Dextran-coated charcoal assay

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. 

Menendez-Botet, C. J., Stankievic, R., Schwartz, D., and Schwartz, M. K., Comparison of the quantitative measurement of human estrogen and progesterone receptor in tissue cytosol by the dextran-coated charcoal assay and the Abbott monoclonal enzyme immunoassay. Clin. Chem. (Winston-Salem, N.C.) 34(6), 1225 (1988). 

Adrenal Tumours The assay-method is entirely based on the Schwartz-Mann Kit. According to this method, cortisol is first extracted from the plasma using CH2C12 (methylene chloride). In the actual radioimmunoassay the cortisol present in the extract competes with Cortisol-H3 i.e., the radioactive tracer) for the common binding sites on transcortin, which is incidently not an antibody but a cortisol-binding protein. Now, the free cortisol is quantitatively removed by adsorption on dextran-coated charcoal from the one bound to the transcortin. Finally, the bound radioactivity (due to Cortisol-H3) is measured which is then employed to calculate exactly the amount of cortisol present in the sample by the help of a Standard Curve (or Calibration Curve). 

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

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. 

Fig. 1. Dose-response curve for amount of dextran-coated charcoal added versus percentage free and bound, adsorbed. These are theoretical curves drawn from generalizations derived from several assays. —, Curve showing binding of free ligand —, curve showing binding of antibody bound ligand. The exact amounts of charcoal giving a particular bound value vary with each assay (e.g., growth hormone assay differs from insulin assay). For significance of arrows A and B, see the section on selection of charcoal dose.

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. 

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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