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BioMed Research International
Volume 2013 (2013), Article ID 793502, 8 pages
http://dx.doi.org/10.1155/2013/793502
Research Article

Detection of Intracellular Factor VIII Protein in Peripheral Blood Mononuclear Cells by Flow Cytometry

1Laboratory of Hemostasis, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, 29 Lincoln Drive, Bethesda, MD 20892, USA
2Department of Pathology and Laboratory Medicine, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, CA 90073, USA

Received 30 November 2012; Accepted 28 January 2013

Academic Editor: Akanchha Kesari

Copyright © 2013 Gouri Shankar Pandey et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.