Sect 6 - Flow Cytometry

Flow Cytometry

method of cell analysis

through use of fluidics, optics, and electronics, the flow cytometer gathers data on various aspects of a cell, including cell size and internal complexity

with addition of fluorescent antibody dyes, cell markers can also be ID

1. Fluidics

2. Optics

3. Electronics

3 major components of Flow Cytometry

Fluidics

carries the sample to the flow cell for analysis

Optics

laser light excitation sources, filters, and lenses to collect light

Electronics

gather data from light sources into a photoconductor and then translate that signal into a digital form for analysis

cell size

granularity

antigens present (bound, fluorescent Abs)

Flow cytometers differentiate cells based on (3):

integration point

Cells suspended in saline flow through a chamber and are measured at the

Sheath Fluid

fluid that surround the sample and provides a physiological barrier for the sample

Laminar Fluid

sample and sheath fluids are kept at different pressures to keep the fluids separated

Hydrodynamic Focusing

the process of narrowing the sample fluid using laminar flow so that the cells can pass single-file through the interrogation point

sample fluid column narrowed (controlled by pressures of the fluid congrol)

the pressures of the fluid

What controls the diameter of the sample fluid?

string of beads

As cells pass through the interrogation point, the resemble

Interrogation Point

the point at which the laser meets the sample fluid

light photons scatter at interrogation point and converted into electrical signal

How are light particles converted to collect data?

photodetector

device used to measure or indicate the presence of light

amplifies electrical signal to be read by the electronics system as an electrical pulse

direct proportional

relationship between number of light particles generated and height of voltage pulse in flow cytometry

Forward Light Scatter (FSC)

light that scatters in the forward direction

reflects the SIZE of the cells

monocytes & neutrophils

Which cells make the most FSC?

**hint: larger cells

Side Light Scatter (SSC)

light that scatters at a 90° angle

reflects GRANULARITY, or internal complexity, of the cell

granulocytes

Which cells have the highest SSC?

lymphocytes

Which cells have the lowest SSC?

populations

FSC and SSC data are used together to create

Principle of fluorescence

Laser light excites fluorescent molecules used as labels (fluorochromes) and the exciting causes fluorochromes to emit light at specific wavelength

Fluorochromes

Labels that are attached to cell antigens using fluorescently tagged antibodies

propidium iodine

used for visibility staining

photomultipler

light emitted by fluorchormes are detected by

true

T or F:

The number of lasers determine the number of fluorochromes that can be used

Compensation

the correction between the excitation wavelength and the detection wavelength

can be done manually by adjusting settings or using a mathematical correction

Histogram

a graph used to differentiate populations of cells

can also use dot plts (provide 2D way to view data

some fluorochromes have the same excitation wavelength but a unique detection wavelength

Why must compensation occur?

Gating

ensures that populations are as pure as possible

- allows for immunophenotyping of cells

immunophenotyping

the detection of Ag that are being expressed on the cell surfaces using detection antibodies

CD markers

cluster of differentiation markers

ags on cell surfaces that can be tagged with abs

CD45

ab that is present on ALL leukocytes

used against SSC to separate populations further

Lymphocyte antigens

B-lymphocyte markers

NK markers

T-large granular

lymphocyte markers

Monocyte/Granulocyte antigens

evaluated for normal cell markers

Blasts antigens

evaluated for all cell markers to determine what type of blasts they are

True, dx Non-Hodgkin's Lymphoma

T or F:

Flow Cytometry CANNOT diagnose Hodgkin's Lymphoma

lymphs look normal

Why can't flow cytometry diagnose Hodgkin's Lymphoma =

CD45

all leukocytes

CD3

T-lymphocytes

CD4

Helper T-lymphocytes (CD3+)

CD8

Cytotoxic T-lymphocytes (CD3+)

CD56

Natural Killer Cells (CD3-)

CD19 & CD20

B-lymphocytes

CD14

Monocytes

CD34

Blasts. Stem cells

CD117

Myeloblasts

CD55

Decay Accelerating Factor (DAF)

CD59

Membrane Inhibitor of Reactive Lysis (MIRL)

Surface Kappa:Lambda

other markers expressed as ratio on B-lymphocytes

looked at against CD19 to confirm they are B cells

0.8 - 2.0

normal Kappa:Lambda ratio

B-lymphs with no K:L chains ("baldies")

Kappa:Lambda ratio

neoplastic process (NHL, CLL/SLL)

Kappa:Lambda ratio >2.0

CD19, CD20, surface Kappa, surface Lambda

What 4 antibodies are expressed on normal, mature B-lymphs?

hematogones

normal immature B-lymphs

2:1

What is a normal CD4:CD8 ratio?

AIDS crisis patient (can be as low as 0.1)

What does a low CD4:CD8 ratio indicate?

CD34+

used to determine the success of a stem cell harvest

CD55, CD59, CD14, CD24

PNH markers

neg for CD3 & CD4

Read the quadrants

CD3 pos

CD4 neg

Read the quadrants

pos for CD3 & CD4

Read the quadrants

CD3 neg

CD4 pos

Read the quadrants