• Document: Principles of Immunophenotyping
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Principles of Immunophenotyping % of Cell types? Immune activation? Changes based on health state? Moving from a heterogeneous population of blood cells to identifying the presence and proportion of different population! -  T cells, B cells, NK Cells, RBCs, monocytes, etc. ! ! -  Can be used clinically to get Complete Blood Counts (CBC)! -  White and red blood cell count, platelet. Proportions of cell types. ! -  Indications of disease state! -  Established clinical antibody panels can be used to provide diagnose, classify or monitor hematological diseases (leukemia) (More in class 7)! -  Can be used in research studies to interrogate different cell populations ! Categories of Cell Parameters: Cell Size/Complexity Michele Black Categories of Cell Parameters: Lineage Markers -  The main way that cells are placed into subsets or identified using FACS is by expression of antigens (usually membrane proteins) found on the cell. ! -  Antibodies directed against these antigens or markers, usually designated by CD or Cluster of Differentiation numbers are used to discriminate cells.! -  Lineage markers uniquely define different cell subsets! -  Often times cells are either + or – for a given maker! ! -  However, not all markers are exclusive to a given cell type and combining additional markers would be needed to make definitive cell distinctions. ! ! ! Categories of Cell Parameters: Lineage Markers Categories of Cell Parameters: Lineage Markers CD45-Leukocyte common antigen -  CD45 is a common marker of hematopoietic cells (absent from mature erythrocytes and platelets)! -  Use of CD45 together with side scatter can be used to distinguish different hematopoietic cells ! -  Use of just this one marker adds an additional parameter to distinguishing the different cell populations. ! Categories of Cell Parameters: Phenotypic or maturation Markers -  White blood cells will express markers that are often associated with their maturation or differentiation state (in addition to their lineage specific marker)! ! -  Cells may often express a continuum of the particular marker, with different populations displaying different levels of positivity. ! -  This type of marker may not always be constant for a given cell or population of cells and can change.! -  Changes in health status of the patient ! -  Removing cells from the body and placing them into culture! -  Supplying the cells with an external stimulus! -  Aberrant expression of maturation markers are often used as an indication of hematologic diseases. ! -  Markers of this type often function in cell metabolism, cell homing, antigen recognition or costimulatory functions. ! Categories of Cell Parameters: Phenotypic or maturation Markers Rev. Bras. Reumatol. vol.50 no.5 São Paulo Sept./Oct. 2010 Categories of Cell Parameters: Phenotypic or maturation Markers Appay et al., Cytometry 2008 Categories of Cell Parameters: Functional Markers -  In addition to markers expressed at the cell surface, leukocytes can also be distinguished by their expression of different functional proteins. ! -  Often these functional proteins are detected intracellularly under special FACS staining conditions. ! -  Examples:! -  Expression (or loss) of a certain cytokine functions! -  Expression of cytotoxic granules within the cell! !! ! Categories of Cell Parameters: Functional Markers CD57 TNFα -  So combining cell size, complexity, lineage markers, maturation markers, and now functional markers gives you a complex but detailed view about a particular cell population. ! Singlet Discrimination -  Data gating schemes often incorporate a gate to eliminate the influence of cell clumps on observed signals. ! -  This is most often done after data acquisition in FlowJo! -  This is based on the principle that on digital flow cytometers, the area and height for the same parameter should be nearly the same for a given cell. ! -

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