Routine and Customized Histological Services
Performance and assistance in routine histological processing of tissues including paraffin embedding, sectioning, H&E and special staining of human and animal tissues. In addition, customized tissue services are routinely performed. Core personnel and pathologists assist the researchers in interpretation of the morphological data and photograph their findings for publication using the core microscope/camera system. One of the specialized services provided to researchers is the preparation of multi-tumor blocks (i.e., tissue arrays). Our experience and technology in this field allows us to provide this unique service.
The Pathology Core offers assistance to all Cancer Center researchers on the application of immunohistochemical techniques to their research projects. This assistance takes various forms. Often, it is of a collaborative nature, where a specific pathologist is in charge of all histological aspects of the project. Alternatively, the Core personnel train researchers in the application of immunohistochemistry and provides laboratory support as needed. Finally, automated immunostaining of cytological or histological preparations is available for those who do not require pathology expertise for the interpretation of results. The Core maintains three Dako automated immunostainers and one Ventana Discovery Ultra immunostainer. The automated procedures permit uniformity of procedures from batch to batch. This equipment, purchased with institutional funds, ensures quick and reproducible immunostains and facilitates quantitative and semi-quantitative analysis of the preparations. The Pathology Core evaluates new experimental antibodies and has the expertise to evaluate IHC in non-routine settings.
The molecular analysis of human disease states has long been complicated by the cellular heterogeneity inherent in tissue samples. For example, vascular components, inflammatory cells and stromal cells are always admixed with malignant cells in a tumor. With the Laser Capture Microdissection (LCM) system, relatively pure cell populations can be obtained from paraffin or frozen tissue samples. A slide with a tissue section is placed on a microscope stage, and the user selects the field of interest. The instrument is designed so that a thermoplastic film is placed over the dry tissue section and a laser beam is fired. This provides enough energy to locally melt the film which then binds to the tissue beneath the laser. When the film is lifted, the cells selected are separated from the tissue section and adhere to the supporting cap. The cells selected are transferred via the cap to a microfuge tube for molecular analysis. The instrument permits examination of the morphology of the cells transferred and the process preserves DNA, RNA and protein for subsequent analysis.
The Core laboratory cuts paraffin blocks and frozen tissue for PCR analysis. These are sectioned with meticulous care to prevent cross contamination. The paraffin sections for PCR analysis are cut using disposable microtome blades on a microtome so that after each block is cut, the microtome can be cleaned with a dilute solution of HCl to destroy residual DNA/RNA. Periodically, a negative control is cut between blocks so that the process can be monitored for potential contamination.
Fresh Tissue Procurement
This service consists of providing tissue and tumor samples, on demand, to diverse researchers within the Cancer Center. Selection of individual tissue samples is performed by a pathologist in order to ensure that representative material is supplied. Control tissue in the case of tumors is chosen to represent as accurately as possible the cell of origin of the tumor itself. For example, in the case of colorectal carcinoma, control tissue is composed only of dissected normal colonic mucosa, free of muscularis and adventitia. Material may include fresh sterile tissue, non-sterile tissue samples, snap-frozen tissue (in isopentane cooled to –55 ºC or in liquid nitrogen), and tissue in any of a variety of fixatives. When necessary, histological examination of an aliquot of the sample provided is available to the researcher to ascertain the viability and overall quality of the specimen.
Frozen Tumor Bank
This facility is of crucial importance to researchers engaged in translational studies and is being actively utilized. In March 2001, the Cancer Center adopted stringent policies on using patient tissues; tissues are released only with a valid patient consent or waiver from the IRB. Our tissue samples can (with IRB approval) be correlated with clinical features and outcomes, making it an extremely valuable resource. We are not requesting funding to specifically support this service from the CCSG, but we consider it to be a vital component of the Pathology Core. This resource consists of the routine collection of excess tumor and normal (matched or unmatched) control tissues and their storage in ultra-low temperature freezers. Tissues are handled to preserve molecular integrity. For over 30 years, tissue has been collected for the tumor bank and is currently active. It is a valuable institutional resource for retrospective studies that are currently not possible on paraffin-embedded-tumor samples. Approximately 19,000 tumor/tissue samples are available from the bank for such studies. We also bank excess tumor and control normal tissues in paraffin blocks that have been fixed in various solutions, including routine formalin and 100% alcohol.
World-renown pathologists at City of Hope are an invaluable, highly rated asset not only for their histopathological and immunohistochemical expertise, but also for their depth of knowledge and experience in the clinical, molecular and etiologic aspects of cancer. The Director and Co-Director of the Core are both practicing diagnostic pathologists and have mature, well-published research programs in the molecular basis of cancer. Both the director and co-director are available to the Cancer Center membership for consultation on the pathological basis of diseases, the histological manifestations of diseases and the use of human tissue for studying disease. They are also available to advise on the development and design of experimental strategies using human pathologic tissue and can assist in the presentation of data for grants and papers. They are also available to interpret immunohistochemical data.
- Seven -80°c freezers are currently used to house the tumor bank. All of these are equipped with alarms and liquid nitrogen back-up
- One -20°c freezer currently used to house DNA and RNA samples isolated form normal and tumor tissues
- Three automated immunostainers (Dako) capable of staining 96 slides simultaneously
- One Ventana Discovery Ultra Immunostainer
- A double-headed microscope with Olympus DP-11 digital camera photo system with qualified pathologist to interpret and photograph results from various studies
- A Micron® cryostat for frozen sectioning
- Perkin-Elmer in situ PCR machine
- PALMS Laser Capture Microdissector
- Frozen Tissue Bank (in operation over 30 years; currently contains approximately 12,000 samples of tumor, many matched with normal tissues)
- Paraffin Tissue Bank (formalin and alcohol-fixed specimens) including numerous multi-tumor tissue arrays (City of Hope holds patents in this area)
- TissueTek VIP Tissue Processing System
- 4 microtomes for sectioning paraffin embedded tissue