Quantification of immunohistochemistry--issues concerning methods, utility and semiquantitative assessment II.

Immunohistochemistry is entering its fourth decade of use on formalin-fixed paraffin-embedded tissues. Over this period the method has evolved to become a major part of the practice of diagnostic surgical pathology worldwide. From the beginning immunohistochemistry has been adapted to provide a range of markers of cell lineage and tissue type, with particular application to the diagnosis and classification of tumours. In this modality immunohistochemical methods were employed simply as 'special stains', the results of which were evaluated qualitatively by the pathologist, as for any other stain. More recently, attention has shifted to the demonstration of prognostic markers in tumour cells, driven by the advent of molecular biology and the discovery of numerous regulatory molecules, coupled with manufacture of the corresponding specific antibodies. Immunohistochemistry has rapidly adapted to this new use, but in so doing the demand for quantification has become paramount; it is no longer enough that the 'stain' is there; rather it is a question of 'How much is there?'. This review explores the limitations of immunohistochemistry when employed in a semiquantitative mode, and explores the possibility of fulfilling the full potential of immunohistochemistry, as a true quantitative immunoassay applied in a tissue section environment.

Low-cost two-dimensional gel densitometry.

A major obstacle to full utilization of the powerful technique of two-dimensional (2-D) gel electrophoresis is the expense and complexity of quantifying the results. Using an analog-to-digital converter already present in the widely available Commodore 64 or Commodore 128 microcomputer, we have developed a 2-D gel densitometer (GELSCAN) which adds only $20.00 to the cost of the Commodore system (currently around $700.00). The system is designed to work with autoradiograms of 2-D gels. Spots of interest are identified visually and then positioned manually over a light source. A pinhole photoelectric sensor mounted in a hand-held, Plexiglas holder, or "mouse," is briefly rubbed over each spot. Maximum density of the spot is determined and its value is converted to counts per minute via an internal calibration curve which corrects for the nonlinear response of film to radiation. Local spot backgrounds can be subtracted and values can be normalized between gels to adjust for variation in amount of radioactivity applied or in exposure time. Reproducibility is excellent and the technique has some practical as well as theoretical advantages over other more complicated approaches to 2-D gel densitometry. In addition, the GELSCAN system can also be used for scanning individual bands in 1-D gels, quantitation of "dot-blot" autoradiograms and other tasks involving transmission densitometry.

Small cell lung cancer: radionuclide bone scans for assessment of tumor extent and response.

Radionuclide bone scans were performed before and during combination chemotherapy in 119 systematically staged patients with small cell carcinoma of the lung. Before therapy, 49 patients (41%) had positive scans. Scan positivity was significantly associated with the presence of metastatic tumor in the bone marrow, positive skeletal radiographs, and elevated serum alkaline phosphatase levels. Nonosseous distant metastases were significantly more likely to be detected as the number of areas of focal abnormalities on bone scan increased. The survival of patients with documented distant metastases in bone and nonosseous sites was significantly inferior to the survival of patients with limited disease, isolated osseous extensive disease, and extensive disease occurring only in nonbony sites. Of 36 patients with initially abnormal scans and tumor regression documented by other methods, scan findings improved in 24 (67%). In 26 (36%) of 72 scans in patients demonstrating disease progression in extraosseous sites, new areas of increased radionuclide uptake appeared. Improvement or worsening in follow-up scans was associated with nonbony tumor response or progression, respectively, 70% of the time. Serial bone scans provide reasonably accurate staging and prognostic information in patients with small cell lung cancer, although they are probably not sufficiently reliable to be used as the sole parameter in therapeutic decision-making.