In any biological experiment, the quality of the data generated is directly related to the quality of the samples and methods used in the study. Ideally, animal studies are designed to include a single variable between the experimental groups. It is essential to have appropriate control groups against which data from treated groups can be compared. In studies that use an animal disease model, naïve animals constitute the usual control group. For toxicology studies, the control group typically receives the vehicle by the same route, frequency, and volume as the test article dosed groups. In medical device studies, two control groups, a group of naïve (non-implanted) animals, and a group subjected to a comparator device are useful.
The Role of Histopathology
Histopathology is the art and science of evaluating tissue sections by light microscopy and recording the observed findings—this is primarily a process of pattern recognition. While the human eye is well adapted to pattern recognition, it is less trained to recognize the quantification of what it sees; therefore, any quantitative scoring of routine histologic sections by a pathologist is semi-quantitative at best.
Quantitative Data from IHC Slides
Immunohistochemistry (IHC) provides highly specific qualitative and quantitative information in relation to the expression of particular proteins within a tissue. While a pathologist can evaluate IHC sections qualitatively, they can only evaluate protein expression semi-quantitatively. While this approach recognizes clear differences between experimental and control groups, more rigorous quantitative comparison of the protein expression requires more precise quantitative data. Fortunately, the technology available today allows for the capture of highly reproducible quantitative data from IHC slides. The study pathologist, using their broad training and expertise, integrates the quantitative IHC data into the discussion and conclusions portion of the pathology report. The quantitative IHC data is included with the histopathology (and other study data) in the context of the biology of the target protein. Evaluation of this data allows the study pathologist to provide a comprehensive analysis of the toxicity of a chemical, pharmaceutical, biopharmaceutical, or medical device, which is necessary in order to make safety assessments.
Because of the inherent variability between animals, and the physiologic differences between different regions of some organs, it is essential that the procedures used to collect and process tissues for both histopathology and IHC are rigorously defined and followed. For example, different lobes of the liver are physiologically different, which can result in varied expression of the target protein in a given animal.
Minimizing variables in a study begins with defining the objective of the study. This will guide the development of the study design. After the draft study protocol has been created, the study pathologist should review and contribute to the protocol—this is especially true in relation to the procedures for tissue collection, preservation, trimming and processing, including IHC procedures. Following these steps will ensure the veracity of the histopathology and quantitative IHC data. While there is no regulatory requirement for the study pathologist to participate in the study design or to sign the study protocol, the pathologist’s signature on the protocol indicates that the methods used to generate the pathology and IHC data are in agreement with the objective of the study and in alignment with scientific current standards.
Histopathological Considerations for IHC-based Studies
Below are a few important factors to consider related to the histopathology of IHC-based studies:
1. Sample Collection
Tissue samples should be collected promptly after euthanasia by properly trained prosectors. The fixative used should be compatible with the procedures that the tissues will later be subjected to, especially with the IHC techniques to be used. Specific regions of larger tissues and the entirety of smaller tissues should be collected, fixed, trimmed, and processed according to accepted standard procedures. The goal is to attain similar tissue representation in the slides for all tissues in all animals. If unfixed frozen sections are required for the IHC procedure, those samples should be collected to ensure that similar representation of each tissue in all animals is achieved.
2. IHC Methods
The goal of the IHC staining procedure is to maximize the contrast between staining by the primary antibody (specific or Fab staining) and the staining by the isotype control (non-specific or FC staining). This requires that the methods used must be optimized by the laboratory performing the staining, regardless of whether the antibody being used is commercially available or is a proprietary therapeutic antibody. Factors to consider are: antigen retrieval methods for fixed tissues, blocking non-specific (FC) staining, and optimal antibody concentration.
The isotype control antibody should be of the same class as the test antibody. Both direct and indirect methods may be used to detect bound antibodies. Because antibodies cannot be seen by the human eye, a method for visualizing the antibody binding must be used. A fluorescent label may be chemically linked to the primary antibody and then directly visualized. Secondary antibodies (against the immunoglobulin of the species from which the primary antibody was derived) are commercially available and are often used. Secondary antibodies may be pre-complexed with the primary antibody or applied after the primary antibody binding has occurred. Avidin-biotin and biotin-streptavidin are commonly used methods for visualizing bound antibodies. The use of two different chromagen systems (with sequential staining by primary antibodies) allows for the staining of one tissue section with two different antibodies.
Because binding sites in a tissue are finite, contrast between primary and isotype control antibodies is not directly related to antibody concentration. Thus, the optimal dilution of a primary antibody must be determined for each study. In tissues in which there is known expression or lack of expression of the target of interest, this is accomplished by testing dilutions of primary antibody in a checker-board titration system.
3. Sample Evaluation
Quantitative IHC data provides a numerical measure of the degree of binding by the primary antibody (measured as chromagen deposition) in the total sample evaluated, but it does not indicate whether the chromagen deposits are the result of on-target or off-target binding of the antibody used in the assay. If the quantitative IHC data is to be accurately interpreted in the context of the study design, a pathologist must evaluate routine (H&E) sections—the purpose of this is to define the pathologic process(s) present and qualitatively/semi-quantitatively evaluate the IHC slides to define the staining as ‘on-target’ or ‘off-target.’
The next step is to scan the slides to capture digital images of the IHC staining in the tissue of interest. The area of interest in each slide to be analyzed is outlined and the total staining in that area is determined by vendor-provided software. The size of the area of interest scanned is calculated to allow the data to be normalized to a standard area. The total amount of staining in the normalized area (in primary and isotype control stained slides) can then be tabulated by group to allow comparison of the degree of specific staining in each treatment group against that in the control group. This data can then be compared by appropriate statistical methods.
The study pathologist can then include the quantitative IHC, qualitative/semi-quantitative IHC data, and the histopathology findings in the discussion and conclusions portion of the pathology report. The semi-quantitative and quantitative IHC data should have similar dose-response relationships between the control and test article groups regardless of the type of study (animal model, toxicology, or medical device).
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About Michael Tomlinson, D.V.M., Ph.D
Dr. Tomlinson, Lead Pathologist at Wax-it Histology Services Inc. and founder of Nova Pathology, brings a wealth of knowledge and over 30 years of experience in Veterinary Pathology and Toxicologic Pathology. He became involved in Medical Product/Device and Toxicologic Pathology in the 80's and has been a Toxicologic Pathologist since 1990. He has held a number of prominent positions over the years, including Staff Pathologist and Laboratory Manager for Pathology Associates Inc., Chicago, IL, Staff Pathologist for Bristol-Myers Squibb, Director of Pathology for SNBL-USA, Ltd., and Staff Pathologist for CRL, Nevada. To learn more about Michael’s work and Nova Pathology, visit www.novapathology.com.
About Wax-it Histology Services, Inc.
Incorporated in 2003, Wax-it Histology Services, Inc. brings a wealth of knowledge to our internationally recognized pharmaceutical, biotechnology, and medical device partners. Wax-it Histology Services, Inc. is a GLP-recognized CRO that offers general histology services (paraffin and frozen), immunohistochemistry services (general IHC, cross-reactivity studies, antibody and biomarker testing/validation), GLP and non-GLP histopathology for preclinical toxicology studies, and resin/plastic sectioning for implantable medical devices and bone.