DCC assay

Biochemical assays such as the dextran-coated charcoal (DCC) assay, certain signal amplification techniques, and other cytosol-based methods have been mostly replaced... 

Another advantage of immunohistochemistry is that tissues of a small size (e.g., biopsies) can be used. This is important because it is better to detect tumors at an early stage, when they are small. The necessity of early detection cannot be overemphasized. Very small tumors and fine-needle aspirates cannot be used for biochemical assays. Although the DCC assay provides quantitative results, it does not take into account the relative amount of the connective tissue in the specimen, the presence of carcinoma in situ lesions, or normal ducts and lobules. These limitations are not encountered when using paraffin sections. In addition, immunohistochemistry allows the use of archival tissues when fresh tissues are not available. This method does not require any special, expensive equipment and can be carried out in any standard laboratory. 

The immunohistochemistry of ERs has been exhaustively compared with the DCC assay. Review of the literature indicates -85% agreement between these two methods (Allred, 1999). This is true when immunohistochemistry is restricted to fresh-frozen sections. Immunohistochemistry of frozen sections compared with paraffin sections is a more specific test to detect ER-positive tumors with very low tumor cellularity the DCC assay gives false-negative results for such tumors. A number of publications have also reported good agreement between the DCC assay and immunohistochemistry of paraffin-embedded... 

The dextran-coated charcoal (DCC) assay measures the hormone-binding capacity of the cytosol fraction of the tumor tissue (Lee and Chan, 1994). Fresh tissue (more than 100 mg) is immediately frozen, homogenized, and the cytosol fraction is extracted. Aliquots of the cytosol are incubated with radiolabeled [3H] estradiol with and without 100-fold molar excess of nonradiolabeled competitor (diethylstilbestrol) to displace radiolabeled steroid from the low-capacity specific binding sites (ER), but not from the high-capacity nonspecific binding sites in the cytosol. The unbound steroid is removed by selective adsorption on DCC. 

The classic quantitative biochemical method for assaying steroid receptors in tumor tissue specimens is the multiple-point dextran-coated charcoal (DCC) titration assay. However, in comparison with the classic DCC assays, enzyme immunoassays are preferred as they cost less and are simpler, require less time, and can be performed using less tissue than DCC titration assays. 

The procedure of the DCC-assay followed the protocol of Lehmann Koolman... 

The DCC assay is dependent upon the capacity of charcoal to adsorb free hormone, whereas the addition of dextran limits the absorption of the hormone-receptor complex by the charcoal (K14). By using increasing concentrations of radiolabeled hormone and a concurrent range of unlabeled hormone competitors, titration of the receptor to a saturation end point occurs. 

Several groups have demonstrated excellent quantitative correlation regarding estrogen receptor analysis between the DCC assay and sucrose density-gradient methods (Al, C2, HI), although Chester et al. (C3) found the DCC assay for estrogen receptor to be more sensitive than the sucrose density-gradient analysis and this difference was more pronounced when molybdate was absent. 

The DCC assay is currently the most popular method for evaluating estrogen receptor and progesterone receptor because it is simple, inexpensive, sensitive, and accurate and because several samples can be processed simultaneously. This is the method that was in use in the Department of Clinical Chemistry at the Memorial Sloan-Kettering Cancer Center from 1973 through 1990. We have replaced this method with a quantitative immunochemical method (ElA) from the Abbott Laboratories (North Chicago, IL). 

In situ hybridization overcomes the problem of cellular localization, but it is difficult to relate the expression of a particular mRNA to the expression of the functional protein. Moreover, this method is difficult to carry out. Immunohistochemistry, on the other hand, is a relatively simple technique that overcomes these problems by identifying the precise cellular localization of the functional protein. This technique, using paraffin sections, provides information on the ER status of tumors very simply and rapidly. In addition, this approach is superior to frozen section immunohistochemistry, the dextran-coated charcoal assay (DCC) (see page 276), or the enzyme-linked immunosorbent assay (ELISA) for predicting the response to endocrine therapy. 

To each assay 405 pi cell homogenate were added and then incubated for 9 hours (+ 4°C). Afterwards aliquots were withdrawn (100 pi each), and 100 pi of DCC-suspension added and well shaken for 30 Particles of charcoal were removed in a short centrifugation step (2 ). 100 pi of the supernatants were pipetted into scintillation vials, 4 ml cocktail were added and the samples were counted. 

Several investigators have utilized fine needle aspirates for hormone receptor evaluation using an immunocytochemical method (ERICA Abbott Laboratories, North Chicago, IL) and compared the data with the quantitative values obtained by chemical methods [dextran-coated charcoal (DCC)] for both ERP and PRP. These data are shown in Table 1. There was a variation of 65 to 100% in concordance in the separate studies. The ERP immunocytochemical assay in fine needle aspirates was also compared with the immunochemical assay in biopsies with a concordance of 87% (Rl). The role of the immunocytochemical assay of receptors is primarily in patients in whom surgical biopsies cannot be obtained or fi om whom the surgical specimen is too small. 

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