Peter A. Humphrey
I. INTRODUCTION. Immunohistochemistry is one of the most powerful and widely used ancillary methods in surgical pathology. The technique makes it possible to simultaneously visualize cell type and differentiation markers in standard tissue sections by light microscopy, and has revolutionized diagnostic surgical pathology.
Antigens in tissue sections were first detected using antibodies via immunofluorescence performed on frozen sections (Proc Soc Exp Biol Med. 1941;47:200). Although immunofluorescence is still used in the evaluation of medical kidney biopsies (see Chap. 19), currently the most common approach for diagnostic detection of antigens uses formalin-fixed paraffin-embedded (FFPE) tissue sections and immunoperoxidase methodology. This enzymatic labeling technique has evolved from simple direct peroxidase conjugation of the primary antibody, to the use of multistep peroxidase-antiperoxidase (PAP), avidin-biotin (and related) conjugate methods, which, along with amplification techniques such as tyramide and polymer-based labeling, allow for much greater sensitivity in antigen detection.
The laboratory utilization of immunohistochemistry (also known as immunohistology and immunostaining) requires appropriate test selection, specimen acquisition and management, methodology, validation, reporting, and interpretation. This chapter provides a concise overview of these elements. Guidelines pertaining to these elements were published in 2011 by the Clinical and Laboratory Standards Institute (CLSI) (www.clsi.org) in a comprehensive document entitled “Quality Assurance for Design Control and Implementation of Immunohistochemistry Assays; Approved Guideline-Second Edition.”
II. TEST SELECTION (PREANALYTICAL PHASE). Immunohistochemical stains are usually ordered after examination of hematoxylin and eosin (H&E)-stained sections. Common indications for immunohistochemistry are the diagnosis and characterization of neoplasms, but there are other indications as well, such as detection of infectious organisms and evaluation of prognostic and/or predictive factors.
The use of specific immunostains is driven by the clinical and morphological context of each individual case. Panels of antibodies are often used, and these panels should be devised on the basis of the anticipated value added to the clinical, radiographic, and pathological differential diagnosis. Panels of antibodies should thus be directed toward a specific question. Various approaches have been used to help construct appropriate immunostain panels, including algorithmic approaches and tabular approaches. Web sites with information on the specificity and sensitivity of various immunostains, and on construction of immunostain panels based on differential diagnosis of specific neoplasms, also exist (see below).
III. SPECIMEN TYPE AND TISSUE MANAGEMENT. Immunostains can also be performed on cytological specimens, although they are usually performed on standard histological tissue sections. Whereas the use of FFPE tissue sections offers obvious logistical advantages, some antigens require the use of fresh tissue or tissue preserved with ethanol-based fixatives. The discussion here focuses on tissues fixed in 10% neutral buffered formalin, because this is the tissue type most commonly available for analysis in routine clinical practice.
Immediate fixation in neutral pH formalin for 12 to 48 hours at room temperature is desirable. However, it must be noted that formalin induces crosslinks that may mask some epitopes, resulting in loss of immunoreactivity. Acid
decalcification of bone samples can also cause loss of immunoreactivity. “Unmasking” of some epitopes from FFPE tissue (and tissue treated with acid decalcification) can be accomplished by antigen retrieval techniques. Enzyme digestion was used for this purpose in the past, but now simple heat treatment (heat-induced antigen retrieval) is the most commonly used approach to optimize antigen detection. Unstained tissue sections cut onto charged slides or poly-L-lysine-coated slides (or gelatin- or albumin-coated slides) are typically used for immunohistochemistry, but it is possible to perform immunostains on sections that have already been stained with H&E (Am J Clin Pathol. 2005;124:708); because success with such restaining protocols is variable, unstained tissue sections remain the best resource for immunostains. Immunostaining should be performed on freshly cut sections from the paraffin block, because unstained sections exposed to air may lose antigen immunoreactivity over the course of days to weeks.
A. The primary antibody is an immunoglobulin molecule that binds to the target antigen in the tissue sections. The primary antibody may be either a monoclonal antibody derived via the hybridoma technique, or a polyclonal antibody from an antiserum. In general, polyclonal antibodies tend to be more sensitive but less specific than monoclonal antibodies. Unlike monoclonal antibodies, polyclonal antibodies are not uniform reagents of unlimited supply; different batches of antisera may result in polyclonal antibody heterogeneity.
Each antibody, whether polyclonal or monoclonal in origin, needs to be tested for sensitivity and specificity in target antigen detection, and the reaction conditions for its use need to be optimized. Titration experiments must be performed to achieve a working dilution of the primary antibody that yields the greatest contrast between specific staining and nonspecific staining. If prediluted reagents and kits are used, it is recommended that the manufacturer protocol be followed because validation was performed with those reaction conditions.
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