Immunohistochemistry Techniques

Immunohistochemistry (IHC) is the use of antibodies to detect proteins in tissue samples. The basis of the test is the antibody-antigen interaction that occurs when monoclonal or polyclonal antibodies are applied to tissue sections cut from patient specimens. This reaction can be identified at the microscope by color on slides; a stain that is usually brown signals the presence of the protein in question. These ancillary stains do not replace standard interpretation of H&E slides but complement the routinely stained material obtained from patient specimens. The stains are named in descriptive terms of the antigen in some cases (for example, gross cystic disease fluid protein, a breast marker) and by cluster designation numbers in others. Some of the antigens that IHC can detect include immunoglobulins, cell surface markers, receptors, enzymes, proto-oncogenes, and pathogens. Thus, IHC can be applied to detect pathogens, to identify the tissue of origin of cancer, and to diagnose disease processes where identification of a cellular protein provides diagnostic clues. IHC is an important tool used by pathologists to establish definitive diagnoses and to provide important prognostic information to aid in clinical decision making.

On rare occasions, one immunohistochemical stain may be enough to support a definitive diagnosis, but most of the time, a panel of immunohistochemical stains must be interpreted and matched to staining patterns reported for a particular disease to make a definitive diagnosis. Interpretations of the immunohistochemical stains are recorded in pathology reports that become part of a patient’s permanent record where they influence clinical decisions.

In cancer, IHC provides both diagnostic and prognostic information. In terms of making a diagnosis, IHC can aid the pathologist in cases where microscopic morphology alone is not determinative or in cases where a tumor phenotype is necessary for selecting the appropriate therapy. An example of IHC’s utility for prognosis is its use to assess the estrogen and progesterone status of adenocarcinoma of the breast, an important step in predicting prognosis and planning therapy.

Custom Immunohistochemistry Services:
Confidential custom target expression-profiling studies are performed on a fee-for-service basis without royalties or milestone payments and include the following:

Study Design
Contract research studies are initiated through customer consultation during which study objectives, available resources and the optimal scientific approach are discussed. Medicilon has conducted successful localization studies for more than 100 companies worldwide, collective experience that will be directly applied to ensure the success of your study.

Antibody Selection and Validation
Antibodies can be provided by the client, acquired from Medicilon’s antibody catalogue or purchased commercially. Should an antibody not be available, Medicilon’s target-specific antibody development and production services might further aid your research needs.

Tissue Selection and Validation
Tissue specimen best suited to address your study objective are chosen from Medicilon’s extensive Tissue Bank comprising normal and diseased, frozen and formalin-fixed, human and non-human tissues or can be supplied by the client. Should a desired tissue not be available in Medicilon’s tissue microarray (TMA) inventory; required specimens can be commercially sourced or collected prospectively by Medicilon for use in your study. Medicilon also offers custom tissue microarray design. All tissue specimens undergo rigorous testing to ensure proper fixation, accurate diagnosis and antigen validation prior to their application in your study.

Assay Optimization
Study-associated antibodies are evaluated in Phase I to ensure optimal performance prior to their application in selected tissues in Phase II. The optimal staining concentration is established by serial dilution of the antibody together with the optimization of antigen-retrieval conditions to enhance the signal-to-noise ratio.

Immunohistochemistry
Phase II entails the use of the antibody under optimized assay conditions to immunolabel selected tissues of interest. Through Medicilon’s extensive experience, the application of either single or multiple antibodies – using the double-labeling technique – can provide additional information on target co-localization and cell type differentiation by specific markers.

Interpretation
Immunolabeled slides are interpreted by Medicilon’s pathologists who have extensive knowledge in the interpretation of the often complex behavior of antibodies in IHC. Cellular localization profiles are correlated during the evaluation of normal and/or diseased tissue specimen.

Detailed Reports
Medicilon offers a flexible report format; facilitating report customization dependent upon the desired level of interpretation. The two most common formats include the Full Text and Tabular report.

Full-Text Reports
Full-Text reports incorporate the detailed description and interpretation of the immunolocalization pattern observed by Medicilon’s pathologists inclusive of 3 to 5 representative images for each tissue sample and is frequently requested for studies involving CNS tissues, co-localization studies or those involving rare events and minor cell types. Patient sample information is included in this format.

Tabular Reports with or without Imaging
Tabular Reports include the pathologist’s quantification of the observed signal present in major cell types within each tissue sample on a scale of 0-4. Results are presented in tabular format and provide practical information during the initial stages of target characterization, screening studies or those involving major cell types or homogeneous tissues. The optional inclusion of selected images for each tissue sample is dependent on the client’s request.

Study Deliverables
Upon conclusion of individual studies, Medicilon will issue a report detailing the assay conditions, the applied antibody dilution specifications and localization results for each assayed tissue specimen. If available, selected patient information including age, sex and cause of death or surgery is provided with each sample. Medicilon offers a variety of reporting formats to meet the varied needs of our customers.

Timeline
Standard IHC studies span the course of 3 to 12 weeks and are dependent on the complexity of the undertaken study; however, Medicilon strives to complete all client-related work in an expeditious manner.