Flow Cytometry

three most common applications of flow cytometry

1. Diagnosis and subclassification of leukemias and lymphomas

2. Detection of minimal residual disease before the overt relapse of acute leukemia

3. Additional information in morphologically challenging cases

how a flow cytometer works with respect to monoclonal antibodies, fluorescent dyes, excitation by a laser, and detection

Fluorescent dyes: how cellular sample is labeled, each one has a different wavelength

Monoclonal antibodies: labeled with fluorescent dyes, attach to different maturation stages and/or lineage

Excitation by a laser: laser detects of dyes

Detection: the dye indicates lineage and/or maturation stage

Hydrodynamic focusing

process by which cells suspended in sheath fluid pass in front of a light source one at a time

Gating

process of selection a population of interest as defined by one or more flow cytometric parameters

• process of drawing circles around the cell population that will be studied in greater detail

The three reasons why (with current technology) we choose to acquire all events by using gating

1. comprehensive analysis of all cells

2. internal positive and negative controls

3. detection of unexpected abnormal populations

Forward vs side scatter

Forward:

• Cell size

Side:

• Cytoplasm

Markers for Immature cells

• CD34

• CD117

• TDT

Markers for Granulocytic/monocytic cells

• CD34

• CD33

• CD15

• CD13

• CD14

Markers for Erythroid precursors

• CD71

• Glycophorin A

Markers for Megakaryocytes

• CD41

• CD42

• CD61

Markers for B cells

• CD19

• CD20

• Kappa light chain

• Lambda light chain

Markers for T cells

• CD3

• CD5

• CD7

• CD4

• CD8

location of granulocytes, monocytes, lymphocytes, erythroid precursors, and blasts on a cd45 v sidescatter scatterplot

Granulocytes: Long top area

Monocytes: right below and slightly right of granulocytes

Lymphocytes: below monocytes

Erythroid precursors: in the bottom left corner

Blasts: Right below Granulocytes (not show in picture)

location of granulocytes, monocytes, lymphocytes, and erythroid precursors on a sidescatter v forward scatter scatterplot

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Minimal Residual Disease is used to detect low incidence of leukemic (or other cells of interest).

• sensitivity of this methodology has dramatically improved therapy, aided in

  predicting recurrence, and provided a more accurate prognosis

• used in

  • Myeloma

  • B and T lymphoblastic leukemia/lymphoma

  • Acute myeloid leukemia

Paroxysmal Nocturnal Hemoglobinuria

• somatic mutation in PIG-A which is responsible for the synthesis of GPI anchors on most hematopoietic cells.

• loss of these GPI anchors results in the loss of GPI-linked complement inhibitors, CD55 and CD59 on red cells

• patients often suffer from severe complement driven intravascular hemolysis due to complement inhibitors

Flow cytometry

The preferred methodology for diagnosing PNH because it can detect the loss of GPI anchors across cell types in a background of transfused and/or normal cells

recommended age of flow cytometry specimens collected in EDTA, Sodium Heparin and body fluids

EDTA: 24 hour stability

Sodium: 48 hour stability

Body fluids: must be processed within hours from procurement

Why must you do flow cytometry quickly?

some cell populations, particularly highly proliferative cells, may be lost if analysis is not timely. In other words, resting/mature cells are selectively retained leading to a inaccurate assessment

Why is washing and gating required?

removal of debris through gating, washing, and staining with 7AAD (viability stain) are each important tools that a MLS can employ to determine the viability of a specimen and increase analytical resolution