title: Detection of intracellular Factor VIII protein in peripheral blood mononuclear cells by flow cytometry.
abstract: Flow cytometry is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. Detection of specific host proteins for diagnosis predominantly uses quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based detection assay for Factor VIII protein in peripheral blood mononuclear cells (PBMCs). An indirect intracellular staining (ICS) method was standardized using monoclonal antibodies to different domains of human Factor VIII protein. The FVIII protein expression level was estimated by calculating the mean and median fluorescence intensities (MFI) values for each monoclonal antibody. ICS staining of transiently transfected cell lines supported the method's specificity. Intracellular FVIII protein expression was also detected by the monoclonal antibodies used in the study in PBMCs of five blood donors. In summary, our data suggest that intracellular FVIII detection in PBMCs of hemophilia A patients can be a rapid and reliable method to detect intracellular FVIII levels.
authors.affiliation: Laboratory of Hemostasis, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, 29 Lincoln Drive, Bethesda, MD 20892, USA. gouri.pandey@fda.hhs.gov
authors.forename: Gouri Shankar
authors.initials: GS
authors.lastname: Pandey
authors.forename: Sandra C
authors.initials: SC
authors.lastname: Tseng
authors.forename: Tom E
authors.initials: TE
authors.lastname: Howard
authors.forename: Zuben E
authors.initials: ZE
authors.lastname: Sauna
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D005169:Factor VIII / Q000096:biosynthesis* / Q000302:isolation & purification; D005434:Flow Cytometry; D015870:Gene Expression; D057809:HEK293 Cells; D006467:Hemophilia A / Q000175:diagnosis* / Q000276:immunology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D017434:Protein Structure, Tertiary
chemical_list: D000911:Antibodies, Monoclonal; C078147:F8 protein, human; D005169:Factor VIII
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