At least two histochemical stains proved extremely useful for such quantitative image analyses. One was Hematoxylin & Eosin (H&E) and the other was Feulgen DNA stain. Hematoxylin was demonstrated to form a dye–metal complex with arginine-rich basic (cationic) nucleoproteins such as histones. Eosin dye is acidic in nature and tends to bind to more eosinophilic cellular structures (cytoplasm, collagen and muscle fibers) producing various shades of pink. The Feulgen reagent binds to DNA stoicimetrically by uncovering the free aldehyde groups in DNA during the acid hydrolysis process, which then reacts with the reagent via a Schiff-Base interaction to form a stable, bluish/purple colored compound that absorbs light at 560 nm. These techniques varied from manual tracing of cancer or benign nuclei with GraphPad-type tools (See Figure 1), which later evolved to software driven programs to measure size, shape, and texture of nuclei by this approach. .
- Diamond et al.in 1981 utilized a manual Graphpad software with a microscope to trace up to 300 malignant and benign nuclei from each CaP patient. The Prostate 3:321-332 (1982)
- Next, they compared nuclear size and shape in a set of prostate organ-confined CaP cases that had long-term follow-up and determined that they could distinguish those with a good prognosis from those with a poor prognosis (metastasis) with high accuracy (p < 0.005). Journal of Urology 139: 1080-1084, 1988; Journal of Urology 139: 1085-1090, 1988.
- Next, Dr. Donald Coffey’s laboratory and Dr. Mitchell Benson compared the use of flow cytometry (where the nuclei were labeled with acridine orange) to measure light scatter (forward and perpendicular) with the nuclear roundness factor performed on the same nuclei to assess tumor aggressiveness and heterogeneity of several well- to poorly-differentiated rat Dunning prostate tumor cell lines in 1984. The Prostate 5: 27-45, 1984.
- Later, commercially available hardware and software validated the clinical value of NRV measurements using a microscope. The images were analyzed with the DynaCell Motility Morphometry Measurement workstation (JAW Associates, Inc., Annapolis, MD, USA). Cancer, 70: 161-168, 1992.
Figure 1 illustrates the early history of nuclear morphometry at how the Johns Hopkins team showed the value of measuring it in Prostate Cancer.