Perspectives Isotype Controls in the Analysis of Lymphocytes and CD341 Stem and Progenitor Cells by Flow Cytometry—Time to Let Go!

The isotype control has long been considered a useful part of both microscopic and flow cytometric immunologic assays, and, consequently, is still routinely used in clinical laboratories. In flow cytometry, the isotype control has traditionally been used to distinguish between fluorescent positive and fluorescent negative cell populations. Additionally, it has been used to estimate the number of cells reacting non-specifically with the target antibody under investigation. Over the past 10 years, the widespread use of directly conjugated monoclonal antibodies (mAb) and multiparameter analysis in clinical flow cytometry has reduced the need for a separate ‘‘negative control’’ tube. This tendency has materialized in guidelines recommending that the isotype control is irrelevant and potentially misleading in commonly used flow cytometric assays (3, 14). This perspective summarizes the rationale for omitting isotype control staining for surface membrane marker analysis, focusing on lymphocyte and CD341 hematopoietic stem/progenitor cell analyses. Consequently, these points also pertain to the immunophenotyping of leukemia/lymphoma samples (14). Prior to the development of directly conjugated mAb, pre-immune sera were used in microscopic and flow cytometric studies to estimate the level of ‘‘non-specific staining’’ of the specific antibody to its target cell, i.e., the binding of that specific antibody by mechanisms other than specific antibody-to-antigen interactions. Such nonspecific binding is usually, but not exclusively, mediated by receptors that bind the Fc portion of the various immunoglobulin subclasses (19). In flow cytometry, an estimate of the number of cells reacting non-specifically is typically determined by placing a cursor at the foot of the isotype control negative population on a fluorescence (FL) histogram such that less than 2% of events are assessed as positive. This cursor position is maintained to determine the ‘‘percent positive cells’’ in the experimental stainings. Currently, many isotype controls are produced by fusion of antibody producing cells with a myeloma-derived cell line to form a hybridoma. By the very nature of mAb production, antibodies produced by hybridomas will differ structurally from each other, even within the same immunoglobulin subclass or isotype. Thus mAb that ‘‘specifically’’ bind to the same antigen on the cells under study might each additionally bind ‘‘non-specifically’’ to other leukocytes, platelets, etc. in an unpredictable manner. Other issues to consider in the use of monoclonal isotypes include differences in protein concentration and FL to protein (F/P) ratio between test antibody and isotype control. Different manufacturers use different protocols to produce, purify and chemically conjugate antibodies with a variety of fluorochromes which almost certainly impact the reliability with which experimental and isotype control mAb can be used to distinguish specific from nonspecific binding. A compounding problem is that in a panel with several surface markers each would need their own isotype control matched for the above criteria. This is rarely done in the clinical laboratory.