FLOW CYTOMETRY

?What is flow cytometry

Flow cytometry is a method of measuring multiple physical

.and chemical characteristics of particles by optical means

Peripheral blood, Bone marrow cells

Bacteria
Yeast
OPTICS (FSC)

Forward Angle Light Scatter

Laser

FALS Sensor
Optics
Side Scatter Channel (SSC)

 The amount of light scattered at right angle to

the incident light beam depends on the internal
complexity of the particle, this known as wide angle or
Side Scatter (SSC) , side scatter detected at 90, to the
laser beam.
)SSC
O ptics (

90 Degree Light Scatter

Laser

FALS Sensor

90LS Sensor
OPTICS
PROPERTIES OF FSC& SSC
PROPERTIES OF FSC& SSC

 As the cell passes through the laser beam, light is

scattered in all directions and that scattered in the
forward direction is proportional to the square of the
radius of a sphere, and so to the size of the cell or
particle.

 The cells may be labeled with fluorochrome-linked

antibodies or stained with fluorescent membrane,
cytoplasmic or nuclear dye.
?What can a Flow Cytometer (FCM) tell us about a cell

 Its relative size (Forward Scatter—FSC)

 Its relative granularity or internal complexity (Side

Scatter—SSC)

 Its relative fluorescence intensity (FL1, FL2, FL3, FL4)

Optics - Fluorescence Channels

 Any fluorescent molecule present in or on the particle

will absorb energy from the laser light and release the
absorbed energy at longer wave length, the emitted
light is collected by lenses and detectors, emitted
fluorescence intensity is proportional to the amount of
fluorescent compound on the particle.
 Fluorescence Detectors

Laser

FALS Sensor

Freq
Fluorescence

Fluorescence detector
(PMT3, PMT4 etc.)
Optical Design

PMT 5

PMT 4

Sample
PMT 3
Dichroic
Flow cell Filters
PMT 2

Scatter PMT 1
Laser
Sensor
Band pass
Filters
Electronics

 The scattered light from particles passing the laser

light is converted to digital values that stored in the
computer for analysis.
Electronics
FLOW CYTOMETRY

 Gating.
 Gating is in essence electronic window that sets upper
and lower limits on the type and amount of material
that passes through.
 It is used to separate a sub-population from
heterogeneous population.
 It permits very specific questions to be asked about a
particular population.
:Gating
.Fluorescence and Fluorochrome

 The coupling of monoclonal antibodies with fluorescent dyes

is necessary for recognition and enumeration by flow
cytometer.
 Each fluorochrome possesses a distinctive spectral pattern of
absorption (excitation) and emission.
 The property of fluorescence is that, the fluorochromes
present on the cell absorb the laser light and re-emit the light
at a lower energy and longer wave length.
 The most popular fluorochrome used in immuno-fluorescent
is fluorescein isothiocyanate (FITC), which has an absorption
maximum between 450 and 550 nm.
.Fluorescence and Fluorochrome

 FITC emits light detected in FL1 on the FCM.

 Another example of an excellent combination is Phycoerythrin
(PE) as a second fluorochrome since it can absorb light at
higher wave lengths than the FITC.
 PE emits light can be detected in FL2 on the FCM
APPLICATION

Applications in Clinical Laboratories

 Immunophenotyping (HIV)
 CD4 absolute counts
 Leukemia and lymphoma immunophenotyping
 Cell cycle and ploidy analysis of tumors
 Reticulocyte enumeration
 Flow cross-matching (organ transplantation)
 Stem cell enumeration
 Residual white blood cell detection
 (QC platelet, red blood cells)
APPLICATION

Research Laboratories
 Immune function studies
 Hematopoietic stem cells
 Multi-drug resistance studies (cancer)
 Kinetics studies (cell function) Platelet analysis
(coronary disease)
 Environmental sample analysis
 Flow and FISH
APPLICATION

Immunophenotyping
 Is the termed used in the identification of cells by
labeling with monoclonal antibodies identified as
cluster designations (CD)

 CDs is a group of antibodies that all recognize the

same antigen
Immunophenotyping

 Is performed by labeling the cells with red, green,

and / or orange labeled monoclonal antibody.

 The cells are then interrogated in the Flow Cytometry,

and the number, percentage of positive cells are
recorded.
CD Markers used for Primary Diagnosis

T-Cell Markers Myeloid Markers

CD 2 CD11b
CD3 CD11c
CD4 CD13
CD5 CD14
CD6 CD33
CD7
CD Markers used for Primary Diagnosis

B-Cell Markers Miscellaneous Marker

CD10 sIgG HAL-DR
CD19 sIgM CD34
CD20 Kappa CD45
CD22 Lambda TdT
CD23 CIgM
CD25 CCd3
TYPES OF MEASUREMENTS

 Intrinsic parameters:
Cell size & Granularity.

 Extrinsic parameters:
Surface Antigens

 Single or multiple surface membrane antigens are

detected with fluorescinated monoclonal antibodies
examples, CD45, CD4, CD8, etc).
TYPES OF MEASUREMENTS

Multi-parametric FCM
 In the multi-parameter approach more than one
feature of the same cell is measured, such as three
color surface and cytoplasmic staining in conjunction
with the cellular DNA content
leukemia and lymphoma.
Distinction of myeloid leukemia from
lymphatic can be precisely determined by
the analysis of surface and cytoplasmic
antigens.