Flow Cytometric Immunophenotyping of Leukocytes and Neutrophils Subsets in Bone Marrow, Peripheral Blood and Liver Tissue

The analysis of cellular constituents in peripheral blood and bone marrow plays a crucial role in detecting hematological diseases and abnormalities in blood cell profiles. Traditional methods for disease identification, such as morphological, phenotypic, and genotypic characterization, are laborious, expensive, and often lack the ability to provide multiparametric characteristics from individual specimens. Flow cytometry-based immunophenotyping has emerged as a gold standard and highly sensitive technique for cell identification based on surface antigens or markers. By utilizing specific antibodies tagged with different fluorochromes, this method allows for the simultaneous analysis of a wide spectrum of over 48 antigens on a single cell. As a result, numerous novel cell populations with distinct characteristics, including immature and mature subsets, have been identified based on the expression of specific surface markers or their combinations. In this chapter, we present a comprehensive overview of the basic processing of blood and bone marrow samples, along with the staining method for immunophenotyping. We will primarily concentrate on examining the myeloid and lymphoid compartments in peripheral blood. To accomplish this, we utilize enrichment and purification techniques specifically designed for leukocyte and neutrophil subsets. These particular cell types are known for their short lifespan and sensitivity, making it crucial to take necessary precautions during the processing to prevent any potential activation or cell death. Furthermore, we thoroughly discuss and implement essential measures to ensure the integrity of the samples and minimize any undesirable effects associated with processing. By addressing these concerns, we can reliably analyze and interpret the data obtained from peripheral blood’s myeloid and lymphoid compartments.