Histopathology - Services
Routine histologic preparation for laboratory investigators: These services include tissue processing such as fixation, embedding, tissue sectioning, and histochemical staining. Central to this service is proper specimen acquisition in coordination with the Biorepository Resource Service Shared Resource, recording, reporting and storage.
Immunohistochemistry: These studies are divided into two major types: (1) localization studies using novel investigator-generated antibodies; or (2) studies using commercially available antibodies. The former generally requires a greater expenditure in time and effort in determining proper antibody dilution, tissue preparation, and defining the conditions for optimal tissue localization. In both cases, the localization is available by immuno-fluorescence or immuno-peroxidase with current development of quantum dots. The automated immunohistochemical stainer equipment allows for testing with a variety of antigen-retrieval methods. Protocols have been developed to perform double and triple staining.
Fluorescent In Situ Hybridization/In Situ Hybridization (FISH, ISH): Quality staining using chromogenic or fluorescent in situ hybridization is offered. These highly sensitive staining techniques use the programmable software purchased as part of an automated immunohistochemistry stainer. This feature allows for protocols that optimize the various procedural steps necessary for in situ hybridization, such as pre-treatments, probe application, denaturation, hybridization and the stringency washes before detection and counterstaining.
Construction of tissue microarrays: Array construction involves retrieval of formalin-fixed paraffin-embedded tissue blocks containing tissue samples of interest, along with the corresponding hematoxylin-&-eosin (H&E)-stained slides, from surgical pathology archives or BRS; preparation of H&E-stained slides from each donor block and identification of areas of interest (e.g., normal tissue, in situ carcinoma, invasive carcinoma, or lymph node metastasis) on the H&E stained slide; array block layout design, taking into consideration the number of cores that will be used to represent each donor block (we typically use 2- to 4-fold redundancy), the spacing between rows and columns (we typically group cores in small squares of 5 x 5), and the size (total cores/cases) of the recipient block. Core biopsies of 0.6 mm in diameter are taken from each donor block and arrayed into a recipient paraffin block (30mmL X 24mmW X 5mmH) using a tissue puncher or automated arrayer (Beecher Instruments) as described by Kononen et al. (Kononen, et al., Nat Med,1998). Once an array block has been constructed, H&E stained sections are re-examined by the reference pathologist to check the histology represented in each tissue disk and to ensure quality control. Although TMAs have traditionally been assembled using paraffin-embedded tissues, the technology is now available to construct arrays from frozen specimens.
The guiding principle in developing TMAs is to design them to be useful for as wide a range of scientific objectives as possible and for biomarkers that might have different underlying biology, mechanisms, or functions. The process is intended to avoid the situation of having to design a new TMA for every new biomarker. To accomplish this goal as guided by the needs of Cancer Institute investigators, we construct four different types of TMAs:
- Test TMAs: General purpose arrays of anonymized de-identified human tissue specimens to optimize utilization of reagents, test new techniques, etc.
- Tumor-type-specific screening TMAs: Anonymized, de-identified, non-annotated collections of human malignant tissue specimens of a particular organ or organ system, with normal adjacent tissues whenever available. These arrays can be used to rapidly test for expression of biomarkers across different tumor types.
- Tumor-specific validation TMAs: Annotated collections of human malignant tissue specimens of a particular organ or organ system, with normal adjacent tissues if available. These arrays might also contain tissues from intermediate stages of tumor development. These arrays can be used to test for expression of biomarkers in particular tumor types, their changes during tumor development, and their validation as prognostic or predictive markers. Depending on the P.I. for the array and the study for which they were constructed, the clinical data may be supplied to the investigator in anonymized and/or de-identified fashion.
- Study-specific TMAs: Collections of tissue samples with full annotation assembled by a P.I. for a specific study.
Histologic preparation of TMA sections for laboratory investigators: These services include array block sectioning and histochemical or immunohistochemistry staining. Typically, 4-5µm sections of tissue microarray blocks are mounted onto glass slides using a paraffin sectioning aid system (adhesive coated slides PSA-CS4x, adhesive tape, UV lamp, Instrumedics Inc) to support the adhesion of the tissue disks to the glass Superfrost Plus slides (Fisher Scientific, Cat# 12-550-15). Microarray sections are processed within 2 weeks of cutting to avoid oxidation of antigens. Alternatively, cut sections can be soaked in xylene (2 min.), oven dried at 60°C for 20-30 min, then air-dried at room temperature for 30 min before being coated with liquid paraffin and stored at room temperature for up to 3 months prior to processing. Central to this service is proper specimen acquisition through the Biorepository Shared Resource.
Expert consultation: Expert consultation is available for developing projects through Drs. Goodell, Chekmareva, and Foran on the proper selection of antibodies, processing, analysis of TMA slides and optimal use of image analysis and archiving.