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American Journal of Pathology, Vol 120, 263-275, Copyright © 1985 by American Society for Investigative Pathology

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Ultrastructural morphometric analysis of normal human lymphocytes stimulated in vitro with mitogens and antigens

CM Payne, MJ Hicks and A Kim

In the present study the authors attempt to experimentally produce the characteristic cerebriform appearance of Sezary cells from normal cells by stimulation in vitro with a variety of mitogens and antigens. Lymphocytes were stimulated with the mitogens phytohemagglutinin (PHA), concanavalin A, and pokeweed mitogen and the antigens spherulin, Candida, and herpes simplex virus Type I. In vitro blast transformation was measured in tritiated thymidine uptake microculture systems. Nuclear contours were morphometrically analyzed with the use of a computerized planimeter. An exhaustive search for Sezary-type cells did not reveal any nuclear profiles that were highly convoluted. The lowest form factor value obtained from the stimulated cultures was 0.14. This value represents 14% of a circle. All stimulated cultures analyzed morphometrically showed significant blast transformation. There was no correlation between the degree of stimulation and the nuclear contour values. An analysis of mean form factor values from ten separate lymphoid populations revealed that neither mitogens nor antigens had any effect on increasing the nuclear irregularity of lymphoid cells. One PHA-stimulated culture, in fact, had less irregular nuclear contours than the 3-day unstimulated control cells. The difference in these means was statistically significant (P less than 0.05). In addition, morphometric analysis of 73 published electron micrographs of human lymphoid cells from a variety of clinical and experimental conditions showing irregular nuclear contours revealed that only 5% had nuclei that were more irregular than the most convoluted nuclei analyzed from lymphocytes stimulated in vitro in the present study. The authors have shown that mitogen- and antigen-stimulated cells do not display the highly irregular nuclear outlines commonly seen in the Sezary syndrome and mycosis fungoides and emphasize the need for more objective analysis of Sezary-like nuclear profiles to determine their specificity.