Quantitative fluorescence flow cytometry: a comparison of the three techniques for direct and indirect immunofluorescence.
Three types of microbead calibrators available for quantitative fluorescence flow cytometry have been studied in parallel using a variety of monoclonal antibodies (MoAbs). The QIFI kit is designed for indirect immunofluorescence (IF), and both the Quantum Simply Cellular (QSC) assay and the Quanti-BRITE assay are designed for direct IF. Because of the different nature of the respective ligands, epitopes on cells versus F'ab-portions on QSC beads, large differences in titration curves for a large number of CD MoAbs were noted between QSC beads and cells. Use of the QSC assay and fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugates of the same CD reagent revealed substantially different numbers of cellular binding sites. Numbers of cellular binding sites as determined by direct IF using the Quanti-BRITE assay and by indirect IF using the QIFI kit were similar. We also found that erythrocyte (RBC)-lysing reagents cause varying and sometimes substantial reduction in the fluorescence intensity (FI) of cells stained directly with CD34 MoAb conjugates, but the RBC-lysing reagents had no effect on the FI of cells stained indirectly with the same CD34 MoAbs. This report defines a number of variables critical for standardized quantitative flow cytometry. We conclude that the choice of calibrators, fluorochrome conjugates, staining methods, and modes of sample processing can effect the determination of cellular binding sites to MoAbs. Direct immunofluorescence using the Quanti-BRITE assay and indirect IF using the QIFI kit appear to yield comparable results for the standardized determination of numbers of cellular binding sites to MoAbs.
Abteilung Innere Medizin, Hämatologie-Onkologie, Charite, Humboldt-Universität, Berlin, Germany.
S
S
Serke
A
A
van Lessen
D
D
Huhn
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D018952:Antigens, CD34 / Q000032:analysis*; D001772:Blood Cell Count / Q000295:instrumentation / Q000379:methods*; D017252:CD3 Complex / Q000032:analysis; D015704:CD4 Antigens / Q000032:analysis; D000069585:Filgrastim; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D019085:Fluorescent Antibody Technique, Direct; D019084:Fluorescent Antibody Technique, Indirect; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D019650:Hematopoietic Stem Cell Mobilization; D006461:Hemolysis; D006801:Humans; D017493:Leukocyte Common Antigens / Q000032:analysis; D051379:Mice; D008863:Microspheres; D011817:Rabbits; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins; D012015:Reference Standards; D012996:Solutions / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D018952:Antigens, CD34; D017252:CD3 Complex; D015704:CD4 Antigens; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins; D012996:Solutions; D016179:Granulocyte Colony-Stimulating Factor; D017493:Leukocyte Common Antigens; D000069585:Filgrastim