Hematopathology Laboratory
Immunohistochemistry
Immunoperoxidase Cell Surface Antigen Profile
Cell surface marker studies using a sensitive immunoperoxidase technique are performed on frozen tissue specimens where cells obtained from fresh tissue are nondiagnostic by flow cytometry or where only frozen tissue is available. This technique may also be used to analyze cytocentrifuge preparations of body fluids or other liquid specimens if an insufficient number of cells are present for flow cytometry. Specimens are evaluated for the presence of malignant lymphoid cells, immature hematopoietic cells, or other hematopoietic cells using a panel of monoclonal antibodies.
The specific panel of monoclonal antibodies used is selected by the Hematopathology Laboratory supervisor and/or the Director based on the patient's clinical history, the referring physician's differential diagnosis, and the appearance of the cells present in the specimen. The standard panel is designed to diagnose B cell lymphomas and includes the T cell markers CD2 and CD5 (also coexpressed on small lymphocytic and mantle cell lymphomas), the B cell marker CD22, kappa and lambda immunoglobulin light chains to determine clonality, and the proliferation marker MiB-1 (used interchangeably with Ki-67) to determine the proliferative fraction. For cases of suspected cutaneous T cell lymphoma, the additional T cell markers CD4, CD8, and CD7 are included in order to demonstrate an abnormal T cell phenotype, as well as CD25 (interleukin 2 receptor alpha). For cases of peripheral T cell lymphoma, CD3, CD4, CD8, CD7, CD30, and CD1 are added to characterize the malignant phenotype. Immunoperoxidase staining for TdT can be performed if lymphoblastic lymphoma is suspected. The laboratory director will issue a written report and interpretation for each case.
Specimen requirements: Fresh tissue specimens in tissue culture medium, tissue frozen in a freezing medium such as OCT (preserved on dry ice during transport), body fluids
Cytoplasmic Immunoglobulin Immunofluorescence
Plasma cells do not express surface membrane immunoglobulin, so conventional flow cytometric techniques used to detect cell surface immunoglobulin are generally of little value. Plasma cells do, however, contain abundant cytoplasmic immunoglobulin. Cells are treated to make their membranes permeable and are then analyzed using a panel of antibodies specific for immunoglobulin light chains and heavy chains. Direct immunofluorescence by fluorescent microscopy is performed on cytocentrifuge preparations of specimens to detect the presence of cytoplasmic immunoglobulin. Flow cytometric techniques are now available to detect cytoplasmic immunofluorescence and will replace most immunofluorescent microscopy in the near future. The detection of a monoclonal pattern is indicative of a malignant plasma cell process such as multiple myeloma, Waldenström's macroglobulinemia, plasmacytoma or lymphoplasmacytoid lymphoma. The laboratory director will issue a written report and interpretation for each case. When flow cytometry is performed, flow histograms will be available upon request.
Specimen requirements: Anticoagulated blood or bone marrow, fresh tissue specimens in tissue culture medium, body fluids
Terminal Deoxynucleotidyl Transferase (TdT)
TdT is an enzyme found in the nucleus of immature lymphocytes and is involved in the rearrangement of immunoglobulin and T cell receptor genes during lymphocyte development. It is present in the nuclei of precursor lymphoid neoplasms such as lymphoblastic leukemia and lymphoma, and is used as a diagnostic marker. The presence of TdT is usually assessed by indirect Immunofluorescence or, occasionally immunoperoxidase techniques. Immunofluorescence is preferred over the immunoperoxidase method due to its greater sensitivity and specificity . Methods for detecting TdT using flow cytometry are now available and will be used for many of the cases where immunofluorescent microscopy was used previously . The laboratory director will issue a written report and interpretation for each case. When flow cytometry is performed, flow histograms will be available upon request.
Specimen requirements: Anticoagulated blood or bone marrow, fresh tissue specimens in tissue culture medium, body fluids, tissue frozen in a freezing medium such as OCT, preserved on dry ice during transport (immunoperoxidase only for frozen tissue)
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Last updated: 6/22/00
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