Immunofluorescence Standards for Flow Cytometry

DESCRIPTION (provided by applicant): The number of receptor/effector molecules expressed by cells can provide important diagnostic, and/or prognostic information These quantitative measurements have been shown to be valuable in treating patients with sepsis, monitoring patients infected with Human Immunodeficiency Virus (HIV), identifying leukemias or lymphomas and in predicting disease occurrence in apparently healthy individuals Nevertheless, there are presently only a limited number of accepted and approved clinical quantitative immunofluorescence tests These limits are not because of shortcomings in instrumentation since current benchtop flow cytometers fulfill the basic requirements of linearity and stability On the other hand, the standardization procedures that are needed for relating fluorescence intensity measurements to the number of antibodies bound are not adequate for more widespread application of these tests The shortcomings in immunofluorescence standards might not be immediately apparent since several types of standards are available For example, there are beads with known numbers of molecules of soluble fluorochromes (MESF'), beads with known numbers of immunoglobulin molecules (Quantitative Indirect Immunofluorescence Intensity, QIFI) and beads with anti-mouse Ig antibodies having a specified antigen binding capacity (ABC) The QIFI beads can provide accurate and precise results, but only in indirect immunofluorescence protocols Because of fluorochrome variability, the use of MESF beads is effectively limited to less variable fluorochromes such as fluorescein or R-phycoerythrin (PE) However, these standards do not address the problem of determining the specific fluorescence of labeled antibodies, even for the limited cases of fluorescein and PE One strategy to solve that problem has been the preparation of antibodies with 1 PE IgG ratios That approach assumes identical fluorescence properties for the PE on standard beads and on antibodies but that assumption is not always correct The ABC beads could solve this problem but do not, most likely because the stated ABC values are incorrect Thus, the existing standards meet only some of the needs for quantitative immunfluorescence measurements The long range goal of the current studies is to develop materials and procedures comprising a comprehensive system of standardization for immunofluorescence measurements The standards will be based on traceable fluorescein standards from NIST The system will include procedures 1) to determine the relationship between fluorescence intensity and MESF, 2) to determine the specific fluorescence of antibodies labeled with any fluorochrome, and 3) to create standards that can be included in flow cytometdc analyses for direct comparison with cells of interest The Specific Aims of the current proposal include experiments to demonstrate that the preparation of the materials needed for these comprehensive standards is feasible.