Photo by Todd McNaught
Bent over a dual-headed microscope, Drs. John Pagel and Julie Randolph-Habecker stare intently at a tumor specimen from a mouse with lymphoma. Pagel, an associate in the Clinical Research Division, is testing new drug treatments that might allow patients with chronic lymphoma to manage their symptoms free of the side effects caused by conventional chemotherapy. As they inspect the tissue sample, Randolph-Habecker, staff scientist and manager of Experimental Histopathology section of the Research Pathology shared resource, directs Pagel's gaze to lymphoma cells that appear to have self-destructed, evidence that the drug is working.
"We need to understand how the drugs we're testing work in living tissue before we can treat cancer patients," Pagel said. "That requires us to examine tissue from tumors to see what effects the drug is having on cells. Having a center pathology resource that is able to help me design and interpret these experiments has been a major asset to these studies. I could not do this easily in my own lab."
Likewise, the Solid Tumor section of the shared resource has been a key research partner of the Seattle Colon Cancer Family Registry (C-CFR), said project manager Allyson Templeton of the Public Health Sciences Division. The Seattle C-CFR, directed by Dr. John Potter and Dr. Polly Newcomb, is one of six recruitment sites collecting data, blood and tissue specimens from individuals diagnosed with colorectal cancer. This international registry serves as a resource for investigators performing research in the genetic epidemiology of colorectal cancer around the world.
"The Solid Tumor resource has done an enormous amount of work for us," Templeton said. "They helped us develop the protocols and the batch-processing form used to process pathology materials, have reviewed and sectioned more than 1,600 tissue samples, and now perform the pathology review of all cases following the CFR-standardized review form. In addition, they helped us locate, purchase and set up capital equipment for the long-term storage of tumor samples sectioned for future protein-based studies. The staff members are professional, knowledgeable and responsive. They've been a tremendous resource to us."
With the recent expansion of the two sections of the Research Pathology shared resource, center investigators — whether their experiments involve a few mice or thousands of human-tumor samples — have many new on-site options for incorporating pathology experiments into their research. The shared resource recently surveyed faculty to identify high-priority areas for further development of pathology services.
Pathology is the study of the nature and causes of disease, with an emphasis on the understanding of the structural and functional changes that take place in cells or tissues. The work typically involves cutting tissue samples into thin sections, staining it with dyes or antibodies to reveal hard-to-see parts of the cells, and visualizing the tissue samples through a microscope. Pathology plays a vital role in the clinic for accurate disease diagnosis. As a research science, pathology enables scientists to understand normal and cancer biology and to identify new tumor characteristics that may later be developed into tests that accurately reveal the subtype, stage and prognosis of cancer.
Both Research Pathology sections, under the overall direction of Dr. Robert Hackman, provide an array of these services for members of the Fred Hutchinson/University of Washington Cancer Consortium. The major focus of Experimental Histopathology, managed by Randolph-Habecker, is analysis of human, mouse and other animal tumor tissue. The section evolved from what was previously known as the Hematopathology section, which primarily focused on the pathology of human blood cancers. The Solid Tumor section, directed Dr. Peggy Porter and managed by Liz Donato, is equipped for large-scale studies with a major focus on human-tumor samples. This section was created in response to the growing number of investigators working on breast, prostate, colon and other solid cancers.
Experimental Histopathology: Enhanced focus on mouse pathology
Mice with conditions that mimic human illnesses, including cancer, have proven to be valuable tools for helping center scientists understand the basic principles of how diseases develop, said Hackman, an investigator in the Clinical Research Division.
"A lot of our investigators are using mouse models to study cancer and other disorders, and that trend is increasing," he said. "That has generated a greater need for mouse pathology support, and in response, we've increased our ability to provide that through Experimental Histopathology."
Users of the facility, which is currently located on the first floor of the Seattle Cancer Care Alliance but will move the first floor of the Weintraub building at the end of 2005, will benefit from Hackman's recent in-depth training in state-of-the-art mouse pathology techniques. For the last two years, Hackman, a clinical pathologist who has been a member of the center's transplant team for 25 years, has attended intensive mouse pathology courses at the Jackson Laboratory in Bar Harbour, Maine, the world's largest center for mouse research. He plans to bring the fifth of the week-long workshops in this series to the center in 2006. This fall, Hackman spent two months at the Jackson Laboratory and recently coauthored a paper with colleagues there that describes the requirements for a first-rate mouse pathology program.
The two major services offered in the section are histology and immunohistochemistry. Histology is the preparation of tissue samples for microscopic analysis. It includes techniques for fixing, or preserving, samples and treating them with stains that react with different parts of tissues or cells and provide color contrast so that they can be visualized.
Instead of stains, immunohistochemistry relies on using antibodies to "light up" different parts of tissue. Antibodies are proteins that attach very specifically to other proteins. When tagged with a dye, they become valuable tools for analyzing proteins made by cancerous cells.
Regardless of the technique that is used, Experimental Histopathology staff will customize the procedure for the investigator, said Randolph-Habecker, who holds a Ph.D. in experimental pathology. Although pathology is a relatively old science, many procedures were originally developed for human tissue and must now be adapted for animals and different tissue types.
"We provide a scientific resource, not just a pair of hands," she said. "We work with investigators to make sure we understand what they need, then collaborate with them to design the experiments, develop the techniques and help them interpret the data." One new tool that will facilitate data interpretation is the section's recent acquisition of a five-headed microscope, which enables five people to view a sample at the same time, with the pathologist walking them through the results. The facility is also staffed by certified histotechnologists Lisa McLemore and Tracy Goodpaster, and is currently adding two positions.
Dr. David Myerson provides technical consultation. In addition, the resource collaborates closely with two other center pathologists, Drs. George Sale and Howard Shulman.
With the center's increasing need for mouse pathology services, Experimental Histopathology is acquiring the stains and commercially available antibodies researchers need. When antibodies are not available for purchase, "we'll collaborate with the investigator to have them made on-site," Randolph-Habecker said.
Solid Tumor: High-throughput analysis for large-scale studies
The Solid Tumor section, located on the fifth floor of the PHS building, grew out of pathology services offered in Porter's laboratory. The resource provides wide variety of histology and immunohistochemistry services as well as customized experimental and work-flow design and data-analysis consultation. The facility's standout feature is its ability to conduct high-throughput work, a term used to describe experiments in which hundreds or even thousands of samples can be analyzed at once with the help of robots or automated equipment.
Although many users of the Solid Tumor section conduct research that involves large numbers of human specimens, the facility is also skilled at handling small-scale pilot projects as well as studies involving animal specimens. For example, the Solid Tumor group performed the tissue sectioning for a recent mouse study by Dr. Valeri Vasioukhin's lab, which identified a protein that drives the spread of prostate cancer.
"Increasingly, researchers at the center are conducting large-scale studies — often in collaboration with other institutions — that involve collection of human-tumor specimens," said Porter, an investigator in the Human Biology and PHS divisions and a board-certified anatomic pathologist. "These investigations are important for the discovery of new markers for predicting cancer prognosis and monitoring progression."
One of the key tools for high-throughput pathology analysis is the tissue microarray, a relatively new technique that mimics high-throughput methods originally developed for large-scale analysis of genes. Researchers construct tissue microarrays by cutting tiny cylinders of tissue and assembling hundreds of them onto a tissue microarray block. Using this method, many patient samples can be analyzed on a few blocks rather than the hundreds, or even thousands, of glass slides that would be needed using conventional methods. Once treated with antibodies or stains, the blocks can be analyzed by automated scanners that capture and store the data. The facility houses a state-of-the-art automated tissue arrayer, known as an ATA 27, manufactured by Beecher Instruments.
"We are one of a handful of institutions around the world with this instrument," Donato said.
Because human biopsy samples can be small, the Solid Tumor section is equipped with instruments that allow tiny amounts of tissue to be extracted for each analysis. Through a technique called laser capture microdissection, abnormal cells are identified visually, under a microscope. With the use of a laser, cells are embedded in the surface of a resin-coated cap and pulled from the tissue for further testing. Once cells of interest are captured, they can be analyzed in more detail for known or suspected mutations.
When larger samples are available, the facility can assist in preparation of cells for analysis by flow cytometry, a technique in which cells are treated with chemicals or antibodies that allow the DNA or specific proteins to fluoresce, allowing the amount of DNA or protein to be quantified.
Donato, a certified medical technologist, said that Solid Tumor technologists all have clinical backgrounds and bring a wealth of skills to the shared resource. Kim Adolphson and Linda Cherepow are both licensed histotechnologists each with more than 20 years of experience. Adolphson is an expert in immunohistochemistry techniques and Cherepow has years of experience in mouse necropsy (autopsy). Cherepow provides necropsy service for individual labs and, in collaboration with the Animal Health Shared Resource, conducts instruction sessions for faculty and staff who would like to learn necropsy skills. In addition to Porter, Drs. Ming-Gang Lin and Xiaopu Yuan serve as pathologists for the facility.
"We can provide consultation at all levels and at all stages of a project," Adolphson said. "We're also happy to answer questions even if an investigator doesn't choose to use our services."
The services of Research Pathology
- Project consultation including fixation and tissue handling, custom tissue processing and embedding, choice of special stains, immunohistochemistry antibody selection, evaluation of novel antibodies and pathologist interpretation.
- Fixation, paraffin embedding and sectioning of tissue
- Optimum cutting temperature (OCT) embedding and sectioning of frozen tissue
- Cytospin preparation of cytology specimens
- Sectioning tissue for molecular analysis
- Histochemical stains such as hematoxylin and eosin (H&E), periodic acid-Schiff (PAS) and trichrome
- Immunohistochemistry utilizing the latest technology in detection, signal amplification and antigen retrieval techniques. Extensive experience performing immunohistochemistry staining on human, mouse and canine tissue. Multiple antibody staining and fluorescence detection also available.
- Customized evaluation of novel antibodies or novel applications of antibodies
- DNA in situ hybridization analysis
- Tissue banks including frozen and paraffin-embedded normal and diseased tissue from human, mouse and canine
- Antibody Cross-Reactivity analysis for FDA approval of monoclonal antibodies for therapeutic use
- Pathologist review of histology and immunohistochemistry, including Mouse Pathology
- Collaborate with other resources including Animal Health, Scientific Imaging, EM and Genomics to provide comprehensive research support
Solid Tumor Research Pathology
- Design of protocols for tissue handling, pathology laboratory testing and optimal workflow design in the analysis of histology specimens from large projects
- Paraffin and frozen embedded tissue sectioning for molecular analysis
- High throughput immunohistochemistry using state-of-the-art Dako Eridan instruments
- Customized evaluation of novel antibodies for immunohistochemistry
- Pathology review and interpretation for immunohistochemistry
- Design, construction, sectioning and immunohistochemistry
- Imaging and image analysis
- Data interpretation using customized Web interface that allows for the secure sharing and interpretation of images by collaborators inside and outside the center
Laser capture microdissection:
- Optimization of specimen handling
- Training for the use of both Pixcell II and Autopix instruments
- Murine necropsy consultation and training (in collaboration with Animal Health Resources)
- Imaging and image analysis of histological preparations