FLOW CYTOMETRY

• Flow cytometry


  Originally designed to measure physical properties of cells based on their ability to deflect light, evolved to include detection of fluorescent signals emitted by dyes bound to specific molecules or attached to proteins through monoclonal antibodies
• Flow cytometry


  • Ability to rapidly and simultaneously analyze multiple parameters in a large number of cells
  • Capability of identifying and quantifying rare-event cells in a heterogeneous cell population
• Flow cytometry is applied to analysis of cell lineage in acute leukemia, detection of clonality in lymphoid populations, discerning abnormal populations in chronic myeloid neoplasms, quantitating minimal residual disease, and monitoring immunodeficiency states
• Immunophenotypes frequently correlate with specific cytogenetic or molecular abnormalities
• Current diagnostic algorithms integrate morphologic, immunophenotypic, and genotypic information, emphasizing the central role of flow cytometry in a hematopathology laboratory
• Life of a flow cytometric specimen

  1. Specimen processing

  2. Flow cytometric instrumentation and analysis

  3. Immunophenotypic features of hematopoietic disorders
• Specimen processing


  • Minimal sample preparation required for specimens collected into a tube or container with an anticoagulant (peripheral blood, bone marrow)
  • Tissue fragments mechanically dissociated to yield a cell suspension (solid tissue specimens)
• Red blood cell lysis


  To obtain a pure population of nucleated cells
• Cellularity and viability assessment


  Before sample staining
• Sample staining


  1. With a cocktail of fluorochrome-conjugated monoclonal antibodies

  2. Additional fixation and permeabilization step for intracytoplasmic markers
• Multicolor/multiparameter flow cytometry


  Simultaneous analysis of multiple markers, facilitates visualization of antigen expression and maturation patterns, allows analysis of complex specimens using fewer tubes and lower total number of cells
• No consensus on standardized panel of antibodies for routine flow cytometric evaluation, comprehensive approach with multiple markers for myeloid and lymphoid lineage recommended
• Monoclonal antibodies


  Significant discovery that led to advancement of flow cytometry, produced and tested for lineage specificity, categorized through workshops on human leukocyte differentiation antigens
• Hematolymphoid antigens commonly used in clinical flow cytometry

  • CD1a
  • CD2
  • CD3
  • CD4
  • CD5
  • CD7
  • CD8
  • CD10
  • CD11b
  • CD13
  • CD14
  • CD15
  • CD16
  • CD18
  • CD19
  • CD20
  • CD22
  • CD31
  • CD33
  • CD34
  • CD36
  • CD38
  • CD41
  • CD42b
  • CD45
  • CD56
  • CD61
  • CD62P
  • CD63
  • CD64
  • CD71
  • CD79a
  • CD117
• Flow cytometer


  Consists of fluidics, a light source (laser), a detection system, and a computer
• Hydrodynamic focusing


  Injecting a cell suspension into a stream of sheath fluid to create a central core of individually aligned cells
• Laser


  Composed of a tube filled with gas (argon or helium-neon) and a power supply, produces a narrow coherent beam of light to illuminate individual cells
• Fluorescence detection


  Fluorochromes attached to antibodies absorb laser light, electrons are raised to a higher energy state, return to ground state emits light of specific wavelength detected by photodetectors
• Forward scatter (FS) and side scatter (SS)

  FS is proportional to particle volume/size, SS reflects surface complexity and internal structures
• Forward scatter (FS or FSC)


  Proportional to particle volume or size
• Side scatter (SS or SSC)

  Reflects surface complexity and internal structures such as granules and vacuoles
• FS, SS, and fluorescence are displayed simultaneously on the instrument screen and registered by the computer
• Gating


  An electronic boundary used by an operator to delineate cell clusters. Gating is a process of selecting a population of interest as defined by one or more flow cytometric parameters.
• Live gate


  Gating applied at the time of data acquisition
• Ungated data collection


  All events detected by the flow cytometer are recorded, allowing comprehensive testing and retention of positive and negative internal controls, and detection of unexpected abnormal populations
• Analysis of Flow Cytometric Data

  Based on an inspection of visual patterns, starting with scanning for abnormal populations and then analysing the antigenic properties of abnormal cells
• CD45/SS display


  Provides information on the relative proportion of specific cell populations, and is useful for detection of blasts
• Pluripotent stem cells


  Morphologically unrecognizable, defined by their functional and antigenic characteristics, usually express CD34, CD117, CD38, and HLA-DR
• Markers expressed by pluripotent stem cells

  • CD34
  • CD117
  • CD38
  • HLA-DR
• Granulocytic lineage maturation


  Myeloblast (CD34+, CD38+, HLA-DR+, CD117+, CD13+, CD33+) -> Promyelocyte (lose CD34, HLA-DR) -> Myelocyte (acquire CD11b, lose CD13) -> Band stage (acquire CD16, increase CD13)
• Monocytic lineage maturation


  Increase CD13, CD33, CD11b -> Acquire CD15, CD14
• Erythroid lineage maturation


  Pronormoblast (CD71+) -> Basophilic normoblast (acquire CD235a) -> Reticulocytes (downregulate CD71)
• Megakaryocytic lineage maturation


  Early megakaryoblasts (CD41+, CD61+) -> Megakaryocytes (acquire CD42, CD62P, CD63)
• B cell lineage maturation

  Lymphoid progenitors (CD34+, TdT+, HLA-DR+) -> Early B cells (CD19+, cytCD22+, cytCD79+) -> Mature B cells (CD20+, lose CD10)
• T cell lineage maturation

  Lymphoid progenitors (CD34+, TdT+) -> Early T cells (CD2+, CD7+, cytCD3+) -> Immature T cells (CD1a+, CD5+, CD4+CD8+) -> Mature T cells (surface CD3+, CD4+ or CD8+)
• Myeloid neoplasms


  • Flow cytometry used for initial diagnosis, follow-up, and prognostication
  • Blasts characterized by low-density CD45 expression
• AML with t(8;21)


  • Immature myeloid immunophenotype with high CD34, coexpression of CD19 and other B cell markers, myeloid antigens CD13, myeloperoxidase, weak CD33, asynchronous CD34/CD15
• AML with inv(16) or t(16;16)

  • Immature cells with CD34, CD117, TdT, subpopulations of maturing cells with monocytic (CD14, CD11b, CD4) and granulocytic (CD15) markers, aberrant CD2 on monocytic population
• Acute promyelocytic leukemia with PML/RARA


  • High side scatter, lack of CD34, HLA-DR, CD11a/b/18, homogeneous strong CD33, myeloperoxidase, CD117, variable CD13, CD15
• AML with t(9;11)


  • Immature myeloid cells with monocytic features, CD34+, CD117+, CD11b+, CD14+, CD64+