Optical Light Scatter
and
Flow Cytometry
CHAPTER 37
MLAB 1415 HEMATOLOGY
JOANNA ELLIS, MLS(ASCP)
Objectives
 Describe the cell anlaysis principles used in flow cytometry.
 Define hydrodynamic focusing
 List and describe the two types of light scatter.
 Identify applications of flow cytometry in the clinical laboratory.
 Define Cluster of Differentiation (CD Marker).
 Define fluorochrome and describe its use in flow cytometry.
 Define immunophenotyping.
Purpose of
Flow
Cytometry:
To detect and
measure
multiple
properties of
particles or
cells for
identification
and
quantitation.
Image from Wikipedia
Introducing the particle or cell into the Flow Cell
Flow Cell
 Flow cytometry is performed on
particles in suspension such as
cells or nuclei.
 The inner column of the flow cell
consists of the liquid sample.
 The outer column of sheath fluid
controls the diameter of the
sample column so that it narrows
and isolates single particles
(hydrodynamic focusing).
 The single particles pass through
a laser beam.
 The light scatter is detected by a
photodetector.
Image from SonyInsider
Light Scatter
 The light from the laser hits the single particle and
scatters in different directions.
Forward
Scatter
LASER
Side Scatter
Types of Light Scatter
 Side light scatter: light scattered at a 90◦ angle
from the particle defines internal complexity
and granularity of the particle.
 Neutrophils
and eosinophils produce a great deal of
side scatter due to their cytoplasmic granules.
 Forward scatter: light that continues in the
forward direction relates the particle size.
 Large
cells such as monocytes and neutrophils
produce more forward scatter than nRBCs, and
normal lymphs.
Forward Scatter
Light Scatter Scatterplot
Side Scatter
Image from Peripheral Blood Smear Evaluation
Fluorochromes
 Fluorochromes are fluorescent compounds that can be
bound by antibodies to the particles in the sample.

Examples: FITC, PE, Propidium iodide, Acridine orange
 Fluorochromes are used in:
 Immunophenotyping
 Cell differentiation and enumeration
 Reticulocyte counting
 DNA analysis
Detection of Fluorochromes
1. Laser beam excites
fluorochrome attached
to particle at one
wavelength
Cell
Photomultiplier
Tube
2. Fluorochrome 3. Light is directed to the
emits light at
photomultiplier tube that
different
detects and quanitifies the
wavelength
light.
C
P
U
4. Computer
software analyzes
data and generates
scatterplot
Immunophenotyping uses Antibodies and Antigens
 Immunophenotyping is the identification of
antigens using detection antibodies.
 Monoclonal antibodies are manufactured using
myeloma cells to bind with a single portion of an
antigen (foreign particle/molecule that illicits an
immune response).
 Polyclonal antibodies are made by injecting
antigen into animals and are directed against
multiple portions of an antigen.
 Most immunophenotyping studies detect cell surface
antigens.
Commercial Antibodies are grouped in
Clusters of Differentiation (CD Markers)
• CD designations or CD Markers: group of antibodies
that recognize the same antigen.

Example: Commercially available Leu-4 and OKT3 are both
designated CD3
• CD marker antibodies labeled with fluorochomes
have been developed to bind with antigens of
identifiable cells. (table 37-3, page 840 in text)
• CD designations are cell markers:

Example: CD4+ cells are helper T-cells, CD8+ cells are
Cytotoxic T-cells
Immunophenotyping with Flow Cytometry
 The manufactured antibody (CD marker) is
attached to a fluorochrome such as FITC and
then added to the sample.
 If the cell has the antigen that the antibody (Ab)
specifically binds (target molecule), the
antibody and fluorochrome attach to the cell.
Fluorochrome
Antibody
Antigen
(target
molecule)
Cell
Immunophenotyping with Flow Cytometry
 When the cell/antibody/fluorochrome
complex passes through the laser beam,
the fluorochrome is excited and fluoresces
at a measureable wavelength detected by
the photomultiplier tube in the flow
cytometer.
 Computer software connected with the
flow cytometer generate a histogram that
visually represent the cells present.
 NO TARGET MOLECULE
FLUORESCENCE
Fluorochrome
Antibody
Antigen
(target
molecule)
NO
Cell
Clinical Applications of Flow Cytometry
 Blood cell enumeration and classification
 Differentials are based on light scatter as well as fluorescent staining in some
analyzers.
 Diagnosis and classification leukemia
 Immunophenotyping of CD markers specific to certain cell populations can
identify the type of leukemia.
 Diagnosis and monitoring of HIV progression
 Immunophenotyping can determine cell deficiencies. The HIV virus infects and
destroys CD4+ cells. Immunophenotyping with Flow Cytometry can determine
how many of these cells are in circulation.
 Reticulocyte enumeration
 Fluorochromes that stain RNA directly are used to more accurately (than manual
method) count immature erythrocytes.
 DNA analysis
 Certain fluorochromes attach to DNA directly. The amount of DNA is measured
to determine if there are numerical chromosomal abnormalities
References
 "Flow Cytometry." Wikipedia. Wikimedia Commons. Web. 17 Aug.
2010. <http://www.wikipedia.org/>. image
 McKenzie, Shirlyn B., and J. Lynne. Williams. "Chapter 37." Clinical
Laboratory Hematology. Boston: Pearson, 2010. Print.
 McManus, Christopher. "Sony Acquires ICyt And Officially Enters
Flow Cytometry Business." Sony Insider. 12 Feb. 2010. Web. 17
Aug. 2010. <http://www.sonyinsider.com/2010/02/12/sonyacquires-icyt-and-officially-enters-flow-cytometry-business/>.
Image.
 Riley, Roger, G. W. James, Sandra Sommer, and Mary Jo Martin.
"Peripheral Blood Smear Evaluation." Virginia Commonwealth
University Department of Pathology. Medical College of Virginia,
18 Sept. 1999. Web. 17 Aug. 2010.
<http://www.pathology.vcu.edu/education/PathLab/pages/hemato
path/pbs.html>. Image.