Review Eur J Pediatr (1992) 151 : 398-406
European Journalof
Pediatrics
9 Springer-Verlag1992
Flow cytometric immunophenotyping: principles and pitfalls M. Othmer and F. Zepp Laboratory for Immunology, Department of Paediatrics, University of Mainz, Langenbeckstrasse 1, W-6500 Mainz, Federal Republic of Germany Received July 10, 1991 / Accepted July 11, 1991
Abstract. Within the last decade flow cytometry (FCM) has become an integral part of basic immunological research. Elaboration of this technology has been intensively stimulated by a rapidly growing sophistication in monoclonal antibody technology and vice versa. At present numerous applications are established that allow an increasingly detailed insight into the immune system, however, automation still must be considered the "cinderella of the arts". Thus, transition of this powerful approach from a basic to a routine clinical procedure is much more difficult than expected. Sufficient usage of flow cytometers still requires some knowledge of physics and its technical applications. Moreover, several problems arise from the complexity of the biological systems investigated. Here we give a brief introduction to immunofluorescence and FCM followed by a discussion of six exemplary pitfalls that we hope will emphasise the general importance of methodological considerations. Key words: Monoclonal antibody - Immunofluorescence - Flow cytometry
Introduction In 1975 K6hler and Milstein described their epoch-making method for the production of monoclonal antibodies (moAb) [20]. With the help of these powerful tools a great variety of cell surface antigens were discovered and it became clear that the diversity of surface molecules was due to their extreme diversity of regulatory and functional properties. Unfortunately, this fascinating new view of the immune system not only gave rise to enOffprint requests to: F. Zepp Abbreviations: FCM = flow cytometry; Fc-receptor = recep-
tor for the fragment crystalline of the immunoglobulin molecule; FITC = fluorescein-isothiocyanate; FL = fluorescence; FSC = forward scatter; IF = immunofluorescence; Ig = immunoglobulin; moAb -- monoclonal antibody; PBMC = peripheral blood mononuclear cells; PE = phycoerythrin; SSC = side scatter; TCR = T-cell receptor
thusiasm but also to much confusion outside the community of experts. In many cases several moAbs were produced that all reacted with the same antigen. Part of the confusion was based on an incongruous nomenclature. To overcome these problems a series of international workshops were held that succeeded in putting the chaos in order [3, 19, 24, 32]. During the last decade another technique has substantially influenced immunology, namely flow cytometry (FCM). In combination with immunofluorescence (IF) this technique allows a detailed study of moAb-biuding patterns even at the level of single cells as well as a precise determination of the phenotype of an individual immune system. Due to this immense potential and its clinical relevance, an increasing number of physicians utilize these techniques in their daily work [29]. However, flowcytometric immunophenotyping is an extremely complex approach. In consequence, there still exists uncertainty among physicians concerning the interpretation of FCM results. Especially those who begin to use flow cytometers may soon become disappointed. Results that on first view indicate a pathological condition, may be distorted by technical problems, and usage of even more intricate approaches such as multi-colour IF may potentiate these problems. After an introduction to immunophenotyping we describe six methodological pitfalls that we hope will exemplify the importance of technical considerations. Principles of cellular immunofluorescence and flow cytometry - a quick course Cellular phenotyping requires the detection of gene products in the context of cells. These molecules may be found on the ceil surface, in the cytoplasm, or retated to intracellular organelles. Here we will restrict our considerations to molecules expressed on the cell-surface. To detect the molecules of interest an ideal reagent should be highly specific and free of contamination. Moreover, it should be available in the same quality over a long period of time. Obviously moAbs fulfil these requirements. So-called hybridoma cells serve as a stable source for these mono-specific antibody preparations
399 sheath
sample
plezo-elect[Ic crystal
LASER
PMT
FL2
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drop-oharglng signal
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European Journalof
Pediatrics
9 Springer-Verlag1992
Flow cytometric immunophenotyping: principles and pitfalls M. Othmer and F. Zepp Laboratory for Immunology, Department of Paediatrics, University of Mainz, Langenbeckstrasse 1, W-6500 Mainz, Federal Republic of Germany Received July 10, 1991 / Accepted July 11, 1991
Abstract. Within the last decade flow cytometry (FCM) has become an integral part of basic immunological research. Elaboration of this technology has been intensively stimulated by a rapidly growing sophistication in monoclonal antibody technology and vice versa. At present numerous applications are established that allow an increasingly detailed insight into the immune system, however, automation still must be considered the "cinderella of the arts". Thus, transition of this powerful approach from a basic to a routine clinical procedure is much more difficult than expected. Sufficient usage of flow cytometers still requires some knowledge of physics and its technical applications. Moreover, several problems arise from the complexity of the biological systems investigated. Here we give a brief introduction to immunofluorescence and FCM followed by a discussion of six exemplary pitfalls that we hope will emphasise the general importance of methodological considerations. Key words: Monoclonal antibody - Immunofluorescence - Flow cytometry
Introduction In 1975 K6hler and Milstein described their epoch-making method for the production of monoclonal antibodies (moAb) [20]. With the help of these powerful tools a great variety of cell surface antigens were discovered and it became clear that the diversity of surface molecules was due to their extreme diversity of regulatory and functional properties. Unfortunately, this fascinating new view of the immune system not only gave rise to enOffprint requests to: F. Zepp Abbreviations: FCM = flow cytometry; Fc-receptor = recep-
tor for the fragment crystalline of the immunoglobulin molecule; FITC = fluorescein-isothiocyanate; FL = fluorescence; FSC = forward scatter; IF = immunofluorescence; Ig = immunoglobulin; moAb -- monoclonal antibody; PBMC = peripheral blood mononuclear cells; PE = phycoerythrin; SSC = side scatter; TCR = T-cell receptor
thusiasm but also to much confusion outside the community of experts. In many cases several moAbs were produced that all reacted with the same antigen. Part of the confusion was based on an incongruous nomenclature. To overcome these problems a series of international workshops were held that succeeded in putting the chaos in order [3, 19, 24, 32]. During the last decade another technique has substantially influenced immunology, namely flow cytometry (FCM). In combination with immunofluorescence (IF) this technique allows a detailed study of moAb-biuding patterns even at the level of single cells as well as a precise determination of the phenotype of an individual immune system. Due to this immense potential and its clinical relevance, an increasing number of physicians utilize these techniques in their daily work [29]. However, flowcytometric immunophenotyping is an extremely complex approach. In consequence, there still exists uncertainty among physicians concerning the interpretation of FCM results. Especially those who begin to use flow cytometers may soon become disappointed. Results that on first view indicate a pathological condition, may be distorted by technical problems, and usage of even more intricate approaches such as multi-colour IF may potentiate these problems. After an introduction to immunophenotyping we describe six methodological pitfalls that we hope will exemplify the importance of technical considerations. Principles of cellular immunofluorescence and flow cytometry - a quick course Cellular phenotyping requires the detection of gene products in the context of cells. These molecules may be found on the ceil surface, in the cytoplasm, or retated to intracellular organelles. Here we will restrict our considerations to molecules expressed on the cell-surface. To detect the molecules of interest an ideal reagent should be highly specific and free of contamination. Moreover, it should be available in the same quality over a long period of time. Obviously moAbs fulfil these requirements. So-called hybridoma cells serve as a stable source for these mono-specific antibody preparations
399 sheath
sample
plezo-elect[Ic crystal
LASER
PMT
FL2
~ ~ ~ flow~ eh=am ber
drop-oharglng signal
dichroic mirror =ilters
'
lenses
m
~
~
