The immunohistochemical evaluation has expanded our knowledge of both the function and pathological processes in a wide range of lymph nodal, extra-nodal tissues, and bone marrow (BM). It is impossible to consider the full range of immunohistochemical markers available for studying hematopoietic tissues as well as the wide variety of ways to approach the evaluation of lymph node, extranodal tissues, or BM.

Without a doubt, immunohistochemistry (IHC) is more extensively used in hematopathology than in any other subspecialty area of pathology. In parallel with our understanding of phenotypical and immunological composition of the hematopoietic system, our armamentarium of paraffin section-reactive markers has grown as well. In some cases, it could be said that we have "too many

From: Methods in Molecular Biology, vol. 378: Monoclonal Antibodies: Methods and Protocols Edited by: M. Albitar © Humana Press Inc., Totowa, NJ

markers," leading to confusion and challenges in choosing and interpreting them wisely.

It is important to recognize that fundament for IHC interpretation is a careful histological analysis of a thin, well-stained H&E section. Simply having the best histology possible can avoid a variety of problems. Too often, poor quality is replaced by a battery of IHC stains. This is unfortunate, not cost effective, and ultimately is poor patient care. Rather than try to make a difficult diagnosis on suboptimal material, it may be best to simply obtain another better quality specimen. Among the technical factors that plague interpretation of IHC are thickness of section, folds or wrinkles in section, interference by fixative (i.e., B5, Bouin's), inadequate blocking of endogenous peroxidase (especially liver or peroxidase-rich cells), ineffective counterstaining (hematoxylin, fast green), and over/understaining (1-4).

Especially when dealing with small biopsy samples, it is crucial to order up front, in addition to the H&E-stained sections, an adequate number of unstained slides, as an insurance policy for the potential need for additional stains. Proper selection of immunostains requires a preliminary differential diagnosis. A careful microscopic interpretation is as important for IHC as it is for conventionally stained sections (5). It is important to know the expected reaction pattern of the immunostain being used because the cellular and tissue localization of the stain reaction is often a critical factor in its interpretation. Surface staining may be especially difficult to evaluate, particularly when numerous cells are closely crowded together. Cytoplasmic staining tends to be easier to interpret. Golgi staining is often very specific (e.g., seen with CD30, CD15), but is seen only in selected cases. Nuclear staining can be also very specific, in the right context, particularly in stains that are expected to have exclusive nuclear reactivity (such as TdT, cyclin D1, and so on). "Edge artifact" is common, resulting in intensification of staining along section edges. Its cause is not fully understood, but it can hamper adequate interpretation.

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