Principles of Immunophenotyping

Transcription

1 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

2 Cell Size/Complexity Michele Black

3 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.

4 Lineage Markers

5 Lineage Markers

6 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.

7 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.

8 Phenotypic or maturation Markers Rev. Bras. Reumatol. vol.50 no.5 São Paulo Sept./Oct. 2010

9 Phenotypic or maturation Markers Appay et al., Cytometry 2008

10 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

11 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.

12 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. - So if the data is graphed it should look like a diagonal line. - Cell clumps or doublets will have a different value because there is a slight difference in the relationship between the cells size and the signal generated for that cell.

13 A Dump Channel - The use of a dump channel or dump gate in your flow panel is intended to remove unwanted cells from your analysis - But this is done without any manipulation to the cells prior to your analysis, preserving their original integrity and the accuracy of the analysis. - This technique also eliminates any contribution of non-specific binding of your antibodies of interest to the undesired cell population. - However, you must be certain the marker that you are dumping is not expressed by any of your cells of interest, regardless of the activation or differentiation state. - The advantage of the dump channel is that several lineage markers can be combined in a single flourochrome color because there is no desire to distinguish between them. - Your population of interest to gate on would then be the cells that are negative for that chosen flourochrome. - Example: The intent of the panel is to focus on only T cells - Dump Channel: - CD14-Moncytes - CD16-Neutrophils/Moncytes - CD56-NK Cells

14 A Viability Stain - The use of a viability dye in your flow panel is intended to remove unwanted dead cells from your analysis - Dead cells have a higher autofluorescence, therefore eliminating them from your analysis will allow more accurate gating. - Dead cells will also non-specifically bind antibodies and can give false positives. Dead cells

15 A Viability Stain Various viability stains are now available for use that use different lasers and channels. Nucleic Acid Dyes: - Propidium Iodine (PI): - Membrane impermeable dye that is excluded from viable cells. - Intercalates with DNA - Excited by 488nm laser and emits at 617nm. - 7AAD (7-Aminoactinomycin D) - Membrane impermeable dye that is excluded from viable cells. - Intercalates with DNA - Excited by 488nm laser and detected with 650nm long-pass filter - Neither of these dyes can be used if staining for intracellular proteins is being performed and cell fixation with these dyes is difficult. - Amine reactive dyes are a new class of viable dyes that can be incorporated with intracellular staining and cell fixation. - These new dyes are available in a variety of colors are are now the most common viability stains. - The dye binds only on the cell surface on viable cells but also binds internally in dead cell, producing a stronger signal in dead cells.

16 T cell Gating Hierarchy

17 Regulatory CD4+ T-cell Gating Strategy SSC-A <PE-A>: CD127 <Alexa647-A>: FoxP3 FSC-H <PE-Texas Red-A>: CD3 <Aqua-A>: Viability