One problem with all counting and image analysis using sections is that cell numbers will vary according to the thickness of the section and the amount of the original volume of tissue contained in that section.
As we are comparing sections, we are assuming that sections that have been cut at 5 microns setting are indeed 5 microns. In essence we are comparing the number of cell counted in a volume of tissue.
Abercrombie in 1946 showed the fallacy of this assumption and introduced the "Abercrombie Correction Factor". This takes into account the size for example of nuclei. He showed that as thickness of sections increases so does the accuracy of the counts of actual cells.
Another problem in histology is the degree of shrinkage especially of soft tissues due to variations in times in solvents, in wax etc. as these may significantly alter the volume of tissue in sections cut at 5 microns even if we assume that they are all the same thickness.
One way to determine thickness is to use a substance embedded with the tissue and determine the relative section thickness by the optical density of this material.
While I realize that virtually no one will be doing this I feel that we are all somewhat complacent regarding the thickness of the section and that tissue processed at different times or by even slightly different methods are not totally comparable as to volume of tissue contained in sections and therefore numbers of cells, even if these sections were all at exactly the same thickness, a dangerous assumption.
While may of us are arrogant enough to feel that we can tell the difference between for example a 5 and a 4 micron section, I am not so sure that our often subjective judgment is appropriate when counting cells is sections.
From: firstname.lastname@example.org on behalf of ???
Sent: Fri 3/9/2007 7:24 AM
Subject: [Histonet] cellular infiltration score
Hello, everyone. I stained the monoclear cells(CD4,CD8,CD20 and CD68) in kidney tissue sections by immunohistochemistry. I want to know how to score the cellular infiltration. If can I set the score as follows: <100/mm3-normal, >200/mm3-mild, >300/mm3-moderate, ...... I am waiting for your kindly reply.
Nanjing University School of Medicine,
Research institute of Nephrology, Jinling Hospital,
Nanjing, China 210002
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