The principles for the computer-assisted image analyses of immunostained target cells are outlined in Figure 1. Stromal cell structures stained with antibodies to CD3, CD8, and CD68 were quantified using ImageJ, a Java image processing application downloaded free of charge from the website of the National Institutes of Health (NIH).23 The computer algorithms for the image analyses were developed according to previously described techniques.24,25 An outline of the measured structures was superimposed on the original photograph to permit manual validation. For the quantification of T lymphocytes, intact lymphocytic cells were counted separately from non-intact cell structures histopathologically identified as apoptotic cell fragments. All photographs and cell counts were controlled by an experienced pathologist and if necessary a second senior pathologist was consulted.
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