biotinyltyramide and Neoplasms

Immunofluorescent staining of mammalian cells has provided a reliable approach for detection of specific antigen expression in situ. An advantage of fluorescent markers has been their applicability to automated, high-throughput cellular analysis by flow cytometry. Flow cytometry has thus become an integral component of clinical laboratory diagnostics, particularly in the areas of immunology and hematology. One of the major drawbacks of traditional immunofluorescent staining, even with flow cytometric detection, has been the difficulty in detecting low abundance cellular antigens, some of which may have clinical and scientific significance. To address these problems, staining techniques have recently been developed to increase the sensitivity of cellular antigen detection by flow cytometry. In this review we will describe a few of these techniques and focus on enzymatic amplification staining as a means to generate a highly augmented antigen-specific signal. We will also discuss practical applications of enzymatic amplification for immunostaining of clinical specimens.

Topics: Biotin; Flow Cytometry; Fluorescent Antibody Technique; Hematopoietic Stem Cells; Horseradish Peroxidase; Humans; Immunophenotyping; Lymphocyte Subsets; Neoplasms; Sensitivity and Specificity; Staining and Labeling; Tyramine

The catalyzed signal amplification (CSA) technique, based on the peroxidase-mediated deposition of haptenized tyramide and also known as tyramide signal amplification and catalyzed reportor deposition systems, is widely accepted as a signal amplification method for immunohistochemistry and in situ hybridization. In this study, we examined the applicability of a new simplified CSA system employing fluorescyl-tyramide (FT) to pathologic testing and research with formalin-fixed, paraffin-embedded tissues. By using the FT, instead of biotinyl-tyramide (BT) that is commonly employed in the CSA system with chromogen, nonspecific staining caused by endogenous biotin was completely avoided. The FT-CSA system loaded on the automated immunostaining equipment also allowed for more reproducible detection in short times. When applied to cyclin D1 immunostaining that is important in differentiation among small B-cell lymphomas, the system was useful in demonstrating its protein expression in mantle cell lymphomas considered negative or equivocally positive for cyclin D1 in a conventional immunodetection. In immunohistochemistry for phosphorylated proteins and murine hematologic markers that often require higher sensitivity than conventional methods, the FT-CSA system provided desirable staining results with intense signal amplification. Our results indicate that the simplified CSA system employing the FT can be useful in enlarging the target range for routine immunohistochemistry due to its high applicability.

Topics: Animals; Antibodies; Antigens, Neoplasm; Biotin; Humans; Immunoenzyme Techniques; Immunohistochemistry; Mice; Mice, SCID; Microscopy, Fluorescence; Neoplasms; Paraffin Embedding; Sensitivity and Specificity; Tyramine