balance adjustments. Object segmentation can be performed in 8-bit
black and white as well as in 24-bit color images with the use of thresholding
adjustable in multiple ways. The application can apply several algorithms for
edge detection, such as Roberts Cross, Sobel and Canny edge detectors. The
basic operators of the Mathematical Morphology “dilation”, “erosion”,
“opening”, “closing” and “hit-and-miss transform” can be performed in binary
images. Finally the program permits manual or automatic object counting and
measurement of distances, angles or areas.
Conclusion: As it has been observed in thorough testing, this application
constitutes a stable, fast, reliable and easy to use tool for image processing and
analysis. Moreover, its open architecture permits the continuing upgrading and
improving with the addition of newer algorithms customized according to the
clinical or researching demands of the user.
212
Microarrays for dummies
B Ylstra , P van den ijssel
VU University Medical Center, Amsterdam, Netherlands
Modern day technique has enabled tens of thousands of genes to be printed on a
single microscope slide: high-density microarrays. The development of these
arrays has boosted genomics research, exploiting the huge amount of data made
available by the human genome project, as all known genes could now be
included. Complete RNA expression profiles can be acquired, which can be
clustered and related to disease status. From these clusters a molecular profile
or ‘signature’ of the disease can be extracted. Thus, research can take full
advantage of developments in microarray technique to find diagnostic and
prognostic profiles for various diseases. A major challenge is to implement
these signatures as diagnostic or prognostic tools in the clinic.
Despite the availability of a number of commercial platforms, many institutes
have raised core facilities providing them with homemade microarrays to
minimize cost and maximize flexibility. Since all these facilities use their own
combination of probes, slides, printing and scanning equipment, hybridization
conditions and sample preparation, this gives rise to a huge variability in
quality. Because regulatory federal and state agencies will allow only excellent
quality microarrays to be used for diagnostics, many of today’s platforms will
not pass.
Many statistical ‘tricks’ are being applied to correct for artifacts in the result of
microarray experiments. In this seminar we will introduce the basics of the
technique and implementation. We will pinpoint essential steps to raise the
quality of microarrays produced in core facilities to the perfection required for
diagnostics or prognostics.
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