CHAPTER 4
Special Procedures
New technologies have contributed to improvements in the diagnosis of infectious agents and tumors as well as to the classification of tumors, especially the non-Hodgkin lymphomas (NHLs), childhood tumors, and sarcomas. Use of a more extensive test menu of paraffin-active monoclonal antibodies for immunohistochemistry; molecular cytogenetic studies, including standard cytogenetics; multicolor fluorescence in situ hybridization (FISH); polymerase chain reaction (PCR) and its many variations; and locus-specific FISH; as well as developments in high-resolution techniques, including microarray gene expression profiling, proteomics, and array comparative genomic hybridization (CGH), allow a more accurate diagnosis and more precise definition of biomarkers of value in risk stratification and prognosis. The ophthalmic surgeon is responsible for appropriately obtaining and submitting tissue for evaluation and consulting with the ophthalmic pathologist. See Table 4-1 for a checklist of important considerations when submitting tissue for pathologic consultation.
Immunohistochemistry
Pathologists making a diagnosis take advantage of the property that a given cell can express specific antigens. The immunohistochemical stains commonly used in ophthalmic pathology work because a primary antibody binds to a specific antigen in or on a cell, and because that antibody is linked to a chromogen, usually through a secondary antibody (Fig 4-1). The color product of the chromogens generally used in ophthalmic pathology is brown or red in tissue sections, depending on the chromogen selected for use (Fig 4-2). Red chromogen is especially helpful in working with ocular pigmented tissues and melanomas, because it differs from the brown melanin pigment (see Fig 4-7).
The precise cell or cells that display the specific antigen can be identified using these methods. Many antibodies are used routinely for diagnosis, treatment, and prognosis:
cytokeratins for lesions composed of epithelial cells (adenoma, carcinoma)
desmin, myoglobin, or actin for lesions with smooth muscle or skeletal muscle features (leiomyoma, rhabdomyosarcoma)
S-100 protein for lesions of neuroectodermal origin (schwannoma, neurofibroma, melanoma) HMB-45 and Melan A for melanocytic lesions (nevus, melanoma)
chromogranin and synaptophysin for neuroendocrine lesions (metastatic carcinoid [see Fig 4-2], small cell carcinoma)
leukocyte common antigen for lesions of hematopoietic origin (leukemia, lymphoma) CD antigens for subtyping white blood cells
Her2Neu and c-Kit for prognosis and treatment (metastatic breast carcinoma, mastocytosis)
Table 4-1
These antibodies vary in their specificity and sensitivity. Specificities and sensitivities of new antibodies are continually being evaluated (for examples, see the online immunohistology query system at www.immunoquery.com). Automated equipment and antigen retrieval techniques are currently used to increase sensitivity and decrease turnaround time.
Figure 4-1 Schematic representation of the general immunohistochemistry method. 1, The cellular antigen is recognized by the specific primary antibody, 2. A secondary antibody, 3, directed against the primary antibody, reacts with the enzymatic complex to create the chromogen, 4. The final product allows the visualization of the cell containing the antigen.
Patricia Chévez-Barrios, MD.)
Figure 4-2 A metastatic carcinoid to the orbit seen by H&E (A) shows bland epithelial characteristics. B, Chromogranin antibody highlights the neuroendocrine nature of the cells. (Courtesy of Patricia Chévez-Barrios, MD.)
Flow Cytometry, Molecular Pathology, and Diagnostic
Electron Microscopy
Flow Cytometry
Flow cytometry is used to analyze the physical and chemical properties of particles or cells moving in single file in a fluid stream (Fig 4-3, a). An example of flow cytometry is immunophenotyping of leukocytes. The cells need to be fresh (unfixed). Fluorochrome-labeled specific antibodies bind to the surface of lymphoid cells, and a suspension of labeled cells is sequentially illuminated by a light source (usually argon laser) for approximately 10−6 second (Fig 4-3, b). As the excited fluorochrome returns to its resting energy level, a specific wavelength of light is emitted (Fig 4-3, c), which is sorted by wavelength stream (Fig 4-3, d) and received by a photodetector (Fig 4-3, e). This signal is then converted to electronic impulses, which are in turn analyzed by computer software. The results may be imaged by a multicolored dot-plot histogram (Fig 4-4). The most common use of flow cytometry in clinical practice is for immunophenotyping hematopoietic proliferations. This procedure may be performed on vitreous, aqueous, or ocular adnexal tissue.