From: Lucille H Kimura (lucille_h.kimura@tamc.chcs.amedd.army.mil)
Date: Mon Apr 13 1998 - 22:56:56 EST
Thanks to Mario Roederer, Dave Coder and others who have taken the time to organize and put in writing some of the critical issues concerning the use of isotype controls. As much as I feel we're wasting money running isotype controls for peripheral blood lymphocyte immunophenotyping of immunodeficient patient samples, there are many times that we are forced to depend upon isotype controls to help us interpret results: 1) Many of us have had to analyze cell lines which have higher autofluorescence and isotype control fluorescence than lymphocytes. The CAP leukemia/lymphoma proficiency survey samples are cell lines, which make interpretation difficult because any positive staining for an antigen appears as one peak, with no negative population for that antigen to help define a negative region. We are required to respond to each antigen analyzed as negative, dimly positive, moderately positive or strongly positive, depending upon the degree of overlap or intensity of the positive peak compared to our isotype control. As such, our interpretations (and misinterpretations) are unfortunately highly dependent upon the nature of the isotype control. 2) Treatment of cells for cytoplasmic antigen detection results in overall increased levels of fluorescence in both negative and positive cells. Isotype controls have been helpful with leukemic samples stained for cytoplasmic antigens but often confuse the interpretation of cytokine analyses, especially with stimulated cells. As biological materials are difficult to standardize, it appears that we must continue to practice the art of flow cytometry with caution and hope that one day someone will figure out a way to block all 'nonspecific' binding and make isotype controls a thing of the past. I'm not holding my breath.
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